Modified anti-TSLP antibodies

Abstract

The present application generally relates to compositions or variants of the anti-TSLP antibody tezepelumab that have increased stability compared to tezepelumab when stored over extended periods of time.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 178,915, filed April 23, 2021, the entirety of which is incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Materials A sequence listing, which is part of this disclosure, is submitted as a text file contemporaneously herewith. The name of the text file containing the sequence listing is "55581_Seqlisting.txt", was created on April 12, 2022, and is 32,649 bytes in size. The contents of the sequence listing are incorporated herein by reference in their entirety.

[0003] The present application generally relates to compositions and variants of the anti-TSLP antibody tezepelumab that have increased stability relative to tezepelumab when stored over extended periods of time. [Background technology]

[0004] Thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine produced in response to environmental and proinflammatory stimuli, leads to activation of multiple inflammatory cells and downstream pathways (Soumelis et al. Nat Immunol 2002;3:673-80; Allakhverdi et al. J Exp Med 2007;204:253-8). TSLP is increased in the airways of asthmatics and correlates with Th2 cytokine and chemokine expression (Shikotra et al. J Allergy Clin Immunol 2012;129:104-11 e1-9) and disease severity (Ying et al. J Immunol 2005;174:8183-90; Ying et al. J Immunol 2008;181:2790-8). Although TSLP is central to the regulation of Th2 immunity, it may also play an important role in other pathways of inflammation and thus may be associated with multiple asthma phenotypes.

[0005] Tezepelumab is a human immunoglobulin G2 (IgG2) monoclonal antibody (mAb) that binds to TSLP and prevents its interaction with the TSLP-receptor complex. In a proof-of-concept study in patients with mild atopic asthma, tezepelumab was shown to inhibit early and late asthmatic responses and suppress biomarkers of Th2 inflammation after inhaled allergen exposure (Gauvreau, et al. N Engl J Med 2014;370:2102-10). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Soumelis et al.Nat Immunol 2002;3:673-80 [Non-Patent Document 2] Allakhverdi et al. J Exp Med 2007;204:253-8 [Non-Patent Document 3] Shikotra et al.J Allergy Clin Immunol 2012;129:104-11 e1-9 [Non-Patent Document 4] Ying et al. J Immunol 2005;174:8183-90 [Non-Patent Document 5] Ying et al. J Immunol 2008;181:2790-8 [Non-Patent Document 6] Gauvreau,et al.N Engl J Med 2014;370:2102-10 Summary of the Invention [Means for solving the problem]

[0007] Monitoring antibody therapeutics in formulations over time is important to determine storage conditions that reduce degradation of the therapeutic and maintain product integrity. The present disclosure provides a study of anti-TSLP antibody attributes that may change over time during storage and that may be beneficial or detrimental to antibody stability.

[0008] In one aspect, the disclosure provides an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof, comprising: (A) a light chain variable domain comprising: (i) a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO: 3, (ii) a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO: 4, and (iii) a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO: 5, and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO: 7 with a mutation at least one of the following residues D54 or G55, and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO: 8. In various embodiments, HCDR2 has the sequence VIWYX1X2SNKHYADSVKG (SEQ ID NO: 13), where X1 is D or E and X2 is G or A. In various embodiments, HCDR2 has the following sequence: VIWYEGSNKHYADSVKG (SEQ ID NO: 14), VIWYDASNKHYADSVKG (SEQ ID NO: 15) or VIWYEASNKHYADSVKG (SEQ ID NO: 16).

[0009] In various embodiments, the mutation in HCDR2 is D54E. In various embodiments, the mutation in HCDR2 is G55A. In various embodiments, the anti-TSLP antigen binding protein or fragment thereof optionally comprises a mutation in at least one of residues LCDR2 D49, D50, or S51 of SEQ ID NO:4. In various embodiments, the mutation in LCDR2 is one or more of D49E, D50E, or S51A. In various embodiments, LCDR2 has the sequence X1X2X3DRPS, where X1 is D or E, X2 is D or E, and X3 is S or A (SEQ ID NO: 17). In various embodiments, LCDR2 has the following sequence: EDSDRPS (SEQ ID NO: 18), DESDRPS (SEQ ID NO: 19), EESDRPS (SEQ ID NO: 20), DDADRPS (SEQ ID NO: 21), DEADRPS (SEQ ID NO: 22), EDADRPS (SEQ ID NO: 23) or EEADRPS (SEQ ID NO: 24).

[0010] In various embodiments, the disclosure provides an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof, comprising: (A) a light chain variable domain comprising: (i) a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 sequence comprising an amino acid sequence having a mutation in at least one of the following residues D49, D50, or S51 of SEQ ID NO:4; (iii) a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7, and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8.

[0011] In various embodiments, LCDR2 has the sequence X1X2X3DRPS, where X1 is D or E, X2 is D or E, and X3 is S or A (SEQ ID NO: 17). Optionally, LCDR2 has the following sequence: EDSDRPS (SEQ ID NO: 18), DESDRPS (SEQ ID NO: 19), EESDRPS (SEQ ID NO: 20), DDADRPS (SEQ ID NO: 21), DEADRPS (SEQ ID NO: 22), EDADRPS (SEQ ID NO: 23), or EEADRPS (SEQ ID NO: 24). In various embodiments, the mutation in LCDR2 is D49E. In various embodiments, the mutation in LCDR2 is D50E. In various embodiments, the mutation in LCDR2 is S51A. In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof optionally comprises a mutation at one of the following residues D54 or G55 of HCDR2 as set forth in SEQ ID NO: 7. In various embodiments, the mutation in HCDR2 is one or more of D54E or G55A of SEQ ID NO: 7. In various embodiments, HCDR2 has the sequence VIWYX1X2SNKHYADSVKG (SEQ ID NO: 13), where X1 is D or E and X2 is G or A. In various embodiments, HCDR2 has the following sequence: VIWYEGSNKHYADSVKG (SEQ ID NO: 14), VIWYDASNKHYADSVKG (SEQ ID NO: 15) or VIWYEASNKHYADSVKG (SEQ ID NO: 16).

[0012] In various embodiments, the disclosure provides an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof, comprising: (A) a light chain variable domain selected from the group consisting of: i. a sequence of amino acids at least 80% identical to SEQ ID NO:12; ii. a sequence of amino acids encoded by a polynucleotide sequence at least 80% identical to SEQ ID NO:11; or iii. a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO:11; or (B) a heavy chain variable domain, i. a sequence of amino acids at least 80% identical to SEQ ID NO:10. a heavy chain variable domain selected from the group consisting of: a sequence of amino acids that is at least 80% identical to SEQ ID NO: 9; ii. a sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 9; or iii. a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO: 9; or (C) a light chain variable domain of (A) and a heavy chain variable domain of (B), wherein the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof retains one or more of the CDRs of the anti-TSLP antigen binding protein or fragment thereof and contains a mutation in one or more of HCDR2 D54 or G55 of SEQ ID NO: 7 or LCDR2 D49, D50 or S51 of SEQ ID NO: 4.

[0013] In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NOs:25-28, and a light chain comprising the amino acid sequence of SEQ ID NO:12 or SEQ ID NOs:29-36.

[0014] In various embodiments, the anti-TSLP immunoglobulin, antigen-binding protein or fragment thereof, or antibody or fragment thereof comprises an anti-TSLP antigen-binding protein selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0015] In various embodiments, the immunoglobulin, antigen binding protein, or antibody is a human antibody. In various embodiments, the antibody is an IgG2 antibody. In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein, or fragment thereof, or antibody, or fragment thereof, specifically binds to a TSLP polypeptide set forth in amino acids 29-159 of SEQ ID NO:2. In various embodiments, the binding sites of both the anti-TSLP immunoglobulin, antigen binding protein, or fragment thereof, or antibody, or fragment thereof, have identical binding to TSLP.

[0016] In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof is greater than or equal to 10 -8 Binds to TSLP with an affinity below the M Kd.

[0017] Further contemplated are compositions comprising an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof described herein and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0018] The disclosure also provides an isolated nucleic acid comprising a polynucleotide sequence encoding the light chain variable domain, the heavy chain variable domain, or both, of the immunoglobulin, antigen binding protein, or antibody described herein.

[0019] The present disclosure further contemplates recombinant expression vectors comprising a nucleic acid encoding an anti-TSLP immunoglobulin, antigen binding protein, or antibody described herein. Host cells comprising the expression vectors are also provided.

[0020] Further contemplated herein is a method of producing an immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof that specifically binds to a TSLP polypeptide comprising amino acids 29-159 of SEQ ID NO:2, comprising incubating a host cell under conditions that permit it to express the immunoglobulin, antigen binding protein or fragment thereof, wherein the host cell comprises (i) a recombinant expression vector encoding a light chain variable domain of an antigen binding protein described herein and a recombinant expression vector encoding a heavy chain variable domain of an antigen binding protein described herein, or (ii) a recombinant expression vector encoding both the light chain variable domain and the heavy chain variable domain of an immunoglobulin, antigen binding protein or antibody described herein.

[0021] In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof has increased stability at 25° C. compared to the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof having the amino acid sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 12. In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof has increased stability at 40° C. after 4 weeks compared to the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof having the amino acid sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 12. In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof has reduced high molecular weight species at 40° C. after 4 weeks compared to the anti-TSLP antigen binding protein or fragment thereof having the amino acid sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 12.

[0022] In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof has reduced isomerization at 50° C. compared to the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof having the amino acid sequences set forth in SEQ ID NO:10 and SEQ ID NO:12.

[0023] In various embodiments, less than 2% of the anti-TSLP immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof, exhibit isomerization as determined by SEC, e.g., SEC of antibody-antigen complexes, after at least 2 weeks (optionally at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months) of storage at about 25° C. In various embodiments, less than 2% of the antigen binding proteins or fragments thereof exhibit isomerization as determined by SEC after about 22 months to about 36 months of storage at 2° C. to 8° C., followed by at least 2 weeks, or at least 1 month, or at least 2 months, or at least 3 months of storage at about 25° C.

[0024] Also provided herein is a method of treating an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of an immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof, or a composition thereof, as described herein. In various aspects, the inflammatory disease is selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis (EoE), nasal polyps, chronic spontaneous urticaria, Ig-driven disease, IgA nephropathy, lupus nephritis, eosinophilic gastritis, chronic sinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IPF). In various embodiments, the asthma is mild, moderate, or severe asthma. In various embodiments, the asthma is severe asthma. In various embodiments, the asthma is eosinophilic or non-eosinophilic asthma.

[0025] In various embodiments, the method comprises administering the composition at intervals of every two weeks or every four weeks, hi various embodiments, the composition is administered for a period of at least four months, six months, nine months, one year or more.

[0026] In various embodiments, the disclosure provides a method of producing a composition comprising a plurality of anti-TSLP monoclonal antibodies or antigen-binding fragments thereof, the plurality of anti-TSLP monoclonal antibodies or antigen-binding fragments thereof comprising: a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8, the method comprising: determining whether or not the L-aspartic acid at HC position 54 is an isoaspartic acid (isoAsp) or cyclic aspartic acid (cAsp) in HCDR2 set forth in SEQ ID NO:7; and concentrating the composition for IgG2 anti-TSLP monoclonal antibodies that contain at least one of: LC W102; an L-aspartic acid at LC position 49 or position 50 compared to an isoAsp or cAsp of LCDR2 as set forth in SEQ ID NO:4; LC N65 compared to a deamidated N65 as set forth in SEQ ID NO:12; or an L-aspartic acid at LC position 91 of LCDR3 as set forth in SEQ ID NO:5 compared to an isoAsp or cAsp. cAsp is also known as succinimide, characterized by a loss of HO compared to Asp or isoAsp.

[0027] In various embodiments, less than 2.0% of the anti-TSLP monoclonal antibodies contain isomerized HC D54. In various embodiments, no more than 0.9% of the anti-TSLP monoclonal antibodies contain isomerized HC D54. In various embodiments, no more than 2% of the anti-TSLP monoclonal antibodies contain oxidized HC W102. In various embodiments, less than 2.0% of the anti-TSLP monoclonal antibodies contain isomerized LC D49 or D50. In various embodiments, no more than 0.9% of the anti-TSLP monoclonal antibodies contain isomerized LC D49 or D50. In various embodiments, no more than 0.5% of the anti-TSLP monoclonal antibodies contain deamidated LC N65. In various embodiments, no more than 0.9% of the anti-TSLP monoclonal antibodies contain isomerized LC D91.

[0028] Further contemplated is a composition comprising an anti-TSLP monoclonal antibody, the anti-TSLP monoclonal antibody comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8, wherein the anti-TSLP monoclonal antibody of the composition is numerically 10 -8 The antibody comprises a limited content of isomeric HC D54 (SEQ ID NO: 7) and / or a limited content of isomeric LCD D49 or D50 (SEQ ID NO: 4) that are effective for binding to TLSP with a Kd of less than or equal to M.

[0029] Also provided is a composition comprising an IgG2 anti-TSLP monoclonal antibody, the IgG2 anti-TSLP monoclonal antibody comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8; wherein no more than 0.9% of the anti-TSLP monoclonal antibody comprises isomerized HC D54; no more than 2% of the anti-TSLP monoclonal antibody comprises oxidized HC W102; no more than 6.7% of the anti-TSLP monoclonal antibody comprises isomerized LC D49 or D50; no more than 0.5% of the anti-TSLP monoclonal antibody comprises deamidated LC N65; or no more than 0.9% of the anti-TSLP monoclonal antibody comprises isomerized LC D49 or D50. In various embodiments, 0.9% or less of the anti-TSLP monoclonal antibodies contain isomerized LC D54. In various embodiments, 2% or less of the anti-TSLP monoclonal antibodies contain oxidized HC W102. In various embodiments, 0.9% or less of the anti-TSLP monoclonal antibodies contain isomerized LC D49 or LC D50. In various embodiments, 0.5% or less of the anti-TSLP monoclonal antibodies contain deamidated LC N65. In various embodiments, 0.9% or less of the anti-TSLP monoclonal antibodies contain isomerized LC D91.

[0030] Also provided is a composition comprising an IgG2 anti-TSLP monoclonal antibody, the IgG2 anti-TSLP monoclonal antibody comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8; wherein no more than 0.9% of the anti-TSLP monoclonal antibody comprises isomerized HC D54; no more than 2% of the anti-TSLP monoclonal antibody comprises oxidized HC W102; no more than 12.9% of the anti-TSLP monoclonal antibody comprises isomerized LC D49 or D50; no more than 0.5% of the anti-TSLP monoclonal antibody comprises deamidated LC N65; or no more than 0.9% of the anti-TSLP monoclonal antibody comprises isomerized LC D49 or D50. In various embodiments, no more than 0.9% of the anti-TSLP monoclonal antibodies contain isomerized HC D54. In various embodiments, no more than 2% of the anti-TSLP monoclonal antibodies contain oxidized HC W102. In various embodiments, no more than 0.9% of the anti-TSLP monoclonal antibodies contain isomerized LC D49 or LC D50. In various embodiments, no more than 0.5% of the anti-TSLP monoclonal antibodies contain deamidated LC N65. In various embodiments, no more than 0.9% of the anti-TSLP monoclonal antibodies contain isomerized LC D91.

[0031] In various embodiments, the anti-TSLP antibodies comprise a combination of L-aspartic acid of HC D54 and L-aspartic acid of LC D49 or D50. In various embodiments, the anti-TSLP antibodies are enriched in L-aspartic acid of HC D54 at least 6-fold over the level of isoAsp.

[0032] In various embodiments, the antibody is an IgG2 antibody. In various embodiments, the anti-TSLP antibody comprises a heavy chain variable region set forth in SEQ ID NO: 10 or SEQ ID NOs: 25-28 and a light chain variable region set forth in SEQ ID NO: 12 or SEQ ID NOs: 29-36, and includes one or more of the sequence modifications described herein.

[0033] The disclosure also provides a composition comprising an anti-TSLP monoclonal antibody, the anti-TSLP monoclonal antibody comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8; greater than 98% of the anti-TSLP monoclonal antibodies of the composition comprise an L-aspartic acid at HC position 54 compared to an isoAsp or cAsp at position 54 (SEQ ID NO:7); or at least 99% of the anti-TSLP monoclonal antibodies of the composition comprise an unoxidized HC compared to an oxidized W102. at least 97% of the anti-TSLP monoclonal antibodies of the composition comprise an L-aspartic acid at LC position 49 or 50 compared to an isoAsp or cAsp at position 49 or 50 (SEQ ID NO: 4); at least 99.1% of the anti-TSLP monoclonal antibodies of the composition comprise an LC N65 compared to a deamidated LC N65 (SEQ ID NO: 12); or at least 99.1% of the anti-TSLP monoclonal antibodies of the composition comprise an L-aspartic acid at LC position 91 compared to an isoAsp or cAsp at position 91 (SEQ ID NO: 5).

[0034] The present disclosure also provides compositions comprising an anti-TSLP antibody or antigen-binding fragment thereof described herein for use in treating an inflammatory disease described herein. In certain embodiments, the present disclosure provides use of a composition comprising an anti-TSLP antibody or antigen-binding fragment thereof described herein in the preparation of a medicament for treating an inflammatory disease.

[0035] Syringes, e.g., single-use or pre-filled syringes, sterile sealed containers, e.g., vials, bottles, containers, and / or kits or packages containing any of the aforementioned antibodies or compositions, optionally together with suitable instructions for use, are also contemplated.

[0036] It is understood that each feature or embodiment or combination described herein is a non-limiting, illustrative example of any of the aspects of the invention and is therefore intended to be combinable with any other feature or embodiment or combination described herein. For example, when a feature is described using terms such as "one embodiment," "an embodiment," "particular embodiment," "further embodiment," "particular exemplary embodiment," and / or "other embodiment," each of these types of embodiments is a non-limiting example of the feature that is intended to be combined with any other feature or combination of features described herein, without the need to recite every possible combination. Such features or combinations of features apply to any of the aspects of the invention. When examples of values ​​falling within a range are disclosed, any of these examples are considered as possible endpoints of the range, and any numerical values ​​between such endpoints are contemplated, and any combination of upper and lower endpoints is contemplated.

[0037] The headings herein are for the convenience of the reader and are not intended to be limiting. Further aspects, embodiments and variations of the invention will become apparent from the detailed description, and / or drawings, and / or claims. [Brief description of the drawings]

[0038] [Figure 1] Figure 1 shows the workflow for characterizing residues and modifications that potentially affect binding as determined by SEC affinity binding of heat-stressed AMG157(40C4W) and TSLP. In silico sequence analysis is shown (top). Mutation of some of the residues with modifications appeared to improve the stability of AMG157 at room temperature (bottom). [Diagram 2]Relative abundance of potential attributes in the T0, 40C4W, and 22M5C+2M25C samples of AMG157 as determined by SEC of antigen-antibody complexes. These attributes were predicted to potentially affect TSLP stability by in silico sequencing (shown in FIG. 1). White, black, and gray bars represent AMG157 T0, 40C4W, and 22M5C+2M25C sample modification rates, respectively. Dashed lines are shown to represent 2%. [Figure 3A] SEC-UV profiles of AMG157 T0, AMG157 40C4W, TSLP, AMG157 T0+TSLP mixture, and AMG157 40C4W+TSLP mixture are shown. The five SEC-UV peak regions are assigned based on peak shape and molecular weight. Peak 3 represents the bound fraction of AMG157 with TSLP, and peak 5 corresponds to the unbound fraction of AMG157. An illustration of each assigned peak is shown above the corresponding peak. [Figure 3B] The modification rates of five attributes in the bound and unbound fractions of SEC binding are shown. [Figure 4] Volcano plot showing how the attributes of AMG157 are statistically distributed to affect TSLP binding. The attributes shown in the upper right corner are modifications of AMG157 that potentially affect TSLP binding. The x-axis is the log2 value of the fold change between non-binding and binding, and the y-axis is the -log10 value of the p-value representing statistical significance. The grey area is considered background. [Figure 5A] The percent modification of bound and unbound AMG157 fractions in 10 residues that were deemed potentially important based on in silico sequence analysis is shown. However, antibody-antigen SEC revealed that the ratio of modifications between unbound and bound fractions was not statistically different. It is assumed that the modifications do not affect the binding measured by SEC of the antibody-antigen method. Figures 5A and 5B have percentage scales of 0-50% and 0-1%, respectively. [Figure 5B]The percent modification of bound and unbound AMG157 fractions in 10 residues that were deemed potentially important based on in silico sequence analysis is shown. However, antibody-antigen SEC revealed that the ratio of modifications between unbound and bound fractions was not statistically different. It is assumed that the modifications do not affect the binding measured by SEC of the antibody-antigen method. Figures 5A and 5B have percentage scales of 0-50% and 0-1%, respectively. [Figure 6] SEC-UV profiles of AMG157 T0, 40°C 4W and 50°C 1W are shown. The SEC-UV profiles of AMG157 T0, 40°C 4W and 50°C 1W are shown by black solid line, blue dotted line and red dashed line, respectively. Based on the elution time and theoretical molecular weight of AMG157, the peak eluting at about 10.5 min is assigned as the high molecular weight species of AMG157 (HMW), and the peak eluting at about 15.5 min is assigned as the monomer of AMG157. According to the integrated peak area, the percentage of HMW species at 40°C 4W and 50°C 1W is about 9% and 67%, respectively, which is shown in the upper left of the figure. [Figure 7A] Volcano plot showing how AMG157 attributes are distributed between HMW and monomeric species after 50C1W stress, indicating statistical significance. Attributes shown in the upper right corner are AMG157 modifications that correlate with the formation of HMW in 50C1W. The x-axis is the log2 value of the fold change between HMW and monomeric species, and the y-axis is the -log10 value of the p-value representing statistical significance. The grey area is considered background noise, but may contain true values ​​with lower confidence. [Figure 7B] The proportion of 20 modifications in HMW species and monomers of the AMG157 50C1W sample is shown. These 20 modifications include the statistically significant modifications from the top right white corner (7 modifications market with asterisks) and the modifications from the adjacent "gray area close to statistical significance". Each of these 20 modifications has relatively high values ​​of fold change and significance. The sum of the -log10 and log2 values ​​of the p-values ​​of the fold change for these 20 modifications was greater than 4.6. [Figure 8A] 1 shows isomerization level measurements by peptide mapping and potency measurements of antibody drug substance and antibody stressed at 40° C. for 4 weeks. [Figure 8B] 4 shows the CEX-HPLC profile of the antibody. [Figure 8C] 1 shows the results of a conventional method of characterization of CEX fractions of AMG157 40° C. 4W sample for chemical modification and relative potency by peptide mapping. [Figure 8D] 1 shows the results of a long-term stability study near the end of shelf life. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] It is further believed that treatment with tezepelumab may eliminate day-to-day disease activity, enabling more patients to become steroid-free or reducing the need for the use of steroids in the treatment of inflammatory diseases such as asthma.

[0040] Unless otherwise stated, the following terms used in this Application, including the specification and claims, have the definitions given below.

[0041] As used in this specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural as well as singular referents unless the context clearly dictates otherwise.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in this disclosure, but are not limited to: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d Ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker Ed., 1988); THE GLOSSARY OF GENETICS, 5th Ed., R. Rieger et al. (Eds.), Springer Verlag (1991); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).

[0043] The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term "about" or "approximately" precedes a first value in a series of two or more values, it is understood that the term "about" or "approximately" applies to each of the values ​​in the series.

[0044] The term "inflammatory disease" refers to a medical condition in which the immune system attacks its own cells or tissues, causing abnormal inflammation, resulting in chronic pain, redness, swelling, stiffness, and damage to normal tissues. Examples of inflammatory diseases include asthma, chronic digestive ulcer, tuberculosis, periodontitis, sinusitis, active hepatitis, ankylosing spondylitis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), Crohn's disease, ulcerative colitis, osteoarthritis, atherosclerosis, systemic lupus erythematosus, atopic dermatitis, eosinophilic esophagitis (EoE), nasal polyps, chronic spontaneous urticaria, Ig-driven diseases (such as IgA nephropathy and lupus nephritis), eosinophilic gastritis, chronic sinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IPF). In an exemplary embodiment, the inflammatory disease is asthma, atopic dermatitis, or COPD. In an exemplary aspect, the inflammatory is asthma, and in some cases the asthma is severe asthma, eosinophilic asthma, non-eosinophilic asthma, or hypoeosinophilic asthma.

[0045] As used herein, the term "asthma" refers to allergic, non-allergic, eosinophilic and non-eosinophilic asthma.

[0046] As used herein, the term "allergic asthma" refers to asthma induced by one or more inhaled allergens. Such patients have positive IgE fluorescent enzyme immunoassay (FEIA) levels against one or more allergens that induce an asthmatic response. Most allergic asthma is usually associated with Th2-type inflammation.

[0047] The term "non-allergic asthma" refers to patients who have low eosinophils, low Th2 or low IgE at the time of diagnosis. Patients with "non-allergic asthma" usually have a negative IgE fluorescent enzyme immunoassay (FEIA) that responds to an allergen panel that includes allergens specific to the region. In addition to low IgE, these patients often have low or no eosinophil counts and low Th2 counts at the time of diagnosis.

[0048] As used herein, the term "severe asthma" refers to asthma that requires intensive treatment (e.g., GINA Steps 4 and 5) to maintain good control or where good control is not achieved despite intensive treatment (GINA, Global Strategy for Asthma Management and Prevention. Global Initiative for Asthma (GINA) December 2012).

[0049] As used herein, the term "eosinophilic asthma" refers to asthma patients with a screening blood eosinophil count of 300 cells / μL or less or 250 cells / μL or less, and "hypoeosinophilic" asthma refers to asthma patients with blood or serum that is less than 250 cells / μL. Alternatively, "hypoeosinophilic" asthma refers to asthma patients with blood or serum that is less than 300 cells / μL.

[0050] "T helper (Th)1 cytokines" or "Th1 specific cytokines" refer to cytokines expressed (intracellularly and / or secreted) by Th1 T cells, including IFN-g, TNF-a, and IL-12. "Th2 cytokines" or "Th2 specific cytokines" refer to cytokines expressed (intracellularly and / or secreted) by Th2 T cells, including IL-4, IL-5, IL-13, and IL-10. "Th17 cytokines" or "Th17 specific cytokines" refer to cytokines expressed (intracellularly and / or secreted) by Th17 T cells, including IL-17A, IL-17F, IL-22, and IL-21. Certain populations of Th17 cells express IFN-g and / or IL-2 in addition to the Th17 cytokines listed herein. Polyfunctional CTL cytokines include IFN-g, TNF-a, IL-2, and IL-17.

[0051] The term "specifically binds" refers to an antibody or polypeptide that is "antigen-specific" and is "specific," "selective binder," "specific binder," "antigen target," or "immunoreactive" to the antigen and binds to the target antigen with higher affinity than other antigens of similar sequence. As used herein, the agent is considered to specifically bind to a target protein that is useful for identifying immune cell types, e.g., surface antigens (e.g., T cell receptor, CD3), cytokines (e.g., TSLP, IL-4, IL-5, IL-13, IL-17, IFN-g, TNF-a), and the like. In various embodiments, the antibody specifically binds to the target antigen but can cross-react with orthologs of closely related species, e.g., the antibody can be a human protein and also bind to a closely related primate protein. In various embodiments, the immunoglobulin, antigen-binding protein or fragment thereof, or antibody or fragment thereof specific for TLSP is numerically 10 -8 In various embodiments, the anti-TSLP antibodies described herein bind with a Kd of at least 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 It binds with an affinity (Kd) of less than or equal to M.

[0052] The term "antibody" refers to a tetrameric glycoprotein consisting of two heavy chains and two light chains, each containing a variable region and a constant region. "Heavy chain" and "light chain" refer to substantially full-length canonical immunoglobulin light and heavy chains (see, e.g., Immunobiology, 5th Edition (Janeway and Travers et al., Eds., 2001)). Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

[0053] Antigen-binding proteins include antibodies, antibody fragments, and antibody-like proteins that may have structural changes in the structure of a canonical tetrameric antibody. Antibody "variants" refer to antigen-binding proteins or fragments thereof that may have structural changes in antibody sequence or function compared to a parent antibody with a known sequence. Antibody variants include V regions with changes in the constant region or alternatively, optionally with the addition of V regions to the constant region in a non-canonical manner. Examples include multispecific antibodies (e.g., bispecific antibodies with extra V regions), antibody fragments capable of binding to antigen (e.g., Fab', F'(ab)2, Fv, single chain antibodies, diabodies), biparatopic peptides and recombinant peptides including the above, so long as they exhibit the desired biological activity.

[0054] Antibody fragments include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), complementarity determining region (CDR) fragments, CDR-grafted antibody binding regions, single chain antibodies (scFv), fragments of single chain antibodies, chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, linear antibodies; chelating recombinant antibodies, tri- or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, single domain antibodies (including camelized antibodies), VHH-containing antibodies or variants or derivatives thereof, as well as polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide, e.g., the antigen-binding portion of an antibody comprising one, two, three, four, five or six CDR sequences, so long as the antibody retains the desired biological activity.

[0055] "Valency" refers to the number of antigen-binding sites on each antibody or antibody fragment that target an epitope. A typical full-length IgG molecule or F(ab)2 is "bivalent" in that it has two identical target binding sites. "Monovalent" antibody fragments such as F(ab)' or scFc have a single antigen-binding site. Trivalent or tetravalent antigen-binding proteins can also be engineered to be multivalent.

[0056] A "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0057] The term "inhibiting TSLP activity" includes inhibiting any one or more of the following: binding of TSLP to its receptor; proliferation, activation, or differentiation of TSLPR-expressing cells in the presence of TSLP; inhibition of Th2 cytokine production in a polarization assay in the presence of TSLP; activation or maturation of dendritic cells in the presence of TSLP; and mast cell cytokine release in the presence of TSLP. See, e.g., U.S. Pat. No. 7,982,016 B2, column 6 and Example 8 and U.S. Patent Application Publication No. 2012 / 0020988 A1, Examples 7-10.

[0058] The term "sample" or "biological sample" refers to a specimen obtained from a subject for use in the present methods, and includes urine, whole blood, plasma, serum, saliva, sputum, tissue biopsy, cerebrospinal fluid, peripheral blood mononuclear cells with in vitro stimulation, peripheral blood mononuclear cells without in vitro stimulation, intestinal lymphoid tissue with in vitro stimulation, intestinal lymphoid tissue without in vitro stimulation, intestinal lavage, bronchioloalveolar lavage, nasal wash, and induced sputum.

[0059] The terms "treat", "treating" and "treatment" refer to the partial or complete elimination, reduction, inhibition or amelioration, either temporarily or permanently, of clinical symptoms, onset or progression of an event, disease or condition associated with an inflammatory disorder as described herein. As recognized in the relevant art, a drug used as a therapeutic agent may reduce the severity of a given pathology, but need not eliminate every symptom of the disease to be considered a useful therapeutic agent. Similarly, a treatment administered prophylactically need not be completely effective in preventing the onset of a condition to constitute a viable prophylactic agent. It is sufficient to merely reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect) or to reduce the likelihood that the disease will develop or worsen in a subject. One embodiment of the present invention relates to a method for determining the efficacy of a treatment, comprising administering a therapeutic agent to a patient in an amount and for a time sufficient to induce a sustained improvement over baseline of an index reflecting the severity of a particular disorder.

[0060] The term "therapeutically effective amount" refers to an amount of a therapeutic agent effective to ameliorate or alleviate a symptom or sign of a disease associated with a disease or disorder.

[0061] TSLP Thymic stromal lymphopoietin (TSLP) is produced in response to proinflammatory stimuli and is expressed primarily by dendritic cells (Gilliet, J Exp Med. 197:1059-1067, 2003; Soumelis, Nat Immunol. 3:673-680, 2002; Reche, J Immunol. 167:336-343, 2001), mast cells (Allakhverdi, J Exp Med. 204:253-258, 2007) and CD34+ progenitor cells (Swedin et al. Pharmacol Ther 2017;169:13-34). TSLP signals through a heterodimeric receptor composed of the interleukin (IL)-7 receptor alpha (IL-7Rα) chain and the common gamma chain-like receptor (TSLPR) (Pandey, Nat Immunol. 1:59-64, 2000; Park, J Exp Med. 192:659-669, 2000).

[0062] Human TSLP mRNA (Brightling et al., J Allergy Clin Immunol 2008;121:5-10;quiz 1-2; Ortega et al. N Engl J Med 2014;371:1198-207) and protein levels (Ortega et al., supra) are increased in the airways of asthmatic individuals compared to controls, and the magnitude of this expression correlates with disease severity (Brightling et al., supra). Recent studies have demonstrated an association between single nucleotide polymorphisms in the human TSLP locus and protection from asthma, atopic asthma, and airway hyperresponsiveness, suggesting that differential regulation of TSLP gene expression may influence disease susceptibility (Ortega et al. N Engl J Med 2014;371:1198-207; To et al. BMC Public Health 2012;12:204). These data suggest that targeting TSLP may inhibit multiple biological pathways involved in asthma.

[0063] Early preclinical studies of TSLP suggested that after TSLP is released from airway epithelial or stromal cells, it activates mast cells, dendritic cells, and T cells to release Th2 cytokines (e.g., IL-4 / 13 / 5).Recently published human data showed good correlation between tissue TSLP gene and protein expression, Th2 gene signature score, and tissue eosinophils in severe asthma.Thus, anti-TSLP targeted therapy may be effective in asthma patients with Th2-type inflammation (Shikotra et al, J Allergy Clin Immunol.129(1):104-11,2012).

[0064] Data from other studies suggest that TSLP may promote airway inflammation through Th2-independent pathways, such as crosstalk between airway smooth muscle and mast cells (Allakhverdi et al, J Allergy Clin Immunol. 123(4):958-60, 2009; Shikotra et al., supra). TSLP may also promote the induction of T cells to differentiate into Th-17-cytokine producing cells, resulting in increased neutrophilic inflammation commonly seen in more severe asthma (Tanaka et al, Clin Exp Allergy. 39(1):89-100, 2009). These data and other emerging evidence suggest that blocking TSLP may help suppress multiple biological pathways, including but not limited to those involving Th2 cytokines (IL-4 / 13 / 5).

[0065] antibody Antibodies or antibody variants or antigen binding proteins specific for TSLP are believed to be useful in the treatment of asthma, including severe asthma, eosinophilic asthma, non-eosinophilic / hypoeosinophilic and other forms of asthma described herein, atopic dermatitis and COPD.

[0066] Specific binding agents, such as antibodies and antibody variants or fragments, that bind to a target antigen, e.g., TSLP, are useful in the methods of the invention. In one embodiment, the specific binding agent is an antibody. The antibody can be monoclonal (MAb); recombinant; chimeric; humanized, such as complementarity determining region (CDR) grafted; human; antibody variants, including single chain; and / or bispecific; as well as fragments, variants, or derivatives thereof. Antibody fragments include those portions of an antibody that bind to an epitope on a polypeptide of interest. Examples of such fragments include Fab and F(ab') fragments generated by enzymatic cleavage of full-length antibodies. Other binding fragments include fragments generated by recombinant DNA techniques, such as expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.

[0067] Monoclonal antibodies can be modified for use as therapeutic or diagnostic agents. One embodiment is a "chimeric" antibody in which a portion of the heavy (H) and / or light (L) chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass. Fragments of such antibodies are also included, so long as they exhibit the desired biological activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985, Proc. Natl. Acad. Sci. 81:6851-55.

[0068] In another embodiment, the monoclonal antibody is a "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art. See U.S. Patent Nos. 5,585,089 and 5,693,762. Generally, a humanized antibody has one or more amino acid residues introduced from a source that is non-human. Humanization can be performed, for example, by replacing at least a portion of a rodent complementarity determining region with the corresponding region of a human antibody using methods described in the art (Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1998, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-36).

[0069] Human antibody variants (including antibody fragments) that bind TSLP are also encompassed by the present invention. Using transgenic animals (e.g., mice) that are capable of producing a repertoire of human antibodies without producing endogenous immunoglobulins, such antibodies are produced by immunization with a polypeptide antigen (i.e., having at least six consecutive amino acids), optionally conjugated to a carrier. See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. 90:2551-55; Jakobovits et al., 1993, Nature 362:255-58; Bruggermann et al., 1993, Year in Immuno. 7:33. See also PCT applications PCT / US96 / 05928 and PCT / US93 / 06926. Further methods are described in U.S. Patent No. 5,545,807, PCT applications PCT / US 91 / 245 and PCT / GB 89 / 01207, and EP 546073B1 and EP 546073A1. Human antibodies can also be produced by expression of recombinant DNA in host cells or in hybridoma cells as described herein.

[0070] Chimeric, CDR-grafted and humanized antibodies and / or antibody variants are usually produced by recombinant methods. Nucleic acid encoding the antibody is introduced into a host cell and expressed using the materials and procedures described herein. In a preferred embodiment, the antibody is produced in a mammalian host cell, such as a CHO cell. Monoclonal (e.g., human) antibodies may be produced by expression of recombinant DNA in a host cell or by expression in hybridoma cells as described herein.

[0071] The anti-TSLP antibody tezepelumab is described in U.S. Patent No. 7,982,016 and U.S. Patent Application Publication No. 15 / 951,602. It has been discovered herein that under stressed storage conditions, such as at 40° C. for 4 weeks (40C4W) or at 50° C. for 1 week (50C1W), residues on the tezepelumab antibody undergo changes such as isomerization, deamidation, or oxidation that are detrimental to antibody stability. Residues identified as causing reduced stability in the anti-TSLP antibody tezepelumab CDRs (SEQ ID NOs: 3-8) or variable regions (SEQ ID NOs: 10 and 12) include CDRH1 M34, CDRH2 W52, CDRH2 D54, CDRH2 N57, CDRH2 D62, CDRH3 W102, FRH1 N25, FRH1 N26, CDRL2 D49, CDRL2 D50, FRL2 N65, CDRL3 W90, CDRL3 D91, CDRL3 S92, S93, S94, CDRL3 D95. The numbering of residues in tezepelumab is based on the heavy and light chain sequences set forth in SEQ ID NOs: 10 and 12, respectively.

[0072] Anti-TSLP antigen-binding proteins (including fragments thereof) useful in the present methods include anti-TSLP antibodies comprising: a. a light chain variable domain comprising: i. a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; ii. a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; iii. a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; and b. a heavy chain variable domain comprising: i. a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; ii. a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and iii. a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8, wherein the antibody or antibody variant specifically binds to the TSLP polypeptide set forth in amino acids 29 to 159 of SEQ ID NO:2.

[0073] Also contemplated are antibodies or antibody variants, the antibodies or antibody variants comprising: a. a light chain variable domain selected from the group consisting of: i. a sequence of amino acids at least 80% identical to SEQ ID NO: 12; ii. a sequence of amino acids encoded by a polynucleotide sequence at least 80% identical to SEQ ID NO: 11; iii. a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO: 11; and b. a heavy chain variable domain selected from the group consisting of: i. an amino acid sequence at least 80% identical to SEQ ID NO: 10; ii. a sequence of amino acids encoded by a polynucleotide sequence at least 80% identical to SEQ ID NO: 9; iii. a heavy chain variable domain selected from the group consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO: 9; or c. a light chain variable domain of (a) and a heavy chain variable domain of (b), wherein the antibody or antibody variant specifically binds to a TSLP polypeptide set forth in amino acids 29 to 159 of SEQ ID NO: 2.

[0074] Tezepelumab is an exemplary anti-TSLP antibody, comprising a light chain variable domain comprising: ai. a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; ii. a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; iii. a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; and bi. a heavy chain variable domain comprising: a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; ii. a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and iii. a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8.

[0075] Tezepelumab also comprises a light chain variable domain having the amino acid sequence set forth in SEQ ID NO:12, which is encoded by the polynucleotide sequence set forth in SEQ ID NO:11; and a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:10, which is encoded by the polynucleotide sequence set forth in SEQ ID NO:9.

[0076] Tezepelumab is an IgG2 antibody. The full-length heavy and light chain sequences of tezepelumab, including the IgG2 chain, are set forth in SEQ ID NOs: 37 and 38, respectively.

[0077] In various embodiments, the anti-TSLP antibody or antibody variant thereof is bivalent and is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0078] In various embodiments, the anti-TSLP antibody variant is selected from the group consisting of a diabody, a triabody, a tetrabody, a Fab fragment, a single domain antibody, and an scFv, and the dosage is adjusted so that the binding sites are equimolar to that administered by a bivalent antibody.

[0079] The antibody or antibody variant is considered to be an IgG2 antibody. An exemplary sequence of a human IgG2 constant region is available from the Uniprot database as Uniprot number P01859, and is incorporated herein by reference. Information, including sequence information, on the heavy and light chain constant regions of other antibodies is also publicly available via the Uniprot database, as well as other databases familiar to those skilled in the art of antibody engineering and production.

[0080] In certain embodiments, antibody derivatives include tetrameric glycosylated antibodies in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, the variants contain more or fewer N-linked glycosylation sites than the native protein. Instead, substitutions that delete this sequence remove existing N-linked glycans. Also, a rearrangement of the N-linked glycans is produced in which one or more N-linked glycosylation sites (typically those that occur naturally) are deleted and one or more new N-linked sites are generated. Further preferred antibody variants include cysteine ​​variants in which one or more cysteine ​​residues are deleted or replaced by another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants may be useful when the antibody must be refolded into a biologically active conformation, such as after isolation of insoluble inclusion bodies. Cysteine ​​variants generally have fewer cysteine ​​residues than the native protein, and typically have an even number of cysteine ​​residues to minimize interactions resulting from unpaired cysteines.

[0081] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by one of skill in the art at the time such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify key residues of antibodies to human TSLP or to increase or decrease the affinity of the antibodies to human TSLP described herein.

[0082] According to certain embodiments, preferred amino acid substitutions (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity, (4) inhibit formation of high molecular weight (HMW) species, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the native sequence (in certain embodiments, in the portion of the polypeptide outside the domains that form intermolecular contacts). In certain embodiments, conservative amino acid substitutions typically may not substantially alter the structural features of the parent sequence (e.g., the substituted amino acid should not tend to disrupt helices present in the parent sequence or other types of secondary structures that characterize the parent sequence). Examples of polypeptide secondary and tertiary structures recognized by those of skill in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991), each of which is incorporated herein by reference.

[0083] Identification of attributes that contribute to stability and protein binding To determine the attributes that contribute to the binding and activity of the protein, the anti-TSLP antigen binding proteins described herein are subjected to conditions that result in a change in its structure, e.g., a change in the structure of an amino acid of the therapeutic protein, resulting in the formation of a species of the therapeutic protein. In exemplary embodiments, the altered structure of an amino acid is referred to as an "attribute" and can be characterized with respect to its chemical identity or attribute type and its position within the amino acid sequence of the antigen binding protein (e.g., the position of the amino acid where the attribute is present). For example, asparagine and glutamine residues exhibit susceptibility to deamidation. A deamidated asparagine at position 10 of the amino acid sequence of a protein is an example of an attribute. A list of exemplary attribute types for specific amino acids is provided in Table A. Thus, a "structure" as used herein can comprise, consist essentially of, or consist of an attribute type listed in Table A or a combination of two or more attribute types listed in Table A. An attribute is an example of a structure, and it will be understood that whenever "structure" is referred to herein, attributes are contemplated as examples of structures, unless otherwise indicated. For example, high molecular weight species (HMW) and fragments are also examples of attributes.

[0084] [Table 1]

[0085] As an immunoglobulin or fragment thereof, an antibody or antigen binding protein comprises multiple amino acids, and an antibody or antigen binding protein described herein may have two or more attributes (e.g., two or more amino acids with altered structures) and may be described in terms of its attribute profile. As used herein, the term "attribute profile" refers to a list of attributes of an antigen binding protein. In various examples, the attribute profile optionally provides the chemical identity or attribute type, e.g., deamidation, relative to the native structure of the therapeutic protein. In various examples, the attribute profile provides the location of the attribute, e.g., the position of the amino acid where the attribute is present. In some embodiments, the attribute profile provides a description of all attributes present in the antigen binding protein. In other embodiments, the attribute profile provides a description of some attributes present in the protein. For example, the attribute profile may only provide attributes present in a particular portion of the protein, e.g., the constant region, variable region, CDRs. A species of therapeutic protein, such as an antibody or antigen binding protein, is characterized by the attributes present on the protein. A species of target binding protein may differ from another species of the same protein by having a different attribute profile. If two therapeutic proteins have different attribute profiles, then the therapeutic proteins represent two different species of therapeutic proteins. If two therapeutic proteins have identical attribute profiles, then the therapeutic proteins are considered to be the same species of therapeutic proteins.

[0086] In various examples, the immunoglobulin, antibody or antigen binding protein is placed in conditions that result in a change in its structure, e.g., the formation of one or more attributes, where the change in structure changes the affinity of the therapeutic protein for its target. In various embodiments, the immunoglobulin, antibody or antigen binding protein is placed in conditions that result in a change in its structure, e.g., the formation of one or more attributes, where the change in structure reduces the affinity of the antigen binding protein for its target. The reduced affinity in some embodiments results in a partial or total loss of the ability of the immunoglobulin, antibody or antigen binding protein to interact (e.g., bind) with the target. In various examples, the partial or total loss of the ability of the immunoglobulin, antibody or antigen binding protein to interact (e.g., bind) with the target ultimately reduces the effectiveness of the antigen binding protein. In an alternative example, the immunoglobulin, antibody or antigen binding protein is placed in conditions that result in a change in its structure, e.g., the formation of one or more attributes, where the change in structure does not change the affinity of the immunoglobulin, antibody or antigen binding protein for its target. In various embodiments, the change in structure does not reduce the affinity of the protein for its target. Without being bound to any particular theory, the methods of the present disclosure advantageously precisely and accurately distinguish attributes of an immunoglobulin, antibody or antigen binding protein that affect the interaction between an immunoglobulin, antibody or antigen binding protein and a target from attributes that do not affect the interaction.

[0087] In various aspects, the compositions herein comprise a population of species of immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof. In various examples, the population is a homogeneous population of immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof, and optionally, each protein present in the composition sample is of the same species. In various examples, the population is a heterogeneous population comprising at least two different species of immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof, having the attributes described herein. In various aspects, the heterogeneous population comprises at least two, at least three, at least four, at least five, at least six or more different species of immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof. Optionally, the heterogeneous population comprises more than seven, more than eight, more than nine, more than ten, more than twenty, more than thirty, more than forty, more than fifty different species of proteins. In some aspects, each species of the population has a unique attribute profile. In illustrative examples, the species of immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof is the only protein present in the composition. In some aspects, the composition comprises (i) a population immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof, and (ii) a pharma- ceutically acceptable carrier, diluent, excipient, or combination thereof. In some embodiments, at least 80%, 85%, 90%, 95%, or 99% of the immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof of the heterogeneous population comprise an attribute described herein. In some embodiments, no more than 20%, 15%, 10%, 5%, or 1% of the immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof of the heterogeneous population comprise an attribute described herein.

[0088] In an exemplary embodiment, the method includes applying a stress to an immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof sample. In various cases, the stress can be any condition that results in at least one change in the structure of an immunoglobulin amino acid, antigen binding protein or fragment thereof, or antibody or fragment thereof, or target, e.g., the stress can be any condition that results in the formation of at least one attribute in an immunoglobulin amino acid, antigen binding protein or fragment thereof, or antibody or fragment thereof, or target. Optionally, the stress results in a change in the structure of two or more amino acids of an immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof, or target, e.g., the stress results in the formation or two or more attributes (e.g., at least or about 2, at least or about 3, at least or about 4, at least or about 5, at least or about 6, at least or about 7, at least or about 8, at least or about 9, at least or about 10 or more attributes). The stress in various examples results in the formation of one or more attributes that are not present in the immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof, or target prior to application of the stress. Thus, in some aspects, the application of stress results in the formation of species of immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof, or targets that were not present in the sample prior to the application of the stress.

[0089] In exemplary embodiments, the stress is exposure to elevated temperatures, e.g., 25° C., 40° C., 50° C., optionally in one or more buffers or formulations. In exemplary examples, such exposure to elevated temperatures mimics an accelerated stress program.

[0090] Optionally, the stress degrades or dissociates about 5% to about 30%, about 10% to about 30%, about 15% to about 30%, about 20% to about 30%, about 25% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10% of the complexes formed between the immunoglobulin, antibody, or antigen binding protein and the target. In various aspects, the stress causes a decrease in the level of interaction between the immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof, and its target. In some embodiments, the stress reduces the interaction by about 10% to about 50% (e.g., about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, or about 10% to about 15%) compared to the interaction under corresponding conditions without stress. In some embodiments, the stress reduces the K D Increase this K D is associated with weaker binding. In some embodiments, the stress increases the amount of unbound antibody or antigen binding protein by 10% to about 50% (e.g., about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, or about 10% to about 15%). Without being bound to a particular theory, the stress applied in the methods disclosed herein results in the generation of antibody or antigen binding protein species in a more rapid and more robust and reproducible manner, yielding abundant and diverse species for enhanced detection of species that may be produced during manufacturing, storage, and in human circulation (intravenous or subcutaneous space in a human subject).

[0091] separation In exemplary embodiments, the disclosed methods include separating a mixture containing different species of antigens into at least two fractions. In some embodiments, the mixture is separated into multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) fractions. In some embodiments, the separation step of the methods disclosed herein preserves the native folding, conformation and binding ability of the antigen binding protein and its target. In various embodiments, the mixture is separated into an unbound fraction, which contains unbound antibody or antigen binding protein or target, and a bound fraction, which contains antibody / antigen binding protein-target complexes.

[0092] Suitable methods and techniques for separating mixtures into fractions are known in the art.See, for example, Coskun, North Clin Istanb 3(2):156-160(2016);Snyder et al., Practical HPLC Method Development, 2nd ed., John Wiley&Sons, Inc.1997;Snyder et al., Introduction to Modern Liquid Chromatography, John Wiley&Sons, Inc., Hoboken, NJ, 2010;Heftmann, Chromatography: Fundamentals and applications of chromatography and related differential migration methods, 6th ed., Volume69A, Elsevier, Amsterdam, Netherlands, 2004;Mori and Barth, Size Exclusion Chromatography, Springer-Verlag, Berlin, 1999. In some aspects, separation is based on charge, e.g., ion exchange chromatography, capillary isoelectric focusing (cIEF) and / or capillary zone electrophoresis (CZE), or based on hydrophobicity, e.g., reversed phase (RP; e.g., RP-HPLC) and hydrophobic interaction chromatography (HIC-HPLC). In various aspects, separation is based on size, e.g., size exclusion chromatography (SEC; e.g., SE-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). The methods described herein are used to detect product oxidation, fragmentation / clipping of Met or Trp residues, isomerization of Asp, deamidation, formation of pyroglutamic acid at the N-terminus. In various embodiments, the mixture is separated into at least two fractions using a technique that separates components of the mixture based on size, charge, hydrophobicity, affinity for a capture molecule, or a combination thereof.In various examples, the technique is size exclusion chromatography (SEC), affinity chromatography, precipitation using beads or cells, free-flow fractionation (FFF), ion exchange chromatography (IEX), cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC) or ultracentrifugation (UC). Optionally, the mixture is separated into at least two fractions using a technique that separates components of the mixture based on size, optionally the technique is size exclusion chromatography (SEC).

[0093] In various embodiments, the mixture is separated into at least two fractions using a technique that separates components of the mixture based on their affinity to a capture molecule bound to a solid support, optionally beads or cells. In various examples, the mixture is separated by (i) adding the mixture to a container, e.g., a tube, containing beads or cells bound to capture molecules, the cells expressing the capture molecules on their surface, (ii) centrifuging the container (e.g., tube) to obtain a supernatant and a pellet, (iii) recovering the supernatant from the pellet to obtain an unbound fraction, (iv) releasing the bound fraction from the pellet with a solution, (v) centrifuging the container (e.g., tube) containing the pellet and solution to obtain a second supernatant containing the bound fraction and a second pellet containing the beads or cells, and (vi) recovering the second supernatant to obtain the bound fraction. In some embodiments, the mixture is separated by (i) adding the mixture containing the beads bound to the capture molecule to a column to obtain a flow-through and a bound fraction, (ii) collecting the flow-through to obtain an unbound fraction, and (iii) releasing the bound fraction from the beads with a solution and collecting a solution containing the bound fraction. Suitable solid supports include, for example, beads, resins, and papers, optionally made of cellulose, silica, alumina, glass, plastic, or combinations thereof. In an exemplary embodiment, the capture molecule bound to the solid support is a protein. The capture molecule can be the same as the target. Advantageously, the capture molecule is not limited to a specific molecule.

[0094] In various embodiments of the method of identifying attributes of an immunoglobulin, antigen binding protein or target that affect the interaction between an antigen binding protein and a target, for each of the unbound and bound fractions, the method comprises identifying and quantifying the abundance of each attribute present in the antigen binding protein or target species, where if the abundance of the attribute in the unbound fraction is greater than the abundance of the attribute in the bound fraction, the attribute adversely affects the interaction between the antigen binding protein and the target. In various aspects, the method comprises using a mass spectrometer to identify and quantify the abundance of each attribute of the antigen binding protein or target species in each of the unbound and bound fractions.

[0095] In various embodiments of the method for determining the effect of a known attribute present in an antigen binding protein or target species on the interaction between the antigen binding protein and the target, the method comprises quantifying the abundance of the known attribute for each of the unbound and bound fractions, where if the abundance of the known attribute in the unbound fraction is greater than the abundance of the known attribute in the bound fraction, the known attribute adversely affects the interaction between the antigen binding protein and the target. In various aspects, the method comprises quantifying the abundance of the known attribute in each of the unbound and bound fractions using a mass spectrometer.

[0096] Stability refers to resistance to chemical modification of amino acid residues and biophysical protein modifications, such as the formation of HMW species during stress conditions that may occur during manufacturing, storage, and / or additional or alternative stress conditions. For the methods of embodiments described herein and immunoglobulins, antigen binding proteins, and fragments thereof, "stability" and / or "HMW" species may be determined using size exclusion chromatography (SEC). Compositions including immunoglobulins, antigen binding proteins, or fragments may be separated by SEC, such as SEC-UV. SEC may use a mobile phase containing 100 mM sodium phosphate and 250 mM NaCl (pH 6.8), the flow rate may be set at 0.5 ml / min, the column temperature may be set at 37° C., the run time may be 35 minutes, and the autosampler may be set at 4° C. Examples of columns suitable for SEC include gel columns containing silica particles containing diol functional groups, with an average diameter of 5 μm, and an average pore size of about 25 nM (e.g., commercially available from TOSOH Bioscience as G3000SWxl columns). For SEC-UV, ultraviolet / visible spectroscopy (UV / VIS) detection can be performed at 214 nm and 280 nm. It will be appreciated that after separation, peaks representing monomer and HMW species can elute at different times in the SEC elution profile.

[0097] For stability measurements, compositions for SEC analysis may include stressed immunoglobulins, antigen binding proteins or fragments, which may be stressed at elevated temperatures for a period of time, for example, 40° C. for 4 weeks. Note that 4 weeks at 40° C. generally provides a sufficient extrapolation to the shelf-life stability of immunoglobulins, antigen binding proteins and fragments thereof (which typically have a shelf-life stability of 2 years (2Y4C) at 2-8° C., followed by 1 month at room temperature, which may be 25° C. or 30° C. depending on geographic location). Additionally or alternatively, immunoglobulins, antigen binding proteins and fragments thereof may be stressed at elevated temperatures for a period of time, for example, 40° C. for 4 weeks. Additionally or alternatively, immunoglobulins, antigen binding proteins and fragments thereof may be stressed at elevated temperatures for a period of time, for example, 40° C. for 4 weeks. Note that 4 weeks at 40° C. generally provide a sufficient extrapolation to the shelf-life stability of immunoglobulins, antigen binding proteins and fragments thereof (which typically have a shelf-life stability of 2 years (2Y4C) at 2-8° C., followed by 1 month at room temperature, which may be 25° C. or 30° C. depending on geographic location). Additionally or alternatively, immunoglobulins, antigen binding proteins and fragments thereof may be stressed at elevated temperatures for 7 days, for example, 40° C. for 2 years ... 2Stressors may include UVA light), extreme pH (pH 8 or above or 3.6 or below), or oxidizing reagents (e.g., 0.1% H2O2, 5 hours at 25°C). Unless otherwise stated herein or required by the scientific context, stress for the purposes of "stability" is understood to refer to 4 weeks at 40°C. Additional information regarding stress factors and SEC analysis can be found, for example, in WO 2020 / 247790, which is incorporated herein by reference in its entirety.

[0098] Following SEC analysis, peptide mapping can optionally be performed to identify peptide modifications associated with bound and unbound species, for example as described herein and / or in WO 2020 / 247790. For peptide mapping, elution fractions can be collected using a filter with a molecular weight cutoff (e.g., greater than 10 kDa) and eluted with 7.5 M guanidine elution buffer. To determine chemical modifications that affect binding to antigen, stressed immunoglobulin (or antigen-binding protein or fragment thereof) and antigen can be mixed together and separated as the antigen-binding complex elutes first and the unbound immunoglobulin (or antigen-binding protein or fragment thereof) elutes later. To determine chemical modifications that affect or correlate with HMW, monomeric and HMW species can be collected. In the described study, thermal forced stress at 40° C. for one month (40C1M) and associated degradation of antibodies were used. The stressed antibody was mixed with its target (TSLP) and the mixture was separated by SEC for antibody-target complexes and unbound antibodies. Two limitations of this applied SEC antibody-antigen method should be noted. 40C1M stress may result in greater degradation compared to room temperature degradation at 25C or 30C. Also, degradation / modification of one residue may be caused by long-range allosteric interactions with modifications of another residue. This effect may increase at higher temperatures because the structure is more suitable for dynamic motion. Also, the described SEC antigen-antibody method analyzes all species in the sample. This differs from conventional methods that involve CEX separation on individual peaks followed by characterization, as the collected species are better defined and "between the main peaks" species with more than one modification per molecule are avoided.

[0099] It will be understood that "affinity" or "binding" may also be determined by surface plasmon resonance (SPR), biolayer interferometry, or SEC binding affinity experiments as described herein. Unless otherwise stated herein or required otherwise by the scientific context, "affinity" is understood to refer to affinity measured by SPR. Kd values ​​can be measured by SPR using a biosensor system such as the BIAcore® system. Analysis by the BIAcore® system may include analyzing the binding and dissociation of an antigen (e.g., TSLP) from a chip having immobilized molecules (e.g., anti-TSLP immunoglobulins, antigen-binding proteins, or fragments thereof, as described herein) on its surface. 10 -6 Binding complexes less than M can be detected using SPR. In various embodiments, SPR can be performed at 20°C, 25°C, 30°C, or 37°C.

[0100] composition In various embodiments, a composition is provided that includes a plurality of anti-TSLP immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof, each comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8, comprising at least one of an L-aspartic acid at HC position 54 of SEQ ID NO:7 that does not contain an isoaspartic acid (isoAsp) or a cyclic aspartic acid (cAsp); a non-oxidized HC W102 of SEQ ID NO:8; an L-aspartic acid at LC position 49 or 50 of SEQ ID NO:7 that does not contain an isoAsp or a cAsp; a LC N65 set forth in SEQ ID NO:12 that does not contain a deamidated N65; or an L-aspartic acid at LC position 91 of SEQ ID NO:5 that does not contain an isoAsp or a cAsp.

[0101] In various embodiments, a composition is provided that includes a plurality of anti-TSLP monoclonal antibodies or antigen-binding fragments thereof, the plurality of anti-TSLP monoclonal antibodies or antigen-binding fragments thereof each comprising: a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8; an L-aspartic acid at HC position 54 of SEQ ID NO:7 that does not contain an isoaspartic acid (isoAsp) or a cyclic aspartic acid (cAsp); an unoxidized HC W102 of SEQ ID NO:8; an L-aspartic acid at LC position 49 or 50 of SEQ ID NO:4 that does not contain an isoAsp or a cAsp; a deamidated LC N65; or at least one L-aspartic acid at LC position 91 of SEQ ID NO:5 that does not contain isoAsp or cAsp.

[0102] In various embodiments, 0.9% or less of the anti-TSLP monoclonal antibody comprises isomerized HC D54. In various embodiments, 2% or less of the anti-TSLP monoclonal antibody comprises oxidized HC W102. In various embodiments, 0.9% or less of the anti-TSLP monoclonal antibody comprises isomerized LC D50. In various embodiments, 0.5% or less of the anti-TSLP monoclonal antibody comprises deamidated LC N65. In various embodiments, 0.9% or less of the anti-TSLP monoclonal antibody comprises isomerized HC D91. In various embodiments, the anti-TSLP antibody comprises a combination of HC54 L-aspartic acid and LC 49 or 50 L-aspartic acid. In various embodiments, the anti-TSLP antibody is enriched in HC54 L-aspartic acid at least 6-fold over the level of isoAsp. In various embodiments, the anti-TSLP antibody is an IgG2 antibody.

[0103] In one aspect, a composition comprises an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof, comprising: (A) a light chain variable domain comprising: (i) a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3, (ii) a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5, and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7 with a mutation at least one of the following residues D54 or G55, and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8. In various embodiments, HCDR2 has the sequence VIWYX1X2SNKHYADSVKG, where X1 is D or E and X2 is G or A (SEQ ID NO:13). Optionally, HCDR2 has the following sequence: VIWYEGSNKHYADSVKG (SEQ ID NO: 14), VIWYDASNKHYADSVKG (SEQ ID NO: 15), or VIWYEASNKHYADSVKG (SEQ ID NO: 16).

[0104] In various embodiments, the mutation in HCDR2 is D54E. In various embodiments, the mutation in HCDR2 is G55A. In various embodiments, the anti-TSLP antigen binding protein or fragment thereof optionally comprises a mutation in at least one of residues LCDR2 D49, D50, or S51 of SEQ ID NO:4. In various embodiments, the mutation in LCDR2 is one or more of D49E, D50E, or S51A. In various embodiments, LCDR2 has the sequence X1X2X3DRPS, where X1 is D or E, X2 is D or E, and X3 is S or A (SEQ ID NO: 17). Optionally, LCDR2 has the following sequence: EDSDRPS (SEQ ID NO: 18), DESDRPS (SEQ ID NO: 19), EESDRPS (SEQ ID NO: 20), DDADRPS (SEQ ID NO: 21), DEADRPS (SEQ ID NO: 22), EDADRPS (SEQ ID NO: 23) or EEADRPS (SEQ ID NO: 24).

[0105] In various embodiments, the composition comprises an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof, comprising: (A) a light chain variable domain comprising: (i) a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 sequence comprising an amino acid sequence having a mutation in at least one of the following residues D49, D50, or S51 of SEQ ID NO:4; (iii) a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; and (B) a heavy chain variable domain comprising: (i) a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7, and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8.

[0106] In various embodiments, LCDR2 has the sequence X1X2X3DRPS (SEQ ID NO: 17), where X1 is D or E, X2 is D or E, and X3 is S or A. Optionally, LCDR2 has the following sequence: EDSDRPS (SEQ ID NO: 18), DESDRPS (SEQ ID NO: 19), EESDRPS (SEQ ID NO: 20), DDADRPS (SEQ ID NO: 21), DEADRPS (SEQ ID NO: 22), EDADRPS (SEQ ID NO: 23), or EEADRPS (SEQ ID NO: 24). In various embodiments, the mutation in LCDR2 is D49E. In various embodiments, the mutation in LCDR2 is D50E. In various embodiments, the mutation in LCDR2 is S51A. In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof optionally comprises a mutation at one of the following residues D54 or G55 of HCDR2 as set forth in SEQ ID NO: 7. In various embodiments, the mutation in HCDR2 is one or more of D54E or G55A of SEQ ID NO: 7. In various embodiments, HCDR2 has the sequence VIWYX1X2SNKHYADSVKG, where X1 is D or E, and X2 is G or A (SEQ ID NO: 13). Optionally, HCDR2 has the following sequence: VIWYEGSNKHYADSVKG (SEQ ID NO: 14), VIWYDASNKHYADSVKG (SEQ ID NO: 15), or VIWYEASNKHYADSVKG (SEQ ID NO: 16).

[0107] In various embodiments, the composition comprises an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof, which comprises: (A) a light chain variable domain selected from the group consisting of: i. a sequence of amino acids at least 80% identical to SEQ ID NO:12; ii. a sequence of amino acids encoded by a polynucleotide sequence at least 80% identical to SEQ ID NO:11; or iii. a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO:11; or (B) i. a sequence of amino acids at least 80% identical to SEQ ID NO:10. a heavy chain variable domain selected from the group consisting of: a sequence of identical amino acids; ii. a sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:9; or iii. a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO:9; or (C) a light chain variable domain of (A) and a heavy chain variable domain of (B), wherein the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof retains one or more of the CDRs of the anti-TSLP antigen binding protein or fragment thereof and contains a mutation in one or more of HCDR2 D54 or G55 of SEQ ID NO:7 or LCDR2 D49, D50 or S51 of SEQ ID NO:4.

[0108] In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof is [ka] (HCDR2 is underlined) wherein X1 is D or E, X2 is G or A, and optionally [ka] or a mixture thereof).

[0109] In various embodiments, the anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof is [ka] (LCDR1~3 are underlined) wherein X1 is D or E, X2 is D or E, and X3 is S or A; and optionally [ka] or a mixture thereof).

[0110] Also provided are compositions comprising anti-TSLP monoclonal antibodies comprising TSLP antibodies having sequences described herein, e.g., one or more CDRs set forth in SEQ ID NOs: 3-8 and 13-24, and one or more variable regions set forth in SEQ ID NOs: 10 and 12, and SEQ ID NOs: 25-36, respectively, and the anti-TSLP monoclonal antibodies of the compositions are preferably 10 to 15% by weight. -8 In various embodiments, the anti-TSLP antibodies described herein comprise a limited content of isomeric HC D54 and / or a limited content of isomeric LCD D49 or D50 effective to bind TLSP with a Kd value of at least 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 It binds with an affinity (Kd) of less than or equal to M.

[0111] Also provided are compositions comprising anti-TSLP monoclonal antibodies comprising TSLP antibodies having sequences described herein, e.g., CDRs set forth in SEQ ID NOs:3-8 or SEQ ID NOs:13-24 and / or variable regions set forth in SEQ ID NOs:10 and 12 or SEQ ID NOs:25-36, respectively, wherein the compositions comprise an IgG2 anti-TSLP monoclonal antibody and at least one of: 0.9% or less of the anti-TSLP monoclonal antibodies contain isomerized HC D54; 2% or less of the anti-TSLP monoclonal antibodies contain oxidized HC W102; 0.9% or less of the anti-TSLP monoclonal antibodies contain isomerized LC D50; 0.5% or less of the anti-TSLP monoclonal antibodies contain deamidated LC N65; or 0.9% or less of the anti-TSLP monoclonal antibodies contain isomerized LC D91.

[0112] In some embodiments, the composition is part of a formulation described herein. In some embodiments, the composition is a drug substance used to manufacture a formulation described herein.

[0113] Method of administration In one aspect, the methods of the disclosure comprise administering a therapeutic anti-TSLP antibody or antibody variant described herein, optionally in a pharma- ceutical carrier or excipient, hi certain embodiments, the pharmaceutical composition is a sterile composition.

[0114] Contemplated herein are methods of treating inflammatory diseases, conditions, or disorders, such as asthma, chronic obstructive pulmonary disease (COPD), atopic dermatitis, eosinophilic esophagitis (EoE), nasal polyps, chronic spontaneous urticaria, Ig-induced disease, IgA nephropathy, lupus nephritis, eosinophilic gastritis, chronic rhinosinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IPF) with an anti-TSLP antibody or antigen binding protein or fragment thereof described herein. In various embodiments, the disease, condition, or disorder is asthma, including severe asthma, eosinophilic or non-eosinophilic asthma, and hypoeosinophilic asthma.

[0115] Asthma is a chronic inflammatory disorder of the airways. Each year, asthma accounts for an estimated 1.1 million outpatient visits, 1.6 million emergency room visits, and 444,000 hospitalizations (Defrances et al, 2008), available at the Centers for Disease Control website, www.cdc.gov / nchs / data / nhsr / nhsr005.pdf, and 3,500 deaths in the United States. In susceptible individuals, asthmatic inflammation causes recurrent episodes of wheezing, shortness of breath, chest tightness, and coughing. The pathogenesis of asthma is thought to be multifactorial, influenced by both genetic and environmental mechanisms (To et al., BMC Public Health 2012;12:204; Chung et al. Eur Respir J 2014;43:343-73), with environmental allergens being an important contributor (Chung et al., supra; Pavord ID, et al., NPJ Prim Care Respir Med 2017;27:17). The majority of cases arise when a person becomes hypersensitive to an allergen (atopy). Atopy is characterized by increased Th2 cell and Th2 cytokine expression and increased IgE production. Allergy-induced asthma is thought to affect approximately 10 million patients in the United States. Despite available treatment options, asthma remains a major health problem. Approximately 300 million people worldwide currently suffer from asthma, and by 2020, it is predicted that 400 million people will suffer from asthma (Partridge, Eur Resp Rev. 16:67-72, 2007).

[0116] Inhalation of allergens by atopic asthmatics induces several of the symptoms of asthma, including reversible airflow obstruction, airway hyperresponsiveness, and eosinophilic and basophilic airway inflammation. Allergen inhalation challenge has become the predominant model of asthma in many species (Bates et al., Am J Physiol Lung Cell Mol Physiol. 297(3):L401-10, 2009; Diamant et al., J Allergy Clin Immunol. 132(5):1045-1055, 2013).

[0117] Various subtypes of asthma have been identified that are refractory to steroid treatment. Eosinophils are key inflammatory cells in allergic asthma that are characteristically mediated by Th2-type CD4+ T cells. Neutrophilic airway inflammation is associated with corticosteroid treatment of severe asthma and can be mediated by Th1- or Th17-type T cells (Mishra et al., Dis. Model. Mech. 6:877-888, 2013).

[0118] Measures for the diagnosis and assessment of asthma include: airway inflammation assessed using a standardized single-breath fraction exhaled nitric oxide (FeNO) (American Thoracic Society; ATS, Am J Respir Crit Care Med. 171(8):912-30, 2005) test. Spirometry is performed according to the ATS / European Respiratory Society (ERS) guidelines (Miller et al, Eur Respir J. 26(1):153-61, 2005). Post-bronchodilator (post-BD) spirometry tests are assessed after subjects have performed pre-BD spirometry. Maximal bronchodilation is induced using a SABA such as albuterol (90 μg metered dose) or salbutamol (100 μg metered dose) or equivalent with a spacer device for up to eight full puffs (Sorkness et al, J Appl. Physiol.104(2):394-403,2008). The highest pre-BD and post-BD FEV1 obtained after 4, 6 or 8 puffs is used for the determination and analysis of reversibility. The Asthma Control Questionnaire (ACQ)6 is a patient-reported questionnaire that assesses asthma symptoms (i.e., night-time awakenings, symptoms on awakening, activity limitations, shortness of breath, wheezing) and daily rescue bronchodilator use and FEV1 (Juniper et al,Oct 1999). The ACQ-6 is a shortened version of the ACQ that omits the FEV1 measurement from the original ACQ score. The mean ACQ score is the average of the responses. A mean score ≤0.75 indicates well-controlled asthma, a score of 0.75 to ≤1.5 indicates partially controlled asthma, and a score >1.5 indicates uncontrolled asthma (Juniper et al,Respir Med. 100(4):616-21,2006). An individual change of at least 0.5 is considered clinically significant (Juniper et al,Respir Med.99(5):553-8,2005). The Asthma Quality of Life Questionnaire, Standardized (AQLQ[S])+12 (AQLQ(S)+12) is a 32-item questionnaire that assesses the HRQoL experienced by asthma patients (Juniper et al,Chest.115(5):1265-70,May 1999). Daily asthma reports are also used in the assessment.

[0119] Related US Patent Publication No. 2018-0296669 (incorporated herein by reference) discloses that treatment with anti-TSLP antibodies is effective in reducing asthma symptoms in eosinophil-free / hypoeosinophilic populations, as well as in hypereosinophilic populations. Also contemplated is a method of reducing the frequency of asthma exacerbations in a subject.

[0120] Also contemplated herein are methods of treating asthma in subjects with high or low Th2 asthma profiles. TSLP antagonists that inhibit the binding of the TSLP protein to its receptor complex would be effective in treating low eosinophilic asthma populations, as would the antibodies described herein. Similarly, TSLP antagonists that inhibit the binding of TSLP to its receptor complex would be effective in treating low Th2 asthma populations. Also contemplated are methods for treating chronic obstructive pulmonary disease (COPD) in a subject, comprising administering an anti-TSLP antibody or antibody variant or antigen binding protein. The subject to be treated may be a human. The subject may be an adult, an adolescent, or a child.

[0121] Therapeutic antibody (or antibody variant) compositions may be delivered to a patient at multiple sites. Multiple administrations may be given simultaneously or over a period of time. In certain cases, it is beneficial to provide a continuous flow of therapeutic composition. Additional treatments may be given on a periodic basis, for example, hourly, daily, weekly, biweekly, triweekly, monthly or over longer intervals.

[0122] In various embodiments, the amount of therapeutic agent in a given dose, such as a bivalent antibody having two TSLP binding sites, can vary depending on the size of the individual being treated and the characteristics of the disorder being treated.

[0123] In an exemplary treatment, the anti-TSLP antibody or antibody variant is administered in a dose range of about 70 mg to about 280 mg per daily dose. For example, doses can be given at about 70 mg, 210 mg, or 280 mg. In various embodiments, the anti-TSLP antibody or antibody variant can be administered in a dose of 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 10, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, or 280 mg per administration. These concentrates can be administered in single dosage form or in repeated administrations. The above doses are given every two weeks or every four weeks. In various embodiments, the anti-TSLP antibody or antibody variant is administered in a single dose of 70 mg every two weeks or every four weeks. In various embodiments, the anti-TSLP antibody or antibody variant is administered in a single dose of 210 mg every two weeks or every four weeks. In various embodiments, the anti-TSLP antibody or antibody variant is administered in a single dose of 280 mg every two weeks or every four weeks.

[0124] For antibody variants, the amount of antibody variant should be such that the number of TSLP binding sites in a dose has an equimolar number of TSLP binding sites relative to the canonical bivalent antibody described above.

[0125] It is contemplated that the anti-TSLP antibody or antibody variant will be administered every two weeks or every four weeks for a period of at least four months, six months, nine months, a year or more. In various embodiments, administration is subcutaneous or intravenous.

[0126] Treatment with an anti-TSLP antibody or antibody variant is believed to reduce eosinophils in the blood, sputum, bronchoalveolar fluid, or lungs of a subject. Administration is also believed to shift the subject's cell count from a high Th2 population to a low Th2 population. Additionally, administration of an anti-TSLP antibody or antibody variant is believed to improve one or more measures of asthma in a subject selected from the group consisting of forced expiratory volume (FEV), FEV1 reversibility, forced vital capacity (FVC), FeNO, Asthma Control Questionnaire-6 score, and AQLQ(S)+12 score.

[0127] Improvement in asthma may be assessed as one or more of the following: reduction in AER (annualized exacerbation rate), reduction in asthma hospitalizations / severe exacerbations, change (increase) from baseline in time to first asthma exacerbation (after initiation of treatment with an anti-TSLP antibody), reduction in the proportion of subjects with 1 or more asthma exacerbations or subjects with severe exacerbations over the course of treatment, e.g., 52 weeks, compared to placebo, change (increase) from baseline in FEV1 and FVC (pre-bronchodilator and post-bronchodilator), change (decrease) in blood or sputum eosinophils (or lung eosinophils if biopsy or BAL fluid is available), change (decrease) in FeNO from baseline, change (decrease) in IgE from baseline, improvement in asthma symptoms and control as assessed by the ACQ and variants, AQLQ and variants, SGRQ, and asthma symptom diary PRO, change (decrease) in SGRQ, rescue medication use, reduction in systemic corticosteroid use, reduction in blood Th2 / Th1 cell ratio. Most / all of these assessments should be in the entire population and subpopulations, including high and low eosinophils (high >250, low <250), allergic and non-allergic, high and low Th2, high and low periostin (relative to the median), and high and low FeNO (high >24 or low <24).

[0128] The disclosure also contemplates administration of multiple agents, such as an antibody composition in combination with a second agent described herein, including, but not limited to, an anti-inflammatory agent or an asthma therapy.

[0129] However, in various embodiments, the administration is expected to reduce the frequency or level of co-administered therapy in the subject. Exemplary co-administered therapy includes, but is not limited to, inhaled corticosteroids (ICS), long-acting beta 2 agonists (LABA), leukotriene receptor antagonists [LTRA], long-acting antimuscarinics [LAMA], chromones, short-acting beta 2 agonists (SABA) and theophylline or oral corticosteroids. In various embodiments, the administration eliminates the need for corticosteroid therapy.

[0130] formulation In some embodiments, the present disclosure contemplates the use of pharmaceutical compositions comprising a therapeutically effective amount of an anti-TSLP antibody or antibody variant, together with a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Additionally, the present disclosure provides methods of treating a subject by administering such a pharmaceutical composition.

[0131] In certain embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations employed. In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or permeability of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, sucrose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colors, flavors, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin). lydone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapar); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.See REMINGTON'S PHARMACEUTICAL SCIENCES, 18'' Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0132] Suitable vehicles or carriers may be water for injection, saline solution or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, and may further comprise sorbitol or a suitable substitute thereof.

[0133] The formulation components are preferably present in concentrations that are acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower pH (typically within a pH range of about 4.5 to about 8), including about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, and about 8.0.

[0134] In various embodiments, the anti-TSLP antibody or antibody variant is present in a formulation containing acetate and one or more of proline, sucrose, polysorbate 20, or polysorbate 80. In various embodiments, the formulation comprises 5-50 mM acetate, less than 3% (w / v) proline, and 0.015% (w / v) ±0.005% (w / v) polysorbate 20 or polysorbate 80 at a pH of 4.9-6.0. Optionally, the antibody or antibody fragment is at a concentration of about 100 to about 150 mg / ml. The formulation may be stored at -20°C to -70°C. Exemplary anti-TSLP formulations containing these excipients are described in International Application No. PCT / US2021 / 018561, which is incorporated herein by reference.

[0135] In an alternative embodiment, the anti-TSLP antibody or antibody variant is in a formulation containing a surfactant and at least one basic amino acid or salt thereof. In an illustrative example, the basic amino acid is arginine or histidine. In various embodiments, the salt is arginine glutamate or histidine glutamate, optionally at a concentration of 10 to 200 mM. Optionally, the formulation further comprises proline. In an alternative embodiment, the anti-TSLP antibody or antibody variant is in a formulation containing a surfactant and calcium or a salt thereof. In various embodiments, the salt is calcium glutamate, optionally at a concentration of 15 mM to about 150 mM. Optionally, the formulation further comprises proline. In various embodiments, the surfactant is polysorbate 20 or polysorbate 80 or a mixture thereof. Optionally, the antibody or antibody fragment is at a concentration of greater than about 110 mg / ml or greater than about 140 mg / ml. Exemplary anti-TSLP formulations containing these excipients are described in International Patent Application No. PCT / US2021 / 017880, which is incorporated by reference herein.

[0136] When parenteral administration is contemplated, the therapeutic composition for use may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired anti-TSLP antibody in a pharma- ceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the antibody is formulated as a properly preserved, sterile, isotonic solution. In certain embodiments, the formulation may include a formulation of the desired molecule with an agent that can provide controlled or sustained release of a product that can be delivered via depot injection, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. In certain embodiments, hyaluronic acid may also be used, which has the effect of enhancing duration in the circulation. In certain embodiments, an implantable drug delivery device may be used to introduce the antibody. In various embodiments, administration may be via a prefilled syringe or an autoinjector. In various embodiments, the autoinjector is the Ypsomed YpsoMate® device. In various embodiments, the autoinjector is disclosed in WO 2018 / 226565, WO 2019 / 094138, WO 2019 / 178151, WO 20120 / 072577, WO 2020 / 081479, WO 2020 / 081480, PCT / US 20 / 70590, PCT / US 20 / 70591, PCT / US 20 / 53180, PCT / US 20 / 53179, PCT / US 20 / 53178, or PCT / US 20 / 53176.

[0137] kit As a further aspect, the present disclosure includes a kit comprising one or more compounds or compositions packaged in a manner that facilitates their use to carry out the methods of the present disclosure. In one embodiment, such a kit comprises a compound or composition described herein, packaged in a container such as a sealed bottle or container, and has a label attached to the container or included in the package that describes the use of the compound or composition in carrying out the method. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further comprise a device suitable for administering the composition according to a specific route of administration or for carrying out a screening assay. Preferably, the kit comprises a label that describes the use of the antibody composition.

[0138] Further aspects and details of the present disclosure will become apparent from the following examples, which are intended to be illustrative and not limiting. EXAMPLES

[0139] Example 1: Tezepelumab (AMG157) was tested for its stability and ability to form HMW species at elevated stress temperatures. Tezepelumab was subjected to temperature stress conditions, where the antibody was placed in the formulation at 37° C. and the temperature was increased to the conditions described below. Attributes that affect binding and stability were determined using size exclusion chromatography and peptide mapping.

[0140] Materials and Methods AMG157 and destabilizing residues potentially affecting binding: The amino acid sequence of AMG157 as sequence A5 (and chain H5, as L5) and several other TSLP-binding antibodies were also previously described in U.S. Pat. No. 7,982,016 B2.

[0141] The antibody with A2G0F / A2G0F glycosylation (C6500 H9998 O2068 N1734 S52) has a molecular weight of 147189.4 Da, contains a heavy chain N-terminal pyroglutamate, and has a deleted C-terminal K. TSLP contained 74% monomeric, 23% dimeric, and 3% tetrameric species.

[0142] Following molecular evaluation, in silico evaluation of the sequence identified several residues in the CDRs that were potentially susceptible to chemical modifications that could affect binding and potency. Those CDR residues as well as several other residues from the framework were considered, which comprise the target area based on current understanding (Figure 1, top). Residues in the CDRs and their common modifications were selected as possible attributes, as they could potentially affect binding to the target and potency.

[0143] Methods for identifying chemical modifications that affect binding; size exclusion chromatography (SEC) and fraction collection: After incubation, the AMG157 mixture was separated by SEC using a G3000SWxl TOSOH Bioscience, 7.8 mm ID x 30 cm column (catalog number 08541, TOSOH Bioscience, San Francisco, CA), the mobile phase included 100 mM sodium phosphate and 250 mM NaCl (pH 6.8). The flow rate was set to 0.5 ml / min, the column temperature was set to 37°C, the run time was set to 35 min, and the autosampler was set to 4°C. Ultraviolet / visible spectroscopy (UV / VIS) detection was performed at 214 nm and 280 nm. Elution fractions were collected using a filter with a molecular weight cutoff of >10 kDa and eluted with 7.5 M guanidine elution buffer. The eluted fractions were subjected to sample preparation for peptide mapping as described below.

[0144] SEC of the antibody with the ligand complex followed by LC-MS / MS characterization determines the ratio of modifications in the unbound and bound fractions of the antibody. This method differs from SEC methods that typically detect protein aggregation, such as differentiation between monomers and dimers, because it detects binding between the antibody and the ligand, not just aggregation of the antibody itself. The SEC binding affinity experiment was initiated by mixing the AMG157 protein with its target. Upon separation of the antibody-antigen mixture by SEC-UV, peaks representing the bound complex of the therapeutic protein, the ligand and the unbound therapeutic protein-containing attribute eluted at different times in the SEC elution profile. This allowed the collection of fractions of bound antibody-antigen complex and unbound antibody. The collected fractions were digested by trypsin and analyzed using the LC-MS / MS method, generating abundance plots of the therapeutic protein attribute in the bound and unbound fractions. A volcano plot was also generated with log2 times on the x-axis and -log10 p-value on the y-axis. The Log2 fold change represents the ratio of the attribute in the unbound / bound fraction, which indicates how much the attribute affects the binding of the therapeutic protein to the ligand. The minus log10 of the p-value represents how confidently the fold change is expressed.

[0145] Method for identification of chemical modifications that affect aggregation at 50°C: A similar approach was used to study the attributes of high molecular weight (HMW) and monomeric species of AMG157 50°C 1W samples. LC-MS / MS characterization of AMG157 50°C 1W after SEC-UV determines the ratio of modifications in the HMW and monomeric fractions of the antibody. Upon SEC separation, peaks representing monomeric and HMW species with the identified attributes can elute at different times in the SEC elution profile. The HMW and monomeric fractions of AMG157 50°C 1W were collected, digested, and analyzed using an LC-MS / MS method. Plots of the presence of attributes of the therapeutic protein in the HMW and monomeric fractions were created. Volcano plots were also created with log2 fold change on the x-axis and -log10 p-value on the y-axis. The log2 fold change represents the ratio of the attribute in the HMW / monomer fractions, which indicates the extent to which the attribute causes the formation of HMW antibodies. The minus log10 of the p-value represents how confident the fold change is.

[0146] Peptide mapping: Peptide mapping of collected fractions was performed using a sample preparation procedure including guanidine refolding, reduction and alkylation of disulfide bonds, buffer exchange and digestion with trypsin to peptides suitable for LC-MS analysis as described in (Ren et al., Anal. Biochem. 392:12-21 (2009)). Briefly, samples containing AMG157 were diluted to approximately 1 mg / ml with 0.5 ml of denaturing buffer (7.5 M guanidine hydrochloride (GdnHCl) and 0.25 M Tris) at pH 7.5. Reduction was achieved by adding 3 μl of 0.5 M dithiothreitol (DTT) followed by incubation at room temperature for 30 minutes. Carboxymethylation was achieved by adding 7 μl of 0.5 M iodoacetic acid (IAA). The reaction was carried out at room temperature for 15 minutes in the dark. Excess IAA was quenched by adding 4 μl of 0.5 M DTT. Reduced and alkylated AMG157 samples were buffer exchanged into digestion buffer (0.1 M Tris or 0.1 M ammonium bicarbonate) at pH 7.5 using a NAP-5 column (GE Healthcare, Piscataway, NJ, USA). Lyophilized trypsin was dissolved in water to a final concentration of 1 mg / ml. Digestion was initiated by adding 1 mg / ml trypsin solution to the reduced, alkylated and buffer exchanged antibody 1 sample to achieve an enzyme / substrate ratio of 1:25. Digestion was carried out for 30 min at 37°C. The final digest was quenched by adding 5 tl of 20% FA. LC-MS / MS peptide mapping analysis of digested antibody samples was performed as described in (Ren et al., Anal. Biochem. 392:12-21, 2009) on an Agilent 1290 UHPLC system coupled to a Thermo Scientific Q-Exactive Biopharma mass spectrometer. The resulting LC-MS / MS raw data as well as the sequence of AMG157 were used to identify and quantitate the modifications by MassAnalyzer software (Zhang, Anal. Chem. 81:8354-8364 (2009)).

[0147] Surface plasmon resonance (SPR) is one of the traditional approaches that can measure binding affinity for binding complexes. 10 -6 Binding complexes of less than 10 M can be detected using SPR. In contrast, SEC binding affinity experiments have shown -8 Antibody / ligand complexes can be measured with a Kd of less than 10 M. Weakly bound complexes (10 -8 The ligand molecules (Kd greater than M) dissociate on the SEC column. As a result, the antibody and ligand molecules elute separately as unbound species.

[0148] Stressing tezepelumab at 50°C for 1 week (1W) produced a very large proportion (~67%) of high molecular weight (HMW) species. The HMW species contained a high proportion of chemical modifications, including isomerization and deamidation on several residues, especially isomerization of the LC D91. D91 isomerization increased dramatically to ~23% in the HMW fraction compared to 1% in the monomer.

[0149] The effect of 4 weeks of stress at 40°C (40C4W) on tezepelumab binding to TSLP was also evaluated. Using SEC affinity binding followed by peptide mapping, five attributes of AMG157 (e.g., chemical modifications) that could affect TSLP binding were selected for analysis: HC D54 isomerization, HC W102 oxidation, LC D49 or D50 isomerization, LC N65 deamidation, and LC D91 isomerization (Figure 3). For the D49 or D50 pair, it was difficult to distinguish the effect on binding between the two residues, since both contribute to binding. The effect on binding was in the order D54>W102>D49 / D50>N65>D91. After completion of testing of established shelf-life conditions as 2 years at 5°C followed by 2 months at 25°C (2Y5C+2M25C), only one of the modifications (LC D49 or D50 isomerization) could exceed the detectable level of 2%. The binding is Kd=10 -10 From the typical antibody-antigen equilibrium dissociation constant of M, Kd = 10 -8M, resulting in dissociation of the antibody-antigen complex on the SEC column and separate elution of the two molecules. This method correlated well with conventional methods for characterization of CEX fractions, revealing that HC D54 isomerization correlated strongly with loss of potency as measured by the cell-based assay.

[0150] To verify the suitability of the peptide mapping method, labile residues in the CDRs and adjacent regions were predicted in silico, suggesting that 16 modifications (attributes) of tezepelumab (AMG157) may occur and may affect the binding of AMG157 to TSLP. Peptide mapping was performed on AMG157 T0 and 40C4W samples, and 16 predicted modifications were identified. HC D62-containing peptides had poor recovery, and the isomerization of HC D62 could not be reliably quantified. The peptide mapping results confirmed that the peptide mapping method was suitable for the study, being able to detect all modifications except for HC D62 (Figure 2).

[0151] Several possible antibody attribute mutations (residues with chemical modifications and subsequent residues) have been proposed to enhance room temperature stability, including HC D54E, HC G55A, LC D49E or D50E, LC S51A. U.S. Patent No. 7,982,016 discloses an anti-TSLP antibody as sequence A5 (also chain H5, L5), which is depicted in the CDRs of SEQ ID NOs: 3-8.

[0152] Tezepelumab 50C1W stress produced a very large proportion (approximately 67%) of HMW species. The HMW species contained a high proportion of chemical modifications, including isomerization and deamidation on several residues, especially isomerization of LC D91 and HC D54, suggesting that the HMW fraction may have lower potency due to chemical modifications that affect binding. Also, the HMW species of the tezepelumab 40C4W sample remained the same after binding, suggesting that they were not involved in TSLP binding.

[0153] result A total of 15 modifications are considered as potential modification attributes in AMG157 binding to TSLP based on in silico sequence analysis (Figure 1). Peptide mapping was applied to measure the percentage of predicted attributes in AMG157 TO and 40°C 4W samples (Figure 2). 40°C 4W corresponds to the shelf life of the liquid formulation (4°C 2Y) and is considered a reasonable condition for manufacturing and storage. The modifications exceeding 2% under reasonable manufacturing and storage conditions are HC D54 isomerization, HC N57 deamidation, HC D62 isomerization, and LC D49D50 isomerization, although not all of these modifications affect binding to TSLP.

[0154] Chemical modifications of AMG157 that affect binding to TSLP: Using SEC affinity binding of AMG157 40 °C 4W and TSLP, the residues and modifications that affect binding were experimentally determined. The SEC-UV profile suggests that unbound AMG157 eluting at 15.5 min constitutes less than 10% after 40 °C 4W stress, indicating that the loss of potency should be less than 10%, which is in good agreement with the potency measurements (Figure 8A). Based on the elution times and theoretical molecular weights of AMG157 (147 kDa) and TSLP (16 kDa), the peak eluting at 14 min was determined to be a complex containing one antibody and two TSLT molecules, and the peak at 15 min was determined to be a complex containing one antibody and one TSLP molecule. Other experiments using the same SEC conditions and multi-angle light scattering (MALS) detector identified similar mass complexes eluting at these times. Large complexes eluting at approximately 10.5-12.5 min were also observed when AMG157 T0 or 40°C 4W bound TSLP (Figure 3A). They can be explained by the fact that TSLP contains 23% dimeric and 3% tetrameric species (see Materials and Methods section) and can potentially crosslink some antibodies, resulting in larger complexes. Note that biological characterization of the CEX basic fraction revealed a decrease in receptor-ligand binding and cell-based reporter gene efficacy (Figure 8C). Biochemical characterization (including peptide mapping) identified CDR aspartate isomerization and several other modifications enriched in the basic CEX fraction, including aggregates of fragmented species (HMW), partially reduced species, high mannose and fucosylated glycans, non-CDR Met oxidation, heavy chain C-terminal lysine and N-terminal signaling peptides, and disulfide isoform A. Methods involving antibody-antigen SEC were utilized as an orthogonal approach to evaluate and distinguish chemical modifications of antibodies that affect binding to TSLP from modifications that do not affect binding.

[0155] By SEC of AMG157 40°C 4W and TSLP complexes followed by LC-MS / MS characterization, the ratios of modifications in the unbound (5) and bound (3) fractions of AMG157 were determined. With statistical significance p<0.03, the following five residues were considered as relevant attributes: HC D54 isomerization, HC W102 oxidation, LC D49 or D50 isomerization, LC N65 deamidation, and LC D91 isomerization. For the D49 D50 pair, it is difficult to distinguish the effect of which of the two residues is mutated, as both contribute to binding. The relative abundance of each modification is summarized in Figure 3B. In the bound and unbound fractions of AMG157, the five attributes are all less than 10%. The largest difference (ratio or fold change) in the modification rate between the bound and unbound fractions is seen in the HC D54 isomerization of AMG157. The abundance of HC D54 isomerization in the bound and unbound fractions is about 0.5% and about 3.5%, respectively. LC D49D50 isomerization shows the highest percentage (about 6.2%) in the unbound fraction of AMG157 40° C. 4W.

[0156] Aspartic acid (D) is susceptible to isomerization in the current DG motif of HCDR2, but the different conformations are less pronounced when one of the residues is changed, for example, from G to A or D to E. Similarly, LCDR2 has the motif DDSDRPS, in which aspartic acid is susceptible to isomerization, and changes to the residues are proposed to improve stability, for example, from D to E or S to A.

[0157] Figure 4 shows a volcano plot to determine relevant attributes in AMG157 40°C 4W binding to TSLP. In the statistical plot, log2(unbound / bound) indicates the strength of binding, D54>W102>D49D50>N65>D91. The equilibrium dissociation constant (Kd) of unbound AMG157 to TSLP is 10 -8M, at which point the degraded antibody dissociated on-column from TSLP. Unbound AMG157 showed much weaker binding compared to the typical AMG157 Kd in the nM range. In the SEC affinity binding method, D54 isomerization showed the largest fold change in % unbound / % bound (value of 6) with high confidence of identification (p-value = 4 × 10-4). In combination with attribute modeling of AMG157, the SEC affinity binding experiment showed that several mutations of related attributes (residues with chemical modifications and subsequent residues) could improve room temperature stability, including HC D54E, HC G55A, LC D49D50E and LC S51A (Figure 1, lower panel).

[0158] Residues and modifications showing a potential impact on binding (HC D54 isomerization, HC W102 oxidation, LC D49D50 isomerization, LC N65 deamidation, and LC D91 isomerization) were listed as possible attributes, whereas several other residues considered as possible attributes did not affect binding by SEC affinity methods. Figure 5 summarizes the relative abundance in the bound and unbound fractions in AMG157 40°C 4W for the 11 modifications that did not change statistically significantly between the bound and unbound fractions (Figures 1 and 4). It also shows that, with the exception of HC M34 oxidation and HC D62 isomerization, all modification rates were below 1% after 4 weeks of stress at 40°C in the formulation, and do not constitute a significant proportion of modifications.

[0159] The attributes of the unbound antibody eluting at 15.6 min (peak 5) versus the complex eluting at 10.5-12.5 min (peaks 1 + 2) were the same as peak 5 versus peak 3, but with inferior statistics, indicating that the larger complex is an antibody-TSLP complex.

[0160] This finding is consistent with the crystal structure of AMG157 Fab bound to TSLP, suggesting that HC D54, HC W102 and LC D49 are in close proximity to TSLP (within 6 Å) and are very likely involved in direct binding. It should be noted that isoaspartic acid formation (HC D54, LC D49D50, D91) results in main chain extension (and shortened side chain), which changes the position and orientation of these and nearby residues. This may result in loss of binding. The nearest atoms in the complex were chosen to measure distances without considering the possible nature of the interaction (hydrophobicity, hydrogen bonds, salt bridges). In summary, long-range allosteric effects may occur after isomerization at LC D49D50, LC N65 and LC D91, resulting in loss of binding to TSLP.

[0161] Correlation with conventional methods: Following the conventional approach, AMG157 40°C 4W samples were separated by CEX on the main fraction and three basic fractions, which were collected and characterized for chemical modification and relative potency by peptide mapping. Relative potency was measured by cell-based and binding assays. The assay results showed that basic fraction 3 contained 39% D54 isomerization and its cell-based potency was only 61%, indicating that this chemical modification affects potency in this cell-based assay. This result is in good agreement with the SEC affinity binding to TSLP, which identified the HC D54 isomerization with the highest ratio of unbound to bound fraction and the most impact on binding.

[0162] The percentage of chemical modifications:HMW species that correlate with aggregation at 50°C was about 9% for the AMG157 40°C 4W sample. 50°C 1W stress of AMG157 produced a very large percentage (about 67%) of HMW species, suggesting partial unfolding of the antibody molecule at this temperature. Upon separation by SEC, HMW and monomer species in AMG157 50°C 1W were collected and analyzed using peptide mapping. By using a similar statistical method used in the SEC affinity binding measurements, a volcano plot was created to evaluate the attributes involved in the formation of HMW species of AMG157 (Figure 6A). In the upper right corner of the volcano plot, seven attributes appear to be related to HMW formation and are statistically significant (indicated by an asterisk in Figure 6B). The presence of these and several other modifications from the "near statistically significant grey area" was plotted for HMW and monomer (Figure 6B). Isomerization, deamidation, and succinimide formation (HO loss) constitute the majority of modifications that strongly correlate with HMW formation. For example, LC D91 isomerization dramatically increased to about 23% in the HMW fraction versus 1% in the monomer. Another modification that may affect binding, HC D54 isomerization, was 10% in the HMW fraction versus 2% in the monomer. That is, LC D91 isomerization and HC D54 isomerization each correlated with the formation of HMW species. The high level of chemical modification affecting binding in the HMW fraction suggests that it may have lower potency compared to the monomer. Also, the HMW species of AMG157, which elutes from the SEC at 10.5 min, remained "unconsumed" after binding to TSLP, further suggesting that the HMW species has weak binding. Note that the AMG157 40C4W HMW species is large in size and elutes at the extremes of the size separation.

[0163] Partial unfolding was suspected for AMG157 after 50 °C 1 W, and dramatic HMW species formation was observed, possibly exposing residues that are not exposed and modified in the typical process. Utilization of 40 °C 4 W stressed material with fewer HMW species and modifications should be more representative of the typical process.

[0164] Overall, the SEC affinity binding method experimentally determined the residues and modifications that affect AMG157 binding to TSLP. HC D54 isomerization, HC W102 oxidation, LC D49 or D50 isomerization, LC N65 deamidation, and LC D91 isomerization appear to be relevant attributes of AMG157 binding to TSLP, with statistical significance at p<0.03. Modifications that affect binding with high statistical significance and greater than 2% after 40°C 4W are HC D54 and LC D50 isomerization. It should be noted that D49 / 50 isomerization does not correlate with loss of potency in other studies such as biological assays, and this finding under high stress conditions may be an artifact of the method. Chemical modifications correlate with the formation of HMW species after 50°C 1W stress, especially LC D91 isomerization. Considering these results, several mutations of AMG157 attributes (residues with chemical modifications and trailing residues) were proposed to improve room temperature stability, including HC D54E, HC G55A, LC D49E or LC D50E, LC S51A.

[0165] All publications, patents and patent applications discussed and cited herein are incorporated herein by reference in their entirety.It is understood that the disclosed invention is not limited to the specific methodology, protocols and materials described, which may vary.It is also understood that the terminology used herein is only for describing specific embodiments, and is not intended to limit the scope of the appended claims.

[0166] Those skilled in the art will recognize, or be able to ascertain, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. An anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof, (A) a light chain variable domain, (i) a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; and (iii) a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:

5. a light chain variable domain comprising: (B) a heavy chain variable domain, (i) a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a heavy chain CDR2 sequence comprising an amino acid sequence having a mutation at one of the following residues D54 or G55 as set forth in SEQ ID NO:7; and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:

8. A heavy chain variable domain comprising An anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof comprising:

2. 2. The anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof of claim 1, wherein said mutation in HCDR2 is D54E or G55A.

3. The HCDR2 has the sequence １ X ２ SNKHYADSVKG (where X １ is D or E, and X ２ 3. The anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof of claim 1 or 2, wherein said HCDR2 has the following sequence: VIWYEGSNKHYADSVKG (SEQ ID NO: 14), VIWYDASNKHYADSVKG (SEQ ID NO: 15), or VIWYEASNKHYADSVKG (SEQ ID NO: 16).

4. Optionally, the LCDR2 comprises a mutation in at least one of the following residues D49, D50 or S51 of SEQ ID NO: 4, preferably said mutation is any one of D49E, D50E and S51A, and / or said LCDR2 has the sequence X1X2X3DRPS, where X1 is D or E, X2 is D or E, and X3 is D or E.

4. The anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof of any one of claims 1 to 3, wherein said LCDR2 has the following sequence: EDSDRPS (SEQ ID NO: 18), DESDRPS (SEQ ID NO: 19), EESDRPS (SEQ ID NO: 20), DDADRPS (SEQ ID NO: 21), DEADRPS (SEQ ID NO: 22), EDADRPS (SEQ ID NO: 23) or EEADRPS (SEQ ID NO: 24).

5. An anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof, (A) a light chain variable domain, (i) a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; (ii) a light chain CDR2 sequence comprising an amino acid sequence having a mutation in at least one of the following residues D49, D50 or S51 of SEQ ID NO:4; and (iii) a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:

5. a light chain variable domain comprising: (B) a heavy chain variable domain, (i) a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:

8. A heavy chain variable domain comprising and optionally said mutation in LCDR2 is D49E, D50E and / or S51A.

6. The LCDR2 has the sequence X １ X ２ X ３ DRPS (where X １ is D or E, and X ２ is D or E, and X ３ 6. The anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof of claim 5, wherein said LCDR1 has the following sequence: EDSDRPS (SEQ ID NO: 18), DESDRPS (SEQ ID NO: 19), EESDRPS (SEQ ID NO: 20), DDADRPS (SEQ ID NO: 21), DEADRPS (SEQ ID NO: 22), EDADRPS (SEQ ID NO: 23) or EEADRPS (SEQ ID NO: 24).

7. 7. The anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof of claim 5 or 6, optionally comprising a mutation at one or more of the following residues D54 or G55 of HCDR2 as set forth in SEQ ID NO:7, optionally wherein the mutation is (i) any one of D54E and / or G55A of HCDR2 as set forth in SEQ ID NO:7, or (ii) said HCDR2 has the sequence VIWYX1X2SNKHYADSVKG, where X1 is D or E and X2 is G or A (SEQ ID NO:13), and optionally said HCDR2 has the following sequence: VIWYEGSNKHYADSVKG (SEQ ID NO:14), VIWYDASNKHYADSVKG (SEQ ID NO:15) or VIWYEASNKHYADSVKG (SEQ ID NO:16).

8. (A) a light chain variable domain, i. an amino acid sequence at least 80% identical to SEQ ID NO:12; ii. an amino acid sequence encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:11; or iii. An amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions with the complement of the polynucleotide consisting of SEQ ID NO:

11. a light chain variable domain selected from the group consisting of: (B) a heavy chain variable domain, i. an amino acid sequence at least 80% identical to SEQ ID NO:10; ii. an amino acid sequence encoded by a polynucleotide sequence that is at least 80% identical to SEQ ID NO:9; or iii. An amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions with the complement of the polynucleotide consisting of SEQ ID NO:

9. A heavy chain variable domain selected from the group consisting of: (C) The light chain variable domain of (A) and the heavy chain variable domain of (B).

8. An anti-TSLP immunoglobulin, antigen binding protein or fragment thereof or antibody or fragment thereof according to any one of claims 1 to 7, which comprises the CDRs of said anti-TSLP antigen binding protein or fragment thereof.

9. 9. The antigen binding protein or fragment thereof according to any one of claims 1 to 8, wherein said anti-TSLP immunoglobulin, antigen binding protein or fragment thereof or antibody or fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NOs: 25 to 28, and a light chain comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NOs: 29 to 36.

10. 10. The anti-TSLP immunoglobulin, antigen-binding protein or fragment thereof, or antibody or fragment thereof according to any one of claims 1 to 9, selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody and an IgG4 antibody.

11. 11. An anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof according to any one of claims 1 to 10, wherein said antigen binding protein is a human antibody, optionally an IgG2 antibody.

12. 12. An anti-TSLP immunoglobulin, antigen binding protein or fragment thereof or antibody or fragment thereof according to any one of claims 1 to 11, wherein both binding sites of the anti-TSLP antigen binding protein or fragment thereof have identical binding to TSLP.

13. 13. A composition comprising an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof according to any one of claims 1 to 12, and a pharma- ceutically acceptable carrier, excipient or diluent.

14. 13. An isolated nucleic acid comprising a polynucleotide sequence encoding the light chain variable domain, the heavy chain variable domain or both of the immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof according to any one of claims 1 to 12.

15. A recombinant expression vector comprising the nucleic acid of claim 14.

16. A host cell comprising the vector of claim 15.

17. 17. A method for producing an immunoglobulin, antigen binding protein or fragment thereof or an antibody or fragment thereof that specifically binds to a TSLP polypeptide comprising amino acids 29-159 of SEQ ID NO:2, comprising incubating a host cell according to claim 16 under conditions that allow it to express said immunoglobulin, antigen binding protein or fragment thereof or antibody or fragment thereof, said host cell comprising (i) a recombinant expression vector encoding the light chain variable domain of the immunoglobulin, antigen binding protein or fragment thereof or antibody or fragment thereof according to any one of claims 1 to 12 and a recombinant expression vector encoding the heavy chain variable domain of the immunoglobulin, antigen binding protein or fragment thereof or antibody or fragment thereof according to any one of claims 1 to 12, or (ii) 13. A method comprising a recombinant expression vector encoding both the light chain variable domain and the heavy chain variable domain of the immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof according to any one of claims 1 to 12. Claim 18: i) having increased stability at 25°C compared to an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof having the amino acid sequence set forth in SEQ ID NO:10 and SEQ ID NO:

12. ii) has increased stability at 40° C. after 4 weeks compared to an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof having the amino acid sequence set forth in SEQ ID NO: 10 and SEQ ID NO: 12; iii) has reduced high molecular weight species after 4 weeks at 40° C. compared to an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof having the amino acid sequence set forth in SEQ ID NO:10 and SEQ ID NO:12; iv) has reduced isomerization at 50° C. compared to an anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or an antibody or fragment thereof having the amino acid sequence set forth in SEQ ID NO: 10 and SEQ ID NO: 12; v) less than 2% of said immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof, exhibit isomerization and / or deamidation as determined by SEC after about 22 months to about 36 months of storage at 2°C to 8°C, followed by storage at about 2 weeks, or at least 1 month, or at least 2 months at about 25°C; or vi) less than 2% of said immunoglobulins, antigen binding proteins or fragments thereof, or antibodies or fragments thereof exhibit isomerization and / or deamidation after about 22 months to about 36 months of storage at 2°C to 8°C, followed by storage at about 2 weeks, or at least 1 month, or at least 2 months at about 25°C, as determined by SEC.

13. An anti-TSLP immunoglobulin, antigen binding protein or fragment thereof, or antibody or fragment thereof according to any one of claims 1 to 12.

19. 14. A pharmaceutical composition for use in treating an inflammatory disease in a subject, comprising an immunoglobulin, antigen binding protein or fragment thereof or an antibody or fragment thereof according to any one of claims 1 to 12 or a composition according to claim 13.

20. 20. The pharmaceutical composition of claim 19, wherein the inflammatory disease is selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis (EoE), nasal polyps, chronic spontaneous urticaria, Ig-driven diseases, IgA nephropathy, lupus nephritis, eosinophilic gastritis, chronic sinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IPF).

21. 21. The pharmaceutical composition of claim 19 or 20, comprising administering the composition at intervals of every two weeks or every four weeks.

22. 22. The pharmaceutical composition of claim 20 or 21, wherein the asthma is severe asthma, optionally eosinophilic or non-eosinophilic asthma.

23. 1. A method for producing a composition comprising a plurality of anti-TSLP monoclonal antibodies or antigen-binding fragments thereof, said plurality of anti-TSLP monoclonal antibodies or antigen-binding fragments thereof comprising: A light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; A light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; A light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; A heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; A heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and Heavy chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO:8 and each of the methods includes the following attributes: L-aspartic acid at HC position 54 compared to isoaspartic acid (isoAsp) or cyclic aspartic acid (cAsp) at HC position 54; Non-oxidized HC W102 compared to oxidized HC W102; L-aspartic acid at LC position 49 or position 50 compared to isoAsp or cAsp at LC position 49 or position 50; LC N65 compared to deamidated LC N65; or L-aspartic acid at LC position 91 compared to isoAsp or cAsp at LC position 91 and enriching said composition for IgG2 anti-TSLP monoclonal antibodies or antigen-binding fragments thereof for at least one of the following:

24. (i) 0.9% or less of said anti-TSLP monoclonal antibodies contain isomerized HCD54; (ii) 2% or less of the anti-TSLP monoclonal antibodies contain oxidized HC W102; (iii) 0.9% or less of the anti-TSLP monoclonal antibodies contain isomerized LC D50 or LC D49; (iv) 0.5% or less of said anti-TSLP monoclonal antibodies contain deamidated LC N65; and / or (v) the method of claim 23, wherein 0.9% or less of said anti-TSLP monoclonal antibodies contain isomerized LC D91.

25. 25. The method of claim 23 or 24, wherein the anti-TSLP antibody is an IgG2 antibody.

26. 26. The method of any one of claims 23-25, wherein the anti-TSLP antibody comprises (i) L-aspartic acid of HC D54, and (ii) L-aspartic acid of LC D49 and / or D50, and optionally the anti-TSLP antibody is enriched in L-aspartic acid of HC D54 at least 6-fold over the level of isoAsp.

27. The method according to any one of claims 23 to 26, wherein the anti-TSLP antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 10 and a light chain variable region as set forth in SEQ ID NO:

12.

28. A light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; A light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; A light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; A heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; A heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and Heavy chain CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO:8 and wherein the anti-TSLP monoclonal antibodies of the composition are numerically greater than 10. －８ A composition comprising a limited content of isomerized HC D54 and / or a limited content of isomerized LC D49 or D50, which are effective to bind to TLSP with a Kd of less than or equal to M.

29. 1. A composition comprising an IgG2 anti-TSLP monoclonal antibody comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8, 0.9% or less of said anti-TSLP monoclonal antibodies contain isomerized HCD54; 2% or less of said anti-TSLP monoclonal antibodies contain oxidized HC W102; 0.9% or less of said anti-TSLP monoclonal antibodies contain isomerized LC D49 or LC D50; 0.5% or less of said anti-TSLP monoclonal antibodies contain deamidated LC N65; or Does not more than 0.9% of the anti-TSLP monoclonal antibody contain isomerized LCD91? The composition, (i) 0.9% or less of the anti-TSLP monoclonal antibody contains isomerized HCD54. (ii) 2% or less of the anti-TSLP monoclonal antibodies contain oxidized HC W102; (iii) 0.9% or less of the anti-TSLP monoclonal antibodies contain isomerized LC D49 or LC D50; (iv) 0.5% or less of said anti-TSLP monoclonal antibodies contain deamidated LC N65; and / or (v) the composition of claim 29, wherein 0.9% or less of said anti-TSLP monoclonal antibodies contain isomerized LC D91.

31. The composition of claim 29 or 30, wherein the anti-TSLP antibody is an IgG2 antibody.

32. 32. The composition of any one of claims 29-31, wherein the anti-TSLP antibody comprises a combination of HC54 L-aspartic acid and LC49 and / or LC50 L-aspartic acid, and optionally the anti-TSLP antibody is enriched in HC54 L-aspartic acid at least 6-fold over isoAsp levels.

33. The anti-TSLP antibody comprises a heavy chain variable region represented by SEQ ID NO: 10 and a light chain variable region represented by SEQ ID NO: 12, 0.9% or less of said anti-TSLP monoclonal antibodies contain isomerized HCD54; 2% or less of said anti-TSLP monoclonal antibodies contain oxidized HC W102; 0.9% or less of said anti-TSLP monoclonal antibodies contain isomerized LC D49 or LC D50; 0.5% or less of said anti-TSLP monoclonal antibodies contain deamidated LC N65; or Does not more than 0.9% of the anti-TSLP monoclonal antibody contain isomerized LCD91? The composition according to any one of claims 29 to 32, which is at least one of:

34. A composition comprising an anti-TSLP monoclonal antibody comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:3; a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:4; a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO:7; and a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:8, greater than 98% of said anti-TSLP monoclonal antibodies of said composition comprise an L-aspartic acid at HC position 54 compared to an isoAsp or cAsp at HC position 54; at least 99% of said anti-TSLP monoclonal antibodies of said composition comprises non-oxidized HC W102 compared to oxidized HC W102; at least 97% of said anti-TSLP monoclonal antibodies of said composition comprise an L-aspartic acid at LC position 49 or 50 compared to an isoAsp or cAsp at LC position 49 or 50; At least 99.1% of the anti-TSLP monoclonal antibodies of the composition comprise LC N65 compared to deamidated LC N65; or At least 99.1% of the anti-TSLP monoclonal antibodies of the composition contain an L-aspartic acid at LC position 91 compared to an isoAsp or cAsp at LC position 91. The composition,

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