WO2017153426A1 - Protéines de liaison à la tslp - Google Patents

Protéines de liaison à la tslp Download PDF

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WO2017153426A1
WO2017153426A1 PCT/EP2017/055356 EP2017055356W WO2017153426A1 WO 2017153426 A1 WO2017153426 A1 WO 2017153426A1 EP 2017055356 W EP2017055356 W EP 2017055356W WO 2017153426 A1 WO2017153426 A1 WO 2017153426A1
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tslp
binding protein
seq
full length
length human
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PCT/EP2017/055356
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English (en)
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Farheen AHMED
Michelle Anne BARTHOLOMEW
Chun-Wa Chung
Pietro Della Cristina
Caroline J Dimech
Peter Joseph MORLEY
Rachana Shailesh SHAH
Paula TILLING
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Glaxosmithkline Intellectual Property Development Limited
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Publication of WO2017153426A1 publication Critical patent/WO2017153426A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to TSLP binding proteins that interact with particular residues of human full length TSLP, or contact particular regions of human full length TSLP.
  • the invention also includes pharmaceutical compositions and medical uses of these TSLP binding proteins.
  • TSLP thymic stromal lymphopoetin
  • Full length TSLP is a short-chain four a-helical bundle cytokine that induces Signal
  • Transducer and Activator of Transcription phosphorylation via the functional TSLP receptor (TSLPR), a heterodimeric receptor complex consisting of the IL-7Ra and the unique TSLPR chain (CRFL2) (Park et al, JEM 192(5):659-682 (2002)).
  • TSLPR functional TSLP receptor
  • CRFL2 unique TSLPR chain
  • sfTSLP short isoform of TSLP expressed from an alternative transcription start site appears to be expressed in human cells, but does not appear to activate STAT5 and may serve a different function to full length TSLP (Bjerkan et al., Mucosal immunology 8(1) 49-56 (2015)).
  • TSLP is most highly produced by epithelial and stromal cells lining the barrier surfaces of the skin, gut, and lungs but is also produced by other cell types implicated in allergic disease ⁇ e.g., dendritic cells, mast cells, smooth muscle cells). Production is induced upon exposure to a number of factors including protease allergens (Kouzaki et a/, J Immunol. 183(2): 1427-34 (2009)), viruses, bacteria, inflammatory mediators, cigarette smoke and environmental particulates (Bleck et al, J Clin Immunol 28(2): 147-156 (2008)).
  • TSLP acts on a broad range of cell types (e.g. dendritic cells, CD4+ T cells, eosinophils, basophils, mast cells and Type 2 innate lymphoid cells (ILC2) (Mjosberg et al, Immunity 37(4):649-59 (2012)) to drive inflammation, and in particular, Type 2 inflammation (characterised by the production of the cytokines IL-5, IL-13 and IL-4).
  • Type 2 inflammation is a feature of asthma and other allergic diseases such as atopic dermatitis and Netherton Syndrome.
  • TSLP has been found to induce fibroblast accumulation and collagen deposition in animals demonstrating an additional role in promoting fibrotic disorders.
  • TSLP TSLP signalling
  • TSLP TSLP signalling
  • neutralisation of TSLP or its receptor with antibodies is efficacious in murine or primate asthma or rhinitis models.
  • blocking TSLP with an anti-TSLPR mAb in a primate asthma model cynomolgus monkeys naturally sensitised to Ascaris suum antigen
  • eosinophilia airway resistance and IL-13 levels Cheng et al, Journal of Allergy and Clinical Immunology 132(2):455-462 (2013).
  • TSLP is over-expressed in the epithelium and lamina intestinal of lungs of asthmatic subjects at both the mRNA and protein level (Ying et al. J Immunol. 181(4): 2790-2798 (2008); Shikotra et al. J Allergy Clin Immunol. 129(1): 104-111 (2012); Kaur et al. Chest. 142(l):76-85 (2012)), even in patients taking high dose inhaled corticosteroids. Strong supportive data for the importance of TSLP in asthma comes from the efficacy of an anti-TSLP monoclonal antibody (AMG-157/MEDI9929) in an allergen challenge study in mild asthmatics (Gavreau et al, N Engl J Med.
  • Asthma is a common chronic disease affecting an estimated 300 million people worldwide, and symptoms can be controlled in many patients, using bronchodilators (e.g. ⁇ 2- aderenergic receptor agonists) and inhaled or oral corticosteroids, depending on the severity of the disease.
  • bronchodilators e.g. ⁇ 2- aderenergic receptor agonists
  • inhaled or oral corticosteroids depending on the severity of the disease.
  • bronchodilators e.g. ⁇ 2- aderenergic receptor agonists
  • PCT/EP2016/055026 discloses a lineage of anti-TSLP domain antibodies that bind TSLP (e.g. human full length TSLP) with an IC50 of less than or equal to 5 nM. These domain antibodies and their amino acid sequences are set out in Table 1.
  • domain antibody DOM-30h-440-81/86 having the amino acid sequence set out in SEQ ID NO: 1 was characterised in PCT/EP2016/055026 and shown to have the following desirable properties:
  • this domain antibody was concluded to be useful in the treatment of diseases associated with TSLP signalling.
  • cross reactivity reflects the fact that the epitope is in a region of TSLP that is conserved between human and cynomolgus monkey.
  • cross reactivity with cynomolgus monkey permits preclinical work to be carried out in this species, facilitating the development of binding proteins having this epitope.
  • the inhibition of binding of TSLP to the TSLP receptor arises because the epitope is in the region of TSLP responsible for receptor binding (see figure 1).
  • binding proteins targeting this epitope should exhibit reduced internalisation compared to binding proteins that allow TSLP to bind to either IL-7Ra or TSLPR.
  • the lack of binding to IL- 7 arises because the epitope on TSLP does not resemble an epitope on IL-7.
  • This selectivity is highly desirable as it avoids interfering with the action of IL-7 on the body (e.g. acting as a growth factor for lymphoid cells).
  • the binding to full length human TSLP and not short form TSLP reflects the fact that the epitope of TSLP is in part in a portion of TSLP not present in the short isoform.
  • Other TSLP binding proteins contacting the same epitope of the human full length TSLP as DOM30h-440-81/86 with suitable affinity may be expected to have similar biological activity. Such binding proteins would be highly desirable.
  • epitope is the part of the antigen that is in contact with an antibody or fragment thereof (see, for example, Essential Immunology, Sixth Edition, Blackwell Scientific Publishing, 1988, Ed. Roitt, Chapter 4). Other definitions refer to the part of the antigen that is bound by the antibody or fragment.
  • contact and bound might imply that an epitope should properly only consist of residues that directly interact with the antibody or fragment via non-covalent interactions such as electrostatics (hydrogen bonding, ionic), Van de Waals forces, n -effects and hydrophobic bonds. On such a strict interpretation, an epitope would not include residues that do not interact, but assist in the interaction between antigen and antibody (or fragment).
  • residues in the antigen are preferably small (e.g. glycine) to permit the close interaction required to facilitate direct interaction between other residues of the antigen and antibody (or fragment).
  • residues e.g. proline
  • certain residues may assist the antigen sequence to adopt the correct conformation to permit binding.
  • DOM-30h-440-81/86 The binding of DOM-30h-440-81/86 to human full length TSLP for binding has been investigated by hydrogen deuterium exchange and X-ray crystallography (see examples 1 and 2). Deuterium uptake by TSLP was reduced in the presence of the DOM-30h-440-81/86 in two non-consecutive regions: Tyr26 to Ser42 and Gln89 to Ala95. This data suggests a discontinuous epitope where two linear peptide regions are brought together by the three dimensional structure of TSLP.
  • Example 2 and Figure 3 show that peptides containing the Tyr26 to Ser42 region were most strongly protected from deuterium uptake.
  • the X ray crystallography data presented in Example 1 shows significantly more direct and indirect interactions in the N-terminal portion of the epitope than in the C- terminal portion. Further evidence of this comes from the inability of DOM-30h-440-81/86 to bind to the short isoform of TSLP which lacks the N-terminal portion of the epitope.
  • the present invention provides a TSLP binding protein that either:
  • a) interacts with the following residues of full length human TSLP (SEQ ID NO: 60): Lys31 and Phe35; or
  • c) binds to full length human TSLP having one or more residues of Lys31, Ser32, Thr33, Phe35, Asn36, Asn37, Ser40, Cys41, Ser42, Argl21 and Argl22 mutated exhibiting altered affinity in comparison with full length human TSLP (SEQ ID NO: 60) with no mutations; or
  • d) binds to a peptide having the amino acid sequence set forth in SEQ ID NO: 17; or e) binds to full length human TSLP (SEQ ID NO: 60) and results in peptides derived from full length human TSLP containing part or the whole of the sequence from Tyr26 and Ser42 being more resistant to deuterium incorporation compared to corresponding peptides derived from uncomplexed full length human TSLP;
  • TSLP binding protein does not have the amino acid sequence set forth in SEQ ID NO: 1.
  • said TSLP binding protein does not have the amino acid sequence set forth in any of SEQ ID NOs: 2 to 16.
  • said TSLP binding protein is not a TSLP-binding protein comprising: a) CDR1, CDR2 and CDR3 of SEQ ID NO: 1 or a variant of any one or all of these CDRs, wherein the CDR variant has 1,2, or 3 amino acid modifications; or b) an amino acid sequence at least 90 % identical to the sequence of SEQ ID NO:
  • TSLP binding protein has an IC50 of less than or equal to 5 nM.
  • said TSLP binding protein is not a TSLP binding protein that binds an epitope comprising the following residues of full length human TSLP (e.g. SEQ ID NO: 60): Tyrl5, Lys31, Ser32, Thr33, Phe35, Asn36, Asn37, Ser40, Cys41, Ser42, SerlH, Glnll5, Glnll7, Glyll8, Argl21, Argl22, Argl25, Prol26, Leul28 and Lys 129.
  • SEQ ID NO: 60 Tyrl5, Lys31, Ser32, Thr33, Phe35, Asn36, Asn37, Ser40, Cys41, Ser42, SerlH, Glnll5, Glnll7, Glyll8, Argl21, Argl22, Argl25, Prol26, Leul28 and Lys 129.
  • said TSLP binding protein does not competes for binding to TSLP with a single variable domain of SEQ ID NO: l.
  • the TSLP used in the competition assay is full length human TSLP (e.g. SEQ ID NO: 60).
  • the present invention provides a TSLP binding protein of the invention for use as a medicament.
  • the invention provides a TSLP binding protein of the invention for use in treating a disease associated with TSLP signalling.
  • the invention also provides a pharmaceutical composition comprising a TSLP binding protein of the invention and optionally one or more pharmaceutically acceptable carriers.
  • Figure 1 is an overlay of the X-ray structure of TSLP- DOM-30h-440-81/86 (dark ribbon) with literature complex TSLP/IL7Ra/TSLPR complex (PDB: 4NN7, in lighter grey shades).
  • Figure 2A shows the % surface area buried on TSLP- DOM30h-440-81/86 complex formation for residues 1-55 of TSLP.
  • Figure 2B shows the % surface area buried on TSLP- DOM30h-440-81/86 complex formation for residues 56-129 of TSLP.
  • Figure 3 is a representative plot showing the fractional difference in deuterium update between uncomplexed full length human TSLP and TSLP- DOM30h-440-81/86 for individual peptides at 30 and 300 seconds after dilution into deuterated buffer.
  • the present invention provides a TSLP binding protein that either: a) interacts with the following residues of full length human TSLP (SEQ ID NO: 60): Lys31 and Phe35; or b) increases the % buried surface area of residues Ser32, Thr33, Asn37, Ser40, Cys41 and Ser42 when complexed with full length human TSLP (SEQ ID NO: 60), as compared to uncomplexed full length human TSLP; or
  • c) binds to full length human TSLP having one or more residues of Lys31, Ser32, Thr33, Phe35, Asn36, Asn37, Ser40, Cys41, Ser42, Argl21 and Argl22 mutated exhibiting altered affinity in comparison with full length human TSLP (SEQ ID NO: 60) with no mutations; or
  • d) binds to a peptide having the amino acid sequence set forth in SEQ ID NO: 17; or e) binds to full length human TSLP (SEQ ID NO: 60) and results in peptides derived from full length human TSLP containing part or the whole of the sequence from Tyr26 and Ser42 being more resistant to deuterium incorporation compared to corresponding peptides derived from uncomplexed full length human TSLP;
  • TSLP binding protein does not have the amino acid sequence set forth in SEQ ID NO: 1.
  • the invention provides a TSLP binding protein that interacts with the following residues of full length human TSLP (SEQ ID NO: 60): Lys31 and Phe35. In another embodiment, the TSLP binding protein interacts with the following residues of full length human TSLP (SEQ ID NO: 60): Lys31, Ser32, Thr33, Phe35 and Asn37. In another embodiment, the TSLP binding protein interacts with the following residues of full length human TSLP (SEQ ID NO: 60): Lys31, Ser32, Thr33, Phe35, Asn36, Asn37, Ser40, Cys41 and Ser42. In an alternative embodiment, the invention provides a TSLP binding protein that interacts with the following residues of full length human TSLP (SEQ ID NO: 60): Lys31, Phe35, Argl21 and Argl22.
  • the interaction may be a direct interaction or an indirect interaction via water.
  • the interaction is a direct interaction.
  • a direct interaction is a hydrogen bond between the TSLP binding protein and the named residue(s) of full length human TSLP.
  • Interacting residues may be identified by any technique capable of atomic level resolution.
  • interacting residues are identified by X-ray crystallography.
  • the invention provides a TSLP binding protein that increases the % buried surface area of residues Ser32, Thr33, Asn37, Ser40, Cys41 and Ser42 when complexed with full length human TSLP (SEQ ID NO: 60), as compared to uncomplexed full length human TSLP.
  • the % buried surface area of a residue is increased if a greater percentage of the surface of the residue is buried in the binding protein-full length human TSLP complex when compared to the % buried in uncomplexed full length human TSLP.
  • the increase in % buried surface area is greater than or equal to 40%, in another embodiment, the increase in % buried surface area is greater than or equal to 50%, and in an additional embodiment, the increase in % buried surface area is greater than or equal to 60%.
  • the change in percentage buried surface area may be calculated by any suitable technique, for example, comparison of the X ray crystal structures of complexed and uncomplexed TSLP.
  • the invention provides a TSLP binding protein that binds to full length human TSLP (SEQ ID NO: 60) having one or more residues of Lys31, Ser32, Thr33, Phe35, Asn36, Asn37, Ser40, Cys41, Ser42 Argl21 and Argl22 mutated exhibiting altered affinity in comparison with full length human TSLP with no mutations. Binding may be assessed by any suitable method, for example, SPR or ELISA.
  • the TSLP may be tagged (e.g. biotinylated) to facilitate the binding assay, but its sequence may not be extended by additional amino acids.
  • altered affinity refers to the situation where the antibody or fragment exhibits substantially reduced or substantially increased affinity for the mutated version when compared with wild type full length human TSLP.
  • a substantial increase in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is less than or equal to the mean KD for wild type full length human TSLP measured on the basis of at least three readings minus one standard deviation (the larger of the standard deviations for the wild type or mutated version should be used).
  • a substantial increase in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is less than or equal to the mean KD for wild type full length human TSLP measured on the basis of at least three readings minus two standard deviations (the larger of the standard deviations for the wild type or mutated version should be used).
  • a substantial increase in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is less than or equal to the mean KD for wild type full length human TSLP measured on the basis of at least three readings minus three standard deviations (the larger of the standard deviations for the wild type or mutated version should be used).
  • a substantial increase in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is at least 3 fold less than the mean KD for wild type full length human TSLP measured on the basis of at least three readings. In another embodiment, a substantial increase in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is at least 5 fold less than the mean KD for wild type full length human TSLP measured on the basis of at least three readings. In yet another embodiment, a substantial increase in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is at least 10 fold less than the mean KD for wild type full length human TSLP measured on the basis of at least three readings.
  • a substantial decrease in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is greater than or equal to the mean KD for wild type full length human TSLP measured on the basis of at least three readings plus one standard deviation (the larger of the standard deviations for the wild type or mutated version should be used).
  • a substantial decrease in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is greater than or equal to the mean KD for wild type full length human TSLP measured on the basis of at least three readings plus two standard deviations (the larger of the standard deviations for the wild type or mutated version should be used).
  • a substantial decrease in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is greater than or equal to the mean KD for wild type full length human TSLP measured on the basis of at least three readings plus three standard deviations (the larger of the standard deviations for the wild type or mutated version should be used). In one embodiment, a substantial decrease in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is at least 3 fold greater than the mean KD for wild type full length human TSLP measured on the basis of at least three readings.
  • a substantial decrease in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is at least 5 fold greater than the mean KD for wild type full length human TSLP measured on the basis of at least three readings. In yet another embodiment, a substantial decrease in affinity is where the mean KD for the mutated version measured on the basis of at least three readings is at least 10 fold greater than the mean KD for wild type full length human TSLP measured on the basis of at least three readings. In one embodiment, altered affinity refers to reduced affinity.
  • the invention provides a TSLP binding protein that binds to full length human TSLP (SEQ ID NO: 60) having one or more residues of Lys31, Ser32, Thr33, Phe35, Asn36, Asn37, Ser40, Cys41 and Ser42 mutated exhibiting altered affinity in comparison with full length human TSLP with no mutations.
  • the invention provides a TSLP binding protein that binds to full length human TSLP (SEQ ID NO: 60)having one or more residues of Lys31, Ser32, Thr33, Phe35 and Asn37 mutated exhibiting altered reduced affinity in comparison with full length human TSLP with no mutations.
  • the invention provides a TSLP binding protein that binds to full length human TSLP (SEQ ID NO: 60) having one or more residues of Lys31 and Phe35 mutated exhibiting altered affinity in comparison with full length human TSLP with no mutations.
  • the invention provides a TSLP binding protein that binds to a peptide having the amino acid sequence set forth in SEQ ID NO: 17.
  • the term "binds to” requires a binding response substantially greater than observed for any non- overlapping peptide of equivalent length derived from full length human TSLP. Binding may be assessed by any suitable method, for example ELISA.
  • the peptides may be tagged (e.g. biotinylated) to facilitate the binding assay, but the sequence may not be extended by additional amino acids.
  • the peptide may have the sequence set forth as SEQ ID NO: 18.
  • the requirement to bind to a peptide having the sequences specified does not necessarily mean that the binding protein may not interact with residues outside of this sequence or, for example, protect them from e.g. deuterium uptake provided that an "all or nothing" binding response is achieved (i.e. the levels of binding achieved by peptides having the sequence set forth in SEQ ID NO: 17 (or 18) being substantially greater than levels achieved by other, non-overlapping TSLP peptides).
  • a substantially greater binding response in the context of this invention refers to the situation where the mean KD for a peptide containing the sequence set forth in SEQ ID NO: 17 (or 18) on the basis of at least three readings is less than or equal to the mean KD for the non-overlapping TSLP peptides measured on the basis of at least three readings minus one standard deviation (the largest standard deviation observed for any peptide being used).
  • the mean KD for a peptide containing the sequence set forth in SEQ ID NO: 17 (or 18) on the basis of at least three readings is less than or equal to the mean KD for the non-overlapping TSLP peptides measured on the basis of at least three readings minus two standard deviations (the largest standard deviation observed for any peptide being used).
  • the mean KD for a peptide containing the sequence set forth in SEQ ID NO: 17 (or 18) on the basis of at least three readings is less than or equal to the mean KD for the non-overlapping TSLP peptides measured on the basis of at least three readings minus three standard deviations (the largest standard deviation observed for any peptide being used).
  • the mean KD for a peptide containing the sequence set forth in SEQ ID NO: 17 (or 18) on the basis of at least three readings is at least 3 fold lower than the mean KD for the non-overlapping TSLP peptides measured on the basis of at least three readings.
  • the mean KD for a peptide containing the sequence set forth in SEQ ID NO: 17 (or 18) on the basis of at least three readings is at least 5 fold lower than the mean KD for the non-overlapping TSLP peptides measured on the basis of at least three readings.
  • the mean KD for a peptide containing the sequence set forth in SEQ ID NO: 17 (or 18) on the basis of at least three readings is at least 10 fold lower than the mean KD for the non-overlapping TSLP peptides measured on the basis of at least three readings.
  • the invention provides a TSLP binding protein that binds to full length human TSLP (SEQ ID NO: 60) and results in peptides derived from full length human TSLP containing part or the whole of the sequence from Tyr26 and Ser42 being more resistant to deuterium incorporation compared to corresponding peptides derived from uncomplexed full length human TSLP.
  • resistance to deuterium incorporation is assessed at a time point of between 30 and 300 seconds after dilution into deuterated buffer (e.g. at one of 30, 100, 120 or 300 seconds).
  • the TSLP binding protein of any of the embodiments referred to above does not have the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 16.
  • the TSLP binding protein of the invention exhibits no significant binding to IL-7 (SEQ ID NO: 63).
  • No significant binding to IL-7 refers to the situation where less than or equal to 10 response units is observed when the TSLP binding protein at a concentration of 160 nM in HBS-EP buffer is passed over human IL-7 (SEQ ID NO: 63; immobilised on a CM4 chip by primary amine coupling to a level of 1400 RU) at 25°C in BIACORETM T200.
  • the method should be run in duplicate within the same BIAcore run and the curve double-referenced using a buffer injection curve.
  • the TSLP binding protein of the invention exhibits cross reactivity with cynomolgus TSLP (SEQ ID NO: 62). This is achieved where there is less than a 5 fold difference in IC50 between human full length TSLP (SEQ ID NO: 60) and cynomolgus TSLP (SEQ ID NO: 62) in inhibiting TSLP stimulated phosphorylation of Signal Transduction and Activator of Transcription 5 in the vaginal carcinoma cell line SW756 (ATCC).
  • a suitable assay utilises SW756 cells seeded into 96-well plates at a density of 25,000 cells/well and incubated overnight at 37°C in 5% CO2 to allow adherence.
  • TSLP binding protein at an EC75 concentration of lng/ml
  • the TSLP/binding protein complex is added to the cells and incubated for 30 minutes at 37°C in 5% CO2, followed by cell lysis. Lysates may be analysed by MesoScale Discovery (MSD) to quantify pSTAT5 as according to the manufacturer's protocol (K15163D-3) using an MSD Sector Imager 6000. Data may be plotted using a 4 parameter logistic fit model to obtain potency values.
  • MSD MesoScale Discovery
  • the TSLP binding protein of the invention exhibits no significant binding to the short isoform of human TSLP (SEQ ID NO: 61). Binding to the short isoform of human TSLP may be assessed using the ForteBio Octet label-free interaction analysis instrument at 25°C. Biotinylated short form TSLP diluted to 10 ⁇ g/ml in IgG-free PBS buffer is loaded onto streptavidin sensor tips to a level of 8-9 nm. Next, sensors are dipped into solutions of TSLP binding proteins at a concentration of 500 nM and the binding response is measured. A value of less than 0.1 nm in this assay when the TSLP binding protein is tested at 500 nM is indicative of no significant binding to the short isoform of human TSLP.
  • the TSLP binding protein may be an antibody or have a so-called alternative antibody format. Fragments of antibodies or fragments of proteins having an alternative antibody format, which fragments retain the specified binding properties are also included.
  • antibody is used herein in the broadest sense to refer to proteins with an immunoglobulin scaffold and includes monoclonal, recombinant, synthetic, polyclonal, chimeric, human, humanised, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies and modified versions of any of the foregoing.
  • Antibodies may have an IgG, IgM, IgA, IgD or IgE scaffold, for example an IgG, or IgA scaffold.
  • an antibody may comprise an IgG scaffold selected from IgGl, IgG2, IgG3, IgG4 or IgG4PE.
  • the scaffold may be IgGl.
  • Proteins having an alternative antibody format have a non-immunoglobulin scaffold having loops connecting elements of secondary structure which can be engineered to include CDR regions, for example CDR regions from antibodies identified as having appropriate binding properties when incorporated into an immunoglobulin scaffold.
  • Non immunoglobulin scaffolds include CTLA-4 (Evibodies; Journal Immunological Methods 248(1-2): 31-45, 2001), lipocalin, Protein A derived molecules such as Z-domain of Protein A (Affibodies, Protein Eng Des Sel 17: 455-462, 2004 and EP1641818), A-domain (Avimer/Maxibody), heat shock proteins such as GroEI and GroES, transferrin (trans-body), ankyrin repeat protein (DARPin), peptide aptamer, C-type lectin domain (Tetranectin), human ⁇ -crystallin and human ubiquitin (affilins), PDZ domains, scorpion toxin kunitz-type domains of human protease inhibitor
  • Antibody fragments may comprise one or more domains of a full length antibody.
  • antibody fragments include Fab, F(ab')2, Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv, diabodies, TANDABTM, and single variable domains that retain the specified binding properties in the absence of other domains are included (single variable domains that are capable of binding antigen in the absence of other domains are sometimes referred to as domain antibodies).
  • Single variable domains include VH, VHH, VL and modified antibody variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain),
  • a single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent, nurse shark and Camelid VHH domains. Single variable domains from other species may be humanised according to standard techniques available in the art.
  • Custom antibody production services could be used to raise antibodies against peptides having the sequence set out in SEQ ID NO: 17 or SEQ ID NO: 18. The resulting antibodies could then be screened for the required binding without undue burden.
  • Antibody fragments, proteins having an alternative antibody format or fragments of proteins having an alternative antibody format may readily be derived from antibodies identified in this way.
  • TSLP binding proteins could be identified by screening for proteins having the required binding properties from libraries according to conventional methods.
  • TSLP binding proteins may be prepared by any of a number of conventional techniques. For example, TSLP binding proteins may be purified from cells that naturally express them (e.g., an antibody can be purified from a hybridoma that produces it), or produced in recombinant expression systems.
  • host cells are transformed with a recombinant expression vector encoding the desired antigen binding protein.
  • a wide range of host cells can be employed, including Prokaryotes (including Gram negative or Gram positive bacteria, for example Escherichia coli, Bacilli sp., Pseudomonas sp., Corynebacterium sp.), Eukaryotes including yeast (for example Saccharomyces cerevisiae, Pichia pastoris), fungi (for example Aspergilus sp.), or higher Eukaryotes including insect cells and cell lines of mammalian origin (for example, CHO, Perc6, HEK293, HeLa).
  • Prokaryotes including Gram negative or Gram positive bacteria, for example Escherichia coli, Bacilli sp., Pseudomonas sp., Corynebacterium sp.
  • Eukaryotes including yeast (for example Saccharomyces cerevisiae, Pichi
  • the host cell may be an isolated host cell.
  • the host cell is usually not part of a multicellular organism (e.g., plant or animal).
  • the host cell may be a non-human host cell.
  • the cells can be cultured under conditions that promote expression of the antigen binding protein, and the polypeptide recovered by conventional protein purification procedures.
  • the antigen binding proteins contemplated for use herein include substantially homogeneous antigen binding proteins substantially free of contaminating materials.
  • post-translational modifications may include the cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation patterns, deamidation (for example at an asparagine or glutamine residue), oxidation (for example at a methionine, tryptophan or free cysteine residue), disulfide bond scrambling, isomerisation (for example at an aspartic acid residue), C- terminal lysine clipping (for example from one or both heavy chains), and N-terminal glutamine cyclisation (for example, in the heavy and/or light chain).
  • the present disclosure encompasses the use of antibodies which have been subjected to, or have undergone, one or more post- translational modifications.
  • the modification may occur in a CDR, the variable framework region, or the constant region.
  • the modification may result in a change in charge of the molecule.
  • TSLP expression and/or function is linked to a number of inflammatory disorders, predominantly those allergic in nature (characterised by immunoglobulin E (IgE)-related immunological responses), but also non-allergic diseases.
  • diseases include, but are not limited to, asthma (including severe asthma), idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome (NS), eosinophilic esophagitis (EoE), food allergy, allergic diarrhoea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, cancer (e.g., breast, pancreas, B-cell acute lymphoblastic leukaemia), rheumatoid arthritis, COPD, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS), and nasal polyposis.
  • asthma
  • TSLP stimulates the production of the type 2 cytokines IL-5, IL-13 and IL-4
  • diseases to which these cytokines have been linked such as, but not limited to, asthma, allergic rhinitis, chronic eosinophilic pneumonia, eosinophilic bronchitis, allergic bronchopulmonary aspergillosis, coeliac disease, eosinophilic gastroenteritis, Churg-Strauss syndrome, eosinophilic myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic esophagitis, and inflammatory bowel disease.
  • the invention provides a TSLP binding protein as described herein for use in treating a disease associated with TSLP signalling.
  • the subject is a human.
  • Use of a TSLP binding protein as defined herein in the manufacture of a medicament for the treatment of a disease associated with TSLP signalling is also provided.
  • the subject is a human.
  • the invention provides a method of treating a disease associated with TSLP signalling in a human patient in need thereof, the method comprising administering a TSLP binding protein as defined herein to the human patient.
  • the disease associated with TSLP signalling is selected from the group consisting of: asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, Netherton syndrome, eosinophilic esophagitis (EoE), food allergy, allergic diarrhoea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, cancer, rheumatoid arthritis, COPD, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS), nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, coeliac disease, Churg-Strauss syndrome, eosinophilic myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis and
  • the disease associated with TSLP signalling is selected from the group consisting of: asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, Netherton syndrome, eosinophilic esophagitis (EoE), food allergy, allergic diarrhoea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, cancer, rheumatoid arthritis, COPD, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyposis.
  • the disease associated with TSLP signalling is asthma.
  • the disease associated with TSLP signalling is severe asthma or mild to moderate asthma.
  • Severe asthma is asthma that can only be controlled using the maximally recommended doses of the most effective combinations of controller medication(s) and bronchodilator(s) recommended by treatment guidelines, or which remains uncontrolled despite this therapy. It will be appreciated that the most effective combinations may change over time, but can be readily ascertained by reference to published treatment guidelines (e.g. GINA). Similarly, maximally recommended doses may be identified by reference to these treatment guidelines.
  • the controller medication may be selected from the group consisting of: inhaled corticosteroid and systemic corticosteroid.
  • a bronchodilator may be selected from the group consisting of: a long acting beta agonist, a leukotriene modifier and theophylline.
  • severe asthma is asthma which requires treatment with high dose inhaled corticosteroid and either long acting beta agonist or leukotriene modifier/theophylline, or treatment with systemic corticosteroid to prevent it from becoming "uncontrolled” or which remains “uncontrolled” despite this therapy.
  • Mild to moderate asthma is asthma that can be controlled using first line treatment options recommended by treatment guidelines (at recommended doses).
  • the first line treatment is regular low dose inhaled corticosteroid /leukotriene receptor antagonist /theophylline plus as needed short acting beta agonist, or medium dose inhaled corticosteroid/long acting beta agonist as maintenance treatment plus as needed short acting beta agonist.
  • treatment can be therapeutic, prophylactic or preventative.
  • Treatment encompasses alleviation, reduction, or prevention of at least one aspect or symptom of a disease and encompasses prevention or cure of the diseases described herein.
  • the TSLP binding protein described herein is used in an effective amount for therapeutic, prophylactic or preventative treatment.
  • a therapeutically effective amount of the TSLP binding protein described herein is an amount effective to ameliorate or reduce one or more symptoms of, or to prevent or cure, a disease.
  • a TSLP binding protein of the invention may be administered alone or in combination with other therapeutic agents.
  • the TSLP binding protein and one or more other therapeutic agents may be administered separately, simultaneously or sequentially.
  • the dose of TSLP binding protein may differ from the dose used for monotherapy.
  • a TSLP binding protein of the invention may be administered in combination with inhaled, intranasal or parenteral corticosteroids such as fluticasone furoate, fluticasone propionate, budesonide, ciclesonide, beclomethasone dipropionate, mometasone furoate, triamcinolone acetonide and prednisolone.
  • a TSLP binding protein of the invention may be administered as a fixed dose combination with an inhaled corticosteroid such as a fixed dose combination with fluticasone furoate or fluticasone propionate.
  • a TSLP binding protein of the present invention may be administered in combination with a bronchodilator such as a beta-2 adrenoreceptor agonist and/or a muscarinic antagonist.
  • a bronchodilator such as a beta-2 adrenoreceptor agonist and/or a muscarinic antagonist.
  • Suitable beta-2 adrenoreceptor agonists include vilanterol, salmeterol, salbutamol, formoterol, salmefamol, fenoterol, carmoterol, etanterol, naminterol, clenbuterol, pirbuterol, flerbuterol, reproterol, bambuterol, indacaterol, terbutaline, and salts thereof.
  • Suitable muscarinic antagonists include umeclidinium, tiotropium, glycopyrrolate, ipratropium, and salts thereof such as the bromide salt of umeclidinium.
  • a TSLP binding protein of the invention may be administered as a fixed dose combination with a beta-2 adrenoreceptor agonist and/or a muscarinic antagonist such as a fixed dose combination with vilanterol trifenatate, umeclidinium bromide, or the dual combination of vilanterol trifenatate and umeclidinium bromide.
  • a TSLP binding protein of the present invention may be administered with a combination of one or more bronchodilators and an inhaled steroid.
  • Such combinations may include dual combinations such as fluticasone furoate and vilanterol trifenatate, fluticasone furoate and umeclidinium bromide, fluticasone propionate and salmeterol, budesonide and formoterol, mometasone and formoterol, and triple therapy such as fluticasone furoate, vilanterol trifenatate and umeclidinium bromide.
  • a TSLP binding protein of the present invention may be administered as a fixed dose combination with an inhaled corticosteroid and one or more bronchodilators, such as a fixed dose combination with fluticasone furoate and vilanterol trifenatate, or fluticasone propionate and salmeterol, or fluticasone furoate and umeclidinium bromide, or fluticasone furoate, vilanterol trifenatate and umeclidinium bromide.
  • bronchodilators such as a fixed dose combination with fluticasone furoate and vilanterol trifenatate, or fluticasone propionate and salmeterol, or fluticasone furoate and umeclidinium bromide, or fluticasone furoate, vilanterol trifenatate and umeclidinium bromide.
  • a TSLP binding protein of the present invention may be administered in combination with anti-leukotriene antagonists such as montelukast, zafirlukast and pranlukast; PDE4 inhibitors such as roflumilast; xanthenes; anti-IgE antibodies such as omalizumab; antagonists of IL-5 such as mepolizumab, benralizumab and reslizumab; antagonists of IL-13 such as lebrikizumab and tralokinumab ; antagonists of IL-4/IL-13 such as dupilumab; antagonists of IL-6 such as sirukumab and antagonists of IL-1, IL-4, IL-33, IL-25 and TNF-a.
  • anti-leukotriene antagonists such as montelukast, zafirlukast and pranlukast
  • PDE4 inhibitors such as roflumilast
  • a TSLP binding protein of the present invention may be administered in combination with an anti-histamine such as cetirizine hydrochloride, levocetirizine, desloratidine, loratidine, fexofenadine hydrochloride or azelastine.
  • an anti-histamine such as cetirizine hydrochloride, levocetirizine, desloratidine, loratidine, fexofenadine hydrochloride or azelastine.
  • a TSLP binding protein of the present invention may be administered in combination with pirfenidone or nintedanib or an avB6 antagonist, for example, those disclosed in WO2014/154725.
  • compositions for use in the treatment of the human diseases described herein.
  • Such compositions optionally comprise a pharmaceutically acceptable carrier as known and called for by acceptable pharmaceutical practice.
  • compositions may be administered by injection or continuous infusion (examples include, but are not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular and intraportal).
  • Pharmaceutical compositions may be suitable for topical administration (which includes, but is not limited to, epicutaneous, inhaled, intranasal or ocular administration) or enteral administration (which includes, but is not limited to, oral or rectal administration).
  • the pharmaceutical composition is inhaled.
  • Pharmaceutical compositions may comprise between ⁇ and 15 mg, for example between ⁇ and 10 mg.
  • the pharmaceutical composition comprises between 500 ⁇ and 2.5mg, for example between 250 ⁇ and 5mg of TSLP binding protein.
  • Effective doses and treatment regimes for administering the TSLP binding protein may be dependent on factors such as the age, weight and health status of the patient, disease to be treated and other concomitant medications. Such factors are within the purview of the attending physician. Guidance in selecting appropriate doses may be found in e.g Bai et al., Clin Pharmacokinet, 51, 119-35 (2012).
  • Table 1 Affinity of DOM30h-440-81/86 for recombinant human and cynomolgus monkey TSLP (Mean data derived from two experiments) Ligand ka (1/Ms) kd (1/s) KD (M) KD (nM)
  • DOM30h-440-81 L/86 The ability for DOM30h-440-81 L/86 to block TSLP binding to its receptor complex was determined using the TSLP receptor binding assay (RBA).
  • RBA TSLP receptor binding assay
  • the concentration of DOM30h-440-81/86 was determined spectrophotometrically by measurement of the absorbance of UV light at 280nM using a Nanodrop 1000 instrument (Thermo Scientific). Example data are shown in Table 2.
  • the extracellular domains of the human TSLPR and IL-7R were coated onto ELISA plates to self associate and form the TSLP receptor heterodimer.
  • DOM30h-440- 81/86 was either tested at a single concentration, or diluted in a concentration range (for example 3.8pM - ⁇ ) and pre-incubated for one hour with either human or cynomolgus monkey TSLP at a predetermined concentration (e.g., 1.5ng/ml).
  • the dAb - TSLP complexes were then added to microwell plates for 2 hours and the amount of bound TSLP was quantified using either a biotinylated TSLP detection antibody and steptavidin:HRP (absorbance measured at 450nM using a SpectraMax plate reader) or with a ruthenylated TSLP detection antibody (electrochemiluminescence measured using an MSD Sector Imager). Data were plotted using a 4 parameter logistic fit model to obtain potency values.
  • SW756 vaginal carcinoma cell line SW756(ATCC). These cells express endogenous TSLP receptors as determined by mRNA analysis and have been shown to respond to TSLP as demonstrated by STAT5 phosphorylation.
  • SW756 cells were seeded into 96-well plates at a density of 25,000 cells/well and incubated overnight at 37°C in 5% CO2 to allow adherence.
  • Table 2 Potency of DOM30h-440-81/86 for inhibition of human and cynomolgus monkey TSLP expressed from E. coli
  • the potency of DOM30h-440-81/86 to inhibit human TSLP expressed from human embryonic kidney (HEK) cells was determined.
  • sfTSLP short form TSLP
  • pAb rabbit anti-TSLP polyclonal antibody
  • streptavidin and anti-histidine (his) sensor tips were pre-incubated in IgG-free PBS buffer.
  • biotinylated sfTSLP and full length TSLP his were diluted to l ⁇ g/mL in IgG-free PBS buffer.
  • the streptavidin sensor tips were then dipped into the biotinylated sfTSLP, and the anti-his sensor tips were dipped into the full length TSLP: his.
  • Blank sensors without biotinylated sf TSLP and full length TSLP his were also prepared by soaking in IgG-free PBS buffer.
  • sensors were dipped into solutions of DOM30h-440-81/86 or the polyclonal antibody at concentrations of 500 or 1000 nM and the binding response was measured. Buffer was also used as a blank control.
  • the binding threshold was set at 0.1 response units and the study was performed at 25°C.
  • the polyclonal antibody (ab47943) bound to both full length TSLP and sfTSLP.
  • DOM30h-440-81/86 bound to full length TSLP protein but did not bind to sfTSLP.
  • Some non-specific binding of the pAb to blank sensors was observed, but this was only when the much higher concentration of 1000 nM was used.
  • Example 2 Crystal Structure of DOM30h-440-81/86 complexed to human
  • the complex was made by mixing 24.6mg of purified recombinant refolded human TSLP (from E. coli) with 20mg recombinant DOM30h-440-81/86 (a molar ratio of 0.91 hTSLP to DOM30h-440-81/86), prior to concentration to a volume of ⁇ 2ml using a centrifugal concentration device fitted with a 5k molecular weight cut off membrane (VivaSpin 20 Sarorious: catalogue no. VS2012). The complex was then purified from uncomplexed material using Superdex S75 size exclusion column (GE Healthcare 17-1180-01) equilibrated with running buffer of PBS containing 0.5M arginine.
  • Superdex S75 size exclusion column GE Healthcare 17-1180-01
  • the resolved complex was again concentrated using a Vivaspin 20 before dialysed into a final buffer of 20 mM HEPES pH 7.0, 150 mM NaCI.
  • the final yield was 53 ⁇ I of protein at 57.78mg/mL, (as measured by absorbance at 280nm).
  • the complex components were validated by protein intact mass spectrometry and SDS-PAGE gel.
  • the structure of the hTSLP- DOM30h-440-81/86 complex can be overlaid on the structure of the rat TSLP/ILRa/TSLP receptor complex ( Figure 1). This shows that DOM30h- 440-81/86 directly interferes with the interaction between TSLP and the TSLPR. As discussed above, this is highly desirable since TSLP antagonists that act by preventing recruitment of the IL-7Ra chain, or by binding directly to TSLPR (or IL-7Ra), may be internalised and processed as antigens more effectively than a TSLP antagonist that binds TSLP and stays in solution as a complex with TSLP.
  • the epitope for DOM30h-440-81/86 can be defined more precisely by identifying residues on human TSLP that become inaccessible to solvent on binding to DOM30h-440-81/86. Accordingly, the anti-TSLP dAb/human TSLP co-crystal structure was analysed using Qt-PISA v2.0.1 (Protein Interfaces, Complexes and Assemblies; Krissinel and Henrick (2007) and the buried surface area (BSA) was calculated for each residue of the human TSLP.
  • Figure 2 shows the % exposed surface area of each residue of TSLP that becomes buried on binding to DOM30h-440-81/86.
  • the epitope for DOM30h-440-81/86 on human TSLP includes the following residues: Tyrl5, Ser20, Ile24, Lys31, Ser32, Thr33, Glu34, Phe35, Asn36, Asn37, Thr38, Val39, Ser40, Cys41, Ser42, Asn43, His46, Serll4, Glnll5, Glnll7, Glyll8, Argl21, Argl22, Asnl24, Argl25, Prol26, Leul27, Leul28 and Lysl29.
  • MOE v2014.09 (Molecular Operating Environment). Protein residues within 7 A of the dAb or TSLP were selected, and then the "Ligand Interaction" tool with the default parameters was used to identify water molecules or residues from the interacting molecule that were deemed to be interacting with these residues. Note that due to this tool being designed for defining small molecule ligand interactions, rather than protein residues, the "Ligand interactions" of each selected residue were calculated individually. The interactions defined by MOE were edited to delete any intrachain interactions, and to delete all water interactions apart from those that formed a bridge between the two chains. The remaining interacting residues are shown below:
  • the interacting residues in the epitope are all residues that become more inaccessible to solvent upon binding DOM-30h-440-81/86.
  • Example 3 Hydrogen-deuterium exchange of DOM30h-440-81/86 complexed to human TSLP
  • TSLP- DOM30h-440-81/86 complex were initially diluted into non-deuterated H2O buffers to give working stock concentration in excess of 10 ⁇ .
  • a typical buffer would be lOmM phosphate H20 pH7.4.
  • the HDX labeling reaction was carried out with a 10-20 fold dilution from non- deuterated into deuterated buffer at 20-23°C. Samples were taken at 4-5 given time points between 0-1800 seconds (e.g. 0, 120, 300 and 1800, or 0, 30, 100, 300 and 1000). The samples were quenched with a pre-chilled low pH and denaturing quench buffer at 4°C, consisting of either 200 mM sodium phosphate, 4 M guanidine HCI, 200 mM tris(2- carboxyethyl)phosphine, pH 2.2 or 7M urea, 0.8 M tris(2-carboxyethyl)phosphine, 0.4 M guanidine HCI, 0.1 % formic acid. Between 1-4 replicates were taken for each timepoint.
  • the denatured quenched samples were injected onto an immobilised pepsin digestion column (Waters Enzymate BEH, 2.1 mm X 30mm, Part no: 186007233) with a digestion time of typically 180-240 seconds, a column flow rate of 70 ⁇ /min and digest buffer of 0.2% aqueous formic acid at 20°C.
  • the released peptides were analysed by a UPLC-MS at 0-5°C.
  • the deuterium incorporation for each time point and state was calculated using DynamX Analysis Software v 3.0 (or HD Examiner) by comparing the undeuterated peptide list imported from PLGS to the data acquired for the deuterated samples.
  • the regions Tyr26 to Ser42 and Gln89 to Ala95 show the most significant changes on

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Abstract

La présente invention concerne des protéines de liaison à la TSLP qui interagissent avec des résidus particuliers de la TSLP humaine pleine longueur, ou qui entrent en contact avec des régions particulières de la TSLP humaine pleine longueur. L'invention concerne également des compositions pharmaceutiques et des utilisations médicales de ces protéines de liaison à la TSLP.
PCT/EP2017/055356 2016-03-09 2017-03-07 Protéines de liaison à la tslp WO2017153426A1 (fr)

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CN117106084B (zh) * 2021-12-02 2024-03-22 北京东方百泰生物科技股份有限公司 一种抗tslp的单克隆抗体、其抗原结合片段及其应用

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