WO2024186635A2 - Anticorps anti-facteur de cellule souche (scf) et anti-lymphopoïétine stromale thymique (tslp) et constructions bispécifiques - Google Patents

Anticorps anti-facteur de cellule souche (scf) et anti-lymphopoïétine stromale thymique (tslp) et constructions bispécifiques Download PDF

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WO2024186635A2
WO2024186635A2 PCT/US2024/018068 US2024018068W WO2024186635A2 WO 2024186635 A2 WO2024186635 A2 WO 2024186635A2 US 2024018068 W US2024018068 W US 2024018068W WO 2024186635 A2 WO2024186635 A2 WO 2024186635A2
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nos
antibody
amino acid
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WO2024186635A3 (fr
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Tibor Keler
Diego ALVARADO
Joel Goldstein
Laura A. Vitale
Michael Murphy
Lawrence J. Thomas
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Celldex Therapeutics, Inc.
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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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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

  • Inflammation plays a key role in many diseases, some of which have become more common and severe. Chronic inflammatory diseases contribute to more than half of deaths worldwide (Furman, D. et al., Nature Medicine, 2019, 25(12): 1822- 1832). Stem cell factor (SCF) and its receptor c-Kit are involved in the perpetuation of chronic inflammation.
  • SCF Stem cell factor
  • SCF is expressed by various structural and inflammatory cells in the airways. Binding of SCF to c-Kit (also known as Kit Ligand) leads to activation of multiple pathways, including phosphatidyl-inositol-3 (PI3)-kinase, phospholipase C (PLC)-gamma, Src kinase, Janus kinase (JAK)/Signal Transducers and Activators of Transcription (STAT) and mitogen activated protein (MAP) kinase pathways (Reber, L. et al., Eur J Pharmacol, 2006, 533(1- 3):327-40).
  • PI3 phosphatidyl-inositol-3
  • PLC phospholipase C
  • COK Janus kinase
  • MAP mitogen activated protein
  • SCF is produced in two transmembrane forms (viz SCF 220 and SCF 248 ) as a result of alternative splicing of exon 6 (Lennartsson and Rbnnstrand, Physiol Rev. 2012 Oct; 92(4): 1619-49).
  • SCF 248 exon 6 is retained and encodes a proteolytic cleavage site, generating the soluble SCF 165 when this is cleaved.
  • SCF 220 which lacks the cleavage site, forms membrane-bound SCF dimers (mSCF).
  • c-Kit is a type III receptor tyrosine kinase encoded by the c-kit gene.
  • c-Kit comprises five extracellular immunoglobulin (Ig)-like domains, a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (see, e.g., Yarden et al., Nature, 1986, 323:226-232; Ullrich and Schlessinger, Cell, 1990, 61 :203-212; Clifford et al., J. Biol. Chem., 2003, 278:31461-31464).
  • Ig immunoglobulin
  • c-Kit is also known as CD117 or stem cell factor receptor ("SCFR"), because it is the receptor for the stem cell factor (also known as Kit Ligand).
  • SCFR stem cell factor receptor
  • SCF ligand binding to the first three extracellular Ig-like domains of c- Kit induces receptor dimerization, and thereby activates intrinsic tyrosine kinase activity through the phosphorylation of specific tyrosine residues in the juxtamembrane and kinase domains (see, e.g., Weiss and Schlessinger, Cell, 1998, 94:277-280; Clifford et al., J. Biol. Chem., 2003, 278:31461- 31464).
  • Members of the Stat, Src, ERK, and AKT signaling pathways have been shown to be downstream signal transducers of c-Kit signaling.
  • the fourth (D4) and fifth (D5) extracellular Ig-like domains of c-Kit are believed to mediate receptor dimerization (see, e.g., International Patent Application Publication No. WO 2008/153926; Yuzawa et al., Cell, 2007, 130:323-334).
  • c-Kit plays an important role in hematopoiesis, melanogenesis, and gametogenesis (see Ueda et al., Blood, 2002, 99:3342-3349).
  • mast cells are long-lived innate immune sentinel cells that reside in tissues across the body, particularly at interfaces with the external environment (Alvarado D, Maurer M, Gedrich R, et al. Allergy. 2022;00:1-11). During normal homeostasis, mast cells can exert protective functions against helminth infections, venoms, and may play a role in wound healing and initiating adaptive responses. However, mast cells are better known for their role in driving or contributing to numerous allergic, inflammatory, and autoimmune disorders.
  • mast cells Upon stimulation, mast cells release pre-formed mediators stored in granules (proteases, histamine, serotonin, and cytokines), followed by a second wave of eicosanoids (leukotrienes and prostaglandin D2) and a wide array of inflammatory cytokines and chemokines through de novo synthesis.
  • mediators stored in granules (proteases, histamine, serotonin, and cytokines)
  • eicosanoids leukotrienes and prostaglandin D2
  • mast cell activation underlies the etiology of allergic reactions and has been strongly implicated in chronic acute and pruritic conditions, neuroinflammatory disorders, pain, fibrosis, and autoimmune diseases.
  • therapies that inhibit specific mast cell triggers such as anti-IgE (omalizumab) or mediators (antihistamines) have been approved by health authorities and recommended by guidelines as therapies, although many patients have limited benefit indicating additional mast cell triggers or mediators are likely involved.
  • therapies that lead to comprehensive mast cell suppression may result in broader efficacy in indications where mast cells contribute to disease pathophysiology (Alvarado D, Maurer M, Gedrich R, et al. Allergy. 2022;00:1-11).
  • c-Kit c-KIT/CDl 17 receptor tyrosine kinase and its only ligand stem cell factor (SCF) are master regulators of mast cell biology.
  • c-Kit is highly expressed throughout the life of a mast cell and is also expressed in hematopoietic stem cells, melanocytes, interstitial cells of Cajal, germ cells, and a subset of taste receptor cells.
  • Mast cells arise from multipotent hematopoietic stem cell progenitors, entering circulation as immature progenitors and influx into tissues, where they reach maturity.
  • stromal cells e.g., fibroblasts, keratinocytes, and endothelial cells
  • exogenous SCF is required to differentiate, mature, and maintain primary mast cells grown in vitro (Alvarado D, Maurer M, Gedrich R, et al. Allergy. 2022;00: 1-11).
  • Thymic stromal lymphopoietin also has been shown to play a critical role in driving inflammation (Rui He and Raif S. Geha, Ann NY Acad Sci, 2010, 1183:13-24).
  • TSLP is expressed by epithelial cells in the thymus, lung, skin, intestine, and tonsils, as well as airway smooth muscle cells, lung fibroblasts, and stromal cells (Reche et al., Journal of Immunology, 2001, 167: 336-343).
  • TSLP TSLP drives allergic inflammatory responses through its activity on a number of innate immune cells, including dendritic cells, monocytes, and mast cells (Soumelis et al., Nature Immunology, 2002, 3:673-680; Reche et al., Journal of Immunology, 2001, 167:336-343; (Allakhverdi et al. (2007) The Journal of Experimental Medicine 204:253-258).
  • TSLP has also been shown to play a role in the pathogenesis of diseases that include fibrosis. For example, TSLP has been shown to be upregulated in both cutaneous and lung fibrotic conditions. (Shin et al., Journal of Investigative Dermatology, 2016, 136(2):360-362).
  • anti-TSLP and anti-SCF antibodies e.g. , fully human, humanized, and chimeric antibodies
  • binding domains thereof i.e., antigen binding fragments
  • Bispecific and multispecific constructs comprising an anti-TSLP and/or anti- SCF antibody (or antigen binding fragments thereof) linked to at least one additional binding agent (e.g. , a ligand or an antibody or antigen binding fragment thereof) also are provided.
  • the bispecific and multispecific constructs comprise the anti-TSLP and/or anti-SCF antibodies (or antigen binding fragments thereof) described herein linked at least one additional binding agent.
  • Compositions comprising the antibodies, bispecific and multispecific constructs also are provided.
  • TSLPR TSLP receptor
  • c-Kit SCF with its receptor
  • the present disclosure provides methods for inhibiting or blocking the binding of TSLP to TSLPR and/or blocking the binding of SCF to c-Kit, inhibiting or preventing activation of immune cells, as well as reducing or preventing the accumulation of immune cells within organs or tissues, thereby treating or preventing various diseases and disorders that involve inflammation.
  • an inflammatory disease or disorder e.g., autoimmune diseases, cardiovascular diseases, gastrointestinal diseases, lung diseases, metabolic diseases (such as Type 2 diabetes), neurodegenerative diseases (such as Parkinson’s disease), certain types of cancer (such as colon cancer) and mental illnesses (such as depression)
  • the anti-TSLP antibody or binding domain thereof comprises the heavy and/or light chain CDRs or variable regions of any one of antibodies 1D10-A, 1D10-B, 1D10-C, 1D10-D, 1D10-E, 1D10-F, 1D10-G, 1D10-H, or 1D10-I (as shown in Table 6 of Example 2).
  • the anti-TSLP antibody or binding domain thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, or 13, and/or the CDR1, CDR2 and CDR3 domains of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, 21, 25, or 29.
  • the anti-TSLP antibody or binding domain thereof comprises heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 5A (of Example 2), or conservative sequence modifications thereof, and/or light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 5B (of Example 2), or conservative sequence modifications thereof.
  • the anti-TSLP antibody or binding domain thereof respectively comprises heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 2, 3, and 4, (b) SEQ ID NOs: 6, 7, and 8, (c) SEQ ID NOs: 10, 11, and 12, (d) SEQ ID NOs: 14, 15, and 16, or conservative sequence modifications thereof, and/or light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 18, 19, and 20, (b) SEQ ID NOs: 22, 23, and 24, (c) SEQ ID NOs: 26, 27, and 28, (d) SEQ ID NOs: 30, 31, and 32, or conservative sequence modifications thereof, as set forth in Table 1A below.
  • the anti-TSLP antibody or binding domain respectively comprises the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 6, 7, and 8 (i.e., the heavy chain CDR1, CDR2 and CDR3 sequences of 1D10-H2) and light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 18, 19, and 20 (i.e., the light chain CDR1, CDR2 and CDR3 sequences of 1D10-L1 ).
  • the anti-TSLP antibody or binding domain thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, or 13, or sequences at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • the antibody or binding domain thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, 21, 25, or 29, or sequences at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • the anti-TSLP antibody or binding domain thereof comprises a combination of heavy and light chain variable regions having the amino acid sequences as set forth in Table IB below.
  • the anti-TSLP antibody or binding domain comprises the heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 (i.e., the heavy chain variable region of 1D10-H2) and the light chain variable region having the amino acid sequence of SEQ ID NO: 17 (i.e., the light chain variable region of 1D10-L1).
  • the anti-TSLP antibody or binding domain thereof comprises a combination of heavy and light chain variable regions having the amino acid sequences as set forth in Table 6 of Example 2.
  • the anti-SCF antibody or binding domain thereof comprises the heavy and/or light chain CDRs or variable regions of any one of antibodies anti-SCF antibody mAbl2-A, mAbl2-B, mAbl2-C, mAbl2-D, mAbl2-E, mAb!2-F, mAbl2-G, mAbl2-H, or mAbl2-I (as shown in Table 9 of Example 9).
  • the anti-SCF antibody or binding domain thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 33, 37, 41, or 45, and/or the CDR1, CDR2 and CDR3 domains of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 49, 53, 57, or 61.
  • the anti-SCF antibody or binding domain thereof comprises heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 8A (of Example 9), or conservative sequence modifications thereof, and/or light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 8B, or conservative sequence modifications thereof.
  • the anti-SCF antibody or binding domain thereof respectively comprises heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 34, 35, and 36, (b) SEQ ID NOs: 38, 39, and 40, (c) SEQ ID NOs: 42, 43, and 44, (d) SEQ ID NOs: 46, 47, and 48, or conservative sequence modifications thereof, and/or light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 50, 51, and 52, (b) SEQ ID NOs: 54, 55, and 56, (c) SEQ ID NOs: 58, 59, and 60, (d) SEQ ID NOs: 62, 63, and 64, or conservative sequence modifications thereof, as set forth in Table 2 A below.
  • the anti- SCF antibody or binding domain comprises the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 46, 47, and 48 (i.e., the heavy chain CDR1, CDR2 and CDR3 sequences of mAbl2-H4) and the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 58, 59, and 60 (i.e., the light chain CDR1, CDR2 and CDR3 sequences of mAbl2-L3).
  • the antibody or binding domain thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 33, 37, 41, or 45, or sequences at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • the antibody or binding domain thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 49, 53, 57, or 61, or sequences at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • the anti-SCF antibody or binding domain thereof comprises a combination of heavy and light chain variable regions having the amino acid sequences as set forth in Table 2B below.
  • the anti-SCF antibody or binding domain comprises the heavy chain variable region having the amino acid sequence of SEQ ID NO: 45 (i.e., the heavy chain variable region of mAb!2-H4) and the light chain variable region having the amino acid sequence of SEQ ID NO: 57 (i.e., the light chain variable region of mAb!2-L3).
  • the anti-SCF antibody or binding domain thereof comprises a combination of heavy and light chain variable regions having the amino acid sequences as set forth in Table 9 of Example 9.
  • the CDR1, 2, and/or 3 regions of the anti-TSLP antibodies or binding domains described herein comprise the amino acid sequences of antibody 1D10 (i.e., heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 2, 3, and 4, (b) SEQ ID NOs: 6, 7, and 8, (c) SEQ ID NOs: 10, 11, and 12, (d) SEQ ID NOs: 14, 15, and 16, and/or light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 18, 19, and 20, (b) SEQ ID NOs: 22, 23, and 24, (c) SEQ ID NOs: 26, 27, and 28, (d) SEQ ID NOs: 30, 31, and 32).
  • antibody 1D10 i.e., heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 2, 3, and 4, (b) SEQ ID NOs
  • the CDR1, 2, and/or 3 regions of the anti-SCF antibodies or binding domains described herein comprise the amino acid sequences of antibody mAbl2 (i.e., heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 34, 35, and 36, (b) SEQ ID NOs: 38, 39, and 40, (c) SEQ ID NOs: 42, 43, and 44, (d) SEQ ID NOs: 46, 47, and 48, and/or light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 50, 51, and 52, (b) SEQ ID NOs: 54, 55, and 56, (c) SEQ ID NOs: 58, 59, and 60, (d) SEQ ID NOs: 62, 63, and 64) disclosed herein.
  • antibody mAbl2 i.e., heavy chain CDR1, CDR2 and C
  • the antibody comprises a derivative of the CDR sequences of 1D10 and mAb!2, yet still retain the ability of to bind either TSLP or SCF effectively.
  • Such derivatives include CDRs that include one or more (e.g., 1, 2, 3, 4, 5, or 6) amino acid additions, deletions, or substitutions, e.g., conservative sequence substitutions.
  • the anti-TSLP or anti-SCF antibodies or binding domains comprise one or more CDRs that are, for example, 90%, 95%, 98% or 99.5% identical to one or more CDRs of antibodies 1D10 and mAbl2, respectively. Ranges intermediate to the above-recited values, e.g. , CDRs that are 90-95%, 95-98%, or 98-100% identical identity to one or more of the above sequence.
  • the antibody or binding domain sequences can also include consensus sequences.
  • the anti-TSLP antibody or binding domain comprises a heavy chain variable region comprising SEQ ID NO: 1, 5, 9, 13, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • the anti-TSLP binding domain comprises a light chain variable region comprising SEQ ID NO: 17, 21, 25, 29, 18, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • the anti-SCF antibody or binding domain comprises a heavy chain variable region comprising SEQ ID NO: 33, 37, 41, 45, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • the anti-SCF antibody or binding domain comprises a light chain variable region comprising SEQ ID NO: 49, 53, 57, 61, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • Anti-TSLP and/or anti-SCF antibodies or binding domains thereof that compete for binding with any of the antibodies or binding domains thereof described herein or that bind the same epitope as any of the antibodies or binding domains described herein also are encompassed.
  • the anti-TSLP antibody or binding domain thereof competes for binding to TSLP with antibody 1D10, as described herein.
  • the anti-TSLP antibody or binding domain binds to the same epitope on TSLP as antibody 1D10, as described herein.
  • the anti-SCF antibody or binding domain thereof competes for binding to SCF with antibody mAb!2, as described herein.
  • the anti-SCF antibody or binding domain thereof binds to the same epitope on SCF as antibody mAbl2, as described herein. In another embodiment, the anti-SCF antibody or binding domain binds to soluble human SCF (hSCF 165 ) in preference to membrane bound human SCF (hSCF 222 ). In another embodiment, the anti-SCF antibody or binding domain binds to an epitope comprising residue K100 of human SCF.
  • the bispecific or multispecific constructs provided herein comprise an anti-TSLP antibody (or binding domain) or anti-SCF antibody (or binding domain), linked to a second binding agent (e.g., a ligand, a second antibody, or antigen binding domain thereof).
  • the construct comprises an anti-TSLP antibody (or binding domain) or anti-SCF antibody (or antigen binding domain) comprising the heavy and light chain CDR and/or full-length variable region sequences respectively set forth in Tables 1A, IB, and Tables 2A, 2B.
  • the second antibody binds to a member of the TNF superfamily (e.g., TNFa), a tumor necrosis factor (TNF) receptor (e.g., TNFRSF4), an interleukin (e.g., IL-23, IL-23 A, IL-17A, IL-5, IL-11, IL-12 or IL-13), an immunoglobulin (e.g., IgE) or an integrin (e.g., integrin a407 ) or OX40L or VEGF.
  • TNF tumor necrosis factor
  • TNFRSF4 tumor necrosis factor receptor
  • an interleukin e.g., IL-23, IL-23 A, IL-17A, IL-5, IL-11, IL-12 or IL-13
  • an immunoglobulin e.g., IgE
  • an integrin e.g., integrin a407
  • OX40L or VEGF vascular endot
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to a member of the TNF superfamily (e.g., TNFa; for example variable domains of adalimumab, golimumab or certolizumab).
  • TNFa a member of the TNF superfamily
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to a tumor necrosis factor (TNF) receptor (e.g., TNFRSF4).
  • TNF tumor necrosis factor
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL- 12 and/or IL-23 (for example variable domains of guselkumab, tildrakizumab or ustekinumab).
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-23A (for example variable domains of risankizumab).
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-17A (for example variable domains of secukinumab).
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL- 13 (for example variable domains of lebrikizumab or tralokinumab).
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IgE (for example variable domains of omalizumab or ligelizumab).
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-5.
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-11.
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to CD40L (for example variable domains of oxelumab; see US Patent 7501496).
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to VEGF (for example variable domains of bevacizumab; see US Patent 7060269).
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to an integrin (e.g., integrin a407; for example variable domains of vedolizumab).
  • an integrin e.g., integrin a407; for example variable domains of vedolizumab.
  • a construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to a member of the TNF superfamily (e.g., TNFa; for example variable domains of adalimumab, golimumab or certolizumab).
  • TNFa a member of the TNF superfamily
  • a construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL- 12 and/or IL-23 (for example variable domains of guselkumab, tildrakizumab or ustekinumab).
  • a construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-23A (for example variable domains of risankizumab).
  • a construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-17A (for example variable domains of secukinumab).
  • a construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL- 13 (for example variable domains of lebrikizumab or tralokinumab).
  • a construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IgE (for example variable domains of omalizumab or ligelizumab).
  • a construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to an integrin (e.g., integrin a4p7; for example variable domains of vedolizumab).
  • an integrin e.g., integrin a4p7; for example variable domains of vedolizumab.
  • a construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to CD40L (for example variable domains of oxelumab; see US Patent 7501496).
  • a construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to VEGF (for example variable domains of bevacizumab; see US Patent 7060269).
  • the anti-TSLP antibody (or antigen binding domain) of the bispecific or multispecific construct comprises the heavy and/or light chain CDR and/or full- length variable region amino acid sequences as respectively set forth in Tables 1A and IB.
  • the anti-SCF antibody (or antigen binding domain) of the bispecific or multispecific construct comprises the heavy and/or light chain CDR and/or full-length variable region amino acid sequences as set forth in Tables 2A and 2B.
  • a bispecific or multispecific construct comprises an anti- TSLP antibody (or binding domain) and an anti-SCF antibody (or binding domain) linked to each other.
  • a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to TSLP.
  • the anti-SCF and/or anti-TSLP antibodies or binding domains are antibodies or binding domains having CDRs and/or variable domain sequences as described herein.
  • the bispecific construct comprises:
  • an anti-TSLP binding domain comprising heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 5 A, or conservative sequence modifications thereof, and light chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 5B, or conservative sequence modifications thereof;
  • an anti-SCF binding domain comprising heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 8 A, or conservative sequence modifications thereof, and/or light chain variable region CDR1 , CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 8B, or conservative sequence modifications thereof.
  • an anti-TSLP binding domain comprising heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 2, 3, and 4, (ii) SEQ ID NOs: 6, 7, and 8, (iii) SEQ ID NOs: 10, 11, and 12, (iv) SEQ ID NOs: 14, 15, and 16, or conservative sequence modifications thereof, and/or light chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 18, 19, and 20, (ii) SEQ ID NOs: 22, 23, and 24, (iii) SEQ ID NOs: 26, 27, and 28, (iv) SEQ ID NOs: 30, 31, and 32, or conservative sequence modifications thereof; and
  • an anti-SCF binding domain comprising heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 34, 35, and 36, (ii) SEQ ID NOs: 38, 39, and 40, (iii) SEQ ID NOs: 42, 43, and 44, (iv) SEQ ID NOs: 46, 47, and 48, or conservative sequence modifications thereof, and/or light chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 50, 51, and 52, (ii) SEQ ID NOs: 54, 55, and 56, (iii) SEQ ID NOs: 58, 59, and 60, (iv) SEQ ID NOs: 62, 63, and 64, or conservative sequence modifications thereof.
  • the bispecific construct comprises:
  • an anti-TSLP binding domain comprising heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 6, 7, and 8, or conservative sequence modifications thereof, and/or light chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 18, 19, and 20, or conservative sequence modifications thereof;
  • an anti-SCF binding domain comprising heavy chain variable region CDR1 , CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 46, 47, and 48, or conservative sequence modifications thereof, and/or light chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 58, 59, and 60, or conservative sequence modifications thereof.
  • the bispecific construct comprises a combination of anti- TSLP heavy and light chain variable region sequences having the amino acid sequences set forth in Table IB, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences), and anti-SCF heavy and light chain variable region sequences having the amino acid sequences set forth in Table 2B, or a sequence at least 90% identical thereto e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • the bispecific construct comprises anti-TSLP heavy and light chain variable region sequences having the amino acid sequences set forth in Table 4, or a sequence at least 90% identical thereto e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences), and anti-SCF heavy and/or light chain variable region sequences having the amino acid sequences set forth in Table 5, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • the bispecific construct comprises:
  • an anti-TSLP binding domain comprising a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table IB, or sequences at least 90% identical thereto;
  • an anti-SCF binding domain comprising heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 34, 35, and 36, (ii) SEQ ID NOs: 38, 39, and 40, (iii) SEQ ID NOs: 42, 43, and 44, (iv) SEQ ID NOs: 46, 47, and 48, or conservative sequence modifications thereof, and/or light chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 50, 51, and 52, (ii) SEQ ID NOs: 54, 55, and 56, (iii) SEQ ID NOs: 58, 59, and 60, (iv) SEQ ID NOs: 62, 63, and 64, or conservative sequence modifications thereof.
  • the bispecific construct comprises:
  • an anti-SCF binding domain comprising a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 2B, or sequences at least 90% identical thereto;
  • an anti-TSLP binding domain comprising heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 2, 3, and 4, (ii) SEQ ID NOs: 6, 7, and 8, (iii) SEQ ID NOs: 10, 11, and 12, (iv) SEQ ID NOs: 14, 15, and 16, or conservative sequence modifications thereof, and/or light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 18, 19, and 20, (ii) SEQ ID NOs: 22, 23, and 24, (iii) SEQ ID NOs: 26, 27, and 28, (iv) SEQ ID NOs: 30, 31, and 32, or conservative sequence modifications thereof.
  • the bispecific construct comprises an anti-TSLP binding domain and an anti-SCF binding domain comprising amino acid sequences set forth in Table 10 of Example 19, e.g., construct 5.7 (1D10 (VH1-L1) - mAbl2 (VH4-L3)), also referred to herein as CDX-622.
  • the anti-TSLP binding domain and the anti-SCF binding domain are genetically fused.
  • the bispecific construct can be, for example, a fusion protein, which can be made by genetic engineering using standard recombinant DNA techniques to operatively link nucleic acid encoding the anti-TSLP7 and anti-SCF binding domains.
  • the anti-TSLP binding domain and the anti-SCF binding domain are chemically conjugated.
  • the bispecific construct can be a conjugate made by chemical conjugation of the anti-TSLP and anti-SCF binding domains.
  • the anti- TSLP binding domain further comprises a human IgGl constant domain.
  • the anti-SCF binding domain is linked to the C-terminus of the heavy chain of the anti-TSLP binding domain.
  • the anti-SCF binding domain is a scFv.
  • the anti-TSLP binding domain further comprises a human IgGl constant domain.
  • the anti-SCF binding domain is linked to the C-terminus of the heavy chain of the anti-TSLP binding domain.
  • the anti-SCF binding domain is a scFv.
  • the anti-SCF binding domain further comprises a human IgGl constant domain.
  • the anti-TSLP binding domain is linked to the C-terminus of the heavy chain of the anti-SCF binding domain.
  • the anti-TSLP binding domain is a scFv.
  • the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, wherein:
  • the anti-TSLP antibody comprises heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 2, 3, and 4, (ii) SEQ ID NOs: 6, 7, and 8, (iii) SEQ ID NOs: 10, 11, and 12, (iv) SEQ ID NOs: 14, 15, and 16, and/or light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 18, 19, and 20, (ii) SEQ ID NOs: 22, 23, and 24, (iii) SEQ ID NOs: 26, 27, and 28, (iv) SEQ ID NOs: 30, 31, and 32;
  • the anti-SCF scFv comprises heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 34, 35, and 36, (ii) SEQ ID NOs: 38, 39, and 40, (iii) SEQ ID NOs: 42, 43, and 44, or (iv) SEQ ID NOs: 46, 47, and 48, and/or light chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 50, 51, and 52, (ii) SEQ ID NOs: 54, 55, and 56, (iii) SEQ ID NOs: 58, 59, and 60, or (iv) SEQ ID NOs: 62, 63, and 64; and and
  • the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, wherein:
  • the anti-TSLP antibody comprises heavy chain variable region CDRL CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 6, 7, and 8, and light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 18, 19, and 20;
  • the anti-SCF scFv comprises heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 46, 47, and 48, and light chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 58, 59, and 60; and and
  • the bispecific construct comprises an anti-SCF antibody linked to an anti-TSLP scFv, wherein:
  • the anti-SCF antibody comprises heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 34, 35, and 36, (ii) SEQ ID NOs: 38, 39, and 40, (hi) SEQ ID NOs: 42, 43, and 44, or (iv) SEQ ID NOs: 46, 47, and 48, and light chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 50, 51, and 52, (ii) SEQ ID NOs: 54, 55, and 56, (iii) SEQ ID NOs: 58, 59, and 60, or (iv) SEQ ID NOs: 62, 63, and 64 and a human IgGl constant domain; and
  • the anti-TSLP scFv comprises heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 2, 3, and 4, (ii) SEQ ID NOs: 6, 7, and 8, (iii) SEQ ID NOs: 10, 11, and 12, (iv) SEQ ID NOs: 14, 15, and 16, and light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (i) SEQ ID NOs: 18, 19, and 20, (ii) SEQ ID NOs: 22, 23, and 24, (iii) SEQ ID NOs: 26, 27, and 28, (iv) SEQ ID NOs: 30, 31, and 32.
  • the bispecific construct comprises an anti-SCF antibody linked to an anti-TSLP scFv, wherein:
  • the anti-SCF antibody comprises heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 46, 47, and 48, and light chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 58, 59, and 60, and a human IgGl constant domain; and
  • the anti-TSLP scFv comprises heavy chain variable region CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 6, 7, and 8, and light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in SEQ ID NOs: 18, 19, and 20.
  • the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, wherein:
  • the anti-TSLP antibody comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table IB and a human IgGl constant domain;
  • the anti-SCF scFv comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 2B.
  • the bispecific construct comprises an anti-SCF antibody linked to an anti-TSLP scFv, wherein:
  • the anti-SCF antibody comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 2B and a human IgGl constant domain;
  • the anti-TSLP scFv comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table IB.
  • the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, wherein:
  • the anti-TSLP antibody comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 4 and a human IgGl constant domain;
  • the anti-SCF scFv comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 5.
  • the bispecific construct comprises an anti-SCF antibody linked to an anti-TSLP scFv, wherein:
  • the anti-SCF antibody comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 5 and a human IgGl constant domain;
  • the anti-TSLP scFv comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 4.
  • the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, wherein:
  • the anti-TSLP antibody comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 6 and a human IgGl constant domain;
  • the anti-SCF scFv comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 6.
  • An exemplary bispecific is construct 5.2 (1D10 H2-mAb 12 VH1VL4 (ds)) having the amino acid sequence as set forth in SEQ ID NO: 65 or encoded by the nucleotide sequence as set forth in SEQ ID NO: 66.
  • Another exemplary bispecific is construct 5.7 (ID 10 H2- mAbl2 VH4VL3 (ds)), also referred to herein as “CDX-622,” having the amino acid sequence as set forth in SEQ ID NO: 67 or encoded by the nucleotide sequence as set forth in SEQ ID NO: 68.
  • compositions including any of the antibodies, antigen binding fragments, or bispecific constructs described herein and a pharmaceutically acceptable carrier.
  • kits or vials comprising any of the antibodies, antigen binding fragments, or bispecific constructs described herein and instructions for use.
  • isolated nucleic acid molecules encoding the antibodies, antigen binding fragments, or bispecific constructs described herein are also provided, as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors.
  • the nucleic acid molecule comprises a nucleotide sequence encoding an antibody variable region, wherein the antibody variable region comprises an amino acid sequence as set forth in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, or an amino acid sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one or more of the aforementioned sequences).
  • the nucleic acid molecule comprises a nucleotide sequence encoding heavy and light chain variable regions of an antibody, wherein the heavy and light chain variable regions comprise the amino acid sequences as shown in Table IB or Table 2B, or amino acids sequences at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical the aforementioned sequences).
  • Also provided herein are in vitro and in vivo methods for blocking or inhibiting TSLP and/or SCF binding to its receptor comprising contacting cells expressing TSLPR or c-Kit with the antibodies, antigen binding fragments, or bispecific constructs (or compositions) described herein, in an amount effective to block TSLP and/or SCF binding to its receptor.
  • methods for inhibiting activation of immune cells, as well as reducing or preventing the accumulation of immune cells within organs or tissues, in a subject comprising administering to the subject any of the antibodies, antigen binding fragments, or bispecific constructs(or compositions) described herein, in an amount effective to inhibit activation of immune cells or reduce accumulation of immune cells within organs or tissues in a subject.
  • such methods result in inhibition or blocking of (a) TSLP-induced activation and/or proliferation of mast cells, DC, and/or NKT cells, (b) TSLP-induced osteoprotegerin (OPG) secretion, (c) TSLP-induced secretion of Th2 cytokines (such as TARC, CCL22, IL-4, IL- 13 or IL-5), and / or (d) SCF-induced secretion of mast cells, eosinophils, type 2 innate lymphoid (1LC2) cells, and/or type 3 innate lymphoid (ILC3) cells.
  • OPG osteoprotegerin
  • methods are provided herein for reducing or inhibiting inflammation, as well as methods of treating an inflammatory disease or disorder in a subject in need thereof e.g., autoimmune diseases, cardiovascular diseases, gastrointestinal diseases, lung diseases, metabolic diseases (such as Type 2 diabetes), vasculitis (including large vessel vasculitis such as Takayasu’s arteritis, polymyalgia rheumatica and temporal arteritis; medium vessel vasculitis such as Buerger’s disease, Kawasaki disease, cutaneous vasculitis and polyarteritis nodosa and small vessel vasculitis such as Behcet’s syndrome, Churg- Strauss syndrome, cutaneous vasculitis, Henoch-Schonlein purpura, granulomatosis with polyangiitis, microscopic polyangiitis and cryoglobulinemia), neurodegenerative diseases (such as Parkinson’s disease), certain types of cancer (such as colon cancer) and mental illnesses (such as depression)), by administering the inflammatory disease
  • methods of for treating a condition or disease in a subject comprising administering to the subject any of the antibodies, antigen binding fragments, or bispecific constructs (or compositions) described herein, in an amount effective to treat the condition or disease.
  • the subject can be, for example, one who suffers from a condition or disease in which reduction of inflammation is desired.
  • the condition or disease is associated with immune cell migration, activation, and/or proliferation via interaction (e.g., binding) of TSLP and/or SCF with is receptor (TSLPR and/or c-Kit, respectively) on immune cells, such as disorders of the immune system, allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma, obstructive airways disease, chronic obstructive pulmonary disease (COPD), and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorders, and rhino-conjunctivitis.
  • TSLPR is receptor
  • c-Kit is receptor
  • immune cells such as disorders of the immune system, allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma, obstructive airways disease, chronic obstructive pulmonary disease (COP
  • fibrotic diseases and maladies associated with tissue remodeling include idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, smoking-induced lung injury, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, sclerodoma, inflammation, liver cirrhosis, renal fibrosis, parenchymal fibrosis, endomyocardial fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, fibrous histiocytoma, fibrothorax, hepatic fibrosis, fibromyalgia, gingival fibrosis, or radiation- induced fibrosis.
  • tissue remodeling e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, smoking-induced lung injury, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis,
  • methods for treating a condition or disease in a subject comprising administering to the subject a combination of any of the anti-TSLP antibodies, or antigen binding fragments thereof, described herein, and anti- SCF antibodies, or antigen binding fragments thereof, described herein.
  • the anti-TSLP antibody, or antigen binding fragment thereof comprises heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 2, 3, and 4, (b) SEQ ID NOs: 6, 7, and 8, (c) SEQ ID NOs: 10, 11, and 12, (d) SEQ ID NOs: 14, 15, and 16, or conservative sequence modifications thereof, and light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 18, 19, and 20, (b) SEQ ID NOs: 22, 23, and 24, (c) SEQ ID NOs: 26, 27, and 28, (d) SEQ ID NOs: 30, 31, and 32, or conservative sequence modifications thereof; and
  • the anti-SCF antibody, or antigen binding fragment thereof comprising heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 34, 35, and 36, (b) SEQ ID NOs: 38, 39, and 40, (c) SEQ ID NOs: 42, 43, and 44, (d) SEQ ID NOs: 46, 47, and 48, or conservative sequence modifications thereof, and light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 50, 51, and 52, (b) SEQ ID NOs: 54, 55, and 56, (c) SEQ ID NOs: 58, 59, and 60, (d) SEQ ID NOs: 62, 63, and 64, or conservative sequence modifications thereof.
  • the anti-TSLP antibody, or antigen binding fragment thereof comprises heavy and light chain variable region amino acid sequences as set forth in Table IB, or sequences at least 90% identical thereto (e.g., at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences); and
  • the anti-SCF antibody, or antigen binding fragment thereof comprises heavy and light chain variable region amino acid sequences as set forth in Table 2B, or sequences at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • the anti-TSLP antibody, or antigen binding fragment thereof, and the anti-SCF antibody, or antigen binding fragment thereof are administered separately.
  • the anti-TSLP antibody, or antigen binding fragment thereof, and the anti- SCF antibody, or antigen binding fragment thereof are administered sequentially.
  • the anti-TSLP antibody, or antigen binding fragment thereof can be administered first followed by e.g., immediately followed by) administration of the anti-SCF antibody, or antigen binding fragment thereof, or vice versa.
  • the anti-TSLP antibody, or antigen binding fragment thereof, and the anti-SCF antibody, or antigen binding fragment thereof are administered together.
  • the anti-TSLP antibody, or antigen binding fragment thereof, and the anti-SCF antibody, or antigen binding fragment thereof are administered simultaneously.
  • the anti-TSLP antibody, or antigen binding fragment thereof, and the anti-SCF antibody, or antigen binding fragment thereof are simultaneously administered in a single formulation.
  • the anti- TSLP antibody, or antigen binding fragment thereof, and the anti-SCF antibody, or antigen binding fragment thereof are formulated for separate administration and are administered concurrently or sequentially. Such concurrent or sequential administration preferably results in both antibodies being simultaneously present in treated patients.
  • an antibody, antigen binding fragment, or bispecific construct is administered in combination with one or more additional therapeutic agent or procedure.
  • FIGs. 1-3 are graphs showing binding of anti-TSLP antibodies to human TSLP, as a function of antibody concentration.
  • FIGs. 4-6 are graphs showing anti-TSLP antibodies block binding of human TSLP to human TSLP receptor (TSLP-R).
  • FIG. 7 is a graph showing anti-TSLP antibodies inhibit BaF3 cell proliferation.
  • FIG. 8 is a graph showing anti-TSLP antibodies inhibit TARC induction in human dendritic cells.
  • FIGs. 9 A and 9B are tables showing the affinity and kinetic parameters (with background subtracted) of anti-TSLP antibodies for human (FIG. 9A) and cynomolgus (FIG. 9B) TSLP.
  • FIGs. 10 and 11 are graphs showing binding of anti-SCF antibodies to human SCF, as a function of antibody concentration.
  • FIGs. 12 and 13 are graphs showing anti-SCF antibodies block binding of human SCF to human c-Kit.
  • FIGs. 14A and 14B are tables showing the affinity and kinetic parameters (with background subtracted) of anti-SCF antibodies for human (FIG. 14A) and cynomolgus (FIG. 14B) SCF.
  • FIG. 15 is a graph showing anti-SCF antibodies inhibit c-Kit phosphorylation in human CHO-KIT cells.
  • FIG. 16 is a graph showing anti-SCF antibodies inhibit human mast cell degranulation.
  • FIG. 17 is a graph showing anti-SCF antibodies inhibit M-07e cell proliferation.
  • FIGs. 18A and 18B are graphs showing how binding of anti-SCF to human SCF is affected by mutating amino acid residues in human SCF.
  • FIG. 18C is a three-dimensional drawing showing the binding of SCF to KIT-D1 including residue K100.
  • FIGs. 19A and 19B are graphs showing binding of anti-SCF antibodies to S1/S14 mouse cells.
  • FIG. 20 is a graph showing anti-SCF antibodies inhibit c-Kit phosphorylation in human M-07e cells.
  • FIGs. 21A and 21B are schematics showing the structure of the bispecific construct having an anti-SCF antibody linked to an anti-TSLP scFV (A) and an anti-TSLP antibody linked to an anti-SCF scFV (B).
  • FIG. 22 is a graph showing binding of bispecific antibody constructs to human TSLP.
  • FIG. 23 is a graph showing binding of bispecific antibody constructs to human SCF.
  • FIG. 24 is a graph showing binding of bispecific antibody constructs to human TSLP and human SCF.
  • FIGs. 25A and 25B are graphs showing bispecific antibody constructs block binding of human TSLP to human TSLP receptor (TSLP-R).
  • FIG. 26 is a graph showing bispecific antibody constructs block binding of human SCF to c-Kit.
  • FIGs. 27A and 27B are graphs showing bispecific antibody constructs inhibit BaF3 cell proliferation.
  • FIGs. 28A and 28B are graphs showing bispecific antibody constructs inhibit TARC induction in human dendritic cells.
  • FIGs. 29A and 29B are tables showing the affinity and kinetic parameters (with background subtracted) of bispecific antibody constructs for human TSLP and SCF and cynomolgus TSLP and SCF.
  • FIG. 30 is a graph showing bispecific antibody constructs inhibit c-Kit phosphorylation in human CHO-KIT cells.
  • FIG. 31 is a graph showing bispecific antibody constructs inhibit human mast cell degranulation.
  • FIG. 32 is a graph showing bispecific antibody constructs inhibit M-07e cell proliferation.
  • FIG. 33 is a graph showing bispecific antibody, CDX-622, blocks KIT phosphorylation in M-07e cells that have been stimulated with soluble SCF with greater potency than KIT stimulated with SCF 220 -expressing cells.
  • FIGs. 34A and 34B are graphs respectively showing downregulation in the expression of mast cell and melanocyte genesassociated with mast cell function in skin biopsies of cynomolgus macaques after dosing with mAh 12.
  • FIGs. 35A and 35B are representative images of the biopsy cross sections from animals treated with mAbl2 before treatment (FIG. 35A) and at day 30 (FIG. 35B).
  • FIGs. 36A and 36B are tables showing the mast cell count with the average data shown in FIG. 36A and the aggregate data shown in FIG. 36B.
  • FIGs. 37A, 37B, and 37C are graphs analyzing Mean Corpuscular Hemoglobin (FIG. 37A), Mean Corpuscular Hemoglobin Concentration (FIG. 37B), and Mean Corpuscular Volume (FIG. 37C).
  • FIG. 38 is a graph showing circulating levels of mAb!2 from serum samples collected from the monkeys over the course of the study.
  • FIG. 39 is a graph showing the presence of anti-drug antibodies (AD As) from the monkeys over the course of the study (the red line indicates the cutpoint of the assay).
  • FIGs. 40A-40F are graphs showing administration of bispecific antibody, CDX-622, did not result in significant decreases in hematological parameters in a study in cynomolgus macaques.
  • FIGs. 41A-41F are graphs showing the decrease in the expression of several selected genes associated with mast cell function in skin biopsies of cynomolgus macaques after dosing with CDX-622.
  • FIG. 42 is a graph showing inhibition by the bispecific antibody, CDX-622, of TSLP - induced CD80 expression on human dendritic cells.
  • FIG. 43 is a graph showing inhibition by the bispecific antibody, CDX-622, of TSLP binding to TSLP-R (ELISA).
  • FIG. 44 is a graph showing inhibition by the bispecific antibody, CDX-622, of TSLP - mediated cell proliferation (BaF3 Cells).
  • FIG. 45 is a graph showing the survival rate of human eosinophils treated with bispecific antibody, CDX-622.
  • FIG. 46 is a graph showing SCF-induced cytokine release from human primary mast cells treated with antibodies mAbl2, 1D10, or bispecific, CDX-622.
  • FIG. 47 is a graph showing MCP-1 induction with SCF and TSLP in LAD2 cells.
  • FIG. 48 is a graph showing simultaneous blockade of SCF and TSLP in LAD2 cells treated with antibodies mAb!2, 1D10, or bispecific, CDX-622.
  • FIG. 49 is a graph showing inhibition of TSLP-induced CD80 expression on human dendritic cells treated with antibody 1D10 or bispecific CDX-622.
  • the term “subject” includes any human or non-human animal.
  • the methods and compositions of the present invention can be used to treat a subject with an immune disorder.
  • non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
  • binding domain and “antigen binding portion” are used interchangeably and refer to the portion of a protein or antibody which comprises the amino acid residues that interact with an antigen. Binding domains include, but are not limited to, antibodies e.g., full length antibodies), as well as antigen-binding portions thereof. The binding domain confers on the binding agent its specificity and affinity for the antigen. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. Such proteins may be derived from natural sources, or partly or wholly synthetically produced.
  • antibody as referred to herein includes whole antibodies.
  • An “antibody” refers, in one preferred embodiment, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding fragment thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • Vn and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from aminoterminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antibody fragment refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (e.g., human TSLP or human SCF).
  • fragments are, for example between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids in length, suitably about 8 to about 300, for example about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.
  • VL and VH an isolated complementarity determining region
  • a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (sFv); see e.g. , Bird et al. (1988) Science 242:423-426; and Huston et al.
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody.
  • antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
  • human monoclonal antibody refers to an antibody that displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to an antibody which displays a single binding specificity and which has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, e.g. , a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • a transgenic non-human animal e.g. , a transgenic mouse
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • variable and constant regions that utilize particular human germline immunoglobulin sequences are encoded by the germline genes, but include subsequent rearrangements and mutations which occur, for example, during antibody maturation.
  • the variable region contains the antigen binding domain, which is encoded by various genes that rearrange to form an antibody specific for a foreign antigen.
  • the variable region can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody to the foreign antigen.
  • the constant region will change in further response to an antigen (i.e., isotype switch).
  • the rearranged and somatically mutated nucleic acid molecules that encode the light chain and heavy chain immunoglobulin polypeptides in response to an antigen may not have sequence identity with the original nucleic acid molecules, but instead will be substantially identical or similar i.e., have at least 80% identity).
  • human antibody includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences.
  • Human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) (see, Lonberg, N. et al. (1994) Nature 368(6474): 856-859); Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y.
  • human antibody does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (/. ⁇ ?., chimeric and humanized antibodies).
  • an “isolated antibody,’’ as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to human TSLP or human SCF is substantially free of antibodies that specifically bind antigens other than human TSLP or human SCF; an isolated antibody that specifically binds to human TSLP is substantially free of antibodies that specifically bind antigens other than human TSLP).
  • An isolated antibody that specifically binds to an epitope may, however, have cross-reactivity to the same antigen from different species.
  • an isolated antibody is typically substantially free of other cellular material and/or chemicals.
  • epitopes refers to a site on an antigen to which an immunoglobulin or antibody specifically binds.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation.
  • epitope mapping Methods for determining what epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from the antigen ( ⁇ ?.g., TSLP or SCF) are tested for reactivity with the given antibody (e.g., an anti-TSLP or anti-SCF antibody.
  • Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).
  • antibody that binds the same epitope is intended to encompass antibodies that interact with, i.e., bind to, the same structural region on human TSLP or human SCF as a reference anti-TSLP antibody or reference anti-SCF antibody.
  • the “same epitope” to which the antibodies bind may be a linear epitope or a conformational epitope formed by tertiary folding of the antigen.
  • the term “competing antibody” refers to an antibody that competes for binding to human TSLP with a reference anti-TSLP antibody or an antibody that competes for binding to human SCF with a reference anti-SCF antibody, i.e., competitively inhibits binding of the reference anti-TSLP antibody to TSLP or competitively inhibits binding of the reference anti- SCF antibody to SCF.
  • a “competing antibody” may bind the same epitope on TSLP or SCF as the reference anti-TSLP antibody or reference anti-SCF antibody, may bind to an overlapping epitope or may sterically hinder the binding of the reference anti-TSLP antibody to TSLP or reference anti-SCF antibody to SCF.
  • Antibodies that recognize the same epitope or compete for binding can be identified using routine techniques. Such techniques include, for example, an immunoassay, which shows the ability of one antibody to block the binding of another antibody to a target antigen, i.e. , a competitive binding assay. Competitive binding is determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as TSLP or SCF.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay see Stahli et al., Methods in Enzymology 9:242 (1983)
  • solid phase direct biotin-avidin EIA see Kirkland el al., J. Immunol. 137:3614 (1986)
  • solid phase direct labeled assay solid phase direct labeled sandwich assay
  • solid phase direct labeled sandwich assay see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)
  • solid phase direct label RIA using 1-125 label see Morel et al., Mol. Immunol.
  • epitope mapping methods such as, x-ray analyses of crystals of antigemantibody complexes which provides atomic resolution of the epitope.
  • Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component.
  • computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library.
  • computational algorithms have also been developed which have been shown to map conformational discontinuous epitopes.
  • the terms “specific binding,’- “selective binding,” “selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen.
  • the antibody binds with an equilibrium dissociation constant (Kp>) of approximately less than 10 7 M, such as approximately less than 10 8 M, 10 9 M or 10 10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 2000 instrument (e.g., using recombinant human TSLP or recombinant human SCF as the analyte and the antibody as the ligand) and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
  • a non-specific antigen e.g., BSA, casein
  • KD is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction.
  • the human antibodies of the invention bind to TSLP or SCF with a dissociation equilibrium constant (KD) of approximately 10’ 8 M or less, such as less than 10' 9 M or 10’ 10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 2000 instrument (e.g., using recombinant human TSLP or recombinant human SCF as the analyte and the antibody as the ligand).
  • SPR surface plasmon resonance
  • Kd is intended to refer to the off rate constant for the dissociation of an antibody from the antibody /antigen complex.
  • ka as used herein, is intended to refer to the on rate constant for the association of an antibody with the antigen.
  • EC50 refers to the concentration of an antibody or an antigen-binding portion thereof, which induces a response, either in an in vitro or an in vivo assay, which is 50% of the maximal response, i.e., halfway between the maximal response and the baseline.
  • isotype refers to the antibody class (e.g. , IgM or IgGl) that is encoded by heavy chain constant region genes.
  • a human monoclonal antibody of the invention is of the IgGl isotype.
  • a human monoclonal antibody of the invention is of the IgG2 isotype.
  • the terms “inhibits” or “blocks” are used interchangeably and encompass both partial and complete inhibition/blocking.
  • the inhibition/blocking preferably reduces or alters the normal level or type of activity that occurs when binding occurs without inhibition or blocking.
  • Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of TSLP-R when in contact with an anti-TSLP antibody as compared to TSLP-R not in contact with an anti-TSLP antibody, e.g., inhibits binding of CD70 by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • inhibition and blocking are also intended to include any measurable decrease in the binding affinity of c-Kit when in contact with an anti-SCF antibody as compared to c-Kit not in contact with an anti-SCF antibody, e.g., inhibits binding of CD70 by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the anti- TSLP antibody inhibits binding of TSLP-R (or c-Kit) by at least about 70%.
  • the anti-TSLP antibody inhibits binding of TSLP-R (or c-Kit) by at least 80%.
  • Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of TSLP or SCF when in contact with an anti-TSLP (or anti-SCF) antibody as compared to TSLP or SCF not in contact with an anti-TSLP (or anti-SCF) antibody, e.g., inhibits binding of TSLP or SCF by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the anti-TSLP antibody inhibits binding of TSLP by at least about 70%. In another embodiment, the anti-TSLP antibody inhibits binding of TSLP by at least 80%. In one embodiment, the anti-SCF antibody inhibits binding of SCF by at least about 70%. In another embodiment, the anti- SCF antibody inhibits binding of SCF by at least 80%.
  • cross-reacts refers to the ability of an anti-TSLP binding domain or an anti-SCF binding domain of the invention to bind to TSLP or SCF, respectively, from a different species. For example, a TSLP binding domain of the invention that binds human TSLP may also bind another species of TSLP.
  • an anti-SCF binding domain of the invention that binds human SCF may also bind another species of SCF.
  • cross-reactivity is measured by detecting a specific reactivity with purified antigen in binding assays (e.g., SPR, ELISA) or binding to, or otherwise functionally interacting with, cells physiologically expressing TSLP or SCF.
  • binding assays e.g., SPR, ELISA
  • Methods for determining cross-reactivity include standard binding assays as described herein, for example, by BiacoreTM surface plasmon resonance (SPR) analysis using a BiacoreTM 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometric techniques.
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally- occurring.
  • the present invention also encompasses “conservative sequence modifications” of any of the sequences set forth in SEQ ID NOs: 1-349, i.e., nucleotide and amino acid sequence modifications which do not abrogate the binding of the VH and VL sequences encoded by the nucleotide sequence or containing the amino acid sequence, to the antigen.
  • conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as, nucleotide and amino acid additions and deletions.
  • modifications can be introduced into SEQ ID NOs: 1-349 by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • conservative amino acid sequence modifications refer to at most 1, 2, 3, 4 or 5 conservative amino acid substitutions to the CDR sequences described herein.
  • each such CDR may contain up to 5 conservative amino acid substitutions, e.g., up to (i.e., not more than) 4 conservative amino acid substitutions, e.g., up to (i.e., not more than) 3 conservative amino acid substitutions, e.g., up to (i.e., not more than) 2 conservative amino acid substitutions, or no more than 1 conservative amino acid substitution.
  • mutations can be introduced randomly along all or part of an anti-TSLP or anti-SCF binding domain coding sequence, such as by saturation mutagenesis, and the resulting modified anti-TSLP or anti-SCF antibodies can be screened for binding activity.
  • nucleic acids For nucleic acids, the term “substantial homology’- indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.
  • substantially homology indicates that two amino acid sequences, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80% of the amino acids, usually at least about 90% to 95%, and more preferably at least about 98% to 99% or 99.5% of the amino acids.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • the nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • an antibody os bispecific antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain comprises at least one (e.g., one, two, three, four, five or six) amino acid modifications (e.g. substitution, deletion or addition) or at least one (e.g., one, two, three, four, five or six) non-naturally occurring amino acid residues.
  • the Fc region or domain comprises at least one (e.g., one, two, three, four, five or six) amino acid modifications (e.g. substitution, deletion or addition) or at least one (e.g., one, two, three, four, five or six) non-naturally occurring amino acid residues.
  • an antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgGl and comprises at least one (e.g., one, two, three, four, five or six) amino acid modifications (e.g.
  • an antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgG2 and comprises at least one (e.g., one, two, three, four, five or six) amino acid modifications (e.g.
  • an antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgG3 and comprises at least one (e.g., one, two, three, four, five or six) amino acid modifications (e.g.
  • an antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgG4 and comprises at least one (e.g., one, two, three, four, five or six) amino acid modifications (e.g.
  • substitution, deletion or addition or at least one (e.g., one, two, three, four, five or six) non-naturally occurring amino acid residues, which are equivalents to the amino acid residue(s) described herein for a human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • an antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgGl and comprises at least one (e.g., one, two, three, four, five or six) amino acid modifications (e.g.
  • substitution, deletion or addition or at least one non-naturally occurring amino acid residue (e.g., one, two, three, four, five or six) selected from the group consisting of 234A, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R.243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T,
  • the Fc region or domain may comprise additional and/or alternative non-naturally occurring amino acid residues known to one skilled in the art (see, e.g., U.S. Patents 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).
  • an antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgG2 and comprises at least one (e.g., one, two, three, four, five or six) amino acid modifications (e.g.
  • an antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgG3 and comprises at least one (e.g., one, two, three, four, five or six) amino acid modifications (e.g.
  • an antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgG4 and comprises at least one (e.g., one, two, three, four, five or six) amino acid modifications (e.g.
  • substitution, deletion or addition or at least one (e.g., one, two, three, four, five or six) non-naturally occurring amino acid residues, which are equivalents to the amino acid residue(s) described herein for a human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • an antibody comprising an Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgGl and comprises at least a non-naturally occurring amino acid at one or more positions selected from the group consisting of 239, 330 and 332, as numbered by the EU index as set forth in Kabat.
  • an antibody comprising an Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgGl and comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat.
  • the Fc region or domain may further comprise additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat.
  • an antibody comprising an Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgGl and comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat and at least one non-naturally occurring amino acid at one or more positions are selected from the group consisting of 252 Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.
  • an antibody comprising an Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgG2, IgG3, or IgG4, and comprises at least one non-naturally occurring amino acid residue that is an equivalent(s) to the amino acid residue(s) described herein for a human IgG 1 Fc region or domain, as can be determined by one of skill in the art.
  • an antibody comprising an Fc region or domain, wherein the Fc region or domain is an Fc region or domain of human IgG2, IgG3, or IgG4, and comprises at least one non-naturally occurring amino acid residue at one or more positions that are equivalent(s) to the positions described herein for a human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • an Fc region or domain comprising such sequence exhibits one or more Fc activity, for example, binding affinity to an Fc receptor or effector function, such as ADCC or CDC.
  • an Fc region or domain comprising such sequence exhibits reduced Fc activity, for example, reduced binding affinity to an Fc receptor or reduced effector function, such as ADCC or CDC.
  • an Fc region or domain comprising such sequence exhibits enhanced FcRn activity, for example, enhanced half-life.
  • Fc region or domain modifications are provided in Ghetie et al., 1997, Nat Biotech.l5:637-40; Duncan et al., 1988, Nature 332:563-564; Lund el al., 1991, J. Immunol 147:2657-2662; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et al., 1994, Transplantation 57: 1537-1543; Hutchins et al., 1995, Proc Natl.
  • the antibody described herein comprises a modified (e.g., mutated) human IgGl Fc region or domain, which comprises non-naturally occurring amino acids 234A, 235Q and 322Q as numbered by the EU index as set forth in Kabat.
  • the modified (e.g., mutated) human IgGl Fc region or domain further comprises non-naturally occurring amino acids 252Y, 254T and 256E as numbered by the EU index as set forth in Kabat.
  • the antibody described herein comprises a modified (e.g., mutated) human IgG2 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q and 322Q as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • a modified (e.g., mutated) human IgG2 Fc region or domain which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q and 322Q as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • the antibody described herein comprises a modified (e.g., mutated) human IgG2 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q, 322Q, 252Y, 254T and 256E as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • a modified (e.g., mutated) human IgG2 Fc region or domain which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q, 322Q, 252Y, 254T and 256E as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • the antibody described herein comprises a modified (e.g., mutated) human IgG3 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q and 322Q as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • a modified (e.g., mutated) human IgG3 Fc region or domain which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q and 322Q as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • the antibody described herein comprises a modified (e.g., mutated) human IgG3 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q, 322Q, 252Y, 254T and 256E as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • a modified (e.g., mutated) human IgG3 Fc region or domain which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q, 322Q, 252Y, 254T and 256E as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • the antibody described herein comprises a modified (e.g., mutated) human IgG4 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q and 322Q as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • a modified (e.g., mutated) human IgG4 Fc region or domain which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q and 322Q as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • the antibody described herein comprises a modified (e.g., mutated) human lgG4 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q, 322Q, 252Y, 254T and 256E as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • a modified (e.g., mutated) human lgG4 Fc region or domain which comprises non-naturally occurring amino acids that are equivalents to 234A, 235Q, 322Q, 252Y, 254T and 256E as numbered by the EU index as set forth in Kabat for human IgGl Fc region or domain, as can be determined by one of skill in the art.
  • the antibody described herein comprises VL and VH CDR sequences set herein and a modified (e.g., mutated) human IgGl Fc region or domain, wherein the modified (e.g., mutated) human IgGl Fc region or domain comprises non- naturally occurring amino acids 234A, 235Q, and 322Q as numbered by the EU index as set forth in Kabat.
  • the antibody described herein comprises VL and VH CDR sequences set forth herein and a modified (e.g., mutated) human IgGl Fc region or domain, wherein the modified (e.g., mutated) human IgGl Fc region or domain comprises non- naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T and 256E as numbered by the EU index as set forth in Kabat.
  • TSLP also referred to as “thymic stromal lymphopoietin’’
  • TSLP refers to a cytokine that is involved in the maturation of T cell populations through activation of antigen-presenting cells.
  • TSLP is produced by non-hematopoietic cells, such as fibroblasts, epithelial cells, and different types of stromal or stromal-like cells.
  • TSLP signals via a TSLP receptor (TSLPR, (also referred to as “CRFL2).
  • TSLPR forms a functional complex with TSLP and IL7R and stimulates cell proliferation by activating STAT3 and STAT5, as well as JAK2.
  • the TSLPR chain is closely related to the common receptor gamma chain that is expressed on a wide range of cell types in the adaptive and innate immune system and is implicated in the development of the haematopoietic system.
  • TSLP also affects the polarization of dendritic cells to drive T helper (Th) 2 cytokine production, directly promotes T-cell proliferation in response to T-cell receptor activation and Th2 cytokine production, and supports B-cell expansion and differentiation.
  • Th helper
  • TSLP further amplifies Th2 cytokine production by mast cells and natural killer T cells. These properties confer on TSLP a critical role in driving Th2-mediated inflammation. Accordingly, TSLP expression is linked to many disease states including asthma, inflammatory arthritis, atopic dermatitis, eczema, eosinophilic esophagitis, and others.
  • TSLP includes any variants or isoforms of TSLP which are naturally expressed by cells (e.g., human TSLP deposited with the UniProt® consortium having accession no. Q969D9-1 as set forth in SEQ ID NO: 70).
  • TSLP antibodies (or binding domains) of the invention may cross-react with TSLP from species other than human.
  • the TSLP antibodies (or binding domains) may be specific for human TSLP and may not exhibit any cross-reactivity with other species.
  • TSLP or any variants and isoforms thereof may either be isolated from cells or tissues that naturally express them or be recombinantly produced using well-known techniques in the art and/or those described herein.
  • the TSLP antibodies (or binding domains) are targeted to human TSLP, which has a normal glycosylation pattern.
  • the anti-TSLP antibody (or binding domain) comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of any one of antibodies 1D10-A, 1D10-B, 1D10-C, 1D10-D, 1D10-E, 1D10-F, 1D10-G, 1D10-H, or 1D10-I (as shown in Tale 5 of Example 2).
  • the anti-TSLP antibody or binding domain thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, or 13, and/or the CDR1, CDR2 and CDR3 domains of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, 21, 25, or 29.
  • the anti-TSLP antibody (or binding domain) comprises heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 5A, or conservative sequence modifications thereof, and/or light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 5B, or conservative sequence modifications thereof.
  • the anti-TSLP antibody or (binding domain ) comprises heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 2, 3, and 4, (b) SEQ ID NOs: 6, 7, and 8, (c) SEQ ID NOs: 10, 1 1 , and 12, (d) SEQ ID NOs: 14, 15, and 16, or conservative sequence modifications thereof, and/or light chain CDR1, CDR2 and CDR3 domains having the sequences respectively set forth in (a) SEQ ID NOs: 18, 19, and 20, (b) SEQ ID NOs: 22, 23, and 24, (c) SEQ ID NOs: 26, 27, and 28, (d) SEQ ID NOs: 30, 31, and 32, or conservative sequence modifications thereof, as set forth in Table 1 A.
  • the anti-TSLP antibody (or binding domain) respectively comprises the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 6, 7, and 8, and the light chain CDR1, CDR3, and CDR3 sequences set forth in SEQ ID NOs: 18, 19, and 20.
  • the anti-TSLP antibody (or binding domain) comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, or 13. In another embodiment, the antibody or binding domain thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, 21, 25, or 29. In another embodiment, the antibody (or binding domain) comprises a combination of heavy and/or light chain variable regions having the amino acid sequences set forth in Table IB. In yet another embodiment, the anti-TSLP antibody (or binding domain) comprises a combination of heavy and light chain variable regions having the amino acid sequences set forth in Table 4. In one embodiment, the anti-TSLP antibody (or binding domain) respectively comprises the heavy and light chain variable region sequences set forth in SEQ ID NOs: 5 and 17.
  • sequences substantially identical to the anti-TSLP antibodies (or binding domains) described herein e.g. at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical (identical) to the aforementioned sequences
  • the anti-TSLP antibody (or binding domain) comprises a heavy chain variable region comprising the combinations set forth in Table IB or Table 4, or sequences at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • Anti-TSLP antibodies (and binding domains thereof) that compete for binding with any of the anti-TSLP antibodies (or binding domains) described herein or that bind the same epitope as any of the anti-TSLP antibodies (or binding domains) described herein, are also suitable for use and provided herein.
  • the anti-TSLP antibody (or binding domain) competes for binding to TSLP with antibody ID 10, as described herein (or an antibody having the heavy and/or light chain CDRs and/or heavy and light chain variable region sequences corresponding to antibody 1D10, as described herein).
  • the antibody or anti-TSLP binding domain thereof binds to the same epitope on TSLP as antibody lD10,as described herein (or an antibody having the heavy and light chain CDRs and/or heavy and light chain variable region sequences corresponding to antibody 1D10, as described herein).
  • stem cell factor As used herein, the terms “stem cell factor,” “SCF,” “Mast cell growth factor,” “MGF,” “KIT-ligand,” “KL,” and “steel factor”) are used interchangeably, and include variants, isoforms, species homologs of human SCF, and analogs having at least one common epitope with SCF.
  • SCF stem cell factor
  • MCF mast cell growth factor
  • KIT-ligand KL
  • Steel factor Steel factor
  • SCF is a ligand for the receptor-type protein-tyrosine kinase KIT (“c-Kit”) and is involved in the regulation of cell survival and proliferation, hematopoiesis, stem cell maintenance, gametogenesis, mast cell development, migration, survival, activation, and in melanogenesis.
  • cKit protein-tyrosine kinase KIT
  • SCF activates several signaling pathways, thus promoting phosphorylation of PIK3R1, the regulatory subunit of phosphatidylinositol 3- kinase, and subsequent activates the kinase AKT1.
  • Binding of SCF to c-Kit also transmits signals via GRB2 and activates RAS, RAFI and the MAP kinases MAPK1/ERK2 and/or MAPK3/ERK1.
  • SCF binding also promotes activation of STAT family members STAT1, STAT3 and STAT5, as well as PLCG1, leading to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate.
  • SCF / c-Kit binding also acts synergistically with other cytokines, such as interleukins.
  • the anti-SCF antibody (or binding domain thereof) comprises the heavy and light chain CDRs or variable regions of any one of anti-SCF antibodies mAbl2-A, mAbl2-B, mAbl2-C, mAbl2-D, mAbl2-E, mAbl2-F, mAbl2-G, mAbl2-H, or mAbl2-I (as shown in Table 9 of Example 9).
  • the anti-SCF antibody (or binding domain) comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region having the sequence set forth in SEQ ID NO: 33, 37, 41, or 45, and/or the CDR1, CDR2 and CDR3 domains of the light chain variable region having the sequence set forth in SEQ ID NO: 49, 53, 57, or 61.
  • the anti-SCF antibody (or binding domain) comprises heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 8 A, or conservative sequence modifications thereof, and/or light chain CDR1 , CDR2 and CDR3 domains having the amino acid sequences respectively set forth in Table 8B, or conservative sequence modifications thereof.
  • the anti-SCF antibody comprises heavy chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 34, 35, and 36, (b) SEQ ID NOs: 38, 39, and 40, (c) SEQ ID NOs: 42, 43, and 44, (d) SEQ ID NOs: 46, 47, and 48, or conservative sequence modifications thereof, and/or light chain CDR1, CDR2 and CDR3 domains having the amino acid sequences respectively set forth in (a) SEQ ID NOs: 50, 51, and 52, (b) SEQ ID NOs: 54, 55, and 56, (c) SEQ ID NOs: 58, 59, and 60, (d) SEQ ID NOs: 62, 63, and 64, or conservative sequence modifications thereof, as set forth in Table 2A.
  • the anti-SCF antibody (or binding domain) respectively comprises the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, and the light chain CDR1, CDR3, and CDR3 sequences set forth in SEQ ID NOs: 58, 59, and 60.
  • the anti-SCF antibody (or binding domain) comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 33, 37, 41, or 45. In another embodiment, the anti-SCF antibody (or binding domain) comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 49, 53, 57, or 61. In another embodiment, the anti-SCF antibody (or binding domain) comprises a combination of heavy and light chain variable regions having the amino acid sequences set forth in Table 2B. In yet another embodiment, the antibody (or binding domain) comprises a combination of heavy and light chain variable regions having the amino acid sequences set forth in Table 5 of Example 9. In one embodiment, the anti-SCF antibody (or binding domain) respectively comprises the heavy and light chain variable region sequences set forth in SEQ ID NOs: 45 and 57.
  • sequences substantially identical to the anti-SCF antibodies (or binding domains) described herein e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical (identical) to the aforementioned sequences
  • the anti-SCF antibody (or binding domain) comprises a heavy chain variable region comprising the combinations set forth in Table 2B or Table 5, or sequences at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned sequences).
  • Anti-SCF antibodies and binding domains thereof that that compete for binding with any of the anti-SCF antibodies (or binding domains thereof) described herein or that bind the same epitope as any of the anti-SCF antibodies (or binding domains thereof) described herein, are also suitable for use and provided herein.
  • the anti-SCF antibody, or binding domain thereof competes for binding to SCF with antibody mAbl2, as described herein (or an antibody having the heavy and light chain CDRs and/or heavy and light chain variable region sequences corresponding to antibody mAbl2, as described herein).
  • the anti-SCF antibody (or binding domain) binds to the same epitope on SCF as antibody mAbl2, as described herein (or an antibody having the heavy and light chain CDRs and/or heavy and light chain variable region sequences corresponding to antibody mAbl2).
  • bispecific constructs comprising an anti-TSLP antibody (or binding domain) or an anti-SCF antibody (or binding domain) linked to a second binding agent(s) (e.g., a ligand, an antibody, or antigen binding portion thereof).
  • Bispecific constructs comprising an anti-TSLP antibody (or binding domain) linked to an anti-SCF antibody (or binding domain) also are provided.
  • the term “bispecific construct,” as used herein, also refers to bispecific constructs linked to one or more additional binding agents (i.e. , a third, fourth, or fifth binding agent) to form a “multispecific construct.”
  • a “bispecific” or “bifunctional” construct is an artificial hybrid having two different binding domain (e.g., heavy/light chain) pairs and two different binding sites.
  • Bispecific constructs can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny el al., J. Immunol. 148, 1547-1553 (1992).
  • a bispecific construct having more than two different binding domains is a multispecific construct.
  • the term “linked” refers to the association of two or more molecules.
  • the linkage can be covalent or non-covalent.
  • the linkage also can be genetic (z.e. , recombinantly fused). Such linkages can be achieved using a wide variety of art recognized techniques, such as chemical conjugation and recombinant protein production.
  • suitable reagents and methods are known in the art for coupling two or more moieties, in particular two or more antibodies, or fragments thereof, together.
  • a variety of coupling or crosslinking agents are commercially available and can be used to conjugate the anti-TSLP binding domain and anti-SCF binding domain.
  • Non-limiting examples include Sulfo-SMCC, protein A, carboiimide, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), and N-succinimidyl-3-(2 -pyridyldithio) propionate (SPDP).
  • Sulfo-SMCC, SPDP and DTNB are preferred agents, with Sulfo-SMCC being particularly preferred.
  • crosslinking components e.g., binding domains
  • cross-linking agents e.g., binding domains
  • Karpovsky, B. et al. (1984) J. Exp. Med. 160:1686; Liu, M. A. et al., (1985) Proc. Natl. Acad. Sci USA 82:8648; Segal, D. M. and Perez, P shake U.S. Pat. No. 4,676,980; and Brennan, M. (1986) Biotechniques 4:424.
  • nucleic acid molecules encoding the anti-TSLP antibody (or binding domain) or anti-SCF antibody (or binding domain) can be inserted into an appropriate expression vector using standard recombinant DNA techniques.
  • a nucleic acid molecule(s) encoding the second binding agent e.g., a ligand, antibody, or binding domain thereof
  • the second binding agent also can be inserted into the same expression vector, such that it is operatively linked (e.g., in-frame cloning) to the anti-TSLP antibody (or binding domain) or anti-SCF-binding antibody (or binding domain), thereby resulting in an expression vector that encodes a fusion protein that is the bispecific construct.
  • Exemplary bispecific constructs include a combination of binding agents that bind to the targets listed in Table 3 A and comprise, e.g., an anti-TSLP antibody (or binding domain) or anti-SCF antibody (or binding domain) linked to a second binding agent that binds to a member of the TNF superfamily (e.g., TNFa), a tumor necrosis factor (TNF) receptor (e.g., TNFRSF4), an interleukin (e.g., IL-23, IL-17A, or IL-13), an immunoglobulin (e.g., IgE), or an integrin (e.g., integrin a 4beta7).
  • TNF tumor necrosis factor
  • TNFRSF4 tumor necrosis factor receptor
  • interleukin e.g., IL-23, IL-17A, or IL-13
  • IgE immunoglobulin
  • integrin e.g., integrin a 4beta7.
  • the bispecific construct comprises the anti-TSLP and anti-SCF antibodies (or antigen binding domains) described herein having the heavy and light chain CDR and/or full-length variable regions sequences respectively set forth in Tables 1A, IB, and Tables 2A, 2B.
  • Other exemplary bispecific constructs comprise an anti-TSLP antibody (or binding domain) linked to an anti-SCF antibody (or binding domain), e.g., the anti-TSLP antibody (or binding domain) 1D10-D linked to the anti-SCF antibody (or binding domain) mAbl2-G.
  • the anti-SCF binding portion is operatively linked to the C-terminal region of the heavy chain of the anti-TSLP binding portion.
  • suitable expression vectors and cloning strategies for preparing the bispecific constructs described herein are known in the art.
  • Binding agents for use with the bispecific constructs include, for example, the antibodies (or binding domains thereof) listed in Table 3B.
  • binding portions e.g., antibodies or binding domains thereof
  • the coding regions of the binding portions are combined with cloned promoter, leader sequence, translation initiation, leader sequence, constant region, 3’ untranslated, poly adenylation, and transcription termination, sequences to form expression vector constructs.
  • These constructs can be used to express, for example, full length human IgGiK or IgG4K antibodies.
  • Fully human, humanized and chimeric antibodies used in the bispecific constructs described herein also include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similar plasmids can be constructed for expression of other heavy chain isotypes, or for expression of antibodies comprising lambda light chains.
  • the bispecific construct can be expressed recombinantly in a host cell using standard transfection methods.
  • nucleic acid encoding the bispecific construct can be ligated into an expression vector, such as a eukaryotic expression plasmid, such as used by GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expression systems well known in the art.
  • the purified plasmid with the cloned bispecific construct gene can be introduced in eukaryotic host cells, such as CHO-cells or NSO-cells or alternatively other eukaryotic cells like a plant derived cells, fungi or yeast cells.
  • the method used to introduce these genes could be methods described in the art, such as electroporation, lipofectin, lipofectamine or other.
  • cells expressing the bispecific construct can be identified and selected. These cells represent the transfectomas that can then be amplified for their expression level and upscaled to produce bispecific constructs.
  • these cloned bispecific constructs can be expressed in other expression systems, such as E. coli or in complete organisms or can be synthetically expressed.
  • Recombinant bispecific constructs can be isolated and purified from these culture supernatants and/or cells.
  • the anti-TSLP antibody (or binding domain) and the anti-SCF antibody (or binding domain) are genetically fused to each other.
  • the anti-TSLP antibody (or binding domain) or the anti-SCF antibody (or binding domain) are chemically conjugated.
  • the anti-TSLP antibody (or binding domain) and the anti-SCF antibody (or binding domain) are chemically conjugated to each other.
  • either one of the binding portions further comprises a human IgGl constant domain.
  • Bispecific constructs comprising an anti-TSLP antibody (or binding domain) linked to an anti-SCF antibody (or binding domain) include the following embodiments: (a) the anti- SCF binding portion is linked to the C-terminus of the heavy chain of the anti-TSLP binding portion; (b) the anti-SCF binding portion is a scFv; (c) the anti-SCF binding portion further comprises a human IgG l constant domain; (d) the anti-TSLP binding portion is linked to the C-terminus of the heavy chain of the anti-SCF binding portion; or (e) the anti-TSLP binding portion is a scFv.
  • Exemplary bispecific constructs include any one of the anti-TSLP antibodies (or binding domain) described herein having the CDR or full-length heavy and light chain variable region sequences set forth in Tables 1 A and IB or any one of the anti-SCF antibodies (or binding domain) described herein having the CDR or full-length heavy and light chain variable region sequences set forth in Tables 2A and 2B linked to a second binding domain (e.g., a ligand, antibody, or antigen binding portion thereof).
  • a second binding domain e.g., a ligand, antibody, or antigen binding portion thereof.
  • Exemplary bispecific constructs also include any one of the anti-TSLP antibodies (or binding domain) described herein having the CDR or full-length heavy and light chain variable region sequences set forth in Tables 1A and IB linked to any one of the anti-SCF antibodies (or binding domain) described herein having the CDR or full-length heavy and light chain variable region sequences set forth in Tables 2 A and 2B.
  • bispecific constructs include a combination of any one of the anti- TSLP antibodies (or antigen binding fragments thereof) of Table 1 A or IB with any one of the anti-SCF antibodies (or antigen binding fragments thereof) of Table 2A or 2B, wherein bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, and wherein the anti-TSLP antibody further comprises a human IgGl constant domain.
  • the bispecific construct includes a combination of an anti-TSLP antibody (or antigen binding fragment thereof) of Table 1 A or IB combined with an anti-SCF antibody (or antigen binding fragment thereof) of Table 2 A or 2B, wherein bispecific construct comprises an anti-SCF antibody linked to an anti-TSLP scFv, and wherein the anti-SCF antibody further comprises a human IgGl constant domain.
  • exemplary bispecific constructs include an anti-TSLP binding domain and an anti-SCF binding domain comprising amino acid sequences set forth in Table 10 of Example 19.
  • compositions e.g., a composition comprising one or a combination of any of the antibodies (or binding domains), or bispecific constructs described herein, formulated together with a carrier e.g., a pharmaceutically acceptable carrier).
  • a carrier e.g., a pharmaceutically acceptable carrier
  • the terms “carrier” and “pharmaceutically acceptable carrier” includes any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration e.g. , by injection or infusion).
  • the active compound i.e. , any of the antibodies (or binding domains), or bispecific constructs described herein
  • adjuvants which may be used with the antibodies (or binding domains), bispecific constructs described here include, but are not limited to : Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatised polysaccharides; polyphosphazenes; biodegradable microspheres; cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like factors; 3D- MPL; CpG oligonucleotide; and monophosphoryl lipid A, for example 3-de-O-acylated monophosphoryl lipid A.
  • MPL adjuvants are available from Corixa Corporation (Seattle, Wash; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).
  • CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated
  • Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996.
  • adjuvants include, for example, saponins, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins; Montanide ISA 720 (Seppic, France); SAF (Chiron, California, United States); ISCOMS (CSL), MF-59 (Chiron); the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium); Detox (EnhanzynTM) (Corixa, Hamilton, Mont.); RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs); polyoxyethylene ether adjuvants such as those described in WO 99/52549A1; synthetic imidazoquinolines such as imiquimod [S-2
  • cytokine, chemokine and co-stimulatory molecules as either protein or peptide, including for example pro-inflammatory cytokines such as Interferon, GM-CSF, IL-1 a, IL-1 beta, TGF-a and TGF-p, Thl inducers such as interferon gamma, IL-2, IL-12, IL-15, IL-18 and IL-21, Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13 and other chemokine and co-stimulatory genes such as MCP-1, MIP-1 a, MIP-1 beta, RANTES, TCA-3, CD80, CD86 and CD40L; immunostimulatory agents targeting ligands such as CTLA-4 and L-selectin, apoptosis stimulating proteins and peptides such as Fas; synthetic lipid based adjuvants, such as vaxfectin, (Reyes), Interferon, GM-CSF, IL-1 a,
  • TLR Toll-like Receptor
  • a “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • a composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatihle polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, poly orthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.
  • an appropriate carrier for example, liposomes, or a diluent.
  • Acceptable diluents include saline and aqueous buffer solutions.
  • Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).
  • Carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the antibodies of the invention may be administered once or twice weekly by subcutaneous or intramuscular injection or once or twice monthly by subcutaneous or intramuscular injection.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, a-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.001 per cent to about ninety percent of active ingredient, preferably from about 0.005 per cent to about 70 per cent, most preferably from about 0.01 per cent to about 30 per cent.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (more preferably, 0.005 to 70%, such as 0.01 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a composition of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target.
  • the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
  • compositions can be administered with medical devices known in the art.
  • a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • a needleless hypodermic injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No.
  • the antibodies (or binding domains) or bispecific constructs of the invention can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier (BBB) excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the invention cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g. , V.V. Ranade (1989) J. Clin. Pharmacol. 29:685).
  • Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low el al.)', mannosides (Umezawa et al. , (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol.
  • the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the tumor or infection.
  • the composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • a compound to inhibit inflammation can be evaluated in an animal model system predictive of efficacy in human asthma patients.
  • this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • the composition must be sterile and fluid to the extent that the composition is deliverable by syringe.
  • the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • isolated nucleic acid molecule as used herein in reference to nucleic acids encoding antibodies (or binding domains, e.g., VH, VL, CDR3) that bind to TSLP and/or SCF, as well as bispecific constructs comprising such antibodies.
  • the term is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibodies (or binding domain) or bispecific are free of other nucleotide sequences encoding the antibodies (or binding domain) or bispecific , e.g., other sequences which may naturally flank the nucleic acid in human genomic DNA.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).
  • nucleic acid molecules of the present invention while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures thereof may be mutated, in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired.
  • DNA sequences substantially identical to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).
  • a nucleic acid is “operably linked” or “operatively linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • operably linked indicates that the sequences are capable of effecting switch recombination.
  • nucleic acid molecules encoding the antibodies (or binding domains) or bispecific constructs described herein are also provided, as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors.
  • a nucleic acid molecule coding for any of the antibodies (or binding domains) or bispecific constructs described herein is provided.
  • the nucleic acid molecule is in the form of an expression vector.
  • the nucleic acid molecule is in the form of an expression vector which expresses the antibody (or binding domain) or bispecific construct when administered to a subject in vivo.
  • the nucleic acid molecule comprises a nucleotide sequence encoding an antibody variable region, wherein the antibody variable region comprises the amino acid sequence depicted in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, or an amino acid sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one or more of the aforementioned sequences).
  • the nucleic acid molecule comprises a nucleotide sequence as set forth in Table 11, or a nucleotide sequence at least 90% identical thereto e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one or more of the aforementioned sequences).
  • the nucleic acid molecule comprises a nucleotide sequence encoding heavy and light chain variable regions of an antibody, wherein the heavy and light chain variable regions comprise a combination of the amino acid sequences depicted in Table IB or Table 2B, or amino acids sequences at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical the aforementioned sequences).
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors”(or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors e.g. , replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • recombinant host cell (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • any of the antibodies (or binding domains) and/or bispecific constructs described herein can be administered in combination with an additional therapy, i.e., combined with other agents.
  • the term “coadministered” as used herein includes any or all of simultaneous, separate, or sequential administration of the antibodies (or binding domains) and/or bispecific constructs described herein with one or more additional therapeutic agent, including administration as part of a dosing regimen.
  • the combination therapy can include administering any of the antibodies (or binding domains) or bispecific constructs constructs described herein with at least one or more additional therapeutic agents selected from, but not limited to: immunosuppressants (for example, corticosteroids, non-steroidal glucocorticoid receptor agonists, leukotriene D4 antagonists, leukotriene B4 antagonists, A2A agonists, A2B antagonists, dopamine receptor agonists, pirfenidone, nintedanib, or avB6 antagonists), anti-IL-6?, cyclophosphamide, methotrexate, mycophenolate mofetil, autologous hematopoietic stem cell transplantation?, B cell depleting agents (rituximab, inebilizumab, belimumab), abatacept, anti-TGFb, anti-IL33, anti-IL25, DMARDs (diseasemodifying anti-rheumatic drugs),
  • Additional agents that delete or inhibit immunosuppressive activities for example, by immune cells (for example regulatory T-cells, NKT cells, macrophages, myeloid-derived suppressor cells, immature or suppressive dendritic cells), that may also be administered with the antibodies (or binding domains) or bispecific constructs described herein include, e.g., antibodies and small molecule drugs such as IDO inhibitors such as 1 methyl tryptophan or derivatives, as well as immunosuppressive agents such as rapamycin, cyclosporin and FK506; anti-TNF agents such as etanercept, adalimumab and infliximab; and steroids.
  • IDO inhibitors such as 1 methyl tryptophan or derivatives
  • immunosuppressive agents such as rapamycin, cyclosporin and FK506
  • anti-TNF agents such as etanercept, adalimumab and infliximab
  • steroids for example, by immune cells (for
  • Examples of specific natural and synthetic steroids include, for example: aldosterone, beclomethasone, betamethasone, budesonide, cloprednol, cortisone, corti vazol, deoxycortone, desonide, desoximetasone, dexamethasone, difluorocortolone, fluclorolone, flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluoromethoIone, flurandrenolone, fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone, methylprednisolone, paramethasone, prednisolone, prednisone, tixocortol and triamcinolone.
  • a disease or condition associated with the expression and/or activity of TSLP and/or SCF by administering the antibodies (or binding domains) or bispecific constructs , or compositions described herein, to a patient in need thereof.
  • the methods provided herein are used to treat a disease or condition associated with an immune response (e.g. immune cell migration, activation, and/or proliferation) via interaction (e.g., binding) of TSLP and/or SCF with its receptor (TSLPR and/or c-Kit, respectively) on immune cells.
  • an immune response e.g. immune cell migration, activation, and/or proliferation
  • TSLPR and/or c-Kit receptor
  • treat refers to therapeutic or preventative measures described herein.
  • the methods of “treatment” employ administration to a subject, in need of such treatment, an antibody (or binding domain), bispecific construct, , or composition as described herein, for example, a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • an effective dose or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect.
  • terapéuticaally effective dose is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient’s own immune system.
  • patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
  • immune cell includes cells that have hematopoietic origins and play a role in an immune response, for example, lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • lymphocytes such as B cells and T cells
  • natural killer cells such as myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • immune response means the action of immune cells (such as lymphocytes, antigen presenting cells, phagocytes or granulocytes) and soluble macromolecules (including antibodies, cytokines and complements) produced by immune cells or liver, which leads to selective damage, destruction or removal from human body of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of an autoimmune or pathological inflammation.
  • antigen-specific T cell response refers to an immune response produced by T cells when the T cells are stimulated by an antigen specific to the T cells.
  • Non-limiting examples of responses produced by T cells in response to antigen-specific stimulation include proliferation of T cells and production of cytokines (e.g., IL-2).
  • the present disclosure provides methods for blocking TSLP and/or SCF binding to its receptor (i.e., TLSPR and c-Kit, respectively) in a subject comprising administering to the subject any one of the antibodies (or binding domains), bispecific constructs, , or compositions described herein, in an amount effective to block TSLP and/or SCF binding to its receptor.
  • its receptor i.e., TLSPR and c-Kit, respectively
  • blocking TSLP and/or SCF binding and “inhibiting binding of TSLP and/or SCF” (e.g. , referring to TLP binding to TLSPR and/or SCF binding to c-Kit) are used interchangeably and are intended to include any measurable decrease in the binding between the ligand (TSLP or SCF) and its receptor (TSLPR or c-Kit), e.g. , the blocking of TSLP and/or SCF binding to TSLPR and/or c-Kit by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
  • Also provided herein are methods for inhibiting activation of immune cells in a subject, as well as reducing or preventing the accumulation of immune cells within organs or tissues, in a subject comprising administering to the subject any one of the antibodies (or binding domains), bispecific constructs, , or compositions described herein, in an amount effective to inhibit activation of immune cells or reduce the accumulation of immune cells within organs or tissues in a subject.
  • the terms “inhibiting activation of immune cells” and “reducing the accumulation of immune cells” are used interchangeably and are intended to include any measurable decrease in the amount of immune cells and/or signaling between immune cells within tissue or organs, e.g., the reduction of immune cell activation and/or accumulation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
  • the reduction of immune cell activation and/or accumulation results in the inhibition or blocking of (a) TSLP-induced activation and/or proliferation of mast cells, DC, and/or NKT cells, (b) TSLP-induced osteoprotegerin (OPG) secretion, (c) TSLP-induced secretion of Th2 cytokines (such as TARC, CCL22, IL-4, IL- 13 or IL-5), and / or (d) SCF-induced secretion of mast cells, eosinophils, type 2 innate lymphoid (ILC2) cells, and/or type 3 innate lymphoid (1LC3) cells.
  • OPG osteoprotegerin
  • autoimmune diseases e.g., autoimmune diseases, cardiovascular diseases, gastrointestinal diseases, lung diseases, metabolic diseases (such as Type 2 diabetes), neurodegenerative diseases (such as Parkinson’s disease), certain types of cancer (such as colon cancer) and mental illnesses (such as depression), as well as allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma, obstructive airways disease, chronic obstructive pulmonary disease, and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorders, and rhino-conjunctivitis.
  • diseases and disorders that involve inflammation, e.g., autoimmune diseases, cardiovascular diseases, gastrointestinal diseases, lung diseases, metabolic diseases (such as Type 2 diabetes), neurodegenerative diseases (such as Parkinson’s disease), certain types of cancer (such as colon cancer) and mental illnesses (such as depression), as well as allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 responses, a
  • fibrotic diseases and maladies associated with tissue remodeling include idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, sclerodoma, inflammation, liver cirrhosis, renal fibrosis, parenchymal fibrosis, endomyocardial fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, fibrous histiocytoma, fibrothorax, hepatic fibrosis, fibromyalgia, gingival fibrosis, or radiation- induced fibrosis.
  • tissue remodeling e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, sclerodoma, inflammation,
  • an inflammatory disease or disorder e.g., autoimmune diseases, cardiovascular diseases, gastrointestinal diseases, lung diseases, metabolic diseases (such as Type 2 diabetes), neurodegenerative diseases (such as Parkinson’s disease), certain types of cancer (such as colon cancer) and mental illnesses (such as depression)
  • the terms “inhibits inflammation” and “reduces inflammation” e.g., referring to body tissues, i.e., muscle tissue, epithelial tissue, connective tissue, and nervous tissue) are used interchangeably and are intended to include any measurable decrease in the response of body tissues to harmful stimuli (such as pathogens, damaged cells, or irritants) involving immune cells, blood vessels, and molecular mediators, e.g., the reduction of inflammation of a body tissue by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
  • harmful stimuli such as pathogens, damaged cells, or irritants
  • methods of reducing inflammation comprise contacting body tissue with any one of the antibodies (or binding domains), bispecific constructs, , or the compositions of described herein.
  • Reducing inflammation can comprise, for example, inhibiting binding of human TSLP to human TSLP receptor (TSLP-R), inhibiting binding of SCF Binding to c-Kit, inhibiting BaF3 Cell Proliferation, inhibiting TARC induction in Human Dendritic cells, inhibiting c-Kit phosphorylation in human CHO- KIT cells, inhibiting human mast cell degranulation, and/or inhibiting M-07e cell proliferation.
  • TSLP-R human TSLP receptor
  • methods of for treating a condition or disease in a subject comprising administering to the subject any one of the antibodies (or binding domains), bispecific constructs, , or the compositions described herein, in an amount effective to treat the condition or disease.
  • the subject can be, for example, one who suffers from a condition or disease in which reduction of inflammation is desired.
  • the condition or disease is associated with immune cell migration, activation, and/or proliferation via interaction (e.g., binding) of TSLP and/or SCF with is receptor (TSLPR and/or c-Kit, respectively) on immune cells, such as disorders of the immune system, allergic inflammation, allergic airway inflammation, DC -mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma, obstructive airways disease, chronic obstructive pulmonary disease, and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorders, and rhino-conjunctivitis.
  • fibrotic diseases and maladies associated with tissue remodeling include idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, sclerodoma, inflammation, liver cirrhosis, renal fibrosis, parenchymal fibrosis, endomyocardial fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, fibrous histiocytoma, fibrothorax, hepatic fibrosis, fibromyalgia, gingival fibrosis, or radiation- induced fibrosis.
  • tissue remodeling e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, sclerodoma, inflammation,
  • methods for treating a condition or disease in a subject comprising administering to the subject any one of the anti- TSLP antibodies, or antigen binding fragments thereof, described herein in combination with any one of the anti-SCF antibodies, or antigen binding fragments thereof, described herein.
  • any one of the anti-TSLP antibodies, or antigen binding fragments thereof, according to those listing Tables 1A or IB can be combined with any one of the anti-SCF antibodies, or antigen binding fragments thereof, according to those listing Tables 2A or 2B for treating a condition or disease in a subject.
  • an antibody (or binding domain), bispecific construct, , or composition described herein is administered in combination with one or more additional therapeutics selected from, but not limited to: immunosuppressants (for example, corticosteroids, non-steroidal glucocorticoid receptor agonists, leukotriene D4 antagonists, leukotriene B4 antagonists, A2A agonists, A2B antagonists, dopamine receptor agonists, pirfenidone, nintedanib, or avB6 antagonists), bronchodilators (for example, P-2 adrenergic receptor agonists, muscarinic antagonists, short-acting 2 receptor agonists, long-acting 2 receptor agonists, short-acting anticholinergic drugs, methyl xanthine drugs, long-acting anticholinergic drugs), other cytokine or cytokine receptor antagonists or antibodies (for example, IL- 13 antagonists, IL-6 antagonists, antagonists of IL
  • kits comprising one or more anti-TSLP antibody (or binding domain), anti-SCF antibody (or binding domain), bispecific construct, or composition as described herein, optionally with instructions for use.
  • Kits may also include informative pamphlets, for example, pamphlets informing one how to use reagents to practice a method disclosed herein.
  • pamphlet includes any writing, marketing materials or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • Mouse anti-TSLP monoclonal antibodies were generated by immunizing BALB/c mice with a soluble human TSLP antigen.
  • the antigen was a soluble fusion protein comprising a TSLP protein with a HIS tag (AcroBiosystems®)) was mixed with MPL plus TDM adjuvant system (Sigma®). 5-20 micrograms soluble recombinant TSLP antigen in PBS were mixed 1 : 1 with the adjuvant. Mice were injected with 200 microliters of the prepared antigen into the peritoneal cavity approximately every 14-30 days. Animals that developed anti-TSLP titers were given an iv injection of 1-10 micrograms soluble recombinant TSLP antigen three to four days prior to fusion. Mouse spleens were harvested, and the isolated splenocytes used for hybridoma preparation.
  • the P3x63Ag8.653 murine myeloma cell line (ATCC® CRL 1580) was used for the fusions.
  • RPMI 1640 (Invitrogen®) containing 10% FBS was used to culture the myeloma cells.
  • Additional media supplements were added to the Hybridoma growth media, which included: up to 10% Hybridoma Cloning Supplement (Sigma®), 10% FBS (Sigma), L- glutamine (Gibco®) 0.1% gentamycin (Gibco), 2-mercaptoethanol (Gibco), with HAT (Sigma; 1.0 x 10 4 M hypoxanthine, 4.0 x 10' 7 M aminopterin, 1.6 x 10' 5 M thymidine media.
  • Hybridoma cells were expanded and cell pellets were frozen for RNA isolation and sequencing.
  • the Vn and VL coding regions of human mAbs were identified using RNA from the corresponding hybridomas.
  • RNA was reverse transcribed to cDNA, the V coding regions were amplified by PCR and the PCR product was sequenced, inserted into human IgGl vector, transiently expressed as chimeric antibodies, and purified by protein A column chromatography.
  • Antibody 1D10 was isolated.
  • Example 2 Generation of Humanized TSLP Antibodies A computer model of the parental heavy and light chain variable region domains (j.e. ,
  • VH and VL domains of antibody 1D10 from Example 1 was produced and used to guide the humanization process.
  • Parental VH and VL sequences were aligned with a panel of human germline sequences which had been filtered to select germline sequences that do not contain unwanted sequence liabilities, particularly N-linked glycosylation sites and free Cysteines. The closest matching germlines from two different VH and VL families were selected.
  • a humanization algorithm was then used to select CDR and framework amino acids to graft from the donor parental sequences onto the human acceptor germline sequence (Table 4).
  • Complementarity determining regions (CDRs) from the parent antibody were grafted onto the appropriate human frameworks and back mutations were introduced (Tables 4A and 4B; underlined amino acids are different from parental amino acids).
  • the 1D10 heavy chain variants were designated: 1D10-H1 (SEQ ID NO: 1), 1D10-H2 (SEQ ID NO: 5), 1D10-H3 (SEQ ID NO: 9) and 1D10-H4 (SEQ ID NO: 13).
  • the 1D10 light chain variants were designated: 1D10-L1 (SEQ ID NO: 17), 1D10-L2 (SEQ ID NO: 21), 1D10-L3 (SEQ ID NO: 25) and 1D10-L4 (SEQ ID NO: 29). Pairing of these variable domain sequences is shown in Table 4. The activities of these antibodies and sequences were investigated further as described below. Table 6: Heavy and Light Chain Pairings
  • Microtiter plates were coated with recombinant human TSLP-kappa (Celldex Therapeutics, Inc.®) in PBS, and then blocked with 5% bovine serum albumin in PBS. Protein A purified chimeric mAb 1D10, its humanized versions and an isotype control were added at various concentrations and incubated at 37°C. The plates were washed with PBS/Tween and then incubated with a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate, and analyzed at OD 450nm using a microtiter plate reader. Representative binding curves are shown in FIGs. 1-3.
  • TSLP-R human TSLP receptor
  • Microtiter plates were coated with recombinant human TSLP-R (AcroBiosystems) in PBS, and then blocked with 5% bovine serum albumin in PBS.
  • Protein A purified chimeric mAb ID 10 was preincubated for 20 minutes at room temperature with biotinylated recombinant human TSLP (AcroBiosystems), then added to the plate and incubated at 37°C.
  • the plates were washed with PBS/Tween and then incubated with strepavidin conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate, and analyzed at OD 450nm using a microtiter plate reader. Representative curves are shown in FIGs. 4-6 showing the ability of anti-TSLP human mAbs to block binding of human TSLP to human TSLP receptor (TSLP-R).
  • TSLP-R human TSLP receptor
  • BaF3 cells were transfected to express human TSLP and human IL-7Ra on their surface.
  • the cells were incubated with recombinant human TSLP (R&D Systems®) in the presence of either media, Protein A purified chimeric mAb 1D10, select humanized versions or an isotype control at 37°C, 6%CO2.
  • CellTiter Gio Promega® was added according to the kit instructions and luminescence as a result of cell proliferation was detected and quantitated on a Perkin Elmer Victor X4 luminometer. Representative curves are shown in FIG. 7 showing the ability of anti-TSLP human mAbs to inhibit BaF3 proliferation.
  • DCs Human dendritic cells
  • BioIVT® peripheral blood mononuclear cell leukopaks
  • MACS Cell separation Pan DC Enrichment Kit from Miltenyi Biotec®.
  • the cells were incubated overnight with recombinant human TSLP (R&D Systems®) in the presence of either media, Protein A purified chimeric mAb 1D10, select humanized versions or an isotype control at 37°C, 6%CO2.
  • Supernatants were harvested and TARC production was quantitated by ELISA (R&D Systems). Representative curves are shown in FIG. 8 showing the ability of anti-TSLP human mAbs to inhibit TARC induction in human DCs.
  • Binding affinity and binding kinetics of various human anti-TSLP antibodies were examined by bio-layer interferometry (BLI®) using an Octet® QK e instrument (Sartorius®) according to the manufacturer’s guidelines.
  • AHC Anti-Human Fc Capture
  • Binding was determined by exposing seven of the antibody loaded biosensors to analyte: either soluble human TSLP-HIS or soluble cynomolgus TSLP (AcroBiosciences). Affinity measurements were determined using 2-fold serial dilutions of analyte ranging from 25 to 0.4nM in dilution buffer at 30°C and lOOOrpm plate shake speed. Association of the antibody loaded biosensors in analyte wells was carried out for 300 seconds, the biosensors were then moved to dilution buffer wells for 900sec for dissociation measurements. Both association and dissociation steps were carried out at 30°C and lOOOrpm plate shake speed.
  • soluble cynomolgus TSLP-HIS (ACROBiosystems) was prepared in dilution buffer (lOmM PO4 + 150mM NaCl + Img/mL BSA + 0.05% Tween 20, pH 7.2) to 2.0 pg/mL and loaded on freshly hydrated and regenerated Anti-Penta-HIS (HIS IK) biosensors (Sartorius) for 300sec at 30°C and lOOOrpm plate shake speed. For one assay, eight biosensors were loaded with the same ligand.
  • Binding of cynomolgus TSLP was determined by exposing seven of the ligand loaded biosensors to analyte: purified Anti-TSLP antibodies. Affinity measurements were determined using 2-fold serial dilutions of analyte ranging from 50 to 3.1nM in dilution buffer. Association of the ligand loaded biosensors in analyte wells was carried out for 180 seconds, the biosensors were then moved to dilution buffer wells for 300 sec for dissociation measurements. Both association and dissociation steps were carried out at 30°C and lOOOrpm plate shake speed.
  • Octet BLI Analysis Software (Sartorius) was used in each case to derive kinetic parameters from the concentration series of analyte in dilution buffer binding to captured ligand.
  • the association and dissociation curves were fitted to a 1: 1 binding model using the data analysis software according to the manufacturer’ s guidelines.
  • mice were immunized and boosted with human SCF protein. Immune responses were tested by ELISA against human SCF protein.
  • Plasma cells from mice with high antibody titer were isolated using a CD 138 B cell enrichment kit, loaded onto a Beacon 14K chip, and cloned using a Beacon instrument from Berkeley Lights®. Bead-based screening on the Beacon chip was performed for human, cyno and mouse SCF specific IgG secreting B cells, and to identify single B cells with blocking activity to the ECD of the human KIT receptor. Single B cells were selected and exported into lysis buffer for single cell sequencing in 96 well plates.
  • VH and VL domains A computer model of the parental heavy and light chain variable region domains (i.e., VH and VL domains) of antibody mAbl2 from Example 8 was produced and used to guide the humanization process.
  • Parental VH and VL sequences were aligned with a panel of human germline sequences which had been filtered to select germline sequences that do not contain unwanted sequence liabilities, particularly N-linked glycosylation sites and free Cysteines. The closest matching germlines from two different VH and VL families were selected.
  • a humanization algorithm was then used to select CDR and framework amino acids to graft from the donor parental sequences onto the human acceptor germline sequence (Table 7).
  • Complementarity determining regions (CDRs) from the parent antibody were grafted onto the appropriate human frameworks and back mutations were introduced as necessary (Tables 7A and 7B; underlined amino acids are different from parental amino acids).
  • the mAbl2 heavy chain variants were designated: mAbl2-Hl (SEQ ID NO: 33), mAb!2-H2 (SEQ ID NO: 37), mAb!2-H3 (SEQ ID NO: 41) and mAbl2-H4 (SEQ ID NO: 45).
  • the mAb!2 light chain variants were designated: mAbl2-Ll (SEQ ID NO: 49), mAbl2-L2 (SEQ ID NO: 53), mAbl2-L3 (SEQ ID NO: 57) and mAbl2-L4 (SEQ ID NO: 61). Pairing of these variable domain sequences is shown in Table 9. The activities of these antibodies and sequences were investigated further as described below. Table 9: Heavy and Light Chain Pairings
  • Microtiter plates were coated with recombinant human SCF-HTS (Celldex) in PBS, and then blocked with 5% bovine serum albumin in PBS.
  • Protein A purified chimeric mAb, designated as mAbl2, its humanized versions and an isotype control were added at various concentrations and incubated at 37°C.
  • the plates were washed with PBS/Tween and then incubated with a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate, and analyzed at OD 450nm using a microtiter plate reader. Representative binding curves are shown in FIGs. 10 and 11.
  • Microtiter plates were coated with recombinant human c-Kit-Fc (Celldex) in PBS, and then blocked with 5% bovine serum albumin in PBS.
  • Protein A purified chimeric mAbl2, its humanized versions and an isotype control at various concentrations were pre-incubated for 20 minutes at room temperature with biotinylated recombinant human SCF (AcroBiosystems), then added to the plate and incubated at 37°C.
  • the plates were washed with PBS/Tween and then incubated with strepavidin conjugated to horseradish peroxidase at 37°C.
  • Binding affinity and binding kinetics of human anti-SCF antibodies were examined by bio-layer interferometry (BLI) using an Octet® QK e instrument (Sartorius) according to the manufacturer’s guidelines. Purified antibodies were captured on Anti-Human Fc Capture (AHC) biosensors (Sartorius). Sensors were preconditioned by running two association and regeneration cycles with an irrelevant HuIgGl antibody.
  • BBI bio-layer interferometry
  • AHC Anti-Human Fc Capture
  • Each anti-SCF antibody was prepared in dilution buffer (lOmM PO 4 + 150mM NaCl + Img/mL BSA + 0.05% Tween 20, pH 7.2) to 0.5pg/mE and loaded on freshly hydrated and preconditioned AHC biosensors for 300sec at 30°C and lOOOrpm plate shake speed to achieve a target response of 0.7nm. For one assay, eight biosensors were loaded with the same antibody.
  • Binding was determined by exposing seven of the antibody loaded biosensors to analyte: soluble human SCF-HIS or soluble cynomolgus SCF. Affinity measurements were determined using 2-fold serial dilutions of analyte ranging from 800 to 6.25nM in dilution buffer at 30°C and lOOOrpm plate shake speed. Association of the antibody loaded biosensors in analyte wells was carried out for 300 seconds, the biosensors were then moved to dilution buffer wells for 900 seconds for dissociation measurements.
  • Octet BEI Analysis Software system (Sartorius) was used in each case to derive kinetic parameters from the concentration series of analyte in dilution buffer binding to captured antibody.
  • the association and dissociation curves were fitted to a 1: 1 binding model using the data analysis software according to the manufacturer’s guidelines.
  • Example 13 Inhibition of c-Kit phosphorylation in human CHO-KIT cells
  • a standard MSD plate was coated with purified anti -human CD 117 (c-Kit) antibody. The plate was sealed and shaken at 500 rpm for 10 minutes, then incubated at 4°C overnight.
  • a 96-well tissue culture treated plate was seeded with 100,000 CHO cells overexpressing human c-Kit/well and incubated at 37°C/5% CO2 overnight. Diluted anti-SCF antibodies were added to appropriate wells and incubated for 2 hours at 37°C/5% CO2. The antibody coating was removed from the MSD plate, washed, then blocked with TBST/5% BSA for 1 hour at room temperature with shaking at 500 RPM.
  • Diluted recombinant human SCF was added to all wells, then incubated for 10 minutes at 37°C/5% CO2. Following incubation, cells were lysed in ice cold PBS/0.1% Triton X-100 plus phosphatase and protease inhibitors and shaken at 4°C for 5 minutes. The blocked MSD plate was washed with TBST and the cell lysates were applied to the MSD plate and shaken at 500 rpm for 1 hour at room temperature. SulfoTag-pY20 detection antibody was added to all wells and shaken at 500 rpm for 1 hour at room temperature. Results were read in lx Read Buffer using MSD Sector Plate Reader. Representative curves are shown in FIG. 15 showing the ability of anti-SCF human mAbs to inhibit c-KIT phosphorylation in human CHO-KIT cells.
  • Mature human mast cells were cytokine starved overnight at 37°C/5% CO2. The following day mast cells were washed, resuspended in warm HEPES buffer and transferred to 96-well tissue culture plate. Anti-SCF antibodies were diluted and incubated with human mast cells at 37°C in air for 1 hour. Next, human TgE was added to human mast cell/antihody mixture for 30 minutes at 37°C in air. Following IgE treatment, human SCF and goat antihuman IgE were diluted and added to appropriate wells to induce crosslinking and incubated with human mast cells for 30 minutes at 37°C in air.
  • M-07e cells were incubated with dilutions of anti-SCF antibodies or an isotype control at 37°C, 5%CO2 for 1 hour. Following incubation, recombinant human SCF (R&D Systems) were added to all wells and incubated at 37°C, 6%CO2 for 6 days. After 6 days, CellTiter Gio (Promega) was added according to the kit instructions and luminescence as a result of cell proliferation was detected and quantitated using a microtiter plate reader. Representative curves are shown in FIG. 17 showing the ability of anti-SCF human mAbs to inhibit M-07e cell proliferation.
  • Example 16 Epitope mapping
  • Microtiter plates were coated with recombinant human wild-type SCF-HIS (Celldex), or mutated versions of SCF in PBS, and then blocked with 5% bovine serum albumin in PBS. Protein A purified humanized mAbl2 were added at various concentrations and incubated at 37°C. The plates were washed with PBS/Tween and then incubated with a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate, and analyzed at OD 450nm using a microtiter plate reader. Representative binding curves are shown in FIGs. 18A and 18B. A three-dimensional drawing showing the binding of SCF to KIT-D1 including residue K100 is provided in FIG. 18C.
  • SCF 220 Mouse S1/S14 cells made to overexpress the membrane bound form of SCF, known as SCF 220 (ATCC) were incubated with diluted humanized mAh 12 or human KIT-ECD for 1 hour at 4°C. Cells were washed and incubated with goat-anti-human IgG Fc-specific polyclonal reagent conjugated to R-Phycoerythrin fluorophore for 1 hour at 4°C. Following incubation, cells were washed and fixed. Mean fluorescence intensity was read using the Bectin Dickonson® (BD) Accuri C6 Plus Personal Flow Cytometer instrument..
  • BD Bectin Dickonson®
  • Mouse S1/S14 cells made to overexpress the soluble SCF isoform known as SCF-248 (ATCC) were incubated with diluted humanized mAbl2 or human KIT-ECD for 1 hour at 4°C. Cells were washed and incubated with goat-anti-human IgG Fc-specific polyclonal reagent conjugated to R-Phycoerythrin fluorophore for 1 hour at 4°C. Following incubation, cells were washed and fixed. Mean fluorescence intensity was read using the BD Accuri C6 Plus Personal Flow Cytometer.
  • FIGs. 19A and 19 B Representative binding curves are shown in FIGs. 19A and 19 B showing humanized mAbl2 preferentially binds and blocks soluble SCF.
  • Example 18 Inhibition of c-Kit phosphorylation in human M-07e cells
  • Soluble and SCF 220 induced phosphorylation were assessed by ELISA.
  • Parental S1/S14 or S1/S14-SCF 220 cells were treated with mitomycin C for 2 hours, washed, and allowed to recover overnight at 37°C, 5%CO .
  • M-07e cells were washed and serum starved overnight at 37°C, 5%CO2.
  • Diluted mAbl2 was preincubated with the source of SCF used to stimulate M- 07e cells; either soluble SCF and parental S1/S14 cells or S1/S14-SCF 220 cells for 1 hour at 37°C, 5%CO2.
  • M-07e cells were added to S1/S14 cells and spun down at 1200 rpm for 5 minutes, followed by incubation at 37°C, 5%CO2for 20 minutes. Following incubation, media was removed, and cells lysed in 2x lysis buffer at 4°C for 10 minutes. Recombinant human c-Kit was used to coat a 96- well high binding plate, followed by blocking with 5% BSA. Cell lysates were added to c-Kit coated plate and incubated at room temperature. The plates were washed with PBS/Tween and then incubated with a HRP conjugated pY20 detection antibody.
  • Tetravalent bispecific antibody constructs were developed using a mutated fully human IgGl backbone for a TSLP monoclonal antibody sequence (1D10 VH1 -LI and 1D10 VH2-L1) and the scFv of the SCF monoclonal antibody genetically linked to the C -terminus of the 1D10 heavy chain through a linker (FIGs. 21A and 21B).
  • the humanized antibody scFv sequences used in the bispecifics were taken from mAbl2 VH1-L4, mAbl2 VH2-L4, and mAbl2 VH4-L3.
  • the Fc domain was mutated (234A, 235Q, 322Q, 252Y, 254T and 256E).
  • Bispecific constructs were expressed in CHO cell lines. Table 10 defines the constructs.
  • the 5.7 construct ((1D1O(VH2-L1) - mAbl2(VH4-L3)) was designated “CDX-622” and further analyzed as described in the following examples.
  • Microtiter plates were coated with recombinant human TSLP-kappa (Celldex) in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of the bispecific antibodies and an isotype control were added and incubated at 37°C. The plates were washed with PBS/Tween and then incubated with a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate, and analyzed at OD 450nm using a microtiter plate reader. Representative curves are shown in FIG. 22 showing the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to bind to human TSLP.
  • the bispecific constructs i.e., the IgG and scFv pairings shown in Table 10.
  • Microtiter plates were coated with recombinant human SCF-HIS (Celldex) in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of the bispecific antibodies and an isotype control were added and incubated at 37°C. The plates were washed with PBS/Tween and then incubated with a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate, and analyzed at OD 450nm using a microtiter plate reader. Representative curves are shown in FIG. 23 showing the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to bind to human SCF.
  • the bispecific constructs i.e., the IgG and scFv pairings shown in Table 10.
  • Microtiter plates were coated with recombinant human TSLP-kappa in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of the bispecific antibodies and an isotype control were allowed to bind to the TSLP before adding biotinylated human SCF
  • TSLP-R human TSLP receptor
  • Microtiter plates were coated with recombinant human TSLP-R (AcroBiosystems) in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of the bispecific antibodies and an isotype control were pre-incubated for 20 minutes at room temperature with biotinylated recombinant human TSLP (AcroBiosystems), then added to the plate and incubated at 37°C. The plates were washed with PBS/Tween and then incubated with strepavidin conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate, and analyzed at OD 450nm using a microtiter plate reader. Representative curves are shown in FIGs. 25A and 25B showing the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to block TSLP binding to its receptor, TSLP-R.
  • the bispecific constructs i.
  • BaF3 cells were transfected to express human TSLP and human IL-7Ra on their surface.
  • the cells were incubated with recombinant human TSLP (R&D Systems) in the presence of either media, dilutions of the bispecific antibodies or an isotype control at 37°C, 6%CC>2.
  • CellTiter Gio Promega was added according to the kit instructions and luminescence as a result of cell proliferation was detected and quantitated on a Perkin Elmer Victor X4® luminometer. Representative curves are shown in FIGs. 27A and 27B showing the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to inhibit BaF3 cell proliferation.
  • DCs Human dendritic cells
  • BioIVT peripheral blood mononuclear cell leukopaks
  • MACS Cell separation Pan DC Enrichment Kit from Miltenyi Biotec®.
  • the cells were incubated overnight with recombinant human TSLP (R&D Systems) in the presence of either media, dilutions of the bispecific antibodies or an isotype control at 37°C, 6%CO2- Supernatants were harvested and thymus- and activation- regulated chemokine (TARC) production was quantitated by ELISA (R&D Systems). Representative curves are shown in FIGs. 28A and 28B showing the ability of constructs 5.2 and 5.7 (CDX-622) to inhibit TARC release.
  • Binding affinity and binding kinetics of the purified bispecific antibodies were examined by bio-layer interferometry (BL1) using an Octet® QK e instrument (Sartorius) according to the manufacturer’s guidelines.
  • bispecific antibodies were captured on Anti-Human Fc Capture (AHC) (Sartorius).
  • AHC Anti-Human Fc Capture
  • Each bispecific antibody was prepared in dilution buffer (lOmM PO4 + 150mM NaCl+lmg/mL BSA + 0.05% Tween 20, pH 7.2) to LOpg/mL and loaded on freshly hydrated biosensors for 300sec at 30°C and lOOOrpm plate shake speed. For one assay, eight biosensors were loaded with the same antibody.
  • Binding of human TSLP was determined by exposing seven of the antibody loaded biosensors to analyte: soluble human TSLP-HIS (ACROBiosystems). Affinity measurements were determined using 2-fold serial dilutions of analyte ranging from 12.5 to 0.8nM in dilution buffer. Association of the antibody loaded biosensors in analyte wells was carried out for 300 seconds, the biosensors were then moved to dilution buffer wells for 1200 seconds for dissociation measurements. Both association and dissociation steps were carried out at 30°C and lOOOrpm plate shake speed.
  • soluble cynomolgus TSLP-HIS (ACROBiosystems) was prepared in dilution buffer (lOmM PO4 + 150mM NaCl + Img/mL BSA + 0.05% Tween 20, pH 7.2) to 2.0pg/mL and loaded on freshly hydrated Anti-Penta- HIS (HIS IK) biosensors (Sartorius) for 300 seconds at 30°C and lOOOrpm plate shake speed. For one assay, eight biosensors were loaded with the same antigen.
  • Binding of cynomolgus TSLP was determined by exposing seven of the ligand loaded biosensors to analyte, purified bispecific antibodies. Affinity measurements were determined using 2-fold serial dilutions of analyte ranging from 200 to 3.1nM in dilution buffer. Association of the ligand loaded biosensors in analyte wells was carried out for 180 seconds, the biosensors were then moved to dilution buffer wells for 300 seconds for dissociation measurements. Both association and dissociation steps were carried out at 30°C and lOOOrpm plate shake speed.
  • bispecific antibodies were captured on Anti-Human Fab-CHl 2nd Generation (FAB2G) biosensors (Sartorius). Each bispecific antibody was prepared in dilution buffer (lOmM PO4 + 150mM NaCl + Img/mL BSA + 0.05% Tween 20, pH 7.2) to l.Opg/mL and loaded on freshly hydrated biosensors for 300 seconds at 30°C and lOOOrpm plate shake speed. For one assay, eight biosensors were loaded with the same antibody.
  • FAB2G Anti-Human Fab-CHl 2nd Generation
  • Binding of human and cynomolgus SCF was determined by exposing seven of the antibody loaded biosensors to analyte: soluble human SCF-H1S or soluble cynomolgus SCF- HIS. Affinity measurements were determined using 2-fold serial dilutions of analyte ranging from 800 to 12.5 nM in dilution buffer. Association of the antibody loaded biosensors in analyte wells was carried out for 300 seconds, the biosensors were then moved to dilution buffer wells for 900 to 1200 seconds for dissociation measurements. Both association and dissociation steps were carried out at 30°C and lOOOrpm plate shake speed.
  • Octet BLI Analysis Software (Sartorius) was used in each case to derive kinetic parameters from the concentration series of analyte in dilution buffer binding to captured ligand.
  • the association and dissociation curves were fitted to a 1 : 1 binding model using the data analysis software according to the manufacturer’ s guidelines.
  • Example 28 Inhibition of c-Kit phosphorylation in human CHO-KIT cells
  • a standard MSD plate was coated with purified anti -human CD117 (c-Kit) antibody, sealed, shaken at 500 rpm for 10 minutes and incubated at 4°C overnight.
  • a 96-well tissue culture treated plate was seeded with 100,000 CHO cells overexpressing human c-Kit/well and incubated at 37°C/5% CO2 overnight.
  • Bispecific antibodies were diluted, then added to appropriate wells and incubated for 2 hours at 37°C/5% CO2.
  • the coating antibody was removed from the MSD plate, washed, then blocked with TBST/5% BSA for 1 hour at room temperature with shaking at 500 RPM. Dilute recombinant human SCF was added to all wells, then incubated for 10 minutes at 37°C/5% CO2.
  • Mature human mast cells were cytokine starved overnight at 37°C/5% CO2. The following day mast cells were washed, resuspended in warm HEPES buffer and transferred to 96-well tissue culture plate. Bispecific antibodies were diluted and incubated with human mast cells at 37°C in air for 1 hour. Next, human IgE was added to human mast cell/antibody mixture for 30 minutes at 37°C in air. Following IgE treatment, human SCF and goat antihuman IgE were diluted and added to appropriate wells to induce crosslinking and incubated with human mast cells for 30 minutes at 37°C in air.
  • M-07e cells were incubated with dilutions of bispecific antibodies at 37°C, 5%CO2for 1 hour. Following incubation, recombinant human SCF (R&D Systems) were added to all wells and incubated at 37°C, 6%CO2for 6 days. After 6 days, CellTiter Gio (Promega) was added according to the kit instructions and luminescence as a result of cell proliferation was detected and quantitated using a microtiter plate reader. Representative inhibition curves are shown in FIG. 32 showing the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to inhibit M-07e cell proliferation.
  • the bispecific constructs i.e., the IgG and scFv pairings shown in Table 10.
  • Example 31 Inhibition of KIT phosphorylation in M-07e cells stimulated with soluble SCF compared to membrane-associated SCF (SCF 222 )
  • M-07e cells were incubated with dilutions of bispecific antibody, CDX-622. Following incubation, soluble SCF and membrane-associated SCF (SCF 222 ) were added and incubated according to the kit instructions (CellTiter Gio; Promega). Luminescence as a result of cell proliferation was detected and quantitated using a microtiter plate reader. Representative inhibition data are shown in FIG. 33. As shown, CDX-622 blocks KIT phosphorylation in M-07e cells that have been stimulated with soluble SCF with greater potency than KIT stimulated with SCF 220 -expressing cells
  • mAbl2 was administered on Day 1 and on Day 8, at a dose level of 75 mg/kg/dose by a slow intravenous push. Additional skin biopsies were collected on 30 and day 57.
  • Nanostring RNA analysis of the biopsies was performed with a non-human primate immunology panel of genes along with selected mast cell and melanocyte genes.
  • FIGs. 34A and 34B shows the decrease in the expression of several selected genes associated with mast cell function, reflecting significantly reduced mast cell activity.
  • Example 33 Pilot Study Histology
  • Serum samples were collected from the monkeys over the course of the study and were assayed for the presence of mAbl2. Circulating levels of the monoclonal antibodies are shown in FIG. 38. Serum samples were also assayed for the presence of anti-drug antibodies (AD As). Results are shown in FIG. 39 (the red line indicates the cutpoint of the assay).
  • CDX-622 A pilot study of bispecific antibody, CDX-622, was conducted in cynomolgus macaques. Four cynomolgus macaques were administered 10 mg/kg of CDX-622 on Day 1 by a slow intravenous injection. Blood samples were taken as indicated for determination of circulating test article levels, anti-drug antibodies and clinical pathology. CDX-622 administration did not result in significant decreases in hematological parameters (see FIGs. 40A-F).
  • RNA from each biopsy was isolated and subjected to RNA quantitation by Nanostring analysis. Normalized mast cell specific gene counts are plotted as a function of baseline values and show mast cell depletion (FIGs. 41A-F). Housekeeping gene b-tubulin is shown as a control.
  • Example 38 Inhibition of TSLP Binding to TSLP-R In Vitro
  • Example 39 Inhibition of TSLP-Mediated Cell Proliferation In Vitro
  • Example 42 MCP-1 Induction with the combination of SCF and TSLP / Simultaneous Blockade of SCF and TSLP
  • CDX-622 the parental monoclonal antibodies and an isotype control were preincubated for 20 minutes at room temperature with either 12.5ng/mL recombinant human SCF (Peprotech), 50ng/mL recombinant human TSLP (R&D Systems) or the combination. The mixture was then added to LAD2 cells and incubated overnight. Supernatants were harvested and MCP-1 production was quantitated by ELISA (R&D Systems). Representative data (FIG. 48) shows that, while addition of anti-SCF or anti-TSLP mAbs inhibited cytokines release elicited by their respective targets, CDX-622 reduced their secretion to a greater extent.
  • DCs Human dendritic cells
  • BioIVT peripheral blood mononuclear cell leukopaks
  • MACS Cell separation Pan DC Enrichment Kit from Miltenyi Biotec.
  • the cells were incubated overnight with recombinant human TSLP (R&D Systems) in the presence of either media, antibodies or an isotype control at 37°C, 6% CO2.
  • Cells were harvested, washed and incubated with FITC labeled CD80 (BD Biosciences). After washing, the cell associated fluorescence was determined by analysis using a FACSCanto IITM instrument (BD Biosciences) according to manufacturer’s directions. Representative curves are shown in FIG. 49 showing CDX-622 potently inhibited TSLP-driven upregulation of dendritic cell CD80 cell surface expression.

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Abstract

L'invention concerne de nouveaux anticorps anti-TSLP et anti-SCF, et des domaines de liaison de ceux-ci, ainsi que des constructions bispécifiques comprenant de tels anticorps et domaines de liaison. L'invention concerne également des procédés de traitement de troubles associés à une réponse immunitaire (par exemple, la migration, l'activation et/ou la prolifération de cellules immunitaires) par l'intermédiaire d'une interaction (par exemple, la liaison) de TSLP et/ou SCF avec son récepteur (TSLPR et/ou c-Kit, respectivement) sur des cellules immunitaires (tels que des troubles du système immunitaire) par l'administration des anticorps (ou de fragments de liaison à l'antigène de ceux-ci), de constructions bispécifiques ou de compositions décrites dans la description à un patient en ayant besoin.
PCT/US2024/018068 2023-03-03 2024-03-01 Anticorps anti-facteur de cellule souche (scf) et anti-lymphopoïétine stromale thymique (tslp) et constructions bispécifiques WO2024186635A2 (fr)

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