Modified thymic stromal lymphopoietin compositions and methods of preparation and use thereof

Abstract

Provided here are thymic stromal lymphopoietin (TSLP) compositions and methods to achieve an improved bloodstream half-life for TSLP. Compositions include a covalently attached polymer conjugate of TSLP. The methods include covalently attaching a polymer conjugate to reduce the rate of proteolysis and renal clearance associated with unmodified TSLP and treating a patient in need of treatment with a TSLP-related condition.

Description

MODIFIED THYMIC STROMAL LYMPHOPOIETIN COMPOSITIONS AND METHODS OF PREPARATION AND USE THEREOF Inventors: Kimberly J. Payne; Marvin A. Payne CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 511,478 filed on June 30, 2023, which is incorporated by reference herein in its entirety. GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under grant number R43CA224723 awarded by National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The disclosure relates to modified thymic stromal lymphopoietin (TSLP) compositions and methods for the modification of TSLP to treat disease. BACKGROUND

[0004] TSLP promotes T helper type 2 (TH2) cell responses that are associated with immunity in various inflammatory diseases, including asthma, allergic inflammation, and chronic obstructive pulmonary disease. The shorter (predominant) isoform is an antimicrobial protein, displaying antibacterial and antifungal activity against a variety of organisms, including B. cereus, E. coli, E. faecalis, S. mitis, S. epidermidis, and C. albicans. Alternative splicing of this TSLP gene results in multiple transcript variants. TSLP forms a ternary signaling complex with cytokine receptor-factor 2 (CLRF2) and interleukin-7 receptor-α (Il-7R^), is capable of stimulating cell proliferation through activation of STAT3, STAT5 and JAK2 pathways, and is implicated in the development of the hematopoietic system. TSLP is considered a potentialtherapeutic agent for the treatment of diseases, such as B cell acute lymphoblastic leukemia, which are linked to activation of the TSLP, CLRF2, IL-7R^ signaling complex, as previously described in US Patent Publication No. 20210077581. Exposing CRLF2 leukemia cells to elevated TSLP levels has been shown to decrease both cell viability as well as surface levels of IL-7R^ and CRLF2 in a dose-dependent manner. Additionally, elevated TSLP levels in patient- derived xenograft mice generated from human CRLF2 B-ALL primary bone marrow (BM) cells resulted in increased survival time and decreased leukemia burden as evidenced by decreased spleen mass in the high-TSLP treatment group. TSLP is a naturally occurring human protein and lacks the pan-cytotoxic effects of standard chemotherapy.

[0005] A limitation of therapeutic strategies using naturally occurring human proteins is the relatively short bloodstream half-life. This limitation reduces bioavailability and overall efficacy of treatment. SUMMARY

[0006] Applicants recognized a need for compositions and methods for improving the bloodstream half-life of TSLP and its substituent and analogous forms. In one aspect, a method is provided for preparing a modified human thymic stromal lymphopoietin (TSLP) to achieve an improved half-life. The method includes providing a TSLP and a polymer conjugate to a reaction vessel, allowing a conjugation reaction to proceed under predetermined reaction conditions to attach the polymer conjugate to the TSLP to produce a modified TSLP, and isolating the modified TSLP. In certain embodiments, TSLP is a naturally occurring or a recombinant protein. The method may include where a bloodstream half-life of the modified TSLP is increased relative to a bloodstream half-life of the TSLP. The increase in the bloodstream half-life of the modified TSLP is at least 200%, in certain embodiments. The TSLP can be a full length protein or afragment thereof. The TSLP can include an intact, ternary TSLP-CRLF2-IL7R complex associated with biological activity. The polymer conjugate can contain one or more of a polyether, a polysaccharide, or a biliprotein. The method may include steps of attaching a substantial amount of the polymer conjugate to a N-terminus of the TSLP. The method may include modified TSLP with a molecular weight of at least 19 kilodaltons (kDa). The method may also include a conjugation reaction, which can be a site selective enzymatic, chemical, an atom transfer radical polymerization, or a reversible addition fragmentation chain-transfer polymerization conditions.

[0007] Embodiments include modified TSLP compositions having an improved half-life. The composition includes a TSLP with a covalently bonded polymer conjugate. The modified TSLP can be present in an amount ranging from about 50% to about 99% by weight, and a covalently bonded polymer conjugate in an amount ranging from about 1% to about 50% by weight. The modified TSLP composition may include a modified TSLP with a molecular weight of at least 19 kDa. The modified TSLP composition may include a full-length TSLP protein or a fragment thereof. The modified TSLP composition may include a fragment of the full-length TSLP protein and an intact complex that provides biological activity. The modified TSLP composition may also include a polymer conjugate containing one or more of a polyether, a polysaccharide, or a biliprotein.

[0008] Embodiments include methods of treating a subject in need of treatment for a TSLP- related condition with a modified TSLP composition. The method includes administering a therapeutically effective amount of a TSLP with a covalently bonded polymer conjugate. The TSLP is present in the modified TSLP compositions in an amount ranging from about 50% to about 99% by weight, and the polymer conjugate is present in an amount ranging from about 1% to about 50% by weight. The modified TSLP compositions can contain a full-length TSLP proteinor a fragment thereof. The method can include modified TSLP compositions further containing an intact, ternary TSLP-CRLF2-IL7R complex. The polymer conjugate of the modified TSLP compositions can contain one or more of a polyether, a polysaccharide, or a biliprotein. The TSLP-related condition can be asthma, allergic inflammation, cancer, or chronic obstructive pulmonary disease. The TSLP-related condition can be a leukemia. The TSLP-related condition can be a solid tumor, such as in a cervical, a lung, or an ovarian cancer. The TSLP-related condition can include an abnormal expression of any ternary receptor complex involving binding of TSLP to cognate receptors.

[0009] Aspects and advantages of these exemplary embodiments and other embodiments, are discussed in detail herein. Moreover, it is to be understood that both the foregoing information and the following detailed description provide merely illustrative examples of various aspects and embodiments, and are intended to provide an overview or framework for understanding the nature and character of the claimed aspects and embodiments. Accordingly, these and other objects, along with advantages and features of the present disclosure, will become apparent through reference to the following description and the accompanying drawings. Furthermore, it is to be understood that the features of the various embodiments described herein are not mutually exclusive and may exist in various combinations and permutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings, which are included to provide a further understanding of the embodiments of the present disclosure, are incorporated in and constitute a part of this specification, illustrate embodiments of the present disclosure, and together with the detailed description, serve to explain principles of the embodiments discussed herein. No attempt is made to show structural details of this disclosure in more detail than may be necessary for afundamental understanding of the embodiments discussed herein and the various ways in which they may be practiced.

[0011] FIG. 1 presents the results of the reaction of TSLP with PEG at various pH and PEG:rhTSLP molar ratios following a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis, according to certain embodiments. As used herein, rhTSLP is human full-length recombinant TSLP.

[0012] FIG.2 presents the results following a SDS-PAGE analysis of the reaction of TSLP with PEG under various pH and reaction times to produce a mono-PEGylated TSLP, according to certain embodiments.

[0013] FIG.3 is a graphical representation of the purification of the reaction products of TSLP with PEG to isolate a mono-PEGylated TSLP fraction, according to certain embodiments.

[0014] FIG. 4 is a graphical representation of the N-terminus sequence analysis of mono- PEGylated rhTSLP, according to certain embodiments.

[0015] FIG. 5 is a graphical representation of the bioactivity of a mono-PEGylated rhTSLP, according to certain embodiments.

[0016] FIG. 6 presents the effect of pH on yield and selectivity of PEGylation for various molecular weight PEGs following a SDS-PAGE analysis, according to certain embodiments.

[0017] FIG. 7 illustrates the isolation of mono-PEGylated rhTSLP using 5 kilodalton (kDa) PEG, according to certain embodiments.

[0018] FIG.8 illustrates the isolation of mono-PEGylated rhTSLP using 10 kDa PEG, according to an embodiment.

[0019] FIG. 9 is a graphical representation of the bioactivity analysis of mono-PEGylated rhTSLP using 10 kDa PEG, according to an embodiment.

[0020] FIG. 10 is a graphical representation of the activation assay of the 10-kDa-PEG-TSLP derivative, according to an embodiment. DETAILED DESCRIPTION

[0021] The relatively short half-life of naturally occurring human proteins provides challenges for their clinical utility as a recombinant biologic therapy. As such, the present disclosure provides more physiologically stable versions or conjugated derivatives of TSLP having a prolonged bloodstream half-life. For example, disclosed herein are compositions and methods for preparing a TSLP to achieve an improved half-life. In an embodiment, a method for preparing a modified TSLP to achieve an improved half-life includes providing a TSLP and a polymer conjugate to a reaction vessel, allowing a conjugation reaction to proceed under predetermined reaction conditions to attach the polymer conjugate to the TSLP to produce a modified TSLP, and isolating the modified TSLP. In an embodiment, the TSLP is a naturally occurring or recombinant protein, where the TSLP is a full-length protein, or a fragment thereof. The method may also include the TSLP containing an intact complex that provides biological activity. Certain benefits of the presently disclosed compositions and methods desirably increase the size or molecular weight of TSLP via derivatization or conjugation reactions to produce a longer bloodstream half-life, improved biological bioavailability, and overall efficacy of treatment, thereby facilitating novel therapeutic strategies.

[0022] The description may use the phrases “in certain embodiments,” “in various embodiments,” “in an embodiment,” or “in embodiments,” which may each refer to one or more of the same or different embodiments. Furthermore, the terms “comprising,” “including,” “having,” and the like, as used with respect to embodiments of the present disclosure, are synonymous. The terms “about” or “approximately” are defined as being close to as understoodby one of ordinary skill in the art. In one non-limiting embodiment, the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. The terms “removing,” “removed,” “reducing,” “reduced,” or any variation thereof, when used in the claims and / or the specification includes any measurable decrease of one or more components in a mixture to achieve a desired result. The use of the words “a” or “an” when used in conjunction with any of the terms “comprising,” “including,” “containing,” or “having,” in the claims or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The term “plurality” as used herein refers to two or more items or components. The terms “wt.%”, “vol.%”, or “mol.%” refers to a weight, volume, or molar percentage of a component, respectively, based on the total weight, the total volume of material, or total moles, that includes the component. In a non-limiting example, 10 grams of component in 100 grams of the material is 10 wt.% of component.

[0023] Disclosed here are compositions and methods for preparing a TSLP composition to achieve an improved half-life. One such method includes the steps of providing a TSLP and a polymer conjugate to a reaction vessel, allowing a conjugation reaction to proceed under predetermined reaction conditions to attach the polymer conjugate to the TSLP to produce a modified TSLP, and isolating the modified TSLP. In an embodiment, the TSLP is a naturally occurring or a recombinant protein, and the TSLP can be a full-length protein, or a fragment thereof. The form of TSLP used in certain derivatization experiments related to this disclosure is human full-length recombinant TSLP (rhTSLP) derived from E. coli. Further details thereof may be found in the Uniprot KB / Swissprot database as Q969D9. The predicted molecular mass of the form is about 15 kilodaltons (kDa). The form used does not contain the human signal sequence (MFPFALLYVL SVSFRKIFIL QLVGLVLT) at the N-terminus. The form used is Tyr29-Gln159 of the above sequence, with an N-terminal Met added in front of the TYR29. Certain benefits ofthe present disclosure desirably increase the size of TSLP via derivatization or conjugation reactions to produce a longer half-life in the bloodstream.

[0024] It should be understood that certain TSLP fragments having sufficient size and / or complexes for retaining biological activity may be derivatized for improved half-life via the present techniques. For example, from analysis of the 3-D structure of the ternary TSLP-CRLF2- IL7R complex in the art, it is likely that fragmenting the protein beyond a threshold may cause loss of biological activity, at least in part because the ternary TSLP-CRLF2-IL7R complex should be intact to support biological activity. However, a form of TSLP with a portion removed therefrom has been shown to be biologically active. As such, the present disclosure may incorporate any biologically active derivatives of any fragments of TSLP as part of the modified TSLP. In an embodiment, the TSLP includes an intact complex that provides or is associated with biological activity.

[0025] In certain embodiments, the polymer conjugate contains one or more of a polyether, a polysaccharide, or a biliprotein. In certain embodiments, the covalently bound prosthetic or polymer conjugate contains a water soluble, biocompatible polymer with low toxicity. In certain embodiments, polyethers, such as polyethylene glycol (PEG), are water-soluble polymers approved by the Food and Drug Administration for use in oral, topical, and intravenous drug formulations. In certain embodiments, the polyethers may include any suitable, biologically compatible polyethers. Some non-limiting examples include linear polyether polyol (PEP) (as described for example in Li Z, Chau Y, Synthesis of linear polyether polyol derivatives as new materials for bioconjugation. Bioconjug Chem. 2009 Apr;20(4):780-9. doi: 10.1021 / bc900036f. PMID: 19275208), linear polyglycerols (as described for example in Thomas A, Müller SS, Frey H. Beyond poly(ethylene glycol): linear polyglycerol as a multifunctional polyether for biomedical and pharmaceutical applications. Biomacromolecules. 2014 Jun 9;15(6):1935-54.doi: 10.1021 / bm5002608. Epub 2014 May 29. PMID: 24813747.), and various linear and branched derivatives of polyglycerols (as described for example in Weinhart M, Grunwald I, Wyszogrodzka M, Gaetjen L, Hartwig A, Haag R. Linear poly(methyl glycerol) and linear polyglycerol as potent protein and cell resistant alternatives to poly(ethylene glycol). Chem Asian J. 2010 Sep 3;5(9):1992-2000. doi: 10.1002 / asia.201000127. PMID: 20602410, and in Zamboulis A, Nakiou EA, Christodoulou E, Bikiaris DN, Kontonasaki E, Liverani L, Boccaccini AR. Polyglycerol Hyperbranched Polyesters: Synthesis, Properties and Pharmaceutical and Biomedical Applications. Int J Mol Sci. 2019 Dec 9;20(24):6210. doi: 10.3390 / ijms20246210. PMID: 31835372; PMCID: PMC6940955.). In certain embodiments, the PEG or other polyether has a linear, branched, multi-arm, or y-branched structure. In certain embodiments, the PEG or other polyether is a homopolymer or copolymer. In certain embodiments, the ratio of co- monomers is altered to achieve desired physical properties such as water solubility, hydrodynamic volume, proteolysis rate, renal clearance, or molecular weight of the resulting PEG or other polyether. Synthetic polymers, such as PEG, or synthetically modified natural polymers have molecular weight distributions. In certain embodiments, the conjugate polymer is monodisperse with a polydispersity index of 1, or polydisperse with a polydispersity index greater than 1. In some embodiments, TSLP can also be reacted with polysaccharide variants such as hydroxyethyl starch (HES) (as described for example in Liebner R, Mathaes R, Meyer M, Hey T, Winter G, Besheer A. Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra. Eur J Pharm Biopharm. 2014 Jul;87(2):378-85. doi: 10.1016 / j.ejpb.2014.03.010. Epub 2014 Mar 27. PMID: 24681396.). In some embodiments, TSLP can also be subject to chemical glycosylation (as described for example in Walther R, Zelikin AN. Chemical (neo)glycosylation of biological drugs. Adv Drug Deliv Rev. 2021 Apr;171:62-76. doi: 10.1016 / j.addr.2021.01.021. Epub 2021 Feb 3. PMID:33548302.). Certain embodiments of polymer conjugate may include one or more of PEG, linear PEP, linear polyglycerols, various linear and branched derivatives of polyglycerols, HES, and chemical glycosylation. The polymer conjugates listed herein are provided as non-limiting examples. Certain embodiments of polymer conjugate may include any suitable polymer that may be attached to TSLP according to the present techniques to increase TSLP half-life in vivo and / or to preserve TSLP biological activity.

[0026] In certain embodiments, a substantial amount of the polymer conjugate is attached to a N-terminus of the TSLP. The derivatized TSLP or modified TSLP can have a molecular weight of at least 19 kDa. In embodiments, the range of molecular weights for the derivatized TSLP or modified TSLP can range from 19 kDa to 85 kDa. In some embodiments, the modified TSLP is a TSLP derivative having a 10 kDa conjugate polymer (such as PEG or another suitable conjugate polymer). The modified TSLP can have a total molecular weight of about 25 kDa. In some embodiments, the modified TSLP includes a 20 kDa conjugate polymer. In embodiments, the modified TSLP includes a conjugate polymer having a weight ranging from 5 kDa to 40 kDa. The method may also include where the conjugation reaction is a site selective enzymatic, chemical, an atom transfer radical polymerization, or a reversible addition fragmentation chain- transfer polymerization. In embodiments, the conjugation reaction is a PEGylation reaction or corollary thereof. The conjugation reaction of certain embodiments is a polymerization reaction that adds polymers or components thereof to the TSLP via any suitable step-growth polymerization and / or chain polymerization mechanisms. In certain embodiments, the modified TSLP has a distribution of linkage points between the N-terminus, amino acid side chain functional groups, or the C-terminus of the protein. In certain embodiments, the conjugate polymer has one or more functional chain ends to facilitate reaction with the N-terminus, C- terminus, or amino acid side chains to TSLP. In certain embodiments, targeting the N-terminusfor reaction with the conjugate polymer is beneficial based on its distance from the regions of the TSLP molecule that interact with cognate receptor regions. Modifications in this region may be less likely to interfere with TSLP interactions with its receptor, which would otherwise make TSLP biologically inactive. Reaction with the conjugate polymer at the N-terminus also facilitates production of higher purity in PEGylated TSLP derivatives. This benefit arises because the polymerization or conjugation reaction may primarily be directed toward free amino groups. The availability of the N-terminal amino group presents a unique chemical environment that can be targeted for addition of the conjugate polymer with specific reaction conditions, as disclosed herein.

[0027] In one aspect, a TSLP composition has an improved half-life. As presently recognized, the TSLP composition produced via the techniques disclosed herein desirably has several properties, such as an improved half-life circulating in the blood, improved biological activity in cell-culture activation assays, and / or anti-cancer properties in leukemia and solid tumors. The increased half-life further facilitates increased serum levels of TSLP for improved therapeutic treatments, which may implement a less frequent dosing schedule. The composition can include a TSLP with a covalently bonded polymer conjugate. The TSLP can be present in an amount ranging from about 50% to about 99% by weight. The covalently bonded polymer conjugate can be present in an amount ranging from about 1% to about 50% by weight. The modified TSLP composition may include a modified TSLP with a molecular weight of at least 19 kDa. The modified TSLP composition may include a full-length protein or a fragment thereof. The modified TSLP composition may also include a polymer conjugate containing one or more of a polyether, a polysaccharide, or a biliprotein. In certain embodiments, the molecular weight increases from that of TSLP, approximately 15 kDa, to greater than 85 kDa with the addition of a conjugate. In certain embodiments, the molecular weight of the modified TSLP composition isselected to meet defined half-life requirements. In certain embodiments, the modified TSLP composition includes a TSLP in an amount ranging from about 1% to about 50% by weight. In embodiments, the modified TSLP composition includes a TSLP in an amount ranging from about 40%, 45%, 50%, 65%, 70%, 75% to about 99% by weight. In embodiments, the modified TSLP composition includes a TSLP in an amount of at least 80%, 85%, 90%, or 95% by weight. In some embodiments, the modified TSLP composition is selectively purified to include a TSLP in an amount of at least 90% by weight. The modified TSLP composition may also include TSLP, its substituent, or analogous forms. In certain embodiments, the modified TSLP composition has a distribution of linkage points with the conjugate polymer, and is one or more of the N-terminus, amino acid side chain functional groups, or the C-terminus of the protein. In certain embodiments, the conjugate polymer has one or more functional chain ends to facilitate reaction with the N-terminus, C-terminus, or amino acid side chains to TSLP. In certain embodiments, the TSLP-containing composition can include a protein similar or identical to SEQ ID NO.1, or a portion thereof. In certain embodiments, the TSLP-containing composition can include a protein similar or identical to SEQ ID NO.2, or a portion thereof. The TSLP Sequence for R&D Systems (Biotechne) Human TSLP produced in E. coli is available as Catalog # 1398-TS. The sequence is Tyr-29 – Gln 159 with an N-terminal Met. This is the mature, exported protein with the signal sequence removed and a Met added at the N-terminus for expression in E. coli.

[0028] SEQ ID NO.1: FASTA Sequence of Q969D9.1 >sp|Q969D9.1|TSLP_HUMAN RecName: Full=Thymic stromal lymphopoietin; Flags: Precursor MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFN NTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMK KRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ

[0029] SEQ ID NO. 2: Amino acid sequence of the recombinant human TSLP as expressed in E. coli. Total is 132 Amino AcidsMYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGC ASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWR RFNRPLLKQQ

[0030] Certain embodiments include methods of treating a subject in need of treatment for a TSLP-related condition with a modified TSLP composition. Such treatment includes administering a therapeutically effective amount of a TSLP with a covalently bonded polymer conjugate composition. The TSLP can be present in an amount ranging from about 50% to about 99% by weight. The covalently bonded polymer conjugate can be present in an amount ranging from about 1% to about 50% by weight. The TSLP can be a full length protein or a fragment thereof. The polymer conjugate can contain one or more of a polyether, a polysaccharide, or a biliprotein. In certain embodiments, the modified TSLP composition has a covalently bonded polymer conjugate in an amount ranging from about 50% to about 99% by weight. In certain embodiments, the TSLP-related condition includes asthma, allergic inflammation, or chronic obstructive pulmonary disease. The TSLP-related condition can be a leukemia. One example is acute lymphoblastic leukemia (ALL). The TSLP-related condition can be a solid tumor, such as in a cervical, a lung, or an ovarian cancer. The TSLP-related condition can include an abnormal expression of any ternary receptor complex involving binding of TSLP to cognate receptors. As a non-limiting example, there are several known genetic mechanisms by which CRLF2 may be overexpressed and cause disease. One such mechanism was identified as the P2RY8-CRLF2 fusion which was present in 7% of individuals with B-progenitor ALL and 53% of individuals with Down syndrome associated ALL. The modified TSLP compositions can be administered as a therapeutic for patients with Down syndrome associated ALL that results from CRLF2 overexpression.

[0031] A composition's bloodstream half-life indicates how long it takes for an administered dose to be reduced by half in the bloodstream, either through proteolysis and elimination or elimination alone. The half-life of TSLP evaluated at different doses is shown in Table 1. No statistically significant difference in the half-life of TSLP was found when given intraperitoneally (i.p.) vs. intravenously (i.v.), indicating that, when given by the i.p. route, there is no “flip-flop” of the absorption and elimination phases of the drug. Absorption does not become rate-limiting. The limited half-life can be attributed to rapid renal clearance of the TSLP or proteolyzed fragments. Values represent the mean, with 95% confidence intervals in parentheses. Here, k represents the first-order elimination rate constant of drug; Vdrepresents the volume of distribution of drug; AUC represents the area under the plasma concentration time curve, integrated from time zero to infinity. Concentration of TSLP was measured by ELISA except for those denoted with “*” which represents an independent experiment with the concentrations of TSLP measured by LUMINEX assay. AUC of the modified TSLP was normalized to the rhTSLP due to differences in detection efficiency.

[0032] Table 1: Pharmacokinetic parameters of TSLP following i.v. and i.p. routes of administration. TSLP Dose Route of Elimination Rate Constant Half-life Vd AUC (ng / g) Admin. k (min-1) (minutes) (^L / g) (ng min / mL)

[0033] To produce the above data, plasma concentrations were fitted using a one-compartment model. Graphs of the natural log of plasma concentrations vs. time were analyzed via linearregression to determine the first order elimination rate constant, half-life, and distribution of recombinant hTSLP (rhTSLP). The first-order elimination rate constant, k, was calculated from the linear regression of semi-log plots of plasma concentration vs. time curves. The corresponding half-life was calculated using k, assuming a first-order elimination process and one compartment pharmacokinetics. Distribution of recombinant hTSLP in the PDX mouse model was assessed by volume of distribution parameter (Vd) and calculated based on the dose and extrapolated value of the y-intercept of the corresponding plasma concentration time curves. Assuming average mouse weight is 25 gm and average blood volume is 1.46 mL (using reported normalized blood volume of 58.5 mL / kg from the National Center for the Replacement and Reduction of Animals in Research), the distribution of recombinant hTSLP in the mouse ranges from 15 μL to 45 μL (based on 95% confidence limits), indicating that distribution is limited to plasma volume.

[0034] Looking to the last two rows of Table 1 having similar experimental conditions, the modified TSLP of certain embodiments may provide an average bloodstream half-life of 91 minutes that is 2.46 times (about 2.5 times) the bloodstream half-life of 37 minutes of the unmodified TSLP. In certain embodiments, the bloodstream half-life may be increased by 50%, 100%, 150%, 200%, 250%, or more according to the present techniques. In certain embodiments, the bloodstream half-life may be increased by at least 200% or 250% according to the present techniques. In some embodiments, conjugation reactions may increase half-lives of circulating molecules as much as 100-fold. Compared to unmodified TSLP, the improved bloodstream half- life of the modified TSLP disclosed herein extends overall time that a targeted, therapeutic level of TSLP may be provided to a subject. The bloodstream half-life of TSLP can be improved by the addition of a prosthetic or conjugate polymer, which increases the hydrodynamic volume of the protein-conjugate complex. Proteolysis rates of proteins are affected by the molecular weightand tertiary or folded structures of the proteins. The protein-conjugate complex exhibits slower proteolysis rates due to the shielding of the protein by the conjugate. Similarly, as the molecular weight increases from that of unmodified TSLP, approximately 14 kDa, to greater than 60 kDa with the addition of a conjugate, the renal clearance rates decrease. The renal clearance threshold has been estimated to be about 60 kDa. The tradeoff is the potential for lower biological activity due to disruption of secondary and tertiary structures of TSLP by the conjugate.

[0035] The primary structure of a protein is determined by its amino acid sequence without any regard for the arrangement of the peptide chain in space. The secondary structure is determined by the spatial arrangement of the main peptide chain without any regard for the conformation of side chains or other segments of the main chain. The tertiary structure is determined by both the side chains and other adjacent segments of the main chain, without regard for neighboring peptide chains. Finally, the term quaternary structure is used for the arrangement of identical or different subunits of a large protein in which each subunit is a separate peptide chain. Embodiments of the present disclosure can be further understood by reference to the following examples. EXAMPLES

[0036] Example 1

[0037] FIG. 1 presents the results of the reaction of TSLP with PEG at various pH and PEG:rhTSLP molar ratios following a SDS-PAGE analysis, as shown in Table 2. Reaction conditions include pH 5.5 with a 1:1 ratio of PEG-NHS to rhTSLP (Lane 5) and pH 6.0 with a 1:1 ratio of PEG-NHS to rhTSLP resulted in mono-PEGylated protein. Additional reaction conditions resulted in a mixture of poly-PEGylated forms. Conjugation efficiency appears low based on band intensity of starting material compared to mono-PEGylated band.

[0038] Table 2: Reaction of TSLP and PEG at various pH and PEG:rhTSLP molar ratios Lane # pH Molar Ratio, PEG:rhTSLP 1 7.4 5:1

[0039] Example 2

[0040] FIG.2 presents the results following a SDS-PAGE analysis of the reaction of TSLP with PEG under various pH and reaction times, as shown in Table 3, to produce a mono-PEGylated TSLP. Conjugation is most efficient at pH 5.5 with mostly mono modification. The reaction time does not appear to affect conjugation efficiency at optimized pH levels.

[0041] Table 3: Various pH and reaction time conditions for reaction of PEG, 10 kDa form, with TSLP. Each reaction had a molar ratio of PEG:TSLP of 2:1. Lane # pH Reaction Time, hr 0 0

[0042] Example 3

[0043] FIG. 3 a graphical representation of the purification of the reaction products of TSLP with PEG to isolate a mono-PEGylated TSLP fraction. Purification of mono-PEGylated rhTSLP from non-substituted and poly-substituted forms was performed using ion-exchange chromatography. Collected fractions were assessed by SDS-PAGE for recovery of mono- PEGylated material. A total of three fractions contained mono-PEGylated material with varying degrees of recovery. Fractions CAF01 and CAF03 had estimated recoverable yields of 16% and 12% respectively. Fraction CAF02 yield was not estimated due to the very low recovery. CAF01, CAF02, and CAF03 all exhibited molecular weight bands below 30 kDa indicating mono- PEGylated material.

[0044] Example 4

[0045] FIG. 4 is a graphical representation of the N-terminus sequence analysis of mono- PEGylated recombinant TSLP. N-terminal PEGylation specificity was determined by N-terminal sequence analysis. A blocking result may indicate a PEG molecule at the N-terminus, as shown in Table 4.

[0046] Table 4: Amino acid recovery relative to IDK08. pmol IDK08 CAF01 CAF02 CAF03 L d d 50 50 50 50

[0047] CAF02 and CAF03 samples resulted in 100% N-terminal blockage. CAF01 resulted in ~50% blockage. Based on this data, it appears that CAF02 and CAF03 preparations are N- terminus specific, while CAF01 is a mixture of lysine and N-terminal PEGylation.

[0048] Example 5

[0049] FIG. 5 a graphical representation of the bioactivity of a mono-PEGylated rhTSLP. CAF01 and CAF03 samples were assessed for bioactivity by the proteins ability to induce cell proliferation of BaF3 mouse pro-B cells co-transfected with human IL-7 R alpha and human TSLP. CAF01 (ED50: 0.000902 ng / ml) appears more active by nearly 2 orders of magnitude compared to CAF03 (ED50: 0.0557 ng / ml) and non-modified starting material (ED50: 0.117 ng / ml). Overall, PEGylation as a conjugation reaction does not appear to lower the proteins bioactivity.

[0050] Example 6

[0051] FIG. 6 presents the effect of pH on yield on the selectivity of PEGylation for various molecular weight PEGs. Reactions were assessed by SDS-PAGE to compare modification levels. All reaction conditions were performed at a molar ratio of 2 PEG-NHS to 1 rhTSLP, as shown in Table 5.

[0052] Table 5: Reaction pH and PEG molecular weight for reaction with TSLP. Lane # pH PEG Mw, kDa 0 0 0

[0053] Moving from pH 5.5 to pH 6.0 may increase poly-PEGylation, but also greatly increases the yield of mono-PEGylation. Reactions conditions pH 6.0, 5 kDa PEG to rhTSLP (Lane 4), and pH 6.0, 10 kDa PEG to rhTSLP (Lane 8) appear optimal for purification attempts. Samples ranging from pH 5.5 to 7.4 were selected to move forward to size exclusion-based purification attempts for both the 5 kDa and 10 kDa forms. The 20 kDa form was not selected to move forward in this example due to low efficiency compared to the 5 kDa and 10 kDa.

[0054] Example 7

[0055] FIG. 7 illustrates the isolation of mono-PEGylated rhTSLP using 5 kDa PEG. Purification of mono-PEGylated rhTSLP from non-substituted and poly-substituted forms was performed using size-exclusion chromatography. Collected fractions were assessed by SDS- PAGE for recovery of mono-PEGylated material. The mono-PEGylation peak in pH 6.0 and pH 7.4 conditions appear to be similar heights, but the non-PEGylated peak in pH 7.4 condition is much smaller, thus pointing towards a higher degree of poly-PEGylation and not a net gain in mono-PEGylated material. pH 5.5 conditions may provide more N-terminal specificity, which results in lower overall yield. Estimated yield for the 5 kDa form is approximately 15% to 25%. The B purified sample from pH 6.0 conditions (CAF04) was assessed for bioactivity.

[0056] Example 8

[0057] FIG. 8 illustrates the isolation of mono-PEGylated rhTSLP using 10 kDa PEG. Purification of mono-PEGylated rhTSLP from non-substituted and poly-substituted forms was performed using size-exclusion chromatography. Collected fractions were assessed by SDS- PAGE for recovery of mono-PEGylated material. The 10 kDa PEGylation shows more gain in the ratio of non-PEGylated to mono-PEGylated protein, but also has significantly more poly- PEGylated protein. Additionally, the poly-PEGylated material overlaps more with the mono- PEGylated sample. The estimated yield for the 10 kDa purification by use of size-exclusionchromatography is 15% to 25% mono-PEGylated material. Overall, the sizing-based purification efforts do not appear to provide a net benefit compared to ion exchange-based purification. Additionally, the sizing-based purification poorly separates poly-PEGylated material from mono- PEGylated material.

[0058] Example 9

[0059] FIG. 9 is a graphical representation of the bioactivity analysis of mono-PEGylated rhTSLP, 10 kDa PEG. CAF04 (5 kDa PEGylated protein purified by size exclusion chromatography (SEC), pH 6.0) was assessed for activity by the proteins ability to induce cell proliferation of BaF3 mouse pro-B cells co-transfected with human IL-7 R alpha and human TSLP R. CAF04 (SEC purified 5 kDa PEGylated protein, pH 6.0) appears equivalent to non- PEGylated rhTSLP when assessed for bioactivity. CAF04 had an ED500.436 ng / mL, while non- PEGylated rhTSLP, Lot IDK08 had an ED50 of 0.404 ng / mL.

[0060] Example 10

[0061] In preliminary experiments, recombinant hTSLP molecules with 3 different lengths of PEG polymer were successfully created. The mono-PEGylated derivatives were purified and assessed for bioactivity using cell culture assays. All PEGylated derivatives exhibited activity equal to much greater than recombinant human TSLP without conjugated polymers. FIG.10 is a graphical representation of the activation assay of the 10-kDa-PEG-TSLP. It is clear that PEGylation of TSLP does not interfere with the interaction of TSLP and its cognate receptors. While certain derivatives were PEGylated at more than one location, successful purification was achieved for a 10 kDa derivative mono-PEGylated at the N-terminus of TSLP. The testing of this derivative was performed via standard activation assays (illustrated in FIG.10) and for residence time in a mouse model. This mod-TSLP exhibits a half-life in subjects 2.5 times longer than natural recombinant TSLP (rhTSLP) (Table 1 discussed above) and is active at 2-fold lowerconcentration, providing clear benefits for potential therapeutic use. Indeed, the 10 kDa PEG derivative of TSLP has an ED50 approximately 2-fold lower than rhTSLP. In certain embodiments, pure, stable, and / or endotoxin-free forms of the modified TSLP may have sufficiently long half-lives so that a dose having suitable efficacy is reduced by 90%.

[0062] In embodiments, modified TSLP products may be purified by chromatography. In some embodiments, reaction efficiency may be determined by product purity and identity, as measured by product concentration, SDS-PAGE, and mass spectrometry analysis. Bioactivity may be assessed using BaF3 mouse pro-B cells that are transduced to express human IL-7Rα and CRLF2. To establish stability, the modified TSLP may be incubated at 4ºC and 37ºC and assessed for stability under ordinary storage conditions by mass spectrometry at multiple time points. Moreover, plasma levels of the modified TSLP may be determined using ELISA and LUMINEX for high-sensitivity experiments. Plasma concentration-time data may be analyzed by compartmental methods using nonlinear regression analysis to obtain values for half-life, area under the plasma concentration time curve, distribution, and clearance of the modified TSLP in subjects.

[0063] When ranges are disclosed herein, ranges from any lower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. Additionally, reference to values stated in ranges includes each and every value within that range, even though not explicitly recited. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.

[0064] Other objects, features and advantages of the disclosure will become apparent from the foregoing figures, detailed description, and examples. It should be understood, however, that the figures, detailed description, and examples, while indicating specific embodiments of the disclosure, are given by way of illustration only and are not meant to be limiting. Additionally, it is contemplated that changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from the detailed description. In further embodiments, features from specific embodiments may be combined with features from other embodiments. For example, features from one embodiment may be combined with features from any of the other embodiments. In further embodiments, additional features may be added to the specific embodiments described herein.

Claims

CLAIMS We claim:

1. A method for preparing a human thymic stromal lymphopoietin (TSLP) to achieve an improved half-life, the method comprising: providing a TSLP and a polymer conjugate to a reaction vessel; allowing a conjugation reaction to proceed under predetermined reaction conditions to attach the polymer conjugate to the TSLP to produce a modified TSLP; and isolating the modified TSLP.

2. The method of claim 1, wherein the TSLP is a naturally occurring or a recombinant protein.

3. The method of claim 1, wherein a bloodstream half-life of the modified TSLP is increased relative to a bloodstream half-life of the TSLP.

4. The method of claim 3, wherein the increase in the bloodstream half-life of the modified TSLP is at least 200%.

5. The method of claim 1, wherein the TSLP is a full-length protein or a fragment thereof.

6. The method of claim 5, wherein the TSLP comprises an intact, ternary TSLP-CRLF2-IL7R complex associated with biological activity.

7. The method of claim 1, wherein the polymer conjugate contains one or more of a polyether, a polysaccharide, or a biliprotein.

8. The method of claim 1, wherein a substantial amount of the polymer conjugate is attached to a N-terminus of the TSLP.

9. The method of claim 1, wherein the modified TSLP has a molecular weight of at least 19 kilodaltons (kDa).

10. The method of claim 1, wherein the conjugation reaction is a site selective enzymatic, chemical, an atom transfer radical polymerization, or a reversible addition fragmentation chain- transfer polymerization conditions.

11. A modified human thymic stromal lymphopoietin (TSLP) composition having an improved half-life, the composition comprising: a TSLP with a covalently bonded polymer conjugate, the TSLP being present in an amount ranging from about 50% to about 99% by weight; and the covalently bonded polymer conjugate in an amount ranging from about 1% to about 50% by weight.

12. The modified TSLP composition of claim 11, wherein the modified TSLP has a molecular weight of at least 19 kDa.

13. The modified TSLP composition of claim 11, wherein the TSLP is a full-length protein or a fragment thereof.

14. The modified TSLP composition of claim 13, wherein the TSLP is the fragment of the full- length protein and includes an intact complex that provides biological activity.

15. The modified TSLP composition of claim 11, wherein the polymer conjugate contains one or more of a polyether, a polysaccharide, or a biliprotein.

16. A method of treating a subject in need of treatment for a TSLP-related condition, such treatment, the method comprising: administering a therapeutically effective amount of a TSLP with a covalently bonded polymer conjugate, the TSLP being present in an amount ranging from about 50% to about 99% by weight, and the polymer conjugate being present in an amount ranging from about 1% to about 50% by weight.

17. The method of claim 16, wherein the TSLP is a full-length protein or a fragment thereof.

18. The method of claim 16, wherein the TSLP comprises an intact, ternary TSLP-CRLF2-IL7R complex.

19. The method of claim 16, wherein the polymer conjugate contains one or more of a polyether, a polysaccharide, or a biliprotein.

20. The method of claim 16, wherein the TSLP-related condition comprises asthma, allergic inflammation, or chronic obstructive pulmonary disease.

21. The method of claim 16, wherein the TSLP-related condition is leukemia.

22. The method of claim 16, wherein the TSLP-related condition is a solid tumor.

23. The method of claim 16, wherein the TSLP-related condition comprises an abnormal expression of any ternary receptor complex involving binding of TSLP to cognate receptors.

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