US20210188965A1 - Polypeptides comprising immunoglobulin single variable domains targeting il-13 and tslp - Google Patents

Polypeptides comprising immunoglobulin single variable domains targeting il-13 and tslp Download PDF

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Publication number
US20210188965A1
US20210188965A1 US17/115,906 US202017115906A US2021188965A1 US 20210188965 A1 US20210188965 A1 US 20210188965A1 US 202017115906 A US202017115906 A US 202017115906A US 2021188965 A1 US2021188965 A1 US 2021188965A1
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amino acid
seq
acid sequence
isvd
difference
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Inventor
Heidi Rommelaere
Ann Brigé
Sigrid Cornelis
Bruno DOMBRECHT
Eric Lorent
Melanie Rieger
Timothy Soos
John Park
Bernd Weigle
Klaus Erb
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Ablynx NV
Ablynx NV
Boehringer Ingelheim International GmbH
Sanofi SA
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Ablynx NV
Sanofi SA
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Application filed by Ablynx NV, Sanofi SA filed Critical Ablynx NV
Priority to US17/208,046 priority patent/US11208476B2/en
Publication of US20210188965A1 publication Critical patent/US20210188965A1/en
Priority to US17/530,727 priority patent/US12384840B2/en
Priority to US17/530,850 priority patent/US12492248B2/en
Priority to US17/530,800 priority patent/US11840566B2/en
Assigned to N.V., ABLYNX reassignment N.V., ABLYNX ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BOEHRINGER INGELHEIM INTERNATIONAL GMBH
Assigned to BOEHRINGER INGELHEIM INTERNATIONAL GMBH reassignment BOEHRINGER INGELHEIM INTERNATIONAL GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BOEHRINGER INGELHEIM PHARMA GMBH & CO. KG
Assigned to SANOFI reassignment SANOFI ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SOOS, Timothy
Assigned to BOEHRINGER INGELHEIM PHARMA GMBH & CO. KG reassignment BOEHRINGER INGELHEIM PHARMA GMBH & CO. KG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ERB, KLAUS, PARK, JOHN, WEIGLE, BERND
Assigned to ABLYNX N.V. reassignment ABLYNX N.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LORENT, Eric, ROMMELAERE, HEIDI, BRIGE, ANN, CORNELIS, SIGRID, DOMBRECHT, BRUNO, RIEGER, Melanie
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present technology relates to polypeptides targeting IL-13 and TSLP. It also relates to nucleic acid molecules encoding the polypeptide and vectors comprising the nucleic acids, and to compositions comprising the polypeptide, nucleic acid or vector.
  • the present technology further relates to these products for use in a method of treating a subject suffering from an inflammatory disease. Moreover, the present technology relates to method of producing these products.
  • a cascade of immune responses mediated by the innate and adaptive arms of the immune system e.g., antigen recognition, antigen processing, antigen presentation, cytokine production, antibody production, target cell killing
  • Inflammatory diseases are often chronic and can even be life-threatening.
  • Allergic and atopic diseases such as asthma and atopic dermatitis are driven predominantly by type 2 immune responses and characterized by salient features of type 2 immunity such as high IgE production and eosinophilia.
  • Thymic stromal lymphopoietin (TSLP) and Interleukin-13 (IL-13) are soluble cytokine targets produced by stromal and/or immune cells (Ziegler & Artis, Nat Rev Immunol (2010) 11:289, Gieseck III et al., Nat Rev Immunol (2016) 18:62).
  • Human TSLP and IL-13 drive distinct, overlapping and synergistic aspects of type 2 immunity, type 2 inflammatory diseases such as asthma and atopic dermatitis as well as a broad array of immunological diseases.
  • TSLP thymic stromal lymphopoietin receptor
  • IL-7Ra IL-7R alpha chain
  • IL-13 signalling starts by binding to a heterodimeric receptor complex consisting of alpha IL-4 receptor (IL-4Ra) and alpha Interleukin-13 receptor (IL-13R1a).
  • IL-4Ra alpha IL-4 receptor
  • IL-13R1a alpha Interleukin-13 receptor
  • TSLP drives the maturation of dendritic cells, development and proliferation of mast cells, as well as activating other immune cells such as basophils and innate lymphoid cells (ILC2).
  • IL-13 exerts a range of immunopathologies such as epithelial barrier disruption, mucus production from mucosal-epithelial surfaces, airway remodeling as well as the induction of eosinophil recruiting chemokines such as eotaxin.
  • immunopathologies such as epithelial barrier disruption, mucus production from mucosal-epithelial surfaces, airway remodeling as well as the induction of eosinophil recruiting chemokines such as eotaxin.
  • Dual targeting of TSLP and IL-13 with a single agent has the potential to confer efficacy in both low-type 2 and high-type 2 asthma as well as atopic dermatitis, with the potential to confer efficacy in sub-populations within these indications where a single monospecific agent therapy may not be fully efficacious. Accordingly, there is still an unmet medical need for the treatment of type 2 inflammatory diseases such as asthma and atopic dermatitis that is not only more efficacious but also conveniently applicable to the patient.
  • Such therapy may comprise targeting multiple disease factors, such as IL-13 and TSLP.
  • Targeting multiple disease factors may be achieved for example by co-administration or combinatorial use of two separate biologicals, e.g. antibodies binding to different therapeutic targets.
  • co-administration or combinatorial use of separate biologicals can be challenging, both from a practical and a commercial point of view.
  • two injections of separate products result in a more inconvenient and more painful treatment regime to the patients which may negatively affect compliance.
  • it can be difficult or impossible to provide formulations that allow for acceptable viscosity at the required concentrations and suitable stability of both products.
  • co-administration and co-formulation requires production of two separate drugs which can increase overall costs.
  • Bispecific antibodies that are able to bind to two different antigens have been suggested as one strategy for addressing such limitations associated with co-administration or combinatorial use of separate biologicals, such as antibodies.
  • bispecific antibody constructs have been proposed in multiple formats.
  • bispecific antibody formats may involve the chemical conjugation of two antibodies or fragments thereof (Brennan, M, et al., Science, 1985. 229(4708): p. 81-83; Glennie, M. J., et al., J lmmunol, 1987. 139(7): p. 2367-2375).
  • bispecific antibody formats include, however, high viscosity at high concentration, making e.g. subcutaneous administration challenging, and in that each binding unit requires the interaction of two variable domains for specific and high affinity binding, comprising implications on polypeptide stability and efficiency of production.
  • Such bispecific antibody formats may also potentially lead to Chemistry, Manufacturing and Control (CMC) issues related to mispairing of the light chains or mispairing of the heavy chains.
  • CMC Chemistry, Manufacturing and Control
  • the present technology relates to a polypeptide targeting specifically IL-13 and TSLP at the same time leading to an increased efficiency of modulating a type 2 inflammatory response as compared to monospecific anti-IL-13 or anti-TSLP polypeptides in vitro.
  • the polypeptides are efficiently produced (e.g. in microbial hosts).
  • such polypeptides have limited reactivity to pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject before the first treatment with the antibody construct).
  • such polypeptides exhibit a half-life in the subject to be treated that is long enough such that consecutive treatments can be conveniently spaced apart.
  • the present technology provides a polypeptide comprising or consisting of at least one immunoglobulin single variable domain (ISVD) that specifically binds to IL-13.
  • the polypeptide of the present technology comprises or consists of at least two ISVDs that specifically bind to IL-13, wherein the two ISVDs are optionally linked via a peptidic linker.
  • the two ISVDs specifically binding to IL-13 are distinct ISVDs.
  • the polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
  • the binding unit can be an ISVD that binds to a (human) serum protein, such as human serum albumin.
  • the polypeptide of the present technology comprises or consists of at least one ISVD that specifically binds to TSLP.
  • the polypeptide of the present technology comprises or consists of at least two ISVDs that specifically bind to TSLP, wherein the two ISVDs are optionally linked via a peptidic linker.
  • the two ISVDs specifically binding to TSLP are distinct ISVDs.
  • the polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
  • the binding unit can be an ISVD that binds to a (human) serum protein, such as human serum albumin.
  • the polypeptide of the present technology comprises or consists of at least four ISVDs, wherein at least two ISVDs specifically bind to IL-13 and at least two ISVDs specifically bind to TSLP.
  • the at least two ISVDs specifically binding to IL-13 specifically bind to human IL-13 and the at least two ISVDs specifically binding to TSLP specifically bind to human TSLP.
  • the at least two ISVDs specifically binding to IL-13 are distinct ISVDs and the at least two ISVDs binding to TSLP are distinct ISVDs.
  • the polypeptide comprising or consisting of at least four ISVDs further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
  • the binding unit can be an ISVD that binds to a (human) serum protein, such as human serum albumin.
  • nucleic acid molecule capable of expressing the polypeptide of the present technology, a nucleic acid or vector comprising the nucleic acid, and a composition comprising the polypeptide, the nucleic acid or the vector.
  • the composition is a pharmaceutical composition.
  • the present technology provides the polypeptide, the composition comprising the polypeptide, or the composition comprising the nucleic acid or vector comprising the nucleotide sequence that encodes the polypeptide, for use as a medicament.
  • the polypeptide or composition is for use in the treatment of an inflammatory disease, such as a type 2 inflammatory disease.
  • the type 2 inflammatory disease is selected from atopic dermatitis and asthma.
  • an inflammatory disease such as a type 2 inflammatory disease
  • said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of the polypeptide or a composition according to the present technology.
  • the type 2 inflammatory disease is selected from atopic dermatitis and asthma.
  • the method further comprises administering one or more additional therapeutic agents.
  • the polypeptide or composition of the present technology in the preparation of a pharmaceutical composition for treating an inflammatory disease, such as a type 2 inflammatory disease.
  • an inflammatory disease such as a type 2 inflammatory disease.
  • the type 2 inflammatory disease is selected from atopic dermatitis and asthma.
  • the present technology provides the following embodiments:
  • Embodiment 1 A polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid comprising a nucleotide sequence that encodes the polypeptide, for use as a medicament, wherein the polypeptide comprises or consists of at least one immunoglobulin single variable domain (ISVD), wherein said ISVD comprises three complementarity determining regions (CDR1 to CDR3, respectively), and wherein the at least one ISVD comprises:
  • ISVD immunoglobulin single variable domain
  • Embodiment 2 The polypeptide or composition for use according to embodiment 1, wherein the at least one ISVD comprises:
  • Embodiment 3 The polypeptide or composition for use according to any of embodiments 1 or 2, wherein the amino acid sequence of the at least one ISVD comprises:
  • Embodiment 4 The polypeptide or composition for use according to any of embodiments 1 to 3, wherein said at least one ISVD comprises:
  • Embodiment 5 The polypeptide or composition for use according to embodiment 1, wherein the polypeptide comprises or consists of at least two ISVDs, wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least two ISVDs are optionally linked via one or more peptidic linkers, and wherein:
  • Embodiment 6 The polypeptide or composition for use according to embodiment 5, wherein:
  • Embodiment 7 The polypeptide or composition for use according to any of embodiments 5 or 6, wherein:
  • Embodiment 8 The polypeptide or composition for use according to any of embodiments 5 to 7, wherein:
  • ISVD comprises the amino acid sequence of SEQ ID NO: 2,
  • Embodiment 9 The polypeptide according to any of embodiments 5 to 8, wherein the polypeptide comprises or consists of:
  • Embodiment 10 The polypeptide or composition for use according to any of embodiments 1 or 5, wherein the polypeptide comprises or consists of at least four ISVDs, wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptidic linkers, and wherein:
  • Embodiment 11 The composition for use according to any one of embodiments 1 to 10, which is a pharmaceutical composition which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprises one or more further pharmaceutically active polypeptides and/or compounds.
  • Embodiment 12 The polypeptide or composition for use according to embodiment 10 or 11, wherein:
  • Embodiment 13 The polypeptide or composition for use according to any of embodiments 10 to 12, wherein:
  • Embodiment 14 The polypeptide or composition for use according to any of embodiments 10 to 13, wherein:
  • Embodiment 15 The polypeptide or composition for use according to any of embodiments 1 to 14, wherein said polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
  • Embodiment 16 The polypeptide or composition for use according to embodiment 15, in which said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life is chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.
  • Embodiment 17 The polypeptide or composition for use according to any one of embodiments 15 to 16, in which said one or more other binding units that provide the polypeptide with increased half-life is chosen from the group consisting of binding units that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).
  • serum albumin such as human serum albumin
  • serum immunoglobulin such as IgG
  • Embodiment 18 The polypeptide or composition for use according to embodiment 17, in which said binding unit that provides the polypeptide with increased half-life is an ISVD that can bind to human serum albumin.
  • Embodiment 19 The polypeptide or composition for use according to embodiment 18, wherein the ISVD binding to human serum albumin comprises
  • Embodiment 20 The polypeptide or composition for use according to any of embodiments 18 to 19, wherein the ISVD binding to human serum albumin comprises a
  • CDR1 that is the amino acid sequence of SEQ ID NO: 10
  • CDR2 that is the amino acid sequence of SEQ ID NO: 15
  • CDR3 that is the amino acid sequence of SEQ ID NO: 20.
  • Embodiment 21 The polypeptide or composition for use according to any of embodiments 18 to 20, wherein the amino acid sequence of said ISVD binding to human serum albumin comprises a sequence identity of more than 90% with SEQ ID NO: 5.
  • Embodiment 22 The polypeptide or composition for use according to any of embodiments 18 to 21, wherein said ISVD binding to human serum albumin comprises the amino acid sequence of SEQ ID NO: 5.
  • Embodiment 23 The polypeptide or composition for use according to any of embodiments 10 to 22, wherein the amino acid sequence of the polypeptide comprises a sequence identity of more than 90% with SEQ ID NO: 1.
  • Embodiment 24 The polypeptide or composition for use according to any of embodiments 10 to 23, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1.
  • Embodiment 25 The polypeptide or composition for use according to any of embodiments 1 to 24, for use in the treatment of an inflammatory disease, such as a type 2 inflammatory disease.
  • Embodiment 26 The polypeptide or composition for use according to embodiment 25, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.
  • Embodiment 27 A polypeptide that comprises or consists of at least one immunoglobulin single variable domain (ISVD), wherein said ISVD comprises three complementarity determining regions (CDR1 to CDR3, respectively); and wherein the at least one ISVD comprises:
  • Embodiment 28 The polypeptide according to embodiment 27, wherein the at least one ISVD comprises:
  • Embodiment 29 The polypeptide according to any of embodiments 27 or 28, wherein the amino acid sequence of the at least one ISVD comprises:
  • Embodiment 30 The polypeptide according to any of embodiments 27 to 29, wherein said at least one ISVD comprises:
  • Embodiment 31 The polypeptide according to embodiment 1 or 27, wherein the polypeptide comprises or consists of at least two ISVDs, wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least two ISVDs are optionally linked via one or more peptidic linkers, and wherein:
  • Embodiment 32 The polypeptide according to embodiment 31, wherein:
  • CDR2 that is the amino acid sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid sequence of SEQ ID NO: 21, and the second ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of SEQ ID NO: 19, or
  • Embodiment 33 The polypeptide according to any of embodiments 31 or 32, wherein:
  • Embodiment 34 The polypeptide according to any of embodiments 31 to 33, wherein:
  • Embodiment 35 The polypeptide according to any of embodiments 31 to 34, wherein the polypeptide comprises or consists of:
  • Embodiment 36 The polypeptide according to any of embodiments 27 or 31, wherein the polypeptide comprises or consists of at least four ISVDs, wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptidic linkers, and wherein:
  • Embodiment 37 The polypeptide according to embodiment 36, wherein:
  • Embodiment 38 The polypeptide according to any of embodiments 36 or 37, wherein:
  • Embodiment 39 The polypeptide according to any of embodiments 36 to 38, wherein:
  • Embodiment 40 The polypeptide according to any of embodiments 27 to 39, wherein said polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
  • Embodiment 41 The polypeptide according to embodiment 40, in which said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life is chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.
  • Embodiment 42 The polypeptide according to any one of embodiments 40 to 41, in which said one or more other binding units that provide the polypeptide with increased half-life is chosen from the group consisting of binding units that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).
  • serum albumin such as human serum albumin
  • serum immunoglobulin such as IgG
  • Embodiment 43 The polypeptide according to embodiment 42, in which said binding unit that provides the polypeptide with increased half-life is an ISVD that can bind to human serum albumin.
  • Embodiment 44 The polypeptide according to embodiment 43, wherein the ISVD binding to human serum albumin comprises
  • Embodiment 45 The polypeptide according to any of embodiments 43 to 44, wherein the ISVD binding to human serum albumin comprises a CDR1 that is the amino acid sequence of SEQ ID NO:10, a CDR2 that is the amino acid sequence of SEQ ID NO: 15 and a CDR3 that is the amino acid sequence of SEQ ID NO: 20.
  • Embodiment 46 The polypeptide according to any of embodiments 43 to 45, wherein the amino acid sequence of said ISVD binding to human serum albumin comprises a sequence identity of more than 90% with SEQ ID NO: 5.
  • Embodiment 47 The polypeptide according to any of embodiments 43 to 46, wherein said ISVD binding to human serum albumin comprises the amino acid sequence of SEQ ID NO: 5.
  • Embodiment 48 The polypeptide according to any of embodiments 36 to 47, wherein the amino acid sequence of the polypeptide comprises a sequence identity of more than 90% with SEQ ID NO: 1.
  • Embodiment 49 The polypeptide according to any of embodiments 36 to 48, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1.
  • Embodiment 50 A nucleic acid comprising a nucleotide sequence that encodes a polypeptide according to any of embodiments 27 to 49, or a polypeptide according to embodiments 36 to 49.
  • Embodiment 51 A host or host cell comprising a nucleic acid according to embodiment 50.
  • Embodiment 52 A method for producing a polypeptide according to any of embodiments 27 to 49, or a polypeptide according to embodiments 36 to 49, said method at least comprising the steps of:
  • Embodiment 53 A composition comprising at least one polypeptide according to any of embodiments 27 to 49, or at least one polypeptide according to embodiments 36 to 49, or a nucleic acid according to embodiment 50.
  • Embodiment 54 The composition according to embodiment 53, which is a pharmaceutical composition which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprises one or more further pharmaceutically active polypeptides and/or compounds.
  • Embodiment 55 A method of treating an inflammatory disease, such as a type 2 inflammatory disease, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of a polypeptide according to any of embodiments 27 to 49, or according to embodiments 36 to 49, or a composition according to any of embodiments 53 to 54.
  • Embodiment 56 The method according to embodiment 55, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.
  • Embodiment 57 Use of a polypeptide according to any of embodiments 27 to 49, or according to embodiments 36 to 49, or a composition according to any of embodiments 53 to 54, in the preparation of a pharmaceutical composition for treating an inflammatory disease, such as a type 2 inflammatory disease.
  • an inflammatory disease such as a type 2 inflammatory disease.
  • Embodiment 58 Use of the polypeptide or a composition according to embodiments 57, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.
  • FIG. 1 Sensorgram showing simultaneous binding of IL13 and HSA to F027400161 captured via TSLP.
  • FIG. 2 Inhibition of human, cyno and rhesus IL13 in the eotaxin release assay by V HH F027400161 and the reference compounds anti-hIL-13 mAb1 and anti-hIL-13 mAb2.
  • FIG. 3 Inhibition of human and cyno IL13 in the SEAP reporter assay by F027400161 and the reference compounds anti-hIL-13 mAb1 and anti-hIL-13 mAb2.
  • FIG. 4 Inhibition of human and cyno TSLP induced BaF3 proliferation by F027400161 and the reference compound anti-hTSLP mAb1.
  • IRR00096 is a negative control Nb.
  • FIG. 5 Box plot showing the binding of pre-existing antibodies present in 96 human serum samples to F027400161, 163 and 164 compared to control F027301186.
  • FIG. 6 Dose-response inhibition profiles of F-27400161 (also referred to as F027400161) and comparator antibody anti-hTSLP reference mAb1 on TSLP-induced CCL17 response in human DCs.
  • Enriched human DCs were treated with 4 ng/mL of TSLP and incubated with 8 concentrations of the ISVD construct and anti-hTSLP reference mAb1 for 36 hours.
  • CCL17 concentration in freshly collected supernatant was measured by ELISA.
  • IC 50 values were calculated by nonlinear regression (log inhibitor vs responses—variable slope four parameters lease squares fit) in Graphpad Prism 8.0 with no constraints. Data are represented as mean ⁇ standard error of mean (SEM) of all 8 donors combined from 8 individual experiments.
  • FIG. 7 Dose inhibition responses of F-27400161 (also referred to as F027400161) and comparators Anti-hIL-13 reference mAb1 and anti-hTSLP reference mAb1 on 0.5 ng/mL IL-13 and TSLP-induced synergistic production of CCL17 in human PBMCs. Healthy donor PBMCs were stimulated with 0.5 ng/mL of recombinant IL-13+TSLP and incubated with 10 doses of the ISVD construct (Nb) and comparators for 20 hours. CCL17 concentration in the cell culture supernatant was measured by MSD V-Plex kit.
  • IC 50 values were calculated by nonlinear regression (log inhibitor vs responses—variable slope four parameters lease squares fit) in Graphpad Prism 8.0 with no constraints. Data are represented as mean ⁇ standard error of mean (SEM) of all donors combined from 8 individual experiments.
  • FIG. 8 Dose inhibition responses of F-27400161 (also referred to as F027400161) and comparator antibodies anti-hlL-13 reference mAb1 and anti-hTSLP reference mAb1 on 5 ng/mL IL-13 and TSLP-induced synergistic production of CCL17 in human PBMCs. Healthy donor PBMCs were stimulated with 5 ng/mL of recombinant IL-13+TSLP and incubated with 10 doses of the ISVD construct (Nb) and comparator antibodies for 20 hours. CCL17 concentration in freshly collected supernatant was measured by MSD V-Plex kit.
  • IC 50 values were calculated by nonlinear regression (log inhibitor vs responses—variable slope four parameters lease squares fit) in Graphpad Prism 8.0 with no constraints. Data are represented as mean ⁇ standard error of mean (SEM) of all donors combined from 8 individual experiments.
  • FIG. 9 Inhibition profiles of F-27400161 (also referred to as F027400161) and comparator antibodies anti-hIL-13 reference mAb1 and ant-hTSLP reference mAb1 on allergen Der P-induced IL-5, CCL17, and CCL26 production by human PBMCs in a triculture assay.
  • Normal donor PBMCs cocultured with MRCS fibroblasts and A549 epithelial cells were stimulated with 3 mg/mL of Der P, and incubated with 11.1 nM of the ISVD, anti-hIL-13 reference mAb1, or anti-hTSLP reference mAb1 in a 24-well plate for 6 days in a 37° C. cell-culture incubator.
  • IL-5, CCL17, and CCL26 concentration in freshly collected supernatant was measured by Human Magnetic Luminex Assays. Percentage of inhibition were calculated relative to unstimulated (min) and stimulated (max) control samples which did not receive either ISVD polypeptides or antibodies. All calculations were performed using GraphPad Prism 8.0. Data are represented as mean ⁇ standard error of mean (SEM) of all donors combined from 3 independent experiments.
  • FIG. 10 F027400161 significantly reduced detectable levels of human TSLP in the plasma of NSG-SGM3 mice.
  • human plasma TSLP levels were reduced with the 0.1 mg/kg F027400161 dose (45.772 pg/ml) and the 10 mg/kg F027400161 dose (0.072 pg/ml), demonstrating that the TSLP arm of F027400161 can bind to human TSLP in the plasma of humanized NSG-SGM3 mice.
  • FIG. 11 F027400161 significantly reduced detectable levels of human IL-13 in the plasma of NSG-SGM3 mice.
  • human IL-13 levels were reduced with the 0.01 mg/kg F027400161 dose (201.286 pg/ml), 0.05 mg/kg F027400161 dose (22.028 pg/ml), 0.1 mg/kg F027400161 dose (40.740 pg/ml), and with the 10 mg/kg F027400161 dose (1.777 pg/ml), demonstrating that the IL-13 arm of F27400161 can bind to human IL-13 in the plasma of humanized NSG-SGM3 mice.
  • FIGS. 12 and 13 F027400161 Significantly reduced mouse Retnla and Clca1 transcript expression in the lungs of NSG-SGM3 mice that received hydrodynamic delivery of hTSLP and hIL-4. Overexpression of human TSLP and IL-4 in the NSG-SGM3 mice induces the production of hIL-13 from human immune cells derived from the precursor CD34 + . Neutralization of the human IL-13 by F027400161 resulted in the inhibition of the mouse Retnla and Clca1 marker genes at the 10 mg/Kg dose.
  • FIG. 14 Schematic presentation of ISVD construct F027400161 showing from the N-terminus to the C-terminus the monovalent building blocks/ISVDs 4B02, 4B06, 501A02, 529F10, as well as the albumin binder ALB23002. Whereas 4B02 and 4B06 are connected via a 35GS linker, the remaining building block are linked via 9GS linkers.
  • the present technology aims at providing a novel type of drug for treating inflammatory diseases, such as atopic dermatitis and asthma.
  • the present technology relates to a polypeptide targeting IL-13 and TSLP at the same time leading to an increased efficiency of modulating a type 2 inflammatory response as compared to monospecific anti-IL-13 or anti-TSLP polypeptides in vitro and/or in vivo.
  • the polypeptides are efficiently produced (e.g. in microbial hosts).
  • such polypeptides could be shown to have limited reactivity to pre-existing antibodies in the subject to be treated (i.e. antibodies present in the subject before the first treatment with the antibody construct).
  • such polypeptides exhibit a half-life in the subject to be treated that is long enough such that consecutive treatments can be conveniently spaced apart.
  • polypeptide of the present technology is monospecific and monovalent.
  • a monospecific polypeptide of the present technology thus specifically binds to IL-13.
  • Another monospecific polypeptide of the present technology specifically binds to TSLP.
  • the term “monovalent” indicates the presence of only one binding units/building block that (specifically) targets a molecule, such as ISVDs.
  • the present technology provides a monospecific-monovalent polypeptide comprising or consisting of one ISVD that specifically binds to IL-13, which comprises three complementarity determining regions (CDR1 to CDR3, respectively).
  • the ISVD can be selected from an ISVD comprising:
  • the ISVD specifically binds to human IL-13.
  • the ISVD specifically binding to IL-13 is selected from an ISVD comprising:
  • the ISVD specifically binding to IL-13 is selected from an ISVD comprising:
  • the ISVD specifically binding to IL-13 is selected from an ISVD comprising the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 3.
  • the present technology provides a monospecific-monovalent polypeptide comprising or consisting of one ISVD that specifically binds to TSLP, which comprises three complementarity determining regions (CDR1 to CDR3, respectively).
  • the ISVD can be selected from an ISVD comprising:
  • the ISVD specifically binds to human TSLP.
  • the ISVD specifically binding to TSLP is selected from an ISVD comprising:
  • the ISVD specifically binding to TSLP is selected from an ISVD comprising:
  • the ISVD specifically binding to TSLP is selected from an ISVD comprising the amino acid sequence of SEQ ID NO: 4; or the amino acid sequence of SEQ ID NO: 6.
  • polypeptide of the present technology is monospecific and at least bivalent, but can also be e.g., trivalent, tetravalent, pentavalent, hexavalent, etc.
  • bivalent “trivalent”, “tetravalent”, “pentavalent”, or “hexavalent” all fall under the term “multivalent” and indicate the presence of two, three, four, five or six binding units/building blocks, respectively, such as ISVDs.
  • the present technology provides a monospecific-bivalent polypeptide comprising or consisting of two ISVDs that specifically bind to IL-13, wherein each of the two ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the two ISVDs are optionally linked via one or more peptidic linkers, and wherein:
  • the ISVDs are linked via one or more peptidic linkers. In one embodiment, the two ISVDs specifically bind human IL13.
  • the monospecific-bivalent polypeptide comprises or consists of two ISVDs that specifically bind to IL-13, wherein:
  • the monospecific-bivalent polypeptide comprises or consists of two ISVDs that specifically bind to IL-13, wherein:
  • the monospecific-bivalent polypeptide comprises or consists of two ISVDs that specifically bind to IL-13, wherein:
  • the present technology provides a monospecific-bivalent polypeptide comprising or consisting of two ISVDs that specifically bind to TSLP, wherein each of the two ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the two ISVDs are optionally linked via one or more peptidic linkers, and wherein:
  • the ISVDs are linked via one or more peptidic linkers. In one embodiment, the two ISVDs specifically bind human TSLP.
  • the monospecific-bivalent polypeptide comprises or consists of two ISVDs that specifically bind to TSLP, wherein:
  • the monospecific-bivalent polypeptide comprises or consists of two ISVDs that specifically bind to TSLP, wherein:
  • the monospecific-bivalent polypeptide comprises or consists of two ISVDs that specifically bind to TSLP, wherein:
  • first ISVD and second ISVD in this regard only indicate the relative position of the specifically recited ISVDs binding to IL-13/TSLP to each other, wherein the numbering is started from the N-terminus of the polypeptide of the present technology.
  • the “first ISVD” is thus closer to the N-terminus than the “second ISVD”.
  • the “second ISVD” is thus closer to the C-terminus than the “first ISVD”. Since the numbering is thus not absolute and only indicates the relative position of the two ISVDs it does not exclude the possibility that additional binding units/building blocks such as ISVDs binding to IL-13 and TSLP, respectively, can be present in the polypeptide.
  • the polypeptide can further comprise another ISVD binding to human serum albumin that can even be located between the “first ISVD” and “second ISVD” (such a construct is then referred to as multispecific as described in the subsequent section).
  • the (at least two) ISVDs of the monospecific-multivalent polypeptides, in particular of the above described monospecific-bivalent polypeptides are linked via peptidic linkers.
  • peptidic linkers to connect two or more (poly)peptides is well known in the art.
  • Exemplary peptidic linkers that can be used with the monospecific-multivalent polypeptides, in particular with the above described monospecific-bivalent polypeptides are shown in Table A-5.
  • One often used class of peptidic linkers is known as the “Gly-Ser” or “GS” linkers.
  • linkers that essentially consist of glycine (G) and serine (S) residues, and usually comprise one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 73) motif (for example, comprising the formula (Gly-Gly-Gly-Gly-Ser) n in which n may be 1, 2, 3, 4, 5, 6, 7 or more).
  • GGGGS GGGGS
  • SEQ ID NO: 73 a peptide motif
  • the ISVDs of the monospecific-multivalent polypeptides, in particular the monospecific-bivalent polypeptides of the present technology are linked via a linker set forth in Table A-5. In one embodiment, the (at least) two ISVDs are linked via a 35GS linker(s).
  • the monospecific-bivalent polypeptide comprises or consists of:
  • polypeptide of the present technology is at least bispecific, but can also be e.g., trispecific, tetraspecific, pentaspecific, etc.
  • polypeptide is at least bivalent, but can also be e.g., trivalent, tetravalent, pentavalent, hexavalent, etc.
  • bispecific “trispecific”, “tetraspecific”, “pentaspecific”, etc., all fall under the term “multispecific” and refer to binding to two, three, four, five, etc., different target molecules, respectively.
  • bivalent “trivalent”, “tetravalent”, “pentavalent”, “hexavalent”, etc. all fall under the term “multivalent” and indicate the presence of two, three, four, five, six, etc., binding units/building blocks, respectively, such as ISVDs.
  • the polypeptide may be bispecific-tetravalent, such as a polypeptide comprising or consisting of at least four ISVDs, wherein at least two ISVD specifically bind to IL-13 and at least two ISVDs specifically bind to TSLP.
  • IL-13 and TSLP are human IL-13 and human TSLP.
  • the polypeptide may be trispecific-pentavalent, such as a polypeptide comprising or consisting of five ISVDs, wherein two ISVDs specifically bind to human IL-13, two ISVDs specifically bind to human TSLP and one ISVD binds to human serum albumin.
  • Such a polypeptide may at the same time be biparatopic, for example if two ISVDs bind two different epitopes on human IL-13 or human TSLP.
  • the term “biparatopic” refers to binding to two different parts (e.g., epitopes) of the same target molecule.
  • the trispecific-pentavalent polypeptide of the present technology is e.g., ISVD construct F027400161, comprising two ISVDs specifically binding to human IL-13, two ISVDs specifically binding to human TSLP, one ISVD binding to human serum albumin, and which is biparatopic for both binding to IL-13 and TSLP.
  • the multispecific-multivalent polypeptide comprises or consists of at least four ISVDs, wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptidic linkers, and wherein:
  • the IL-13 and TSLP bound by said polypeptide is human IL-13 and human TSLP, respectively.
  • first ISVD “first ISVD”, “second ISVD”, “third ISVD”, etc., in this regard only indicate the relative position of the ISVDs to each other, wherein the numbering is started from the N-terminus of the polypeptide of the present technology.
  • the “first ISVD” is thus closer to the N-terminus than the “second ISVD”, whereas the “second ISVD” is closer to the N-terminus than the “third ISVD”. Accordingly, the ISVD arrangement is inverse when considered from the C-terminus.
  • the polypeptide can further comprise another ISVD binding to human serum albumin that can even be located between e.g. the “third ISVD” and “fourth ISVD”.
  • the present technology provides a bispecific-bivalent polypeptide comprising an ISVD that specifically binds to IL-13 or TSLP as described in detail for the monospecific-monovalent polypeptides above (section 5.1; “Monospecific-monovalent polypeptides”) and an ISVD binding to human serum albumin as described in detail below (section 5.4; “(In vivo) half-life extension”).
  • the present technology provides a bispecific-trivalent polypeptide comprising the monospecific-bivalent polypeptides above (section 5.1; “monospecific-bivalent polypeptides”) and an ISVD binding to human serum albumin as described in detail below (section 5.4; “(In vivo) half-life extension”).
  • the components, such as the ISVDs, of said multispecific-multivalent polypeptide may be linked to each other by one or more suitable linkers, such as peptidic linkers.
  • linkers to connect two or more (poly)peptides is well known in the art. Exemplary peptidic linkers are shown in Table A-5. One often used class of peptidic linker are known as the “Gly-Ser” or “GS” linkers. These are linkers that essentially consist of glycine (G) and serine (S) residues, and usually comprise one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 73) motif (for example, comprising the formula (Gly-Gly-Gly-Gly-Ser) n in which n may be 1, 2, 3, 4, 5, 6, 7 or more).
  • GGGGS SEQ ID NO: 73
  • 9GS linkers GGGGSGGGS, SEQ ID NO: 76
  • SEQ ID NO: 78 35GS linkers
  • the polypeptide comprising or consisting of at least four ISVDs comprises the at least two ISVDs specifically binding to IL-13 and at least two ISVDs specifically binding to TSLP.
  • the at least two ISVDs binding to IL-13 are linked via a 35GS linker
  • the at least two ISVDs specifically binding to TSLP are linked via a 9GS linker.
  • the at least two ISVDs specifically binding to TSLP are separated by an ISVD binding to albumin (9GS-Alb-9GS) (as described in section 5.4 “(In vivo) half-life extension” below). The inventors surprisingly found that such a configuration can increase the production yield of the polypeptide.
  • the polypeptide comprises or consists of the following, in the order starting from the N-terminus of the polypeptide: a first ISVD specifically binding to IL-13, a second ISVD specifically binding to IL-13, a first ISVD specifically binding to TSLP, an optional binding unit providing the polypeptide with increased half-life as defined herein, and a second ISVD specifically binding to TSLP.
  • the binding unit providing the polypeptide with increased half-life is an ISVD.
  • the polypeptide comprises or consists of the following, in the order starting from the N-terminus of the polypeptide: an ISVD specifically binding to IL-13, a linker, a second ISVD specifically binding to IL-13, a linker, a first ISVD specifically binding to TSLP, a linker, an ISVD binding to human serum albumin, a linker, and a second ISVD specifically binding to TSLP.
  • the linker between the two ISVDs binding to IL-13 is a 35GS linker, whereas the other linkers are 9GS linkers.
  • Such configurations of the polypeptide can provide for increased production yield, good CMC characteristics, such as sufficient solubility and biophysical stability, strong potencies with regard to modulation of a type 2 immune response as well as low binding to pre-existing antibodies.
  • the multispecific-multivalent polypeptide of the present technology exhibits reduced binding by pre-existing antibodies in human serum.
  • the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD.
  • the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD.
  • the polypeptide comprises an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C-terminal ISVD.
  • the C-terminus of an ISVD is normally VTVSS (SEQ ID NO: 138).
  • the polypeptide comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD.
  • the ISVD comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) in at least on ISVD.
  • the C-terminus of the ISVD is VKVSS (SEQ ID NO: 139), VQVSS (SEQ ID NO: 140), VTVKS (SEQ ID NO:166), VTVQS (SEQ ID NO:167), VKVKS (SEQ ID NO:168), VKVQS (SEQ ID NO:169), VQVKS (SEQ ID NO:170), or VQVQS (SEQ ID NO:171) such that after addition of a single alanine the C-terminus of the polypeptide for example comprises the sequence VTVSSA (SEQ ID NO: 141), VKVSSA (SEQ ID NO: 142), VQVSSA (SEQ ID NO: 143), VTVKSA (SEQ ID NO:172), VTVQSA (SEQ ID NO:173), VKVKSA (SEQ ID NO:174), VKVQSA (SEQ ID NO:175), VQVKSA (SEQ ID NO:176), or VQVQSA (SEQ ID NO:
  • the C-terminus comprises VTVSSA (SEQ ID NO: 141).
  • the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD, optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD and comprises an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C-terminal ISVD (such that the C-terminus of the polypeptide for example has the sequence VTVSSA (SEQ ID NO: 141), VKVSSA (SEQ ID NO: 142) or VQVSSA (SEQ ID NO: 143), such as VTVSSA (SEQ ID NO: 141)). See e.g. WO2012/175741 and WO2015/173325 for further information in this regard.
  • the multispecific-multivalent polypeptide of the present technology comprises or consists of an amino acid sequence comprising a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 1, wherein the CDRs of the five ISVDs are as defined in items A to E (or A′ to E′ if using the Kabat definition) set forth in sections “5.2 Immunoglobulin single variable domains” and “5.4 (In vivo) half-life extension” below, respectively, wherein in particular:
  • polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide of the present technology has at least half the binding affinity, or at least the same binding affinity, to human IL-13 and to human TSLP as compared to a polypeptide consisting of the amino acid of SEQ ID NO: 1 wherein the binding affinity is measured using the same method, such as Surface Plasmon Resonance (SPR).
  • SPR Surface Plasmon Resonance
  • immunoglobulin single variable domain defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets ISVDs apart from “conventional” immunoglobulins (e.g. monoclonal antibodies) or their fragments (such as Fab, Fab′, F(ab′) 2 , scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site.
  • conventional immunoglobulins e.g. monoclonal antibodies
  • fragments such as Fab, Fab′, F(ab′) 2 , scFv, di-scFv
  • V H heavy chain variable domain
  • V L light chain variable domain
  • CDRs complementarity determining regions
  • the antigen-binding domain of a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
  • a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
  • a Fab fragment, a F(ab′) 2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody would normally not be regarded as an ISVD, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a V H -V L pair of immunoglobulin domains, which jointly bind to an epitope of the respective anti
  • ISVDs are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain.
  • the binding site of an ISVD is formed by a single V H , a single V HH or single V L domain.
  • the single variable domain may be a light chain variable domain sequence (e.g., a V L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a V H -sequence or V HH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).
  • a light chain variable domain sequence e.g., a V L -sequence
  • a heavy chain variable domain sequence e.g., a V H -sequence or V HH sequence
  • An ISVD can for example be a heavy chain ISVD, such as a V H , V HH , including a camelized V H or humanized V HH . In one embodiment, it is a V HH , including a camelized V H or humanized V HH .
  • Heavy chain ISVDs can be derived from a conventional four-chain antibody or from a heavy chain antibody.
  • the ISVD may be a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a “dAb” or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody® (as defined herein, and including but not limited to a V HH ); other single variable domains, or any suitable fragment of any one thereof.
  • the ISVD may be a Nanobody® (such as a V HH , including a humanized V HH or camelized V H ) or a suitable fragment thereof.
  • Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V.
  • V HH domains also known as V HH S, V HH antibody fragments, and V HH antibodies, have originally been described as the antigen binding immunoglobulin variable domain of “heavy chain antibodies” (i.e., of “antibodies devoid of light chains”; Hamers-Casterman et al. Nature 363: 446-448, 1993).
  • V HH domain has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “V H domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “V L domains”).
  • V H domains heavy chain variable domains that are present in conventional 4-chain antibodies
  • V L domains light chain variable domains that are present in conventional 4-chain antibodies
  • immunoglobulins typically involve the immunization of experimental animals, fusion of immunoglobulin producing cells to create hybridomas and screening for the desired specificities.
  • immunoglobulins can be generated by screening of na ⁇ ve or synthetic libraries e.g. by phage display.
  • Antigens can be purified from natural sources, or in the course of recombinant production.
  • Immunization and/or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.
  • the present technology may use immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences.
  • the present technology also includes fully human, humanized or chimeric sequences.
  • the present technology comprises camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g. camelized dAb as described by Ward et al (see for example WO 94/04678 and Davies and Riechmann (1994 and 1996)).
  • the present technology also uses fused immunoglobulin sequences, e.g.
  • a multivalent and/or multispecific construct for multivalent and multispecific polypeptides containing one or more V HH domains and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001, as well as to for example WO 96/34103 and WO 99/23221), and immunoglobulin sequences comprising tags or other functional moieties, e.g. toxins, labels, radiochemicals, etc., which are derivable from the immunoglobulin sequences of the present technology.
  • a “humanized V HH ” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V HH domain, but that has been “humanized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring V HH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a V H domain from a conventional 4-chain antibody from a human being (e.g. indicated above).
  • This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art (e.g. WO 2008/020079).
  • humanized V HH s can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.
  • a “camelized V H ” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V H domain, but that has been “camelized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring V H domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V HH domain of a heavy chain antibody.
  • This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art (e.g. WO 2008/020079).
  • the V H sequence that is used as a starting material or starting point for generating or designing the camelized V H is a V H sequence from a mammal, or the V H sequence of a human being, such as a V H 3 sequence.
  • camelized V H can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V H domain as a starting material.
  • the structure of an ISVD sequence can be considered to be comprised of four framework regions (“FRs”), which are referred to in the art and herein as “Framework region 1” (“FR1”); as “Framework region 2” (“FR2”); as “Framework region 3” (“FR3”); and as “Framework region 4” (“FR4”), respectively; which framework regions are interrupted by three complementary determining regions (“CDRs”), which are referred to in the art and herein as “Complementarity Determining Region 1” (“CDR1”); as “Complementarity Determining Region 2” (“CDR2”); and as “Complementarity Determining Region 3” (“CDR3”), respectively.
  • CDRs complementary determining regions
  • the amino acid residues of an ISVD can be numbered according to the general numbering for V H domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to V HH domains from Camelids in the article of Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for example FIG. 2 of this publication).
  • the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).
  • the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
  • the total number of amino acid residues in a V H domain and a V HH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.
  • FR1 comprises the amino acid residues at positions 1-25
  • CDR1 comprises the amino acid residues at positions 26-35
  • FR2 comprises the amino acids at positions 36-49
  • CDR2 comprises the amino acid residues at positions 50-58
  • FR3 comprises the amino acid residues at positions 59-94
  • CDR3 comprises the amino acid residues at positions 95-102
  • FR4 comprises the amino acid residues at positions 103-113.
  • CDR regions may also be done according to different methods.
  • FR1 of an ISVD comprises the amino acid residues at positions 1-30
  • CDR1 of an ISVD comprises the amino acid residues at positions 31-35
  • FR2 of an ISVD comprises the amino acids at positions 36-49
  • CDR2 of an ISVD comprises the amino acid residues at positions 50-65
  • FR3 of an ISVD comprises the amino acid residues at positions 66-94
  • CDR3 of an ISVD comprises the amino acid residues at positions 95-102
  • FR4 of an ISVD comprises the amino acid residues at positions 103-113.
  • the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.
  • the framework sequences are a suitable combination of immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization).
  • the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a V L -sequence) and/or from a heavy chain variable domain (e.g. a V H -sequence or V HH sequence).
  • the framework sequences are either framework sequences that have been derived from a V HH -sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences that have been camelized (as defined herein).
  • the framework sequences present in the ISVD sequence used in the present technology may contain one or more of hallmark residues (as defined herein), such that the ISVD sequence is a Nanobody®, such as a V HH , including a humanized V HH or camelized V H .
  • suitable fragments or combinations of fragments of any of the foregoing, such as fragments that contain one or more CDR sequences, suitably flanked by and/or linked via one or more framework sequences (for example, in the same order as these CDR's and framework sequences may occur in the full-sized immunoglobulin sequence from which the fragment has been derived).
  • the present technology is not limited as to the origin of the ISVD sequence (or of the nucleotide sequence used to express it), nor as to the way that the ISVD sequence or nucleotide sequence is (or has been) generated or obtained.
  • the ISVD sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences.
  • the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to “humanized” (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized V HH sequences), “camelized” (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.
  • “humanized” as defined herein
  • immunoglobulin sequences such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized V HH sequences
  • nucleotide sequences may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.
  • a suitable naturally occurring template e.g. DNA or RNA isolated from a cell
  • nucleotide sequences that have been isolated from a library and in particular, an expression library
  • nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence using any suitable technique known per
  • an ISVD may be a Nanobody® or a suitable fragment thereof.
  • V H 3 class i.e. Nanobodies with a high degree of sequence homology to human germline sequences of the V H 3 class such as DP-47, DP-51 or DP-29.
  • V H 4 class i.e. Nanobodies with a high degree of sequence homology to human germline sequences of the V H 4 class such as DP-78
  • Nanobodies in particular V HH sequences, including (partially) humanized V HH sequences and camelized V H sequences
  • V HH sequences including (partially) humanized V HH sequences and camelized V H sequences
  • a Nanobody can be defined as an immunoglobulin sequence with the (general) structure
  • Nanobody can be an immunoglobulin sequence with the (general) structure
  • Nanobody can be an immunoglobulin sequence with the (general) structure
  • one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-0 below.
  • sequences such as TERE (for example TEREL), TQRE (for example TQREL), KECE (for example KECEL or KECER), KQCE (for example KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREF or RQREW), QERE (for example QEREG), QQRE, (for example QQREW, QQREL or QQREF), KGRE (for example KGREG), KDRE (for example KDREV) are possible.
  • Some other possible, but less preferred sequences include for example DECKL and NVCEL. (4) With both GLEW at positions 44-47 and KERE or KQRE at positions 43-46.
  • positions 44-47 are GLEW, position 108 is always Q in (non-humanized) V HH sequences that also contain a W at 103.
  • the GLEW group also contains GLEW-like sequences at positions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and ELEW.
  • the present technology inter alio uses ISVDs that can bind to IL-13 or TSLP.
  • binding to a certain target molecule has the usual meaning in the art as understood in the context of antibodies and their respective antigens.
  • the multispecific-multivalent polypeptide of the present technology may comprise two or more ISVDs specifically binding to IL-13 and two or more ISVDs specifically binding to TSLP.
  • the polypeptide may comprise two ISVDs that specifically bind to IL-13 and two ISVDs that specifically bind to TSLP.
  • At least one ISVD can functionally block its target molecule.
  • targeting moieties can block the interaction between IL-13 and IL-13R ⁇ 1 (Interleukin 13 receptor, alpha 1) and/or the interaction between IL-13/IL-13R ⁇ 1 complex and IL-4R ⁇ (alpha interleukin-4 receptor), or can block the interaction between TSLP and TSLPR (TSLP receptor) and/or TSLP/TSLPR complex and IL-7R ⁇ (Interleukin-7 receptor subunit alpha).
  • the polypeptide of the present technology comprises at least two ISVDs that specifically bind to IL-13 and functionally block its interaction with IL-13R ⁇ 1 and/or the interaction between IL-13/IL-13R ⁇ 1 complex and IL-4R ⁇ , and two ISVDs that specifically bind to TSLP and functionally block its interaction with TSLPR and/or the interaction between TSLP/TSLPR complex and IL-7R ⁇ .
  • the ISVDs used in the present technology form part of a polypeptide of the present technology, which comprises or consists of at least four ISVDs, such that the polypeptide can specifically bind to IL-13 and TSLP.
  • the target molecules of the at least four ISVDs as used in the polypeptide of the present technology are IL-13 and TSLP.
  • Examples are mammalian IL-13 and TSLP.
  • human IL-13 Uniprot accession P35225
  • human TSLP Uniprot accession Q969D9
  • the versions from other species are also amenable to the present technology, for example IL-13 and TSLP from mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates, such as cynomolgus monkeys (also referred to herein as “cyno”), or camelids, such as llama or alpaca.
  • ISVDs specifically binding to IL-13 that can be used in the present technology are as described in the following items A and B:
  • Examples of such an ISVD that specifically binds to human IL-13 have one or more, or all, framework regions as indicated for construct 4B02 or 4B06, respectively, in Table A-2 (in addition to the CDRs as defined in the preceding items A and B, respectively).
  • it is an ISVD comprising or consisting of the full amino acid sequence of construct 4B02 or construct 4B06 (SEQ ID NOs: 2 and 3, respectively; see Table A-1 and A-2).
  • the amino acid sequence of the ISVD(s) specifically binding to human IL-13 may have a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 2 or 3 respectively, wherein the CDRs are as defined in the preceding item A or B, respectively.
  • the ISVD specifically binding to IL-13 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 3.
  • the ISVD has at least half the binding affinity, or at least the same binding affinity to human IL-13 as the construct 4B02 or 4B06 set forth in SEQ ID NO: 2 or 3, respectively, wherein the binding affinity is measured using the same method, such as SPR.
  • ISVDs specifically binding to TSLP that can be used in the present technology are as described in the following items C and D:
  • Examples of such an ISVD that specifically binds to human TSLP have one or more, or all, framework regions as indicated for construct 501A02 and 529F10, respectively, in Table A-2 (in addition to the CDRs as defined in the preceding items C and D).
  • it is an ISVD comprising or consisting of the full amino acid sequence of construct 501A02 or 529F10 (SEQ ID NOs: 4 or 6, see Table A-1 and A-2).
  • the amino acid sequence of an ISVD(s) specifically binding to human TSLP may have a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 4 or 6, respectively, wherein the CDRs are as defined in the preceding item C or D.
  • the ISVD binding to human TSLP comprises or consists of the amino acid sequence of SEQ ID NOs: 4 or 6.
  • the ISVD has at least half the binding affinity, or at least the same binding affinity to human TSLP as construct 501A02 or 529F10 set forth in SEQ ID NO: 4 and 6, respectively, wherein the binding affinity is measured using the same method, such as SPR.
  • each of the ISVDs as defined under items A to D above is comprised in the polypeptide of the present technology.
  • Such a polypeptide of the present technology comprising each of the ISVDs as defined under items A to D above has at least half the binding affinity, or at least the same binding affinity, to human IL-13 and to human TSLP as a polypeptide consisting of the amino acid of SEQ ID NO: 1, wherein the binding affinity is measured using the same method, such as SPR.
  • the SEQ ID NOs referred to in the above items A to D and item E below are based on the CDR definition according to the AbM definition (see Table A-2). It is noted that the SEQ ID NOs defining the same CDRs according to the Kabat definition (see Table A-2.1) can likewise be used in the above items A to D and item E below (see section 5.4 “(In vivo) half-life extension”).
  • ISVDs specifically binding to IL-13 or TSLP that can be used in the present technology are as described above using the AbM definition can be also described using the Kabat definition as set forth in items A′ to D′ below:
  • An ISVD that specifically binds to human IL-13 and comprises
  • An ISVD that specifically binds to human IL-13 and comprises
  • Examples of such an ISVD(s) that specifically binds to human IL-13 have one or more, or all, framework regions as indicated for construct 4B02 or 4B06, respectively, in Table A-2-1 (in addition to the CDRs as defined in the preceding items A′ and B′, respectively).
  • it is an ISVD comprising or consisting of the full amino acid sequence of construct 4B02 or construct 4B06 (SEQ ID NOs: 2 and 3, respectively; see Table A-1 and A-2-1).
  • Examples of such an ISVD(s) that specifically binds to human TSLP have one or more, or all, framework regions as indicated for construct 501A02 and 529F10, respectively, in Table A-2-1 (in addition to the CDRs as defined in the preceding items C′ and D′).
  • it is an ISVD comprising or consisting of the full amino acid sequence of construct 501A02 or 529F10 (SEQ ID NOs: 4 or 6, see Table A-1 and A-2-1).
  • the percentage of “sequence identity” between a first amino acid sequence and a second amino acid sequence may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence—compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (i.e. at a single position).
  • amino acid sequence with the greatest number of amino acid residues will be taken as the “first” amino acid sequence, and the other amino acid sequence will be taken as the “second” amino acid sequence.
  • amino acid difference refers to a deletion, insertion or substitution of a single amino acid residue vis-á-vis a reference sequence. In one embodiment, an “amino acid difference” is a substitution.
  • amino acid substitutions are conservative substitutions.
  • conservative substitutions are substitutions in which one amino acid within the following groups (a)-(e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
  • conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val;
  • Lys into Arg into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.
  • binding specifically refers to the number of different target molecules, such as antigens, from the same organism to which a particular binding unit, such as an ISVD, can bind with sufficiently high affinity (see below). “Specificity”, “binding specifically” or “specific binding” are used interchangeably herein with “selectivity”, “binding selectively” or “selective binding”. Binding units, such as ISVDs, specifically bind to their designated targets.
  • the specificity/selectivity of a binding unit can be determined based on affinity.
  • the affinity denotes the strength or stability of a molecular interaction.
  • the affinity is commonly given as by the KD, or dissociation constant, which has units of mol/liter (or M).
  • the affinity can also be expressed as an association constant, KA, which equals 1/KD and has units of (mol/liter) ⁇ 1 (or M ⁇ 1 ).
  • the affinity is a measure for the binding strength between a moiety and a binding site on the target molecule: the lower the value of the KD, the stronger the binding strength between a target molecule and a targeting moiety.
  • binding units used in the present technology will bind to their targets with a dissociation constant (KD) of 10 ⁇ 5 to 10 ⁇ 12 moles/liter or less, or 10 ⁇ 7 to 10 ⁇ 12 moles/liter or less, or 10 ⁇ 8 to 10 ⁇ 12 moles/liter (i.e. with an association constant (KA) of 10 5 to 10 12 liter/ moles or more, or 10 ⁇ to 10 12 liter/moles or more, or 10 8 to 10 12 liter/moles).
  • KD dissociation constant
  • KA association constant
  • Any KD value greater than 10 ⁇ 4 mol/liter (or any KA value lower than 10 4 liters/mol) is generally considered to indicate non-specific binding.
  • the KD for biological interactions such as the binding of immunoglobulin sequences to an antigen, which are considered specific are typically in the range of 10 ⁇ 5 moles/liter (10000 nM or 10 ⁇ M) to 10 ⁇ 12 moles/liter (0.001 nM or 1 pM) or less.
  • specific/selective binding may mean that—using the same measurement method, e.g. SPR—a binding unit (or polypeptide comprising the same) binds to IL13 and/or TSLP with a KD value of 10 ⁇ 5 to 10 ⁇ 12 moles/liter or less and binds to related cytokines with a KD value greater than 10 ⁇ 4 moles/liter.
  • a binding unit or polypeptide comprising the same
  • IL13 and/or TSLP with a KD value of 10 ⁇ 5 to 10 ⁇ 12 moles/liter or less and binds to related cytokines with a KD value greater than 10 ⁇ 4 moles/liter.
  • IL13 related targets are human IL4.
  • related cytokines for TSLP are human IL7.
  • At least two ISVDs comprised in the polypeptide binds to IL13 with a KD value of 10 ⁇ 5 to 10 ⁇ 12 moles/liter or less and binds to IL4 of the same species with a KD value greater than 10 ⁇ 4 moles/liter, and at least two ISVDs comprised in the polypeptide bind to TSLP with a KD value of 10 ⁇ 5 to 10 ⁇ 12 moles/liter or less and binds to human IL7 of the same species with a KD value greater than 10 ⁇ 4 moles/liter.
  • the polypeptide of the present technology has at least half the binding affinity, or at least the same binding affinity, to human IL13 and to human TSLP as compared to a polypeptide consisting of the amino acid of SEQ ID NO: 1, wherein the binding affinity is measured using the same method, such as SPR.
  • binding unit can also specifically bind to the analogous target from a different species.
  • specific binding to human IL13 does not exclude that the binding unit (or a polypeptide comprising the same) can also specifically bind to IL13 from cynomolgus monkeys.
  • specific binding to human TSLP does not exclude that the binding unit (or a polypeptide comprising the same) can also specifically bind to TSLP from cynomolgus monkeys (“cyno”).
  • Specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
  • Scatchard analysis and/or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
  • the dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned below. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more than 10 ⁇ 4 moles/liter or 10 ⁇ 3 moles/liter (e.g. of 10 ⁇ 2 moles/liter).
  • the affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see for example Ober et al. 2001, Intern. Immunology 13: 1551-1559).
  • SPR surface plasmon resonance
  • surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding k on , k off measurements and hence K D (or K A ) values.
  • bio-layer interferometry refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam).
  • reference beam an internal reference layer
  • signal beam a layer of immobilized protein on the biosensor tip
  • BLI can for example be performed using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).
  • affinities can be measured in Kinetic Exclusion Assay (KinExA) (see for example Drake et al. 2004, Anal. Biochem., 328: 35-43), using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA).
  • KinExA Kinetic Exclusion Assay
  • Equilibrated solutions of an antibody/antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).
  • the GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).
  • the polypeptide may further comprise one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased (in vivo) half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
  • In vivo half-life extension means, for example, that the polypeptide has an increased half-life in a mammal, such as a human subject, after administration.
  • Half-life can be expressed for example as t1/2beta.
  • the type of groups, residues, moieties or binding units is not generally restricted and may for example be chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.
  • said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life can be chosen from the group consisting of binding units that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG.
  • said one or more other binding units that provide the polypeptide with increased half-life is a binding unit that can bind to human serum albumin.
  • the binding unit is an ISVD.
  • WO 04/041865 describes Nanobodies® binding to serum albumin (and in particular against human serum albumin) that can be linked to other proteins (such as one or more other Nanobodies binding to a desired target) in order to increase the half-life of said protein.
  • Nanobodies® against (human) serum albumin. These Nanobodies® include the Nanobody® called Alb-1 (SEQ ID NO: 52 in WO 06/122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 06/122787). Again, these can be used to extend the half-life of therapeutic proteins and polypeptide and other therapeutic entities or moieties.
  • WO2012/175400 describes a further improved version of Alb-1, called Alb-23.
  • the polypeptide comprises a serum albumin binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 and Alb-23.
  • the serum albumin binding moiety is Alb-8 or Alb-23 or its variants, as shown in pages 7-9 of WO2012/175400 and the albumin binders described in WO 2012/175741, WO2015/173325, WO2017/080850, WO2017/085172, WO2018/104444, WO2018/134235, WO2018/134234.
  • Some serum albumin binders are also shown in Table A-4.
  • a further component of the polypeptide of the present technology is as described in item E:
  • Examples of such an ISVD that binds to human serum albumin have one or more, or all, framework regions as indicated for construct ALB23002 in Table A-2 (in addition to the CDRs as defined in the preceding item E). In one embodiment, it is an ISVD comprising or consisting of the full amino acid sequence of construct ALB23002 (SEQ ID NO: 5, see Table A-1 and A-2).
  • Item E can be also described using the Kabat definition as:
  • Examples of such an ISVD that binds to human serum albumin have one or more, or all, framework regions as indicated for construct ALB23002 in Table A-2-1 (in addition to the CDRs as defined in the preceding item E′). In one embodiment, it is an ISVD comprising or consisting of the full amino acid sequence of construct ALB23002 (SEQ ID NO: 5, see Table A-1 and A-2-1).
  • the amino acid sequence of an ISVD binding to human serum albumin may have a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 5, wherein the CDRs are as defined in the preceding item E or E′.
  • the ISVD binding to human serum albumin has the amino acid sequence of SEQ ID NO: 5.
  • the ISVD has at least half the binding affinity, or at least the same binding affinity to human serum albumin as construct ALB23002 set forth in SEQ ID NO: 5, wherein the binding affinity is measured using the same method, such as SPR.
  • such an ISVD binding to human serum albumin when such an ISVD binding to human serum albumin has a C-terminal position it exhibits a C-terminal alanine (A) or glycine (G) extension.
  • A alanine
  • G glycine
  • such and ISVD is selected from SEQ ID NOs: 59, 60, 62, 64, 65, 66, 67, 68, 69 and 71 (see table A-4 below).
  • the ISVD binding to human serum albumin has another position than the C-terminal position (i.e. is not the C-terminal ISVD of the polypeptide of the present technology).
  • such an ISVD is selected from SEQ ID NOs: 5, 57, 58, 61, and 63 (see table A-4 below).
  • nucleic acid molecule encoding the polypeptide of the present technology.
  • a “nucleic acid molecule” (used interchangeably with “nucleic acid”) is a chain of nucleotide monomers linked to each other via a phosphate backbone to form a nucleotide sequence.
  • a nucleic acid may be used to transform/transfect a host cell or host organism, e.g. for expression and/or production of a polypeptide.
  • Suitable hosts or host cells for production purposes will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.
  • a host or host cell comprising a nucleic acid encoding the polypeptide of the present technology is also encompassed by the present technology.
  • a nucleic acid may be for example DNA, RNA, or a hybrid thereof, and may also comprise (e.g. chemically) modified nucleotides, like PNA. It can be single- or double-stranded. In one embodiment, it is in the form of double-stranded DNA.
  • the nucleotide sequences of the present technology may be genomic DNA, cDNA.
  • nucleic acids of the present technology can be prepared or obtained in a manner known per se, and/or can be isolated from a suitable natural source.
  • Nucleotide sequences encoding naturally occurring (poly)peptides can for example be subjected to site-directed mutagenesis, so as to provide a nucleic acid molecule encoding polypeptide with sequence variation.
  • site-directed mutagenesis so as to provide a nucleic acid molecule encoding polypeptide with sequence variation.
  • nucleic acid also several nucleotide sequences, such as at least one nucleotide sequence encoding a targeting moiety and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner.
  • nucleic acids may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more “mismatched” primers.
  • restriction sites e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes
  • a vector comprising the nucleic acid molecule encoding the polypeptide of the present technology.
  • a vector as used herein is a vehicle suitable for carrying genetic material into a cell.
  • a vector includes naked nucleic acids, such as plasmids or mRNAs, or nucleic acids embedded into a bigger structure, such as liposomes or viral vectors.
  • Vectors generally comprise at least one nucleic acid that is optionally linked to one or more regulatory elements, such as for example one or more suitable promoter(s), enhancer(s), terminator(s), etc.).
  • the vector is an expression vector, i.e. a vector suitable for expressing an encoded polypeptide or construct under suitable conditions, e.g. when the vector is introduced into a (e.g. human) cell.
  • a vector suitable for expressing an encoded polypeptide or construct under suitable conditions e.g. when the vector is introduced into a (e.g. human) cell.
  • this usually includes the presence of elements for transcription (e.g. a promoter and a polyA signal) and translation (e.g. Kozak sequence).
  • said at least one nucleic acid and said regulatory elements are “operably linked” to each other, by which is generally meant that they are in a functional relationship with each other.
  • a promoter is considered “operably linked” to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being “under the control of” said promotor).
  • two nucleotide sequences when operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.
  • any regulatory elements of the vector are such that they are capable of providing their intended biological function in the intended host cell or host organism.
  • a promoter, enhancer or terminator should be “operable” in the intended host cell or host organism, by which is meant that for example said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence - e.g. a coding sequence - to which it is operably linked.
  • the present technology also provides a composition comprising at least one polypeptide of the present technology, at least one nucleic acid molecule encoding a polypeptide of the present technology or at least one vector comprising such a nucleic acid molecule.
  • the composition may be a pharmaceutical composition.
  • the composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • the present technology also pertains to host cells or host organisms comprising the polypeptide of the present technology, the nucleic acid encoding the polypeptide of the present technology, and/or the vector comprising the nucleic acid molecule encoding the polypeptide of the present technology.
  • Suitable host cells or host organisms are clear to the skilled person, and are for example any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris . In one embodiment, the host is Pichia pastoris.
  • the present technology also provides a method for producing the polypeptide of the present technology.
  • the method may comprise transforming/transfecting a host cell or host organism with a nucleic acid encoding the polypeptide, expressing the polypeptide in the host, optionally followed by one or more isolation and/or purification steps.
  • the method may comprise:
  • Suitable host cells or host organisms for production purposes will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris . In one embodiment, the host is Pichia pastoris.
  • polypeptide of the present technology a nucleic acid molecule or vector as described, or a composition comprising the polypeptide of the present technology, nucleic acid molecule or vector are useful as a medicament.
  • the present technology provides the polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide of the present technology, nucleic acid molecule or vector for use as a medicament.
  • polypeptide of the present technology a nucleic acid molecule or vector as described, or a composition comprising the polypeptide of the present technology, nucleic acid molecule or vector for use in the (prophylactic or therapeutic). treatment of an inflammatory disease.
  • a (prophylactic and/or therapeutic) method of treating an inflammatory disease comprising administering, to a subject in need thereof, a pharmaceutically active amount of the polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide of the present technology, nucleic acid molecule or vector.
  • the polypeptide of the present technology is a nucleic acid molecule or vector as described, or a composition comprising the polypeptide of the present technology, nucleic acid molecule or vector in the preparation of a pharmaceutical composition.
  • the prepared pharmaceutical composition is for treating an inflammatory disease.
  • the inflammatory disease is a type 2 inflammatory disease such as atopic dermatitis and asthma.
  • a “subject” as referred to in the context of the present technology can be any animal, and more specifically a mammal. Among mammals, a distinction can be made between humans and non-human mammals.
  • Non-human animals may be for example companion animals (e.g. dogs, cats), livestock (e.g. bovine, equine, ovine, caprine, or porcine animals), or animals used generally for research purposes and/or for producing antibodies (e.g. mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates, such as cynomolgus monkeys, or camelids, such as llama or alpaca).
  • companion animals e.g. dogs, cats
  • livestock e.g. bovine, equine, ovine, caprine, or porcine animals
  • animals used generally for research purposes and/or for producing antibodies e.g. mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human prima
  • the subject can be any animal, and more specifically any mammal. In one embodiment, the subject is a human subject.
  • Substances may be administered to a subject by any suitable route of administration, for example by enteral (such as oral or rectal) or parenteral (such as epicutaneous, sublingual, buccal, nasal, intra-articular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, or transmucosal) administration.
  • enteral such as oral or rectal
  • parenteral such as epicutaneous, sublingual, buccal, nasal, intra-articular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, or transmucosal
  • substances are administered by parenteral administration, such as intramuscular, subcutaneous or intradermal administration.
  • subcutaneous administration is used.
  • An effective amount of a polypeptide, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector can be administered to a subject in order to provide the intended treatment results.
  • One or more doses can be administered. If more than one dose is administered, the doses can be administered in suitable intervals in order to maximize the effect of the polypeptide, composition, nucleic acid molecule or vector.
  • ID refers to the given SEQ ID NO) ID
  • WFRQAPGK 12 ALSGDG 29 TANSVKGRFTIS 17 KLQYG 34 WGQGT VGGGVQP
  • YADSVKGRFTIS 18 VPFGY 35 RGQGTL GGGVVQP AMK GLEWVS GSTD
  • ID refers to the given SEQ ID NO) ID VHH ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 2 4B02 47 DVQLVES 37 SYRMG 24 WFRQAPGK 42 ALSGDG 52 RFTISRDN 17 KLQYV 34 WGQGT GGGVVQP EREFVA YSTYTAN SKNTVYLQ SGWSY LVTVSS GGSLRLS SVKG MNSLRPED DYPY CAASGRT TALYYCAA FS 3 4B06 48 EVQLVES 38 NYAMK 25 WVRQAPGK 43 SITTGGG 53 RFTISRDN 18 VPFGY 35 RGQGTL GGGVVQP GLEWVS STDYADS SKNTLYLQ YSEHF VTVSS GGSLRLS VKG MNSLRPED SGLSF CAASGFT TALYY
  • ID refers to the given SEQ ID NO
  • ID refers to the given SEQ ID NO
  • Amino acid sequence F027400161 1 DVQLVESGGGVVQPGGSLRLSCAASGRTFSSYR MGWFRQAPGKEREFVAALSGDGYSTYTANSVKG RFTISRDNSKNTVYLQMNSLRPEDTALYYCAAK LQYVSGWSYDYPYWGQGTLVTVSSGGGGSGGGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGGVVQPGGSLRLSCAASGFTFNNYAMKWVRQA PGKGLEWVSSITTGGGSTDYADSVKGRFTISRD NSKNTLYLQMNSLRPEDTALYYCANVPFGYYSE HFSGLSFDYRGQGTLVTVSSGGGGSGGGSEVQL VESGGGVVQPGGSLRLSCAASGSGFGVNILYWY RQAAGIERELIASITSGGITNYVDSVKGRFTIS R
  • ID refers to the SEQ ID NO as used herein
  • ID Amino acid sequence Alb8 57 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDN AKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS Alb23 58 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWV RQAPGKGPEWVSSISGSDTLYADSVKGRFTISRDN SKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS Alb129 59 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWV RQAPGKGLEWVSSISGSDTLYADSVKGRFTISRDN AKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLV TVSSA Alb132 60 EVQLVES
  • llamas Three llamas were immunized with recombinant human IL-13 (Peprotech, cat nr 200-13, E. coli -derived) according to standard protocols, with the aim to induce a heavy-chain antibody dependent humoral immune response. In addition, these llamas were boosted with human/cyno IL-13 from another source (Sino Biological, cat nrs 10369-HNAC and 11057-CNAH, mammalian cell derived).
  • Another three llamas were immunized with recombinant hTSLP-Fc. In addition, these llamas were boosted with cyno TSLP-Fc. Two additional llamas were immunized with hTSLP alternated with cyno TSLP-Fc.
  • Immune blood (PBL) samples were taken at regular intervals, and total RNA was prepared from the isolated B-cells.
  • the humoral immune response was monitored during the immunization process by comparing the antigen specific serum titers of a sample collected prior to initiation of immunization and a serum sample typically collected after multiple antigen administrations.
  • 96-well Maxisorp plates were coated with human IL-13 (Sino Biological, cat nr 10369-HNAC) or human TSLP.
  • Recombinant human TSLP is commercially available, such as from R&D Systems (cat nr 1398-TS) .
  • anti-IL-13 and anti-TSLP ISVDs were demonstrated by using HRP (horseradish peroxidase) conjugated goat anti-llama immunoglobulin (Bethyl Laboratories Inc.) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine).
  • HRP horseradish peroxidase conjugated goat anti-llama immunoglobulin
  • Peripheral blood mononuclear cells were prepared from the blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Total RNA extracted from these cells and from lymph nodes was used as starting material for RT-PCR to amplify ISVD encoding gene fragments. These fragments were cloned into phagemid vector pAX212. Phage was prepared according to standard protocols (Antibody Phage Display: Methods and Protocols (First Edition, 2002, O'Brian and Aitken eds., Humana Press, Totowa, N.J.) and stored after filter sterilization at 4° C. until further use.
  • phage libraries were constructed for IL13, with library sizes between 6.1 ⁇ 10 8 and 1.3 ⁇ 10 9 , and a percentage of insert ranging from 87 to 96%.
  • Five phage libraries were constructed for TSLP, with library sizes between 4.2 ⁇ 10 8 and 9.8 ⁇ 10 8 , and a percentage of insert ranging from 91 to 100%.
  • the phage libraries were incubated with 50 nM soluble biotinylated hIL13-Fc in presence of IgG from human serum (Sigma, I4506). Complexes of hIL-13-Fc and phage were captured from solution on streptavidin coated magnetic beads. After extensive washing with PBS/0.05% Tween20, bound phage were eluted by addition of trypsin (1 mg/ml). Outputs of these round 1 selections were incubated with 0.05, 0.5 or 5 nM soluble biotinylated hIL-13-Fc or cyno IL-13-Fc.
  • Not enriched outputs from the round 2 selections were further incubated with a 0.005, 0.05, 0.5 or 5 nM soluble biotinylated human or cyno IL-13-Fc in round 3. Individual clones from enriched round 2 and round 3 selections were picked.
  • the phage libraries were incubated with 50 nM soluble biotinylated hTSLP-Fc in presence of IgG from human serum (Sigma, 14506) or with 500 nM biotinylated hTSLP or with 500 nM biotinylated cyno TSLP.
  • the human and cyno TSLP sequences are known (Uniprot accession Uniprot accession Q969D9 and NCBI RefSeq XP_005557555.1, respectively). Recombinant protein was used to perform the assay. Complexes of TSLP and phage were captured from solution on streptavidin coated magnetic beads.
  • periplasmic extracts were screened in an ELISA for binding to human and cyno IL13-Fc, respectively TSLP-Fc.
  • the assessment was performed in a Spectraplate 384-HB (PerkinElmer) in a 25 ⁇ l format. The antigens were coated overnight at 1 ⁇ g/ml in PBS at 4° C. Wells were blocked with a casein solution (1%). After addition of a 5-fold dilution of peri plasmic extracts, ISVD binding was detected using mouse anti-Flag-HRP (Sigma) and a subsequent enzymatic reaction in the presence of substrate esTMB (3,3′,5,5′-tetramentylbenzidine).
  • hIL-13:hIL-13R ⁇ 1 binary complex AlphaScreen it was investigated if ISVDs could block the interaction between hIL-13 and the hIL-13R ⁇ 1 extracellular domain.
  • dilutions of the periplasmic extracts were pre-incubated with biotinylated hIL-13 (Peprotech cat nr 200-13).
  • hIL-13R ⁇ 1-hFc R&D systems; cat nr 146-IR
  • anti hFc-coupled Acceptor beads were added and further incubated for 1 hour at room temperature, followed by addition of streptavidin-coupled Donor beads and an additional 1-hour incubation.
  • hIL-13:hIL-13R ⁇ 1:hIL4R ⁇ ternary complex AlphaScreen it was screened if ISVDs could block the recruitment of hIL4R ⁇ to the hIL13:hIL13R ⁇ 1 binary complex.
  • hIL-13 binds to hIL-13R ⁇ 1 and this binary complex recruits hIL4Ra, resulting in formation of the ternary complex hIL-13:hIL-13R ⁇ 1:hIL4Ra.
  • Dilutions of the periplasmic extracts were pre-incubated with hIL-13 (Peprotech cat nr 200-13) and biotinylated huIL4R ⁇ (R&D Systems; cat nr 230-4R/CF).
  • hIL-13R ⁇ 1-hFc R&D systems; cat nr 146-IR
  • anti hFc-coupled Acceptor beads were added and further incubated for 1 hour at room temperature, followed by addition of streptavidin-coupled Donor beads and an additional 1-hour incubation.
  • Acceptor and Donor beads are brought into proximity and upon laser excitation a detectable signal is generated. Decrease in the AlphaScreen signal indicates that the formation of the ternary complex is blocked by the ISVD present in the periplasmic extract.
  • hIL13-Fc, cyno IL13-Fc, hTSLP-Fc and cyno TSLP-Fc were immobilized on ProteOn GLC Sensor Chips by amine coupling using EDC and NHS at flow rate 30 ⁇ l/min for activation.
  • IL13 proteins were injected at 10 ⁇ g/ml in ProteOn Acetate buffer at pH5.0.
  • TSLP proteins were injected at 5 ⁇ g/mL in ProteOn Acetate Buffer at pH5.5. After immobilization, surfaces were deactivated with ethanolamine.
  • Periplasmic extracts of the ISVD candidates were diluted 10 times in PBS-Tween20 (0.1%) and injected for 2 minutes at 45 ⁇ l/min and allowed to dissociate for 900 seconds. Between different samples, the surfaces were regenerated with a 2 minute injection of Phosphoric Acid (0.425%) at 45 ⁇ I/min, in case of IL13, or with a 1 minute injection of Glycine pH3.0 (5 mM)/SDS(0.25%) at 4 ⁇ L/min, in case of TSLP. From the sensorgrams obtained for the different periplasmic extracts off-rates were calculated.
  • Anti-IL-13 ISVDs and anti-TSLP ISVDs were selected for expression and purification, based on their blocking capacity in AlphaScreen assays and off-rate values. Sequences are shown in Tables 1 and 2.
  • ISVDs were expressed in E. coli TG1 cells as c-myc, His6-tagged proteins. Expression was induced by addition of 1 mM IPTG and allowed to continue for 3 h at 37° C. After spinning the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellets and resuspension in dPBS. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC) using Nickel Sepharose TM 6 FF columns (Atoll). ISVDs were eluted from the column with 250 mM imidazole and subsequently desalted towards dPBS.
  • IMAC immobilized metal affinity chromatography
  • endotoxins were removed by gel filtration in the presence of 50 mM Octyl ⁇ -D-glucopyranoside (OGP, Sigma). Endotoxin levels were determined using a standard LAL-assay.
  • the binary and ternary complex AlphaScreen assays as described in example 3, were used to determine the IC50 values of the anti-IL-13 and anti-TSLP ISVDs, purified as described in Example 5. Instead of dilutions of the periplasmic extracts, a dilution series of each purified ISVD starting from 500 nM down to 1.8 pM was pre-incubated with IL-13 or TSLP.
  • the tested anti-IL-13 ISVDs inhibited the formation of the ternary complex and partially blocked the binary complex with IC50 values as shown in Table 5.
  • IC50 values for the blocking anti-IL13 ISVDs as determined in the binary and ternary complex AlphaScreen assays IL-13-IL-13R ⁇ 1 IL-13-IL-13R ⁇ 1-IL4R ⁇ AlphaScreen AlphaScreen ISVD ID IC50 (M) % Inhibition IC50 (M) % Inhibition F0107004B02 4.0E ⁇ 09 82 1.7E ⁇ 09 100 F0107004B06 6.1E ⁇ 08 55 6.7E ⁇ 09 100
  • the tested anti-TSLP ISVDs fully inhibited the formation of the ternary complex, and also inhibited the formation of the binary complex, with IC50 values as shown in Table 6.
  • the inhibitory potency of the anti-IL-13 ISVDs was determined in a cell-based assay monitoring IL-13 mediated proliferation of TF-1 cells.
  • TF-1 cells were cultured in RPMI 1640 medium with the addition of 1/5 HEPES, 1/500 Na-Pyruvate, 1/500 Glutamax and 2 ng/mL recombinant human GM-CSF.
  • TF-1 cells were seeded at 40.000 cells per well in growth medium w/o GM-CSF.
  • a dilution series of the purified anti-IL-13 ISVDs or reference compounds was added. After 15 min incubation at 37° C., 200 pM of IL-13 (Peprotech cat nr 200-13) was added. After 96 hours, proliferation of the TF-1 cells was determined with Cell Titer 96 Aqueous One Solution (Promega #G3580) on an EnVision Multilabel Reader (Perkin Elmer).
  • the ISVDs shown in Table 7 inhibit IL-13-induced TF1 proliferation.
  • the blocking potency of the anti-TSLP ISVDs was determined in a cell-based assay monitoring TSLP mediated proliferation of BaF3 cells transfected with plasmids encoding hTSLPR and hIL7Ra. Cells were seeded at a density of 20.000 cells/well in RPMI 1640 growth medium in cell culture treated white 96 well plates. A dilution series of anti-TSLP ISVDs were added, followed by addition of 50 pM hTLSP-Fc or 50 pM cyno TSLP-Fc for stimulation of the cells.
  • the human and cyno TSLP sequences are known (Uniprot accession Uniprot accession Q969D9 and NCBI RefSeq XP_005557555.1, respectively). Recombinant protein was used to perform the assay.
  • IL-13 For IL-13, around 2000 RU of hIL13-Fc or 4000 RU cyno IL13-Fc was immobilized directly on a CM5 sensor chip. The ISVDs were then injected at different concentrations (between 3 ⁇ M and 12 nM) for 120 s and allowed to dissociate for 900s. Regeneration of the hIL-13-Fc and cyno IL-13-Fc surfaces were performed using a 47 s injection of 0.85% H 3 PO 4 :MilliQ (1:1).
  • TSLP For TSLP, around 8000 RU of anti-hulgG antibody (GE Healthcare) was immobilized directly on a CM5 sensor chip.
  • hTSLP-Fc at 1 ⁇ g/mL
  • cyno TSLP-Fc at 0.75 ⁇ g/mL
  • the ISVDs were then injected at different concentrations (between 0.4 nM and 3000 nM) for 120 s and allowed to dissociate for 900s.
  • the selected anti-IL-13 ISVDs (F0107004B02 and F0107004B06) were formatted into biparatopic and bivalent ISVD constructs.
  • the building blocks in the constructs are genetically linked by a flexible 35GS (GlySer) linker.
  • ISVDs were expressed as FLAG3-HIS6-tagged protein in Pichia pastoris (amino acid sequences are shown in Table 14).
  • Induction of ISVD construct expression occurred by stepwise addition of methanol. Clarified medium with secreted ISVD construct was used as starting material for immobilized metal affinity chromatography (IMAC) followed by desalting resulting in 90% purity as assessed by SDS-PAGE.
  • IMAC immobilized metal affinity chromatography
  • the biparatopic and bivalent IL-13 constructs were characterised in the binary and ternary complex blocking AlphaScreen assays (as described in example 6) as well as in the TF-1 proliferation assay (as described in example 7).
  • An overview of the generated constructs and their blocking potencies in the binary and ternary AlphaScreen assay and in the TF-1 proliferation assay is shown in Table 11.
  • the biparatopic construct displays excellent potencies against hIL-13 and cylL-13, similar to the potency of anti-hlL-13 reference mAb1.
  • bivalent constructs improve in potency, but not to the same extent as the biparatopic construct F010700029 in the TF1 proliferation assay.
  • the bivalent constructs do not reach full inhibition in the IL13-IL13R ⁇ 1 Alphascreen.
  • the most potent anti-IL-13 biparatopic ISVD construct was tested in the IL-13 induced A549 eotaxin release assay.
  • A549 suspension cells were cultured in Ham's F12K medium supplemented with 10% FCS. Cells were seeded into a 96 well plate at 200.000 cells/well.
  • 200pM hIL-13 (Sino Biological cat nr 10369-HNAC) or cyno IL-13 (Sino Biological cat nr 11057-CNAH) were added, followed by a dilution series of the ISVD constructs.
  • Eotaxin-3 was determined in the supernatants using the MSD ELISA (Human Eotaxin-3 Tissue Culture Kit (Meso Scale, K151ABB-1)). Results are shown in Table 12.
  • IL13R ⁇ 2 (SinoBiological cat nr 10350-H08H) was coated on a 384 well Spectraplate (Perkin Elmer).
  • hIL13-Fc (SinoBiological, cat nr 10369-H01H) was mixed with serial dilutions of ISVD constructs or positive control compound IL-13R ⁇ 2 and incubated for 1 hour. After washing and blocking, the coated receptor was incubated with the IL13-Fc ISVD/control mix and incubated for 1 hour.
  • ISVD ISVD construct construct ID description Sequence F010700003 F0107004B06- EVQLVESGGGLVQPGGSLRLS (SEQ ID 35GS- CAASGFTFNNYAMKWVRQAPG NO: 148) F0107004B02 KGLEWVSSITTGGGSTDYADS VKGRFTISRDNRKNTLYLQMN SLKPEDTAVYYCANVPFGYYS EHFSGLSFDYRGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSG GGGSGGGGSG GGGSEVQLVES GGGLVQAGGSLRLSCAASGRT FSSYRMGWFRQAPGKEREFVA ALSGDGYSTYTANSVNSRFTI SRDNAKNTVYLQMNSLKPEDT AIYYCAAKLQYVSGWSYDYPY WGQGTLVTVSS F010700014 F0107004B02-
  • the selected TSLPR blockers (F0107501A02 and F0107529F10) were combined into biparatopic ISVD constructs.
  • the constructs were expressed as FLAG3-HIS6-tagged protein in Pichia pastoris (amino acid sequences are shown in Table 16).
  • Induction of ISVD construct expression occurred by stepwise addition of methanol.
  • Clarified medium with secreted ISVD construct was used as starting material for immobilized metal affinity chromatography (IMAC) followed by desalting resulting in 90% purity as assessed by SDS-PAGE.
  • IMAC immobilized metal affinity chromatography
  • the biparatopic constructs were titrated as purified proteins against 50 or 5 pM hTSLP and 50 or 5 pM cyno TSLP in the BaF3 proliferation assay (as described in example 7) and compared to different anti-TSLP reference compounds (anti-hTSLP reference mAb2 and anti-hTSLP reference mAb1).
  • the data is summarized in Table 15 (hTSLP and cyno TSLP).
  • the biparatopic constructs clearly outperform the reference mAbs.
  • the biparatopic construct with F0107501A02 at the N-terminus shows improved reactivity towards cyno TSLP compared to the construct with F0107501A02 at the C-terminus.
  • ISVD ISVD construct construct ID description Sequence F010703842 F0107529F10- EVQLVESGGGLVQAGGSLRLS (SEQ ID 35GS- CAASGFTFADYDYDIGWFRQA NO: 152) F0107501A02 PGKEREGVSCISNRDGSTYYT DSVKGRFTISSDNAKNTVSLQ MNSLKPEDTAVYYCAVEIHCD DYGVENFDFDPWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSEVQLVE SGGGLVQAGESLRLSCAASGS GFGVNILYWYRQAAGIERELI ASITSGGITNYVDSVKGRFTI SRDNAENTMYLQMNSLKAEDT GVYYCASRNIFDGTTEWGQGT LVTVSS F027400016 F0107501A02 DVQ
  • Anti-IL-13 ISVDs F0107004B02 and F0107004B06 and anti-TSLP ISVDs F0107501A02 and F0107529F10 were further sequence optimized.
  • Sequence optimization involves replacing one or more specific amino acid residues in the sequence in order to improve one or more (desired) properties of the ISVDs.
  • Sequence optimisation of anti-IL-13 ISVD F0107004B02 resulted in a final sequence optimised variant F027100019, which comprises 8 amino acid substitutions (i.e. E1D, L11V, A14P, N64K, S65G, A74S, K83R, I89L) compared to the parental ISVD F107004B02.
  • Sequence optimisation of anti-IL-13 ISVD F0107004B06 resulted in a final sequence optimised variant F027100183, which comprises 4 amino acid substitutions (i.e. L11V, R74S, K83R, V89L) compared to the parental ISVD F107004B06.
  • the sequence optimised variants were assembled from oligonucleotides using a PCR overlap extension method.
  • the variants were expressed in E.coli and purified by IMAC and desalting.
  • F027100019 and F027100183 were evaluated for their hIL-13 binding capacity by surface plasmon resonance, using hIL-13 from Peprotech (cat nr 200-13). Additionally, F027100019 was tested for its neutralizing activity in the eotaxin release assay. Monomeric behavior of both variants was monitored by Size Exclusion-HPLC (SE-HPLC). Thermal stability of the variants was tested in a thermal shift assay (TSA) using the Lightcycler (Roche).
  • the parental ISVDs and their variants are incubated at different pH's in the presence of sypro orange and a temperature gradient is applied.
  • sypro orange binds and the measured fluorescence increases suddenly, as such a melting temperature can be determined for a certain pH. Results are summarized in Table 17 and Table 18.
  • F027100019 exhibited good potency in the Eotaxin release assay and its affinity to hIL-13 was determined to be 34 nM in SPR.
  • the Tm of F027100019 is 3° C. higher than for the parental ISVD F0107004B2.
  • the % framework identity in the framework regions for F027100019 is 85% based on the AbM definition (see Antibody Engineering, Volt by Kontermann & Dübel (Eds), Springer Verlag Heidelberg Berlin, 2010) and 86% based on the Kabat definition.
  • Affinity of F027100183 is similar compared to the WT sequence and the variant has a Tm of 61° C.
  • the variant elutes as a 100% monomeric peak on SE-HPLC.
  • the % framework identity in the framework regions for F027100183 is 94.4% based on the AbM definition and 93.1% based on the Kabat definition.
  • Sequence optimisation of anti-TSLP ISVD F0107529F10 resulted in a final sequence optimised variant F027400021, which comprises 8 amino acid substitutions (i.e., Lily, A14P, T60A, S71R, A74S, S79Y, K83R, V89L) compared to the parental ISVD F0107529F10.
  • Sequence optimisation of anti-TSLP ISVD F0107501A02 resulted in a final sequence optimised variant F027400160, which comprises 6 amino acid substitutions (i.e., L11V, A14P, E16G, A74S, K83R, V89L) compared to the parental ISVD F0107501A02.
  • the sequence optimised variants were assembled from oligonucleotides using a PCR overlap extension method.
  • the constructs were expressed in E.coli and purified by IMAC and desalting.
  • the variants were evaluated for their binding capacity to human and cyno TSLP by surface plasmon resonance. Monomeric behaviour of all variants was monitored by Size Exclusion-HPLC (SE-HPLC) and the thermal stability in a thermal shift assay (TSA). Additionally, the variants of F0107501A02 were tested for their blocking activity on hTSLP in the ternary complex Alphascreen. Results are summarized in Table 19 and Table 20.
  • the % framework identity in the framework regions for F027400160 is 83.1% based on the AbM definition and 80.5% based on the Kabat definition.
  • ISVD ISVD ID description Sequence F010704076 F0107501A02 EVQLVESGGGLVQPGGSLRLS (SEQ ID (A14P, E16G, CAASGSGFGVNILYWYRQAAG NO: 160) A74S, K83R)* IERELIASITSGGITNYVDSV KGRFTISRDNSENTMYLQMNS LRAEDTGVYYCASRNIFDGTT EWGQGTLVTVSS F010704099 F0107529F10 EVQLVESGGGLVQPGGSLRLS (SEQ ID (A14P, T60A, CAASGFTFADYDYDIGWFRQA NO: 161) S71R, A74S, PGKEREGVSCISNRDGSTYYA S79Y, K83R)* DSVKGRFTISRDNSKNTV
  • the above identified optimized ISVDs F027100019 (optimized variant of F0107004B02), F027100183 (optimized variant of F0107004B06), F027400021 (optimized variant of F0107529F10), and F027400160 (optimized version of F0107501A02) were used for the generation of multispecific ISVD construct F027400161.
  • the optimized monovalent building blocks used in F027400161 are designated in the following in an abbreviated form according to their ISVD origin as 04B02, 04B06, 529F10, and 501A02, respectively.
  • ISVD-containing polypeptide F027400161 (SEQ ID NO: 1) binding to IL-13 and TSLP resulted from a data-driven bispecific engineering and formatting campaign in which several anti-TLSP building blocks, several anti-IL13 building blocks and the anti-HSA building block ALB23002 were included. Different positions/orientations of the building blocks and different linker lengths (9GS vs 35GS) were applied and proofed to be critical for different parameters (potency, cross-reactivity, expression yield, etc. . . . ).
  • ISVD constructs were transformed in Pichia Pastoris for small scale productions. Induction of ISVD expression occurred by stepwise addition of methanol. Clarified medium with secreted ISVD was used as starting material for purification via Protein A affinity chromatography followed by desalting. The purified samples were used for functional characterisation and expression evaluation.
  • Table 23 illustrates that different yields ranging from low to high titer were obtained for six constructs comprising the same building blocks but ordered in different ways and connected with different linker lengths.
  • Highest expression titers are obtained for constructs comprising the IL-13 ISVDs 4B02 and 4B06 linked via a 35GS linker (F027400161 and F027400163).
  • the solubility of F027400161 and F027400163 was much higher than their respective counterparts F027400296 and 298, of which building blocks are linked with four 9GS linkers and ALB is positioned at the C-terminus.
  • the large bispecific panel was trimmed down to a panel of 2 bispecific constructs, consisting of ISVD constructs F027400161 and F027400163 proven to be potent on both targets (human and cyno) and having the potential of high expression levels, based on preliminary yield estimates.
  • Table 24 and example 22 demonstrate that pre-existing antibody reactivity is driven by the orientation of the building blocks and the linker lengths.
  • ISVD construct F027400161 was selected based on potency, reduced binding to pre-existing antibodies and superior expression levels and CMC characteristics
  • Recombinant protein was used to perform the assay, human IL-13 (Sino Biological cat nr 10369-HNAC), cyno IL-13 (Sino Biological cat nr 11057-CNAH) and rhesus IL-13 (R&D systems cat nr 2674-RM) and human (Sigma Aldrich, cat nr A8763), cyno and mouse (Albumin Bioscience cat nr N1204H1CM) serum albumin was quantified by means of in-solution affinity measurements on a Gyrolab xP Workstation (Gyros).
  • TSLP or IL-13 ranging from 1 ⁇ M-0.25 fM
  • serum albumin ranging from 100 ⁇ M-320 pM
  • F027400161 250 pM in case of TSLP, 10 nM in case of IL-13 and 1 ⁇ M in case of serum albumin
  • 48 or 72 hours in case of TSLP and IL-13
  • 2 hours in case of serum albumin
  • Biotinylated human TSLP/IL-13/serum albumin was captured in the microstructures of a Gyrolab Bioaffy 1000 CD, which contains columns of beads and is used as a molecular probe to capture free F027400161 from the equilibrated solution.
  • the mixture of TLSP/IL-13/serum albumin and F027400161 (containing free TLSP/IL-13/serum albumin, free F027400161 and TLSP/IL-13/serum albumin—F027400161 complexes) was allowed to flow through the beads, and a small percentage of free F027400161 was captured, which is proportional to the free ISVD concentration.
  • Cytokines were immobilized on a Proteon GLC sensor chip at 25 ⁇ g/mL for 600 s using amine coupling, with 80 seconds injection of EDC/NHS for activation and a 150 seconds injection of 1 M Ethanolamine HCl for deactivation (ProteOn Amine Coupling Kit. cat. 176-2410). Flow rate during activation and deactivation was set to 30 ⁇ l/min and during ligand injection to 25 ⁇ l/min. The pH of the 10 mM Acetate immobilization buffer was 6.0 for IL13 and IL4 (Peprotech cat nr 200-07) and 5.5 for TSLP and IL7 (R&D systems cat nr 204-IL/CF).
  • F027400161 1 ⁇ M was injected for 2 minutes and allowed to dissociate for 600 s at a flow rate of 45 ⁇ lL/min.
  • As running buffer PBS (pH7.4) +0.005% Tween 20 was used.
  • TSLP and the short form of TSLP were immobilized on a proteon GLC sensor chip at 10 ⁇ g/mL, respectively 5 ⁇ g/ml for 150 s using amine coupling as described above.
  • the pH of the 10 mM Acetate immobilization buffer was 5.5 for TSLP and 4.0 for the short form of TSLP.
  • 500 nM of F027400161 was injected for 2 minutes and allowed to dissociate for 600 s at a flow rate of 45 ⁇ L/min.
  • As running buffer PBS (pH7.4) +0.005% Tween 20 was used.
  • 500 nM anti-hTSLP reference mAb1 was injected and as positive control 500 nM ⁇ -hTSLP pAb (Abcam ab47943).
  • hTSLP recombinant human TSLP is commercially available, such as from R&D Systems (cat nr 1398-TS) was immobilized on a CM5 Sensor chip via amine coupling. 100 nM of F027400161 was injected for 2 min at 10 ⁇ l/min over the TSLP surface in order to capture the ISVD construct via the TSLP building blocks 501A02-529F10.
  • hIL13 PeproTech, cat nr 200-13
  • HSA or hOX40L or 1000 nM of HSA were injected or mixtures of 100 nM IL13+100 nM HSA , 100 nM IL13+1000 nM HSA, 100 nM OX40L+100 nM HSA , 100 nM OX40L+1000 nM HSA or 100nM IL13+100nM OX40L, at a flow rate of 10 ⁇ l/min for 2 min followed by a subsequent 300 seconds dissociation step.
  • the TSLP surfaces are regenerated with a 1 minute injection of 0.5% SDS+10 mM glycine pH3 at 45 ⁇ I/min.
  • the sensorgram ( FIG. 1 ) demonstrates that F027400161 can bind human IL13, human TSLP and HSA simultaneously as shown by the increase in response units after capture on TSLP: ⁇ 130 RU increase from IL13 only, ⁇ 60 RU increase from 100 nM HSA and ⁇ 350 RU from 1000 nM HSA only, ⁇ 180 RU increase for the IL13 and 100 nM HSA mixture, and ⁇ 500 RU for the IL13 and 1000 nM HSA mixture. Higher concentrations of HSA were needed to see decent RU increase levels, due to the lower affinity of F027400161 for HSA (see example 16).
  • A549 suspension cells were cultured in Ham's F12K medium, supplemented with 10% FCS, and seeded into a 96 well plate at 400.000 cells/well. After 24 hours incubation, a dilution series of F027100161 or reference compounds (anti-hlL-13 reference mAb1 and anti-hlL-13 reference mAb2) were added. After 20 min incubation, human IL13 (Sino Biological cat nr 10369-HNAC), cyno IL13 (Sino Biological cat nr 11057-CNAH) or rhesus IL13 (R&D Systems, cat nr 2674-RM-025) is added to a final concentration of 160 pM.
  • heparin is added at a final concentration of 50 ⁇ g/ml, to enhance the eotaxin expression.
  • eotaxin-3 secreted in the cell supernatant was quantified by use of the Human CCL26/Eotaxin-3 DuoSet ELISA (R&D systems, DY346).
  • F027400161 inhibited human, cyno and rhesus IL13-induced eotaxin-3 release in a concentration-dependent manner with an IC50 of 194 pM (for human IL13), 1040 pM (for cyno IL13) and 713 pM (for rhesus IL13), comparable to the reference compound anti-hlL-13 reference mAb1, and better than the reference compound anti-hlL-13 reference mAb2 (Table 26, FIG. 2 ).
  • HEK-BlueTM IL-4/IL-13 cells were generated by stable transfection of HEK293 cells with the human STAT6 gene and a STAT6-inducible SEAP reporter gene. Upon IL-4 and IL-13 stimulation, the cells produce STAT6-induced SEAP secreted in the supernatant quantified by QUANTI-BlueTM.
  • HEK-BIueTM cells were cultured DMEM, supplemented with 10% FBS and seeded into a 96 well plate at 50.000 cells/well.
  • a dilution series of F027100161 or reference compounds (anti-hlL-13 reference mAb1 and anti-hlL-13 reference mAb2) was pre-incubated with 10 pM hIL13 (Sino Biological cat nr 10369-HNAC) or cyno IL-13 (Sino Biological cat nr 11057-CNAH) for 1 hour at room temperature and added to the cells.
  • F027400161 inhibited human and cyno IL-13 induced SEAP secretion in a concentration-dependent manner with an IC50 of 32.8 pM (for human IL-13) and 53.4 pM (for cyno IL-13), better than the reference compound anti-hlL-13 reference mAb2 (Table 27, FIG. 3 ).
  • Functional activity of soluble TSLP from the different species of interest was studied using a cell-based assay investigating proliferation of BaF3 cells transfected with plasmids encoding hTSLPR and hIL7Ra.
  • Cells were seeded at a density of 15000 cells/well in RPMI 1640 growth medium in cell culture treated white 96 well plates. A dilution series of F027100161 or reference compounds (anti-hTSLP reference mAb1) were added, followed by addition of 5 pM human or cyno TLSP for stimulation of the cells.
  • the human and cyno TSLP sequences are known (Uniprot accession Uniprot accession Q969D9 and NCBI RefSeq XP_005557555.1, respectively). Recombinant protein was used to perform the assay.
  • F027400161 inhibited human and cyno TSLP dependent proliferation of Ba/F3 cells in a concentration-dependent manner with an IC50 of 7.8 pM (for human TSP) and 24 pM (for cyno TSLP), performing hence much better than the reference compound anti-hTSLP reference mAb1 (Table 28, FIG. 4 ).
  • ISVDs are captured on the chip via binding of the ALB23002 building block to HSA, which is immobilized on the chip.
  • HSA the ligand lanes of a ProteOn GLC Sensor Chip are activated with EDC/NHS (flow rate 30 ⁇ l/min) and HSA is injected at 100 ⁇ l/ml in ProteOn Acetate buffer pH4.5 to render immobilization levels of approximately 2900 RU.
  • surfaces are deactivated with ethanolamine HCl (flow rate 30 ⁇ l/min).
  • ISVD constructs are injected for 2 min at 45 ⁇ I/min over the HSA surface to render an ISVD capture level of approximately 800 RU.
  • the samples containing pre-existing antibodies are centrifuged for 2 minutes at 14,000 rpm and supernatant is diluted 1:10 in PBS-Tween20 (0.005%) before being injected for 2 minutes at 45 ⁇ I/min followed by a subsequent 400 seconds dissociation step.
  • the HSA surfaces are regenerated with a 2 minute injection of HCl (100 mM) at 45 ⁇ I/min.
  • Sensorgrams showing pre-existing antibody binding are obtained after double referencing by subtracting 1) ISVD-HSA dissociation and 2) non-specific binding to reference ligand lane. Binding levels of pre-existing antibodies are determined by setting report points at 125 seconds (5 seconds after end of association). Percentage reduction in pre-existing antibody binding is calculated relative to the binding levels at 125 seconds of a reference ISVD.
  • the pentavalent ISVD construct F027400161 optimized for reduced pre-existing antibody binding by introduction of mutations L11V and V89L in each building block and a C-terminal alanine, shows substantially less binding to pre-existing antibodies compared to a control non-optimized pentavalent ISVD construct F027301186, (Table 24 and FIG. 5 ).
  • Pre-existing antibody binding depends on the orientation of the building blocks and the linker lengths present in the multispecific constructs.
  • Table 24 and FIG. 5 demonstrate that construct F027400161 shows lower pre-existing antibody reactivity than construct F027400163, due to its specific orientation, but that F027400164 shows lower reactivity than F027400161, due to short linkers all over.
  • the type 2 inflammation cascade is initiated and propagated by a concerted action of epithelial cells, dendritic cells, type 2 helper T cells (Th2 cells), mast cells and innate lymphoid cells in a context-dependent manner.
  • the cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the initiation and progression of allergic inflammation through its ability to activate dendritic cells (DCs).
  • DCs dendritic cells
  • human DCs Upon activation by TSLP, human DCs produce CCL17, a Th2-associated chemokine, and drive Th2 cell differentiation from na ⁇ ve CD4+ T cells.
  • F027400161 targets both TSLP and IL-13, can block the interaction between TSLP and DCs, and thereby, reduce CCL-17 production and is projected to confer efficacy in type 2 inflammatory diseases and beyond.
  • F027400161 to inhibit TSLP-induced CCL17 production was assessed in human DCs isolated from 8 individual healthy donors in comparison to the reference monospecific antibody, anti-hTSLP reference mAb1.
  • Human DCs (CD3 ⁇ CD14 ⁇ CD11c + HLA-DR high ) were isolated and enriched from healthy human PBMCs in buffy coat (human leucocyte pack) samples.
  • a total of 0.5-0.8 ⁇ 10 6 DCs/well were incubated with eight 3-fold serially diluted concentrations of F027400161 (400 ng/mL or 5.714 nM top concentration) or eight 4-fold serially diluted concentrations of anti-hTSLP reference mAb1 (4000 ng/mL or 27 nM top concentration) prior to stimulation with 4 ng/mL of recombinant human TSLP for 36 hours in 37° C. cell culture incubator.
  • Recombinant human TSLP is commercially available, such as from R&D Systems (Catalog number 1398-TS) .
  • CCL17 production in freshly collected cell culture supernatant was measured by ELISA and IC 50 values of the ISVD construct and benchmark antibody were calculated in Graphpad Prism.
  • the reference monospecific antibody anti-hTSLP reference mAb1 inhibited TSLP induced CCL17 production at a mean IC 50 concentration of 793.4 pM, while F027400161 inhibited TSLP induced CCL17 production with a mean IC 50 of 53.26 pM.
  • Type 2 cytokines such as thymic stromal lymphopoietin (TSLP) and Interleukin-13 (IL-13) exert unique, additive and synergistic response to drive asthma and atopic dermatitis (AD) pathophysiology.
  • TSLP thymic stromal lymphopoietin
  • IL-13 Interleukin-13
  • AD atopic dermatitis
  • F027400161 to inhibit 0.5 ng/mL IL-13 and TSLP-induced synergistic production of CCL17 (TARC) was evaluated in human PBMCs from 8 individual healthy donors. The study was designed to evaluate non-inferiority of the ISVD construct versus monospecific biologics, anti-hTSLP reference mAb1 and anti-hlL-13 reference mAb1.
  • recombinant human TSLP is commercially available, such as from R&D Systems (cat nr 1398-TS) plus IL-13 (R&D Systems, cat nr 213-ILB-005/CF) and incubated with ten 3-fold serially diluted concentrations of the ISVD construct (10 nM top concentration), anti-hlL-13 reference mAb1 (10 nM top concentration) and anti-hTSLP reference mAb1 (100 nM top concentration) in a 96-well plate for 20 hours in 37° C. cell-culture incubator.
  • the assays were performed with technical triplicates within each donor for F027400161.
  • the concentrations of the cytokines used were within 2-fold of the standard error of reported literature values from sera, BAL fluid, sputum and skin of normal humans, asthmatics and atopic dermatitis patients (Berra ⁇ es A et al, Immunol Letter 178: 85-91, 2016; Bellini A et al, Mucosal Immunology 5(2):140-9, 2012; Davoodi P et al, Cytokine 60(2):431-7, 2012; Szegedi K et al, J Eur Acad Dermatol Venereol 29(11):2136-44, 2015).
  • CCL17 production in freshly collected cell culture supernatant was measured by Meso Scale Diagnostics (MSD) V-PLEX Human TARC Kit.
  • F027400161 demonstrated 100% inhibition of 0.5 ng/mL IL-13+TSLP-induced synergistic CCL17 production with a mean IC 50 of 0.0061 nM.
  • the reference antibody anti-hIL-13 mAb1 despite having a lower mean IC 50 of 0.0028 nM, was unable to fully block the synergistic CCL17 response at equimolar doses of F027400161 and plateaued at approximately 80% inhibition.
  • anti-hTSLP reference mAb1 was only able to block approximately 50% of the CCL17 production with a mean IC 50 of 2.932 nM.
  • F027400161 is more potent than anti-hTSLP reference mAb1 and is superior compared to anti-hIL-13 reference mAb1 for blocking pathophysiological relevant concentrations of IL-13 and TSLP-induced synergistic response in human PBMCs, highlighting its therapeutic potential for the treatment of type 2 inflammatory diseases such as asthma and atopic dermatitis.
  • F027400161 The ability of F027400161 to inhibit 5 ng/mL IL-13+TSLP-induced CCL17 production was evaluated in human PBMCs from 8 healthy individual donors. The study was designed to evaluate non-inferiority of the ISVD construct versus monospecific biologics, anti-hTSLP reference mAb1 and anti-IL-13 reference mAb1.
  • the concentrations of the cytokines used were ⁇ 10 fold over the upper end of the pathophysiological ranges of TSLP and IL-13 that have been reported in the literature in normal humans, asthmatics and atopic dermatitis patients (Berra ⁇ es A et al, Immunol Letter 178: 85-91, 2016; Bellini A et al, Mucosal Immunology 5(2):140-9, 2012; Davoodi P et al, Cytokine 60(2):431-7, 2012; Szegedi K et al, J Eur Acad Dermatol Venereol 29(11):2136-44, 2015) as hypothetical concentrations during transient inflammatory state.
  • F027400161 demonstrated 100% inhibition of 5 ng/mL IL-13+TSLP-induced synergistic CCL17 production with a mean IC 50 of 0.0387 nM. While PBMCs treated with equimolar doses of the comparator antibody anti-hlL-13 reference mAb1 showed a lower mean IC 50 of 0.01339 nM, the inhibition response never reached 100% and plateaued at approximately 90% inhibition. On the other hand, anti-hTSLP reference mAb1 only partially blocked approximately 40% of the CCL17 production with a mean IC 50 of 19 nM.
  • TSLP drives type 2 immune response by inducing CCL17, IL-5, and IL-13 production. Subsequently, IL-13 triggers CCL26 production by local epithelial cells, leading to the ramification of type 2 immune response mediated inflammatory diseases and beyond.
  • F027400161 to inhibit TSLP-induced IL-5 and CCL17, and IL-13-induced CCL26 production was evaluated in a triculture assay system using MRCS fibroblasts and A549 epithelial cells coculturing with Der P-stimulated human PBMCs from 6 individual normal donors for 6 days.
  • the study was designed to evaluate non-inferiority of the ISVD versus monospecific biologics, anti-hTSLP reference mAb1 and anti-hIL-13 reference mAb1.
  • MRCS fibroblasts produced ⁇ 100 pg/mL endogenous TSLP constitutively, and A549 epithelial cells produced CCL26 in response to IL-13 produced by PBMCs with Der P and endogenous TSLP stimulation.
  • One day prior to coculture with human PBMCs seventy-five thousand MRC5 fibroblasts and A549 epithelial cells per well were plated.
  • One million cells per well of human PBMCs were added into plated MRC5 fibroblasts and A549 epithelial cells, stimulated with 3 ⁇ g/mL of Der P, and incubated with 11.1 nM of ISVD, anti-hlL-13 reference mAb1, or anti-hTSLP reference mAb1 in a 24-well plate for 6 days in a 37oC cell-culture incubator.
  • the assays were performed with technical triplicates within each donor for F027400161.
  • the production of IL-5, CCL17, and CCL26 in freshly collected cell culture supernatant was measured by Human Magnetic Luminex Assays from RnD System.
  • FIG. 9 The collective results of the inhibition responses of F027400161 and benchmark antibodies, anti-hlL-13 reference mAb1 and anti-hTSLP reference mAb1, are shown in FIG. 9 .
  • F027400161 demonstrated 60% inhibition of CCL17, 50% inhibition of IL-5, and 95% inhibition of CCL26 production.
  • the reference antibody anti-hTSLP reference mAb1 displayed 50% inhibition of CCL17, 50% inhibition of IL-5, and approximately 55% inhibition of CCL26 production.
  • anti-hlL-13 reference mAb1 demonstrate comparable 95% inhibition of CCL26 production, this reference antibody was only able to block approximately 35% of the CCL17 production and less than 10% of IL-5 production ( FIG. 9 ). Lack of complete inhibition of IL-5 and CCL17 by these tested molecules may suggest that Der P stimulation triggers PBMCs to elicit pathways other than TSLP and IL-13 to drive the production of IL-5 and CCL17.
  • F027400161 targets both human TSLP and IL-13 and does not cross react with the murine orthologs. Hence, to evaluate the biological activities of F027400161, a xenografted, humanized model system was used.
  • Female NSG-SGM3 (NOD/SCID-IL2R ⁇ / ⁇ , NOD.Cg-PrkdcscidlI2r ⁇ tm1Wjl/SzJ) were obtained from Jackson Labs, Bar Harbor, Me., USA.
  • mice express human hematopoietic cytokines: stem cell factor (SCF), granulocyte/macrophage stimulating factor (GM-CSF), and interleukin-3 (IL-3), all driven by a human cytomegalovirus promoter/enhancer sequence.
  • SCF stem cell factor
  • GM-CSF granulocyte/macrophage stimulating factor
  • IL-3 interleukin-3
  • the triple transgenic mouse produces above cytokines constitutively, providing cell proliferation and survival signals, supporting the stable engraftment of CD33+myeloid lineages, and several types of lymphoid cells. Briefly, the protocol followed for engraftment is as follows:
  • mice On day 0 of the study, mice were irradiated with 150 centiGray at a rate of 120 rads/minute for 1 minute and 15 seconds. Mice were engrafted with 1 ⁇ 10 5 cord blood CD34+stem/progenitor cells by the intravenous (IV) route in 200 ⁇ I of Dulbecco's phosphate buffered saline (DPBS) approximately 6 hours post-engraftment. One group of mice were irradiated in the same manner, but not engrafted. These mice are considered irradiated na ⁇ ve mice. On day 88 post engraftment, mice received a hydrodynamic (HDD) i.v.
  • HDD hydrodynamic
  • mice that received minicircle DNA and showed plasma TSLP levels 1 standard deviation below the mean were also removed from the study.
  • Mice that received minicircle DNA by HDD i.v. injection received subcutaneous doses of either vehicle (20 mM phosphate, 125 mM L-arginine HCL, and 0.01% tween 20 pH 7.0) or F027400161 (0.01, 0.05, 0.1, or 10 mg/kg) on days 95, 97, 99, and 101.
  • Plasma levels of human TSLP from the day 103 plasma samples were determined by MSD kit evaluation (Cat# K15067-L-2, Meso Scale Diagnostics, Rockville, Md., USA). Plasma levels of human IL-13 from the day 103 plasma samples were determined by ELISA (Cat# 88-7439-88Human IL-13 ELISA kit, Invitrogen/Thermo-Fischer, Waltham, Mass., USA). Internal assay validation experiments demonstrated that both the human TSLP and IL-13 detection kits were not able to detect F027400161 bound hTSLP and hIL-13.
  • RNAs were processed for quantification by TaqMan and the data analyzed as described in the protocols.
  • lungs harvested from the mice were stored in RNALater, processed and purified according to standard protocols to generate high quality RNA.
  • Purified lung RNA was then reverse transcribed to cDNA using Quanta 0-Script 5X master mix according to manufacturer's protocol.
  • Obtained lung cDNA was used to quantify the transcript expression levels of the human IL-13 responsive mouse target genes (mouse Retnla and mouse Clca1) and an endogenous control (Rpl37a) using a TaqMan assay according to manufacturer's protocols.
  • Data analysis was performed in Quantstudio 6&7 flex software. For each probe, C T values and delta C T values (against RpI37a) were exported into excel and relative expression values for each gene were calculated using the following formula:
  • Normalized relative expression (Power(2, ⁇ (delta CT ))*1000.
  • the two human IL-13 regulated mouse genes evaluated were Retnla (Resistin like alpha), that plays a role in pulmonary vascular remodeling and Clca1 (chloride channel accessory 1) (Lewis CC, 2009, J Allergy Clin Immunol.;123(4):795-804).
  • Lungs were harvested from the mice and stored in RNA later. Lung lobes were then dried and transferred to fastprep lysing Matrix A tubes (for homogenizing lungs) containing 1 mL RLT+2-ME. Samples were homogenized in MP-Bio homogenizer using program 1 (Two 40 second cycles separated by 5 minutes to avoid sample heatup). Samples were then spun at 10,000g for 3 minutes. Collected 350ul of lysate [in RLT+2ME (1% v/v)]. Pipette using a multi-channel multiple times ( ⁇ 20x) to lyse the cells.
  • RNA purification 350ul homogenate was used. 1 ⁇ volume (350 ul) of 70% Ethanol was added and the homogenate mixed thoroughly and transferred to a 96 well RNeasy spin plate placed in elution plate and RNA was prepared using the Qiagen RNA mini tissue RNA extraction protocol with following modifications.
  • the 96 well plate was covered with sealable aluminum foil and centrifuged for 2 minutes at 4000 ⁇ g. To wash the column, 400 ⁇ l Buffer RWT was added to the RNeasy spin columns and the spin-column plate was centrifuged for 2 minutes at RT at 4000 ⁇ g. DNAse I digestion was carried out by adding 80 ul of 1 ⁇ DNAse I mix and incubating at RT for 15 minutes.
  • Quanta Q-Script 5 ⁇ master mix was used and cDNA was synthesized using manufacturer's protocol. Final concentration of cDNA was 25 ng/ul. cDNA was stored at ⁇ 20 Celsius till the TaqMan assay was performed.
  • TaqMan multiplex master mix was prepared in a 1.5 ml microcentrifuge tube by adding the components in the following order. Three separate master mixes made for each probe-set along with the internal Rpl37a control. Each multiplex qPCR reaction was conducted in a 10 ⁇ l reaction volume.
  • thermo cycler pre-denaturation at 95° C. for 3 min, 40 cycles of denaturation at 95° C. for 2 s, and annealing and extension at 60° C. for 5 s. Fluorescent measurements were carried out during the extension step.
  • CT values and delta CT values were exported into excel and relative expression values for each gene were calculated using the following formula:
  • Thymic stromal lymphopoietin master switch for allergic inflammation.
  • polypeptides, nucleic acid molecules encoding the same, vectors comprising the nucleic acids and compositions described herein may be used for example in the treatment of subjects suffering from inflammatory diseases.

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JP2023504914A (ja) 2023-02-07
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IL293548A (en) 2022-08-01
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MY208649A (en) 2025-05-22
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CA3163910A1 (en) 2021-06-17
CO2022008813A2 (es) 2022-07-19
KR20220123237A (ko) 2022-09-06
CN114980974A (zh) 2022-08-30
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US11840566B2 (en) 2023-12-12
US20220177566A1 (en) 2022-06-09
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AU2020400938A1 (en) 2022-07-28
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