Compositions and methods for detecting and modulating fibronectin-integrin interactions and signaling - Patents.com

Abstract

Provided are antibodies comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 14, 16, or 18, or an amino acid sequence that is about 95% identical thereto, and paratope-containing fragments thereof. Also provided are V fragments comprising, consisting essentially of, or consisting of SEQ ID NO:4. H Segment, SEQ ID NO: 16, comprising, consisting essentially of, or consisting of L The present invention relates to nucleic acids encoding the FN conformational states, or combinations thereof; methods for using the same to detect and / or target FN conformational states in a sample; methods for treating diseases and / or disorders and / or ameliorating at least one symptom resulting from a disease or disorder associated with abnormal expression of force-induced conformational states of FN in a subject; and methods for screening for compounds having selective binding activity for FN conformational states.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] The subject matter of this disclosure claims the benefit of U.S. Provisional Patent Application No. 63 / 213,492, filed June 22, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] [Reference to electronically submitted sequence listing] The contents of the electronically submitted Sequence Listing in an ASCII text file submitted with this application (Name: 3062_161_PCT_ST25.txt; Size: 29 KB; and Creation Date: June 22, 2022) are hereby incorporated by reference in their entirety.

[0003] [Technical field] The presently disclosed subject matter relates to compositions comprising modified antibodies and fragments thereof, and methods of using the same, for detecting and modulating fibronectin-integrin interactions and signaling. In particular, the presently disclosed subject matter relates to compositions and methods useful for targeting mechanically exposed cryptic sites within the integrin binding domain of fibronectin. Summary of the Invention

[0004] This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of those embodiments. This summary is merely illustrative of many different embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such embodiments may typically exist with or without the mentioned feature; likewise, the feature may be applicable to other embodiments of the presently disclosed subject matter, whether or not it is listed in this summary. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.

[0005] In some embodiments, the presently disclosed subject matter relates to providing an isolated and purified antibody comprising, consisting essentially of, or consisting of SEQ ID NO:2, 4, 6, 14, 16, or 18, or a fragment thereof, or an antibody having an amino acid sequence that is about 95% identical to one of SEQ ID NO:2, 4, 6, 14, 16, or 18. In some embodiments, the antibody or fragment thereof is humanized.

[0006] In some embodiments, the isolated and purified antibody or fragment thereof comprises a heavy chain CDR1 comprising the amino acid sequence SYAMS (SEQ ID NO:8), a heavy chain CDR2 comprising the amino acid sequence DIYDGGGTNYADSVKG (SEQ ID NO:10), a heavy chain CDR3 comprising the amino acid sequence TADNFDY (SEQ ID NO:12), a light chain CDR1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO:20), a light chain CDR2 comprising the amino acid sequence AASTLQS (SEQ ID NO:22), and a light chain CDR3 comprising the amino acid sequence QQANSAPTT (SEQ ID NO:24); and / or the isolated and purified antibody comprises a heavy chain framework region 1 comprising EVQLLESGGGLVQPGGSLRLSCAAS (SEQ ID NO:40), a light chain CDR2 comprising the amino acid sequence WVRQAPGKGLEWV (SEQ ID NO:41), a light chain CDR3 comprising the amino acid sequence WVRQAPGKGLEWV (SEQ ID NO:42), and a light chain CDR3 comprising the amino acid sequence WVRQAPGKGLEWV (SEQ ID NO:43). and / or the isolated and purified antibody further comprises a heavy chain framework region 1 comprising DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 44), a light chain framework region 2 comprising WYQQKPGKAPKLLIY (SEQ ID NO: 45), a light chain framework region 3 comprising GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 46), and a light chain framework region 4 comprising FGQGTKVEIK (SEQ ID NO: 47).

[0007] In some embodiments, the isolated and purified antibody, or fragment or homolog thereof, comprises a modification at its N-terminus, its C-terminus, or both. In some embodiments, the modification comprises the addition of a peptide tag, a SARAH domain, or a combination thereof. In some embodiments, the tag comprises a His tag (e.g., HHHHHH; SEQ ID NO:35), a myc tag (e.g., EQKLISEEDL; SEQ ID NO:33), a VSV tag (e.g., YTDIEMNRLGK; SEQ ID NO:34), an HA tag (e.g., YPYDVPDYA; SEQ ID NO:36), a SortaseA tag (e.g., LPTEGG (SEQ ID NO:37) and / or LPXTG; SEQ ID NO:48), a PelB sequence (e.g., MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO:38) or MKYLLPTAEAGLLLLLAAPQIA (SEQ ID NO:49)), or any combination of one or more thereof. In some embodiments, the SARAH domain comprises a sequence selected from the group consisting of SEQ ID NOs:28-32.

[0008] The presently disclosed subject matter also provides, in some embodiments, isolated and purified nucleic acid sequences encoding the antibodies and fragments disclosed herein.

[0009] The subject matter of the present disclosure also provides, in some embodiments, a method for targeting the conformational state of fibronectin (FN) in a sample, optionally a biological sample isolated from a subject or present in a subject.In some embodiments, the method comprises contacting the sample with a composition having selective binding activity for the conformational state of FN, including FnIII9-4G-10 (4G), thereby targeting the conformational state.In some embodiments, the sample comprises or is suspected to comprise tissue undergoing tissue repair, diseased tissue, tissue suffering from a disorder, or any combination thereof.

[0010] The subject matter of the present disclosure also provides, in some embodiments, a method for detecting the conformational state of fibronectin (FN) in a sample. In some embodiments, the method includes contacting the sample with a composition having selective binding activity for a conformational state of FN, including FnIII9-4G-10 (4G); and detecting binding of the composition, thereby detecting the conformational state of FN. In some embodiments, the sample includes or is suspected to include tissue undergoing tissue repair, diseased tissue, tissue suffering from a disorder, or a combination thereof. In some embodiments, the sample includes or is suspected to include a pathological extracellular matrix (ECM). In some embodiments, the sample includes or is suspected to include a fibrotic ECM. In some embodiments, detecting binding of the composition includes detecting a binding ratio of the composition to FN. In some embodiments, detecting binding of the composition includes distinguishing between normal and diseased tissue. In some embodiments, detecting binding of the composition includes determining the severity of fibrosis in the sample. In some embodiments, detecting binding of the composition comprises detecting a transient force-induced conformational change in FN. In some embodiments, detecting binding of the composition comprises extracting structural information of the ECM in the sample. In some embodiments, extracting structural information of the ECM in the sample comprises delineating regions of high ECM strain. In some embodiments, high ECM strain is associated with enhanced αv integrin binding properties.

[0011] In some embodiments, the method further comprises determining the type of treatment to administer to the subject based on detecting binding of the composition.

[0012] The subject matter of the present disclosure also provides, in some embodiments, a method for treating a disease and / or disorder in a subject. In some embodiments, the method comprises administering to a subject in need of treatment a therapeutically effective amount of a composition having selective binding activity to a conformational state of FN, including FnIII9-4G-10 (4G), thereby achieving treatment. In some embodiments, the disease and / or disorder has a characteristic selected from the group consisting of tissue undergoing tissue repair, diseased tissue, tissue suffering from a disorder, and any combination thereof. In some embodiments, the characteristic is a pathological extracellular matrix (ECM). In some embodiments, the characteristic is a fibrotic ECM.

[0013] In some embodiments of the disclosed method, the composition having selective binding activity for the conformational state of FN, including FnIII9-4G-10 (4G), is an isolated and purified antibody or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 14, 16, or 18, or an antibody or fragment thereof having about 95% identity to the sequence of SEQ ID NO: 2, 4, 6, 14, 16, or 18. In some embodiments, the amino acid sequence comprises at least one modification selected from the group consisting of amino acid deletion, amino acid addition, amino acid substitution, and combinations thereof. In some embodiments, the antibody or fragment thereof is humanized.

[0014] The subject matter of the present disclosure also provides, in some embodiments, a method for screening for an antibody and / or its fragment and / or derivative having selective binding activity for a conformational state of FN comprising FnIII9-4G-10 (4G). In some embodiments, the method includes providing a sample comprising a conformational state of FN comprising FnIII9-4G-10 (4G); contacting the sample with a candidate antibody and / or its fragment and / or derivative; and detecting binding of the candidate antibody and / or its fragment and / or derivative to the sample. In some embodiments, the candidate antibody and / or its fragment and / or derivative is a member of a library of antibodies and / or its fragments and / or derivatives. In some embodiments, the candidate antibody and / or its fragment and / or derivative is an intact antibody. In some embodiments, the conformational state of FN is a force-induced conformational change in Fn.

[0015] The presently disclosed subject matter also provides, in some embodiments, antibodies and / or fragments and / or derivatives thereof identified by the methods of the present disclosure.

[0016] The presently disclosed subject matter also provides, in some embodiments, a method for treating a disease and / or disorder in a subject comprising administering to a subject in need of treatment a therapeutically effective amount of a composition comprising an antibody or fragment thereof according to the presently disclosed subject matter, thereby achieving treatment.

[0017] The presently disclosed subject matter also provides, in some embodiments, a method for ameliorating at least one symptom of a disease or disorder associated with abnormal expression of a force-induced conformational state of FN, including FnIII9-4G-10 (4G), in a subject. In some embodiments, the method comprises administering to a subject in need of amelioration a therapeutically effective amount of a composition comprising an antibody or fragment thereof according to the presently disclosed subject matter, whereby at least one symptom of a disease or disorder associated with abnormal expression of a force-induced conformational state of FN, including FnIII9-4G-10 (4G), is ameliorated.

[0018] In some embodiments of the methods of treatment, the disease or disorder is associated with tissue undergoing tissue repair, diseased tissue, tissue suffering from a disorder, or any combination thereof. In some embodiments, the disease or disorder is associated with a pathological extracellular matrix (ECM). In some embodiments, the disease or disorder is associated with a fibrotic ECM.

[0019] Accordingly, it is an object of the presently disclosed subject matter to provide compositions and methods for detecting and modulating fibronectin-integrin interactions and signaling. This and other objects are achieved in whole or in part by the presently disclosed subject matter. Moreover, while the objects of the presently disclosed subject matter have been described above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art upon review of the following description, drawings, and examples. Additionally, various aspects and embodiments of the presently disclosed subject matter are described in further detail below. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 is a graph showing the results of an in vitro ELISA binding assay to determine the binding potency of H5 antibody and its derivatives to diseased (diseased) and healthy (normal) forms of fibronectin (Fn) at various dosages. The data represent a combination of two independent experiments: the first comparing H5-scFv produced in E. coli (eH5) versus N. benthamiana (nicoH5), and the second comparing H5-scFv (H5(scFv)) versus H5-IgG1 (H5(IgG))). eH5 vs. Fn (affected): open rectangle with thick black line; eH5 vs. Fn (normal): open circle with thin black line; nicoH5 vs. Fn (affected): filled rectangle with large dashed line; nicoH5 vs. Fn (normal): filled circle with small dashed line; H5(scFv) vs. Fn (affected): open triangle with thick black line; H5(scFv) vs. Fn (normal): inverted open triangle with dashed line; H5(IgG) vs. Fn (affected): filled triangle with thin dashed line; H5(IgG) vs. Fn (normal): inverted filled triangle with thick dashed line.

[0021] [Brief explanation of sequence listing] SEQ ID NOs:1 and 2 are the nucleic acid and amino acid sequences, respectively, of the heavy chain of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:3 and 4 are the nucleic acid and amino acid sequences, respectively, of the heavy chain variable region of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:5 and 6 are the nucleic acid and amino acid sequences, respectively, of the heavy chain constant region of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:7 and 8 are the nucleic acid and amino acid sequences, respectively, of the heavy chain CDR1 of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:9 and 10 are the nucleic acid and amino acid sequences, respectively, of the heavy chain CDR2 of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:11 and 12 are the nucleic acid and amino acid sequences, respectively, of the heavy chain CDR3 of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:13 and 14 are the nucleic acid and amino acid sequences, respectively, of the light chain of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:15 and 16 are the nucleic acid and amino acid sequences, respectively, of the light chain variable region of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:17 and 18 are the nucleic acid and amino acid sequences, respectively, of the light chain constant region of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:19 and 20 are the nucleic acid and amino acid sequences, respectively, of the light chain CDR1 of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:21 and 22 are the nucleic acid and amino acid sequences, respectively, of the light chain CDR2 of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:23 and 24 are the nucleic acid and amino acid sequences, respectively, of the light chain CDR3 of an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NO:25 is the amino acid sequence of the pentapeptide motif PHSRN found in the ninth type III repeat of fibronectin. SEQ ID NOs:26 and 27 are the amino acid sequences of an exemplary tetrapeptide linker consisting of four glycine residues and an exemplary pentapeptide linker consisting of a serine residue followed by four glycine residues. Note that to create a linker peptide, one, two, three or more copies of SEQ ID NO:26 can be combined (i.e., concatemerized), one, two, three or more copies of SEQ ID NO:27 can be combined (i.e., concatemerized), or one, two, three or more copies of SEQ ID NO:26 can be combined with one, two, three or more copies of SEQ ID NO:27. SEQ ID NOs:28-32 are amino acid sequences of exemplary SARAH domains that can be added to the N-terminus, C-terminus, or both, of an antibody or fragment thereof of the subject matter of the present disclosure. SEQ ID NOs:33-38 are amino acid sequences of exemplary tags that can be added to the N-terminus, C-terminus, or both of the subject antibodies or fragments thereof of the present disclosure. SEQ ID NO:31 is an exemplary myc tag, SEQ ID NO:32 is an exemplary VSV tag, SEQ ID NO:33 is an exemplary His tag, SEQ ID NO:34 is an exemplary HA tag, SEQ ID NO:35 is an exemplary Sortase A tag, and SEQ ID NO:36 is an exemplary PelB tag. SEQ ID NO:39 is an exemplary linker sequence. SEQ ID NOs:40-43 are exemplary heavy chain framework regions 1-4, respectively, that can be used in an exemplary H5-IgG1 antibody of the subject matter of this disclosure. SEQ ID NOs:44-47 are exemplary light chain framework regions 1-4, respectively, that can be used in an exemplary H5-IgG1 antibody of the presently disclosed subject matter. SEQ ID NOs:48 and 49 are additional amino acid sequences of various exemplary tags, including a SortaseA tag (e.g., LPXTG; SEQ ID NO:48) and a PelB sequence (e.g., MKYLLPTAEAGLLLLLAAPQIA; SEQ ID NO:49). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Fibronectin (Fn) is an extracellular matrix protein that orchestrates complex cell adhesion and signaling through cell surface integrin receptors during tissue development, tissue repair, and diseases such as fibrosis. Fn is susceptible to mechanical forces at its tandem type III repeats, resulting in significant elongation of the molecule. Thus, it has long been hypothesized that cell- and tissue-derived forces can activate an "integrin switch" within the critical integrin-binding type III repeats 9 and 10, conferring differential integrin binding specificity leading to differential cellular responses. However, no direct evidence exists to prove this hypothesis or demonstrate the physiological existence of the switch. Provided in accordance with the subject matter of the present disclosure is direct experimental evidence of the Fn integrin switch, using antibodies engineered to detect transient force-induced conformational changes both in vitro and ex vivo, providing an opportunity to detect and target early molecular signatures of cell contractile forces in tissue repair and disease.

[0023] The extracellular matrix (ECM) forms a complex microenvironment of structural elements that surrounds cells in vivo. Cells interact with and receive guidance from the ECM through cellular structures known as focal adhesions, which are large protein complexes composed of transmembrane receptors (integrins) and intracellular adaptor proteins that mechanically link the cell's cytoskeleton to fibrillar ECM proteins such as fibronectin (Fn). The interactions between proteins within focal adhesions are dynamic; mechanical forces play a key role for focal adhesion maturation and development, as well as for force-sensitive cell signaling via mechanosensitive proteins. Recent studies have shown that the conformation of both intracellular focal adhesion components (e.g., vinculin, integrins; see Zhu et al., 2008; Grashoff et al., 2010; Carisey et al., 2013) and extracellular components (e.g., Fn; see Smith et al., 2007; Lemmon et al., 2011; Cao et al., 2012) can be altered by forces transmitted to and from the ECM. In the latter case, previous studies have demonstrated that Fn within the ECM exhibits characteristic, yet undetermined, altered structural states in response to cellular forces both in vitro and in vivo (see Chandler et al., 2011 ; Cao et al., 2012 ).

[0024] Fn contains three types of tandem repeat units, each containing two antiparallel β-sheets. While type I and type II repeats are structurally stabilized by disulfide bonds, type III repeats are stabilized only by hydrogen bonds and van der Waals forces and are susceptible to unfolding by physiologically relevant forces (Krammer et al., 1999; Craig et al., 200; Craig et al., 2004; Li et al., 2005; Gee et al., 2008). These findings, combined with the active role of type III repeats 9 and 10 of Fn (FnIII9-10) in mediating integrin-specific interactions, have inspired the theory that mechanical forces may actuate a "switch" in the integrin-binding profile of Fn (Krammer et al., 1999). Fn-integrin interactions are known to drive important cellular behaviors and are primarily mediated through the canonical and promiscuous integrin binding sequence Arg-Gly-Asp (RGD) within type III repeat 10 (Ruoslahti and Pierschbacher, 1987). A subset of integrins, including integrin α5β1, further depends on the sequence motif PHSRN (SEQ ID NO:25) within the adjacent type III repeat 9 (Aota et al., 1994; Mardon and Grant, 1994; Mould, 1997; Garcia et al., 2002). Integrin specificity for Fn can be modulated in vitro by altering the structural stability of the integrin-binding domain (i.e., the 9th and 10th type III repeats) by directed mutations (van der Walle et al., 2002), resulting in the modulation of developmentally and pathologically relevant cell differentiation pathways (Martino et al., 2009; Brown et al., 2011) and, importantly, cellular responses to microenvironmental dynamics (e.g., stiffening; Markowski et al., 2012). Despite these findings, the integrin switch theory and its potential relevance to biological processes in vivo remained undetermined prior to the subject matter of the present disclosure.

[0025] Reports suggest that the relative separation distance between the "synergy" PHSRN sequence (SEQ ID NO:25) in the 9th FnIII repeat and the RGD site in the 10th FnIII repeat is important for the binding and activation of integrins α5β1 (Martino et al., 2009) and α3β1 (Brown et al., 2015), with the optimal PHSRN (SEQ ID NO:25)-RGD distance being 3.7 nm for high affinity integrin α5β1 binding (Craig et al., 2008). Furthermore, recent findings have demonstrated that elongation of Fn fibrils reduces cell spreading and adhesion (Hubbard et al., 2016).

[0026] Development of conformation-specific antibodies by phage display is well established, as the work of Lefkowitz and coworkers used phage display to isolate conformation-specific Fabs against activated β-arrestin-1 (Shukla et al., 2013). However, particular challenges in the development of the conformation-specific antibodies of this disclosure were that (1) conformational changes in the integrin-binding domain were due to the application of force, (2) application of force to the Fn fiber resulted in multiple conformational changes along the length of the 440 kDa protein; and (3) the conformational changes were highly unstable due to the ability of Fn type III repeats to refold in the absence of force. Here, we utilized structures predicted from steering molecular dynamics simulations combined with molecular engineering to generate distorted integrin-binding domain mimics to perform phage display and discover parental H5 clones. The two model Fn fragments may differ not only in the separation between RGD and PHSRN (SEQ ID NO:25), but also in their relative conformational stability. FnIII9*10 is stabilized by a Leu1408Pro mutation between FnIII9 and FnIII1018, whereas FnIII-4G-10 is separated by a four-glycine linker between the two domains.

[0027] An exemplary use of the modified H5-IgG1 antibodies and fragments thereof of the presently disclosed subject matter is to probe pathological ECM, in some embodiments fibrotic ECM, containing highly contractile myofibroblasts. Recent reports suggest that αv integrins on myofibroblasts are involved in fibrogenesis in a wide range of fibrotic diseases, and that pharmacological blockade of αv integrins ameliorates liver and lung fibrosis (Henderson et al., 2013). The presently disclosed subject matter is the use of the modified H5-IgG1 antibodies disclosed herein in the context of idiopathic pulmonary fibrosis (IPF), a fatal form of progressive pulmonary fibrosis in humans. The lungs of IPF patients are mechanically and biochemically heterogeneous, with areas of soft normal lung tissue and stiffer areas of mature fibrosis. The modified H5-IgG1 antibodies and fragments thereof of the presently disclosed subject matter can be used to delineate areas of high ECM distortion that also exhibit enhanced αv integrin binding properties due to the conformation of the integrin binding domain, likely indicative of ongoing fibrosis.

[0028] The ability of modified H5-IgG1 antibodies to extract structural information from the ECM can also be demonstrated in a model of retinal angiogenesis, a process in which new blood vessels are formed by endothelial sprouting (Patan, 2004). In mouse tissue sections, areas of high modified H5-IgG1:Fn ratios can be found in the extensions of endothelial tip cells, suggesting that Fn is unfolded in these regions. Fn is known to be a mediator of retinal angiogenesis, as astrocytes deposit fibronectin prior to differentiation of angioblasts into endothelial cells (Jiang et al., 1994). The results described here suggest that forces from endothelial tip cells unfold Fn, exhibiting αvβ3 binding properties within the provisional matrix that may affect the formation of new blood vessels.

[0029] In some embodiments, described herein is a conformation-sensitive single chain antibody based on the modified H5-IgG1 of the subject of the present disclosure against the integrin-binding FnIII9-10 domain of Fn, demonstrating its mechanosensitive binding to Fn in multiple model systems in vitro and ex vivo. Without wishing to be bound by any particular theory of action, these force-sensitive conformational changes observed in the integrin-binding domain of Fn are seen as evidence of a long-theorized Fn "integrin-switch" that likely controls integrin-specific cellular responses in vivo and in engineered settings based on controlling the presentation and accessibility of Fn epitopes. It is also provided herein that an exemplary modified H5-IgG1 antibody specifically detects force-induced conformational changes in proteins. As tissue mechanics become increasingly implicated in the pathogenesis of fibrotic diseases, opportunities are emerging to explore targeting the mechanochemical features of ECM as a paradigm for tissue imaging and disease diagnosis.

[0030] I. Abbreviations and Definitions Certain abbreviations used in this disclosure and / or claims are summarized in Table 1. TIFF2024526154000002.tif126170

[0031] Headings are included herein for reference and to facilitate locating certain sections. These headings are not intended to limit the scope of the concepts described therein, and these concepts may have applicability in other sections throughout the entire specification.

[0032] In describing and claiming the subject matter of the present disclosure, the following terminology will be used in accordance with the definitions set forth below. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the subject matter of the present disclosure.

[0033] In describing the subject matter of this disclosure, it will be understood that a number of techniques and steps are disclosed, each of which has separate advantages and each of which can also be used in combination with one or more, or in some cases all, of the other disclosed techniques.

[0034] Thus, for the sake of clarity, this specification refrains from unnecessarily repeating every possible combination of the individual steps, but the specification and claims should nevertheless be read with the understanding that such combinations are fully within the subject matter and scope of the present disclosure.

[0035] The term "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. "Comprising" is a term used in claim language to mean that the specified elements are required, but that other elements may still be added to form a structure within the scope of the claim.

[0036] As used herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When the phrase "consisting of" appears in a characterizing clause of a claim rather than immediately following a preamble, it limits only the elements recited in that clause; other elements are not excluded from the claim as a whole.

[0037] As used herein, the phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps and additionally those that do not materially affect the basic and novel characteristics of the claimed subject matter.

[0038] With respect to the terms "comprising," "consisting of," and "consisting essentially of," when one of these three terms is used herein, the claimed subject matter of this disclosure may include the use of either of the other two terms.

[0039] As used herein, the term "and / or" when used in the context of a list of entities refers to those entities either alone or in any combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, but also any and all combinations and subcombinations of A, B, C, and D.

[0040] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, unless expressly stated otherwise.

[0041] The term "about" as used herein means approximately, in the region of, roughly, or approximately. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify a numerical value above and below a variance of 10% of the stated value. In some embodiments, the term "about" means plus or minus 10% of the numerical value with which it is used. Thus, about 50% means a range of 45% to 55%. Numerical ranges described herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all numbers and fractions thereof are presumed to be modified by the term "about".

[0042] As used herein, the term "adjuvant" refers to a substance that elicits an enhanced immune response when used in combination with a particular antigen.

[0043] As used herein, the terms "administration" of a composition and / or "administering" a composition should be understood to mean providing a composition of the presently disclosed subject matter to a subject in need of treatment.

[0044] As used herein, "agent" is meant to include drugs, proteins, antibodies and / or fragments or derivatives thereof, that are contacted with a cell, tissue, or organ to elicit an effect.

[0045] The term "additional therapeutically active compound" or "additional therapeutic agent" as used in the context of the subject matter of the present disclosure refers to the use or administration of a compound for an additional therapeutic use for the particular injury, disease, or disorder being treated. Such compounds may include, for example, those that are used to treat unrelated diseases or disorders, or diseases or disorders that may not be responsive to the primary treatment for the injury, disease, or disorder being treated. Diseases and disorders that are being treated by additional therapeutically active agents include, for example, cancer, fibrosis, and the like. Additional compounds may also be used to treat symptoms associated with the injury, disease, or disorder, including, but not limited to, pain and inflammation.

[0046] As used herein, an "agonist" is a composition of matter that, when administered to a mammal, such as a human, enhances or prolongs a biological activity resulting from the level or presence of a target biologically active molecule of interest in the mammal.

[0047] An "antagonist" is a composition of matter that, when administered to a mammal, such as a human, inhibits a biological activity resulting from the level or presence of a biologically active molecule of interest in the mammal.

[0048] As used herein, "alleviating the symptoms of a disease or disorder" means reducing the severity of the symptoms or the frequency with which such symptoms are experienced by the patient, or both.

[0049] As used herein, "analogs" of a compound are, by way of example, compounds that are structurally similar to each other but are not necessarily isomers (eg, 5-fluorouracil is an analog of thymine).

[0050] As used herein, amino acids are represented by their full name, their corresponding three letter code, and / or their corresponding one letter code, as summarized in Table 2: TIFF2024526154000003.tif123170

[0051] The term "amino acid" as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. "Standard amino acid" means any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid residue" means any amino acid other than the standard amino acids, whether prepared synthetically or derived from a natural source. As used herein, "synthetic amino acid" also encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within peptides (e.g., antibodies, fragments, and derivatives of the presently disclosed subject matter), particularly those at the carboxy or amino termini, can be modified by methylation, amidation, acetylation, or substitution with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting its activity. In addition, disulfide bonds may or may not be present in the peptides of the presently disclosed subject matter.

[0052] The term "amino acid" is used interchangeably with "amino acid residue" and can refer to free amino acids and to amino acid residues of peptides. Whether the term refers to a free amino acid or a residue of a peptide will be clear from the context in which the term is used. Amino acids have the following general structure: TIFF2024526154000004.tif33170

[0053] Amino acids can be classified into seven groups based on the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxyl (OH) group, (3) side chains containing a sulfur atom, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, which is an imino acid with a side chain fused to an amino group.

[0054] The nomenclature used to describe the antibodies, fragments, and derivatives thereof of the presently disclosed subject matter follows the conventional practice of showing the amino group to the left and the carboxy group to the right of each amino acid residue. In formulae representing selected specific embodiments of the presently disclosed subject matter, the amino- and carboxy-terminal groups are not specifically shown, but are understood to be in the form they will assume at physiological pH values, unless otherwise indicated.

[0055] The term "basic" or "positively charged" amino acid, as used herein, refers to an amino acid in which the R group carries a net positive charge at pH 7.0, and includes, but is not limited to, the standard amino acids lysine, arginine, and histidine.

[0056] The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically or selectively binding to a particular epitope on an antigen. An antibody may be an intact immunoglobulin derived from natural or recombinant sources, or may be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. Antibodies in the presently disclosed subject matter may exist in various forms. The term "antibody" refers to polyclonal and monoclonal antibodies and derivatives thereof, including chimeric antibodies, synthetic antibodies, humanized antibodies, and human antibodies, and includes whole immunoglobulins or antibodies, or any functional fragment of an immunoglobulin molecule that binds to a target antigen and / or combinations thereof. Examples of such functional entities include intact antibody molecules, antibody fragments (Fs), and antibody fragments (Fs). v , single chain F v , complementarity determining region (CDR), V L (light chain variable region), V H (heavy chain variable region), Fab, F(ab')2, and any combination thereof, or any other functional portion of an immunoglobulin peptide capable of binding to a target antigen.

[0057] Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bonds in the hinge region, itself splitting the V H -C H1The F(ab')2 dimer is a dimer of Fab, a light chain bound to a Fab1 light chain. F(ab')2 can be reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby converting the F(ab')2 dimer into a Fab1 monomer. A Fab1 monomer is essentially a Fab with part of the hinge region (see Paul, 1993). Although various antibody fragments are defined in terms of the digestion of an intact antibody, one skilled in the art will appreciate that such fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody as used herein also includes antibody fragments produced by the modification of a whole antibody or those synthesized de novo using recombinant DNA methodology.

[0058] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in all antibody molecules.

[0059] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in all antibody molecules.

[0060] The term "single-chain antibody" refers to an antibody in which the genetic information encoding a functional fragment of an antibody is located in a single contiguous length of DNA. For a detailed description of single-chain antibodies, see Bird et al., 1988; Huston et al., 1988.

[0061] The term "humanized" refers to an antibody whose constant region has at least about 80% or more homology to human immunoglobulins. In addition, some of the amino acid residues of the non-human variable region, such as mouse, can be modified to contain amino acid residues of human origin. Humanized antibodies are also called "reshaped" antibodies. Engineering of the complementarity determining regions (CDRs) is a method to achieve humanized antibodies. See, for example, Jones et al., 1986; Riechmann et al., 1988, both of which are incorporated herein by reference. For a review article on humanized antibodies, see Winter and Milstein, 1991, which are incorporated herein by reference. See also U.S. Patent Nos. 4,816,567; 5,482,856; 6,479,284; 6,677,436; 7,060,808; 7,906,625; 8,398,980; 8,436,150; 8,796,439; and 10,253,111; and U.S. Patent Application Publication Nos. 2003 / 0017534, 2018 / 0298087, 2018 / 0312588, 2018 / 0346564, and 2019 / 0151448, each of which is incorporated by reference in its entirety.

[0062] The term "synthetic antibody" as used herein refers to an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence which specifies the antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0063] The term "antigen" as used herein is defined as a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunocompetent cells, or both. Antigens may be derived from organisms, protein / antigen subunits, killed or inactivated whole cells or lysates.

[0064] The term "antimicrobial agent" as used herein refers to any natural, synthetic, or semi-synthetic compound, or composition or mixture thereof, that is safe for human or animal use and is effective in killing or substantially inhibiting the growth of microorganisms when practiced in the methods of the presently disclosed subject matter. As used herein, "antimicrobial agent" includes antibacterial agents, antifungal agents, and antiviral agents.

[0065] As used herein, the term "biocompatible" refers to a material that does not elicit a substantial adverse response in the host.

[0066] As used herein, the term "biologically active fragment" or "bioactive fragment" of a polypeptide includes natural or synthetic portions of a full-length protein that are capable of specifically or selectively binding to its natural ligand or performing a function of the protein.

[0067] The term "biological sample" as used herein refers to a sample obtained from a subject, including, but not limited to, skin, hair, tissue, blood, plasma, cells, sweat and urine.

[0068] As used herein, the terms "cell" and "cell line" can be used interchangeably. All of these terms include their progeny, any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.

[0069] As used herein, the terms "cell culture" and "culture" refer to the maintenance of cells in an artificial in vitro environment. However, it is understood that the term "cell culture" is a general term and can be used to encompass the culture of not only individual cells, but also tissues, organs, organ systems, or whole organisms, and that the terms "tissue culture," "organ culture," "organic system culture," or "organotypic culture" may sometimes be used interchangeably with the term "cell culture."

[0070] The terms "cell culture medium," "culture medium" (in each case the plural is "media"), and "media formulation" refer to a nutrient solution for culturing cells and can be used interchangeably.

[0071] The "coding region" of a gene includes nucleotide residues of the coding strand of the gene and nucleotides of the non-coding strand of the gene that are homologous or complementary, respectively, to the coding region of an mRNA molecule generated by transcription of the gene.

[0072] As used herein, "compound" refers to any type of substance or agent generally considered a drug, or a candidate for use as a drug, combinations, and mixtures of the above, as well as the polypeptides and antibodies of the presently disclosed subject matter.

[0073] As used herein, the term "conservative amino acid substitution" is defined herein as a group of amino acid exchanges within the five groups summarized in Table 3. TIFF2024526154000005.tif81170

[0074] A "control" cell, tissue, sample, or subject is a cell, tissue, sample, or subject of the same type as a test cell, tissue, sample, or subject. A control can, for example, be tested at exactly or about the same time that a test cell, tissue, sample, or subject is tested. A control can also, for example, be tested at a time separated from the time that a test cell, tissue, sample, or subject is tested, and the results of testing the control can be recorded so that the recorded results can be compared to the results obtained by testing the test cell, tissue, sample, or subject. A control can also be obtained from a separate or similar source than the test group or test subject, where the test sample is obtained from a subject suspected of having the disease or disorder for which the test is being performed.

[0075] A "test" cell, tissue, sample, or subject is one that is being examined or treated.

[0076] A tissue "normally contains" a cell when one or more cells are present in the tissue in an animal not afflicted with a disease or disorder.

[0077] As used herein, a "derivative" of a compound refers to a compound that can be produced from another compound of similar structure in one or more steps, such as replacement of an H with an alkyl, acyl, or amino group.

[0078] Use of the word "detect" and grammatical variations thereof is meant to refer to the measurement of a species without quantification, while use of the words "determine" or "measure" and grammatical variations thereof is meant to refer to the measurement of a species with quantification. The terms "detect" and "identify" are used interchangeably herein.

[0079] As used herein, a "detectable marker" or "reporter molecule" is an atom or molecule that allows for the specific detection of a compound containing the marker in the presence of similar compounds that do not contain the marker. Detectable markers or reporter molecules include, for example, radioisotopes, antigenic determinants, enzymes, nucleic acids available for hybridization, chromophores, fluorophores, chemiluminescent molecules, electrochemically detectable molecules, and molecules that produce altered fluorescence polarization or altered light scattering.

[0080] A "disease" is a condition in the health of an animal in which the animal is unable to maintain homeostasis, and if the disease is not ameliorated, the animal's health will continue to deteriorate.

[0081] In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the animal's health status is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further deterioration in the animal's health.

[0082] As used herein, the term "domain" refers to a portion of a molecule or structure that shares common physicochemical characteristics, such as, but not limited to, hydrophobic, polar, globular, and helical domains, or properties such as ligand binding, signal transduction, cell penetration, etc. Examples of binding domains include, but are not limited to, DNA binding domains, ATP binding domains, and the integrin binding domain of fibronectin.

[0083] As used herein, "effective amount" or "therapeutically effective amount" means an amount sufficient to produce a selected effect, such as alleviating the symptoms of a disease or disorder. In the situation where a compound is administered in the form of a combination, such as multiple compounds, the amount of each compound when administered in combination with another compound may be different than when that compound is administered alone. Thus, the effective amount of a combination of compounds refers collectively to the combination as a whole, and the actual amount of each compound may vary. The term "more effective" means that the selected effect is alleviated to a greater extent by one treatment compared to the second treatment being compared.

[0084] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis in biological processes of other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, may be referred to as encoding a protein or other product of that gene or cDNA.

[0085] An "enhancer" is a DNA regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.

[0086] The term "epitope" as used herein is defined as a small chemical group on an antigen molecule that can elicit and react with an antibody. An antigen can have one or more epitopes. Most antigens have many epitopes; that is, they are multivalent. In general, epitopes are about five amino acids or sugars in size. Those skilled in the art will understand that in general, the overall three-dimensional structure of the molecule, rather than the specific linear sequence, is the primary criterion for antigen specificity.

[0087] A "fragment" or "segment" is a portion of an amino acid sequence comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide. The terms "fragment" and "segment" are used interchangeably herein.

[0088] As used herein, the term "fragment" as applied to a protein or peptide (e.g., an antibody of the presently disclosed subject matter or a fragment or derivative thereof) can typically be at least about 3-15 amino acids in length, at least about 15-25 amino acids in length, at least about 25-50 amino acids in length, at least about 50-75 amino acids in length, at least about 75-100 amino acids in length, and more than 100 amino acids in length. In some embodiments, a fragment of an antibody of the presently disclosed subject matter comprises a paratope.

[0089] As used herein, the term "fragment" as applied to nucleic acids will usually be at least about 20 nucleotides in length, typically at least about 50 nucleotides, more typically from about 50 to about 100 nucleotides, and in some embodiments at least about 100 to about 200 nucleotides, in some embodiments at least about 200 nucleotides to about 300 nucleotides, and in some embodiments at least about 300 to about 350, in some embodiments at least about 350 nucleotides to about 500 nucleotides, and in some embodiments at least about 500 to about 600, in some embodiments at least about 600 nucleotides to about 620 nucleotides, and in some embodiments at least about 620 to about 650, and in most embodiments the nucleic acid fragment will be greater than about 650 nucleotides in length.

[0090] As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property by which it is characterized. For example, a functional enzyme is an enzyme that exhibits the characteristic catalytic activity by which it is characterized.

[0091] "Homology" as used herein refers to the similarity of subunit sequences between two polymer molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules or two RNA molecules, or between two polypeptide molecules. If both subunit positions of two molecules are occupied by the same monomeric subunit, e.g., if each position of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a linear function of the number of matching or homologous positions, e.g., if half of the positions of two compound sequences (e.g., 5 positions in a polymer 10 subunits long) are homologous, then the two sequences are 50% homologous, and if 90% of the positions, e.g., 9 out of 10, are matching or homologous, then the two sequences share 90% homology. As an example, the DNA sequences 3'-ATTGCC-5' and 3'-TATGGC-5' share 50% homology.

[0092] As used herein, "homology" is used synonymously with "identity."

[0093] The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, which can be accessed, for example, at the National Center for Biotechnology Information (NCBI) World Wide Web site. BLAST nucleotide searches can be performed with the NBLAST program (named "blastn" on the NCBI Web site) to obtain nucleotide sequences homologous to the nucleic acids described herein, using the following parameters: gap penalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1; expectation value=10.0; and word size=11. BLAST protein searches can be performed with the XBLAST program (called "blastn" on the NCBI website) or the NCBI "blastp" program using the following parameters to obtain amino acid sequences homologous to the protein molecules described herein: expectation score of 10.0, BLOSUM62 scoring matrix. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997. Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search that detects distant relationships between molecules (ibid.) and relationships between molecules that share common patterns. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0094] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, exact matches are typically counted.

[0095] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of binding between nucleic acids) are affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the associated conditions, the length of the hybrid formed, and the G:C ratio within the nucleic acids.

[0096] The term "ingredient" refers to any compound, regardless of chemical or biological origin, that may be used in cell culture media to maintain or promote cell growth, survival, or differentiation. The terms "component," "nutrient," "nutritional supplement," and "ingredient" may be used interchangeably and are all intended to refer to such compounds. Typical non-limiting ingredients used in cell culture media include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, and the like. Other ingredients that promote or maintain the culture of cells ex vivo may be selected by the skilled artisan depending on the particular needs.

[0097] The term "inhibit" as used herein means to suppress or block an activity or function so that it is lower compared to a control value. Inhibition can be through direct or indirect mechanisms. In some embodiments, activity is suppressed or blocked by at least 10% compared to a control value, in some embodiments by at least 25%, and in some embodiments by at least 50%.

[0098] The term "inhibitor" as used herein refers to any compound or agent, such as but not limited to the antibodies, fragments, and derivatives thereof, of the subject matter of the present disclosure, whose application results in the inhibition of a process or function of interest, including but not limited to expression, level, activity. Inhibition may be inferred if the activity or function of interest is decreased.

[0099] The term "protein inhibiting" as used herein refers to any method or technique that inhibits the synthesis, level, activity, or function of a protein, as well as methods that inhibit the induction or stimulation of the synthesis, level, activity, or function of a protein of interest. The term also refers to any metabolic or regulatory pathway that can modulate the synthesis, level, activity, or function of a protein of interest. The term includes binding and complex formation with other molecules. Thus, the term "protein inhibitor" refers to any agent or compound whose application results in the inhibition of a protein function or protein pathway function. However, the term does not imply that each and every one of these functions must be inhibited simultaneously. In some embodiments, the antibodies, fragments, and derivatives thereof of the presently disclosed subject matter are protein inhibitors.

[0100] As used herein, "injection or application" includes administration of the compositions of the presently disclosed subject matter by any number of routes and means, including, but not limited to, intravitreal, topical, oral, buccal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular (intraventricular), transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal, ocular, pulmonary, or rectal means.

[0101] "Injury" refers to any injury to the body caused by violence, accident, trauma, fracture, etc., as well as injury resulting from surgery.

[0102] As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that may be used to communicate the utility of the peptides (e.g., antibodies, fragments, and derivatives thereof) of the presently disclosed subject matter in the kits to provide relief from various diseases or disorders described herein. Optionally, or alternatively, the instructional material may describe one or more methods of alleviating a disease or disorder in a mammalian cell or tissue. The instructional material of the kits of the presently disclosed subject matter may, for example, be affixed to a container that contains the identified antibodies, fragments, and / or derivatives of the presently disclosed subject matter or may be shipped together with a container that contains the identified antibodies, fragments, and / or derivatives thereof. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the antibodies, fragments, and / or derivatives of the presently disclosed subject matter be used in conjunction by the recipient.

[0103] Used interchangeably herein are the terms 1) "isolate" and "select"; 2) "detect" and "identify."

[0104] The term "isolated," when used with respect to compositions and cells, refers to a particular composition or cell of interest, or a population of cells of interest, that is at least partially isolated from other cell types or other cellular material with which it naturally coexists in the tissue of origin. A composition or cell sample is "substantially pure" when it is at least 60%, or at least 75%, or at least 90%, and in some cases at least 99% free of material, compositions, or cells other than the composition or cell of interest. Purity can be measured by any suitable method, such as fluorescence activated cell sorting (FACS) or other assays that distinguish cell types. Exemplary isolation techniques for antibodies and fragments thereof are disclosed herein.

[0105] An "isolated nucleic acid" refers to a nucleic acid segment or fragment that is separated from sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment, e.g., from sequences that flank the fragment in the genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, e.g., RNA, DNA, or proteins that naturally accompany the nucleic acid in a cell. Thus, the term includes recombinant DNA that is incorporated into, for example, a vector, an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or exists as a separate molecule independent of other sequences (e.g., as a cDNA or genomic or cDNA fragment generated by PCR or restriction enzyme digestion). It also includes recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0106] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may contain introns.

[0107] As used herein, a "ligand" is a molecule that specifically or selectively binds to a target molecule. A ligand (e.g., an antibody) "specifically binds to," "is specifically immunoreactive with," "has selective binding activity with," "selectively binds to," or "is selectively immunoreactive with" a molecule if the ligand functions in a binding reaction that determines the presence of the molecule in a sample of heterologous molecules. Thus, under specified assay (e.g., immunoassay) conditions, the ligand preferentially binds to a particular molecule and does not bind to a significant degree to other molecules present in the sample. For example, an antibody specifically or selectively binds, under immunoassay conditions, to an antigen that bears the epitope to which the antibody was raised. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive with an antigen. See Harlow and Lane, 1988, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0108] A "receptor" is a molecule that specifically or selectively binds to a ligand.

[0109] As used herein, the term "bond" refers to a connection between two groups. The connection can be either covalent or non-covalent, including but not limited to ionic bonds, hydrogen bonds, and hydrophobic / hydrophilic interactions.

[0110] As used herein, the term "linker" refers to a molecule that joins two other molecules together, either covalently or non-covalently, for example through ionic or hydrogen bonds or van der Waals interactions.

[0111] The term "modulate" as used herein refers to changing the level of an activity, function, or process. The term "modulate" encompasses both inhibiting and stimulating an activity, function, or process. The term "modulate" is used interchangeably herein with the term "regulate."

[0112] The term "nucleic acid" typically refers to large polynucleotides. "Nucleic acid" refers to any nucleic acid, whether it is composed of deoxyribonucleosides or ribonucleosides, and whether it is composed of phosphodiester bonds or modified bonds, such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone bonds, and combinations of such bonds. The term "nucleic acid" also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0113] As used herein, the term "nucleic acid" includes RNA, as well as single- and double-stranded DNA and cDNA. Furthermore, the terms "nucleic acid", "DNA", "RNA" and similar terms also include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, so-called "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered to be within the scope of the subject matter of this disclosure. "Nucleic acid" also refers to any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester bonds or modified bonds, such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone bonds, and combinations of such bonds. The term "nucleic acid" specifically also includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil). Common notation is used herein to describe polynucleotide sequences: the left end of a single-stranded polynucleotide sequence is the 5' end; the left end of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nucleotides to the nascent RNA transcript is referred to as the transcription direction. The DNA strand with the same sequence as the mRNA is referred to as the "coding strand;" the sequence on the DNA strand that is 5' of a reference point on the DNA is referred to as the "upstream sequence;" the sequence on the DNA strand that is 3' of a reference point on the DNA is referred to as the "downstream sequence."

[0114] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may contain introns.

[0115] The term "oligonucleotide" typically refers to short polynucleotides, generally less than about 50 nucleotides. Where a nucleotide sequence is represented as a DNA sequence (i.e., A, T, G, C), it is understood that this also includes RNA sequences in which "T" is replaced by "U" (i.e., A, U, G, C).

[0116] Describing two polynucleotides as "operably linked" means that a single-stranded or double-stranded nucleic acid segment contains two polynucleotides arranged within the nucleic acid segment such that at least one of the two polynucleotides is capable of exerting the physiological effect by which it is characterized by the other. By way of example, a promoter operably linked to the coding region of a gene can promote transcription of the coding region.

[0117] As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by the physical disruption of a subject's tissue and administration of the pharmaceutical composition via tissue disruption. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is believed to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialysis infusion.

[0118] The term "peptide" typically refers to a short polypeptide. However, in some embodiments, the term "peptide" refers to an antibody of the presently disclosed subject matter, or a fragment or derivative thereof. Thus, in some embodiments, the term "peptide" refers to an intact antibody.

[0119] As used herein, the term "by application" refers to the administration of any molecule (eg, the antibodies, fragments, and derivatives thereof, of the presently disclosed subject matter) to a subject.

[0120] The term "pharmaceutical composition" means a composition that includes at least one active ingredient, whereby the composition is suitable for study in a mammal (such as, but not limited to, a human) for a particular efficacious outcome. Those of skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient will produce a desired efficacious outcome based on the needs of the artisan.

[0121] As used herein, the term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the U.S. Federal government or listed in the United States Pharmacopeia for use in animals, including humans.

[0122] As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient that is compatible with any other ingredients of the pharmaceutical composition and is not harmful to the subject to which the composition is administered.

[0123] "Plurality" means at least two.

[0124] "Polynucleotide" means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide can be a single-stranded or double-stranded nucleic acid.

[0125] "Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic, non-natural analogues thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic, non-natural analogues thereof.

[0126] "Synthetic peptide or polypeptide" means a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. A variety of solid phase peptide synthesis methods are known to those skilled in the art.

[0127] The term "prevent" as used herein means to keep something from happening or to take precautions against something possibly or likely to happen. In the medical context, "prevention" generally refers to measures taken to reduce the chances of contracting a disease or condition.

[0128] A "preventive" or "prophylactic" treatment is a treatment administered to a subject who does not show signs, or who shows only early signs, of a disease or disorder. Preventive or prophylactic treatment is administered for the purpose of reducing the risk of developing a condition associated with the development of the disease or disorder.

[0129] "Primer" refers to a polynucleotide that can specifically hybridize to a designated polynucleotide template and provide a point of initiation of synthesis of a complementary polynucleotide. Such synthesis occurs when a polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and a polymerizing agent such as DNA polymerase. Primers are typically single-stranded, but can also be double-stranded. Primers are typically deoxyribonucleic acids, although a wide variety of synthetic and natural primers are useful for many applications. Primers are complementary to the template to which they are designed to hybridize to serve as a site for initiation of synthesis, but need not reflect the exact sequence of the template. In such cases, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled, for example, with chromogenic, radioactive, or fluorescent moieties, and used as detectable moieties.

[0130] The term "proliferate" means to reproduce or produce.

[0131] As used herein, "protecting group" with respect to a terminal amino group refers to the terminal amino group of a peptide, which is combined with any of a variety of amino-terminus protecting groups traditionally used in peptide synthesis. Such protecting groups include, for example, acyl protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxycarbonyl; and aliphatic urethane protecting groups such as tert-butoxycarbonyl or adamantyloxycarbonyl. For suitable protecting groups, see Gross and Mienhofer, 1981.

[0132] As used herein, "protecting group" with respect to a terminal carboxyl group refers to the terminal carboxyl group of a peptide which is coupled with any of a variety of carboxyl terminus protecting groups, including, for example, tert-butyl, benzyl, or other acceptable groups coupled to the terminal carboxyl group via an ester or ether bond.

[0133] The term "protein" typically refers to a large polypeptide. A common notation is used herein to depict a polypeptide sequence: the left end of a polypeptide sequence is the amino terminus; the right end of a polypeptide sequence is the carboxyl terminus. In some embodiments of the presently disclosed subject matter, the protein is an antibody or a fragment or derivative thereof.

[0134] As used herein, the term "protein regulatory pathway" refers to both the upstream regulatory pathway that regulates a protein and the downstream events that the protein regulates, including, but not limited to, the transcription, translation, levels, activity, post-translational modifications, and function of the protein of interest, as well as the downstream events that the protein regulates.

[0135] The terms "protein pathway" and "protein regulatory pathway" are used interchangeably herein.

[0136] As used herein, the term "purified" and similar terms refer to the enrichment of a molecule or compound relative to other components that normally accompany the molecule or compound in its natural environment. The term "purified" does not necessarily indicate that complete purity of a particular molecule has been achieved during the process. As used herein, a "highly purified" molecule or compound refers to a molecule or compound that is greater than 90% pure. Exemplary purification techniques for antibodies and fragments and derivatives thereof are disclosed herein.

[0137] "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not joined together in nature. The amplified or constructed recombinant polynucleotide can be included in an appropriate vector, and the vector can be used to transform a suitable host cell.

[0138] A recombinant polynucleotide may serve a non-coding function as well, (eg, promoter, origin of replication, ribosome binding site, etc.).

[0139] A host cell containing a recombinant polynucleotide is referred to as a "recombinant host cell." A gene expressed in a recombinant host cell, which contains the recombinant polynucleotide, produces a "recombinant polypeptide."

[0140] A "recombinant polypeptide" is one produced by expression of a recombinant polynucleotide.

[0141] The term "modulate" refers to stimulating or inhibiting a desired function or activity.

[0142] As used herein, the term "regulatory element" is used interchangeably with "regulatory sequence" and refers to promoters, enhancers and other expression control elements, or any combination of such elements.

[0143] A "reversibly implantable" device is one that can be inserted into an animal's body (e.g., surgically or by insertion into a natural orifice of the animal) and then removed without significant harm to the animal's health.

[0144] As used herein, "sample" in some embodiments refers to a biological sample from a subject, including, but not limited to, a normal tissue sample, a diseased tissue sample, a biopsy, blood, saliva, feces, semen, tears, and urine. A sample may also be any other source of material obtained from a subject that contains cells, tissues, or bodily fluids of interest. Samples may also be obtained from cell or tissue cultures.

[0145] As used herein, the term "secondary antibody" refers to an antibody that binds to the constant region of another antibody (the primary antibody).

[0146] The term "signal sequence" refers to a polynucleotide sequence that encodes a peptide that directs the path that a polypeptide takes within a cell, i.e., directs the cellular processing of the polypeptide within the cell, including, but not limited to, the eventual secretion of the polypeptide from the cell. A signal sequence is typically, but not exclusively, a sequence of amino acids found at the amino terminus of a polypeptide that targets the synthesis of the polypeptide to the endoplasmic reticulum. In some cases, the signal peptide is proteolytically removed from the polypeptide and is therefore not present in the mature protein.

[0147] "Small interfering RNA (siRNA)" refers inter alia to an isolated dsRNA molecule composed of both sense and antisense strands. In some embodiments, it is more than 10 nucleotides in length. siRNA also refers to a single transcription product, such as a hairpin, that has both a sense sequence and a complementary antisense sequence from a target gene. siRNA also includes any form of dsRNA (proteolytically cleaved product of a larger dsRNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides.

[0148] As used herein, the term "solid support" when used in reference to a substrate that forms bonds with molecules refers to a solvent-insoluble substrate that is capable of forming bonds (in some embodiments, covalent bonds) with a variety of molecules. The support can be biological in nature, such as, but not limited to, a cell or bacteriophage particle, or synthetic, such as, but not limited to, acrylamide derivatives, agarose, cellulose, nylon, silica, or magnetic particles.

[0149] The term "solid support suitable for maintaining cells in a tissue culture environment" refers to any surface, such as a tissue culture dish or plate, or even a cover, to which medium containing cells can be added and the support placed in a suitable environment, such as a tissue culture incubator, for maintaining or growing the cells. Of course, this should be a sterile or sterilizable solid support. The support does not have to be suitable for cell attachment.

[0150] The term "solid support is a low-attachment, ultra-low-attachment, or non-attachment support for cell culture purposes" refers to a vehicle such as a bacteriological plate or tissue culture dish or plate that has not been treated or prepared to enhance the ability of mammalian cells to attach to the surface. For example, dishes to which a layer of agar has been added to prevent cell attachment may be included. Those skilled in the art will know that bacteriological plates are generally used with agar, where bacteria are suspended in the agar and grown in the agar, and therefore bacterial plates are not treated to promote attachment of mammalian cells.

[0151] The term "standard" as used herein refers to something that is used for comparison. For example, a standard can be a known standard agent or molecule that is administered or added to a control sample and used to compare the results when measuring a molecule in a test sample. A standard can also refer to an "internal standard," such as an agent or molecule that is added to a sample in a known amount to help determine purification or recovery rates, etc., when the sample is processed or subjected to a purification or extraction procedure prior to measuring a marker of interest.

[0152] As used herein, the term "stimulate" refers to inducing or increasing the level of an activity or function such that it is higher compared to a control value. Stimulation can be by direct or indirect mechanisms. In some embodiments, the activity or function is stimulated by at least 10% compared to a control value, and in some embodiments by at least 25%, and in some embodiments by at least 50%.

[0153] The term "stimulatory agent" as used herein refers to any composition, molecule or agent that results in the stimulation of a process or function of interest, including, but not limited to, wound healing, angiogenesis, bone healing, osteoblast production and function, and osteoclast production, differentiation, and activity.

[0154] A "subject" of diagnosis or treatment is an animal, including humans. It also includes pets and livestock.

[0155] As used herein, a "subject in need thereof" is a patient, animal, mammal, or human who would benefit from the methods of the presently disclosed subject matter.

[0156] Describing two polynucleotides as "operably linked" means that a single-stranded or double-stranded nucleic acid segment contains two polynucleotides arranged within the nucleic acid segment such that at least one of the two polynucleotides is capable of exerting the physiological effect by which it is characterized by the other. By way of example, a promoter operably linked to the coding region of a gene can promote transcription of the coding region.

[0157] As used herein, a "substantially homologous amino acid sequence" includes an amino acid sequence having at least about 95% homology to the amino acid sequence of a reference antibody chain, in some embodiments at least about 96% homology, in some embodiments at least about 97% homology, in some embodiments at least about 98% homology, and in most embodiments at least about 99% or more homology. Amino acid sequence similarity or identity can be calculated using the BLASTP and TBLASTN programs using the BLAST (Basic Local Alignment Search Tool) 2.0.14 algorithm. The default settings used in these programs are suitable for identifying substantially similar amino acid sequences for the purposes of the presently disclosed subject matter.

[0158] "Substantially homologous nucleic acid sequence" refers to a nucleic acid sequence that corresponds to a reference nucleic acid sequence, which encodes an amino acid sequence that has substantially the same structure and function as the amino acid sequence encoded by the reference nucleic acid sequence; for example, only amino acid changes that do not significantly affect the function of the amino acid sequence are made. In some embodiments, a substantially identical nucleic acid sequence encodes the same amino acid sequence encoded by a reference nucleic acid sequence. The percentage of identity between a substantially similar nucleic acid sequence and a reference nucleic acid sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more. The substantial identity of nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example, by physical / chemical methods (i.e., hybridization) or sequence alignment by computer algorithms. Suitable nucleic acid hybridization conditions for determining whether a nucleotide sequence is substantially similar to a reference nucleotide sequence include 7% sodium dodecyl sulfate SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 2x standard citrate solution (SSC), 0.1% SDS at 50°C; in some embodiments, 7% (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 1x SSC, 0.1% SDS at 50°C; in some embodiments, 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.5x SSC, 0.1% SDS at 50°C; and in some embodiments, 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1x SSC, 0.1% SDS at 65°C. Suitable computer algorithms for determining substantial similarity between two nucleic acid sequences include the GCS program package (Devereux et al., 1984), and the BLASTN or FASTA programs (Altschul et al., 1990a; Altschul et al., 1990b; Altschul et al., 1997). The default settings provided with these programs are suitable for determining substantial similarity of nucleic acid sequences for purposes of the presently disclosed subject matter.

[0159] The term "substantially pure" describes a molecule, such as a protein or polypeptide, that has been separated from components that naturally accompany it. Typically, a molecule is substantially pure when at least 10%, and in some embodiments at least 20%, and in some embodiments at least 50%, and in some embodiments at least 60%, and in some embodiments at least 75%, and in some embodiments at least 90%, and in most embodiments at least 99% (by volume, wet or dry weight, or mole percent or mole fraction) of the total material in a sample is the molecule of interest. Purity can be measured by any suitable method, such as, in the case of polypeptides, by column chromatography, gel electrophoresis, or HPLC analysis. A molecule, such as a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from natural contaminants that naturally accompany it in the natural state.

[0160] A "surface active agent" or "surfactant" is a substance that has the ability to reduce the surface tension of a material, allowing penetration into and through the material.

[0161] The term "symptom" as used herein refers to any deviation from the normal or pathological phenomenon in structure, function, or sensation experienced by a patient that is indicative of disease. In contrast, a "sign" is objective evidence of disease. For example, a nosebleed is a sign; it is obvious to the patient, doctor, nurse, or other observer.

[0162] A "therapeutic" treatment is a treatment administered to a subject exhibiting pathological signs with the intent of reducing or eliminating those signs.

[0163] A "therapeutically effective amount" of a molecule is that amount of the molecule sufficient to confer a beneficial effect on the subject to which it is administered.

[0164] Use of the phrase "tissue culture dish or plate" refers to any type of container that can be used to seed cells for proliferation or differentiation.

[0165] The term "thermal injury" is used interchangeably herein with "thermal burn."

[0166] "Tissue" means (1) a group of similar cells combined to perform a specific function; (2) a part of an organism consisting of a collection of cells having similar structure and function; or (3) a group of cells similarly characterized by their structure and function, such as muscle or nervous tissue.

[0167] The term "topical application" as used herein refers to administration to a surface such as the skin. The term is used interchangeably with "application to the skin" in the case of the skin. "Topical application" is "direct application".

[0168] "Transdermal" delivery means delivery whereby a drug passes through the skin or mucosal tissue and into the bloodstream. Transdermal also refers to the skin as a portal of entry for administration of a drug or molecule by topical application of the drug or molecule. "Transdermal" is used interchangeably with "percutaneous."

[0169] The term "transfection" is used interchangeably with the terms "gene transfer," "transformation," and "transduction" and refers to the introduction of a polynucleotide into a cell. "Transfection efficiency" refers to the relative amount of a transgene taken up by a transfected cell. In practice, transfection efficiency is estimated by the amount of reporter gene product expressed after the transfection procedure.

[0170] As used herein, the term "transgene" means an exogenous nucleic acid sequence comprising a nucleic acid encoding a promoter / regulatory sequence operably linked to a nucleic acid encoding an amino acid sequence, which exogenous nucleic acid is encoded by a transgenic mammal.

[0171] As used herein, the term "transgenic mammal" means a mammal whose germ cells contain an exogenous nucleic acid.

[0172] As used herein, a "transgenic cell" is any cell that contains a nucleic acid sequence that has been introduced into the cell in a manner that permits expression of a gene encoded by the introduced nucleic acid sequence.

[0173] As used herein, the term "treat" means reducing the frequency with which a patient or subject experiences a symptom, or administering a drug or molecule to reduce the frequency with which a symptom is experienced.

[0174] A "prophylactic" treatment is a treatment administered to a subject who does not show signs of a disease, or who shows only early signs of a disease, for the purpose of reducing the risk of developing pathology associated with the disease.

[0175] As used herein, the term "treating" includes prophylaxis of a particular disorder or condition, or alleviation of symptoms associated with a particular disorder or condition, and / or prevention or elimination of said symptoms. A "prophylactic" treatment is a treatment administered to a subject who does not show signs of a disease or who shows only early signs of a disease, for the purpose of reducing the risk of developing pathology associated with the disease.

[0176] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic molecules, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral molecules that facilitate the transfer or delivery of nucleic acids into cells, such as, for example, polylysine molecules, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, recombinant viral vectors, etc. Examples of non-viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, etc.

[0177] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate a recombinant polynucleotide.

[0178] As used herein, the term "wound" refers to the physical tearing, breaking, or rupture of a tissue or cell layer. A wound may occur due to any physical injury, including a surgical procedure, or as a result of a disease, injury, or condition.

[0179] Methods useful for carrying out the subject matter of the present disclosure that are not described herein are also known in the art. Useful methods include those described in PCT International Patent Application Publication No. WO 2007 / 019107; WO 2007 / 030652; WO 2007 / 089798; WO 2008 / 060374, which are incorporated herein by reference.

[0180] II. Exemplary Implementations In some embodiments, the subject matter of the present disclosure provides compositions and methods useful for targeting mechanically exposed cryptic sites in the integrin binding domain of fibronectin. Thus, in some embodiments, the subject matter of the present disclosure provides a method for targeting the conformational state of fibronectin (FN) in a sample, optionally a biological sample isolated from a subject or present in a subject. In some embodiments, the method comprises contacting the sample with a composition having selective binding activity for the conformational state of FN, including FnIII9-4G-10 (4G), thereby targeting the conformational state. In some embodiments, the sample comprises or is suspected to comprise tissue undergoing tissue repair, diseased tissue, tissue suffering from a disorder, or any combination thereof.

[0181] The subject matter of the present disclosure provides detection of an integrin-binding mechanoswitch within fibronectin (Fn) during tissue formation and fibrosis. The disclosed results demonstrate the presence and activation in vivo of a long-theorized Fn conformational switch within the integrin-binding domain and suggest its influence in skewing integrin specificity during both developmental processes and pathological tissue fibrosis. Thus, antibodies such as the modified H5-IgG1 antibody of the present disclosure represent an attractive approach for detecting and targeting important developmental and disease processes.

[0182] Disclosed herein are the binding kinetics and activity of modified H5 antibodies that recognize FnIII9, also referred to herein as FnIII9-4G-10 (4G), and selectively inhibit αvβ3 binding to Fn-absorbed surfaces.

[0183] In particular, the modified H5 antibody of the presently disclosed subject matter was produced by combining the variable region of the H5 antibody with the universal constant region of the IgG1 subclass. The nucleic acid sequence encoding the heavy chain variable region of the H5 antibody was combined with a sequence encoding the IgG1 subclass constant region of IgG1, including CH1, CH2, and CH3. Similarly, the nucleotide sequence encoding the light chain variable region of the H5 antibody was combined with a nucleic acid sequence encoding the constant light (CL) chain-kappa. To express the modified H5 antibody in full-length IgG1 format, donor constructs encoding the H5-light chain variable region and the H5-heavy chain variable region were stably integrated into the immunoglobulin locus (IgG1) of the genome of hybridoma cells by Cas9 targeting. Culture supernatants of cells expressing the modified H5-IgG1 antibody were analyzed for the presence of IgG, quantified, and used in the ELISA binding assay described in Example 1. For routine large-scale production of H5-IgG1 in mammalian cells, the H5-heavy chain variable region and H5-light chain variable region sequences can be separately cloned into two independent IgG1 expression vector backbones by Gibson Assembly® Cloning, NEBUILDER® HiFi DNA Assembly (New England Biolabs, Ipswich, Massachusetts, United States of America), IN-FUSION® Snap Assembly (Takara Bio USA, Inc., Mountain View, California, United States of America), or any other strategy. These plasmids encode the constant regions of human IgG1 heavy and kappa light chains, respectively, and provide a framework for inserting the variable regions of choice. For transient expression of H5-IgG1, plasmids containing the H5-heavy and H5-light chains are co-transfected into selected mammalian suspension cells, such as 293-6E or Chinese Hamster Ovary (CHO) cells. Culture supernatants are collected between 48 and 96 hours post-transfection and used for isolation of full-length H5-IgG1 by protein L or protein G chromatography.

[0184] The modified H5-IgG1 antibodies and antigen-binding fragments thereof are characterized by a much higher affinity for the FN fragment, exhibiting at least 50-fold higher binding when compared to the parent H5 antibody at equimolar concentrations. Furthermore, the modified H5-IgG1 antibodies exhibited a significantly higher affinity for the FN fragment, compared to the parent H5 antibody at equimolar concentrations. * showed higher affinity for FN-4G-10 (i.e., diseased) serotype compared with -10 (healthy) serotype.

[0185] Thus, the presently disclosed subject matter provides compositions and methods useful for detecting distinct conformational states of Fn, in some embodiments, the binding of modified H5-IgG1 antibodies and antigen-binding fragments thereof to Fn can detect distinct conformational states of Fn, such as FnIII9-4G-10 (4G).

[0186] The subject matter of the present disclosure provides compositions and methods useful in some embodiments for detecting distinct conformational states of Fn, and the binding ratio is useful for distinguishing normal tissue from diseased or disordered tissue. In some embodiments, the severity of fibrosis in tissue can be determined. In some embodiments, the subject matter of the present disclosure is useful for detecting transient force-induced conformational changes in Fn. The compositions and methods are useful for targeting early molecular signatures of cellular contractile forces during tissue repair. The compositions and methods are useful for targeting early molecular signatures of cellular contractile forces in diseases and disorders.

[0187] In some embodiments, the presently disclosed subject matter provides compositions and methods useful for detecting and comparing pathological ECM, hi some embodiments, the presently disclosed subject matter provides compositions and methods useful for detecting and distinguishing fibrotic ECM.

[0188] In some embodiments, the presently disclosed subject matter provides compositions and methods useful for delineating areas of high ECM distortion, which in some embodiments is associated with enhanced αv integrin binding properties due to the conformation of the integrin binding domain, possibly indicative of ongoing fibrosis.

[0189] In some embodiments, the antibodies of the presently disclosed subject matter are useful for extracting structural information from the ECM.

[0190] In some embodiments, the compositions and methods are useful for determining the antibody:Fn ratio, which can be further used to diagnose or distinguish between normal and diseased tissue, and to determine the type of treatment to administer if a subject is diagnosed with a disease or disorder.

[0191] The subject matter of the present disclosure provides other antibodies and biologically active fragments and homologs thereof, as well as methods for preparing and testing new antibodies for the properties disclosed herein.

[0192] In some embodiments, the antibodies or biologically active fragments or homologs thereof are useful for treating diseases or disorders associated with the fibronectin interacting signaling pathways disclosed herein, in some embodiments, the pathways are regulated by mechanoswitches.

[0193] In some embodiments, the presently disclosed subject matter employs a biologically active antibody, or a biologically active fragment or homolog thereof. In some embodiments, the isolated polypeptide comprises a mammalian molecule that is at least about 30% homologous to a polypeptide having at least one amino acid sequence of the sequences disclosed herein. In some embodiments, the isolated polypeptide is at least about 35% homologous, and in some embodiments about 40% homologous, and in some embodiments about 45% homologous, and in some embodiments about 50% homologous, and in some embodiments about 55% homologous, and in some embodiments about 60% homologous, and in some embodiments about 65% homologous, and in some embodiments about 70% homologous, and in some embodiments about 75% homologous, and in some embodiments about 80% homologous, and in some embodiments about 85% homologous, and in some embodiments about 90% homologous, and in some embodiments about 95% homologous, and in some embodiments about 96% homologous, and in some embodiments about 97% homologous, and in some embodiments about 98% homologous, and in most embodiments about 99% homologous to at least one of the peptide sequences disclosed herein.

[0194] The subject matter of the present disclosure further encompasses modifications of the antibodies and fragments thereof disclosed herein, including amino acid deletions, additions, and substitutions, particularly conservative substitutions. The subject matter of the present disclosure also encompasses modifications to increase in vivo half-life and decrease in vivo degradation. Substitutions, additions, and deletions can include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 changes, so long as the activity disclosed herein is substantially the same.

[0195] The subject matter of the present disclosure includes isolated nucleic acids comprising a nucleic acid sequence encoding an antibody of the subject matter of the present disclosure, or a fragment or homolog thereof, In some embodiments, the nucleic acid sequence encodes a peptide comprising a biologically active fragment of the antibody sequence of the subject matter of the present disclosure, or a homolog thereof.

[0196] In some embodiments, a homolog of a peptide (antibody or fragment) of the disclosed subject matter has one or more amino acid substitutions, deletions, or additions and has sequence identity as described herein. In some embodiments, the substitutions, deletions, or additions are conservative. In some embodiments, a cysteine ​​residue in a peptide of the disclosed subject matter is substituted with a serine or alanine.

[0197] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0198] The subject matter of this disclosure encompasses the use of purified isolated peptides, recombinant peptides, and synthetic peptides.

[0199] The subject matter of the present disclosure further includes the use of drugs or other molecules that can target the exposed cryptic sites disclosed herein, e.g., recognize the structural changes disclosed herein, and have the same activity as disclosed herein.

[0200] Thus, the subject matter of the present disclosure provides, in some embodiments, a method for detecting the conformational state of fibronectin (FN) in a sample. In some embodiments, the method includes contacting the sample with a composition having selective binding activity for the conformational state of FN, including FnIII9-4G-10 (4G), and detecting binding of the composition, thereby detecting the conformational state of FN. In some embodiments, the sample includes or is suspected to include tissue undergoing tissue repair, diseased tissue, tissue suffering from a disorder, or a combination thereof. In some embodiments, the sample includes or is suspected to include a pathological extracellular matrix (ECM). In some embodiments, the sample includes or is suspected to include a fibrotic ECM.

[0201] In some embodiments, detecting binding of the composition comprises distinguishing between normal and diseased tissue. In some embodiments, detecting binding of the composition comprises determining the severity of fibrosis in the sample, for example, by using a binding ratio of the composition to FN. In some embodiments, detecting binding of the composition comprises detecting a transient force-induced conformational change in FN. In some embodiments, detecting binding of the composition comprises extracting structural information of the ECM in the sample. In some embodiments, extracting structural information of the ECM in the sample comprises delineating regions of high ECM strain. In some embodiments, high ECM strain is associated with enhanced αv integrin binding properties.

[0202] In some embodiments, the method further comprises determining the type of treatment to administer to the subject based on detecting binding of the composition.

[0203] The subject matter of the present disclosure also provides, in some embodiments, a method for treating a disease and / or disorder in a subject. In some embodiments, the method comprises administering to a subject in need of treatment a therapeutically effective amount of a composition having selective binding activity to a conformational state of FN, including FnIII9-4G-10 (4G), thereby achieving treatment. In some embodiments, the disease and / or disorder has a characteristic selected from the group consisting of tissue undergoing tissue repair, diseased tissue, tissue suffering from a disorder, and any combination thereof. In some embodiments, the characteristic is a pathological extracellular matrix (ECM). In some embodiments, the characteristic is a fibrotic ECM.

[0204] In some embodiments of the disclosed method, the composition having selective binding activity for the conformational state of FN, including FnIII9-4G-10 (4G), is an isolated and purified antibody, or fragment thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 14, 16, or 18, or an antibody having a sequence that is about 95% identical to the sequence of SEQ ID NO: 2, 4, 6, 14, 16, or 18, or a fragment thereof. In some embodiments, the amino acid sequence comprises at least one modification selected from the group consisting of amino acid deletion, amino acid addition, amino acid substitution, and combinations thereof. In some embodiments, the isolated antibody is present in multiple copies (i.e., multimerized), with each copy being linked by a linker. The multiple copies may include two copies in some embodiments, three copies in some embodiments, four copies in some embodiments, five copies in some embodiments, and more than five copies in some embodiments. In some embodiments, the individual members of a multimeric composition are identical to one another, and in some embodiments, one or more individual members of a multimeric composition are different from at least one other individual member of the multimeric composition.

[0205] The subject matter of the present disclosure also provides, in some embodiments, a method for screening molecules that have selective binding activity for a conformational state of FN, including FnIII9-4G-10 (4G). In some embodiments, the method includes providing a sample that includes a conformational state of FN, including FnIII9-4G-10 (4G); contacting the sample with a candidate molecule; and detecting binding of the candidate molecule to the sample. In some embodiments, the candidate molecule is a member of a molecular library. In some embodiments, the candidate compound is a small molecule or an antibody. In some embodiments, the conformational state of FN is a force-induced conformational change in Fn.

[0206] The presently disclosed subject matter also provides, in some embodiments, compounds identified by the methods of the present disclosure.

[0207] The presently disclosed subject matter also provides, in some embodiments, a method for treating a disease and / or disorder in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an antibody or fragment thereof according to the presently disclosed subject matter, thereby achieving treatment.

[0208] The presently disclosed subject matter also provides, in some embodiments, a method for ameliorating at least one symptom resulting from a disease or disorder associated with abnormal expression of a force-induced conformational state of FN, including FnIII9-4G-10 (4G), in a subject. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising an antibody or fragment thereof according to the presently disclosed subject matter, whereby at least one symptom resulting from a disease or disorder associated with abnormal expression of a force-induced conformational state of FN, including FnIII9-4G-10 (4G), is ameliorated.

[0209] In some embodiments of the methods of treatment, the disease or disorder is associated with tissue undergoing tissue repair, diseased tissue, tissue suffering from a disorder, or any combination thereof. In some embodiments, the disease or disorder is associated with a pathological extracellular matrix (ECM). In some embodiments, the disease or disorder is associated with a fibrotic ECM.

[0210] III. Exemplary Sequences of the Subject Matter of the Disclosure The presently disclosed subject matter provides for the use of various antibodies, and biologically active fragments and homologs thereof, having the activities described herein.

[0211] In some embodiments, the presently disclosed subject matter provides an isolated and purified antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, 4, 6, 14, 16, or 18, a fragment thereof, an antibody having an amino acid sequence that is about 95% identical to any one of SEQ ID NO:2, 4, 6, 14, 16, or 18, a fragment thereof, and an amino acid sequence substantially homologous to any of the foregoing sequences. In some embodiments, the amino acid sequence comprises at least one modification selected from the group consisting of an amino acid deletion, an amino acid addition, an amino acid substitution, and a combination thereof. In some embodiments, the antibody or fragment thereof is humanized.

[0212] In some embodiments, the isolated and purified antibody, or fragment or homolog thereof, comprises a heavy chain CDR1 of sequence SYAMS (SEQ ID NO:8), a heavy chain CDR2 of sequence DIYDGGGTNYADSVKG (SEQ ID NO:10), a heavy chain CDR3 of sequence TADNFDY (SEQ ID NO:12), a light chain CDR1 of sequence RASQSISSYLN (SEQ ID NO:20), a light chain CDR2 of sequence AASTLQS (SEQ ID NO:22), and a light chain CDR3 of sequence QQANSAPTT (SEQ ID NO:24).

[0213] In some embodiments, the isolated and purified antibody, or fragment or homolog thereof, comprises a heavy chain framework region 1 comprising EVQLLESGGGLVQPGGSLRLSCAAS (SEQ ID NO:40), a heavy chain framework region 2 comprising WVRQAPGKGLEWV (SEQ ID NO:41), a heavy chain framework region 3 comprising RFTTSRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:42), and a heavy chain framework region 4 comprising WGQGTLVTVSS (SEQ ID NO:43); and / or the isolated and purified antibody further comprises a light chain framework region 1 comprising DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:44), a light chain framework region 2 comprising WYQQKPGKAPKLLIY (SEQ ID NO:45), a light chain framework region 3 comprising GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:46), and a light chain framework region 4 comprising FGQGTKVEIK (SEQ ID NO:47).

[0214] In some embodiments, the isolated and purified antibody, or fragment or homolog thereof, comprises a modification at its N-terminus, its C-terminus, or both. In some embodiments, the modification comprises the addition of a peptide tag, a SARAH domain, or a combination thereof. In some embodiments, the tag comprises a his tag, a myc tag, a VSV tag, an HA tag, a SortaseA tag, a PelB sequence, or any combination of one or more thereof. In some embodiments, the His tag comprises, consists essentially of, or consists of the amino acid sequence HHHHHH (SEQ ID NO:35). In some embodiments, the myc tag comprises, consists essentially of, or consists of the amino acid sequence EQKLISEEDL (SEQ ID NO:33). In some embodiments, the VSV tag comprises, consists essentially of, or consists of the amino acid sequence YTDIEMNRLGK (SEQ ID NO:34). In some embodiments, the HA tag comprises, consists essentially of, or consists of the amino acid sequence YPYDVPDYA (SEQ ID NO:36). In some embodiments, the SortaseA tag comprises, consists essentially of, or consists of the amino acid sequence LPXTG (SEQ ID NO:48), where X at amino acid 3 of SEQ ID NO:48 can be any amino acid. In some embodiments, the PelB sequence comprises, consists essentially of, or consists of the amino acid sequence MKYLLPTAEAGLLLLLAAPQIA (SEQ ID NO:49). In some embodiments, the isolated and purified antibodies, or fragments or homologs thereof, of the presently disclosed subject matter comprise any combination of one or more of a His tag, a myc tag, a VSV tag, an HA tag, a SortaseA tag, and a PelB sequence, including any combination of one or more of these sequences or even more than one of one or more of the listed sequences. In some embodiments, the SARAH domain comprises a sequence selected from the group consisting of SEQ ID NOs:28-32.

[0215] The presently disclosed subject matter also provides, in some embodiments, isolated and purified nucleic acid sequences encoding the antibodies and fragments disclosed herein, and nucleic acid sequences substantially homologous thereto.

[0216] The presently disclosed subject matter also provides, in some embodiments, recombinant nucleic acids and nucleic acid sequences substantially homologous thereto. In some embodiments, the recombinant nucleic acid comprises a V sequence comprising a first amino acid sequence comprising SEQ ID NO:4. H Segment V comprising a second amino acid sequence comprising SEQ ID NO:16 L In some embodiments, the recombinant nucleic acid further comprises a first nucleic acid segment encoding one or more subsequences of an intact antibody, such that the recombinant nucleic acid encodes a recombinant intact antibody.

[0217] In some embodiments, the antibodies, fragments, and homologs of the present disclosure may include tag sequences, linker sequences, spacer sequences, and / or other additional sequences that can be used to facilitate expression, stability, purification, isolation, or other desired characteristics or aspects. Multiple copies of such sequences may be used. Such sequences may be added to the N-terminus, C-terminus, or both of the antibodies, fragments, or homologs of the present disclosure. Representative such sequences include SARAH sequences. SEQ ID NOs:28-32 are exemplary amino acid sequences of SARAH domains that may be added to the N-terminus, C-terminus, or both of the antibodies, fragments, or homologs of the present disclosure. Representative such sequences also include tag sequences such as myc, VSV, His, HA, SortaseA, and PelB tags. As specific, non-limiting examples, SEQ ID NOs:33-38 are amino acid sequences of exemplary tags that can be added to the N-terminus, C-terminus, or both of an antibody, fragment, or homologue of the subject matter of the present disclosure. EQKLISEEDL (SEQ ID NO:33) is an exemplary myc tag, YTDIEMNRLGK (SEQ ID NO:34) is an exemplary VSV tag, HHHHHH (SEQ ID NO:35) is an exemplary His tag, YPYDVPDYA (SEQ ID NO:36) is an exemplary HA tag, LPTEGG (SEQ ID NO:37) is an exemplary SortaseA tag, and MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO:38) is an exemplary PelB tag.

[0218] In some embodiments, the antibody or fragment thereof of the presently disclosed subject matter is modified with a SARAH domain. Exemplary approaches for incorporating a SARAH domain are described in European Patent Application No. 17868682.0; PCT International Patent Application WO 2018 / 088403; U.S. Patent Application No. 16 / 345639; and Arimori et al., 2017, each of which is incorporated herein by reference in its entirety. In some embodiments, the N-terminus of the SARAH domain is linked to the heavy chain domain (V H region) and / or the C-terminus of the light chain domain (V LThe SARAH domain is linked to the C-terminus of a SARAH domain (region). A SARAH domain is a domain (peptide) that comprises a short N-terminal helix (h1) and a long C-terminal helix (h2), which in some embodiments typically comprises 42 to 54, optionally 43 to 49, further optionally 47 to 49, and further optionally 49 amino acid residues, and has the property of forming an antiparallel coiled coil between h2 and another SARAH domain. Note that h1 may comprise 5 to 7 amino acid residues and h2 may comprise 38 to 42 amino acid residues.

[0219] A SARAH domain as used herein may be any SARAH domain deemed appropriate by one of skill in the art upon review of this disclosure. In a further non-limiting example, when two SARAH domains form an antiparallel coiled coil between h2, the distance between the N-termini of the two SARAH domains (two h1) is optionally about 35A to 45A, further optionally about 39A to 41A, further optionally about 40A.Specific examples of such SARAH domains include the SARAH domain of human mammalian sterile 20-like kinase 1 polypeptide (hMST1; DYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQSKRQPILDAIEAK, SEQ ID NO: 28; corresponding to amino acids 432-480 of GENBANK® biological sequence database accession number NP_006273.1), the SARAH domain of human mammalian sterile 20-like kinase 2 polypeptide (hMST2; DFDFLKNLSLEELQMR LKALDPMMEREIEELRQRYTAKRQPILDAMDAK, SEQ ID NO: 29; corresponding to amino acids 325 to 373 of GENBANK® Biological Sequence Database accession number NP_001243242.1), SARAH domain of human ras-related domain-containing protein 5 isoform C polypeptide (hRAF5; GEVEWDAFSIPELQNFLTILEKEEQDKIQQVQKKYDKFRQKLEEALRES, SEQ ID NO: 30; corresponding to amino acids 325 to 373 of GENBANK® Biological Sequence Database accession number NP_001243242.1), corresponding to amino acids 212-260 of the GENBANK® Biological Sequence Database accession number NP_872606.1), the SARAH domain of human ras-related domain-containing protein 1 isoform B polypeptide (hRAF1; GEVNWDAFSMPELHNFLRILQREEEEHLRQILQKYSYSRQKIQEALHAS, SEQ ID NO:31; showing 47 / 49 amino acid identity with amino acids 138-186 of the GENBANK® Biological Sequence Database accession number NP_001193886.1), the human protein salvador The SARAH domain of Homolog 1 polypeptide (hSAV1; HILKWELFQLADLDTYQGMLKLLFMKELEQIVKMYEAYRQALLTELENR, SEQ ID NO: 32; corresponding to amino acids 320 to 368 of GENBANK® Biological Sequence Database Accession Number NP_068590.1) and further includes those having at least 85% sequence identity, in some embodiments at least 90%, and in some embodiments at least 95% sequence identity to one of the aforementioned SARAH domains.For the aforementioned representative SARAH domains, a general design approach may include the heavy and light chains of a given antibody being individually fused to the 49-residue SARAH domain via a two-residue (Gly-Ser) linker. In some embodiments of the aforementioned representative SARAH domains, two Cys residues (38 and 49) in the hRAF1 SARAH domain were substituted with Ser to avoid the formation of undesired disulfide bonds. In some embodiments, residues 24 and 35 were mutated to Cys to form an asymmetric interchain disulfide bond based on the homodimeric hMST1 structure.

[0220] One skilled in the art will understand that, based on the sequences of the components of the antibodies disclosed herein, they can be modified independently of each other by conservative amino acid changes, including insertions, deletions, and substitutions, and that the valency can also be altered. Amino acid changes (fragments and homologs) can also be made independently within antibodies when they are used in combination therapy.

[0221] In some embodiments, the proteins or peptides of the presently disclosed subject matter, or combinations thereof, can be administered by a route selected, including, but not limited to, intravenous, intrathecal, locally, intramuscular, topically, orally, intraarterially, parenterally, and the like. Administration can be more than once. One of skill in the art can determine how often the molecules are administered, the dosages used, and in what combinations they can be administered with other agents, such as therapeutic agents and / or other drugs or molecules, such as antimicrobial agents, anti-inflammatory agents, etc. One of skill in the art will be able to determine when or if additional agents and routes of administration are used.

[0222] In some embodiments, the protein or polypeptide is administered by injection. Parenteral administration routes for polypeptides follow known methods, for example, injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial, subcutaneous, or intralesional routes. Proteins or polypeptides can be administered continuously by infusion or bolus injection. A typical composition for intravenous infusion may be configured to contain 10 to 50 ml of sterile 0.9% NaCl or 5% glucose, optionally supplemented with 20% albumin solution, and between 10 μg and 50 mg, and in some embodiments between 50 μg and 10 mg, of the polypeptide. A typical pharmaceutical composition for intramuscular injection would, for example, be configured to contain 1-10 ml of sterile buffered water and between 10 μg and 50 mg, and in some embodiments between 50 μg and 10 mg, of the polypeptide of the presently disclosed subject matter. Methods for preparing parenterally administrable compositions are well known in the art and are described in more detail in various sources, including, for example, Genaro 1985, which is incorporated herein by reference in its entirety for all purposes.

[0223] When used in vivo for therapy, the antibodies of the presently disclosed subject matter are administered to a subject in a therapeutically effective amount (i.e., an amount having the desired therapeutic effect). They are usually administered parenterally. The dose and administration regimen will depend on the extent of the disease or disorder, the characteristics of the particular antibody or immunotoxin used, e.g., its therapeutic index, the patient, and the patient's medical history. Advantageously, the antibody or fragment thereof is administered continuously over a period of 1-2 weeks. Optionally, administration is during the course of an adjunct therapy, e.g., antimicrobial therapy, or administration of tumor necrosis factor, interferon, or other cytoprotective or immunomodulatory agents.

[0224] For parenteral administration, the antibodies and fragments thereof can be combined with a pharma- ceutically acceptable parenteral vehicle to be formulated into a unit dosage injectable form (solution, suspension, emulsion). Such vehicles are essentially non-toxic and non-therapeutic. Examples of such vehicles include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes can be used as carriers. The vehicle may contain small amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. Antibodies are typically formulated in such vehicles at a concentration of about 1.0 mg / ml to about 10 mg / ml.

[0225] The antibody compositions used will be formulated and dosed in a manner consistent with good medical practice, taking into account the condition or disorder being treated, the condition of the individual patient, the site of delivery of the composition, the method of administration, and other factors known to the practitioner. The antibody compositions are prepared for administration as described below in Preparation of Polypeptides for Administration.

[0226] Hybrid antibodies and hybrid antibody fragments, in some embodiments, include complete antibody molecules having full-length heavy and light chains, or any fragment thereof, including but not limited to, antigen-binding fragments (e.g., including paratopes). Chimeric antibodies having variable regions as described herein and constant regions from various species are also suitable. See, e.g., U.S. Patent Application Publication No. 2003 / 0022244.

[0227] The peptides of the presently disclosed subject matter can be readily prepared by standard, well-established techniques, e.g., solid phase peptide synthesis (SPPS) as described in Stewart et al., 1984; Bodanszky and Bodanszky, 1984. First, an appropriately protected amino acid residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin.

[0228] "Suitably protected" refers to the presence of protecting groups on both the α-amino group of the amino acid and any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis and can be removed under conditions that do not affect the final peptide product. The stepwise synthesis of oligopeptides is carried out by removing the N-protecting group from an initial amino acid and attaching to it the carboxyl terminus of the next amino acid in the sequence of the desired peptide, which is also suitably protected. The carboxyl of the incoming amino acid can be activated to react with the N-terminus of the support-bound amino acid by formation of a reactive group, such as a carbodiimide, a symmetrical anhydride, or an "active ester" group such as a hydroxybenzotriazole or pentafluorophenyl ester.

[0229] Examples of solid phase peptide synthesis methods include the BOC method, which utilizes tert-butyloxycarbonyl as the α-amino protecting group, and the FMOC method, which utilizes 9-fluorenylmethyloxycarbonyl to protect the α-amino of amino acid residues, both of which are well known to those skilled in the art.

[0230] To confirm that a protein or peptide obtained by chemical or biological synthetic techniques is the desired peptide, an analysis of the peptide composition should be performed. Such amino acid composition analysis can be performed using high resolution mass spectrometry to determine the molecular weight of the peptide. Alternatively, or in addition, the amino acid content of a peptide can be confirmed by hydrolyzing the peptide in an acidic aqueous solution and using HPLC or an amino acid analyzer to separate, identify and quantify the components of the mixture. Protein sequenators, which sequentially degrade a peptide and identify the amino acids in order, can also be used to accurately determine the sequence of a peptide.

[0231] Prior to use, the peptides can be purified to remove contaminants. In this regard, it is understood that the peptides are purified to meet standards set by appropriate regulatory authorities. To achieve the required level of purity, any one of a number of common purification procedures can be used, including, for example, reversed-phase high performance liquid chromatography (HPLC) using alkylated silica columns, such as C4-, C8-, or C18-silica. Purification is generally accomplished using a gradient mobile phase of increasing organic content, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid. Ion exchange chromatography can also be used to separate peptides based on their charge.

[0232] Substantially pure peptides obtained as described herein can be purified according to known procedures for protein purification, using immunological, enzymatic, or other assays to monitor the purification at each step of the procedure. Protein purification methods are well known in the art and are described, for example, in Deutscher et al., 1990.

[0233] [Peptide modification and preparation] Methods for producing, modifying, and purifying peptides are known. It will be understood that the proteins and peptides of the presently disclosed subject matter can incorporate modified amino acid residues without affecting activity. For example, the termini can be derivatized to include blocking groups, i.e., chemical substituents suitable for protecting and / or stabilizing the N-terminus and C-terminus from "undesirable degradation", i.e., sequential degradation at the termini of the molecule, a term that encompasses any kind of enzymatic, chemical, or biochemical degradation of the molecule at the termini that may affect the function of the molecule.

[0234] Blocking groups include protecting groups conventionally used in the field of peptide chemistry that do not adversely affect the in vivo activity of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, and substituted forms thereof such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful as N-terminal blocking groups and can be attached to the N-terminus of the peptide or used in place of the N-terminal residue. Suitable C-terminal blocking groups, with or without the incorporation of the C-terminal carboxyl group, include esters, ketones, or amides. Ester- or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl, and propyl, and amide-forming amino groups such as primary amines (-NH2), and mono- and dialkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino, and the like, are examples of C-terminal blocking groups. Descarboxylated amino acid analogs, such as agmatine, are also useful C-terminal blocking groups and can be attached to or used in place of the C-terminal residue of the peptide. Furthermore, it will be appreciated that the terminal free amino and carboxyl groups can be removed en masse from a peptide to yield its desamino and descarboxylated forms without affecting the peptide's activity.

[0235] Acid addition salts of the presently disclosed subject matter are also contemplated as functional equivalents. Thus, peptides according to the presently disclosed subject matter that have been treated with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, to provide water-soluble salts of the peptides are suitable for use in the presently disclosed subject matter.

[0236] The subject matter of the present disclosure also provides analogs of proteins. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences, or modifications that do not affect sequence, or both. For example, conservative amino acid changes can be made, which change the primary sequence of a protein or peptide, but do not usually change its function. Thus, 10 or more conservative amino acid changes typically do not affect peptide function.

[0237] Modifications (which do not normally alter the primary sequence) include in vivo or in vitro chemical derivatization of the polypeptide, e.g., acetylation or carboxylation. Also included are glycosylation modifications, e.g., modifications made during polypeptide synthesis and processing, or during further processing, by modifying the glycosylation pattern of the polypeptide, e.g., by exposing the polypeptide to enzymes that affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also included are sequences that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0238] Also included are polypeptides that have been modified using routine molecular biology techniques to improve their resistance to proteolysis, or to optimize solubility properties, or to make them more suitable as therapeutic agents. Analogs of such polypeptides include those that contain residues other than naturally occurring L-amino acids, such as D-amino acids, or non-natural or non-standard synthetic amino acids. The peptides of the disclosed subject matter are not limited to the products of any of the specific exemplary processes listed herein.

[0239] The subject matter of the present disclosure includes a compound having the structure: This includes the use of beta-alanine (also called β-alanine, β-Ala, bA, and βA) having TIFF2024526154000006.tif35170.

[0240] Although sequences are provided herein using the designation "βA," in the sequence listing submitted herewith, "βA" is provided as "Xaa," and references in the text of the sequence listing indicate that Xaa is beta-alanine.

[0241] It will be appreciated that peptides or antibodies, derivatives or fragments thereof, can incorporate amino acid residues that are modified without affecting activity. For example, the termini can be derivatized to include blocking groups, i.e. chemical substituents suitable for protecting and / or stabilizing the N- and C-termini from "undesirable degradation", i.e. sequential degradation of the compound at its termini, a term that encompasses any kind of enzymatic, chemical or biochemical degradation of the compound at its termini that may affect the function of the compound.

[0242] Blocking groups include protecting groups conventionally used in the field of peptide chemistry that do not adversely affect the in vivo activity of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, and substituted forms thereof such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful as N-terminal blocking groups and can be attached to the N-terminus of the peptide or used in place of the N-terminal residue. Suitable C-terminal blocking groups, with or without the incorporation of the C-terminal carboxyl group, include esters, ketones, or amides. Ester- or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl, and propyl, and amide-forming amino groups such as primary amines (-NH2), and mono- and dialkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino, and the like, are examples of C-terminal blocking groups. Descarboxylated amino acid analogs, such as agmatine, are also useful C-terminal blocking groups and can be attached to or used in place of the C-terminal residue of the peptide. Furthermore, it will be appreciated that the terminal free amino and carboxyl groups can be removed en masse from a peptide to obtain its desamino and descarboxylated forms without affecting the peptide's activity.

[0243] Other modifications can also be incorporated without adversely affecting activity, including, but not limited to, substituting one or more of the naturally occurring L-isomer forms of amino acids with the D-isomer forms of the amino acids. Thus, the peptides can contain one or more D-amino acid residues or can contain all amino acids in the D-form. Retro-inverso forms of the peptides of the presently disclosed subject matter are also contemplated, e.g., reverse peptides in which all amino acids are substituted with D-amino acid forms.

[0244] Substantially pure proteins obtained as described herein can be purified according to known procedures for protein purification, using immunological, enzymatic, or other assays to monitor the purification at each step of the procedure. Protein purification methods are well known in the art and are described, for example, in Deutscher et al., 1990.

[0245] As discussed, modification or optimization of the peptide ligands of the presently disclosed subject matter is within the scope of this application. Modified or optimized peptides are included in the definition of peptide binding ligands. Specifically, the identified peptide sequences can be modified to optimize their potency, pharmacokinetic behavior, stability, and / or other biological, physical, and chemical properties.

[0246] [Amino acid substitution] In certain embodiments, the disclosed methods and compositions may involve preparing peptides with one or more substituted amino acid residues.

[0247] In various embodiments, the structural, physical, and / or therapeutic characteristics of a peptide sequence can be optimized by substituting one or more amino acid residues.

[0248] Other modifications can also be incorporated without adversely affecting activity, including, but not limited to, substituting one or more of the naturally occurring L-isomer forms of amino acids with the D-isomer forms of the amino acids. Thus, the peptides can contain one or more D-amino acid residues or can contain amino acids that are all in the D form. Retro-inverso forms of the peptides of the presently disclosed subject matter are also contemplated, e.g., reverse peptides in which all amino acids are substituted with the D-amino acid form.

[0249] Those skilled in the art will recognize that, in general, amino acid substitutions in peptides typically involve replacing one amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions). The properties of various amino acids and the effects of amino acid substitutions on protein structure and function have been the subject of extensive study and knowledge in the art.

[0250] For example, the following isosteric and / or conservative amino acid changes can be made to the parent polypeptide sequence with the expectation that the resulting polypeptide will have a similar or improved profile of properties as described above:

[0251] Alkyl-substituted hydrophobic amino acid substitutions: include alanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid, S-cyclohexylalanine, or other simple α-amino acids substituted with C1-10 carbon aliphatic side chains, including branched, cyclic, and straight chain alkyl, alkenyl, or alkynyl substitutions.

[0252] Aromatic substituted hydrophobic amino acid substitutions: phenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy substituted forms of the aromatic amino acids listed above, specific examples of which are 2-, 3-, or 4-aminophenylalanine, 2-, 3-, or 4-chlorophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2'-, 3'- or 4'-amino-, 2'-, 3'- or 4'-chloro-, 2, 3 or 4-biphenylalanine, 2'-, 3'- or 4'-methyl-2, 3 or 4-biphenylalanine, and 2- or 3-pyridylalanine.

[0253] Substitution of amino acids containing basic functional groups: arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, alkyl, alkenyl, or aryl substitutions of the above amino acids (C1-C 10 Branched, straight chain, or cyclic) derivatives, including those in which the substitution is on a heteroatom (e.g., the alpha nitrogen, or the distal nitrogen) or on the alpha carbon, for example, in the pro-R position. Illustrative example compounds include N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine, N,N-gamma,gamma'-diethyl-homoarginine. Also included are compounds in which an alkyl group occupies the pro-R position of the alpha carbon, such as alpha methyl arginine, alpha methyl 2,3-diaminopropionic acid, alpha methyl histidine, alpha methyl ornithine. Also included are amides formed from alkyl, aromatic, heteroaromatic (wherein the heteroaromatic group has one or more nitrogen, oxygen, or sulfur atoms, alone or in combination) carboxylic acids, or any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives, and lysine, ornithine, or 2,3-diaminopropionic acid.

[0254] Substitutions for acidic amino acids: include alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of aspartic acid, glutamic acid, homoglutamic acid, tyrosine, 2,4-diaminopropionic acid, ornithine or lysine, and tetrazole substituted alkyl amino acids.

[0255] Substitutions of side chain amide residues: include asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine.

[0256] Substitutions for hydroxyl-containing amino acids: serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic substituted derivatives of serine or threonine. It is also understood that an amino acid within each of the above listed categories can be substituted with another amino acid from the same group.

[0257] For example, the hydropathic index of amino acids can be considered (Kyte and Doolittle, 1982). The relative hydropathic properties of amino acids contribute to the secondary structure of a resulting protein, which in turn determines the interactions of the protein with other molecules. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), which are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). When making conservative substitutions, it is preferred to use amino acids whose hydropathic index is within + / -2, more preferably within + / -1, and even more preferably within + / -0.5.

[0258] In amino acid substitutions, the hydrophilicity of the amino acid residues can also be taken into consideration (e.g., U.S. Pat. No. 4,554,101). Hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0); glutamic acid (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±0.1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is preferred to replace an amino acid with another amino acid having a similar hydrophilicity.

[0259] Other considerations include the size of the amino acid side chain. For example, it is generally not preferred to substitute an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, such as tryptophan or tyrosine. The effect of various amino acid residues on the secondary structure of the protein is also considered. Through empirical studies, the effect of different amino acid residues on the tendency of protein domains to adopt alpha-helices, beta-sheets, or reverse turns secondary structures has been determined and is known in the art (see, for example, Chou and Fasman, 1974; Chou and Fasman, 1978; Chou and Fasman, 1979).

[0260] Based on these considerations and extensive empirical studies, conservative amino acid substitution tables have been compiled and are known in the art, e.g., arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Alternatively, Ala(A)Leu, Ile, Val; Arg(R)Gln, Asn, Lys; Asn(N)His, Asp, Lys, Arg, Gln; Asp(D)Asn, Glu; Cys(C)Ala, Ser; Gln(Q)Glu, Asn; Glu(E)Gln, Asp; Gly(G)Ala; His(H)Asn, Gln, Lys, Arg; Ile(I)Val, Met, Ala, Phe, Leu; Leu(L)Val, Met, Ala, Phe, Ile;Lys(K)Gln, Asn, Arg;Met(M)Phe, Ile, Leu;Phe(F)Leu, Val, Ile, Ala, Tyr;Pr o(P)Ala;Ser(S), Thr;Thr(T)Ser;Trp(W)Phe, Tyr;Tyr(Y)Trp, Phe, Thr, Ser;Val(V)Ile, Leu, Met, Phe, Ala.

[0261] Other considerations for amino acid substitutions include whether the residue is located in the interior of the protein or exposed to solvent. For internal residues, conservative substitutions include Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp. See, for example, the PROWL Rockefeller University website. For solvent exposed residues, conservative substitutions include Asp and Asn; Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr. Various matrices have been constructed to aid in the selection of amino acid substitutions, such as the PAM250 score matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix, and Risler matrix (see, e.g., PROWL Rockefeller University website).

[0262] When determining amino acid substitutions, the presence of inter- or intramolecular bonds, such as ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or the formation of disulfide bonds between nearby cysteine ​​residues, may also be taken into consideration.

[0263] Methods for substituting any amino acid for any other amino acid in an encoded peptide sequence are well known and are a matter of routine experimentation for those skilled in the art, for example by the technique of site-directed mutagenesis or by synthesis and assembly of oligonucleotides encoding the amino acid substitution and splicing into an expression vector construct.

[0264] [Antibody formats and their preparation] Antibodies against the proteins, polypeptides, or peptide fragments thereof of the subject matter of the present disclosure can be generated using methods well known in the art. For example, U.S. Patent No. 5,436,157, incorporated herein by reference in its entirety, discloses a method for producing antibodies against peptides. For the production of antibodies, various host animals, including but not limited to rabbits, mice, and rats, can be immunized by injecting the polypeptide or a peptide fragment thereof. To enhance the immunological response, various adjuvants can be used depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels, such as aluminum hydroxide, surfactants, such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

[0265] In some embodiments, one or more antibodies or fragments thereof are used. In some embodiments, one or both antibodies are single chain, monoclonal, bispecific, synthetic, polyclonal, chimeric, human, or humanized, or active fragments or homologs thereof. In some embodiments, the antibody binding fragment is a F(ab')2, F(ab)2, Fab', or Fab fragment.

[0266] For preparation of monoclonal antibodies, any technique which provides for the production of antibody molecules by continuous cell lines in culture can be utilized. For example, human monoclonal antibodies can be produced using the hybridoma technique originally developed by Kohler and Milstein, the trioma technique, the human B-cell hybridoma technique (Kozbor and Roder, 1983), and the EBV hybridoma technique (Cole et al., 1985). In some embodiments, monoclonal antibodies are produced in germ-free animals.

[0267] In accordance with the subject matter of the present disclosure, human antibodies may be used by utilizing human hybridomas (Cote et al., 1983) or by transforming human B cells in vitro with the EBV virus (Cole et al., 1985). In addition, techniques developed for the production of "chimeric antibodies" by splicing genes from a mouse antibody molecule specific for an epitope of an SLLP polypeptide together with genes from a human antibody molecule of appropriate biological activity (Morrison et al., 1984; Neuberger et al., 1984; Takeda et al., 1985) may be used; such antibodies are within the scope of the subject matter of the present disclosure. Once a specific monoclonal antibody has been developed, preparation of mutants and variants thereof by conventional techniques is also available.

[0268] Various techniques have been developed for the production of antibody fragments of humanized antibodies. Traditionally, these fragments were obtained by proteolytic digestion of full-length antibodies (see, for example, Morimoto and Inouye, 1992; Brennan et al., 1985). However, these fragments can now be produced directly by recombinant host cells. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992a). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. See PCT International Patent Application WO 1993 / 16185; US Pat. Nos. 5,571,894, 5,587,458. Antibody fragments can also be "linear antibodies", as described, for example, in US Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0269] A humanized (chimeric) antibody is an immunoglobulin molecule that contains human and non-human portions. More specifically, the antigen-binding region (or variable region) of a humanized chimeric antibody is derived from a non-human source (e.g., mouse), and the constant region of the chimeric antibody (which confers biological effector functions to the immunoglobulin) is derived from a human source. A humanized chimeric antibody must have the antigen-binding specificity of the non-human antibody molecule and the effector functions conferred by the human antibody molecule. Numerous methods of producing chimeric antibodies are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 4,975,369, 5,075,431, 5,081,235, 5,169,939, 5,202,238, 5,204,244, 5,231,026, 5,292,867, 5,354,847; 5,472,693; 5,482,856; 5,491,088; 5,500,362; and 5,502,167). Detailed methods for preparing chimeric (humanized) antibodies can be found in U.S. Pat. No. 5,482,856. "Humanized" antibodies are human / non-human chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR residues being those of a human immunoglobulin sequence. The humanized antibody may optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.For further details, see, e.g., Jones et al., 1986; Riechmann et al., 1988; Presta, 1992, PCT International Patent Application Publication No. WO 92 / 02190, U.S. Patent Application Publication No. 2006 / 0073137, and U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,761; 5,693,762; 5,714,350; 5,766, See Nos. 886; 5770196; 5777085; 5821123; 5821337; 5869619; 5877293; 5886152; 5895205; 5929212; 6054297; 6180370; 6407213; 6548640; 6632927; 6639055; and 6750325.

[0270] In some embodiments, the presently disclosed subject matter provides fully human antibodies. Human antibodies are composed entirely of human characteristic polypeptide sequences. Human antibodies of the presently disclosed subject matter can be produced using a wide variety of methods (see, e.g., U.S. Pat. No. 5,001,065 for review).

[0271] Typically, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable domain. Humanization can be performed essentially according to the method of Winter and coworkers (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988), by substituting hypervariable region sequences of a human "acceptor" antibody for the corresponding sequences. Such "humanized" antibodies are thus chimeric antibodies in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species (see, e.g., U.S. Pat. Nos. 4,816,567 and 5,482,856). In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0272] Another method for making humanized antibodies is described in U.S. Patent Application Publication No. 2003 / 0017534, in which humanized antibodies and antibody preparations are produced from transgenic non-human animals that are genetically engineered to contain one or more humanized immunoglobulin loci that can undergo gene rearrangement and gene conversion in the transgenic non-human animal to produce a variety of humanized immunoglobulins.

[0273] In some embodiments, the choice of human variable domains (both light and heavy chains) used to generate a humanized antibody is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against a library of known human variable domain sequences or a library of human germline sequences. The human sequence that is closest to the rodent sequence can then be accepted as the human framework region of the humanized antibody (Sims et al., 1993; Chothia and Lesk, 1987). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., 1992b; Presta et al., 1993). Other approaches designed to reduce the immunogenicity of antibody molecules in human patients include veneered antibodies (see, e.g., U.S. Patent Application Publication No. 2002 / 0034765 and U.S. Patent Application Publication No. 2004 / 0253645) and antibodies modified by T-cell epitope analysis and removal (see, e.g., U.S. Patent Application Publication No. 2003 / 0153043 and U.S. Patent No. 5,712,120).

[0274] When an antibody is humanized, it is important that the antibody retains high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues from the recipient and import sequences can be selected and combined to achieve the desired antibody characteristic, such as increased affinity for the target antigen. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0275] The antibody portion of the presently disclosed subject matter can be a single chain antibody.

[0276] Fragments within the scope of the term "antibody" include those produced by digestion with various proteases, those produced by chemical cleavage and / or chemical dissociation, and those produced recombinantly, so long as the fragment is capable of specifically binding to a target molecule. Such fragments include Fab, Fab', Fv, and F(ab')2.

[0277] Antibody fragments containing the idiotype of an antibody molecule can be generated by known techniques. For example, such fragments include, but are not limited to, F(ab')2 fragments which can be generated by pepsin digestion of the antibody molecule, Fab' fragments which can be generated by reducing the disulfide bridges of F(ab')2 fragments, Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.

[0278] Monoclonal antibodies against full-length or peptide fragments of a protein or peptide can be prepared using any well-known procedure for preparing monoclonal antibodies, such as those described in Harlow and Lane, 1988; Tuszynski et al., 1988. Chemical synthesis techniques can also be used to synthesize quantities of a desired peptide. Alternatively, DNA encoding the desired peptide can be cloned and expressed from an appropriate promoter sequence in a suitable cell for the production of large amounts of peptide. Monoclonal antibodies against the peptide can be produced from mice immunized with the peptide, using standard procedures referenced herein.

[0279] For example, sites for amino acid substitutions in exemplary complementarity determining region (CDR) residues or sequences and / or framework regions (FR) of such humanized antibodies that have improved properties, such as lower immunogenicity, improved antigen binding or other functional properties, and / or improved physicochemical properties, such as, for example, greater stability, are provided.

[0280] The subject matter of the present disclosure encompasses more than the specific fragments and humanized fragments disclosed herein. In some embodiments, the antibody is selected from the group consisting of a single chain antibody, a monoclonal antibody, a bispecific antibody, a chimeric antibody, a synthetic antibody, a polyclonal antibody, or a humanized antibody, or an active fragment or homolog thereof.

[0281] Nucleic acids encoding the antibodies obtained using the procedures described herein can be cloned and sequenced using techniques available in the art, for example, as described in Wright et al., 1992 and the references cited therein. Further, antibodies of the disclosed subject matter can be "humanized" using the techniques described in Wright et al., 1992 and the references cited therein, and Gu et al., 1997.

[0282] To generate a phage antibody library, a cDNA library is first obtained from mRNA isolated from a cell, e.g., a hybridoma, expressing a desired protein, e.g., a desired antibody, expressed on the phage surface. A cDNA copy of the mRNA is generated using reverse transcriptase. cDNA specifying immunoglobulin fragments is obtained by PCR, and the resulting DNA is cloned into an appropriate bacteriophage vector to generate a bacteriophage DNA library containing DNA specifying immunoglobulin genes. Procedures for generating bacteriophage libraries containing heterologous DNA are well known in the art and are described, for example, in Green and Sambrook, 2012.

[0283] Bacteriophage encoding a desired antibody can be engineered to display the protein on its surface in such a way that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed. Thus, when bacteriophage expressing a particular antibody are incubated in the presence of a cell expressing the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophage that do not express the antibody will not bind to the cell. Such panning techniques are well known in the art.

[0284] A process such as the one described above was developed to produce human antibodies using M13 bacteriophage display (Burton and Barbas, 1994). Essentially, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes, and thus the cDNA encodes the same. The amplified cDNA is cloned into an M13 expression vector, creating a library of phages expressing human Fab fragments on their surface. Phages displaying the antibody of interest are selected by antigen binding and propagated in bacteria to produce soluble human Fab immunoglobulins. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes the DNA encoding the human immunoglobulins, rather than the cells expressing the human immunoglobulins.

[0285] The procedure presented here describes the generation of phage encoding the Fab portion of an antibody molecule. However, the subject matter of this disclosure should not be construed as being limited to the generation of phages encoding Fab antibodies only. A Fab molecule contains an entire Ig light chain, i.e., both the variable and constant regions of the light chain, but only the variable region and the first constant region domain (CH1) of the heavy chain. A single-chain antibody molecule contains a single chain of protein that contains an Ig Fv fragment. An Ig Fv fragment contains only the variable regions of the heavy and light chains of an antibody, without the constant regions. Panning of the phage so generated to isolate the desired antibodies is performed in a similar manner as described for a phage library containing Fab DNA.

[0286] The subject matter of this disclosure should also be construed to include synthetic phage display libraries in which heavy and light chain variable regions can be synthesized to encompass almost any possible specificity (Barbas, 1995; de Kruif et al., 1995).

[0287] In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, such as ELISA (enzyme-linked immunosorbent assay). Antibodies produced in accordance with the presently disclosed subject matter may include, but are not limited to, polyclonal, monoclonal, chimeric (i.e., "humanized"), and single chain (recombinant) antibodies, Fab fragments, and fragments produced by a Fab expression library.

[0288] In some embodiments, the antibodies, fragments, and variants of the present disclosure can be purified after expression in E. coli. By way of example and not limitation, E. coli, like mammalian cells, can be used as a host for recombinant protein production, including immunoglobulin fragments. E. coli can be used to produce large amounts of the antibodies and fragments of the presently disclosed subject matter (see, e.g., Verma et al., 1998).

[0289] In some embodiments, the E. coli strain used can be BL21 DE3, which expresses T7 polymerase, which transcribes DNA sequences up to 5 times faster than native E. coli RNA polymerase and allows for robust induction of protein expression with isopropyl β-D-1-thiogalactopyranoside (IPTG). In addition, BL21 DE3 lacks Lon protease (Gottesman, 1996) and the outer membrane protease OmpT (Grodberg and Dunn, 1988).

[0290] The antibodies, fragments, and derivatives thereof of the present disclosure can also be purified by affinity HPLC using a Protein L column, which preferentially binds the antibody light chain.

[0291] Substantially pure peptides obtained as described herein can be purified according to known procedures for protein purification, using immunological, enzymatic, or other assays to monitor the purification at each step of the procedure. Protein purification methods are well known in the art and are described, for example, in Deutscher et al., 1990.

[0292] In the field of recombinant humanized antibodies, it is common to graft murine CDR sequences onto well-established human immunoglobulin frameworks previously used in human therapy, such as the framework regions of Herceptin (Trastuzumab). In some embodiments, expression cassettes are used that encode one or more of heavy chain framework regions 1-4, including but not limited to those set forth as SEQ ID NOs:40-43, respectively, and / or light chain framework regions 1-4, including but not limited to those set forth as SEQ ID NOs:44-47, respectively. Nucleotide sequences encoding the CDRs disclosed herein (i.e., SEQ ID NOs:7, 9, and 11, which encode SEQ ID NOs:8, 10, and 12 for heavy chain CDRs 1 to 3, respectively, and SEQ ID NOs:19, 21, and 23, which encode SEQ ID NOs:20, 22, and 24 for light chain CDRs 1 to 3, respectively) can be introduced into such expression cassettes, such that full-length heavy chain variable regions and light chain variable regions, including but not limited to those encoded by SEQ ID NO:3 (encoding SEQ ID NO:4) and SEQ ID NO:15 (encoding SEQ ID NO:16), respectively, can be produced in host cells.

[0293] In some embodiments, when used in vivo for therapy, the antibodies or fragments and / or derivatives thereof of the presently disclosed subject matter are administered to a subject in a therapeutically effective amount (i.e., an amount that has the desired therapeutic effect). They are usually administered parenterally. The dose and administration schedule will depend on the extent of the infection, the characteristics of the particular antibody or immunotoxin used, e.g., its therapeutic index, the patient, and the patient's medical history. Advantageously, the antibody or immunotoxin is administered continuously over a period of 1-2 weeks. Optionally, administration is during the course of antimicrobial therapy or adjunctive therapy, such as administration of tumor necrosis factor, interferon, or other cytoprotective or immunomodulatory agents.

[0294] In some embodiments, for parenteral administration, the antibody or its fragment and / or derivative can be combined with a pharma- ceutically acceptable parenteral vehicle and formulated into a unit dose injectable form (solution, suspension, emulsion). Such vehicles are essentially non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes can be used as carriers. The vehicle may contain small amounts of additives such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives. The antibody is typically formulated in such vehicles at a concentration of about 1.0 mg / ml to about 10 mg / ml.

[0295] Pharmaceutical Compositions and Administration The presently disclosed subject matter is also directed to methods of administering an antibody, fragment, and / or derivative thereof of the presently disclosed subject matter to a subject.

[0296] Pharmaceutical compositions comprising the antibodies, fragments, and / or derivatives thereof of the present disclosure may be administered to a subject in need thereof by any number of routes, including, but not limited to, topical, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular (intraventricular), transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0297] According to some embodiments, methods of treating a subject in need of such treatment are provided. The methods include administering to a subject in need thereof a pharmaceutical composition comprising at least one antibody, fragment, and / or derivative of the presently disclosed subject matter. The antibodies, fragments, and / or derivatives of the presently disclosed subject matter can also be administered together with other known biologically active agents and / or other pharmaceutical agents.

[0298] Pharmaceutical compositions useful in practicing the presently disclosed subject matter may be administered to deliver a dose between 1 ng / kg / day and 100 mg / kg / day.

[0299] The subject matter of the present disclosure includes the preparation and use of pharmaceutical compositions that include as an active ingredient at least one antibody, fragment, and / or derivative thereof useful for the treatment of diseases and disorders disclosed herein. Such pharmaceutical compositions may consist solely of the active ingredient in a form suitable for administration to a subject, or the pharmaceutical composition may include the active ingredient and one or more pharma- ceutically acceptable carriers, one or more additional ingredients, or some combination thereof. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, e.g., in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0300] As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient that is compatible with any other ingredients of the pharmaceutical composition and is not harmful to the subject to which the composition is administered.

[0301] The compositions of the presently disclosed subject matter may include at least one active antibody, fragment, and / or derivative thereof, one or more acceptable carriers, and optionally other antibodies, fragments, and / or derivatives thereof, and / or therapeutic agents.

[0302] For in vivo applications, the antibodies, fragments, and / or derivatives of the presently disclosed subject matter may include pharma- ceutically acceptable salts. Suitable acids capable of forming such salts with the antibodies, fragments, and / or derivatives of the presently disclosed subject matter include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid, and the like.

[0303] Pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents, additives, solvents, and / or adjuvants. In some embodiments, the composition is sterile and non-pyrogenic. Examples of suitable carriers include, but are not limited to, water, normal saline, glucose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine ​​hydrochloride, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol, etc.), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, kaolin, agar and tragacanth, or mixtures of these substances.

[0304] The pharmaceutical compositions may also contain minor amounts of non-toxic auxiliary pharmaceutical substances or excipients and / or additives, such as wetting agents, emulsifying agents, pH buffering agents, antimicrobial and antifungal agents (parabens, chlorobutanol, phenol, sorbic acid, etc.). Suitable additives include, but are not limited to, physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additives (e.g., 0.01 to 10 mole percent) of chelating agents (e.g., DTPA or DTPA-bisamide, etc.), or additives (e.g., 1 to 50 mole percent) of calcium chelate complexes (e.g., calcium DTPA or CaNaDTPA-bisamide, etc.), or optionally additives of calcium or sodium salts (e.g., calcium chloride, calcium ascorbate, calcium gluconate, or calcium lactate). Absorption enhancers or absorption delayers (e.g., liposomes, aluminum monostearate, or gelatin) can be used, if desired. The compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension prior to injection, or as emulsions. Preparation of pharmaceutical compositions according to the presently disclosed subject matter can be by methods well within the purview of those skilled in the art.

[0305] The antibodies, fragments, and / or derivatives, pharma- ceutically acceptable salts, or pharmaceutical compositions comprising the antibodies, fragments, and / or derivatives of the presently disclosed subject matter can be administered such that the antibodies, fragments, and / or derivatives provide the desired physiological effect. Administration can be enteral or parenteral; for example, orally, rectally, intracisternally, intravaginally, intraperitoneally, topically (e.g., using powders, ointments, or drops), or as a buccal or intranasal spray or aerosol. In some embodiments, administration is parenteral. Exemplary parenteral administration methods include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intraarterial bolus injection, intraarterial infusion, and catheter infusion into the vascular system), injection around and into a target tissue, deposition including subcutaneous injection or infusion (such as by osmotic pumps), intramuscular injection, and direct application to a target area, for example, by a catheter or other placement device.

[0306] When administration of the antibodies, fragments, and / or derivatives thereof is by injection or direct application, the injection or direct application may be a single dose or multiple doses. When administration of the antibodies, fragments, and / or derivatives thereof is by infusion, the infusion may be a single continuous dose or multiple infusions over an extended period of time.

[0307] The formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or hereafter developed. In general, such preparation methods include the step of combining the active ingredient with the carrier or one or more other accessory ingredients and then, if necessary or desirable, shaping or packaging the product into the desired single or multiple dosage unit.

[0308] Those skilled in the art will appreciate that such pharmaceutical compositions are generally suitable for administration to any type of animal. Subjects to which administration of the pharmaceutical compositions of the presently disclosed subject matter is contemplated include, but are not limited to, humans and other primates, mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, cats, and dogs, and birds, including commercially relevant birds such as chickens, ducks, geese, and turkeys.

[0309] The pharmaceutical compositions of the presently disclosed subject matter may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, or a convenient fraction of such a dosage, for example, one-half or one-third of such a dosage.

[0310] The relative amounts of active ingredient, pharma- ceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the presently disclosed subject matter will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is to be administered. By way of example, the composition may contain from 0.1% to 100% (w / w) active ingredient.

[0311] In addition to the active ingredient, the pharmaceutical compositions of the presently disclosed subject matter may further comprise one or more additional pharma- ceutical active agents. Particularly contemplated additional agents include antiemetic agents and scavengers, such as cyanide and cyanate scavengers.

[0312] Controlled- or sustained-release formulations of the pharmaceutical compositions of the presently disclosed subject matter can be prepared using conventional techniques.

[0313] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: additives; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharma-ceutically acceptable polymers or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the presently disclosed subject matter are known in the art and are described, for example, in Genaro, 1985, which is incorporated herein by reference.

[0314] Typically, the dosage of the compound of the presently disclosed subject matter that may be administered to an animal, in some embodiments a human, ranges from 1 μg to about 100 g per kg of animal body weight. The exact dosage administered will vary depending on many factors, including, but not limited to, the type of animal and the type of condition being treated, the age of the animal, and the route of administration. In some embodiments, the dosage of the compound will vary from about 1 mg to about 10 g per kg of animal body weight. In another embodiment, the dosage will vary from about 10 mg to about 1 g per kilogram of animal body weight.

[0315] The compound can be administered to the animal as frequently as several times per day, or can be administered once a day, once a week, once every two weeks, once a month, etc., or less frequently, such as once every few months or once a year or less. The frequency of administration will be readily apparent to one of skill in the art and will depend on any number of factors, including, but not limited to, the type of cancer being diagnosed, the type and severity of the condition or disease being treated, the species and age of the animal, etc.

[0316] Suitable preparations include injectables as liquid solutions or suspensions, although solid forms suitable for dissolving or suspending in liquid prior to injection can also be prepared. Preparations can also be emulsified, or the polypeptides can be encapsulated in liposomes. The active ingredient is often mixed with additives that are pharma-ceutically acceptable and compatible with the active ingredient. Suitable additives are, for example, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, vaccine preparations can also contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants.

[0317] The presently disclosed subject matter also includes kits that include a composition of the presently disclosed subject matter and instructional materials that describe the extracellular administration of the composition to a cell or tissue of a subject. In some embodiments, the kits include a suitable (in some embodiments, sterile) solvent for dissolving or suspending a composition of the presently disclosed subject matter prior to administering the compound to a subject.

[0318] [Idiopathic pulmonary fibrosis] Fibronectin (Fn) has been identified as a potential target in early-onset idiopathic pulmonary fibrosis (IPF). IPF causes scarring and tissue thickening similar to that seen in other forms of respiratory disease, making it difficult to pinpoint the exact cause of the disease. A possible pathway in the progression of fibrosis involves a distorted conformation of Fn. The subject matter of the present disclosure provides an antibody called H5 that binds to a disease model, a distorted conformation of Fn called FnIII9-4G-10 (4G). A representative antibody that binds better to 4G than H5 is also provided herein. By way of example and not limitation, a diverse library of antibodies was developed by introducing mutations into the H5 sequence by mutagenesis and amplifying by error-prone PCR. This library was then screened using phage display and multiple rounds of ELISA. Clonal strength was quantified by absorbance ratio, with higher ratios indicating better binding performance. Testing allowed the identification of 23 clones that outperformed H5; however, upon sequencing of the clones, four were identified as distinct from H5.

[0319] Pulmonary fibrosis (PF) is a disease in which lung tissue thickens, stiffens, and scars over time. Scarred lung tissue impedes the transfer of oxygen from the lungs into the bloodstream, thereby reducing the amount of available oxygen circulating through the body (see, e.g., National Heart, Lung, and Blood Institute website; King et al., 2011). PF is further characterized by the deposition of extracellular matrix (ECM) proteins, such as fibronectin (Fn), which contributes to the destruction of functional alveolar units, leading to respiratory failure (Datta et al., 2011). Controllable amounts of ECM production can benefit the body, such as through scar tissue for injury healing, but uncontrolled amounts can be fatal, such as in idiopathic pulmonary fibrosis (IPF; Raghu and Mitocenic, 2018). IPF is one of at least 200 different forms of PF and is a chronic fibrotic condition that causes an irreversible decline in lung function. IPF affects 1 in 200 adults over the age of 65, with 50,000 adults diagnosed each year and a further 40,000 deaths from IPF (Pulmonary Fibrosis Foundation, 2018). Immediate treatment is needed to slow the progression of IPF. Therefore, it is essential that research continues to better detect and understand the pathways of IPF.

[0320] The representative H5 antibody of the present disclosure binds to a distorted conformation of Fn. Two different fragments were designed to model the distorted and normal conformations of the relevant region of Fn. The distorted form of Fn, FnIII9-4G-10 (4G), contains the addition of four glycines between the ninth and tenth type III repeats, a mutation that reduces the binding affinity of Fn to α5β1 integrin. The engineered Fn was used in experiments to model changes in response to mechanical forces in pathological conditions and to demonstrate the existence of an integrin switch in Fn. Normal FnIII9 * 10(9 *10) expresses the normal folding of Fn in the ninth and tenth type III repeats. Thus, modifications of the H5 antibody to improve binding to distorted Fn and to inhibit or limit binding to normal Fn are provided in accordance with the subject matter of the present disclosure through directed evolution, phage display, and ELISA.

[0321] As disclosed herein, an exemplary H5 antibody of the presently disclosed subject matter has been identified as having a "maximal" binding affinity to 4G. However, the binding did not reach the "absolute maximum", i.e., the best possible binding state. The basis of directed evolution is to create a library with the greatest genetic diversity to reach a fitness peak. Thus, also disclosed herein are experiments to develop a library of H5 antibody clones sufficiently diverse to reach the "absolute maximum" binding affinity to 4G. In some embodiments, random mutagenesis is used to introduce random mutations into the present H5 antibody DNA sequence (Cadwell and Joyce, 1992).

[0322] Phage display technology improved the binding affinity of the H5 clone to 4G,9 * 10. Phage display is a process used to display polypeptides on the surface of temperate filamentous bacteriophage through manipulation of the phage genotype (Bazan et al., 2012). Its use in antibody generation was developed within a larger framework called directed evolution, which uses a trial-and-error approach to introduce genetic mutations into antibodies to enhance at least one antibody's target-binding ability (Trafton, 2010). This technique was recently made famous by its contributors receiving the Nobel Prize in Chemistry (Offord and Grens, 2018). Through this framework, in some embodiments, modified antibodies and antigen-binding fragments thereof are provided through optimization of the diversity of antibody libraries by mutagenesis through error-prone PCR, while also ensuring that protein adaptations can be successfully generated on a faster timescale.

[0323] Finally, ELISA was performed (see Example 1) and clones identified as superior to H5 were isolated from the phage for further antibody characterization. The clones were sequenced as disclosed herein. EXAMPLES

[0324] The following examples provide illustrative embodiments. In view of this disclosure and the general level of skill of those skilled in the art, those skilled in the art will understand that the following examples are for illustrative purposes only, and that numerous changes, modifications, and variations can be made without departing from the scope of the subject matter of the present disclosure.

[0325] Example 1 ELISA Analysis of Various Exemplary Antibodies and scFvs Unmodified H5 antibodies were produced in either E. coli (shown as eH5 and H5(scFv) in FIG. 1) or Nicotiana benthamiana (nicoH5). Modified H5-IgG1 (H5-IgG) antibodies of the presently disclosed subject matter were produced in a mammalian hybridoma cell line known as plug-and-(dis)play hybridoma (PnP).

[0326] An in vitro ELISA binding assay was used to determine the potency of binding of the H5 antibody and its derivatives to diseased and healthy forms of fibronectin. Maleimide-activated ELISA plates were used to determine the binding potency of FN-4G-10 and FN-9. * The recombinant fibronectin fragments, named H5-10, were linked to mimic the diseased and normal forms of FN, respectively. -5 Starting with M and 10 -11 Different molar concentrations ending in M ​​were used. Bound antibody was detected by colorimetric method.

[0327] The results are shown in Figure 1. Data represent a combination of two independent experiments, one comparing H5-scFv produced in E. coli (eH5) and N. benthamiana (nicoH5), and the other comparing H5-scFv (scFv) and H5-IgG1 (H5-IgG). Between the two H5 formats tested, the modified IgG1 format of H5 showed even higher affinity for the FN fragment, with at least 50-fold higher binding compared to when equimolar concentrations of antibody were used. Furthermore, FN-9 * Two fragments were identified that showed higher affinity for FN-4G-10 (diseased) compared to -10 (healthy).

[0328] [References] All references cited in this disclosure, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (including but not limited to UniProt, EMBL, and GENBANK® biological sequence database entries and all available annotations therein), are hereby incorporated by reference in their entirety to the extent that they supplement, explain, provide background for, and / or teach the methodologies, techniques, and / or compositions used herein. The discussion of the references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or any portion of any reference) is relevant prior art. Applicant reserves the right to challenge the accuracy and pertinence of the cited references.

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[0330] Although the presently disclosed subject matter has been disclosed with reference to particular embodiments, it will be apparent that other embodiments and variations of the presently disclosed subject matter may be devised by those skilled in the art without departing from the true spirit and scope of the presently disclosed subject matter.

Claims

【Claim 1】 One invention described in the specification.

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