WO2017102835A1 - Molécule de liaison comprenant un polypeptide-alphacorps et un anticorps - Google Patents

Molécule de liaison comprenant un polypeptide-alphacorps et un anticorps Download PDF

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Publication number
WO2017102835A1
WO2017102835A1 PCT/EP2016/080986 EP2016080986W WO2017102835A1 WO 2017102835 A1 WO2017102835 A1 WO 2017102835A1 EP 2016080986 W EP2016080986 W EP 2016080986W WO 2017102835 A1 WO2017102835 A1 WO 2017102835A1
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alphabody
antibody
polypeptide
binding
target
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PCT/EP2016/080986
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English (en)
Inventor
Yvonne MCGRATH
Stefan Loverix
Sophie THIOLLOY
Franky BAATZ
Eric LORENT
Karen VANDENBROUCKE
Maria Paulina HENDERIKX
Sabrina DEROO
Ignace Lasters
Johan Desmet
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Complix Nv
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Publication of WO2017102835A1 publication Critical patent/WO2017102835A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the present invention relates to multispecific targeting molecules comprising an alphabody- polypeptide and an antibody and their use in treating diseases, in particular inflammatory diseases such as autoimmune diseases.
  • multispecific targeting in which two or more targets are tackled simultaneously by means of a single multispecific compound, has been adopted as an attractive approach by several pharma companies.
  • CMC Chremistry, Manufacturing and Control
  • These difficulties primarily relate to the inherent complexity of the molecular structures of multispecific compounds.
  • the vast majority of multispecific targeting molecules involve the use of multispecific antibodies, i.e., antibody variants comprising different binding sites, each directed towards a different target. This is an appealing approach, as it involves the use of natural targeting molecules in a substantially natural configuration (i.e., the overall antibody architecture is largely maintained).
  • such approaches are complicated by the requirement of efficient binding of two or more dissimilar targets at two or more dissimilar sites.
  • bi- or multispecific compounds should also permit simultaneous binding to their respective distinct target molecules.
  • a major problem to obtain such multispecific molecular constructs lies in the fact that the distinct binding functionalities are usually selected or optimized independently.
  • Such independently optimized intermediate constructs must then be combined into a final multispecific construct without a priori guarantee of success. For example, optimization of one site can cause a reduction in affinity or potency of a second site, or an independently optimized site can itself be reduced in affinity or potency when combined with a second site.
  • such multispecific product may have undesirable physical characteristics such as inferior solubility, stability, pi, etc., as a result of the combination step.
  • the present inventors have surprisingly found that multispecific binding molecules can be obtained which comprise at least two alphabody-polypeptides and one antibody, while at the same time some of the drawbacks of existing multispecific antibody constructs are avoided.
  • the multispecific binding molecules according to the present invention are essentially fusion proteins comprising at least an alphabody- polypeptide that is covalently coupled (fused) to the N-terminus of an antibody light chain or an alphabody-polypeptide that is covalently coupled (fused) to the N-terminus of an antibody heavy chain.
  • the multispecific binding molecules according to the invention comprise at least an intact antibody, i.e., a properly folded immunoglobulin structure consisting of two light chains and two heavy chains that are covalently connected by disulfide bonds.
  • the multispecific binding molecules according to the invention essentially comprise at least an antibody structure and two alphabody-polypeptide structures which are covalently fused to the N-termini of either the antibody light chains or the heavy chains.
  • Both the alphabody-polypeptide and antibody structures comprised within the binding molecules according to the invention, possess a distinct target binding specificity. This feature renders the molecules of the invention at least bispecific. It will be understood that both the alphabody-polypeptide and antibody molecules may individually possess more than one distinct binding functionality, in which case these molecules are multispecific in the sense of 'more than bispecific'. However, bispecificity is herein also considered a form of multispecificity. However, in particular embodiments, the application provides bispecific binding molecules comprising an alphabody-polypeptide and an antibody binding function.
  • the present inventors have surprisingly found that the alphabody-polypeptides, when fused to the N-terminus of either the light or heavy chains in an intact antibody, but not to the C-terminus of the light nor the heavy chain, retain a target binding strength that is essentially unaltered compared to the binding strength as measured for the isolated, non-coupled alphabody-polypeptide.
  • the present inventors have surprisingly found that the alphabody-polypeptides, when fused to the N-terminus of either the light or heavy chains in an intact antibody, but not to the C-terminus of the light nor the heavy chain, obtain a significantly increased potency in cellular assays, this increased potency being referred to hereinafter as a 'potency jump'.
  • This potency jump is manifested as a several fold higher ratio in cellular inhibition potency over target binding affinity for the molecules according to the invention than for the same alphabody-polypeptides when not coupled to an antibody.
  • the molecules according to the invention have a several fold lower IC50/KD than the same alphabody- polypeptides in these molecules when measured in a non-coupled state.
  • Such several fold lower IC50/KD is referred to as a jump in potency (relative to affinity), and could not have been a priori anticipated on the basis of the mere fusion step.
  • the present inventors have surprisingly found that the multispecific molecules according to the invention, comprising alphabody-polypeptides that are fused to the N-termini of either the light or heavy chains of an antibody, can be produced at high yields in conventional mammalian host cells.
  • Such high yields are surprising in view of the alpha- helical nature of the fused alphabody-polypeptides, whereas the antibody to which they are fused is an immunoglobulin composed of immunoglobulin domains having a beta-stranded structure.
  • an alpha-helical domain fused to an all-beta immunoglobulin chain represents a non-natural element in an antibody context, which could have impeded any step in the recombinant production process.
  • the obtained high production yields imply that the synthesis, folding and assembly of the immunoglobulin part within the molecules according to the invention is essentially not affected despite the presence of an N-terminally fused alphabody-polypeptide.
  • fusion constructs between an alpha-helical structure, in particular an alphabody-polypeptide which essentially consists of a 3-stranded coiled coil structure have never been made or contemplated before.
  • the present inventors have surprisingly found that the multispecific molecules according to the invention can be extremely potent when, on the one hand the alphabody- polypeptides contained within these molecules are directed against a given cytokine and, on the other hand the antibody part is directed against a target different from this given cytokine.
  • the present inventors have surprisingly found that the multispecific molecules according to the invention can be conveniently optimized through independent optimization of the constituting binding components.
  • the alphabody- polypeptides contained within these molecules are directed against the cytokine human IL- 23, and the antibody part is directed against a different cytokine, human TNFa.
  • the said anti- IL-23 alphabody-polypeptides have been optimized in the form of isolated, soluble and monovalent proteins as described in WO2014064080. Some of these alphabody- polypeptides, and a number of variants thereof, are herein disclosed as fusion constructs to the anti-TNFa antibody adalimumab.
  • alphabody- polypeptides such as alphabody-polypeptides directed against human IL-23
  • independently selected antibodies such as the antibody adalumimab directed against human TNFa
  • fusion constructs as presently claimed, thereby obtaining multispecific molecules having beneficial characteristics with regard to production, physical behaviour (affinity, solubility, stability, pi) and activity in cellular assays (target neutralizing potency).
  • the multispecific binding molecules according to the invention have the additional advantage that two targets which are involved for instance in the development or progression of diseases can be targeted simultaneously using only a single binding molecule.
  • a lower dose may be applied compared to single targeting of either compound. Lower doses may obviously result in less off-target effects.
  • one target may be located on a diseased cell, such as an infected cell or a cancer cell, whereas another target may be an immunomodulatory target, which may be recruited to the diseased cell and specifically act thereon.
  • a fusion protein comprising an antibody and two alphabody-polypeptides is capable of simultaneously binding the respective targets. Contrary to expectations, fusing alphabody-polypeptides to an antibody in the manner as claimed does not interfere with either alphabody-polypeptide or antibody functionality and thereby permits simultaneous binding to distinct target molecules.
  • the multispecific binding molecule comprises, consists of, or consists essentially of a fusion protein or chimeric protein comprising, consisting of, or consisting essentially of an alphabody-polypeptide specifically binding a first target; and an antibody specifically binding a first target, or an antigen binding fragment of said antibody.
  • the application provides a multispecific binding molecule characterized in that it comprises at least two Alphabody-polypeptides and an antibody, wherein said Alphabody-polypeptides are capable of binding to a first target molecule and said antibody is capable of binding to at least a second target molecule different from said first target molecule, and said Alphabody-polypeptides are covalently fused to the N-terminus of the light chains or the heavy chains of said antibody.
  • said alphabody-polypeptide or said antibody is a neutralizing alphabody-polypeptide or antibody.
  • said alphabody-polypeptide and said antibody is a neutralizing alphabody-polypeptide and antibody.
  • the multispecific binding molecule provided herein binds said first and second target simultaneously, or is capable of binding said first and second target simultaneously.
  • a linker connects said alphabody-polypeptide with said antibody or antibody fragment, whereby said linker can be a peptide linker.
  • said linker is a flexible linker. More particularly said linker has an amino acid composition comprising at least 50% amino acids selected from the group consisting of glycine, alanine, serine, threonine, proline or derivatives thereof.
  • said linker has a sequence length of between 3 and 50 amino acids, preferably between 3 and 35 amino acids, more preferably between 3 and 20 amino acids.
  • said linker comprises, consists of, or consists essentially of a sequence as set forth in any of SEQ ID NOs: 47 to 51 , or combinations thereof.
  • said first target is a cytokine.
  • said second target is a cytokine.
  • said first and/or second target is a growth factor
  • said first and/or second target is a growth factor.
  • said first target is a cytokine and said second target is a growth factor or said first target is a growth factor and said second target is a cytokine.
  • both said first target and said second target is a cytokine.
  • said first target is IL-23 and said second target is TNF-a.
  • said alphabody-polypeptide is additionally connected to said antibody through a disulphide bond.
  • the sequence of the alphabody-polypeptide is adjusted to optimize the pH of the multispecific binding molecule. In particular embodiments of the multispecific binding molecule provided herein.
  • multispecific binding molecules or fusion protein or chimeric protein comprising at least an alphabody-polypeptide and an antibody wherein said alphabody- polypeptide is capable of binding to a first target and said antibody is capable of binding to at least a second target, wherein said first and second target are identical or different targets.
  • said alphabody-polypeptide and said antibody (fragment) bind the same epitope of the same target, bind different epitopes of the same target, or bind different epitopes of different targets.
  • said fragment of said antibody comprises, consist of, or consists essentially of the three CDRs of the antibody VL and/or the three CDRs of the antibody VH, preferably the three CDRs of the antibody VH and the three CDRs of the antibody VL.
  • said fragment comprises, consists of, or consists essentially of the antibody VH and/or the antibody VL, preferably the antibody VH and the antibody VL.
  • said first and/or second target is selected from: a protein, glycoprotein, lipid, glycolipid, or polysaccharide; a transmembrane protein and/or an extracellular protein.
  • said first and/or said second target is a receptor.
  • said fragment comprises, consists of, or consists essentially of a VH, VL, sdAb, dAb, scFv, Fv, Fab, Fab', F(ab)2, F(ab')2, diabody, minibody, triabody.
  • said alphabody-polypeptide is fused to the C-terminus of the antibody or antibody fragment H- chain;
  • the C-terminus of the antibody or antibody fragment L-chain the N-terminus of the antibody or antibody fragment H-chain, or the N-terminus of the antibody or antibody fragment L- chain.
  • said alphabody-polypeptide is fused to the C-terminus of the antibody or antibody fragment H- chain, the C-terminus of the antibody or antibody fragment L chain, the N-terminus of the antibody or antibody fragment H-chain, and/or the N-terminus of the antibody or antibody fragment L chain.
  • said alphabody-polypeptide is connected or further connected to said antibody or antibody fragment through a disulphide bond.
  • said second alphabody-polypeptide specifically binds, or is capable of specifically binding to the same or a different epitope of the same or a different antigen.
  • said second alphabody-polypeptide is identical to or different from the first alphabody-polypeptide.
  • the application further provides polynucleic acids encoding the multispecific binding molecule or fusion protein or chimeric protein according to any of the embodiments described herein above or combinations thereof.
  • the polynucleic acid comprises, consists of, or consists essentially of a sequence encoding the multispecific binding molecule or fusion protein or chimeric protein according to any of the embodiments above, or combinations thereof.
  • the application further provides vectors comprising the polynucleic acid according to any of the embodiments above.
  • these are expression vectors, more particularly eukaryotic expression vectors.
  • the vector is a recombination vector.
  • the application further provides host cells comprising the multispecific binding molecule or fusion protein or chimeric protein according to any of the embodiments above, the polynucleic acid according to any of the embodiments above, or the vector according to any of the embodiments above.
  • the host cell is a prokaryotic or eukaryotic host cell.
  • the host cell expresses or is capable of expressing the multispecific binding molecule or fusion protein or chimeric protein according to any of the embodiments described above, or the polynucleic acid according to any of the embodiments described above.
  • the application further provides pharmaceutical compositions comprising the multispecific binding molecule or fusion protein or chimeric protein according to any of the embodiments above, the polynucleic acid according to any of the embodiments above, or the vector according to any of the embodiments above, and a pharmaceutically acceptable carrier.
  • the application further provides the multispecific binding molecule or fusion protein or chimeric protein according to any of the embodiments above, the polynucleic acid according to any of the embodiments above, or the vector according to any of the embodiments above, or the pharmaceutical composition according to the embodiments described above for use as a medicament.
  • these are envisaged for use in preventing, treating, and/or alleviating a disease, preferably a human disease.
  • these are envisaged for use in preventing, treating, and/or alleviating cancer, acute or chronic inflammation, an infectious disease, or an autoimmune disease.
  • the terms "one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6 or ⁇ 7 etc. of said members, and up to all said members.
  • the present invention relates to multispecific binding molecules, also referred to herein as "multispecifics” comprising, consisting of, or consisting essentially of the alphabody-polypeptide specifically binding to a first target and an antibody or antibody fragment specifically binding to at least a second target.
  • multispecific binding molecules also referred to herein as "multispecifics” comprising, consisting of, or consisting essentially of the alphabody-polypeptide specifically binding to a first target and an antibody or antibody fragment specifically binding to at least a second target.
  • bispecific binding molecules also referred to herein as "bispecifics” comprising, consisting of, or consisting essentially of an alphabody-polypeptide specifically binding to a first target and an antibody or antibody fragment specifically binding to a second target.
  • first target and second target refer to molecules or structures which can be bound by the alphabody-polypeptide or antibody (fragment).
  • the first and/or second target is a protein, glycoprotein, polysaccharide, lipid, or glycolipid.
  • the targets as referred to herein can be any type of target, but preferably is a target which is involved in (e.g. causally involved in) the development , maintenance, or progression of a disease; or alternatively which is involved in resolving, clearing, treating, preventing, or otherwise alleviating a disease.
  • Suitable first and/or second targets include pathogen antigens, cancer antigens, cytokines, etc.
  • the first and/or second target is a cytokine or growth factor.
  • the first and/or second target are selected from cardiotrophin 1 , cardiotrophin- like cytokine factor 1 , cardiotrophin-like cytokine factor 1 , ciliary neurotrophic factor, interleukin 1 1 , interleukin 6 (interferon, beta 2), leukemia inhibitory factor (cholinergic differentiation factor), oncostatin M, interleukin 4, isoform 1 , interleukin 13, interleukin 12A, interleukin 23, alpha subunit p19, colony stimulating factor 2, interleukin 3, interleukin 5, interleukin 2, interleukin 4 isoform 1 , interleukin 7, interleukin 9, interleukin 15, preproprotein, interleukin 21 , colony stimulating factor 3 isoform a, erythropoietin, growth hormone 1 isoform 1 , growth hormone 2 isoform 1 , leptin, pro
  • the first and or second target is a receptor, such as a cytokine receptor or a growth factor receptor.
  • said first target is a cytokine and said second target is a receptor.
  • the first and/or second target is a cytokine, whereby said first target is interleukin 23 (IL23).
  • the first target is IL23 and the second target is tumor necrosis factor alpha.
  • the first (or second) target is a pathogen target, which may be a surface-exposed antigen.
  • the second (or first) target may be a target involved in pathogen clearance, such as a specific cytokine, or receptor target of a specific leukocyte, or a target of the complement system or activating the complement system.
  • the first and/or second target may alternatively also be an intracellular target.
  • the disease is a disease caused by a virus, such as AIDS (caused by HIV), AIDS Related Complex (caused by HIV), Aseptic meningitis (caused by HSV-2), Bronchiolitis (caused by e.g. RSV), Common cold (caused by e.g. RSV or Parainfluenza virus), Conjunctivitis (caused by e.g. Herpes simplex virus), Croup (caused by e.g.
  • AIDS caused by HIV
  • AIDS Related Complex caused by HIV
  • Aseptic meningitis caused by HSV-2
  • Bronchiolitis caused by e.g. RSV
  • Common cold caused by e.g. RSV or Parainfluenza virus
  • Conjunctivitis caused by e.g. Herpes simplex virus
  • Croup caused by e.g.
  • parainfluenza viruses 1 to 3 Dengue fever (caused by dengue virus), Eastern equine encephalitis (caused by EEE virus), Ebola hemorrhagic fever (caused by Ebola virus), encephalitis and chronic pneumonitis in sheep (caused by Visna virus), encephalitis (caused by Semliki Forest virus), Gingivostomatitis (caused by HSV-I), Genital herpes (caused by HSV-2), Herpes labialis (caused by HSV-I), neonatal herpes (caused by HSV-2), Genital HSV (caused by Herpes simplex virus), Influenza (Flu) (caused by influenza viruses A, B and C), Japanese encephalitis virus (caused by J EE virus), Keratoconjunctivitis (caused by HSV- I), Lassa fever, Leukemia and lymphoma (caused by e.g.
  • Human T cell leukemia virus or Moloney murine leukemia virus Lower respiratory tract infections (caused by e.g. RSV or Sendai virus), Measles (caused by rubeola virus), Marburg hemorrhagic fever (caused by Marburg virus), Molluscum contagiosum (caused by Molluscum), Mononucleosis-like syndrome (caused by CMV), mumps (caused by mumps virus), Newcastle disease (caused by avian paramoxyvirus 1 ), Norovirus, Orf (caused by Orf virus), Pharyngitis (caused by e.g.
  • RSV Influenza virus, Parainfluenza virus and Epstein- Barr virus
  • Pneumonia viral
  • RSV or CMV Progressive multifocal leukencephalopathy
  • Rabies caused by Rabies virus
  • Roseola caused by HHV-6
  • Rubella caused by rubivirus
  • SARS caused by a human coronavirus
  • Shingles caused by Varicella zoster virus
  • Smallpox caused by Variola virus
  • St. Louis encephalitis caused by SLE virus
  • Strep Throat caused by e.g.
  • the first and/or second target is an antigen, preferably a surface-exposed antigen on these viruses.
  • the first (or second) target is a cancer-specific or cancer-associated target, which may be a cell surface-exposed antigen.
  • the second (or first) target may be a target involved in cancer cell removal (e.g. through apoptosis, phagocytosis, etc.), such as a specific cytokine, or receptor target of a specific leukocyte.
  • the first and second target are both involved in the development, maintenance, or progression of a disease, such as an inflammatory disease or an autoimmune disease.
  • the alphabody-polypeptide and antibody (fragment) preferably neutralize their respective target.
  • nucleic acid molecule As used herein, the terms “nucleic acid molecule”, “polynucleotide”, “polynucleic acid”, “nucleic acid” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
  • the nucleic acid molecule may be linear or circular.
  • polypeptide As used herein, the terms “polypeptide”, “protein”, “peptide”, and “amino acid sequence” are used interchangeably, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. As used herein, amino acid residues will be indicated either by their full name or according to the standard three-letter or one-letter amino acid code.
  • nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question, to a particular amino acid sequence, e.g., the amino acid sequence of a desired polypeptide or protein.
  • nucleic acids “encoding” a particular polypeptide or protein may encompass genomic, hnRNA, pre- mRNA, mRNA, cDNA, recombinant or synthetic nucleic acids.
  • a nucleic acid encoding a particular polypeptide or protein may comprise an open reading frame (ORF) encoding said polypeptide or protein.
  • ORF open reading frame
  • An "open reading frame” or “ORF” refers to a succession of coding nucleotide triplets (codons) starting with a translation initiation codon and closing with a translation termination codon known per se, and not containing any internal in-frame translation termination codon, and potentially capable of encoding a polypeptide.
  • the term may be synonymous with "coding sequence” as used in the art.
  • vector is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant/animal virus, into which a polynucleotide can be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell, or can be integrated within the genome of the defined host such that the cloned sequence is reproducible.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the expression construct is an expression vector, suitable for transformation into host organisms/cells, and suitable for maintenance and/or expression of the polynucleic acid according to the present invention as described herein in a transformed host cell.
  • the term "multispecific" as an adjective means that the binding molecule or fusion/chimeric protein referred to is capable of binding at least two different targets or structures, or alternatively two different domains or epitopes or the same target molecule.
  • the term multispecific refers to the capability of binding at least the first and second target.
  • the binding molecules according to the invention are bi-specific in that they only bind the first and second target, and hence do not or at least not intentionally bind other molecules or structures. More particularly, in the context of the present invention at least one target binding function is generated by the alphabody-polypeptide component and a different target binding function is generated by the antibody component.
  • the binding molecules of the invention may be trispecific, tertraspecific, or more in general multispecific, i.e. these can be designed to bind more molecules or structures than the first or second target alone.
  • a trispecific binding molecule may be generated by coupling a multispecific antibody (fragment) capable of binding the second target and also capable of binding another molecule or target, and which antibody (fragment) is coupled to an alphabody-polypeptide capable of binding the first target.
  • a trispecific binding molecule may be generated which contains an antibody (fragment) capable of binding the second target which is coupled to an alphabody-polypeptide capable of binding the first target and further coupled to one or more alphabody-polypeptide capable of binding a third, fourth, ... target.
  • both the alphabody-polypeptide and the antibody (fragment) specifically bind their respective target.
  • the term “specifically” refers to an essentially exclusive binding to the target, i.e. (essentially) only the target is or can be bound.
  • the binding molecule or fusion/chimeric protein according to the invention therefore selectively binds a first and a second target.
  • the binding molecule or fusion/chimeric protein of the invention is said to specifically bind to a particular target when the binding molecule or fusion/chimeric protein of the invention has affinity for, specificity for, and/or is specifically directed against that target (or against at least one part or fragment thereof).
  • the specificity of a binding molecule or fusion/chimeric protein of the invention as used herein can be determined based on affinity and/or avidity.
  • the 'affinity' of a binding molecule or fusion/chimeric protein of the invention is represented by the equilibrium constant for the dissociation of the binding molecule and the target protein of interest to which it binds. The lower the KD value, the stronger the binding strength between the binding molecule or fusion/chimeric protein and the target protein of interest to which it binds.
  • the affinity can also be expressed in terms of the affinity constant (KA), which corresponds to 1/KD.
  • KA affinity constant
  • the binding affinity of an binding molecule of the invention can be determined in a manner known to the skilled person, depending on the specific target protein of interest.
  • the KD can be expressed as the ratio of the dissociation rate constant of a complex, denoted as kOff (expressed in seconds-1 or s-1 ), to the rate constant of its association, denoted kOn (expressed in molar-1 seconds-1 or M 1 s-1 ).
  • kOff Expressed in seconds-1 or s-1
  • kOn Expressed in molar-1 seconds-1 or M 1 s-1
  • a KD value greater than about 1 millimolar is generally considered to indicate non-binding or non-specific binding.
  • the 'avidity' of an alphabody-polypeptide of the invention is the measure of the strength of binding between a binding molecule or fusion/chimeric protein of the invention and the pertinent target protein of interest.
  • Avidity is related to both the affinity between a binding site on the target protein of interest and a binding site on the binding molecule or fusion/chimeric protein of the invention and the number of pertinent binding sites present on the binding molecule of the invention.
  • a binding molecule or fusion/chimeric protein of the invention is said to be 'specific for a first target protein of interest as opposed to a second target protein of interest' when it binds to the first target protein of interest with an affinity that is at least 5 times, such as at least 10 times, such as at least 100 times, and preferably at least 1000 times higher than the affinity with which that binding molecule or fusion/chimeric protein of the invention binds to the second target protein of interest.
  • a binding molecule or fusion/chimeric protein when said to be 'specific for' a first target protein of interest as opposed to a second target protein of interest, it may specifically bind to (as defined herein) the first target protein of interest, but not to the second target protein of interest.
  • epitope includes any molecular or structural determinant (such as a peptide, or a specific proteinaceous 3D structure) capable of specific binding to an antibody (fragment) or alphabody-polypeptide.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an antibody (fragment) or alphabody-polypeptide.
  • an antibody (fragment) or alphabody-polypeptide is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • the binding molecules or fusion/chimeric protein according to the invention is capable of binding a human first and/or second target. It is to be understood that in this context when referring to "specifically" binding the first and/or second target this means that at least a human first and/or second target are specifically bound. As may or may not be the case, the orthologous first and/or second target of other species may or may not be bound.
  • the term "specifically” in this context may refer to specificity for respectively the first or second target as opposed to other molecules or structures derived from of the same or a different species, but excluding the orthologous first or second target of one or more different species.
  • the term "specifically" in this context may refer to specificity for respectively the first or second target as opposed to other molecules or structures derived from of the same species, as well as the first or second target from different species. Accordingly, in certain embodiments, the binding molecules or fusion/chimeric proteins according to the invention specifically bind to the first and/or second target, and bind to the human first and/or second target.
  • the binding molecule or fusion/chimeric protein according to the invention is capable of simultaneously binding the first and the second target.
  • Simultaneous binding means that both the first and the second target are or can be both bound to the binding molecule at the same time. This means that steric hindrance is minimized or absent.
  • the dissociation constant of the alphabody-polypeptide for the first target and/or the dissociation constant of the antibody (fragment) for the second target in the binding molecule is comparable or identical to the respective dissociation constants for the alphabody-polypeptide and/or antibody (fragment) when not fused or present in a single binding molecule. It will however be appreciated that also a less favourable dissociation constant (i.e. lower affinity) of the alphabody-polypeptide and/or antibody (fragment) need not preclude simultaneous binding of both the first and the second target.
  • the binding molecule according to the invention in essence comprises one or more particularly two Alphabody-polypeptides capable of binding a first target and an antibody or antibody fragment capable of binding a second target.
  • the alphabody-polypeptide and antibody are comprised in a single molecule, and hence are covalently linked or coupled. Such covalent linkage may be obtained by chemical coupling of the individual alphabody-polypeptide and antibody (fragment), by techniques known in the art.
  • a linker may or may not be present between the alphabody-polypeptide and the antibody (fragment), but is preferably present. Suitable linkers are described herein elsewhere.
  • the one or more alphabody-polypeptide are linked or coupled to the antibody (fragment) at the level of a polynucleic acid encoding both the alphabody- polypeptide and the heavy and/or light chain of the antibody, or an antigen-binding fragment of the heavy and/or light chain of the antibody.
  • a chimeric polynucleic acid comprises a polynucleic acid encoding the one or more Alphabody-polypeptides and a polynucleic acid encoding the heavy chain or the light chain of the antibody, or encoding an antigen-binding fragment of the heavy chain or the light chain of the antibody.
  • the alphabody-polypeptide and antibody (fragment) encoding sequences may be arranged such that the alphabody-polypeptide encoding sequence is positioned 5' of the antibody (fragment) encoding sequence, or alternatively the alphabody-polypeptide and antibody (fragment) encoding sequences may be arranged such that the antibody (fragment) encoding sequence is positioned 5' of the alphabody-polypeptide encoding sequence.
  • a nucleic acid sequence may or may not be present between the alphabody-polypeptide and antibody (fragment) encoding sequences, but is preferably present. Suitable linker sequences are described herein elsewhere.
  • a suitable antibody light chain may or may not need to be provided, which light chain in itself also may or may not be coupled to an alphabody- polypeptide, such as to result in a binding molecule comprising an alphabody-polypeptide linked to an antibody (fragment) light chain and further optionally comprising an antibody (fragment) heavy chain.
  • a suitable antibody (fragment) heavy chain may or may not need to be provided, which light chain in itself also may or may not be coupled to an alphabody-polypeptide, such as to result in a binding molecule comprising an alphabody- polypeptide linked to an antibody (fragment) heavy chain and further optionally comprising an antibody (fragment) light chain.
  • the binding molecule according to the invention comprises an alphabody-polypeptide linked to an antibody (fragment) heavy or light chain, wherein the antibody (fragment) further comprises the complementary chain (i.e. light or heavy chain).
  • the binding molecule according to the invention as described herein may thus for example contain a fusion/chimeric protein according to the invention as described herein, which may be an alphabody-polypeptide fused to a heavy or light chain, and may further comprise a light or heavy chain (depending on whether the alphabody- polypeptide is fused to respectively the heavy chain or the light chain.
  • the heavy and light chain may be comprised in a single binding molecule, or may alternatively be provided in two separate entities, which will combine under suitable conditions (i.e. reducing conditions allowing the formation of intermolecular disulphide bonds).
  • a classical antibody comprises two (identical) light chains and two (identical) heavy chains, such that the binding molecule according to the invention comprises in preferred embodiments two (identical) alphabody-polypeptides. (as for instance illustrated in Figure 2).
  • the application particularly envisages multispecific binding proteins comprising or consisting of an antibody and two Alphabody-polypeptides, wherein the Alphabody-polypeptides are capable of binding a first target and the antibody is capable of binding at least a second target, and whereby the Alphabody-polypeptides are covalently linked to either the N- terminus of the antibody light chain or the N-terminus of the antibody heavy chain.
  • both Alphabody-polypeptides are linked to the antibody heavy chains.
  • both Alphabody-polypeptides are linked to the antibody light chains.
  • the antibodies in the binding proteins comprise both heavy and light chains.
  • Alphabody-polypeptides linked to the N-terminal end of an antibody heavy or light chain has been found to ensure a surprising increase in potency of the binding of the alphabody-polypeptide to its target, compared to the alphabody-polypeptide by itself.
  • fusion protein or “chimeric protein”, which terms are used interchangeably, reference is made to a molecule comprising the alphabody- polypeptide and at least the antibody (fragment) heavy or light chain.
  • binding molecule or fusion/chimeric protein according to the invention may consist or consist essentially of the alphabody-polypeptide and antibody (fragment) as described herein further, but that additional domains, structures, or modifications may also be present (e.g. domains designed to increase half-life, markers or labels, other binding domains, etc.), as long as these do not interfere with first and/or second target binding.
  • the multispecific binding molecules of the invention comprise an alphabody-polypeptide or antibody (fragment) of which one of both is directed to (i.e. specifically binds with) a molecule or structure, binding of which results in an increased half- life.
  • the antibody (fragment) or alphabody-polypeptide may be directed to serum albumin.
  • Alphabody-polypeptides are known in the art and are for instance extensively described in WO2010066740, WO2013102659, WO2014064092, WO2012093013, WO2012092970, WO2012093172, WO2012092971 , WO2014064080, WO201 1003936, and WO201 1003935 (each of which is hereby incorporated by reference in their entirety).
  • alphabody-polypeptides for use in the binding molecules according to the present invention are those as described in WO2010066740, WO2013102659, WO2014064092, WO2012093013, WO2012092970, WO2012093172, WO2012092971 , WO2014064080, WO201 1003936, and WO201 1003935.
  • alphabody-polypeptide as used herein, can generally be defined as sequences of amino acids which are single-chain, triple-stranded, predominantly alpha-helical, coiled coil amino acid sequences. More particularly, an alphabody-polypeptide structure as used in the context of the present invention can be defined as an amino acid sequences having the general formula HRS1 -L1 -HRS2-L2-HRS3, wherein
  • each of HRS1 , HRS2 and HRS3 is independently a heptad repeat sequence (HRS) comprising or consisting of 2 to 7 consecutive but not necessarily identical heptad repeat units, at least 50% of all heptad a- and d-positions are occupied by isoleucine residues, each HRS starts with an aliphatic or aromatic amino acid residue located at a heptad a-position, particularly with an Isoleucine;
  • HRS heptad repeat sequence
  • each of L1 and L2 are independently a linker fragment, as further defined hereinafter, which covalently connect HRS1 to HRS2 and HRS2 to HRS3, respectively.
  • HRS1 , HRS2 and HRS3 will together form a triple-stranded, alpha-helical, coiled coil structure.
  • a 'parallel alphabody-polypeptide' refers to an alphabody-polypeptide as defined above further characterized in that the alpha-helices of the triple-stranded, alpha- helical, coiled coil structure together form a parallel coiled coil structure, i.e., a coiled coil wherein all three alpha-helices are oriented in parallel.
  • an 'antiparallel alphabody-polypeptide' refers to an alphabody-polypeptide as defined above further characterized in that the alpha-helices of the triple-stranded, alpha- helical, coiled coil structure together form an antiparallel coiled coil structure, i.e., a coiled coil wherein two alpha-helices are parallel and the third alpha-helix is antiparallel with respect to these two helices.
  • polypeptides comprising an amino acid sequence with the general formula HRS1 -L1 -HRS2- L2-HRS3, but which in certain particular embodiments may comprise additional residues, moieties and/or groups, which are covalently linked, more particularly N- and/or C-terminal covalently linked, to a basic alphabody-polypeptide sequence structure having the formula HRS1 -L1 -HRS2-L2-HRS3.
  • HRS1 -L1 -HRS2-L2-HRS3 a basic alphabody-polypeptide sequence structure having the formula HRS1 -L1 -HRS2-L2-HRS3.
  • '(alphabody- polypeptide) polypeptides' which comprise or consist of an alphabody-polypeptide as defined above, which may be covalently linked to additional sequences.
  • the binding features described for an alphabody-polypeptide herein can generally also be applied to polypeptides comprising said alphabody-polypeptide.
  • 'heptad', 'heptad unit' or 'heptad repeat unit' are used interchangeably herein and shall herein have the meaning of a 7-residue (poly)peptide motif that is repeated two or more times within each heptad repeat sequence of an alphabody-polypeptide structure, and is represented as 'abcdefg' or 'defgabc', wherein the symbols 'a' to 'g' denote conventional heptad positions.
  • heptad positions are assigned to specific amino acid residues within a heptad, a heptad unit, or a heptad repeat unit, present in an alphabody-polypeptide structure, for example, by using specialized software such as the COILS method of Lupas et al. (Science 1991 , 252:1 162-1 164; http://www.russell.embl-heidelberg.de/cgi-bin/coils-svr.pl).
  • the heptads or heptad units as present in the alphabody alphabody alphabody- polypeptide structure are not strictly limited to the above-cited representations (i.e.
  • 'heptad a-positions', 'heptad b-positions', 'heptad c-positions', 'heptad d- positions', 'heptad e-positions', 'heptad f-positions' and 'heptad g-positions' refer respectively to the conventional 'a', 'b', 'c', 'd', 'e', 'f and 'g' amino acid positions in a heptad, heptad repeat or heptad repeat unit.
  • a heptad motif (as defined herein) of the type 'abcdefg' is typically represented as ⁇ '
  • a 'heptad motif of the type 'defgabc' is typically represented as ⁇ '
  • the symbol 'H' denotes an apolar or hydrophobic amino acid residue
  • the symbol ' ⁇ ' denotes a polar or hydrophilic amino acid residue.
  • Typical hydrophobic residues located at a- or d-positions include aliphatic (e.g., leucine, isoleucine, valine, methionine) or aromatic (e.g., phenylalanine) amino acid residues.
  • Heptads within coiled coil sequences do not always comply with the ideal pattern of hydrophobic and polar residues, as polar residues are occasionally located at ⁇ ' positions and hydrophobic residues at ' ⁇ ' positions.
  • the patterns ⁇ ' and ⁇ ' are to be considered as ideal patterns or characteristic reference motifs.
  • the characteristic heptad motif is represented as 'HPPHCPC or 'HxxHCxC wherein ⁇ ' and ' ⁇ ' have the same meaning as above, 'C denotes a charged residue (lysine, arginine, glutamic acid or aspartic acid) and ' ⁇ ' denotes any (unspecified) natural amino acid residue.
  • a heptad can equally well start at a d-position, the latter motifs can also be written as 'HCPCHPP' or 'HCxCHxx'. It is noted that single-chain Alphabody-polypeptides are intrinsically so stable that they do not require the aid of ionic interactions between charged ('C') residues at heptad e- and g-positions.
  • a 'heptad repeat sequence' ('HRS') as used herein shall have the meaning of an amino acid sequence or sequence fragment comprising or consisting of n consecutive heptads, where n is a number equal to or greater than 2.
  • a heptad repeat sequence can thus generally be represented by (abcdefg)n or (defgabc)n in notations referring to conventional heptad positions, or by (HPPHPPP)n or (HPPPHPP)n in notations referring to the heptad motifs, with the proviso that a) the amino acids at positions a-g or H and P need not be identical amino acids in the different heptads b) not all amino acid residues in a HRS should strictly follow the ideal pattern of hydrophobic and polar residues and c) the HRS may end with an incomplete or partial heptad motif.
  • the HRS may contain an additional sequence "abc", “abed”, “abede”, or “abedef” following c-terminally the (abcdefg)n sequence.
  • a 'heptad repeat sequence' is an amino acid sequence or sequence fragment comprising n consecutive (but not necessarily identical) heptads generally represented by abcdefg or defgabc, where n is a number equal to or greater than 2, wherein at least 50% of all heptad a- and d-positions are occupied by isoleucine residues, each HRS starting with a full heptad sequence abcdefg or defgabc, and ending with a partial heptad sequence abed or defga, such that each HRS starts and ends with an aliphatic or aromatic amino acid residue located at either a h
  • heptad repeat sequences comprising amino acids or amino acid sequences that deviate from the consensus motif, and if only amino acid sequence information is at hand, then the COILS method of Lupas et al. (Science 1991 , 252:1 162-1 164) is a suitable method for the determination or prediction of heptad repeat sequences and their boundaries, as well as for the assignment of heptad positions.
  • the heptad repeat sequences can be resolved based on knowledge at a higher level than the primary structure (i.e., the amino acid sequence).
  • heptad repeat sequences can be identified and delineated on the basis of secondary structural information (i.e.
  • HRS alpha-helicity
  • tertiary structural i.e., protein folding
  • a typical characteristic of a putative HRS is an alpha-helical structure.
  • Another (strong) criterion is the implication of a sequence or fragment in a coiled coil structure. Any sequence or fragment that is known to form a regular coiled coil structure, i.e., without stutters or stammers as described in Brown et al. Proteins 1996, 26:134-145, is herein considered a HRS.
  • the identification of HRS fragments can be based on high-resolution 3-D structural information (X-ray or NMR structures).
  • the boundaries to an HRS fragment may be defined as the first a- or d-position at which a standard hydrophobic amino acid residue (selected from the group valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan) is located.
  • the boundaries to an HRS fragment can be defined by the presence of an isoleucine amino acid residue.
  • the terms 'linker', 'linker fragment' or 'linker sequence' are used interchangeably herein and refer to an amino acid sequence fragment that is part of the contiguous amino acid sequence of a single-chain alphabody-polypeptide, and which covalently interconnect the HRS sequences of that alphabody-polypeptide structure.
  • the linkers within a single-chain structure of the Alphabody-polypeptides thus interconnect the HRS sequences, and more particularly the first to the second HRS, and the second to the third HRS in an alphabody-polypeptide structure.
  • Each linker sequence in an alphabody-polypeptide structure commences with the residue following the last heptad residue of the preceding HRS and ends with the residue preceding the first heptad residue of the next HRS.
  • a linker fragment in an alphabody- polypeptide structure is preferably flexible in conformation to ensure relaxed (unhindered) association of the three heptad repeat sequences as an alpha-helical coiled coil structure.
  • 'L1 ' shall denote the linker fragment one, i.e., the linker between HRS1 and HRS2, whereas 'L2' shall denote the linker fragment two, i.e., the linker between HRS2 and HRS3.
  • Suitable linkers for use in the polypeptides envisaged herein will be clear to the skilled person, and may generally be any linker used in the art to link amino acid sequences, as long as the linkers are structurally flexible, in the sense that they do not affect the characteristic three dimensional coiled coil structure of the alphabody-polypeptide.
  • the two linkers L1 and L2 in a particular alphabody-polypeptide structure may be the same or may be different. Based on the further disclosure herein, the skilled person will be able to determine the optimal linkers, optionally after performing a limited number of routine experiments.
  • the linkers L1 and L2 are amino acid sequences consisting of at least 4, in particular at least 8, more particularly at least 12 amino acid residues, with a non-critical upper limit chosen for reasons of convenience being about 30 amino acid residues.
  • preferably at least 50% of the amino acid residues of a linker sequence are selected from the group proline, glycine, and serine.
  • the linker sequences essentially consist of polar amino acid residues; in such particular embodiments, preferably at least 50%, such as at least 60%, such as for example 70% or 80% and more particularly 90% or up to 100% of the amino acid residues of a linker sequence are selected from the group consisting of glycine, serine, threonine, alanine, proline, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine and arginine. It is envisaged that the nature of the linker is not critical with regard to the binding properties of the alphabody-polypeptides envisaged herein.
  • a 'solvent-oriented' or 'solvent-exposed' region of an alpha-helix of an alphabody-polypeptide structure shall herein have the meaning of that part on an alphabody-polypeptide structure which is directly exposed or which comes directly into contact with the solvent, environment, surroundings or milieu in which it is present.
  • the solvent-oriented region is largely formed by b-, c- and f-residues. There are three such regions per single-chain alphabody-polypeptide, i.e., one in each alpha-helix. Any part of such solvent-oriented region is also considered a solvent-oriented region. For example, a sub-region composed of the b-, c- and f-residues from three consecutive heptads in an alphabody-polypeptide alpha-helix will also form a solvent-oriented surface region.
  • an alphabody-polypeptide' shall herein have the meaning of that part on an alphabody-polypeptide of a polypeptide as envisaged herein which corresponds to the concave, groove-like local shape, which is formed by any pair of spatially adjacent alpha- helices within said alphabody-polypeptide.
  • Residues implicated in the formation of (the surface of) a groove between two adjacent alpha-helices in an alphabody-polypeptide are generally located at heptad e- and g-positions, but some of the more exposed b- and c- positions as well as some of the largely buried core a- and d-positions may also contribute to a groove surface; such will essentially depend on the size of the amino acid side-chains placed at these positions. If the said spatially adjacent alpha-helices run parallel, then one half of the groove is formed by b- and e-residues from a first helix and the second half by c- and g-residues of the second helix.
  • both halves of the groove are formed by b- and e-residues.
  • both halves of the groove are formed by c- and g-residues.
  • the three types of possible grooves are herein denoted by their primary groove-forming (e- and g-) residues: if the helices are parallel, then the groove is referred to as an e/g-groove; if the helices are antiparallel, then the groove is referred to as either an e/e-groove or a g/g-groove.
  • Parallel Alphabody-polypeptides have three e/g- grooves, whereas antiparallel Alphabody-polypeptides have one e/g-groove, one e/e-groove and one g/g-groove. Any part of an alphabody-polypeptide groove is also considered a groove region.
  • the alphabody-polypeptide and/or antibody (fragment) (and hence the binding molecule or fusion/chimeric protein according to the invention) is capable of inhibiting or neutralizing the respective target or inhibiting, preventing or decreasing the activity or function of the target, and/or of inhibiting, preventing or decreasing the signalling and biological mechanisms and pathways in which the target plays a role.
  • the alphabody-polypeptide and/or antibody (fragment) is capable of inhibiting one or more function of the respective target.
  • 'inhibiting', 'reducing' and/or 'preventing' using the polypeptides envisaged herein may mean for instance inhibiting, reducing and/or preventing the interaction between the target and a natural binding partner thereof and/or preventing one or more biological or physiological mechanisms, effects, responses, functions pathways or activities in which the target is involved, such as by at least 10%, but preferably at least 20%, for example by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more, as measured using a suitable in vitro, cellular or in vivo assay, compared to the activity of the target in the same assay under the same conditions but without using the alphabody-polypeptide and/or antibody (fragment) (and hence the binding molecule or fusion/chimeric protein according to the invention).
  • the alphabody-polypeptide and/or antibody may interfere with protein-protein interactions (or interactions between the target and other molecules or structures in general), such as binding of the target to a receptor, dimerization of the target, etc, or may for instance bind to catalytic domains of enzymes to thereby inhibit catalytic activity.
  • the pi (isoelectric point) of the alphabody-polypeptide and/or antibody (fragment) may be adjusted.
  • the pi of the alphabody-polypeptide or antibody (fragment) refers to the pi of the uncoupled alphabody-polypeptide or antibody (fragment), i.e. the pi of the alphabody-polypeptide or antibody (fragment) per se, when present in the fusion protein as described herein.
  • the isoelectric point of therapeutic proteins should not be too close to this value.
  • the normal blood pH is tightly regulated around 7.4. In instances where the isoelectric point of the binding molecule or fusion/chimeric protein according to the invention (i.e.
  • the alphabody-polypeptide or antibody (fragment) may be engineered such that the isoelectric point of the resulting binding molecule or fusion/chimeric protein becomes substantially different from the pH of the blood stream, e.g. higher than for instance 8.0 or lower than for instance 7.0.
  • the pi of the alphabody-polypeptide or antibody (fragment) may be adjusted by substituting certain amino acid residues. For instance introduction of more positive charges or reduction of the number of negative charges will increase the pi, whereas introduction of more negative charges or reduction of the number of positive charges will decrease the pi.
  • the pi of the alphabody- polypeptide or antibody (fragment) may be increased by replacing uncharged or negatively charged amino acids with positively charged amino acids, such as lysine or arginine, or by replacing negatively charged amino acids with uncharged (polar) amino acids, such as glutamine or asparagine.
  • amino acid residues which are to be replaced are not critical for alphabody-polypeptide or antibody (fragment) structure (i.e. substitution does not (substantially) interfere with or alter the coiled coil structure or the alphabody-polypeptide or the Ig domain structure of the antibody (fragment)) and target binding (i.e. substitution does not (substantially) interfere with binding affinity for the respective targets).
  • the alphabody-polypeptide is cross-linked to another molecule, preferably to another alphabody-polypeptide or the antibody (fragment). It is to be understood that such "cross-linking" does not encompass the normal covalent linkage of the alphabody-polypeptide with the antibody (fragment) in the primary protein structure (i.e. the fusion protein arrangement where alphabody-polypeptide and antibody (fragment) are respectively covalently attached through there respective N- and C-terminus).
  • the cross- linking referred to here is preferably internal cross-linking via a sulphur bridge (i.e. disulphide bond).
  • Such cross-linking may for instance be obtained by introducing one or more cysteine residues in the alphabody-polypeptide (i.e. replacing one or more amino acids with cysteine residues).
  • the amino acid residues which are to be replaced are not critical for alphabody-polypeptide structure (i.e. substitution does not (substantially) interfere with or alter the coiled coil structure) and target binding (i.e. substitution does not (substantially) interfere with binding affinity for the first target), and also, in case cross-linking with the antibody (fragment), does not interfere with binding of the antibody (fragment) to the second target.
  • cysteine residues may be introduced at suitable positions (i.e.
  • cross-linking as described herein between alphabody-polypeptides may encompass cross-linking between two alphabody-polypeptides which bind the first target, and which may be the same or different alphabody-polypeptides.
  • cross-linking as described herein between alphabody-polypeptides may encompass cross-linking between an alphabody-polypeptide which binds the first target and an alphabody-polypeptide which does not bind the first target.
  • Cross-linking of alphabody-polypeptides is particularly advantageous when the alphabody- polypeptide is fused to the C-terminus of the antibody (fragment), preferably to the C- terminus of the heavy chain of the antibody (fragment).
  • Cross-linking of alphabody- polypeptides to the antibody (fragment) is particularly advantageous when the alphabody- polypeptide is fused to the C-terminus of the antibody (fragment), preferably to the C- terminus of the light chain of the antibody (fragment).
  • antibody broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.
  • antibody fragment preferably refers to a functional fragment of the antibody, i.e. a fragment of the antibody which is still capable of (specifically) binding its antigen, i.e. the second target preferably without loss of or without compromising binding strength or affinity.
  • Such fragment retains the essential epitope binding features of the antibody, and typically comprises the antigen-binding portion of an antibody.
  • antigen-binding portion of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (i.e. the second target). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be multispecific, dual specific, or multi- specific formats; specifically binding to two or more different antigens.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab or Fab' fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 or F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb (or sdAb) fragment (Ward et al.
  • VH heavy chain
  • VL light chain
  • CDR complementarity determining region
  • single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • the binding molecule or fusion/chimeric protein according to the invention comprises a full length antibody.
  • the antibody fragment as referred to herein comprises all CDR sequences of the heavy and/or light chain, preferably of both.
  • the antibody fragment as referred to herein comprises the VH and or VL domains, such as a dAb, or comprises both VH and VL, which may or may not be linked as an scFv.
  • the antibody fragment comprises a Fab, Fab', F(ab)2, or Fab')2.
  • the antibody fragment comprises all three CDRs of the heavy and/or light chain.
  • CDR refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions.
  • CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et ah , Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.
  • CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.
  • the methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs, preferably Kabat defined CDRs.
  • framework or “framework sequence” refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations.
  • the six CDRs also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1 , FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.
  • a framework region represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain.
  • a FR represents one of the four sub-regions
  • FRs represents two or more of the four sub-regions constituting a framework region.
  • the antibody fragments as a minimal requirement comprise four FRs and three CDRs, arranged FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4, of the heavy and/or light chain.
  • the antibody (fragment) as described herein may be any type of antibody (fragment), e.g. IgA, IgG, IgD, IgM, or IgE, and any isotype, including without limitation IgG 1 , lgG2, lgG3 and lgG4.
  • the antibody (fragment) as described herein is preferably a monoclonal antibody.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al. , in: Monoclonal Antibodies and T- Cell Hybridomas 563-681 (Elsevier, N.Y., 1981 ) (said references incorporated by reference in their entireties).
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • the antibody (fragment) as described herein may originate from any organism, such as without limitation human, mouse, rabbit, rat, llama, etc. preferably, the antibody (fragment) as used herein is a human antibody (fragment) or a humanized antibody (fragment).
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • humanized antibody is an antibody or a variant, derivative, analog or fragment thereof which immune-specifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody.
  • FR framework
  • CDR complementary determining region
  • substantially in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of a non- human antibody CDR.
  • humanized antibody may refer to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e. , donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CH1 , hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • a humanized antibody only contains a humanized light chain.
  • a humanized antibody only contains a humanized heavy chain.
  • a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG 1 , lgG2, lgG3 and lgG4.
  • the humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well-known in the art.
  • CDR-grafted antibody which comprises heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having human heavy and light chain variable regions in which one or more of the human CDRs (e.g., CDR3) has been replaced with for instance murine CDR sequences.
  • the alphabody-polypeptide and the antibody fragment are coupled to each other by a linker, which is preferably a flexible peptide linker.
  • a linker which is preferably a flexible peptide linker.
  • the alphabody-polypeptide-Antibody (AA) linker is different from the linker described above in the context of the alphabody-polypeptide structure.
  • the AA linker comprises or consists of the amino acids Glycine, Serine, Alanine and Proline.
  • the linker is selected from (GGGGS) 3 , (GGGGS) 4, GSG, GPSPG, GGS(GGGGS) 2 GG and GASPAG.
  • the alphabody-polypeptide may be coupled to the antibody (fragment) at the C-terminus or the N-terminus of the antibody (fragment) heavy chain and/or light chain (or fragment thereof). It is to be understood that additional cross-linking may be effected, such as by disulphide bonds.
  • the invention provides (poly)nucleic acid sequences encoding the binding molecules or fusion/chimeric proteins according to the invention as described herein, as well as vectors and host cells comprising such nucleic acid sequences.
  • the nucleic acid sequences of the invention may be synthetic or semi-synthetic sequences, nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.
  • the (poly) nucleic acids of the invention may be DNA or RNA, and are preferably double-stranded DNA.
  • the polynucleic acids of the invention may also be in a form suitable for transformation of an intended host cell or host organism in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism.
  • the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon.
  • the vector may be an expression vector, i.e., a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).
  • the genetic constructs of the invention may comprise a suitable leader sequence to direct the expressed alphabody-polypeptide to an intended intracellular or extracellular compartment.
  • the genetic constructs of the invention may be inserted in a suitable vector at a pelB leader sequence site to direct the expressed binding molecule or fusion/chimeric protein to the bacterial periplasmic space.
  • the vector may be equipped with a suitable promoter system to, for example, optimize the yield of the binding molecule or fusion/chimeric protein.
  • vector is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant/animal virus, into which a polynucleotide can be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell, or can be integrated with the genome of the defined host such that the cloned sequence is reproducible.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the expression construct is an expression vector, suitable for transformation into host organisms, preferably bacteria, and suitable for maintenance and/or expression of the polynucleic acid according to the present invention as described herein in a transformed host cell.
  • Vectors of the present invention can be operable as cloning vectors or expression vectors in the selected host strain. Numerous vectors are known to practitioners skilled in the art, and selection of an appropriate vector is a matter of choice.
  • the vectors may, for example, be the pASK-IBA3C expression vector (IBA-life sciences), pUR5750 transformation vector (de Groot et al. 1998 Nature Biotechnology 16, 839 - 842), the pCGHT3 transformation vector (Chambers et al. 1988 Gene, Volume 68, Issue 1 : 15; Scholtmeyer etal. 2001 Appl. Environ. Microbiol. 67(1 ): 481 ).
  • an "expression vector” is a construct that can be used to transform a selected host cell and provides for expression of a coding sequence in the selected host.
  • Expression vectors can for instance be cloning vectors, binary vectors or integrating (e.g. recombination vectors, including homologous recombination, or random integration) vectors.
  • the invention thus also relates to a vector comprising any of the polynucleic acids described herein.
  • Said vector may further comprise regulatory sequences for controlling expression of the polynucleic acid in said host cell.
  • expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired product encoded by the expression vector.
  • regulatory sequences and “control sequence” used herein are to be taken in a broad context and refer to regulatory nucleic acid sequences capable of driving and/or regulating expression of the sequences to which they are ligated (covalently linked) and/or operably, linked.
  • the control sequences differ depending upon the intended host organism and upon the nature of the sequence to be expressed.
  • the control sequences generally include a promoter, a ribosomal binding site, and a terminator.
  • control sequences In eukaryotes, control sequences generally include promoters, terminators and, in some instances, enhancers, and/or 5' and 3' untranslated sequences.
  • control sequence' is intended to include, at a minimum, all components necessary for expression, and may also include additional advantageous components. According to a preferred embodiment of the present invention, the control sequence is operable in a host cell as defined herein elsewhere.
  • control sequence encompasses a promoter or a sequence capable of activating or enhancing expression of a nucleic acid molecule in a host cell.
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is (covalently and) operably linked to the nucleic acid encoding the polypeptide of interest. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of a particular nucleic acid sequence, such as that encoding a fusion protein as defined herein, to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive.
  • Inducible promoters are promoters that initiate increased levels of transcription from nucleic acid under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. At this time, a large number of promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to nucleic acid encoding the polypeptide of interest by removing the promoter from the source nucleic acid by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the naturally occurring promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the polypeptide of interest.
  • plasmid vectors containing promoters and control sequences that are derived from species compatible with the host cell are used with these hosts.
  • the vector ordinarily carries one or more replication sites as well as marker sequences, which are capable of providing phenotypic selection in transformed cells.
  • the vectors comprise a constitutive promoter.
  • constitutive promoters suitable for the constructs and methods according to the present invention include but are not limited to the CaMV35S promoter, GOS2, actin promoter, ubiquitin promoter, thiolase promoter.
  • the vectors comprise an inducible promoter.
  • inducible promoters suitable for the constructs and methods according to the present invention include but are not limited to the lac promoter or xylose inducible promoter
  • the present expression vectors will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA, and may thus contain one or more transcription termination sequences.
  • transcription termination sequence encompasses a control sequence at the end of a transcriptional unit, which signals 3' processing and termination of transcription. Additional regulatory elements, such as transcriptional or translational enhancers, may be incorporated in the expression construct.
  • the expression constructs of the invention may further include an origin of replication that is required for maintenance and/or replication in a specific cell type.
  • an origin of replication that is required for maintenance and/or replication in a specific cell type.
  • an expression construct is required to be maintained in a cell as an episomal genetic element (e.g. plasmid or cosmid molecule).
  • Preferred origins of replication include, but are not limited to the f1 -ori, colE1 ori, and Gram+ bacteria origins of replication.
  • the expression construct may optionally comprise a selectable marker gene.
  • selectable marker gene includes any gene, which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with an expression construct of the invention.
  • Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins such as chloramphenicol, zeocin (sh ble gene from Streptoalloteichus hindustanus), genetecin, melibiase (MEL5), hygromycin (aminoglycoside antibiotic resistance gene from E.
  • auxotrophic deficiencies are the amino acid leucine deficiency (e.g. LEU2 gene) or uracil deficiency (e.g. URA3 gene).
  • LEU2 gene amino acid leucine deficiency
  • URA3 gene uracil deficiency
  • Cells that are orotidine-5'-phosphate decarboxylase negative (ura3-) cannot grow on media lacking uracil.
  • ura3- cannot grow on media lacking uracil.
  • a functional URA3 gene can be used as a marker on a cell having a uracil deficiency, and successful transformants can be selected on a medium lacking uracil.
  • URA3 Only cells transformed with the functional URA3 gene are able to synthesize uracil and grow on such medium. If the wild-type strain does not have a uracil deficiency (as is the case with I. orientalis, for example.), an auxotrophic mutant having the deficiency must be made in order to use URA3 as a selection marker for the strain. Methods for accomplishing this are well known in the art. Preferred selection makers include the zeocin resistance gene, G418 resistance gene, hygromycin resistance gene.
  • the selection marker cassette typically further includes a promoter and terminator sequence, operatively linked to the selection marker gene, and which are operable in the host strain. Visual marker genes may also be used and include for example beta-glucuronidase (GUS), luciferase and Green Fluorescent Protein (GFP).
  • GUS beta-glucuronidase
  • GFP Green Fluorescent Protein
  • Plasmids containing one or more of the above listed components and including the desired coding and control sequences employs standard ligation techniques. Isolated plasmids or nucleic acid fragments are cleaved, tailored, and re-ligated in the form desired to generate the plasmids required.
  • the vector as described herein does not comprise regulatory sequences responsible for expression of the protein encoded by the polynucleic acid as described herein. This may for instance be the case for integration or recombination vectors, as is known in the art. By means of example, and without limitation, through recombination, an endogenous FAS gene, or part thereof may be replaced by the polynucleic acid according to the invention as described herein.
  • the host cell which originally comprised a naturally occurring FAS gene now comprises a recombinant FAS gene comprising a TE domain, under control of the endogenous promoter.
  • random integration may be performed, in which case, the polynucleic acid according to the invention as described herein, preferably is operably linked to one or more regulatory sequences, as described herein elsewhere, such as for instance a constitutive or inducible promoter.
  • Genetic modification of the host strains is accomplished in one or more steps via the design and construction of appropriate vectors and transformation of the host strain with those vectors. Electroporation and/or chemical (such as calcium chloride- or lithium acetate-based) transformation methods or Agrobacterium tumefaciens-medlated transformation methods as known in the art can be used.
  • the vectors can either be cut with particular restriction enzymes or used as circular DNA.
  • the vector used for genetic modification of the host strains may be any vector so long as it can integrate in the genome of the host strain.
  • Successful transformants can be selected for in known manner, by taking advantage of the attributes contributed by the marker gene, or by other characteristics (such as ability to produce fatty acids, inability to produce lactic acid or lactate, inability to produce acetic acid or acetate, or ability to grow on specific substrates) contributed by the inserted genes. Screening can be performed by PCR or Southern analysis to confirm that the desired insertions and deletions have taken place, to confirm copy number and to identify the point of integration of genes into the host strain's genome. Activity of the enzyme encoded by the inserted gene and/or lack of activity of enzyme encoded by the deleted gene can be confirmed using known assay methods.
  • the present invention provides host cells comprising nucleic acids or vectors as described herein.
  • a particular embodiment of the invention is a host cell transfected or transformed with a vector comprising the nucleic acid sequence or vector as described herein.
  • Suitable examples of hosts or host cells for expression of the binding molecules or fusion/chimeric proteins of the invention will be clear to the skilled person and include any suitable eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
  • the host cells produce the binding molecule or fusion/chimeric protein according to the invention.
  • the binding molecule or fusion/chimeric protein is secreted.
  • Suitable means for obtaining secretion are well known in the art, and by means of example include the use of (cleavable) signal peptides.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention.
  • Pharmaceutical compositions typically comprise one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions of the invention may further optionally comprise at least one other pharmaceutically active compound.
  • compositions of the present invention can be used in the diagnosis, prevention, alleviation and/or treatment of diseases and disorders, as defined herein elsewhere.
  • the present invention provides pharmaceutical compositions comprising the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention that are suitable for prophylactic, therapeutic and/or diagnostic use in a warmblooded animal, and in particular in a mammal, and more in particular in a human being.
  • the present invention also provides pharmaceutical compositions comprising the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention that can be used for veterinary purposes in the prevention and/or treatment of one or more diseases, disorders or conditions as described herein elsewhere.
  • the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention may be formulated as a pharmaceutical preparation or compositions comprising at least one the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may be suitable for oral, parenteral, topical administration or for administration by inhalation.
  • the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention and/or the compositions comprising the same can for example be administered orally, intraperitoneally, intravenously, subcutaneously, intramuscularly, transdermally, topically, by means of a suppository, by inhalation, again depending on the specific pharmaceutical formulation or composition to be used.
  • the clinician will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used in such administration.
  • compositions may also contain suitable binders, disintegrating agents, sweetening agents or flavoring agents, tablets, pills, or capsules may be coated for instance with gelatin, wax or sugar and the like.
  • suitable binders disintegrating agents, sweetening agents or flavoring agents, tablets, pills, or capsules may be coated for instance with gelatin, wax or sugar and the like.
  • the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention may be incorporated into sustained-release preparations and devices.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • Antibacterial and antifungal agents and the like can optionally be added.
  • Useful dosages of the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention can be determined by determining their in vitro activity, and/or in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the skilled person.
  • the amount of the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention required for use in prophylaxis and/or treatment may vary not only with the particular alphabody-polypeptide or polypeptide selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention may vary depending on the target cell, tumor, tissue, graft, or organ.
  • the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention and/or the compositions comprising the same are administered according to a regimen of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented, alleviated or treated.
  • the clinician will generally be able to determine a suitable treatment regimen.
  • the treatment regimen will comprise the administration of one or more the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses.
  • the desired dose may conveniently be presented in a single dose or as divided doses (which can again be sub-dosed) administered at appropriate intervals.
  • An administration regimen could include long-term (i.e., at least two weeks, and for example several months or years) or daily treatment.
  • the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention will be administered in an amount which will be determined by the medical practitioner based inter alia on the severity of the condition and the patient to be treated.
  • an optical dosage will be determined specifying the amount to be administered per kg body weight per day, either continuously (e.g. by infusion), as a single daily dose or as multiple divided doses during the day.
  • the clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment.
  • binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained.
  • examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.
  • a further aspect relates to the use of the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention for the preparation of a medicament for the prevention and/or treatment of at least one disease and/or disorder in which the first and/or second target is characteristic of, involved in or associated with or which is mediated by the first and/or second target.
  • the invention provides compositions comprising the polypeptides envisaged herein for use in the prevention, alleviation and/or treatment of at least disease and/or disorder mediated by the first and/or second target.
  • the first or the second target is characteristic of, involved in or mediates the disease or disorder.
  • the other target may for instance mediate or otherwise interfere with immune system activation.
  • the first or second target may be a tumor-specific target (i.e. a target only expressed in tumor cells, overexpressed in tumor cells versus normal cells, or inappropriately expressed or localized in tumor cells), whereas the other target may be located on one or more cells of the immune system (e.g. T-cells, dendritic cells, macrophages, etc.), such that the binding molecule according to the invention as described herein recruits the immune system cells to the tumor site.
  • a particular advantageous target on immune cells is for instance CD3.
  • the multispecific binding molecules according to the invention are agonizing molecules. They may for instance serve to increase or enhance the interaction between the first and second target.
  • the multispecific binding molecules according to the invention may specifically bind to a cytokine or growth factor and a cytokine or growth factor receptor thereby enhancing, increasing and/or activating the interaction between that cytokine or growth factor and/or its receptor.
  • the methods for the prevention and/or treatment of a disease and/or disorder mediated by the first and/or second target comprises administering to a subject in need thereof, a pharmaceutically active amount of one or more polypeptides and/or pharmaceutical compositions as envisaged herein.
  • the subject or patient to be treated is in particular a mammal, and more in particular a human suffering from or at risk for a disease or disorder.
  • diseases and disorders include infectious diseases, such as viral, bacterial, or parasitic diseases; cancer; inflammatory diseases, such as chronic or acute inflammatory diseases or disorders; disorders of the immune system and immune-mediated inflammatory disorders such as psioriasis, bowel diseases (colitis, Crohn's disease, IBD), infectious diseases, and other autoimmune diseases (such as rheumatoid arthritis, Multiple Sclerosis, Spondylarthritis, Sarcoidosis, Lupus, Behcet's disease), transplant rejection, cystic fibrosis, asthma, chronic obstructive pulmonary disease, cancer, viral infection, common variable immunodeficiency.
  • the invention relates to the use of the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention for preventing, alleviating and/or treating such diseases or disorders.
  • the invention relates to methods for preventing, alleviating, and/or treating such diseases or disorders, comprising administering the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention to a subject in need thereof.
  • a subject in need thereof can be a subject who is diagnosed with such disease or disorder or who is at risk of developing such disease or disorder.
  • the efficacy of the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person.
  • the disease or disorder which can be prevented, alleviated, or treated with the binding molecule or fusion/chimeric protein, polynucleic acid, vector or host cell according to the invention is an infectious disease, cancer, an inflammatory disease, or an autoimmune disease; preferably a human disease.
  • the autoimmune disease is selected from the group comprising or consisting of psoriasis, rheumatoid arthritis, adult or pediatric Crohn's disease, pediatric or adult ulcerative colitis, ankylosing spondylitis, psoriatic arthritis, juvenile idiopathic arthritis, and plaque psoriasis.
  • the invention relates to methods for preventing, treating, or alleviating the above autoimmune diseases by administering the multispecific binding molecules as described herein; or alternatively relates to the multispecific binding molecules as described herein for use in preventing, treating, or alleviating the above autoimmune diseases.
  • the cancer is selected from the group comprising or consisting of solid tumors or non solid tumors, and include primary tumors and metastasis.
  • Tumors may in certain embodiments be selected from leukemia, such as ALL, CML, DLBCL, NHL, AML; lymphoma; melanoma; breast cancer; colorectal cancer; gastric cancer; oesophageal cancer; head and neck cancer; prostate cancer; carcinoma, such as adenocarcinoma; osteosarcoma; neuroblastoma; pancreatic cancer; lung cancer.
  • the invention relates to methods for treating, alleviating, or preventing or reducing the progression of, reducing proliferation, or reducing growth and/or metastasis of the above cancers by administering the multispecific binding molecules as described herein; or alternatively relates to the multispecific binding molecules as described herein for use in treating, alleviating, preventing or reducing the progression of, reducing proliferation, or reducing growth and/or metastasis of the above cancers.
  • the infectious disease is selected from bacterial, fungal, viral (such as HIV), or parasitic diseases. Accordingly, in certain embodiments, the invention relates to methods for preventing, treating, alleviating, or dissemination of the above infectious diseases by administering the multispecific binding molecules as described herein; or alternatively relates to the multispecific binding molecules as described herein for use in preventing, treating, alleviating, or dissemination of the above infections diseases.
  • the inflammatory disease is selected from chronic or acute (e.g. sepsis) inflammatory disease, such as asthma, multiple sclerosis, inflammatory bowel disease, transplant rejection, cystic fibrosis, chronic obstructive pulmonary disease, or common variable immunodeficiency.
  • the invention relates to methods for preventing, treating, or alleviating the above inflammatory diseases by administering the multispecific binding molecules as described herein; or alternatively relates to the multispecific binding molecules as described herein for use in preventing, treating, or alleviating the above inflammatory diseases.
  • Figure 1 Amino acid sequences of alphabody-polypeptide-antibody fusion constructs according to various embodiments of the invention.
  • Alphabody-polypeptides were fused to either heavy or light chain of adalimumab.
  • Different linkers were employed to space antibody and alphabody-polypeptide.
  • the different building blocks (antibody, alphabody-polypeptide, linker, special amino acids) are indicated in the sequence as follows: Antibody heavy or light chain (standard), alphabody-polypeptide (bold), Linker (white lettering), Special amino acids (underlined)
  • Figure 2 Graphical representation of the fusion constructs of Figure 1 .
  • FIG. 3 Determining the affinity to TNFa, ELISA principle. Multispecific constructs or adalimumab bound to immobilized biotinolyted TNFa are detected with an HRP-labeled anti- human antibody.
  • Figure 4 Determining the affinity to IL-23, ELISA principle. Binding of multispecific constructs to IL-23 was measured indirectly by detection of residual, non-bound IL-23 that remained in solution after incubation with the multispecific. Therefore, the multispecific constructs were pre-incubated with IL-23. The free, non-bound IL-23 was then captured by an immobilized sensor and detected by antibody staining.
  • FIG. 5 Demonstrating simultaneous binding to TNFa and IL-23, ELISA principle. Multispecifics were pre-incubated with varying concentrations of IL-23 and then added to immobilized TNFa. The TNFa-multispecific-IL-23 complex was then detected with a primary mouse-anti-IL-23 and a secondary HRP labeled anti-mouse antibody.
  • the multispecific construct can bind TNFa and IL-23 simultaneously.
  • MB23_DI4- VL_ADA, MB23_DI4-VH_ADA and CL_ADA-MB23_DI4 were tested in ELISA for simultaneous binding to TNFa and IL-23.
  • 10 nM IL-23 was incubated with 3.3nM of multispecific.
  • Adalimumab was treated in parallel and served as a negative control. The mix was then applied to TNFa that was immobilized on an ELISA plate.
  • the TNFa/multispecifics/IL-23 complex was detected with an antibody against the p40 subunit of IL-23 and a secondary HRP-labeled antibody.
  • FIG. 7 Potency of the multispecifics in the IL-23 inhibition assay (splenocyte assay).
  • the multispecifics were tested for their IL-23 inhibition potency in a splenocyte assay.
  • mouse splenocytes respond with the production of IL-17 to treatment with IL-23.
  • IL-17 production drops when IL-23 is blocked by an IL-23 inhibitor.
  • FIG. 8 Potency of the multispecifics in the TNFa inhibition assay (NF- ⁇ activation assay). The multispecifics were tested for their potency to inhibit TNFa in a NF- ⁇ activation assay.
  • TNFa drives the activation of NF- ⁇ , which in turn induces production of a Luciferase. Luciferase production drops if TNFa is blocked by an inhibitor.
  • Example 1 Generation of bispecific alphabody-polypeptide-antibody fusion molecules alphabody-polypeptide-antibody bispecific molecules were generated by fusion of a gene encoding an alphabody-polypeptide to the gene encoding an antibody heavy or light chain. To yield different bispecific constructs, the anti-IL-23 alphabody-polypeptide MB23_DI4 was fused at different positions of the anti-TNFa antibody adalimumab (see Figure 1 for sequences of the fusion proteins and Figure 2 for a graphical representation of the fusion proteins).
  • the alphabody-polypeptide was either fused to the N-terminus of the light chain of adalimumab, the N-terminus of the heavy chain, the C-terminus of the light chain, or the C- terminus of the heavy chain.
  • the adalimumab sequence was taken from the public domain.
  • Binding agents were formed by joining of two heavy and two light chains such that the binding agents in all below described examples contained one antibody and two Alphabody- polypeptides (fused to either the two light chains or the two heavy chains of the antibody).
  • Different linkers were used to space the alphabody-polypeptide from the antibody. These cover the following amino acid sequences (in 1 -letter code): (GGGGS) 3 , GSG, GPSPG, GGS(GGGGS) 2 GG and GASPAG.
  • the architecture of the fusion construct was designed so as to determine optimal affinity and bi-functional activity of the binding to both cytokines (TNFa and IL-23).
  • the target binding sites of both fusion partners alphabody-polypeptide and antibody, are accessible simultaneously without steric hindrance from the domains of the bispecific molecule itself or through target binding. It was investigated if steric hindrance of the two proteins forming the bispecific may be influenced by the fusion site of the Alphabody- polypeptide to the antibody as well as by the employed linkers. It was further investigated if, and to what extent, both factors (i.e., the fusion site and linker type) impact other characteristics such as producibility, solubility and stability.
  • Table 1 Constructs having different fusion topologies and linkers types.
  • fusion proteins listed in Table 1 represent embodiments according to the present invention which allow the following design operable to be investigated, in particular in connection with expressability, solubility, and functionality (such as binding strength and potency): influence of topology of the alphabody-polypeptide and antibody with respect to each other, influence of the type of linker, influence of forced orientation of the alphabody- polypeptide vis-a-vis the antibody and influence of the isoelectric point of the alphabody- polypeptide.
  • linker design To evaluate the effect of linker design, different types of linkers were tested. We designed and tested constructs with linkers that differed in length and flexibility. The tested linkers involved the amino acid sequences (GGGGS) 3 , GSG, GPSPG, GAS PAG and GGS(GGGGS) 2 GG (see Table 1 ).
  • the binding interfaces of both the antibody and alphabody- polypeptide need to be freely accessible.
  • the target binding site of the alphabody-polypeptide may not be fully accessible for IL-23 when the alphabody-polypeptide is fused to the C-terminus of the light chain (as in construct CL_Ada-MB23_DI4), because of its close proximity to the antibody hinge region at this fusion position.
  • the alphabody-polypeptide was in an alternative variant covalently 'back-coupled' to the light chain of the antibody through a disulfide bond between a cysteine at position B2fC in the alphabody-polypeptide and a non- native cysteine at position 152 in the light chain of the antibody (as in construct Ada_CL_152C-MB23_DI4_B2fC). Formation of a disulfide bond between the two engineered cysteines was intended to fix the orientation of the alphabody-polypeptide and facilitate IL-23 binding.
  • the C-terminus of the heavy chain is an attractive point for alphabody-polypeptide fusion, because it is freely accessible and far away from the target binding interface of the antibody.
  • the C-termini of the antibody heavy chains are in close proximity in the assembled antibody. It was reasoned that fusion of alphabody-polypeptides at this point could be risky, because the chance that the alphabody-polypeptides would self-associate and occlude each other's IL-23 binding site could be high.
  • the alphabody-polypeptides were designed to hold a cysteine which allows formation of a disulfide bridge between the two alphabody-polypeptides in the fusion construct. By this approach, the two alphabody-polypeptides are forced into a position that exposes their IL-23 binding interfaces.
  • the isoelectric point of adalimumab itself is 8.1 and that of the alphabody-polypeptide MB23_DI4 is 4.1 .
  • the isoelectric point of the alphabody-polypeptide- antibody fusion construct MB23_DI4-VL_Ada was calculated to be 7.4. Therefore, in an alternative construct, the pi was moved up to 8.3 in certain test constructs by the introduction of positive charges (arginines) to the alphabody-polypeptide.
  • Vectors encoding the respective fusion constructs were transiently transfected into HEK293- E253 or CHO cells.
  • Bispecific proteins consisting of the alphabody-polypeptide fused to adalimumab were harvested from the cell culture supernatant.
  • expression yields (titers) were analyzed at this point. Fusion constructs were then purified from the cell culture supernatant by protein A affinity chromatography. Final yields were determined spectrophotometrically (OD at 280 nm).
  • Example 2 Expression yields of the fusion proteins of Example 1
  • CMC manufacturing and controls'
  • Bispecific antibodies and antibody fusion constructs often express poorly (personal communication with CMC companies). Furthermore, they are often heterogeneous and sometimes lose affinity to protein A, so that complex purification protocols need to be developed.
  • the fusion constructs the naked alphabody-polypeptide MB23_DI4 and free adalimumab were expressed in CHO cells after transient transfection of plasmid DNA encoding heavy and light chain of the antibody fusion constructs.
  • Adalimumab on its own expressed at 591 mg/l and the naked alphabody-polypeptide expressed at 100 mg/l (see Table 2).
  • the alphabody-polypeptide was fused to the C-terminus of the light chain (CL_Ada-MB23_DI4), it expressed at 459 mg/l which corresponds to 78 % of the expression level of free adalimumab.
  • MB23_DI4 CHO cells transient 100 mg/l NA
  • adalimumab CHO cells transient 591 mg/l 130 mg/l
  • MB23_DI4-VL_Ada CHO cells transient 259 mg/l 173 mg/l
  • MB23_DI4-VH_Ada CHO cells transient 235 mg/l 120 mg/l
  • VH_Ada_sl_pl8 expression Ada CL 152C- HEK cells, transient ND 106 mg/l
  • Ada CH3- HEK cells Ada CH3- HEK cells, transient ND 106 mg/l
  • Protein A binds with strong affinity to the Fc portion of immunoglobulins such as adalimumab and is therefore conveniently used for affinity purification.
  • adalimumab yielded 130 mg/l, meaning a loss of 78 % of production yield and a recovery of 22%.
  • the variants having increased isoelectric point (MB23_DI4-VL_Ada_pl8 and MB23_DI4-VH_Ada_sl_pl8) scored the poorest (with yields of only 47 and 57 mg/ml, respectively).
  • Example 3 Quantification of binding to IL-23 and TNFa of the fusion proteins of Example 1
  • Target affinity is a major determinant for the functionality and potency of therapeutic compounds.
  • the fusion of a protein to an antibody without losing target affinity for any of the two binding parts is not obvious, and the topology of the fusion partners can be expected to influence the respective target affinities.
  • the fusion of an alphabody-polypeptide to the N-terminus of the adamimumab light or heavy chain brings the alphabody-polypeptide in close proximity to the TNFa binding site on the antibody. This may hamper TNFa binding at the antibody, but also disturb IL-23 binding at the alphabody-polypeptide domain.
  • C-terminal fusions at the light chain it was a priori also possible that the alphabody-polypeptide would have a reduced accessibility due to its proximity to the antibody hinge region. Hence, these possible effects had to be determined experimentally.
  • IL-23 and TNFa affinity of the different fusion constructs was tested in ELISA to understand the influence of antibody- alphabody-polypeptide fusion and fusion topology on target affinity.
  • Binding affinities were calculated from the resulting sigmoidal binding curves.
  • ELISA quantifying binding affinity to IL-23
  • the bispecific construct was incubated with varying concentrations of IL-23 in individual wells of an ELISA plate and allowed to bind, as per the detailed protocol below.
  • an IL-23 sensor i.e. an alphabody-polypeptide with IL-23 binding specificity
  • the IL-23 inhibitor/IL-23 mix was then applied to the sensor plate. Free IL-23 that was not bound by the IL-23 inhibitor could then bind to the sensor molecule on the ELISA plate.
  • IL-23 bound to the sensor was detected with a biotinylated antibody directed against the p40 subunit of IL-23. The biotinylated antibody was then detected by addition of HRP-coupled streptavidin followed by HRP staining.
  • affinity to IL-23 was sub-nanomolar (0.4 nM), when the alphabody- polypeptide was fused to the N-terminus of the light or heavy chain of adalimumab. Importantly, affinity dropped 10-fold to 4 nM, when the alphabody-polypeptide was fused to the C-terminus of the light chain.
  • the differences in IL-23 affinity are attributed to different accessibilities of the IL-23 binding site in different constructs, depending on the fusion topology.
  • alphabody-polypeptides may be well accessible to IL-23 when fused to the N-termini of the antibody chains, accessibility was found to be hindered in the fusion construct with the alphabody-polypeptides coupled to the C-termini of the antibody light chains.
  • the fusion topology appeared to have only a minor influence on TNFa binding in ELISA (see Table 3). Fusion of an alphabody-polypeptide to the N-termini of the antibody heavy and light chains could a priori be expected to affect accessibility for TNFa. Fusion of the alphabody- polypeptide to the C-terminus of the light chain, far away from the TNFa binding site, was expected to result in unhindered access of TNFa. However, the results showed that there was not any significant difference in TNFa affinity between the three fusion constructs.
  • biotinylated TNFa was coupled to a streptavidin-coated ELISA plate.
  • varying concentrations of bispecifics or adalimumab were incubated with a fixed concentration of IL-23, as per the protocol below.
  • the mixes were then applied to the TNFa-coated plate and incubated to allow binding to TNFa.
  • a mouse-anti-IL-23 antibody was added.
  • the IL-23 antibody used here binds IL-23 at its p40 subunit, leaving the p19-subunit of IL-23 free for binding to the alphabody-polypeptide in the bispecific.
  • mice Pre-incubate mouse anti-human-p40 (10 nM) and goat anti-mouse-HRP in excess (12 nM) in PBS+0.1 %BSA, 1 h at RT, shaking
  • Example 4 Potency assay of the fusion proteins of Example 1 in cell based assays
  • Fusion topology could be expected to have an influence on the potency of the fusion constructs, especially in those instances where target affinity was affected. Steric hindrance resulting in reduced accessibility of target binding sites in the alphabody-polypeptide or antibody may compromise construct potency. On the other hand, fusion constructs may gain activity in functional assays, e.g. from avidity, when binding more than one target at a time.
  • the potency of the fusion constructs in inhibiting the stimulatory effect of IL-23 in a cellular assay was measured in an IL-23 inhibition assay in mouse splenocytes.
  • Splenocytes respond with induction of IL-17 to treatment with IL-23, which can be measured in the cell supernatant.
  • IL-17 production is reduced if IL-23 is inhibited.
  • the two constructs comprising N- terminal fusions of the alphabody-polypeptides to either the light or heavy chains of the adalimumab antibody performed significantly better than the construct comprising alphabody- polypeptides C-terminally fused to the light chains (see Figure 7 and Table 4; binding affinities (KDs) calculated from the binding curves).
  • MB23_DI4-VL_ADA was the best performing construct with an IC50 of 0.05 nM, followed by MB23_DI4-VH_ADA with an IC50 of 0.08 nM.
  • the C-terminal fusion CL_ADA-MB23_DI4 was about 10 times less potent, having an IC50 of 0.65 nM. As explained above, these differences in potencies may be due to differences in the accessibility of the IL-23 binding site in the alphabody-polypeptide depending on the fusion topology. Importantly, and unexpectedly, these results show that the IC50s in this cellular assay were much lower than the respective binding affinities (as shown in Table 5). This phenomenon shows up as a 'potency jump' and can be considered a most preferable feature of the present fusion constructs. It indicates that N-terminal fusion constructs as claimed have an extreme 'picomolar-range' neutralization capacity.
  • TNFa inhibition potency was measured in a reporter cell line that responds to TNFa by activating Nf- ⁇ which in turn drives the expression of a luciferase. Luciferase production is reduced if TNFa is inhibited.
  • the C-terminal fusion construct CL_ADA-MB23_DI4 was the best performing construct in the TNFa inhibition assay with an IC50 of 0.29 nM (see Figure 8 and Table 5; binding affinities (KDs) calculated from the binding curves).
  • the N-terminal fusion constructs were about a factor 2-3 less potent than the C-terminal fusion construct, with an IC50 of 0.70 nM for MB23_DI4-VL_ADA and 0.77 nM for MB23_DI4-VH_ADA, compared to 0.29 nM for CL_ADA-MB23_DI4. This difference was unexpected, because ELISA had shown similar binding affinities for all three constructs.
  • TNFa may be presented in different ways in ELISA and in cell-based assays, leading to differences in accessibility of the binding sites of the fusion construct. Moreover, the potency jump observed in the IL-23 based assay for N- terminal fusions may be more beneficial than the relatively small potency loss in this TNFa based assay, which still shows IC50 values well below 1 nM.
  • Example 5 Generation of further bispecific alphabody-polypeptide constructs alphabody-polypeptide-antibody bispecifics were generated as described in example 1 by fusion of a gene encoding an alphabody-polypeptide to the gene encoding an antibody heavy or light chain.
  • Binding agents were formed by joining of two heavy and two light chains such that the binding agents in all below described examples contained one antibody and two Alphabody- polypeptides (fused to either the two light chains or the two heavy chains of the antibody).
  • the anti-IL-23 alphabody-polypeptide MB23_DI4 was fused to different anti-TNFa antibodes Certolizumab, Golimumab or Adalimumab using different linkers. The sequences of these antibodies was taken from the public domain.
  • the alphabody-polypeptide can be either fused to the N-terminus of the light chain of the antibody, the N-terminus of the heavy chain, the C-terminus of the light chain, or the C- terminus of the heavy chain.
  • the relative activity of the N-terminal fusions compared to the C-terminal fusion counterparts can be determined.
  • N-terminal fusions being of interest for the present invention, are summarized in Table 6
  • Table 6 Constructs having different fusion topologies and linkers types.
  • SEQ ID NO: 1 1 IL-23 alphabody- SIEQIQKEITTIQEVISAIEKYIQTMTGGPN polypeptide pi GGSGGGMSIEQIQKQIRRIQCQIRRIQKQ adjusted+cysteine IYAMTGGGPGGSGGMSIEQIQKQISAIQ
  • VGTNVAWYQQKPGKAPKALIYSASFLYS GVPYRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYNIYPLTFGQGTKVEIK
  • GSSIHWYQQRTNGSPRLLIKYASESMS GIPSRFSGSGSGTDFTLSINTVESEDIAD YYCQQSHSWPFTFGSGTNLEVK

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Abstract

La présente invention concerne des molécules de liaison multispécifiques comprenant un polypeptide-alphacorps et un anticorps ou un fragment d'anticorps. L'invention concerne en outre des acides polynucléiques codant ces molécules de liaison, ainsi que des vecteurs comprenant de tels acides polynucléiques, des cellules hôtes comprenant les acides polynucléiques ou les vecteurs, et des compositions pharmaceutiques comprenant les acides polynucléiques, les vecteurs ou les cellules hôtes. L'invention concerne en outre l'utilisation des molécules de liaison, des acides polynucléiques, des vecteurs, des cellules hôtes ou des compositions pharmaceutiques pour le diagnostic, la prophylaxie ou le traitement de maladies.
PCT/EP2016/080986 2015-12-14 2016-12-14 Molécule de liaison comprenant un polypeptide-alphacorps et un anticorps WO2017102835A1 (fr)

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