US20200283512A1 - Anti-serum albumin binding variants - Google Patents

Anti-serum albumin binding variants Download PDF

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US20200283512A1
US20200283512A1 US16/879,238 US202016879238A US2020283512A1 US 20200283512 A1 US20200283512 A1 US 20200283512A1 US 202016879238 A US202016879238 A US 202016879238A US 2020283512 A1 US2020283512 A1 US 2020283512A1
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dom7h
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Elena De Angelis
Carolyn Enever
Haiqun Liu
Oliver Schon
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Glaxo Group Ltd
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • 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
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the invention relates to improved variants of the anti-serum albumin immunoglobulin single variable domain DOM7h-11, as well as ligands and drug conjugates comprising such variants, compositions, nucleic acids, vectors and hosts.
  • SA binding moieties such as anti-SA immunoglobulin single variable domains (dAbs), which have therapeutically-useful half-lives.
  • dAbs anti-serum albumin binding moieties
  • monomer anti-SA dAbs as well as multi-specific ligands comprising such dAbs, eg, ligands comprising an anti-SA dAb and a dAb that specifically binds a target antigen, such as TNFR1.
  • Binding moieties are disclosed that specifically bind serum albumins from more than one species, eg human/mouse cross-reactive anti-SA dAbs.
  • WO05118642 and WO2006/059106 disclose the concept of conjugating or associating an anti-SA binding moiety, such as an anti-SA immunoglobulin single variable domain, to a drug, in order to increase the half-life of the drug.
  • Protein, peptide and NCE (new chemical entity) drugs are disclosed and exemplified.
  • WO2006/059106 discloses the use of this concept to increase the half-life of insulintropic agents, eg, incretin hormones such as glucagon-like peptide (GLP)-1.
  • GLP glucagon-like peptide
  • WO2008/096158 discloses DOM7h-11, which is a good anti-SA dAb. It would be desirable to provide improved dAbs that are variants of DOM7h-11 and that specifically bind serum albumin, preferably albumins from human and non-human species, which would provide utility in animal models of disease as well as for human therapy and/or diagnosis. It would also be desirable to provide for the choice between relatively modest- and high-affinity anti-SA binding moieties (dAbs). Such moieties could be linked to drugs, the anti-SA binding moiety being chosen according to the contemplated end-application. This would allow the drug to be better tailored to treating and/or preventing chronic or acute indications, depending upon the choice of anti-SA binding moiety.
  • anti-dAbs that are monomeric or substantially so in solution. This would especially be advantageous when the anti-SA dAb is linked to a binding moiety, eg, a dAb, that specifically binds a cell-surface receptor, such as TNFR1, with the aim of antagonizing the receptor.
  • a binding moiety eg, a dAb
  • the monomeric state of the anti-SA dAb is useful in reducing the chance of receptor cross-linking, since multimers are less likely to form which could bind and cross-link receptors (eg, TNFR1) on the cell surface, thus increasing the likelihood of receptor agonism and detrimental receptor signaling.
  • the present inventors surprisingly found that beneficial mutations can be targeted to the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) of DOM7h-11.
  • the invention therefore, provides an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11, wherein the variant comprises at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) compared to DOM7h-11, and wherein the variant has from 2 to 8 changes compared to the amino acid sequence of DOM7h-11.
  • SA anti-serum albumin
  • the invention provides an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11, wherein the variant comprises a Met at position 32 (numbering according to Kabat) compared to DOM7h-11, and wherein the variant has from 0 to 4 further changes compared to the amino acid sequence of DOM7h-11.
  • SA anti-serum albumin
  • Embodiments of either aspect of the invention provide DOM7h-11 variants of good anti-serum albumin affinities.
  • the choice of variant can allow for tailoring of half-life according to the desired therapeutic and/or prophylactic setting.
  • the affinity of the variant for serum albumin is relatively high, such that the variant would be useful for inclusion in products that find utility in treating and/or preventing chronic or persistent diseases, conditions, toxicity or other chronic indications.
  • the affinity of the variant for serum albumin is relatively modest, such that the variant would be useful for inclusion in products that find utility in treating and/or preventing acute diseases, conditions, toxicity or other acute indications.
  • the affinity of the variant for serum albumin is intermediate, such that the variant would be useful for inclusion in products that find utility in treating and/or preventing acute or chronic diseases, conditions, toxicity or other acute or chronic indications.
  • any fused therapeutic agent to the ALBUDABTM variant (an ALBUDABTM is an anti-serum albumin dAb or immunoglobulin single variable domain), be it NCE, peptide or protein, consequently would be able to act longer on its target and exhibit a longer lasting therapeutic effect. This would allow for targeting chronic or persistent diseases without the need of frequent dosing.
  • a variant with moderate affinity would only stay in serum circulation for a short time (eg, for a few hours or a few days) allowing for the specific targeting of therapeutic targets involved in acute diseases by the fused therapeutic agent.
  • An aspect of the invention provides a multispecific ligand comprising any anti-SA variant as described above and a binding moiety that specifically binds a target antigen other than SA.
  • An aspect of the invention provides a fusion product, eg, a fusion protein or fusion with a peptide or NCE (new chemical entity) drug, comprising a polypeptide, protein, peptide or NCE drug fused or conjugated (for an NCE) to any variant as described above, wherein the variant is DOM7h-11-15 or DOM7h-11-15 S12P (or a variant having an amino acid that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of DOM7h-11-15) or DOM7h-11-12 (or a variant having an amino acid that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of DOM7h-11-12).
  • a fusion product eg, a fusion protein or fusion with a peptide or NCE (new chemical entity) drug, comprising a polypeptide, protein, peptide or NCE drug fused or conjugated (for an NCE) to any variant as described above, where
  • DOM7h-11-15 and DOM7h-11-12 give only a modest drop in affinity when fused or conjugated to partner making them useful in fusion products.
  • DOM7h-11-15 S12P is identical to DOM7h-11-15, with the exception that position 12 (numbering according to Kabat) is a proline instead of a serine.
  • position 12 numbering according to Kabat
  • WO08052933 provides advantages set out in WO08052933, including to reduce binding to Protein-L of fusion proteins containing this domain antibody and to facilitate purification.
  • the entire disclosure of WO08052933 is incorporated herein by reference.
  • the invention provides a DOM7h-11 variant as disclosed herein wherein the variant comprises an amino acid sequence as set out below with the exception that position 12 (numbering according to Kabat) is a proline.
  • the invention also provides fusion proteins, conjugates or composition comprising such DOM7h-11 variants.
  • One aspect of the invention provides a variant of DOM7h-11 that comprises an amino acid sequence that is identical to the amino acid sequence of DOM7h-11-15 S12P or has up to 4 changes compared to the amino acid sequence of DOM7h-11-15 S12P , provided that the amino acid sequence of the variant has at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat).
  • An aspect of the invention provides a composition comprising a variant, fusion protein or ligand of any preceding aspect and a pharmaceutically acceptable diluent, carrier, excipient or vehicle.
  • An aspect of the invention provides a method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of a variant according to any aspect or embodiment of the invention to said patient.
  • An aspect of the invention provides a polypeptide fusion or conjugate comprising an anti-serum albumin dAb as disclosed herein (eg, DOM7h-11-15 or DOM7h-11-3 or DOM7h-11-15 S12P or DOM7h-11-15 S12P with up to 4 changes compared to the amino acid sequence of DOM7h-11-15 S12P ) and an incretin or insulinotropic agent, eg, exendin-4, GLP-1(7-37), GLP-1(6-36) or any incretin or insulinotropic agent disclosed in WO06/059106, these agents being explicitly incorporated herein by reference as though written herein for inclusion in the present invention and claims below.
  • an anti-serum albumin dAb as disclosed herein (eg, DOM7h-11-15 or DOM7h-11-3 or DOM7h-11-15 S12P or DOM7h-11-15 S12P with up to 4 changes compared to the amino acid sequence of DOM7h-11-15 S12P ) and an in
  • FIG. 1 Amino-acid sequence alignment for DOM7h-11 (SEQ ID NO: 421) variant dAbs.
  • a “.” at a particular position indicates the same amino as found in DOM7h-11 at that position.
  • the CDRs are indicated by underlining and bold text (the first underlined sequence is CDR1, the second underlined sequence is CDR2 and the third underlined sequence is CDR3).
  • the figure comprises the following variants: DOM 7h-11-12 (SEQ ID NO:1), DOM 7h-11-15 (SEQ ID NO:2), DOM 7h-11-18 (SEQ ID NO:3), DOM 7h-11-19 (SEQ ID NO:4), and DOM 7h-11-3 (SEQ ID NO: 5).
  • FIG. 2 Kinetic parameters of DOM7h-11 variants.
  • the affinity in this respect is the affinity for serum albumin.
  • Various example anti-serum dAbs and fusion proteins are listed, and these support the ranges disclosed. Many of the examples have favourable kinetics in human and one or more non-human animals (eg, in human and Cynomolgus monkey and/or mouse). Choice of dAb or product comprising this can be tailored, according to the invention, depending on the setting (eg, chronic or acute) to be treated therapeutically.
  • TNFR1 Tumor Necrosis Factor Receptor 1
  • anti-TNFR1 antagonist refers to an agent (e.g., a molecule, a compound) which binds TNFR1 and can inhibit a (i.e., one or more) function of TNFR1.
  • an antagonist of TNFR1 can inhibit the binding of TNF ⁇ to TNFR1 and/or inhibit signal transduction mediated through TNFR1.
  • TNFR1-mediated processes and cellular responses e.g., TNF ⁇ -induced cell death in a standard L929 cytotoxicity assay
  • TNF ⁇ -induced cell death in a standard L929 cytotoxicity assay can be inhibited with an antagonist of TNFR1.
  • a “patient” is any animal, eg, a mammal, eg, a non-human primate (such as a baboon, rhesus monkey or Cynomolgus monkey), mouse, human, rabbit, rat, dog, cat or pig. In one embodiment, the patient is a human.
  • a mammal eg, a non-human primate (such as a baboon, rhesus monkey or Cynomolgus monkey), mouse, human, rabbit, rat, dog, cat or pig.
  • the patient is a human.
  • peptide refers to about two to about 50 amino acids that are joined together via peptide bonds.
  • polypeptide refers to at least about 50 amino acids that are joined together by peptide bonds. Polypeptides generally comprise tertiary structure and fold into functional domains.
  • an antibody refers to IgG, IgM, IgA, IgD or IgE or a fragment (such as a Fab, F(ab′) 2 , Fv, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, diabody) whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
  • a fragment such as a Fab, F(ab′) 2 , Fv, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, diabody
  • antibody format refers to any suitable polypeptide structure in which one or more antibody variable domains can be incorporated so as to confer binding specificity for antigen on the structure.
  • suitable antibody formats are known in the art, such as, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy chains and/or light chains, antigen-binding fragments of any of the foregoing (e.g., a Fv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment), a single antibody variable domain (e.g., a dAb, V H , V HH , V L ), and modified versions of any of the foregoing (e.g., modified by the covalent attachment of polyethylene glycol or other suitable polymer or a humanized V HH
  • immunoglobulin single variable domain refers to an antibody variable domain (V H , V HH , V L ) that specifically binds an antigen or epitope independently of different V regions or domains.
  • An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other variable regions or variable domains where the other regions or domains are not required for antigen binding by the single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds antigen independently of the additional variable domains).
  • a “domain antibody” or “dAb” is the same as an “immunoglobulin single variable domain” as the term is used herein.
  • a “single immunoglobulin variable domain” is the same as an “immunoglobulin single variable domain” as the term is used herein.
  • a “single antibody variable domain” or an “antibody single variable domain” is the same as an “immunoglobulin single variable domain” as the term is used herein.
  • An immunoglobulin single variable domain is in one embodiment a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004, the contents of which are incorporated herein by reference in their entirety), nurse shark and Camelid V HH dAbs.
  • Camelid V HH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains.
  • the V HH may be humanized.
  • a “domain” is a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
  • a “single antibody variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.
  • prevention and “preventing” involves administration of the protective composition prior to the induction of the disease or condition.
  • Treatment and “treating” involves administration of the protective composition after disease or condition symptoms become manifest.
  • suppression or “suppressing” refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease or condition.
  • dose refers to the quantity of ligand administered to a subject all at one time (unit dose), or in two or more administrations over a defined time interval.
  • dose can refer to the quantity of ligand (e.g., ligand comprising an immunoglobulin single variable domain that binds target antigen) administered to a subject over the course of one day (24 hours) (daily dose), two days, one week, two weeks, three weeks or one or more months (e.g., by a single administration, or by two or more administrations).
  • the interval between doses can be any desired amount of time.
  • pharmaceutically effective when referring to a dose means sufficient amount of the ligand, domain or pharmaceutically active agent to provide the desired effect.
  • the amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, the particular drug or pharmaceutically active agent and the like. Thus, it is not always possible to specify an exact “effective” amount applicable for all patients. However, an appropriate “effective” dose in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • Half lives (t1 ⁇ 2 alpha and t1 ⁇ 2 beta) and AUC can be determined from a curve of serum concentration of ligand against time.
  • the WinNonlin analysis package eg version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA) can be used, for example, to model the curve.
  • a second phase (beta phase) is the phase when the ligand has been distributed and the serum concentration is decreasing as the ligand is cleared from the patient.
  • the t alpha half life is the half life of the first phase and the t beta half life is the half life of the second phase.
  • variable domain, fusion protein or ligand has a t ⁇ half-life in the range of (or of about) 15 minutes or more.
  • the lower end of the range is (or is about) 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours.
  • the variable domain, fusion protein or ligand according to the invention will have a t ⁇ half life in the range of up to and including 12 hours (or about 12 hours).
  • the upper end of the range is (or is about) 11, 10, 9, 8, 7, 6 or 5 hours.
  • An example of a suitable range is (or is about) 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.
  • the present invention provides the variable domain, fusion protein or ligand according to the invention has a t ⁇ half-life in the range of (or of about) 2.5 hours or more.
  • the lower end of the range is (or is about) 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours, or 12 hours.
  • the t ⁇ half-life is (or is about) up to and including 21 or 25 days.
  • the upper end of the range is (or is about) 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days, 19 days, 20 days, 21 days or 22 days.
  • variable domain, fusion protein or ligand according to the invention will have a t ⁇ half life in the range 12 to 60 hours (or about 12 to 60 hours). In a further embodiment, it will be in the range 12 to 48 hours (or about 12 to 48 hours). In a further embodiment still, it will be in the range 12 to 26 hours (or about 12 to 26 hours).
  • terminal half-life means a terminal half-life determined using non-compartmental modeling.
  • the WinNonlin analysis package eg version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA) can be used, for example, to model the curve in this way.
  • the single variable domain, fusion protein or ligand has a terminal half life of at least (or at least about) 8 hours, 10 hours, 12 hours, 15 hours, 28 hours, 20 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days or 25 days.
  • the upper end of this range is (or is about) 24 hours, 48 hours, 60 hours or 72 hours or 120 hours.
  • the terminal half-life is (or is about) from 8 hours to 60 hours, or 8 hours to 48 hours or 12 to 120 hours, eg, in man.
  • variable domain, fusion protein or ligand according to the invention has an AUC value (area under the curve) in the range of (or of about) 1 mg ⁇ min/ml or more.
  • the lower end of the range is (or is about) 5, 10, 15, 20, 30, 100, 200 or 300 mg ⁇ min/ml.
  • variable domain, fusion protein or ligand according to the invention has an AUC in the range of (or of about) up to 600 mg ⁇ min/ml.
  • the upper end of the range is (or is about) 500, 400, 300, 200, 150, 100, 75 or 50 mg ⁇ min/ml.
  • variable domain, fusion protein or ligand will have an AUC in (or about in) the range selected from the group consisting of the following: 15 to 150 mg ⁇ min/ml, 15 to 100 mg ⁇ min/ml, 15 to 75 mg ⁇ min/ml, and 15 to 50 mg ⁇ min/ml.
  • “Surface Plasmon Resonance” Competition assays can be used to determine if a specific antigen or epitope, such as human serum albumin, competes with another antigen or epitope, such as cynomolgus serum albumin, for binding to a serum albumin binding ligand described herein, such as a specific dAb. Similarly competition assays can be used to determine if a first ligand such as dAb, competes with a second ligand such as a dAb for binding to a target antigen or epitope.
  • a specific antigen or epitope such as human serum albumin
  • another antigen or epitope such as cynomolgus serum albumin
  • competition assays can be used to determine if a first ligand such as dAb, competes with a second ligand such as a dAb for binding to a target antigen or epitope.
  • the term “competes” as used herein refers to substance, such as a molecule, compound, preferably a protein, which is able to interfere to any extent with the specific binding interaction between two or more molecules.
  • the phrase “does not competitively inhibit” means that substance, such as a molecule, compound, preferably a protein, does not interfere to any measurable or significant extent with the specific binding interaction between two or more molecules.
  • the specific binding interaction between two or more molecules preferably includes the specific binding interaction between a single variable domain and its cognate partner or target.
  • the interfering or competing molecule can be another single variable domain or it can be a molecule that is structurally and/or functionally similar to a cognate partner or target.
  • binding moiety refers to a domain that specifically binds an antigen or epitope independently of a different epitope or antigen binding domain.
  • a binding moiety may be a domain antibody (dAb) or may be a domain which is a derivative of a non-immunoglobulin protein scaffold, eg, a scaffold selected from the group consisting of CTLA-4, lipocalin, SpA, an adnectin, affibody, an avimer, GroEl, transferrin, GroES and fibronectin, which binds to a ligand other than the natural ligand (in the case of the present invention, the moiety binds serum albumin).
  • WO2008/096158 discloses examples of protein scaffolds and methods for selecting antigen or epitope-specific binding domains from repertoires (see Examples 17 to 25). These specific disclosures of WO2008/096158 are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein).
  • the invention provides an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11, wherein the variant comprises at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) compared to DOM7h-11, and wherein the variant has from 2 to 8 changes compared to the amino acid sequence of DOM7h-11.
  • position 49 (according to Kabat) is Leu.
  • position 50 (according to Kabat) is optionally Ala or Trp.
  • position 51 (according to Kabat) is optionally Phe or Asn.
  • the variant comprises a mutation at each of positions 49, 50 and 51 (numbering according to Kabat) compared to DOM7h-11.
  • the variant comprises a LFG motif, where L is at position 49 (numbering according to Kabat), wherein L, F and G are Leu, Phe and Gly respectively.
  • the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-3, DOM7h-11-15, DOM7h-11-12 and DOM7h-11-19 or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has at least one mutation in the FW2/CDR2 junction as defined above.
  • the variant comprises an amino acid sequence that is identical to the amino acid sequence of DOM7h-11-15 S12P or has up to 4 changes compared to the amino acid sequence of DOM7h-11-15 S12P , provided that the amino acid sequence of the variant has at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat).
  • the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-3, or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has L at position 49, W at position 50 and N at position 51.
  • the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-12, or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has M at position 32 and L at position 49.
  • the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-15 or DOM7h-11-15 S12P , or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has M at position 32, L at position 49, A at position 50 and F at position 51.
  • the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-18, or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has M at position 32 and H at position 87.
  • the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-19, or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has M at position 32, L at position 49 and T at position 91. All numbering in this paragraph is according to Kabat.
  • An aspect of the invention provides an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11, wherein the variant comprises a Met at position 32 (numbering according to Kabat) compared to DOM7h-11, and wherein the variant has from 0 to 4 further changes compared to the amino acid sequence of DOM7h-11.
  • the variant comprises at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) compared to DOM7h-11.
  • the variant comprises at least one mutation compared to DOM7h-11 selected from the following
  • the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-12, DOM7h-11-15, DOM7h-11-15 S12P , DOM7h-11-18 and DOM7h-11-19 or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has Met at position 32.
  • the variant comprises one or more of the following kinetic characteristics:—
  • the invention also provides a ligand comprising a variant of any preceding aspect or embodiment of the invention.
  • the ligand can be a dual-specific ligand (see WO04003019 for examples of dual-specific ligands).
  • the invention provides a multispecific ligand comprising an anti-SA variant of any preceding aspect or embodiment of the invention and a binding moiety that specifically binds a target antigen other than SA.
  • the binding moiety can be any binding moiety that specifically binds a target, eg, the moiety is an antibody, antibody fragment, scFv, Fab, dAb or a binding moiety comprising a non-immunoglobulin protein scaffold.
  • non-immunoglobulin scaffolds are CTLA-4, lipocallin, staphylococcal protein A (spA), AffibodyTM AvimersTM, adnectins, GroEL and fibronectin.
  • a linker is provided between the anti-target binding moiety and the anti-SA single variant, the linker comprising the amino acid sequence AST, optionally ASTSGPS.
  • Alternative linkers are described in WO2007085814 (incorporated herein by reference) and WO2008/096158 (see the passage at page 135, line 12 to page 140, line 14, which disclosure and all sequences of linkers are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein).
  • the target antigen may be, or be part of, polypeptides, proteins or nucleic acids, which may be naturally occurring or synthetic.
  • the ligand of the invention may bind the target antigen and act as an antagonist or agonist (e.g., EPO receptor agonist).
  • EPO receptor agonist e.g., EPO receptor agonist
  • One skilled in the art will appreciate that the choice is large and varied. They may be for instance, human or animal proteins, cytokines, cytokine receptors, where cytokine receptors include receptors for cytokines, enzymes, co-factors for enzymes or DNA binding proteins.
  • Suitable cytokines and growth factors include, but are preferably not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF- ⁇ 1, insulin, IFN- ⁇ , IGF-I, IGF-II, IL-1 ⁇ , IL-113, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin ⁇ , Inhibin ⁇ , IP-10,
  • EGFR epidermal growth factor receptor
  • ErBb2 receptor on tumor cells
  • an internalising cellular receptor LDL receptor
  • FGF2 receptor ErbB2 receptor
  • transferrin receptor PDGF receptor
  • VEGF receptor VEGF receptor
  • PsmAr an extracellular matrix protein
  • elastin fibronectin
  • laminin al-antitrypsin
  • tissue factor protease inhibitor PDK1, GSK1, Bad
  • caspase-9 Forkhead
  • an antigen of Helicobacter pylori an antigen of Mycobacterium tuberculosis
  • an antigen of influenza virus an antigen of influenza virus. It will be appreciated that this list is by no means exhaustive.
  • the multispecific ligand comprises an anti-SA dAb variant of the invention and an anti-TNFR1 binding moiety, eg, an anti-TNFR1 dAb.
  • the ligand has only one anti-TNFR1 binding moiety (eg, dAb) to reduce the chance of receptor cross-linking.
  • the anti-SA dAb variant is DOM7h-11-3 or DOM7h-11-15 or DOM7h-11-15 S12P .
  • the anti-TNFR1 binding moiety is DOM1h-131-206 disclosed in WO2008149148 (the amino acid sequence of which and the nucleotide sequence of which, as disclosed in that PCT application, are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein).
  • the multispecific ligand comprises or consists of the amino acid sequence of DOM1h-131-206 and the amino acid sequence of DOM7h-11-3 or DOM7h-11-15 or DOM7h-11-15 S12P .
  • the anti-TNFR1 binding moiety or dAb is any such moiety or dAb disclosed in co-pending application U.S. Ser. No. 61/153,746, the disclosure of which is incorporated herein by reference.
  • the anti-TNFR1 binding moiety comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of DOM1h-574-156, DOM1h-574-72, DOM1h-574-109, DOM1h-574-138, DOM1h-574-162 or DOM1h-574-180 or the amino acid sequence of any anti-TNFR1 dAb disclosed in Table 3.
  • the multispecific ligand comprises or consists of the amino acid sequence of DOM1h-574-156 and the amino acid sequence of DOM7h-11-3 or DOM7h-11-15 or DOM7h-11-15 S12P .
  • the ligand of the invention is a fusion protein comprising a variant of the invention fused directly or indirectly to one or more polypeptides.
  • the fusion protein can be a “drug fusion” as disclosed in WO2005/118642 (the disclosure of which is incorporated herein by reference), comprising a variant of the invention and a polypeptide drug as defined in that PCT application.
  • drug refers to any compound (e.g., small organic molecule, nucleic acid, polypeptide) that can be administered to an individual to produce a beneficial, therapeutic or diagnostic effect through binding to and/or altering the function of a biological target molecule in the individual.
  • the target molecule can be an endogenous target molecule encoded by the individual's genome (e.g. an enzyme, receptor, growth factor, cytokine encoded by the individual's genome) or an exogenous target molecule encoded by the genome of a pathogen (e. g. an enzyme encoded by the genome of a virus, bacterium, fungus, nematode or other pathogen).
  • Suitable drugs for use in fusion proteins and conjugates comprising an anti-SA dAb variant of the invention are disclosed in WO2005/118642 and WO2006/059106 (the entire disclosures of which are incorporated herein by reference, and including the entire list of specific drugs as though this list were expressly written herein, and it is contemplated that such incorporation provides disclosure of specific drugs for inclusion in claims herein).
  • the drug can be glucagon-like peptide 1 (GLP-1) or a variant, interferon alpha 2b or a variant or exendin-4 or a variant.
  • the invention provides a drug conjugate as defined and disclosed in WO2005/118642 and WO2006/059106, wherein the conjugate comprises a variant of the invention.
  • the drug is covalently linked to the variant (eg, the variant and the drug are expressed as part of a single polypeptide).
  • the drug is non-covalently bonded or associated with the variant.
  • the drug can be covalently or noncovalently bonded to the variant directly or indirectly (e.g., through a suitable linker and/or noncovalent binding of complementary binding partners (e.g., biotin and avidin)).
  • one of the binding partners can be covalently bonded to the drug directly or through a suitable linker moiety, and the complementary binding partner can be covalently bonded to the variant directly or through a suitable linker moiety.
  • the drug is a polypeptide or peptide
  • the drug composition can be a fusion protein, wherein the polypeptide or peptide, drug and the polypeptide binding moiety are discrete parts (moieties) of a continuous polypeptide chain.
  • the polypeptide binding moieties and polypeptide drug moieties can be directly bonded to each other through a peptide bond, or linked through a suitable amino acid, or peptide or polypeptide linker.
  • a ligand which contains one single variable domain (monomer) variant of the invention or more than one single variable domain (multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin can further comprise one or more entities selected from, but preferably not limited to a label, a tag, an additional single variable domain, a dAb, an antibody, an antibody fragment, a marker and a drug.
  • entities selected from, but preferably not limited to a label, a tag, an additional single variable domain, a dAb, an antibody, an antibody fragment, a marker and a drug.
  • One or more of these entities can be located at either the COOH terminus or at the N terminus or at both the N terminus and the COOH terminus of the ligand comprising the single variable domain, (either immunoglobulin or non-immunoglobulin single variable domain).
  • One or more of these entities can be located at either the COOH terminus, or the N terminus, or both the N terminus and the COOH terminus of the single variable domain which specifically binds serum albumin of the ligand which contains one single variable domain (monomer) or more than one single variable domains (multimer, fusion protein, conjugate, and dual specific ligand as defined herein).
  • Non-limiting examples of tags which can be positioned at one or both of these termini include a HA, his or a myc tag.
  • the entities can be bound to the ligand which contains one single variable domain (monomer) or more than one single variable domain (multimer, fusion protein, conjugate, and dual specific ligand as defined herein), which binds serum albumin, either directly or through linkers as described above.
  • An aspect of the invention provides a fusion product, eg, a fusion protein or fusion with a peptide or conjugate with an NCE (new chemical entity) drug, comprising a polypeptide drug fused or conjugated (for an NCE) to any variant as described above, optionally wherein the variant is DOM7h-11-15 or DOM7h-11-15 S12P (or a variant having an amino acid that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of DOM7h-11-15 or DOM7h-11-15 S12P ) or DOM7h-11-12 (or a variant having an amino acid that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of DOM7h-11-15 or DOM7h-11-15 S12P ).
  • DOM7h-11-15, DOM7h-11-15 S12P and DOM7h-11-12 give only a modest drop in affinity when fused or conjugated to partner, making them useful in fusion products.
  • the invention provides a composition comprising a variant, fusion protein, conjugate or ligand of any aspect of the invention and a pharmaceutically acceptable diluent, carrier, excipient or vehicle.
  • nucleic acid encoding any of the variants, fusion proteins, conjugates or ligands described herein, e.g., a ligand which contains one single variable domain (monomer) variant of the invention or more than one single variable domain (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) variant which specifically binds to serum albumin, or which specifically binds both human serum albumin and at least one non-human serum albumin, or functionally active fragments thereof.
  • a ligand which contains one single variable domain (monomer) variant of the invention or more than one single variable domain (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) variant which specifically binds to serum albumin, or which specifically binds both human serum albumin and at least one non-human serum albumin, or functionally active fragments thereof.
  • a vector and/or an expression vector a host cell comprising the vector, e.g., a plant or animal cell and/or cell line transformed with a vector, a method of expressing and/or producing one or more variants, fusion proteins or ligands which contains one single variable domain (monomer) variant or more than one single variable domain variants (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, or fragment(s) thereof encoded by said vectors, including in some instances culturing the host cell so that the one or more variants, fusion proteins or ligands or fragments thereof are expressed and optionally recovering the ligand which contains one single variable domain (monomer) or more than one single variable domain (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, from the host cell culture medium.
  • the vector e.g., a plant or
  • Vectors encoding the nucleic acid molecules are contemplated herein, as are host cells from a cell line or organism containing one or more of these expression vectors. Also contemplated are methods of producing any variant, fusion protein or ligand, including, but preferably not limited to any of the aforementioned nucleic acids, vectors and host cells.
  • An aspect of the invention provides a nucleic acid comprising a nucleotide sequence encoding a variant according to the invention or a multispecific ligand of the invention or fusion protein of the invention.
  • An aspect of the invention provides a nucleic acid comprising the nucleotide sequence of a DOM7h-11 variant selected from DOM7h-11-3, DOM7h-11-15, DOM7h-11-15 S12P , DOM7h-11-12, DOM7h-11-18 and DOM7h-11-19 or a nucleotide sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said selected sequence.
  • An aspect of the invention provides a vector comprising the nucleic acid of the invention.
  • An aspect of the invention provides an isolated host cell comprising the vector.
  • DOM7h-14 variant sequences which are not according to the invention, are disclosed in a co-pending US provisional patent application entitled IMPROVED ANTI-SERUM ALBUMIN BINDING VARIANTS, filed on the same day as the present application. These sequences of DOM7h-14 variants (SEQ ID NO:s 1-10 in the co-pending application) are incorporated herein by reference as though explicitly written herein.
  • Nucleotide sequence (SEQ ID NO: 320) encoding DOM7h14-10/G4SC GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC CGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCT TGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGGCGT TCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCTACG TACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAAGGGACC AAGGTGGAAATCAAACGGGGTGGCGGAGGGGGTTCCTGT DOM7h14-10/TVAAPSC fusion Amino acid sequence (SEQ ID NO: 321) DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLL
  • Nucleotide sequence (SEQ ID NO: 322) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC CGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCT TGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGGCGT TCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCTACG TACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAAGGGACC AAGGTGGAAATCAAACGGACCGTCGCTGCTCCATCTTGT (used in mouse studies):- DOM7h-11/DOM1m-21-23 fusion DMS number 5515 Amino acid sequence (SEQ ID NO: 323) EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVS
  • DOM7h-11 and DOM7h-14 variants are not according to the invention.
  • HSA Human Serum Albumin
  • RSA Raster Serum Albumin antigens
  • Biotinylated products of above two antigens were made by using EZ Link Sulfo-NHS-SS-Biotin (Pierce, Cat. No. 21331). Free biotin reagent was removed by passing the samples twice through PD10 desalting column followed by overnight dialysis against 1000 ⁇ excess volume of PBS at 4° C. Resulting product was tested by mass spec and 1-2 biotins per molecule were observed.
  • Vector pDOM4 is a derivative of the Fd phage vector in which the gene III signal peptide sequence is replaced with the yeast glycolipid anchored surface protein (GAS) signal peptide. It also contains a c-myc tag between the leader sequence and gene III, which puts the gene III back in frame.
  • pDOM33 is a modified version of the pDOM4 vector where the c-myc tag has been removed which renders the dAb-phage fusion resistant to the protease trypsin. This allows the use of trypsin within the phage selection to select for dAbs that are more protease stable (see WO2008149143).
  • plasmid DNA encoding the dAb to be matured was amplified by PCR, using the GENEMORPH® II RANDOM MUTAGENESIS KIT (random, unique mutagenesis kit, Stratagene). The product was digested with Sal I and Not I and used in a ligation reaction with cut phage vector pDOM33.
  • PCR reactions were performed using degenerate oligonucleotides containing NNK or NNS codons to diversify the required positions in the dAb to be affinity matured. Assembly PCR was then used to generate a full length diversified insert.
  • the insert was digested with Sal I and Not I and used in a ligation reaction with pDOM4 for mutagenesis of multiple residues and pDOM5 for mutagenesis of single residues.
  • the pDOM5 vector is a pUC119-based expression vector where protein expression is driven by the LacZ promoter.
  • a GAS1 leader sequence (see WO 2005/093074) ensures secretion of isolated, soluble dAbs into the periplasm and culture supernatant of E.
  • dAbs are cloned SalI/NotI in this vector, which appends a myc tag at the C-terminus of the dAb.
  • This protocol using SalI and Not I results in inclusion of an ST amino acid sequence at the N-terminus.
  • the ligation produced by either method was then used to transform E. coli strain TB1 by electroporation and the transformed cells plated on 2 ⁇ TY agar containing 15 ⁇ g/ml tetracycline, yielding library sizes of >5 ⁇ 10 7 clones.
  • the error-prone libraries had the following average mutation rate and size: DOM7h-11 (2.5 mutations per dAb), size: 6.1 ⁇ 10 8 , DOM7h-14 (2.9 mutations per dAb), size: 5.4 ⁇ 10 8 .
  • Each CDR library has four amino acid diversity. Two libraries were generated for each of CDRs 1 and 3, and one library for CDR2. The positions diversified within each library are as follows (amino acids based on VK dummy DPK9 sequence):
  • V ⁇ ALBUDABTM anti-serum albumin dAb
  • HSA soluble selection
  • biotinylated RSA concentrations of 1 ⁇ M for round 1, 100 nm for round 2 and 20 nM, 10 nM or 1 nM for round 3.
  • a pool of phage DNA from the appropriate round of selection is prepared using a QIAfilter midiprep kit (Qiagen), the DNA is digested using the restriction enzymes Sal1 and Not1 and the enriched V genes are ligated into the corresponding sites in pDOM5 the soluble expression vector which expresses the dAb with a myc tag (see PCT/EP2008/067789).
  • the ligated DNA is used to electro-transform E. coli HB 2151 cells which are then grown overnight on agar plates containing the antibiotic carbenicillin. The resulting colonies are individually assessed for antigen binding.
  • the culture supernatant containing soluble dAb was centrifuged and analysed by BiaCoreTM for binding to high density HSA, CSA, MSA and RSA CM5 chips. Clones were found to bind to all these species of serum albumin by off-rate screening. The clones were sequenced revealing unique dAb sequences.
  • the minimum identity to parent (at the amino acid level) of the clones selected was 97.2% (DOM7h-11-3: 97.2%, DOM7h-11-12: 98.2%, DOM7h11-15: 96.3%, DOM7h-11-18: 98.2%, DOM7h-11-19: 97.2%)
  • the minimum identity to parent (at the amino acid level) of the clones selected was 96.3% (DOM7h-14-10: 96.3%, DOM7h-14-18: 96.3%, DOM7h-14-19: 98.2%, DOM7h-14-28: 99.1%, DOM7h-14-36: 97.2%)
  • dAbs were expressed as bacterial supernatants in 2.5 L shake flasks in Onex media at 30° C. for 48 hrs at 250 rpm. dAbs were purified from the culture media by absorption to protein L agarose followed by elution with 10 mM glycine pH2.0. Binding to HSA, CSA, MSA and RSA by BiaCoreTM was confirmed using purified protein at 3 concentrations 1 ⁇ M, 500 nM and 50 nM.
  • K D binding affinity
  • DOM7h-14 derived variants are cross-reactive to mouse, rat, human and cyno serum albumin.
  • DOM7h-14-10 has improved affinity to rat, cyno and human serum albumin compared to parent.
  • DOM7h-14-28 has an improved affinity to RSA.
  • DOM7h-14-36 has an improved affinity to RSA, CSA and MSA.
  • DOM7h-11-3 has improved affinity to CSA and HSA.
  • DOM7h-11-12 has improved affinity to RSA, MSA and HSA.
  • DOM7h-11-15 has improved affinity to RSA, MSA, CSA and HSA.
  • DOM7h-11-18 and DOM7h-11-19 have improved affinity to RSA, MSA and HSA.
  • DOM7h-11-3 From affinity maturation performed against HSA using the CDR2 library (Y49, A50, A51, S53), round 3 output 10 nM HSA
  • DOM7h-11-12 From affinity maturation performed against HSA using the error prone library, round 3 outputs (100 nM, HSA) with 100 ug/ml trypsin.
  • DOM7h-11-15 From cross-over selections performed against HSA as round 1 followed by additional 3 rounds of selections against RSA using the CDR2 library (Y49, A50, A51, S53) at round 3 selection with 1 nM of RSA.
  • DOM7h-14-19 From affinity maturation performed against HSA using the error prone library, round 3 outputs (100 nM, HSA) with 100 ug/ml trypsin.
  • DOM7h-14-10, DOM7h-14-18, DOM7h-14-28, DOM7h-14-36 From affinity maturation performed against HSA using CDR3 library (Y92, Y93, T94, N96), round 3 output.
  • the routine bacterial expression level in 2.5 L shake flasks was determined following culture in Onex media at 30° C. for 48 hrs at 250 rpm.
  • the biophysical characteristics were determined by SEC MALLS and DSC.
  • SEC MALLS size exclusion chromatography with multi-angle-LASER-light-scattering
  • RI refractive index
  • DSC Different Scanning calorimetry: briefly, the protein is heated at a constant rate of 180° C./hrs (at 1 mg/mL in PBS) and a detectable heat change associated with thermal denaturation measured.
  • the transition midpoint ( app T m ) is determined, which is described as the temperature where 50% of the protein is in its native conformation and the other 50% is denatured.
  • DSC determined the apparent transition midpoint (appTm) as most of the proteins examined do not fully refold. The higher the Tm, the more stable the molecule. Unfolding curves were analysed by non-2-state equations.
  • the software package used was Origin R v7.0383.
  • ALBUDABsTM DOM7h-14-10, DOM7h-14-18, DOM7h-14-19, DOM7h-11, DOM7h11-12 and DOM7h-11-15 were cloned into the pDOM5 vector.
  • ALBUDABTM 20-50 mg quantities were expressed in E. coli and purified from bacterial culture supernatant using protein L affinity resin and eluted with 100 mM glycine pH2. The proteins were concentred to greater than 1 mg/ml, buffer exchanged into PBS and endotoxin depleted using Q spin columns (Vivascience).
  • PK pharmacokinetic
  • ALBUDABsTM were dosed as single i.v injections at 2.5 mg/kg using 3 rats per compound. Serum samples were taken at 0.16, 1, 4, 12, 24, 48, 72, 120, 168 hrs. Analysis of serum levels was by anti-myc ELISA as per the method described below.
  • DOM7h-11, DOM7h11-12 and DOM7h-11-15 were dosed as single i.v injections at 2.5 mg/kg per dose group of 3 subjects and serum samples taken at 10 mins; 1 h; 8 h; 24 h; 48 h; 72 h; 96 h. Analysis of serum levels was by anti-myc ELISA as per the method described below.
  • Cynomolgus monkey PK DOM7h-14-10 and DOM7h-11-15 were dosed as single i.v injections at 2.5 mg/kg into 3 female Cynomolgus monkeys per dose group and serum samples taken at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, 144, 192, 288, 336, 504 hrs. Analysis of serum levels was by anti-myc ELISA as per the method described below.
  • the ALBUDABTM concentration in serum was measured by anti-myc ELISA. Briefly, goat anti-myc polyclonal antibody (1:500; Abcam, catalogue number ab9132) was coated overnight onto Nunc 96-well Maxisorp plates and blocked with 5% BSA/PBS+1% tween. Serum samples were added at a range of dilutions alongside a standard at known concentrations. Bound myc-tagged ALBUDAB′ was then detected using a rabbit polyclonal anti-Vk (1:1000; in-house reagent, bleeds were pooled and protein A purified before use) followed by an anti-rabbit IgG HRP antibody (1:10,000; Sigma, catalogue number A2074).
  • TMB Standard TMB 1-Component Microwell Peroxidase Substrate, KPL, catalogue number 52-00-00
  • the concentration of unknown samples was established by interpolation against the standard curve taking into account dilution factors.
  • the mean concentration result from each time point was determined from replicate values and entered into WinNonLin analysis package (eg version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA).
  • the data was fitted using a non-compartmental model, where PK parameters were estimated by the software to give terminal half-lives. Dosing information and time points were selected to reflect the terminal phase of each PK profile.
  • AUC Area under the curve from dosing time extrapolated to infinity
  • CL clearance
  • t1 ⁇ 2 is the time during which the blood concentration is halved
  • Vz volume of distribution based on the terminal phase.
  • DOM7h-11 12 and DOM7h-11-15 have an improved AUC and t1/2 in rat and mouse compared to parent.
  • DOM7h-11-15 also has an improved AUC and t1/2 in cyno compared to parent. This improvement in AUC/t1/2 correlates with an improved in vitro KD to serum albumin.
  • the affinity matured Vk ALBUDABsTM were linked to Interferon alpha 2b (IFN ⁇ 2b) to determine whether a useful PK of the ALBUDABTM was maintained as a fusion protein.
  • IFN ⁇ 2b Interferon alpha 2b
  • Interferon alpha 2b amino acid sequence (SEQ ID NO: 374) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKA ETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVI QGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRS FSLSTNLQESLRSKE Interferon alpha 2b nucleotide sequence: (SEQ ID NO: 375) TGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCT CCTGGCACAGATGAGGAAAATCTCTTTTCTCCTGCTTGAAGGACAGAC ATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCT GAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTT CAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACC
  • IFNa2b was linked to the ALBUDABTM via a TVAAPS (SEQ ID NO: 422) linker region (see WO2007085814).
  • the constructs were cloned by SOE-PCR (single overlap extension according to the method of Horton et al. Gene, 77, p 61 (1989)).
  • PCR amplification of the ALBUDABTM and IFN sequences were carried out separately using primers with a ⁇ 15 base pair overlap at the TVAAPS (SEQ ID NO: 422) linker region.
  • the primers used are as follows:—
  • IFN ⁇ 2b SOE fragment 5′ (SEQ ID NO: 376) GCCCGGATCCACCGGCTGTGATCTG IFN ⁇ 2b SOE fragment 3′ (SEQ ID NO: 377) GGAGGATGGAGACTGGGTCATCTGGATGTC Vk SOE fragment 5′ (SEQ ID NO: 378) GACATCCAGATGACCCAGTCTCCATCCTCC Vk SOE fragment 3′ to also introduce a myc tag (SEQ ID NO: 379) GCGCAAGCTTTTATTAATTCAGATCCTCTTC TGAGATGAGTTTTTGTTCTGCGGCCGCCCGT TTGATTTCCACCTTGGTCCC
  • the fragments were purified separately and subsequently assembled in a SOE (single overlap extension PCR extension) reaction using only the flanking primers.
  • SOE single overlap extension PCR extension
  • IFN ⁇ 2b SOE fragment 5′ (SEQ ID NO: 380) GCCCGGATCCACCGGCTGTGATCTG Vk SOE fragment 3′ to also introduce a myc tag (SEQ ID NO: 381) GCGCAAGCTTTTATTAATTCAGATCCTCTTC TGAGATGAGTTTTTGTTCTGCGGCCGCCCGT TTGATTTCCACCTTGGTCCC
  • the assembled PCR product was digested using the restriction enzymes BamHI and HindIII and the gene ligated into the corresponding sites in the pDOM50, a mammalian expression vector which is a pTT5 derivative with an N-terminal V-J2-C mouse IgG secretory leader sequence to facilitate expression into the cell media.
  • Plasmid DNA was prepared using QIAfilter megaprep (Qiagen). 1 ⁇ g DNA/ml was transfected with 293-Fectin into HEK293E cells and grown in serum free media. The protein is expressed in culture for 5 days and purified from culture supernatant using protein L affinity resin and eluted with 100 mM glycine pH2. The proteins were concentred to greater than 1 mg/ml, buffer exchanged into PBS and endotoxin depleted using Q spin columns (Vivascience).
  • Interferon alpha 2b-ALBUDAB TM sequences with and without myc-tag (as amino acid- and nucleotide sequence)
  • the Interferon alpha 2b is N-terminal to the ALBUDAB TM in the following fusions.
  • K D binding affinity of the ALBUDABTM-IFN ⁇ 2b fusion proteins to each serum albumin
  • purified fusion proteins were analysed by BiaCoreTM over albumin (immobilised by primary-amine coupling onto CM5 chips; BiaCoreTM) using fusion protein concentrations from 5000 nM to 39 nM (5000 nM, 2500 nM, 1250 nM, 625 nM, 312 nM, 156 nM, 78 nM, 39 nM) in HBS-EP BiaCoreTM buffer.
  • Biophysical Characterisation was carried out by SEC MALLS and DSC as described above for the single ALBUDABs TM Biophysical parameters DMS SEC DSC ALBUDAB TM Fusion number MALLS Tm(° C.) DOM 7h-14 IFN ⁇ 2b DMS7321 M/D 58-65 DOM 7h-14-10 IFN ⁇ 2b DMS7322 M/D 55-65 DOM 7h-14-18 IFN ⁇ 2b DMS7323 M/D 55-65 DOM 7h-14-19 IFN ⁇ 2b DMS7324 M/D 59-66 DOM 7h-11 IFN ⁇ 2b DMS7325 M/D 65.8-66.2 DOM 7h-11-12 IFN ⁇ 2b DMS7326 M/D 67-67.3 DOM 7h-11-15 IFN ⁇ 2b DMS7327 M/D 56.3-66.2 M/D indicates a monomer/dimer equilibrium as detected by SEC MALLS We observed expression for all clones in Tabale 13 in the range of 17.5 to
  • ALBUDABsTM IFN ⁇ 2b fusions DMS7321 (IFN ⁇ 2b-DOM7h-14) DMS7322 (IFN ⁇ 2b-DOM7h-14-10) DMS7323 (IFN ⁇ 2b-DOM7h-14-18), DMS7324 (IFN ⁇ 2b-DOM7h-14-19), DMS7325 (IFN ⁇ 2b-DOM7h-11), DMS7326 (IFN ⁇ 2b-DOM7h-11-12), DMS7327 (IFN ⁇ 2b-DOM7h-11-15) were expressed with the myc tag at 20-50 mg quantities in HEK293 cells and purified from culture supernatant using protein L affinity resin and eluted with 100 mM glycine pH2. The proteins were concentrated to greater than 1 mg/ml, buffer exchanged into Dulbecco's PBS and endotoxin depleted using Q spin columns (Vivascience).
  • IFN-ALBUDABsTM were dosed as single i.v injections at 2.0 mg/kg using 3 rats per compound. Serum samples were taken at 0.16, 1, 4, 8, 24, 48, 72, 120, 168 hrs. Analysis of serum levels was by EASY ELISA according to manufacturers instructions (GE Healthcare, catalogue number RPN5960).
  • DMS7322 (IFN2b-DOM7h-14-10) DMS7325 (IFN2b-DOM7h-11), DMS7326 (IFN2b-DOM7h-11-12), DMS7327 (IFN2b-DOM7h-11-15) all with myc tags were dosed as single i.v injections at 2.0 mg/kg per dose group of 3 subjects and serum samples taken at 10 mins; 1 h; 8h; 24h; 48h; 72h; 96h. Analysis of serum levels was by EASY ELISA according to manufacturers instructions (GE Healthcare, catalogue number RPN5960).
  • AUC Area under the curve from dosing time extrapolated to infinity
  • CL clearance
  • t1 ⁇ 2 is the time during which the blood concentration is halved
  • Vz volume of distribution based on the terminal phase.
  • IFN ⁇ 2b -ALBUDABs TM were tested in rat and mouse.
  • t1 ⁇ 2 is improved compared to parent.
  • the improvement in t1 ⁇ 2 correlates with the improved in vitro K D to serum albumin.
  • IFN ⁇ 2b-DOM7h-14-10 variants the improvement in in vitro K D to serum albumin also correlated to an improvement in t1 ⁇ 2 in rat.
  • NCE, NCE-GGGGSC and NCE-TVAAPSC are described below.
  • ABUDAB TM albumin-binding dAb
  • exendin-4-linker-ALBUDAB′ sequence was cloned into the pTT-5 vector (obtainable from CNRC, Canada).
  • the exendin-4 was at the 5′ end of the construct and the dAb at the 3′ end.
  • the linker was a (G4S)3 linker.
  • Endotoxin-free DNA was prepared in E. coli using alkaline lysis (using the endotoxin-free plasmid Giga kit, obtainable from Qiagen CA) and used to transfect HEK293E cells (obtainable from CNRC, Canada).
  • Transfection was into 250 ml/flask of HEK293E cells at 1.75 ⁇ 10 6 cells/ml using 333 ul of 293 fectin (Invotrogen) and 250 ug of DNA per flask and expression was at 30° C. for 5 days.
  • the supernatant was harvested by centrifugation and purification was by affinity purification on protein L.
  • Protein was batch bound to the resin, packed on a column and washed with 10 column volumes of PBS. Protein was eluted with 50 ml of 0.1M glycine pH2 and neatralised with Tris pH8. Protein of the expected size was identified on an SDS-PAGE gel.
  • NCE new chemical entity
  • ALBUDABTM fusion was tested.
  • the NCE, a small molecule ADAMTS-4 inhibitor was synthesised with a PEG linker (PEG 4 linker (ie 4 PEG molecules before the maleimide) and a maleimide group for conjugation to the ALBUDABTM.
  • PEG 4 linker ie 4 PEG molecules before the maleimide
  • maleimide group for conjugation to the ALBUDABTM.
  • Conjugation of the NCE to the ALBUDABTM is via an engineered cystine residue at amino acid position R108C, or following a 5 amino acid (GGGGSC) or 6 amino acid (TVAAPSC (SEQ ID NO: 419)) spacer engineered at the end of the ALBUDABTM.
  • the AALBUDABTM was reduced with TCEP (Pierce, Catalogue Number 77720), desalted using a PD10 column (GE healthcare) into 25 mM Bis-Tris, 5 mM EDTA, 10% (v/v) glycerol pH6.5.
  • TCEP Triplicate, Catalogue Number 77720
  • PD10 column GE healthcare
  • a 5 fold molar excess of maleimide activated NCE was added in DMSO not to exceed 10% (V/V) final concentration.
  • the reaction was incubated over night at room temperature and dialysed extensively into 20 mM Tris pH7.4
  • DOM7h-14 R108C (SEQ ID NO: 412) DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ GTKVEIKC Nucleotide: (SEQ ID NO: 413) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA GGGACCAAGGTGGAAATCAAATGC
  • NCE-ALBUDABsTM DOM7h-14-10 GGGGSC(SEQ ID NO: 62) and DOM7h14-10 TVAAPSC exhibit a 5 to 10 fold decrease in in vitro affinity (K D ) to RSA as determined by BiaCoreTM when fused to the chemical entity. PK data are not available for these molecules yet.
  • dAb-ALBUDABTM fusion the 2 DOM7h-11 ALBUDABsTM with the highest affinity to RSA experience a 2-fold decrease in affinity to RSA as on BiaCoreTM when fused to a therapeutic domain antibody (DOM1m-21-23) compared to the unfused ALBUDABTM.
  • the DOM7h-11 clone shows a micromolar K D when fused (2.8 uM) as well as when unfused ( ⁇ 5 uM).
  • Exendin 4-ALBUDABTM fusion the effect of fusing the ALBUDABsTM to a peptide on the binding ability to RSA is about 10-fold, apart from DOM7h-14-10, which only shows a 4-fold decrease in binding. The effect, however, is more pronounced for the DOM7h-14 series (except DOM7h-14-10) than it appears to be for the DOM7h-11 series.
  • the T1/2 of the fusion increased with improved affinity to the species' SA.
  • ALBUDABTM-therapeutics are therapeutically amenable (for treatment and/or prophylaxis of diseases, conditions or indications) when the ALBUDABTM-drug fusions show an affinity range (K D ) of from 0.1 nM to 10 mM for serum albumin binding.
  • ALBUDABsTM and ALBUDABTM fusions Provide the therapeutic ranges of ALBUDABsTM and ALBUDABTM fusions (Protein-ALBUDABsTM for example IFN ⁇ 2b-DOM7h-14-10; Peptide-ALBUDABsTM for example Exendin-4-DOM7h-14-10; dAb-ALBUDABsTM for example DOM1m21-23-DOM7h11-15; NCE-ALBUDABTM for example ADAMTS-4-DOM7h-14-10) as follows: Affinity (K D ) ranges that are useful for therapy of chronic or acute conditions, diseases or indictions are shown. Also shown are affinity ranges marked as “intermediate”. ALBUDABsTM and fusions in this range have utility for chronic or acute diseases, conditions or indications.
  • the affinity of the ALBUDABTM or fusion for serum albumin can be tailored or chosen according to the disease, condition or indication to be addressed.
  • the invention provides ALBUDABsTM with affinities that allow for each ALBUDABTM to be categorised as “high affinity”, “medium affinity” or “low affinity”, thus enabling the skilled person to select the appropriate ALBUDABTM of the invention according to the therapy at hand. See FIG. 2 .
  • An aspect of the invention provides a nucleic acid comprising the nucleotide sequence of DOM7h-11-15 S12P or a nucleotide sequence that is at least 80% identical to said selected sequence.
  • DOM7h-11-15 S12P was produced using the following nucleic acid sequence (the underlined C denotes the change (versus the nucleic acid encoding DOM7h-11-15) leading to a proline at position 12):—
  • DOM7h-11-15 S12P was constructed by using DOM7h-11-15 as a template in a PCR where a primer was used to introduce the S12P mutation.
  • the primer sequence is:—
  • An alternative aspect of the invention provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 415 or a nucleotide sequence that is at least 80% identical to said selected sequence.
  • DOM7h-11-15 S12P is encoded by, and expressed from, a vector that contains a linker region and a C-terminal sequence encoding a protein or peptide drug or a single variable domain or other antibody fragment to make the in-line protein fusion product.
  • the linker in one embodiment, comprises the amino acid sequence TVA, eg, TVAAPS (SEQ ID NO: 422).
  • Other aspects of the invention are a vector comprising the nucleic acid; and an isolated host cell comprising the vector.
  • the invention also provides a method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of DOM7h-11-15 S12P to said patient.

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Abstract

The invention relates to improved variants of the anti-serum albumin immunoglobulin single variable domain DOM7h-11, as well as ligands and drug conjugates comprising such variants, compositions, nucleic acids, vectors and hosts.

Description

  • The invention relates to improved variants of the anti-serum albumin immunoglobulin single variable domain DOM7h-11, as well as ligands and drug conjugates comprising such variants, compositions, nucleic acids, vectors and hosts.
    • BACKGROUND OF THE INVENTION
  • WO04003019 and WO2008/096158 disclose anti-serum albumin (SA) binding moieties, such as anti-SA immunoglobulin single variable domains (dAbs), which have therapeutically-useful half-lives. These documents disclose monomer anti-SA dAbs as well as multi-specific ligands comprising such dAbs, eg, ligands comprising an anti-SA dAb and a dAb that specifically binds a target antigen, such as TNFR1. Binding moieties are disclosed that specifically bind serum albumins from more than one species, eg human/mouse cross-reactive anti-SA dAbs.
  • WO05118642 and WO2006/059106 disclose the concept of conjugating or associating an anti-SA binding moiety, such as an anti-SA immunoglobulin single variable domain, to a drug, in order to increase the half-life of the drug. Protein, peptide and NCE (new chemical entity) drugs are disclosed and exemplified. WO2006/059106 discloses the use of this concept to increase the half-life of insulintropic agents, eg, incretin hormones such as glucagon-like peptide (GLP)-1.
  • Reference is also made to Holt et al, “Anti-Serum albumin domain antibodies for extending the half-lives of short lived drugs”, Protein Engineering, Design & Selection, vol 21, no 5, pp 283-288, 2008.
  • WO2008/096158 discloses DOM7h-11, which is a good anti-SA dAb. It would be desirable to provide improved dAbs that are variants of DOM7h-11 and that specifically bind serum albumin, preferably albumins from human and non-human species, which would provide utility in animal models of disease as well as for human therapy and/or diagnosis. It would also be desirable to provide for the choice between relatively modest- and high-affinity anti-SA binding moieties (dAbs). Such moieties could be linked to drugs, the anti-SA binding moiety being chosen according to the contemplated end-application. This would allow the drug to be better tailored to treating and/or preventing chronic or acute indications, depending upon the choice of anti-SA binding moiety. It would also be desirable to provide anti-dAbs, that are monomeric or substantially so in solution. This would especially be advantageous when the anti-SA dAb is linked to a binding moiety, eg, a dAb, that specifically binds a cell-surface receptor, such as TNFR1, with the aim of antagonizing the receptor. The monomeric state of the anti-SA dAb is useful in reducing the chance of receptor cross-linking, since multimers are less likely to form which could bind and cross-link receptors (eg, TNFR1) on the cell surface, thus increasing the likelihood of receptor agonism and detrimental receptor signaling.
    • SUMMARY OF THE INVENTION
  • Aspects of the present invention solve these problems.
  • To this end, the present inventors surprisingly found that beneficial mutations can be targeted to the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) of DOM7h-11.
  • In one aspect the invention, therefore, provides an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11, wherein the variant comprises at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) compared to DOM7h-11, and wherein the variant has from 2 to 8 changes compared to the amino acid sequence of DOM7h-11.
  • In one aspect the invention provides an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11, wherein the variant comprises a Met at position 32 (numbering according to Kabat) compared to DOM7h-11, and wherein the variant has from 0 to 4 further changes compared to the amino acid sequence of DOM7h-11.
  • Embodiments of either aspect of the invention provide DOM7h-11 variants of good anti-serum albumin affinities. The choice of variant can allow for tailoring of half-life according to the desired therapeutic and/or prophylactic setting. For example, in one embodiment, the affinity of the variant for serum albumin is relatively high, such that the variant would be useful for inclusion in products that find utility in treating and/or preventing chronic or persistent diseases, conditions, toxicity or other chronic indications. In one embodiment, the affinity of the variant for serum albumin is relatively modest, such that the variant would be useful for inclusion in products that find utility in treating and/or preventing acute diseases, conditions, toxicity or other acute indications. In one embodiment, the affinity of the variant for serum albumin is intermediate, such that the variant would be useful for inclusion in products that find utility in treating and/or preventing acute or chronic diseases, conditions, toxicity or other acute or chronic indications.
  • It is conceivable that a molecule with an appropriately high affinity and specificity for serum albumin would stay in circulation long enough to have the desired therapeutic effect (Tomlinson, Nature Biotechnology 22, 521-522 (2004)). Here, a high affinity anti-SA variant would stay in serum circulation matching that of the species' serum albumin (WO2008096158). Once in circulation, any fused therapeutic agent to the ALBUDAB™ variant (an ALBUDAB™ is an anti-serum albumin dAb or immunoglobulin single variable domain), be it NCE, peptide or protein, consequently would be able to act longer on its target and exhibit a longer lasting therapeutic effect. This would allow for targeting chronic or persistent diseases without the need of frequent dosing.
  • A variant with moderate affinity (but specificity to SA) would only stay in serum circulation for a short time (eg, for a few hours or a few days) allowing for the specific targeting of therapeutic targets involved in acute diseases by the fused therapeutic agent.
  • This way it is possible to tailor the anti-SA-containing product to the therapeutic disease area by choosing an anti-SA variant with the appropriate albumin binding affinity and/or serum half-life.
  • An aspect of the invention provides a multispecific ligand comprising any anti-SA variant as described above and a binding moiety that specifically binds a target antigen other than SA.
  • An aspect of the invention provides a fusion product, eg, a fusion protein or fusion with a peptide or NCE (new chemical entity) drug, comprising a polypeptide, protein, peptide or NCE drug fused or conjugated (for an NCE) to any variant as described above, wherein the variant is DOM7h-11-15 or DOM7h-11-15S12P (or a variant having an amino acid that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of DOM7h-11-15) or DOM7h-11-12 (or a variant having an amino acid that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of DOM7h-11-12). DOM7h-11-15 and DOM7h-11-12 give only a modest drop in affinity when fused or conjugated to partner making them useful in fusion products. DOM7h-11-15S12P is identical to DOM7h-11-15, with the exception that position 12 (numbering according to Kabat) is a proline instead of a serine. This provides advantages set out in WO08052933, including to reduce binding to Protein-L of fusion proteins containing this domain antibody and to facilitate purification. The entire disclosure of WO08052933 is incorporated herein by reference. Similarly, the invention provides a DOM7h-11 variant as disclosed herein wherein the variant comprises an amino acid sequence as set out below with the exception that position 12 (numbering according to Kabat) is a proline. The invention also provides fusion proteins, conjugates or composition comprising such DOM7h-11 variants.
  • One aspect of the invention provides a variant of DOM7h-11 that comprises an amino acid sequence that is identical to the amino acid sequence of DOM7h-11-15S12P or has up to 4 changes compared to the amino acid sequence of DOM7h-11-15S12P, provided that the amino acid sequence of the variant has at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat).
  • An aspect of the invention provides a composition comprising a variant, fusion protein or ligand of any preceding aspect and a pharmaceutically acceptable diluent, carrier, excipient or vehicle.
  • An aspect of the invention provides a method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of a variant according to any aspect or embodiment of the invention to said patient.
  • An aspect of the invention provides a polypeptide fusion or conjugate comprising an anti-serum albumin dAb as disclosed herein (eg, DOM7h-11-15 or DOM7h-11-3 or DOM7h-11-15S12P or DOM7h-11-15S12P with up to 4 changes compared to the amino acid sequence of DOM7h-11-15S12P) and an incretin or insulinotropic agent, eg, exendin-4, GLP-1(7-37), GLP-1(6-36) or any incretin or insulinotropic agent disclosed in WO06/059106, these agents being explicitly incorporated herein by reference as though written herein for inclusion in the present invention and claims below.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1: Amino-acid sequence alignment for DOM7h-11 (SEQ ID NO: 421) variant dAbs. A “.” at a particular position indicates the same amino as found in DOM7h-11 at that position. The CDRs are indicated by underlining and bold text (the first underlined sequence is CDR1, the second underlined sequence is CDR2 and the third underlined sequence is CDR3). The figure comprises the following variants: DOM 7h-11-12 (SEQ ID NO:1), DOM 7h-11-15 (SEQ ID NO:2), DOM 7h-11-18 (SEQ ID NO:3), DOM 7h-11-19 (SEQ ID NO:4), and DOM 7h-11-3 (SEQ ID NO: 5).
  • FIG. 2: Kinetic parameters of DOM7h-11 variants. KD units=nM; Kd units=sec−1; Ka units=M−1 sec−1. The notation A e-B means A×10−B and C e D means C×10D. The overall kinetic ranges in various species, as supported by the examples below, are indicated. Optional ranges are also provided for use in particular therapeutic settings (acute or chronic indications, conditions or diseases and “intermediate” for use in both chronic and acute settings). High affinity dAbs and products comprising these are useful for chronic settings. Medium affinity dAbs and products comprising these are useful for intermediate settings. Low affinity dAbs and products comprising these are useful for acute settings. The affinity in this respect is the affinity for serum albumin. Various example anti-serum dAbs and fusion proteins are listed, and these support the ranges disclosed. Many of the examples have favourable kinetics in human and one or more non-human animals (eg, in human and Cynomolgus monkey and/or mouse). Choice of dAb or product comprising this can be tailored, according to the invention, depending on the setting (eg, chronic or acute) to be treated therapeutically.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • Within this specification the invention has been described, with reference to embodiments, in a way which enables a clear and concise specification to be written. It is intended and should be appreciated that embodiments may be variously combined or separated without parting from the invention.
  • Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods.
  • As used herein, the term “antagonist of Tumor Necrosis Factor Receptor 1 (TNFR1)” or “anti-TNFR1 antagonist” or the like refers to an agent (e.g., a molecule, a compound) which binds TNFR1 and can inhibit a (i.e., one or more) function of TNFR1. For example, an antagonist of TNFR1 can inhibit the binding of TNFα to TNFR1 and/or inhibit signal transduction mediated through TNFR1. Accordingly, TNFR1-mediated processes and cellular responses (e.g., TNFα-induced cell death in a standard L929 cytotoxicity assay) can be inhibited with an antagonist of TNFR1.
  • A “patient” is any animal, eg, a mammal, eg, a non-human primate (such as a baboon, rhesus monkey or Cynomolgus monkey), mouse, human, rabbit, rat, dog, cat or pig. In one embodiment, the patient is a human.
  • As used herein, “peptide” refers to about two to about 50 amino acids that are joined together via peptide bonds.
  • As used herein, “polypeptide” refers to at least about 50 amino acids that are joined together by peptide bonds. Polypeptides generally comprise tertiary structure and fold into functional domains.
  • As used herein an antibody refers to IgG, IgM, IgA, IgD or IgE or a fragment (such as a Fab, F(ab′)2, Fv, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, diabody) whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
  • As used herein, “antibody format” refers to any suitable polypeptide structure in which one or more antibody variable domains can be incorporated so as to confer binding specificity for antigen on the structure. A variety of suitable antibody formats are known in the art, such as, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy chains and/or light chains, antigen-binding fragments of any of the foregoing (e.g., a Fv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment), a single antibody variable domain (e.g., a dAb, VH, VHH, VL), and modified versions of any of the foregoing (e.g., modified by the covalent attachment of polyethylene glycol or other suitable polymer or a humanized VHH).
  • The phrase “immunoglobulin single variable domain” refers to an antibody variable domain (VH, VHH, VL) that specifically binds an antigen or epitope independently of different V regions or domains. An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other variable regions or variable domains where the other regions or domains are not required for antigen binding by the single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds antigen independently of the additional variable domains). A “domain antibody” or “dAb” is the same as an “immunoglobulin single variable domain” as the term is used herein. A “single immunoglobulin variable domain” is the same as an “immunoglobulin single variable domain” as the term is used herein. A “single antibody variable domain” or an “antibody single variable domain” is the same as an “immunoglobulin single variable domain” as the term is used herein. An immunoglobulin single variable domain is in one embodiment a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004, the contents of which are incorporated herein by reference in their entirety), nurse shark and Camelid VHH dAbs. Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. The VHH may be humanized.
  • A “domain” is a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain. A “single antibody variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.
  • In the instant application, the term “prevention” and “preventing” involves administration of the protective composition prior to the induction of the disease or condition. “Treatment” and “treating” involves administration of the protective composition after disease or condition symptoms become manifest. “Suppression” or “suppressing” refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease or condition.
  • As used herein, the term “dose” refers to the quantity of ligand administered to a subject all at one time (unit dose), or in two or more administrations over a defined time interval. For example, dose can refer to the quantity of ligand (e.g., ligand comprising an immunoglobulin single variable domain that binds target antigen) administered to a subject over the course of one day (24 hours) (daily dose), two days, one week, two weeks, three weeks or one or more months (e.g., by a single administration, or by two or more administrations). The interval between doses can be any desired amount of time. The term “pharmaceutically effective” when referring to a dose means sufficient amount of the ligand, domain or pharmaceutically active agent to provide the desired effect. The amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, the particular drug or pharmaceutically active agent and the like. Thus, it is not always possible to specify an exact “effective” amount applicable for all patients. However, an appropriate “effective” dose in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • Methods for pharmacokinetic analysis and determination of ligand (eg, single variable domain, fusion protein or multi-specific ligand) half-life will be familiar to those skilled in the art. Details may be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinetc analysis: A Practical Approach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. ex edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta half lives and area under the curve (AUC). Optionally, all pharmacokinetic parameters and values quoted herein are to be read as being values in a human. Optionally, all pharmacokinetic parameters and values quoted herein are to be read as being values in a mouse or rat or Cynomolgus monkey.
  • Half lives (t½ alpha and t½ beta) and AUC can be determined from a curve of serum concentration of ligand against time. The WinNonlin analysis package, eg version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA) can be used, for example, to model the curve. When two-compartment modeling is used, in a first phase (the alpha phase) the ligand is undergoing mainly distribution in the patient, with some elimination. A second phase (beta phase) is the phase when the ligand has been distributed and the serum concentration is decreasing as the ligand is cleared from the patient. The t alpha half life is the half life of the first phase and the t beta half life is the half life of the second phase. Thus, in one embodiment, in the context of the present invention, the variable domain, fusion protein or ligand has a tα half-life in the range of (or of about) 15 minutes or more. In one embodiment, the lower end of the range is (or is about) 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, or alternatively, the variable domain, fusion protein or ligand according to the invention will have a tα half life in the range of up to and including 12 hours (or about 12 hours). In one embodiment, the upper end of the range is (or is about) 11, 10, 9, 8, 7, 6 or 5 hours. An example of a suitable range is (or is about) 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.
  • In one embodiment, the present invention provides the variable domain, fusion protein or ligand according to the invention has a tβ half-life in the range of (or of about) 2.5 hours or more. In one embodiment, the lower end of the range is (or is about) 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours, or 12 hours. In addition, or alternatively, the tβ half-life is (or is about) up to and including 21 or 25 days. In one embodiment, the upper end of the range is (or is about) 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days, 19 days, 20 days, 21 days or 22 days. For example, the variable domain, fusion protein or ligand according to the invention will have a tβ half life in the range 12 to 60 hours (or about 12 to 60 hours). In a further embodiment, it will be in the range 12 to 48 hours (or about 12 to 48 hours). In a further embodiment still, it will be in the range 12 to 26 hours (or about 12 to 26 hours).
  • As an alternative to using two-compartment modeling, the skilled person will be familiar with the use of non-compartmental modeling, which can be used to determine terminal half-lives (in this respect, the term “terminal half-life” as used herein means a terminal half-life determined using non-compartmental modeling). The WinNonlin analysis package, eg version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA) can be used, for example, to model the curve in this way. In this instance, in one embodiment the single variable domain, fusion protein or ligand has a terminal half life of at least (or at least about) 8 hours, 10 hours, 12 hours, 15 hours, 28 hours, 20 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days or 25 days. In one embodiment, the upper end of this range is (or is about) 24 hours, 48 hours, 60 hours or 72 hours or 120 hours. For example, the terminal half-life is (or is about) from 8 hours to 60 hours, or 8 hours to 48 hours or 12 to 120 hours, eg, in man.
  • In addition, or alternatively to the above criteria, the variable domain, fusion protein or ligand according to the invention has an AUC value (area under the curve) in the range of (or of about) 1 mg·min/ml or more. In one embodiment, the lower end of the range is (or is about) 5, 10, 15, 20, 30, 100, 200 or 300 mg·min/ml. In addition, or alternatively, the variable domain, fusion protein or ligand according to the invention has an AUC in the range of (or of about) up to 600 mg·min/ml. In one embodiment, the upper end of the range is (or is about) 500, 400, 300, 200, 150, 100, 75 or 50 mg·min/ml. Advantageously the variable domain, fusion protein or ligand will have an AUC in (or about in) the range selected from the group consisting of the following: 15 to 150 mg·min/ml, 15 to 100 mg·min/ml, 15 to 75 mg·min/ml, and 15 to 50 mg·min/ml.
  • “Surface Plasmon Resonance”: Competition assays can be used to determine if a specific antigen or epitope, such as human serum albumin, competes with another antigen or epitope, such as cynomolgus serum albumin, for binding to a serum albumin binding ligand described herein, such as a specific dAb. Similarly competition assays can be used to determine if a first ligand such as dAb, competes with a second ligand such as a dAb for binding to a target antigen or epitope. The term “competes” as used herein refers to substance, such as a molecule, compound, preferably a protein, which is able to interfere to any extent with the specific binding interaction between two or more molecules. The phrase “does not competitively inhibit” means that substance, such as a molecule, compound, preferably a protein, does not interfere to any measurable or significant extent with the specific binding interaction between two or more molecules. The specific binding interaction between two or more molecules preferably includes the specific binding interaction between a single variable domain and its cognate partner or target. The interfering or competing molecule can be another single variable domain or it can be a molecule that is structurally and/or functionally similar to a cognate partner or target.
  • The term “binding moiety” refers to a domain that specifically binds an antigen or epitope independently of a different epitope or antigen binding domain. A binding moiety may be a domain antibody (dAb) or may be a domain which is a derivative of a non-immunoglobulin protein scaffold, eg, a scaffold selected from the group consisting of CTLA-4, lipocalin, SpA, an adnectin, affibody, an avimer, GroEl, transferrin, GroES and fibronectin, which binds to a ligand other than the natural ligand (in the case of the present invention, the moiety binds serum albumin). See WO2008/096158, which discloses examples of protein scaffolds and methods for selecting antigen or epitope-specific binding domains from repertoires (see Examples 17 to 25). These specific disclosures of WO2008/096158 are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein).
  • In one aspect, the invention provides an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11, wherein the variant comprises at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) compared to DOM7h-11, and wherein the variant has from 2 to 8 changes compared to the amino acid sequence of DOM7h-11. Optionally, position 49 (according to Kabat) is Leu. Additionally or alternatively, position 50 (according to Kabat) is optionally Ala or Trp. Additionally or alternatively, position 51 (according to Kabat) is optionally Phe or Asn. In one embodiment, the variant comprises a mutation at each of positions 49, 50 and 51 (numbering according to Kabat) compared to DOM7h-11. In one embodiment, the variant comprises a LFG motif, where L is at position 49 (numbering according to Kabat), wherein L, F and G are Leu, Phe and Gly respectively.
  • In one embodiment, the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-3, DOM7h-11-15, DOM7h-11-12 and DOM7h-11-19 or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has at least one mutation in the FW2/CDR2 junction as defined above. In one embodiment, the variant comprises an amino acid sequence that is identical to the amino acid sequence of DOM7h-11-15S12P or has up to 4 changes compared to the amino acid sequence of DOM7h-11-15S12P, provided that the amino acid sequence of the variant has at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat). In one embodiment, the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-3, or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has L at position 49, W at position 50 and N at position 51. In one embodiment, the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-12, or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has M at position 32 and L at position 49. In one embodiment, the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-15 or DOM7h-11-15S12P, or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has M at position 32, L at position 49, A at position 50 and F at position 51. In one embodiment, the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-18, or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has M at position 32 and H at position 87. In one embodiment, the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-19, or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has M at position 32, L at position 49 and T at position 91. All numbering in this paragraph is according to Kabat.
  • An aspect of the invention provides an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11, wherein the variant comprises a Met at position 32 (numbering according to Kabat) compared to DOM7h-11, and wherein the variant has from 0 to 4 further changes compared to the amino acid sequence of DOM7h-11. Optionally, the variant comprises at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) compared to DOM7h-11.
  • In one embodiment of any aspect of the invention, the variant comprises at least one mutation compared to DOM7h-11 selected from the following
    • Position 49=L, Position 50=A or W, Position 51=F or N, Position 87=H, and Position 91=T.
  • In one embodiment, the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-12, DOM7h-11-15, DOM7h-11-15S12P, DOM7h-11-18 and DOM7h-11-19 or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has Met at position 32.
  • In one embodiment, the variant comprises one or more of the following kinetic characteristics:—
      • (a) The variant comprises a binding site that specifically binds human SA with a dissociation constant (KD) from (or from about) 0.1 to (or to about) 10000 nM, optionally from (or from about) 1 to (or to about) 6000 nM, as determined by surface plasmon resonance;
      • (b) The variant comprises a binding site that specifically binds human SA with an off-rate constant (Kd) from (or from about) 1.5×10−4 to (or to about) 0.1 sec−1, optionally from (or from about) 3×10−4 to (or to about) 0.1 sec−1 as determined by surface plasmon resonance;
      • (c) The variant comprises a binding site that specifically binds human SA with an on-rate constant (Ka) from (or from about) 2×106 to (or to about) 1×104M−1 sec−1, optionally from (or from about) 1×106 to (or to about) 2×104M−1 sec−1 as determined by surface plasmon resonance;
      • (d) The variant comprises a binding site that specifically binds Cynomolgus monkey SA with a dissociation constant (KD) from (or from about) 0.1 to (or to about) 10000 nM, optionally from (or from about) 1 to (or to about) 6000 nM, as determined by surface plasmon resonance;
      • (e) The variant of any preceding claim, wherein the variant comprises a binding site that specifically binds Cynomolgus monkey SA with an off-rate constant (Kd) from (or from about) 1.5×10−4 to (or to about) 0.1 sec−1, optionally from (or from about) 3×10−4 to (or to about) 0.1 sec−1 as determined by surface plasmon resonance;
      • (f) The variant of any preceding claim, wherein the variant comprises a binding site that specifically binds Cynomolgus monkey SA with an on-rate constant (Ka) from (or from about) 2×106 to (or to about) 1×104M−1 sec−1, optionally from (or from about) 1×106 to (or to about) 5×103M−1 sec−1 as determined by surface plasmon resonance;
      • (g) The variant comprises a binding site that specifically binds rat SA with a dissociation constant (KD) from (or from about) 1 to (or to about) 10000 nM, optionally from (or from about) 20 to (or to about) 6000 nM, as determined by surface plasmon resonance;
      • (h) The variant comprises a binding site that specifically binds rat SA with an off-rate constant (Kd) from (or from about) 2×10−3 to (or to about) 0.15 sec−1, optionally from (or from about) 9×10−3 to (or to about) 0.14 sec−1 as determined by surface plasmon resonance;
      • (i) The variant comprises a binding site that specifically binds rat SA with an on-rate constant (Ka) from (or from about) 2×106 to (or to about) 1×104 M−1 sec−1, optionally from (or from about) 1×106 to (or to about) 3×104 M−1 sec−1 as determined by surface plasmon resonance;
      • (j) The variant comprises a binding site that specifically binds mouse SA with a dissociation constant (KD) from (or from about) 1 to (or to about) 10000 nM as determined by surface plasmon resonance;
      • (k) The variant comprises a binding site that specifically binds mouse SA with an off-rate constant (Kd) from (or from about) 2×10−3 to (or to about) 0.15 sec−1 as determined by surface plasmon resonance; and/or
      • (l) The variant comprises a binding site that specifically binds mouse SA with an on-rate constant (Ka) from (or from about) 2×106 to (or to about) 1×104 M−1 sec−1, optionally from (or from about) 2×106 to (or to about) 1.5×104 M−1 sec−1 as determined by surface plasmon resonance.
      • Optionally, the variant has
      • I: a KD according to (a) and (d), a Kd according to (b) and (e), and a Ka according to (c) and (f); or
      • II: a KD according to (a) and (g), a Kd according to (b) and (h), and a Ka according to (c) and (i); or
      • III: a KD according to (a) and (j), a Kd according to (b) and (k), and a Ka according to (c) and (1); or
      • IV: kinetics according to I and II; or
      • V: kinetics according to I and III; or
      • VI: kinetics according to I, II and III.
  • The invention also provides a ligand comprising a variant of any preceding aspect or embodiment of the invention. For example, the ligand can be a dual-specific ligand (see WO04003019 for examples of dual-specific ligands). In one aspect, the invention provides a multispecific ligand comprising an anti-SA variant of any preceding aspect or embodiment of the invention and a binding moiety that specifically binds a target antigen other than SA. The binding moiety can be any binding moiety that specifically binds a target, eg, the moiety is an antibody, antibody fragment, scFv, Fab, dAb or a binding moiety comprising a non-immunoglobulin protein scaffold. Such moieties are disclosed in detail in WO2008/096158 (see examples 17 to 25, which disclosure is incorporated herein by reference). Examples of non-immunoglobulin scaffolds are CTLA-4, lipocallin, staphylococcal protein A (spA), Affibody™ Avimers™, adnectins, GroEL and fibronectin.
  • In one embodiment, a linker is provided between the anti-target binding moiety and the anti-SA single variant, the linker comprising the amino acid sequence AST, optionally ASTSGPS. Alternative linkers are described in WO2007085814 (incorporated herein by reference) and WO2008/096158 (see the passage at page 135, line 12 to page 140, line 14, which disclosure and all sequences of linkers are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein).
  • In one embodiment of the multispecific ligand, the target antigen may be, or be part of, polypeptides, proteins or nucleic acids, which may be naturally occurring or synthetic. In this respect, the ligand of the invention may bind the target antigen and act as an antagonist or agonist (e.g., EPO receptor agonist). One skilled in the art will appreciate that the choice is large and varied. They may be for instance, human or animal proteins, cytokines, cytokine receptors, where cytokine receptors include receptors for cytokines, enzymes, co-factors for enzymes or DNA binding proteins. Suitable cytokines and growth factors include, but are preferably not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-β1, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-113, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumour necrosis factor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4, CD4, human chemokine receptors CXCR4 or CCR5, non-structural protein type 3 (NS3) from the hepatitis C virus, TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12, internalizing receptors that are over-expressed on certain cells, such as the epidermal growth factor receptor (EGFR), ErBb2 receptor on tumor cells, an internalising cellular receptor, LDL receptor, FGF2 receptor, ErbB2 receptor, transferrin receptor, PDGF receptor, VEGF receptor, PsmAr, an extracellular matrix protein, elastin, fibronectin, laminin, al-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1, Bad, caspase-9, Forkhead, an antigen of Helicobacter pylori, an antigen of Mycobacterium tuberculosis, and an antigen of influenza virus. It will be appreciated that this list is by no means exhaustive.
  • In one embodiment, the multispecific ligand comprises an anti-SA dAb variant of the invention and an anti-TNFR1 binding moiety, eg, an anti-TNFR1 dAb. Optionally, the ligand has only one anti-TNFR1 binding moiety (eg, dAb) to reduce the chance of receptor cross-linking. In one embodiment, the anti-SA dAb variant is DOM7h-11-3 or DOM7h-11-15 or DOM7h-11-15S12P.
  • In one embodiment, the anti-TNFR1 binding moiety is DOM1h-131-206 disclosed in WO2008149148 (the amino acid sequence of which and the nucleotide sequence of which, as disclosed in that PCT application, are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein). In one embodiment, the multispecific ligand comprises or consists of the amino acid sequence of DOM1h-131-206 and the amino acid sequence of DOM7h-11-3 or DOM7h-11-15 or DOM7h-11-15S12P.
  • In one embodiment, the anti-TNFR1 binding moiety or dAb is any such moiety or dAb disclosed in co-pending application U.S. Ser. No. 61/153,746, the disclosure of which is incorporated herein by reference. In one embodiment, the anti-TNFR1 binding moiety comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of DOM1h-574-156, DOM1h-574-72, DOM1h-574-109, DOM1h-574-138, DOM1h-574-162 or DOM1h-574-180 or the amino acid sequence of any anti-TNFR1 dAb disclosed in Table 3. In one embodiment, the multispecific ligand comprises or consists of the amino acid sequence of DOM1h-574-156 and the amino acid sequence of DOM7h-11-3 or DOM7h-11-15 or DOM7h-11-15S12P.
  • In one embodiment, the ligand of the invention is a fusion protein comprising a variant of the invention fused directly or indirectly to one or more polypeptides. For example, the fusion protein can be a “drug fusion” as disclosed in WO2005/118642 (the disclosure of which is incorporated herein by reference), comprising a variant of the invention and a polypeptide drug as defined in that PCT application.
  • As used herein, “drug” refers to any compound (e.g., small organic molecule, nucleic acid, polypeptide) that can be administered to an individual to produce a beneficial, therapeutic or diagnostic effect through binding to and/or altering the function of a biological target molecule in the individual. The target molecule can be an endogenous target molecule encoded by the individual's genome (e.g. an enzyme, receptor, growth factor, cytokine encoded by the individual's genome) or an exogenous target molecule encoded by the genome of a pathogen (e. g. an enzyme encoded by the genome of a virus, bacterium, fungus, nematode or other pathogen). Suitable drugs for use in fusion proteins and conjugates comprising an anti-SA dAb variant of the invention are disclosed in WO2005/118642 and WO2006/059106 (the entire disclosures of which are incorporated herein by reference, and including the entire list of specific drugs as though this list were expressly written herein, and it is contemplated that such incorporation provides disclosure of specific drugs for inclusion in claims herein). For example, the drug can be glucagon-like peptide 1 (GLP-1) or a variant, interferon alpha 2b or a variant or exendin-4 or a variant.
  • In one embodiment, the invention provides a drug conjugate as defined and disclosed in WO2005/118642 and WO2006/059106, wherein the conjugate comprises a variant of the invention. In one example, the drug is covalently linked to the variant (eg, the variant and the drug are expressed as part of a single polypeptide). Alternatively, in an example, the drug is non-covalently bonded or associated with the variant. The drug can be covalently or noncovalently bonded to the variant directly or indirectly (e.g., through a suitable linker and/or noncovalent binding of complementary binding partners (e.g., biotin and avidin)). When complementary binding partners are employed, one of the binding partners can be covalently bonded to the drug directly or through a suitable linker moiety, and the complementary binding partner can be covalently bonded to the variant directly or through a suitable linker moiety. When the drug is a polypeptide or peptide, the drug composition can be a fusion protein, wherein the polypeptide or peptide, drug and the polypeptide binding moiety are discrete parts (moieties) of a continuous polypeptide chain. As described herein, the polypeptide binding moieties and polypeptide drug moieties can be directly bonded to each other through a peptide bond, or linked through a suitable amino acid, or peptide or polypeptide linker.
  • A ligand which contains one single variable domain (monomer) variant of the invention or more than one single variable domain (multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, can further comprise one or more entities selected from, but preferably not limited to a label, a tag, an additional single variable domain, a dAb, an antibody, an antibody fragment, a marker and a drug. One or more of these entities can be located at either the COOH terminus or at the N terminus or at both the N terminus and the COOH terminus of the ligand comprising the single variable domain, (either immunoglobulin or non-immunoglobulin single variable domain). One or more of these entities can be located at either the COOH terminus, or the N terminus, or both the N terminus and the COOH terminus of the single variable domain which specifically binds serum albumin of the ligand which contains one single variable domain (monomer) or more than one single variable domains (multimer, fusion protein, conjugate, and dual specific ligand as defined herein). Non-limiting examples of tags which can be positioned at one or both of these termini include a HA, his or a myc tag. The entities, including one or more tags, labels and drugs, can be bound to the ligand which contains one single variable domain (monomer) or more than one single variable domain (multimer, fusion protein, conjugate, and dual specific ligand as defined herein), which binds serum albumin, either directly or through linkers as described above.
  • An aspect of the invention provides a fusion product, eg, a fusion protein or fusion with a peptide or conjugate with an NCE (new chemical entity) drug, comprising a polypeptide drug fused or conjugated (for an NCE) to any variant as described above, optionally wherein the variant is DOM7h-11-15 or DOM7h-11-15S12P (or a variant having an amino acid that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of DOM7h-11-15 or DOM7h-11-15S12P) or DOM7h-11-12 (or a variant having an amino acid that is at least 95, 96, 97, 98 or 99% identical to the amino acid sequence of DOM7h-11-15 or DOM7h-11-15S12P). DOM7h-11-15, DOM7h-11-15S12P and DOM7h-11-12 give only a modest drop in affinity when fused or conjugated to partner, making them useful in fusion products.
  • The invention provides a composition comprising a variant, fusion protein, conjugate or ligand of any aspect of the invention and a pharmaceutically acceptable diluent, carrier, excipient or vehicle.
  • Also encompassed herein is an isolated nucleic acid encoding any of the variants, fusion proteins, conjugates or ligands described herein, e.g., a ligand which contains one single variable domain (monomer) variant of the invention or more than one single variable domain (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) variant which specifically binds to serum albumin, or which specifically binds both human serum albumin and at least one non-human serum albumin, or functionally active fragments thereof. Also encompassed herein is a vector and/or an expression vector, a host cell comprising the vector, e.g., a plant or animal cell and/or cell line transformed with a vector, a method of expressing and/or producing one or more variants, fusion proteins or ligands which contains one single variable domain (monomer) variant or more than one single variable domain variants (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, or fragment(s) thereof encoded by said vectors, including in some instances culturing the host cell so that the one or more variants, fusion proteins or ligands or fragments thereof are expressed and optionally recovering the ligand which contains one single variable domain (monomer) or more than one single variable domain (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, from the host cell culture medium. Also encompassed are methods of contacting a ligand described herein with serum albumin, including serum albumin and/or non-human serum albumin(s), and/or one or more targets other than serum albumin, where the targets include biologically active molecules, and include animal proteins, cytokines as listed above, and include methods where the contacting is in vitro as well as administering any of the variants, fusion proteins or ligands described herein to an individual host animal or cell in vivo and/or ex vivo. Preferably, administering ligands described herein which comprises a single variable domain (immunoglobulin or non-immunoglobulin) directed to serum albumin and/or non-human serum albumin(s), and one or more domains directed to one or more targets other than serum albumin, will increase the half life, including the T beta and/or terminal half life, of the anti-target ligand. Nucleic acid molecules encoding the variants, fusion proteins or single domain containing ligands or fragments thereof, including functional fragments thereof, are contemplated herein. Vectors encoding the nucleic acid molecules, including but preferably not limited to expression vectors, are contemplated herein, as are host cells from a cell line or organism containing one or more of these expression vectors. Also contemplated are methods of producing any variant, fusion protein or ligand, including, but preferably not limited to any of the aforementioned nucleic acids, vectors and host cells.
  • An aspect of the invention provides a nucleic acid comprising a nucleotide sequence encoding a variant according to the invention or a multispecific ligand of the invention or fusion protein of the invention.
  • An aspect of the invention provides a nucleic acid comprising the nucleotide sequence of a DOM7h-11 variant selected from DOM7h-11-3, DOM7h-11-15, DOM7h-11-15S12P, DOM7h-11-12, DOM7h-11-18 and DOM7h-11-19 or a nucleotide sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said selected sequence.
  • An aspect of the invention provides a vector comprising the nucleic acid of the invention. An aspect of the invention provides an isolated host cell comprising the vector.
  • Reference is made to WO2008/096158 for details of library vector systems, combining single variable domains, characterization of dual specific ligands, structure of dual specific ligands, scaffolds for use in constructing dual specific ligands, uses of anti-serum albumin dAbs and multispecific ligands and half-life-enhanced ligands, and compositions and formulations of comprising anti-serum albumin dAbs. These disclosures are incorporated herein by reference to provide guidance for use with the present invention, including for variants, ligands, fusion proteins, conjugates, nucleic acids, vectors, hosts and compositions of the present invention.
  • DOM7h-14 variant sequences, which are not according to the invention, are disclosed in a co-pending US provisional patent application entitled IMPROVED ANTI-SERUM ALBUMIN BINDING VARIANTS, filed on the same day as the present application. These sequences of DOM7h-14 variants (SEQ ID NO:s 1-10 in the co-pending application) are incorporated herein by reference as though explicitly written herein.
    • SEQUENCES
  • TABLE 1
    Amino Acid Sequences of DOM7h-11 Variant dAbs
    DOM7h-11-12 (SEQ ID NO: 1)
    DIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLILF
    GSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQ
    GTKVEIKR
    DOM7h-11-15 (SEQ ID NO: 2)
    DIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLILA
    FSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQ
    GTKVEIKR
    DOM7h-11-18 (SEQ ID NO: 3)
    DIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLIWF
    GSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYHCAQAGTHPTTFGQ
    GTKVEIKR
    DOM7h-11-19 (SEQ ID NO: 4)
    DIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLILF
    GSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQTGTHPTTFGQ
    GTKVEIKR
    DOM7h-11-3 (SEQ ID NO: 5)
    DIQMTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLLILW
    NSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQ
    GTKVEIKR
  • TABLE 2
    Nucleotide Sequences of DOM7h-11 Variant dAbs
    DOM7h-11-12 (SEQ ID NO: 6)
    GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT
    CTGTAGGAGA CCGTGTCACC ATCACTTGCC GGGCAAGTCG
    TCCGATTGGG ACGATGTTAA GTTGGTACCA GCAGAAACCA
    GGGAAAGCCC CTAAGCTCCT GATCTTGTTT GGTTCCCGGT
    TGCAAAGTGG GGTCCCATCA CGTTTCAGTG GCAGTGGATC
    TGGGACAGAT TTCACTCTCA CCATCAGCAG TCTGCAACCT
    GAAGATTTTG CTACGTACTA CTGTGCGCAG GCTGGGACGC
    ATCCTACGAC GTTCGGCCAA GGGACCAAGG TGGAAATCAA
    ACGG
    DOM7h-11-15 (SEQ ID NO: 7)
    GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT
    CTGTAGGAGA CCGTGTCACC ATCACTTGCC GGGCAAGTCG
    TCCGATTGGG ACGATGTTAA GTTGGTACCA GCAGAAACCA
    GGGAAAGCCC CTAAGCTCCT GATCCTTGCT TTTTCCCGTT
    TGCAAAGTGG GGTCCCATCA CGTTTCAGTG GCAGTGGATC
    TGGGACAGAT TTCACTCTCA CCATCAGCAG TCTGCAACCT
    GAAGATTTTG CTACGTACTA CTGCGCGCAG GCTGGGACGC
    ATCCTACGAC GTTCGGCCAA GGGACCAAGG TGGAAATCAA
    ACGG
    DOM7h-11-18 (SEQ ID NO: 8)
    GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT
    CTGTAGGAGA CCGTGTCACC ATCACTTGCC GGGCAAGTCG
    TCCGATTGGG ACGATGTTAA GTTGGTACCA GCAGAAACCA
    GGGAAAGCCC CAAAGCTCCT GATCTGGTTT GGTTCCCGGT
    TGCAAAGTGG GGTCCCATCA CGTTTCAGTG GCAGTGGATC
    TGGGACAGAT TTCACTCTCA CCATCAGCAG TCTGCAACCT
    GAAGATTTTG CTACGTACCA CTGTGCGCAG GCGGGGACGC
    ATCCTACGAC GTTCGGCCAA GGGACCAAGG TGGAAATCAA
    ACGG
    DOM7h-11-19 (SEQ ID NO: 9)
    GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT
    CTGTAGGAGA CCGTGTCACC ATCACTTGCC GGGCAAGTCG
    TCCGATTGGG ACGATGTTAA GTTGGTACCA GCAGAAACCA
    GGGAAAGCCC CTAAGCTCCT GATCTTGTTT GGTTCCCGGT
    TGCAAAGTGG GGTCCCATCA CGTTTCAGTG GCAGTGGATC
    TGGGACGGAT TTCACTCTCA CCATCAGCAG TCTGCAACCT
    GAAGATTTTG CTACGTACTA CTGTGCGCAG ACTGGGACGC
    ATCCCACGAC GTTCGGCCAA GGGACCAAGG TGGAAATCAA
    ACGG
    DOM7h-11-3 (SEQ ID NO: 10)
    GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT
    CTGTAGGAGA CCGTGTCACC ATCACTTGCC GGGCAAGTCG
    TCCGATTGGG ACGACGTTAA GTTGGTACCA GCAGAAACCA
    GGGAAAGCCC CTAAGCTCCT GATCCTTTGG AATTCCCGTT
    TGCAAAGTGG GGTCCCATCA CGTTTCAGTG GCAGTGGATC
    TGGGACAGAT TTCACTCTCA CCATCAGCAG TCTGCAACCT
    GAAGATTTTG CTACGTACTA CTGTGCGCAG GCTGGGACGC
    ATCCTACGAC GTTCGGCCAA GGGACCAAGG TGGAAATCAA
    ACGG
  • TABLE 3
    Amino Acid Sequences of anti-TNFR1 dAbs
    >DOM1h-509 (SEQ ID NO: 11)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYRMHWVRQAPGKSLEWVSSIDTRGSST
    YYADPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAVTMFSPFFDYWGQGTLV
    TVSS
    >DOM1h-510 (SEQ ID NO: 12)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFADYGMRWVRQAPGKGLEWVSSITRTGRVT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWRNRHGEYLADFDYWGQG
    TLVTVSS
    >DOM1h-543 (SEQ ID NO: 13)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFMRYRMHWVRQAPGKGLEWVSSIDSNGSST
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRTERSPVFDYWGQGTLV
    TVSS
    >DOM1h-549 (SEQ ID NO: 14)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVT
    VSS
    >DOM1h-574 (SEQ ID NO: 15)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGGHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYTGHWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-1 (SEQ ID NO: 16)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGGHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYTGRWEPYDYWGQGTLVT
    VSS
    >DOM1h-574-2 (SEQ ID NO: 17)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGGHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-7 (SEQ ID NO: 18)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGGHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-8 (SEQ ID NO: 19)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGGHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-9 (SEQ ID NO: 20)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGGHT
    YYADSVKGRFTISRDNSKNTLYMQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-10 (SEQ ID NO: 21)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFGKYSMGWVRQAPGKDLEWVSQISNTGGHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-11 (SEQ ID NO: 22)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGGHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYTGRWEPFDHWGQGTLVT
    VSS
    >DOM1h-574-12 (SEQ ID NO: 23)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-13 (SEQ ID NO: 24)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-14 (SEQ ID NO: 25)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-15 (SEQ ID NO: 26)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-16 (SEQ ID NO: 27)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-17 (SEQ ID NO: 28)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-18 (SEQ ID NO: 29)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFGKYSMGWVRQAPGKDLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-19 (SEQ ID NO: 30)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFGKYSMGWVRQAPGKDLEWVSQISNTGDHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-25 (SEQ ID NO: 31)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-26 (SEQ ID NO: 32)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFEYWGQGTLVT
    VSS
    >DOM1h-574-27 (SEQ ID NO: 33)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWKPFEYWGQGTLVT
    VSS
    >DOM1h-574-28 (SEQ ID NO: 34)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-29 (SEQ ID NO: 35)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWRPFEYWGQGTLVT
    VSS
    >DOM1h-574-30 (SEQ ID NO: 36)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTGDRR
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAAYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-31 (SEQ ID NO: 37)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFNYWGQGTLVT
    VSS
    >DOM1h-574-32 (SEQ ID NO: 38)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-33 (SEQ ID NO: 39)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCAIYTGRWVPFDNWGQGTLVT
    VSS
    >DOM1h-574-35 (SEQ ID NO: 40)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFITYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFQYWGQGTLVT
    VSS
    >DOM1h-574-36 (SEQ ID NO: 41)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFGKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-37 (SEQ ID NO: 42)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-38 (SEQ ID NO: 43)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-39 (SEQ ID NO: 44)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRR
    YYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-40 (SEQ ID NO: 45)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFKYWGQGTLVT
    VSS
    >DOM1h-574-53 (SEQ ID NO: 46)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYSMGWVRQAPGKGLEWVSQISNTGERR
    YYADSVKGRFTISRDNPKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFEYWGQGTLVT
    VSS
    >DOM1h-574-54 (SEQ ID NO: 47)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVNYSMGWVRQAPGKGLEWVSQISNTGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPYEYWGQGTLVT
    VTS
    >DOM1h-574-65 (SEQ ID NO: 48)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTGDRR
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-66 (SEQ ID NO: 49)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTGDRR
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWKPFEYWGQGTLVT
    VSS
    >DOM1h-574-67 (SEQ ID NO: 50)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTGDRR
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-68 (SEQ ID NO: 51)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTGDRR
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWRPFEYWGQGTLVT
    VSS
    >DOM1h-574-69 (SEQ ID NO: 52)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTGDRR
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-70 (SEQ ID NO: 53)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-71 (SEQ ID NO: 54)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWKPFEYWGQGTLVT
    VSS
    >DOM1h-574-72 (SEQ ID NO: 55)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-73 (SEQ ID NO: 56)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWRPFEYWGQGTLVT
    VSS
    >DOM1h-574-74 (SEQ ID NO: 57)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-75 (SEQ ID NO: 58)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-76 (SEQ ID NO: 59)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWKPFEYWGQGTLVT
    VSS
    >DOM1h-574-77 (SEQ ID NO: 60)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-78 (SEQ ID NO: 61)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWRPFEYWGQGTLVT
    VSS
    >DOM1h-574-79 (SEQ ID NO: 62)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-84 (SEQ ID NO: 63)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRR
    YYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-85 (SEQ ID NO: 64)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRR
    YYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWKPFEYWGQGTLVT
    VSS
    >DOM1h-574-86 (SEQ ID NO: 65)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRR
    YYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-87 (SEQ ID NO: 66)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRR
    YYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWRPFEYWGQGTLVT
    VSS
    >DOM1h-574-88 (SEQ ID NO: 67)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRR
    YYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-90 (SEQ ID NO: 68)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKFSMGWVRQAPGKGLEWVSQIANTGDRR
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-91 (SEQ ID NO: 69)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-92 (SEQ ID NO: 70)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-93 (SEQ ID NO: 71)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-94 (SEQ ID NO: 72)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTGDRR
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAAYYCAIYTGRWPDFDYWGQGTLVT
    VSS
    >DOM1h-574-95 (SEQ ID NO: 73)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTGDRR
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAAYYCAIYTGRWPDFEYWGQGTLVT
    VSS
    >DOM1h-574-96 (SEQ ID NO: 74)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWPDFDYWGQGTLVT
    VSS
    >DOM1h-574-97 (SEQ ID NO: 75)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWPDFEYWGQGTLVT
    VSS
    >DOM1h-574-98 (SEQ ID NO: 76)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWPDFDYWGQGTLVT
    VSS
    >DOM1h-574-99 (SEQ ID NO: 77)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWPDFEYWGQGTLVT
    VSS
    >DOM1h-574-100 (SEQ ID NO: 78)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISAWGDRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-101 (SEQ ID NO: 79)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISDGGQRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-102 (SEQ ID NO: 80)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISDSGYRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-103 (SEQ ID NO: 81)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISDGGTRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-104 (SEQ ID NO: 82)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISDKGTRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-105 (SEQ ID NO: 83)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISETGRRT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-106 (SEQ ID NO: 84)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQINNTGSTT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVT
    VSS
    >DOM1h-574-107 (SEQ ID NO: 85)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-108 (SEQ ID NO: 86)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-109 (SEQ ID NO: 87)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-110 (SEQ ID NO: 88)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-111 (SEQ ID NO: 89)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWRPFEYWGQGTLVT
    VSS
    >DOM1h-574-112 (SEQ ID NO: 90)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYTHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-113 (SEQ ID NO: 91)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRR
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-114 (SEQ ID NO: 92)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQILNTADRT
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-115 (SEQ ID NO: 93)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-116 (SEQ ID NO: 94)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRR
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-117 (SEQ ID NO: 95)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRR
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-118 (SEQ ID NO: 96)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVYTGRWVSFEYWGQGTLVT
    VSS
    >DOM1h-574-119 (SEQ ID NO: 97)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALYTGRWVSFEYWGQGTLVT
    VSS
    >DOM1h-574-120 (SEQ ID NO: 98)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-121 (SEQ ID NO: 99)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-122 (SEQ ID NO: 100)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTADRR
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-123 (SEQ ID NO: 101)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRR
    YYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-124 (SEQ ID NO: 102)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGDRR
    YYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-125 (SEQ ID NO: 103)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTADRR
    YYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-126 (SEQ ID NO: 104)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTGDRR
    YYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-127 (SEQ ID NO: 105)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTADRR
    YYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-128 (SEQ ID NO: 106)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTADRR
    YYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-129 (SEQ ID NO: 107)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIVNTGDRR
    YYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-130 (SEQ ID NO: 108)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIANTGDRR
    YYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-131 (SEQ ID NO: 109)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-132 (SEQ ID NO: 110)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWRPFEYWGQGTLVT
    VSS
    >DOM1h-574-133 (SEQ ID NO: 111)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-134 (SEQ ID NO: 112)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYSHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-135 (SEQ ID NO: 113)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYTHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-137 (SEQ ID NO: 114)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYTDAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-138 (SEQ ID NO: 115)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-139 (SEQ ID NO: 116)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-140 (SEQ ID NO: 117)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQIADTGDRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-141 (SEQ ID NO: 118)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTADRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-142 (SEQ ID NO: 119)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-143 (SEQ ID NO: 120)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDDAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-144 (SEQ ID NO: 121)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQIADTADRR
    YYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-145 (SEQ ID NO: 122)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQIADTGDRR
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-146 (SEQ ID NO: 123)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQIADTGDRR
    YYDDAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-147 (SEQ ID NO: 124)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWGPFVYWGQGTLVT
    VSS
    >DOM1h-574-14 8 (SEQ ID NO: 125)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFAYWGQGTLVT
    VSS
    >DOM1h-574-149 (SEQ ID NO: 126)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWGPFQYWGQGTLVT
    VSS
    >DOM1h-574-150 (SEQ ID NO: 127)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFQYWGQGTLVT
    VSS
    >DOM1h-574-151 (SEQ ID NO: 128)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-152 (SEQ ID NO: 129)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFQYWGQGTLVT
    VSS
    >DOM1h-574-153 (SEQ ID NO: 130)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFQYWGQGTLVT
    VSS
    >DOM1h-574-154 (SEQ ID NO: 131)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-155 (SEQ ID NO: 132)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-156 (SEQ ID NO: 133)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-157 (SEQ ID NO: 134)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWRPFEYWGQGTLVT
    VSS
    >DOM1h-574-158 (SEQ ID NO: 135)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWRPFEYWGQGTLVT
    VSS
    >DOM1h-574-159 (SEQ ID NO: 136)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-160 (SEQ ID NO: 137)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-161 (SEQ ID NO: 138)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYSHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-162 (SEQ ID NO: 139)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYSHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-163 (SEQ ID NO: 140)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYTHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-164 (SEQ ID NO: 141)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYTHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-165 (SEQ ID NO: 142)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-166 (SEQ ID NO: 143)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-167 (SEQ ID NO: 144)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFLKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-169 (SEQ ID NO: 145)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIADTADRT
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-170 (SEQ ID NO: 146)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-171 (SEQ ID NO: 147)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIADTADRT
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-172 (SEQ ID NO: 148)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIADTADRT
    YYDHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-173 (SEQ ID NO: 149)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIADTADRR
    YYAHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-174 (SEQ ID NO: 150)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRR
    YYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-175 (SEQ ID NO: 151)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIADTADRR
    YYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-176 (SEQ ID NO: 152)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRR
    YYDHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-177 (SEQ ID NO: 153)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIADTADRR
    YYDHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-178 (SEQ ID NO: 154)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQIADTADRR
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
    >DOM1h-574-179 (SEQ ID NO: 155)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTADRR
    YYDDAVKGRFTITRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFVYWGQGTLVT
    VSS
    >DOM1h-574-180 (SEQ ID NO: 156)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISDTADRT
    YYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVT
    VSS
    >DOM1h-574-4 (SEQ ID NO: 157)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGLEWVSQISNTGGHT
    YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYTGRWEPFEYWGQGTLVT
    VSS
    >DOM1h-574-168 (SEQ ID NO: 158)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFFKYSMGWVRQAPGKGLEWVSQISDTGDRR
    YYDHSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWAPFEYWGQGTLVT
    VSS
  • TABLE 4
    Nucleotide sequences of anti-TNFR1 dAbs
    >DOM1h-509 (SEQ ID NO: 157)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTAGTCAGTATAGGATGCATTGGGTCCGCCA
    GGCTCCAGGGAAGAGTCTAGAGTGGGTCTCAAGTATTGATACTAGGGGTTCGTCTACA
    TACTACGCAGACCCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAAGCTGTGACGATGTTTTCTCCTTTTTTTGACTACTGGGGTCAGGGAACCCTGGTC
    ACCGTCTCGAGC
    >DOM1h-510 (SEQ ID NO: 158)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGCTGATTATGGGATGCGTTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCATCTATTACGCGGACTGGTCGTGTTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAATGGCGGAATCGGCATGGTGAGTATCTTGCTGATTTTGACTACTGGGGTCAGGGA
    ACCCTGGTCACCGTCTCGAGC
    >DOM1h-543 (SEQ ID NO: 159)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTATGAGGTATAGGATGCATTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCATCGATTGATTCTAATGGTTCTAGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAAGATCGTACGGAGCGTTCGCCGGTTTTTGACTACTGGGGTCAGGGAACCCTGGTC
    ACCGTCTCGAGC
    >DOM1h-549 (SEQ ID NO: 160)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTGATTATGAGATGCATTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCATCTATTAGTGAGAGTGGTACGACGACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAACGTCGTTTTTCTGCTTCTACGTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574 (SEQ ID NO: 161)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGGTCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAATATACGGGTCATTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-1 (SEQ ID NO: 162)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGGTCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAATATACGGGTCGTTGGGAGCCTTATGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-2 (SEQ ID NO: 163)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGGTCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-4 (SEQ ID NO: 164)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGGTCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAATATACGGGTCGTTGGGAGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-180 (SEQ ID NO: 165)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACGCGGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-7 (SEQ ID NO: 166)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGGTCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-8 (SEQ ID NO: 167)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGATGGGTCCGCCA
    GGCTCCAGGGAAAGGTCCAGAGTGGGTCTCACAGATTTCGAATACGGGTGGTCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACA
    GTCTCGAGC
    >DOM1h-574-9 (SEQ ID NO: 168)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGGTCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATATCCCGCGACAATTCCAAGAACA
    CGCTGTATATGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-10 (SEQ ID NO: 169)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGGTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGATCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGGTCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-11 (SEQ ID NO: 170)
    GAGGTGCAGCTGTTGGAGTCAGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGGTCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAATATACGGGTCGTTGGGAGCCTTTTGACCACTGGGGTCAGGGGACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-12 (SEQ ID NO: 171)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-13 (SEQ ID NO: 172)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GAAATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-14 (SEQ ID NO: 173)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-15 (SEQ ID NO: 174)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-16 (SEQ ID NO: 175)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGATGGGTCCGCCA
    GGCTCCAGGGAAAGGTCCAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACA
    GTCTCGAGC
    >DOM1h-574-17 (SEQ ID NO: 176)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGATGGGTCCGCCA
    GGCTCCAGGGAAAGGTCCAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACA
    GTCTCGAGC
    >DOM1h-574-18 (SEQ ID NO: 177)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGGTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGATCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-19 (SEQ ID NO: 178)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGGTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGATCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCATACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-25 (SEQ ID NO: 179)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-26 (SEQ ID NO: 180)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-27 (SEQ ID NO: 181)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCGGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAAGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-28 (SEQ ID NO: 182)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-29 (SEQ ID NO: 183)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAGGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-30 (SEQ ID NO: 184)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGCATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-31 (SEQ ID NO: 185)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTAACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-32 (SEQ ID NO: 186)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-33 (SEQ ID NO: 187)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACT
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGTGCCTTTTGACAACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-35 (SEQ ID NO: 188)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTATTACGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTCAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-36 (SEQ ID NO: 189)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGGTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCGGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-37 (SEQ ID NO: 190)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAAGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-38 (SEQ ID NO: 191)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-39 (SEQ ID NO: 192)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTAGA
    TACTACGCAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-40 (SEQ ID NO: 193)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTAAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-53 (SEQ ID NO: 194)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTAGTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGAGCGTAGA
    TACTACGCAGACTCAGTGAAGGGCCGGTTCACCATCTCCCGCGACAATCCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGAGCCTTTTGAATACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-54 (SEQ ID NO: 195)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAACTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTACA
    TACTACGCGGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTATGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCACGAGC
    >DOM1h-574-65 (SEQ ID NO: 196)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGATAATTCCAAGAACA
    CACTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-66 (SEQ ID NO: 197)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAAGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-67 (SEQ ID NO: 198)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-68 (SEQ ID NO: 199)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAGGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-69 (SEQ ID NO: 200)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-70 (SEQ ID NO: 201)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GGTATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-71 (SEQ ID NO: 202)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAAGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-72 (SEQ ID NO: 203)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-73 (SEQ ID NO: 204)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAGGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-74 (SEQ ID NO: 205)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-75 (SEQ ID NO: 206)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-76 (SEQ ID NO: 207)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCCCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAAGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-77 (SEQ ID NO: 208)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-78 (SEQ ID NO: 209)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAGGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-79 (SEQ ID NO: 210)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-84 (SEQ ID NO: 211)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTAGA
    TACTACGCAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-85 (SEQ ID NO: 212)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTAGA
    TACTACGCAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAAGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-86 (SEQ ID NO: 213)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCCCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTAGA
    TACTACGCAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAAGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-87 (SEQ ID NO: 214)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTAGA
    TACTACGCAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAGGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-88 (SEQ ID NO: 215)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGTGATCGTAGA
    TACTACGCAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-90 (SEQ ID NO: 216)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTTTTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-91 (SEQ ID NO: 217)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-92 (SEQ ID NO: 218)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-93 (SEQ ID NO: 219)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-94 (SEQ ID NO: 220)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGCATATTACTGTGC
    GATATATACGGGTCGGTGGCCCGACTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-95 (SEQ ID NO: 221)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGCATATTACTGTGC
    GATATATACGGGTCGGTGGCCCGACTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-96 (SEQ ID NO: 222)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGCCCGACTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-97 (SEQ ID NO: 223)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGCCCGACTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-98 (SEQ ID NO: 224)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGCCCGACTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-99 (SEQ ID NO: 225)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGCCCGACTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-100 (SEQ ID NO: 226)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGATGGGTCCGCCA
    GGCTCCAGGGAAAGGTCCAGAGTGGGTCTCACAGATTTCGGCCTGGGGTGACAGGACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-101 (SEQ ID NO: 227)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAAGGTCCAGAGTGGGTCTCACAGATTTCGGACGGCGGTCAGAGGACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-102 (SEQ ID NO: 228)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGATGGGTCCGCCA
    GGCTCCAGGGAAAGGTCCAGAGTGGGTCTCACAGATTTCGGACTCCGGTTACCGCACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-103 (SEQ ID NO: 229)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCCAGAGTGGGTCTCACAGATTTCGGACGGGGGTACGCGGACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-104 (SEQ ID NO: 230)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGATGGGTCCGCCA
    GGCTCCAGGGAAAGGTCCAGAGTGGGTCTCACAGATTTCGGACAAGGGTACGCGCACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-105 (SEQ ID NO: 231)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGATGGGTCCGCCA
    GGCTCCAGGGAAAGGTCCAGAGTGGGTCTCACAGATTTCGGAGACCGGTCGCAGGACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-106 (SEQ ID NO: 232)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTAACAATACGGGTTCGACCACA
    TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-107 (SEQ ID NO: 233)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCCAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-108 (SEQ ID NO: 234)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCCAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-109 (SEQ ID NO: 235)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-110 (SEQ ID NO: 236)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-111 (SEQ ID NO: 237)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAGGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-112 (SEQ ID NO: 238)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACACACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-113 (SEQ ID NO: 239)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGCAGA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-114 (SEQ ID NO: 240)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTTGAATACTGCTGATCGTACA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-115 (SEQ ID NO: 241)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-116 (SEQ ID NO: 242)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTAGA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-117 (SEQ ID NO: 243)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTAGA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-118 (SEQ ID NO: 244)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GGTATATACTGGGCGTTGGGTGTCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-119 (SEQ ID NO: 245)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GCTATATACTGGGCGTTGGGTGTCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-120 (SEQ ID NO: 246)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTTACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GGTATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-121 (SEQ ID NO: 247)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GCTATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-122 (SEQ ID NO: 248)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACTGCTGATCGTAGA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-123 (SEQ ID NO: 249)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTAGA
    TACTACGCAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-124 (SEQ ID NO: 250)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCGGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACGGGCGATCGTAGA
    TACTACGCACACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-125 (SEQ ID NO: 251)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACTGCTGATCGTAGA
    TACTACGCAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-126 (SEQ ID NO: 252)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCACACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-127 (SEQ ID NO: 253)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGAATACTGCTGATCGTAGA
    TACTACGCACACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-128 (SEQ ID NO: 254)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGCTGATCGTAGA
    TACTACGCACACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-129 (SEQ ID NO: 255)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGTGAATACGGGTGATCGTAGA
    TACTACGCAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-130 (SEQ ID NO: 256)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGAATACGGGTGATCGTAGA
    TACTACGCAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-131 (SEQ ID NO: 257)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-132 (SEQ ID NO: 258)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAGGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-133 (SEQ ID NO: 259)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-134 (SEQ ID NO: 260)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACTCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-135 (SEQ ID NO: 261)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACACACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-137 (SEQ ID NO: 262)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACACAGACGCGGTGAAGGGGCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-138 (SEQ ID NO: 263)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-139 (SEQ ID NO: 264)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-140 (SEQ ID NO: 265)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACGGGTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-141 (SEQ ID NO: 266)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-142 (SEQ ID NO: 267)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATCACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAACCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-143 (SEQ ID NO: 268)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACGGGTGATCGTAGA
    TACTACGATGACGCGGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-144 (SEQ ID NO: 269)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACTGCTGATCGTAGA
    TACTACGATGACTCTGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-145 (SEQ ID NO: 270)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACGGGTGATCGTAGA
    TACTACGATCACTCTGTGAAGGGCCGGTTCACTATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-146 (SEQ ID NO: 271)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACGGGTGATCGTAGA
    TACTACGATGACGCGGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-147 (SEQ ID NO: 272)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGGGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-14 8 (SEQ ID NO: 273)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGTGCCTTTTGCCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-149 (SEQ ID NO: 274)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGGACCTTTTCAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-150 (SEQ ID NO: 275)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTCAGTACTGGGGTCAGGGAACTCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-151 (SEQ ID NO: 276)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-152 (SEQ ID NO: 277)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGCGCCTTTTCAGTACTGGGGTCAGGGAACTCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-153 (SEQ ID NO: 278)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGTGCCTTTTCAGTACTGGGGTCAGGGCACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-154 (SEQ ID NO: 279)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACCGGTGATCGTAGA
    TACTACGATCACTCTGTGAAGGGCCGGTTCACTATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-155 (SEQ ID NO: 280)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-156 (SEQ ID NO: 281)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-157 (SEQ ID NO: 282)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAGGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-158 (SEQ ID NO: 283)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGAGGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-159 (SEQ ID NO: 284)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-160 (SEQ ID NO: 285)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-161 (SEQ ID NO: 286)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACTCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-162 (SEQ ID NO: 287)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACTCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-163 (SEQ ID NO: 288)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACACACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-164 (SEQ ID NO: 289)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACACACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-165 (SEQ ID NO: 290)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-166 (SEQ ID NO: 291)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-167 (SEQ ID NO: 292)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTGAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACCGGTGATCGTAGA
    TACTACGATCACTCTGTGAAGGGCCGGTTCACTATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-168 (SEQ ID NO: 293)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACCGGTGATCGTAGA
    TACTACGATCACTCTGTGAAGGGCCGGTTCACTATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-169 (SEQ ID NO: 294)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACTGCTGATCGTACA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGCGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-170 (SEQ ID NO: 295)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTACA
    TACTACGCACACGCGGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-171 (SEQ ID NO: 296)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACTGCTGATCGTACA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-172 (SEQ ID NO: 297)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACTGCTGATCGTACA
    TACTACGATCACGCGGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCTGAGGACACCGCGGTATATTACTGTGC
    GATATATACTGGGCGTTGGGTGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-173 (SEQ ID NO: 298)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACTGCTGATCGTAGA
    TACTACGCACACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-174 (SEQ ID NO: 299)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTAGA
    TACTACGCACACGCGGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-175 (SEQ ID NO: 300)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACTGCTGATCGTAGA
    TACTACGCACACGCGGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-176 (SEQ ID NO: 301)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTAGA
    TACTACGATCACGCGGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-177 (SEQ ID NO: 302)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACTGCTGATCGTAGA
    TACTACGATCACGCGGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGGACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-178 (SEQ ID NO: 303)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTGCGGATACTGCTGATCGTAGA
    TACTACGATCACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGGTGGGCGCCTTTTGAGTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
    >DOM1h-574-179 (SEQ ID NO: 304)
    GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC
    TCTCCTGTGCAGCCTCCGGATTCACCTTTTTCAAGTATTCGATGGGGTGGGTCCGCCA
    GGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACAGATTTCGGATACTGCTGATCGTAGA
    TACTACGATGACGCGGTGAAGGGCCGGTTCACCATCACCCGCGACAATTCCAAGAACA
    CGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGC
    GATATATACGGGTCGTTGGGAGCCTTTTGTCTACTGGGGTCAGGGAACCCTGGTCACC
    GTCTCGAGC
  • TABLE 5
    Anti-serum albumin dAb (DOM7h) fusions
    (used in Rat studies):-
    DOM7h-14/Exendin-4 fusion
    DMS number 7138
    Amino acid sequence (SEQ ID NO: 305)
    HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSG
    GGGSDIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLL
    IMWRSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGAALPRTF
    GQGTKVEIKR
    Nucleotide sequence (SEQ ID NO: 306)
    CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAG
    GCAGTGCGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGG
    GCACCTCCGCCATCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGC
    GGTGGCGGGTCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
    TCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGG
    TCTCAGTTATCTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTG
    ATCATGTGGCGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGC
    AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAA
    GATTTTGCTACGTACTACTGTGCTCAGGGTGCGGCGTTGCCTAGGACGTTC
    GGCCAAGGGACCAAGGTGGAAATCAAACGG
    DOM7h-14-10/Exendin-4 fusion
    DMS number 7139
    Amino acid sequence (SEQ ID NO: 307)
    HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSG
    GGGSDIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLL
    IMWRSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTF
    GQGTKVEIKR
    Nucleotide sequence (SEQ ID NO: 308)
    CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAG
    GCAGTGCGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGG
    GCACCTCCGCCATCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGC
    GGTGGCGGGTCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
    TCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGG
    TCTCAGTTATCTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTG
    ATCATGTGGCGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGC
    AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAA
    GATTTTGCTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTC
    GGCCAAGGGACCAAGGTGGAAATCAAACGG
    DOM7h-14-18/Exendin-4 fusion
    DMS number 7140
    Amino acid sequence (SEQ ID NO: 309)
    HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSG
    GGGSDIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLL
    IMWRSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLMKPMTF
    GQGTKVEIKR
    Nucleotide sequence (SEQ ID NO: 310)
    CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAG
    GCAGTGCGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGG
    GCACCTCCGCCATCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGC
    GGTGGCGGGTCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
    TCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGG
    TCTCAGTTATCTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTG
    ATCATGTGGCGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGC
    AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAA
    GATTTTGCTACGTACTACTGTGCTCAGGGTCTTATGAAGCCTATGACGTTC
    GGCCAAGGGACCAAGGTGGAAATCAAACGG
    DOM7h-14-19/Exendin-4 fusion
    DMS number 7141
    Amino acid sequence (SEQ ID NO: 311)
    HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSG
    GGGSDIQMTQSPSSLSASVGDRVTISCRASQWIGSQLSWYQQKPGEAPKLL
    IMWRSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGAALPRTF
    GQGTKVEIKR
    Nucleotide sequence (SEQ ID NO: 312)
    CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAG
    GCAGTGCGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGG
    GCACCTCCGCCATCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGC
    GGTGGCGGGTCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
    TCTGTAGGAGACCGTGTCACCATCTCTTGCCGGGCAAGTCAGTGGATTGGG
    TCTCAGTTATCTTGGTACCAGCAGAAACCAGGGGAAGCCCCTAAGCTCCTG
    ATCATGTGGCGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGC
    AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAA
    GATTTTGCTACGTACTACTGTGCTCAGGGTGCGGCGTTGCCTAGGACGTTC
    GGCCAAGGGACCAAGGTGGAAATCAAACGG
    DOM7h-11/Exendin-4 fusion
    DMS number 7142
    Amino acid sequence (SEQ ID NO: 313)
    HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSG
    GGGSDIQMTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLL
    IWFGSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTF
    GQGTKVEIKR
    Nucleotide sequence (SEQ ID NO: 314)
    CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAG
    GCAGTGCGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGG
    GCACCTCCGCCATCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGC
    GGTGGCGGGTCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
    TCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGTCGTCCGATTGGG
    ACGACGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTG
    ATCTGGTTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGC
    AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAA
    GATTTTGCTACGTACTACTGTGCGCAGGCTGGGACGCATCCTACGACGTTC
    GGCCAAGGGACCAAGGTGGAAATCAAACGG
    DOM7h-11-12/Exendin-4 fusion
    DMS number 7147
    Amino acid sequence (SEQ ID NO: 315)
    HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSG
    GGGSDIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLL
    ILFGSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTF
    GQGTKVEIKR
    Nucleotide sequence (SEQ ID NO: 316)
    CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAG
    GCAGTGCGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGG
    GCACCTCCGCCATCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGC
    GGTGGCGGGTCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
    TCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGTCGTCCGATTGGG
    ACGATGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTG
    ATCTTGTTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGC
    AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAA
    GATTTTGCTACGTACTACTGTGCGCAGGCTGGGACGCATCCTACGACGTTC
    GGCCAAGGGACCAAGGTGGAAATCAAACGG
    DOM7h-11-15/Exendin-4 fusion
    DMS number 7143
    Amino acid sequence (SEQ ID NO: 317)
    HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSG
    GGGSDIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLL
    ILAFSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTF
    GQGTKVEIKR
    Nucleotide sequence (SEQ ID NO: 318)
    CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAG
    GCAGTGCGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGG
    GCACCTCCGCCATCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGC
    GGTGGCGGGTCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
    TCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGTCGTCCGATTGGG
    ACGATGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTG
    ATCCTTGCTTTTTCCCGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGC
    AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAA
    GATTTTGCTACGTACTACTGCGCGCAGGCTGGGACGCATCCTACGACGTTC
    GGCCAAGGGACCAAGGTGGAAATCAAACGG
    DOM7h14-10/G4SC-NCE fusion
    Amino acid sequence (SEQ ID NO: 319) encoding
    DOM7h14-10/G4SC
    DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMWR
    SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQGT
    KVEIKRGGGGSC
    The C-terminal cysteine can be linked to a new
    chemical entity (pharmaceutical chemical compound,
    NCE), eg using maleimide linkage.
    Nucleotide sequence (SEQ ID NO: 320) encoding
    DOM7h14-10/G4SC
    GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
    CGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCT
    TGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGGCGT
    TCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATCTGGG
    ACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCTACG
    TACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAAGGGACC
    AAGGTGGAAATCAAACGGGGTGGCGGAGGGGGTTCCTGT
    DOM7h14-10/TVAAPSC fusion
    Amino acid sequence (SEQ ID NO: 321)
    DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMWR
    SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQGT
    KVEIKRTVAAPSC
    The C-terminal cysteine can be linked to a new
    chemical entity (pharmaceutical chemical compound,
    NCE), eg using maleimide linkage.
    Nucleotide sequence (SEQ ID NO: 322)
    GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
    CGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCT
    TGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGGCGT
    TCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATCTGGG
    ACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCTACG
    TACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAAGGGACC
    AAGGTGGAAATCAAACGGACCGTCGCTGCTCCATCTTGT
    (used in mouse studies):-
    DOM7h-11/DOM1m-21-23 fusion
    DMS number 5515
    Amino acid sequence (SEQ ID NO: 323)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRI
    DSYGRGTYYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQF
    GSNAFDYWGQGTQVTVSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRAS
    RPIGTTLSWYQQKPGKAPKLLIWFGSRLQSGVPSRFSGSGSGTDFTLTISS
    LQPEDFATYYCAQAGTHPTTFGQGTKVEIKR
    Amino acid plus nucleotide plus myc tag sequence
    (SEQ ID NO: 324)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRI
    DSYGRGTYYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQF
    GSNAFDYWGQGTQVTVSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRAS
    RPIGTTLSWYQQKPGKAPKLLIWFGSRLQSGVPSRFSGSGSGTDFTLTISS
    LQPEDFATYYCAQAGTHPTTFGQGTKVEIKRAAAEQKLISEEDLN
    Nucleotide sequence (SEQ ID NO: 325)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
    CTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATG
    GGGTGGCTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATT
    GATTCTTATGGTCGTGGTACATACTACGAAGACCCCGTGAAGGGCCGGTTC
    AGCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGC
    CTGCGTGCCGAGGACACCGCCGTATATTACTGTGCGAAAATTTCTCAGTTT
    GGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCAGGTCACCGTCTCG
    AGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTCTCCATCC
    TCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
    CGTCCGATTGGGACGACGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCC
    CCTAAGCTCCTGATCTGGTTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCA
    CGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT
    CTGCAACCTGAAGATTTTGCTACGTACTACTGTGCGCAGGCTGGGACGCAT
    CCTACGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG
    Nucleotide plus myc tag sequence (SEQ ID NO: 326)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
    CTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATG
    GGGTGGCTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATT
    GATTCTTATGGTCGTGGTACATACTACGAAGACCCCGTGAAGGGCCGGTTC
    AGCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGC
    CTGCGTGCCGAGGACACCGCCGTATATTACTGTGCGAAAATTTCTCAGTTT
    GGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCAGGTCACCGTCTCG
    AGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTCTCCATCC
    TCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
    CGTCCGATTGGGACGACGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCC
    CCTAAGCTCCTGATCTGGTTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCA
    CGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT
    CTGCAACCTGAAGATTTTGCTACGTACTACTGTGCGCAGGCTGGGACGCAT
    CCTACGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGCA
    GAACAAAAACTCATCTCAGAAGAGGATCTGAATTAA
    DOM7h-11-12/DOM1m-21-23 fusion
    DMS number 5516
    Amino acid sequence (SEQ ID NO: 327)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRI
    DSYGRGTYYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQF
    GSNAFDYWGQGTQVTVSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRAS
    RPIGTMLSWYQQKPGKAPKLLILFGSRLQSGVPSRFSGSGSGTDFTLTISS
    LQPEDFATYYCAQAGTHPTTFGQGTKVEIKR
    Amino acid plus nucleotide plus myc tag sequence
    (SEQ ID NO: 328)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRI
    DSYGRGTYYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQF
    GSNAFDYWGQGTQVTVSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRAS
    RPIGTMLSWYQQKPGKAPKLLILFGSRLQSGVPSRFSGSGSGTDFTLTISS
    LQPEDFATYYCAQAGTHPTTFGQGTKVEIKRAAAEQKLISEEDLN
    Nucleotide sequence (SEQ ID NO: 329)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
    CTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATG
    GGGTGGCTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATT
    GATTCTTATGGTCGTGGTACATACTACGAAGACCCCGTGAAGGGCCGGTTC
    AGCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGC
    CTGCGTGCCGAGGACACCGCCGTATATTACTGTGCGAAAATTTCTCAGTTT
    GGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCAGGTCACCGTCTCG
    AGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTCTCCATCC
    TCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
    CGTCCGATTGGGACGATGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCC
    CCTAAGCTCCTGATCTTGTTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCA
    CGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT
    CTGCAACCTGAAGATTTTGCTACGTACTACTGTGCGCAGGCTGGGACGCAT
    CCTACGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG
    Nucleotide plus myc tag sequence (SEQ ID NO: 330)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
    CTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATG
    GGGTGGCTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATT
    GATTCTTATGGTCGTGGTACATACTACGAAGACCCCGTGAAGGGCCGGTTC
    AGCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGC
    CTGCGTGCCGAGGACACCGCCGTATATTACTGTGCGAAAATTTCTCAGTTT
    GGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCAGGTCACCGTCTCG
    AGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTCTCCATCC
    TCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
    CGTCCGATTGGGACGATGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCC
    CCTAAGCTCCTGATCTTGTTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCA
    CGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT
    CTGCAACCTGAAGATTTTGCTACGTACTACTGTGCGCAGGCTGGGACGCAT
    CCTACGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGCA
    GAACAAAAACTCATCTCAGAAGAGGATCTGAATTAA
    DOM7h-11-15/DOM1m-21-23 fusion
    DMS number 5517
    Amino acid sequence (SEQ ID NO: 331)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRI
    DSYGRGTYYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQF
    GSNAFDYWGQGTQVTVSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRAS
    RPIGTMLSWYQQKPGKAPKLLILAFSRLQSGVPSRFSGSGSGTDFTLTISS
    LQPEDFATYYCAQAGTHPTTFGQGTKVEIKR
    Amino acid plus nucleotide plus myc tag sequence
    (SEQ ID NO: 332)
    EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRI
    DSYGRGTYYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQF
    GSNAFDYWGQGTQVTVSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRAS
    RPIGTMLSWYQQKPGKAPKLLILAFSRLQSGVPSRFSGSGSGTDFTLTISS
    LQPEDFATYYCAQAGTHPTTFGQGTKVEIKRAAAEQKLISEEDLN
    Nucleotide sequence (SEQ ID NO: 333)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
    CTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATG
    GGGTGGCTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATT
    GATTCTTATGGTCGTGGTACATACTACGAAGACCCCGTGAAGGGCCGGTTC
    AGCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGC
    CTGCGTGCCGAGGACACCGCCGTATATTACTGTGCGAAAATTTCTCAGTTT
    GGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCAGGTCACCGTCTCG
    AGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTCTCCATCC
    TCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
    CGTCCGATTGGGACGATGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCC
    CCTAAGCTCCTGATCCTTGCTTTTTCCCGTTTGCAAAGTGGGGTCCCATCA
    CGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT
    CTGCAACCTGAAGATTTTGCTACGTACTACTGCGCGCAGGCTGGGACGCAT
    CCTACGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG
    Nucleotide plus myc tag sequence (SEQ ID NO: 334)
    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
    CTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATG
    GGGTGGCTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATT
    GATTCTTATGGTCGTGGTACATACTACGAAGACCCCGTGAAGGGCCGGTTC
    AGCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGC
    CTGCGTGCCGAGGACACCGCCGTATATTACTGTGCGAAAATTTCTCAGTTT
    GGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCAGGTCACCGTCTCG
    AGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTCTCCATCC
    TCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
    CGTCCGATTGGGACGATGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCC
    CCTAAGCTCCTGATCCTTGCTTTTTCCCGTTTGCAAAGTGGGGTCCCATCA
    CGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT
    CTGCAACCTGAAGATTTTGCTACGTACTACTGCGCGCAGGCTGGGACGCAT
    CCTACGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGCA
    GAACAAAAACTCATCTCAGAAGAGGATCTGAATTAA

    Where a myc-tagged molecule is indicated in this table, this was the version used in PK studies in the examples. Where no myc-tagged sequences are given, the PK studies in the examples were not done with myc-tagged material, ie, the studies were done with the non-tagged constructs shown.
    • EXEMPLIFICATION
  • All numbering in the experimental section is according to Kabat (Kabat, E. A. National Institutes of Health (US) & Columbia University. Sequences of proteins of immunological interst, edn 5 (US Dept. Of Health and Human Services Public Health Service, National Institues of Health, Bethesda, Md., 1991)).
  • Derivation of DOM7h-11 and DOM7h-14 variants is described. DOM7h-14 variants are not according to the invention.
    • Example 1: Vk Affinity Maturation Selections:
  • HSA (Human Serum Albumin) and RSA (Rat Serum Albumin) antigens were obtained from Sigma (essentially fatty acid free, ˜99% (agarose gel electrophoresis), lyophilized powder Cat. No. A3782 and A6414 respectively)
  • Biotinylated products of above two antigens were made by using EZ Link Sulfo-NHS-SS-Biotin (Pierce, Cat. No. 21331). Free biotin reagent was removed by passing the samples twice through PD10 desalting column followed by overnight dialysis against 1000× excess volume of PBS at 4° C. Resulting product was tested by mass spec and 1-2 biotins per molecule were observed.
    • Affinity Maturation Libraries:
  • Both error-prone and CDR libraries were created using DOM7h-11 and DOM7h-14 parental dAbs (see WO2008/096158 for the sequences of DOM7h-11 and DOM7h-14). The CDR libraries were generated in the pDOM4 vector and the error prone libraries were generated in the pDOM33 vector (to allow for selection with or without protease treatment). Vector pDOM4, is a derivative of the Fd phage vector in which the gene III signal peptide sequence is replaced with the yeast glycolipid anchored surface protein (GAS) signal peptide. It also contains a c-myc tag between the leader sequence and gene III, which puts the gene III back in frame. This leader sequence functions well both in phage display vectors but also in other prokaryotic expression vectors and can be universally used. pDOM33 is a modified version of the pDOM4 vector where the c-myc tag has been removed which renders the dAb-phage fusion resistant to the protease trypsin. This allows the use of trypsin within the phage selection to select for dAbs that are more protease stable (see WO2008149143).
  • For error-prone maturation libraries, plasmid DNA encoding the dAb to be matured was amplified by PCR, using the GENEMORPH® II RANDOM MUTAGENESIS KIT (random, unique mutagenesis kit, Stratagene). The product was digested with Sal I and Not I and used in a ligation reaction with cut phage vector pDOM33.
  • For the CDR libraries, PCR reactions were performed using degenerate oligonucleotides containing NNK or NNS codons to diversify the required positions in the dAb to be affinity matured. Assembly PCR was then used to generate a full length diversified insert. The insert was digested with Sal I and Not I and used in a ligation reaction with pDOM4 for mutagenesis of multiple residues and pDOM5 for mutagenesis of single residues. The pDOM5 vector is a pUC119-based expression vector where protein expression is driven by the LacZ promoter. A GAS1 leader sequence (see WO 2005/093074) ensures secretion of isolated, soluble dAbs into the periplasm and culture supernatant of E. coli. dAbs are cloned SalI/NotI in this vector, which appends a myc tag at the C-terminus of the dAb. This protocol using SalI and Not I results in inclusion of an ST amino acid sequence at the N-terminus.
  • The ligation produced by either method was then used to transform E. coli strain TB1 by electroporation and the transformed cells plated on 2×TY agar containing 15 μg/ml tetracycline, yielding library sizes of >5×107 clones.
  • The error-prone libraries had the following average mutation rate and size: DOM7h-11 (2.5 mutations per dAb), size: 6.1×108, DOM7h-14 (2.9 mutations per dAb), size: 5.4×108.
  • Each CDR library has four amino acid diversity. Two libraries were generated for each of CDRs 1 and 3, and one library for CDR2. The positions diversified within each library are as follows (amino acids based on VK dummy DPK9 sequence):
  • Library size
    DOM7h-11 DOM7h-14
    1 - Q27, S28, S30, S31 (CDR1) 8.8 × 107 5.8 × 107
    2 - S30, S31, Y32, N34 (CDR1) 4.6 × 108 4.2 × 108
    3 - Y49, A50, A51, S53 (CDR2) 3.9 × 108 2.4 × 108
    4 - Q89, S91, Y92, S93 (CDR3) 1.8 × 108 2.5 × 108
    5 - Y92, Y93, T94, N96 (CDR3) 4.0 × 108 3.3 × 108
    • Example 2: Selection Strategies
  • Three phage selection strategies were adopted for Vκ ALBUDAB™ (anti-serum albumin dAb) affinity maturation:
      • 1) Selections against HSA only:
      • Three rounds of selection against HSA were carried out. The error prone libraries and each CDR library were selected as an individual pool in all rounds. The first round of selection was performed against HSA passively coated onto an immunotube at 1 mg/ml. Round 2 was performed against 100 nM HSA and round 3 against 10 nM (CDR selections) or 20 or 100 nM (Error prone selections) HSA, both as soluble selections followed by a fourth round of selection with the error prone libraries against 1.5 nM HSA as a soluble selection. The error prone libraries were eluted with 0.1M glycine pH 2.0 before neutralisation with 1M Tris pH 8.0 and the CDR libraries were eluted with 1 mg/ml trypsin before infection into log phase TG1 cells. The third round of each selection was subcloned into pDOM5 for screening. Soluble selections used biotinylated HSA.
      • 2) Trypsin selections against HSA:
      • In order to select dAbs with increased protease resistance compared to the parental clone and with potentially improved biophysical properties, trypsin was used in phage selections (see WO2008149143). Four rounds of selection were preformed against HSA. The first round of selection of error prone libraries was performed against passively coated HSA at 1 mg/ml without trypsin; the second round against passively coated HSA at 1 mg/ml with 20 μg/ml trypsin for 1 hour at 37° C.; the third round selection was performed by soluble selection using biotinylated HSA against 100 nM HSA with 20 μg/ml or 100 μg/ml trypsin for 1 hour at 37° C. The final round of selection was performed by soluble selection using biotinylated HSA against 100 nM HSA with 100 μg/ml trypsin overnight at 37° C.
      • 3) Cross-over selections against HSA (round 1) and RSA (rounds 2-4):
      • The first round selection was carried out against 1 mg/ml passively coated HSA or 1
  • HSA (soluble selection), followed by a further three rounds of soluble selections against biotinylated RSA at concentrations of 1 μM for round 1, 100 nm for round 2 and 20 nM, 10 nM or 1 nM for round 3.
    • Screening Strategy and Affinity Determination:
  • In each case after selection a pool of phage DNA from the appropriate round of selection is prepared using a QIAfilter midiprep kit (Qiagen), the DNA is digested using the restriction enzymes Sal1 and Not1 and the enriched V genes are ligated into the corresponding sites in pDOM5 the soluble expression vector which expresses the dAb with a myc tag (see PCT/EP2008/067789). The ligated DNA is used to electro-transform E. coli HB 2151 cells which are then grown overnight on agar plates containing the antibiotic carbenicillin. The resulting colonies are individually assessed for antigen binding. In each case at least 96 clones were tested for binding to HSA, CSA (Cynomlgus monkey Serum Albumin), MSA (mouse serum albumin) and RSA by BIACORE™ (surface plasmon resonance). MSA antigen was obtained from Sigma (essentially fatty acid free, ˜99% (agarose gel electrophoresis), lyophilized powder Cat. No. A3559) and CSA was purified from Cynomolgus serum albumin using prometic blue resin (Amersham). Soluble dAb fragments were produced in bacterial culture in ONEX culture media (Novagen) overnight at 37° C. in 96 well plates. The culture supernatant containing soluble dAb was centrifuged and analysed by BiaCore™ for binding to high density HSA, CSA, MSA and RSA CM5 chips. Clones were found to bind to all these species of serum albumin by off-rate screening. The clones were sequenced revealing unique dAb sequences.
  • The minimum identity to parent (at the amino acid level) of the clones selected was 97.2% (DOM7h-11-3: 97.2%, DOM7h-11-12: 98.2%, DOM7h11-15: 96.3%, DOM7h-11-18: 98.2%, DOM7h-11-19: 97.2%)
  • The minimum identity to parent (at the amino acid level) of the clones selected was 96.3% (DOM7h-14-10: 96.3%, DOM7h-14-18: 96.3%, DOM7h-14-19: 98.2%, DOM7h-14-28: 99.1%, DOM7h-14-36: 97.2%)
  • Unique dAbs were expressed as bacterial supernatants in 2.5 L shake flasks in Onex media at 30° C. for 48 hrs at 250 rpm. dAbs were purified from the culture media by absorption to protein L agarose followed by elution with 10 mM glycine pH2.0. Binding to HSA, CSA, MSA and RSA by BiaCore™ was confirmed using purified protein at 3 concentrations 1 μM, 500 nM and 50 nM. To determine the binding affinity (KD) of the ALBUDABs™ to each serum albumin; purified dAbs were analysed by BiaCore™ over albumin concentration range from 5000 nM to 39 nM (5000 nM, 2500 nM, 1250 nM, 625 nM, 312 nM, 156 nM, 78 nM, 39 nM).
  • TABLE 6
    Affinity (KD)
    ALBUDAB ™ to SA (nM) Kd Ka
    Rat
    DOM7h-14 60 2.095E−01 4.00E+06
    DOM7h-14-10 4 9.640E−03 4.57E+06
    DOM7h-14-18 410 2.275E−01 5.60E+05
    DOM 7h-14-19 890 2.870E−01 3.20E+05
    DOM 7h-14-28 45 (140) 7.0E−02 (1.141e−1) 2.10E+06 (8.3e5)
    DOM 7h-14-36 30 (6120) 2.9E−02 (5.54e−2) 1.55E+06 (9e3)
    DOM 7h-11 2100 1.00E−01 4.80E+04
    DOM 7h-11-3 10000 (88000) (7.18e−1) (8.11e3)
    DOM 7h-11-12 200 5.22E−01 2.76E+06
    DOM 7h-11-15 20 2.10E−02 1.10E+06
    DOM 7h-11-18 80 (29000) 6.0E−02 (3.7e−1) 1.64E+06 (1.3e4)
    DOM 7h-11-19 28 (17000) 9.1e−02 (1.4e−1) 9.80E+05 (8.1e3)
    Cyno
    DOM 7h-14 66 9.65E−02 1.50E+06
    DOM 7h-14-10 9 1.15E−02 1.60E+06
    DOM 7h-14-18 180 1.05E−01 6.30E+5 
    DOM 7h-14-19 225 1.56E−01 7.00E+05
    DOM 7h-14-28 66 (136) 1.3E−01 (1.34e−1) 2.50E+06 (9.8e5)
    DOM 7h-14-36 35 (7830) 1.9E−02 (1.1e−1) 9.80E+06 (1.43e4)
    DOM 7h-11 1000 6.82E−01 8.00E+05
    DOM 7h-11-3 670 (200) 9.6E−02 (1.5e−1) 2.90E+05 (7.26e5)
    DOM 7h-11-12 >6000
    DOM 7h-11-15 3 5.57E−03 5.80E+06
    DOM 7h-11-18 10000 (65000) 1.36 (4.8e−1) 2.25E+05 (7.3e3)
    DOM 7h-11-19 >10000 (375000) (6.2e−1) (1.7e3)
    Mouse
    DOM 7h-14 12 4.82E−02 4.10E+06
    DOM 7h-14-10 30 3.41E−02 1.29E+06
    DOM 7h-14-18 65 9.24E−02 2.28E+06
    DOM 7h-14-19 60 5.76E−02 1.16E+06
    DOM 7h-14-28 26 (31) 3.4E−02 (7.15e−2) 1.60E+06 (2.28e6)
    DOM 7h-14-36 35 (33) 2.3E−02 (7.06e−2) 8.70E+05 (2.11e6)
    DOM 7h-11 5000 9.00E−01
    DOM 7h-11-3 >10000 (36000) (6.12e−1) (1.67e4)
    DOM 7h-11-12 130 1.89E−01 1.53E+06
    DOM 7h-11-15 10 9.40E−03 1.10E+06
    DOM 7h-11-18 150 (1600) 2.4E−02 (6.23e−2) 4.40E+05 (4e4)
    DOM 7h-11-19 100 (18000) 3.7E−02 (8.8e−2) 1.40E+06 (4.9e3)
    Human
    DOM 7h-14 33 4.17E−02 1.43E+06
    DOM 7h-14-10 12 1.39E−02 1.50E+06
    DOM 7h-14-18 280 3.39E−02 1.89E+05
    DOM 7h-14-19 70 5.25E−02 8.26E+05
    DOM 7h-14-28 30 (8260) 3.3E−02 (5.6e−2) 1.24E+06 (6.78e3)
    DOM 7h-14-36 28 (1260) 2.4E−02 (6.7e−2) 1.23E+06 (5.4e4)
    DOM 7h-11 2800 6.41E−01 7.00E+05
    DOM 7h-11-3 32 (130) 1.6E−02 (2.35e−2) 6.50E+05 (1.86e5)
    DOM 7h-11-12 350 4.13E−01 1.26E+06
    DOM 7h-11-15 1 1.84E−03 2.00E+06
    DOM 7h-11-18 36 (32000) 5.1E−02 (2.7e−1) 3.40E+06 (8.39e3)
    DOM 7h-11-19 65 (38000) 1.1E−01 (2.09e−1) 1.80E+06 (5.4e3)
    *: values in brackets were derived from a second, independent SPR experiment.
  • All DOM7h-14 derived variants are cross-reactive to mouse, rat, human and cyno serum albumin. DOM7h-14-10 has improved affinity to rat, cyno and human serum albumin compared to parent. DOM7h-14-28 has an improved affinity to RSA. DOM7h-14-36 has an improved affinity to RSA, CSA and MSA.
  • DOM7h-11-3 has improved affinity to CSA and HSA. DOM7h-11-12 has improved affinity to RSA, MSA and HSA. DOM7h-11-15 has improved affinity to RSA, MSA, CSA and HSA. DOM7h-11-18 and DOM7h-11-19 have improved affinity to RSA, MSA and HSA.
    • Example 3: Origins of Key DOM7h-11 Lineage Clones
  • DOM7h-11-3: From affinity maturation performed against HSA using the CDR2 library (Y49, A50, A51, S53), round 3 output 10 nM HSA
  • DOM7h-11-12: From affinity maturation performed against HSA using the error prone library, round 3 outputs (100 nM, HSA) with 100 ug/ml trypsin.
  • DOM7h-11-15: From cross-over selections performed against HSA as round 1 followed by additional 3 rounds of selections against RSA using the CDR2 library (Y49, A50, A51, S53) at round 3 selection with 1 nM of RSA.
  • DOM7h-11-18 From cross-over selections performed against HSA as round 1 followed by additional 3 rounds of selections against RSA using the error prone library, round 3 output at 20 nM of RSA
  • DOM7h-11-19 From cross-over selections performed against HSA as round 1 followed by additional 3 rounds of selections against RSA using the error prone library, round 3 output at 5 nM of RSA
  • TABLE 7
    CDR sequences (according to Kabat;
     ref. as above)
    CDR
    ALBUDAB ™ CDR1 CDR2 CDR3
    DPK9 Vk SQSISSYLN YAASSLQS QQSYSTPNT
    dummy (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    335) 336) 337)
    DOM7h-11 SRPIGTTLS WFGSRLQS AQAGTHPTT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    338) 339) 340)
    DOM7h-11-12 SRPIGTMLS LFGSRLQS AQAGTHPTT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    341) 342) 343)
    DOM 7h-11-15 SRPIGTMLS LAFSRLQS AQAGTHPTT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    344) 345) 346)
    DOM 7h-11-18 SRPIGTMLS WFGSRLQS AQAGTHPTT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    347) 348) 349)
    DOM 7h-11-19 SRPIGTMLS LFGSRLQS AQTGTHPTT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    350) 351) 352)
    DOM 7h-11-3 SRPIGTTLS LWFSRLQS AQAGTHPTT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    353) 354) 355)
    • Example 4: Origins of Key DOM7h-14 Lineage Clones
  • DOM7h-14-19: From affinity maturation performed against HSA using the error prone library, round 3 outputs (100 nM, HSA) with 100 ug/ml trypsin.
  • DOM7h-14-10, DOM7h-14-18, DOM7h-14-28, DOM7h-14-36: From affinity maturation performed against HSA using CDR3 library (Y92, Y93, T94, N96), round 3 output.
  • TABLE 8
    CDR sequences (according to Kabat;
    ref. as above)
    CDR
    ALBUDAB ™ CDR1 CDR2 CDR3
    DPK9 Vk SQSISSYLN YAASSLQS QQSYSTPNT
    dummy (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    335) 336) 337)
    DOM 7h-14 SQWIGSQLS MWRSSLQS AQGAALPRT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    356) 357) 358)
    DOM 7h-14-10 SQWIGSQLS MWRSSLQS AQGLRHPKT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    359) 360) 361)
    DOM 7h-14-18 SQWIGSQLS MWRSSLQS AQGLMKPMT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    362) 363) 364)
    DOM 7h-14-19 SQWIGSQLS MWRSSLQS AQGAALPRT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    365) 366) 367)
    DOM 7h-14-28 SQWIGSQLS MWRSSLQS AQGAALPKT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    368) 369) 370)
    DOM 7h-14-36 SQWIGSQLS MWRSSLQS AQGFKKPRT
    (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:
    371) 372) 373)
    • Example 5: Expression and Biophysical Characterisation
  • The routine bacterial expression level in 2.5 L shake flasks was determined following culture in Onex media at 30° C. for 48 hrs at 250 rpm. The biophysical characteristics were determined by SEC MALLS and DSC.
  • SEC MALLS (size exclusion chromatography with multi-angle-LASER-light-scattering) is a non-invasive technique for the characterizing of macromolecules in solution. Briefly, proteins (at concentration of 1 mg/mL in buffer Dulbecco's PBS at 0.5 ml/min are separated according to their hydrodynamic properties by size exclusion chromatography (column: TSK3000 from TOSOH Biosciences; 5200 from Pharmacia). Following separation, the propensity of the protein to scatter light is measured using a multi-angle-LASER-light-scattering (MALLS) detector. The intensity of the scattered light while protein passes through the detector is measured as a function of angle. This measurement taken together with the protein concentration determined using the refractive index (RI) detector allows calculation of the molar mass using appropriate equations (integral part of the analysis software Astra v.5.3.4.12).
  • DSC (Differential Scanning calorimetry): briefly, the protein is heated at a constant rate of 180° C./hrs (at 1 mg/mL in PBS) and a detectable heat change associated with thermal denaturation measured. The transition midpoint (appTm) is determined, which is described as the temperature where 50% of the protein is in its native conformation and the other 50% is denatured. Here, DSC determined the apparent transition midpoint (appTm) as most of the proteins examined do not fully refold. The higher the Tm, the more stable the molecule. Unfolding curves were analysed by non-2-state equations. The software package used was OriginR v7.0383.
  • TABLE 9
    Biophysical parameters
    ALBUDAB ™ SEC MALLS DSC Tm(° C.)
    DOM7h-14 M 60
    DOM 7h-14-10 M 59
    DOM 7h-14-18 M 58
    DOM 7h-14-19 M 59
    DOM 7h-14-28 M 58.3/60.2
    DOM 7h-14-36 M   59.2
    DOM 7h-11 M 66.9-72.2
    DOM 7h-11-3 M (95%)* 66.6/70.5
    DOM 7h-11-12 M (<2% D)   71.7
    DOM 7h-11-15 M (<5% D) 58.5-60.5
    DOM 7h-11-18 M (98%) 58.9/65.8
    DOM 7h-11-19 M 71.8/76.6
    *in one other trial, monomer was primarily seen by SEC MALLS, although lower than 95%
    We observed expression levels for all clones in Table 9 in the range from 15 to 119 mg/L in E coli.
  • For DOM7h-14 and DOM7h-11 variants, favorable biophysical parameters (monomeric in solution as determined by SEC MALLs and appTm of >55° C. as determined by DSC) and expression levels were maintained during affinity maturation. Monomeric state is advantageous because it avoids dimerisation and the risk of products that may cross-link targets such as cell-surface receptors.
    • Example 6: Determination of Serum Half Life in Rat, Mouse and Cynomolgus Monkey
  • ALBUDABs™ DOM7h-14-10, DOM7h-14-18, DOM7h-14-19, DOM7h-11, DOM7h11-12 and DOM7h-11-15 were cloned into the pDOM5 vector. For each ALBUDAB™, 20-50 mg quantities were expressed in E. coli and purified from bacterial culture supernatant using protein L affinity resin and eluted with 100 mM glycine pH2. The proteins were concentred to greater than 1 mg/ml, buffer exchanged into PBS and endotoxin depleted using Q spin columns (Vivascience). For Rat pharmacokinetic (PK) analysis, ALBUDABs™ were dosed as single i.v injections at 2.5 mg/kg using 3 rats per compound. Serum samples were taken at 0.16, 1, 4, 12, 24, 48, 72, 120, 168 hrs. Analysis of serum levels was by anti-myc ELISA as per the method described below.
  • For Mouse PK, DOM7h-11, DOM7h11-12 and DOM7h-11-15 were dosed as single i.v injections at 2.5 mg/kg per dose group of 3 subjects and serum samples taken at 10 mins; 1 h; 8 h; 24 h; 48 h; 72 h; 96 h. Analysis of serum levels was by anti-myc ELISA as per the method described below.
  • For Cynomolgus monkey PK DOM7h-14-10 and DOM7h-11-15 were dosed as single i.v injections at 2.5 mg/kg into 3 female Cynomolgus monkeys per dose group and serum samples taken at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, 144, 192, 288, 336, 504 hrs. Analysis of serum levels was by anti-myc ELISA as per the method described below.
    • Anti-Myc ELISA Method
  • The ALBUDAB™ concentration in serum was measured by anti-myc ELISA. Briefly, goat anti-myc polyclonal antibody (1:500; Abcam, catalogue number ab9132) was coated overnight onto Nunc 96-well Maxisorp plates and blocked with 5% BSA/PBS+1% tween. Serum samples were added at a range of dilutions alongside a standard at known concentrations. Bound myc-tagged ALBUDAB′ was then detected using a rabbit polyclonal anti-Vk (1:1000; in-house reagent, bleeds were pooled and protein A purified before use) followed by an anti-rabbit IgG HRP antibody (1:10,000; Sigma, catalogue number A2074). Plates were washed between each stage of the assay with 3×PBS+0.1% Tween20 followed by 3×PBS. TMB (SureBlue TMB 1-Component Microwell Peroxidase Substrate, KPL, catalogue number 52-00-00) was added after the last wash and was allowed to develop. This was stopped with 1M HCl and the signal was then measured using absorbance at 450 nm.
  • From the raw ELISA data, the concentration of unknown samples was established by interpolation against the standard curve taking into account dilution factors. The mean concentration result from each time point was determined from replicate values and entered into WinNonLin analysis package (eg version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA). The data was fitted using a non-compartmental model, where PK parameters were estimated by the software to give terminal half-lives. Dosing information and time points were selected to reflect the terminal phase of each PK profile.
  • TABLE 10
    Single ALBUDAB ™ PK
    PK parameters
    Albumin AUC CL t½ Vz
    Species ALBUDAB ™ KD (nM) h × μg/ml ml/h/kg h ml/kg
    Rat DOM7h-14* 60
    DOM7h-14-10 4 2134.6 1.2 42.1 71.2
    DOM7h-14-18 410 617.3 4.1 38.4 228.1
    DOM 7h-14-19 890 632.6 4.1 36.3 213.3
    DOM 7h-11 2100 320.1 7.8 23.3 263.9
    DOM 7h-11-12 200 398.7 6.4 35.5 321.2
    DOM 7h-11-15 20 843.4 3.0 30.3 130.7
    mouse DOM 7h-11 5000 304.7 8.2 18.3 216.8
    DOM 7h-11-12 130 646.6 3.9 43.9 244.8
    DOM 7h-11-15 10 499.2 5.0 33.7 243.4
    Cyno DOM 7h-14* 66 217.5
    DOM 7h-14-10 9 6174.6 0.4 200.8 117.8
    DOM 711-11* 3300 135.1
    DOM 7h-11-15 3 4195 0.6 198.1 170.3
    *Historical data
  • Pharmacokinetic parameters derived from rat, mouse and cynomolgus monkey studies were fitted using a non-compartmental model. Key: AUC: Area under the curve from dosing time extrapolated to infinity; CL: clearance; t½: is the time during which the blood concentration is halved; Vz: volume of distribution based on the terminal phase.
  • DOM7h-11 12 and DOM7h-11-15 have an improved AUC and t1/2 in rat and mouse compared to parent. DOM7h-11-15 also has an improved AUC and t1/2 in cyno compared to parent. This improvement in AUC/t1/2 correlates with an improved in vitro KD to serum albumin.
    • Example 7: ALBUDAB™ IFN Fusions Cloning and Expression
  • As well as single ALBUDABs™, the affinity matured Vk ALBUDABs™ were linked to Interferon alpha 2b (IFNα2b) to determine whether a useful PK of the ALBUDAB™ was maintained as a fusion protein.
  • Interferon alpha 2b amino acid sequence:
    (SEQ ID NO: 374)
    CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKA
    ETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVI
    QGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRS
    FSLSTNLQESLRSKE
    Interferon alpha 2b nucleotide sequence:
    (SEQ ID NO: 375)
    TGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCT
    CCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGAC
    ATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCT
    GAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTT
    CAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAAT
    TCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATA
    CAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCT
    GGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGA
    AATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCT
    TTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAA
  • IFNa2b was linked to the ALBUDAB™ via a TVAAPS (SEQ ID NO: 422) linker region (see WO2007085814). The constructs were cloned by SOE-PCR (single overlap extension according to the method of Horton et al. Gene, 77, p 61 (1989)). PCR amplification of the ALBUDAB™ and IFN sequences were carried out separately using primers with a ˜15 base pair overlap at the TVAAPS (SEQ ID NO: 422) linker region. The primers used are as follows:—
  • IFNα2b SOE fragment 5′
    (SEQ ID NO: 376)
    GCCCGGATCCACCGGCTGTGATCTG
    IFNα2b SOE fragment 3′
    (SEQ ID NO: 377)
    GGAGGATGGAGACTGGGTCATCTGGATGTC
    Vk SOE fragment 5′
    (SEQ ID NO: 378)
    GACATCCAGATGACCCAGTCTCCATCCTCC
    Vk SOE fragment 3′ to also introduce a myc tag
    (SEQ ID NO: 379)
    GCGCAAGCTTTTATTAATTCAGATCCTCTTC
    TGAGATGAGTTTTTGTTCTGCGGCCGCCCGT
    TTGATTTCCACCTTGGTCCC
  • The fragments were purified separately and subsequently assembled in a SOE (single overlap extension PCR extension) reaction using only the flanking primers.
  • IFNα2b SOE fragment 5′
    (SEQ ID NO: 380)
    GCCCGGATCCACCGGCTGTGATCTG
    Vk SOE fragment 3′ to also introduce a myc tag
    (SEQ ID NO: 381)
    GCGCAAGCTTTTATTAATTCAGATCCTCTTC
    TGAGATGAGTTTTTGTTCTGCGGCCGCCCGT
    TTGATTTCCACCTTGGTCCC
  • The assembled PCR product was digested using the restriction enzymes BamHI and HindIII and the gene ligated into the corresponding sites in the pDOM50, a mammalian expression vector which is a pTT5 derivative with an N-terminal V-J2-C mouse IgG secretory leader sequence to facilitate expression into the cell media.
  • Leader sequence (amino acid):
    (SEQ ID NO: 382)
    METDTLLLWVLLLWVPGSTG
    Leader sequence (nucleotide):
    (SEQ ID NO: 383)
    ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGG
    ATCCACCGGGC
  • Plasmid DNA was prepared using QIAfilter megaprep (Qiagen). 1 μg DNA/ml was transfected with 293-Fectin into HEK293E cells and grown in serum free media. The protein is expressed in culture for 5 days and purified from culture supernatant using protein L affinity resin and eluted with 100 mM glycine pH2. The proteins were concentred to greater than 1 mg/ml, buffer exchanged into PBS and endotoxin depleted using Q spin columns (Vivascience).
  • TABLE 11
    Interferon alpha 2b-ALBUDAB ™ sequences with and without myc-tag
    (as amino acid- and nucleotide sequence)
    The Interferon alpha 2b is N-terminal to the ALBUDAB ™ in the following fusions.
    aa + myc nt + myc aa no tag nt no tag
    DMS7321 CDLPQTHSLGSRRT TGCGACTTGCC CDLPQTHSLG TGCGACTTGCC
    (IFNα2b- LMLLAQMRRISLFS ACAGACACAT SRRTLMLLA ACAGACACAT
    DOM7h- CLKDRHDFGFPQE AGTTTGGGATC QMRRISLFSC AGTTTGGGATC
    14) EFGNQFQKAETIPV AAGAAGAACA LKDRHDFGFP AAGAAGAACA
    LHEMIQQIFNLFST TTGATGTTATT QEEFGNQFQ TTGATGTTATT
    KDSSAAWDETLLD AGCACAAATG KAETIPVLHE AGCACAAATG
    KFYTELYQQLNDL CGTAGAATTTC MIQQIFNLFS CGTAGAATTTC
    EACVIQGVGVTETP TTTGTTCTCTT TKDSSAAWD TTTGTTCTCTT
    LMKEDSILAVRKY GTCTAAAGGAC ETLLDKFYTE GTCTAAAGGA
    FQRITLYLKEKKYS CGTCACGACTT LYQQLNDLE CCGTCACGACT
    PCAWEVVRAEIMR CGGATTCCCTC ACVIQGVGV TCGGATTCCCT
    SFSLSTNLQESLRS AGGAAGAGTTT TETPLMKEDS CAGGAAGAGT
    KETVAAPSDIQMT GGAAACCAATT ILAVRKYFQR TTGGAAACCA
    QSPSSLSASVGDRV CCAAAAAGCA ITLYLKEKKY ATTCCAAAAA
    TITCRASQWIGSQL GAAACTATTCC SPCAWEVVR GCAGAAACTA
    SWYQQKPGKAPKL TGTCTTGCACG AEIMRSFSLS TTCCTGTCTTG
    LIMWRSSLQSGVPS AAATGATCCAG TNLQESLRSK CACGAAATGA
    RFSGSGSGTDFTLT CAAATATTCAA ETVAAPSDIQ TCCAGCAAATA
    ISSLQPEDFATYYC TTTGTTTTCTA MTQSPSSLSA TTCAATTTGTT
    AQGAALPRTFGQG CAAAGGACTC SVGDRVTITC TTCTACAAAGG
    TKVEIKR ATCAGCCGCTT RASQWIGSQL  ACTCATCAGCC
    AAAEQKLISEEDL GGGATGAAAC SWYQQKPGK GCTTGGGATGA
    N* (SEQ ID NO: 384) TCTGTTAGATA APKLLIMWR AACTCTGTTAG
    AATTCTACACT SSLQSGVPSR ATAAATTCTAC
    GAACTATATCA FSGSGSGTDF ACTGAACTATA
    ACAACTGAAC TLTISSLQPED TCAACAACTGA
    GATCTAGAGGC FATYYCAQG ACGATCTAGA
    TTGCGTTATTC AALPRTFGQ GGCTTGCGTTA
    AGGGTGTAGG GTKVEIKR TTCAGGGTGTA
    AGTTACTGAAA (SEQ ID GGAGTTACTGA
    CTCCCCTAATG NO: 386) AACTCCCCTAA
    AAAGAAGATT TGAAAGAAGA
    CAATTCTAGCC TTCAATTCTAG
    GTTAGAAAATA CCGTTAGAAA
    CTTTCAGCGTA ATACTTTCAGC
    TCACATTGTAT GTATCACATTG
    TTAAAGGAAA TATTTAAAGGA
    AGAAATACTCC AAAGAAATAC
    CCATGTGCATG TCCCCATGTGC
    GGAGGTGGTTA ATGGGAGGTG
    GAGCAGAAAT GTTAGAGCAG
    TATGAGGTCCT AAATTATGAG
    TCTCTCTTTCT GTCCTTCTCTC
    ACGAATTTGCA TTTCTACGAAT
    AGAATCTTTGA TTGCAAGAATC
    GATCTAAGGA TTTGAGATCTA
    AACCGTCGCTG AGGAAACCGT
    CTCCATCTGAC CGCTGCTCCAT
    ATCCAGATGAC CTGACATCCAG
    CCAGTCTCCAT ATGACCCAGTC
    CCTCCCTGTCT TCCATCCTCCC
    GCATCTGTAGG TGTCTGCATCT
    AGACCGTGTCA GTAGGAGACC
    CCATCACTTGC GTGTCACCATC
    CGGGCAAGTC ACTTGCCGGGC
    AGTGGATTGGG AAGTCAGTGG
    TCTCAGTTATC ATTGGGTCTCA
    TTGGTACCAGC GTTATCTTGGT
    AGAAACCAGG ACCAGCAGAA
    GAAAGCCCCTA ACCAGGGAAA
    AGCTCCTGATC GCCCCTAAGCT
    ATGTGGCGTTC CCTGATCATGT
    CTCGTTGCAAA GGCGTTCCTCG
    GTGGGGTCCCA TTGCAAAGTGG
    TCACGTTTCAG GGTCCCATCAC
    TGGCAGTGGAT GTTTCAGTGGC
    CTGGGACAGAT AGTGGATCTGG
    TTCACTCTCAC GACAGATTTCA
    CATCAGCAGTC CTCTCACCATC
    TGCAACCTGAA AGCAGTCTGCA
    GATTTTGCTAC ACCTGAAGATT
    GTACTACTGTG TTGCTACGTAC
    CTCAGGGTGCG TACTGTGCTCA
    GCGTTGCCTAG GGGTGCGGCG
    GACGTTCGGCC TTGCCTAGGAC
    AAGGGACCAA GTTCGGCCAAG
    GGTGGAAATC GGACCAAGGT
    AAACGGGCGG GGAAATCAAA
    CCGCAGAACA CGG (SEQ ID
    AAAACTCATC NO: 387)
    TCAGAAGAGG
    ATCTGAATTA
    A (SEQ ID
    NO: 385)
    DMS732 CDLPQTHSLGSRRT TGCGACTTGCC CDLPQTHSLG TGCGACTTGCC
    (IFNα2b- LMLLAQMRRISLF SACAGACACAT SRRTLMLLA ACAGACACAT
    DOM7h- CLKDRHDFGFPQE AGTTTGGGATC QMRRISLFSC AGTTTGGGATC
    14-10) EFGNQFQKAETIPV AAGAAGAACA LKDRHDFGFP AAGAAGAACA
    LHEMIQQIFNLFST TTGATGTTATT QEEFGNQFQ TTGATGTTATT
    KDSSAAWDETLLD AGCACAAATG KAETIPVLHE AGCACAAATG
    KFYTELYQQLNDL CGTAGAATTTC MIQQIFNLFS CGTAGAATTTC
    EACVIQGVGVTETP TTTGTTCTCTT TKDSSAAWD TTTGTTCTCTT
    LMKEDSILAVRKY GTCTAAAGGAC ETLLDKFYTE GTCTAAAGGA
    FQRITLYLKEKKYS CGTCACGACTT LYQQLNDLE CCGTCACGACT
    PCAWEVVRAEIMR CGGATTCCCTC ACVIQGVGV TCGGATTCCCT
    SFSLSTNLQESLRS AGGAAGAGTTT TETPLMKEDS CAGGAAGAGT
    KETVAAPSDIQMT GGAAACCAATT ILAVRKYFQR TTGGAAACCA
    QSPSSLSASVGDRV CCAAAAAGCA ITLYLKEKKY ATTCCAAAAA
    TITCRASQWIGSQL GAAACTATTCC SPCAWEVVR GCAGAAACTA
    SWYQQKPGKAPKL TGTCTTGCACG AEIMRSFSLS TTCCTGTCTTG
    LIMWRSSLQSGVPS AAATGATCCAG TNLQESLRSK CACGAAATGA
    RFSGSGSGTDFTLT CAAATATTCAA ETVAAPSDIQ TCCAGCAAATA
    ISSLQPEDFATYYC TTTGTTTTCTA MTQSPSSLSA TTCAATTTGTT
    AQGLRHPKTFGQG CAAAGGACTC SVGDRVTITC TTCTACAAAGG
    TKVEIKR ATCAGCCGCTT RASQWIGSQL ACTCATCAGCC
    AAAEQKLISEEDL GGGATGAAAC SWYQQKPGK GCTTGGGATGA
    N* (SEQ ID NO: 388) TCTGTTAGATA APKLLIMWR AACTCTGTTAG
    AATTCTACACT SSLQSGVPSR ATAAATTCTAC
    GAACTATATCA FSGSGSGTDF ACTGAACTATA
    ACAACTGAAC TLTISSLQPED TCAACAACTGA
    GATCTAGAGGC FATYYCAQG ACGATCTAGA
    TTGCGTTATTC LRHPKTFGQ GGCTTGCGTTA
    AGGGTGTAGG GTKVEIKR TTCAGGGTGTA
    AGTTACTGAAA (SEQ ID GGAGTTACTGA
    CTCCCCTAATG NO: 390) AACTCCCCTAA
    AAAGAAGATT TGAAAGAAGA
    CAATTCTAGCC TTCAATTCTAG
    GTTAGAAAATA CCGTTAGAAA
    CTTTCAGCGTA ATACTTTCAGC
    TCACATTGTAT GTATCACATTG
    TTAAAGGAAA TATTTAAAGGA
    AGAAATACTCC AAAGAAATAC
    CCATGTGCATG TCCCCATGTGC
    GGAGGTGGTTA ATGGGAGGTG
    GAGCAGAAAT GTTAGAGCAG
    TATGAGGTCCT AAATTATGAG
    TCTCTCTTTCT GTCCTTCTCTC
    ACGAATTTGCA TTTCTACGAAT
    AGAATCTTTGA TTGCAAGAATC
    GATCTAAGGA TTTGAGATCTA
    AACCGTCGCTG AGGAAACCGT
    CTCCATCTGAC CGCTGCTCCAT
    ATCCAGATGAC CTGACATCCAG
    CCAGTCTCCAT ATGACCCAGTC
    CCTCCCTGTCT TCCATCCTCCC
    GCATCTGTAGG TGTCTGCATCT
    AGACCGTGTCA GTAGGAGACC
    CCATCACTTGC GTGTCACCATC
    CGGGCAAGTC ACTTGCCGGGC
    AGTGGATTGGG AAGTCAGTGG
    TCTCAGTTATC ATTGGGTCTCA
    TTGGTACCAGC GTTATCTTGGT
    AGAAACCAGG ACCAGCAGAA
    GAAAGCCCCTA ACCAGGGAAA
    AGCTCCTGATC GCCCCTAAGCT
    ATGTGGCGTTC CCTGATCATGT
    CTCGTTGCAAA GGCGTTCCTCG
    GTGGGGTCCCA TTGCAAAGTGG
    TCACGTTTCAG GGTCCCATCAC
    TGGCAGTGGAT GTTTCAGTGGC
    CTGGGACAGAT AGTGGATCTGG
    TTCACTCTCAC GACAGATTTCA
    CATCAGCAGTC CTCTCACCATC
    TGCAACCTGAA AGCAGTCTGCA
    GATTTTGCTAC ACCTGAAGATT
    GTACTACTGTG TTGCTACGTAC
    CTCAGGGTTTG TACTGTGCTCA
    AGGCATCCTAA GGGTTTGAGGC
    GACGTTCGGCC ATCCTAAGACG
    AAGGGACCAA TTCGGCCAAGG
    GGTGGAAATC GACCAAGGTG
    AAACGGGCGG GAAATCAAAC
    CCGCAGAACA GG (SEQ ID
    AAAACTCATC NO: 391)
    TCAGAAGAGG
    ATCTGAATTA
    A (SEQ ID
    NO: 389)
    DMS7323 CDLPQTHSLGSRRT TGCGACTTGCC CDLPQTHSLG TGCGACTTGCC
    (IFNα2b- LMLLAQMRRISLF SACAGACACAT SRRTLMLLA ACAGACACAT
    DOM7h- CLKDRHDFGFPQE AGTTTGGGATC QMRRISLFSC AGTTTGGGATC
    14-18) EFGNQFQKAETIPV AAGAAGAACA LKDRHDFGFP AAGAAGAACA
    LHEMIQQIFNLFST TTGATGTTATT QEEFGNQFQ TTGATGTTATT
    KDSSAAWDETLLD AGCACAAATG KAETIPVLHE AGCACAAATG
    KFYTELYQQLNDL CGTAGAATTTC MIQQIFNLFS CGTAGAATTTC
    EACVIQGVGVTETP TTTGTTCTCTT TKDSSAAWD TTTGTTCTCTT
    LMKEDSILAVRKY GTCTAAAGGAC ETLLDKFYTE GTCTAAAGGA
    FQRITLYLKEKKYS CGTCACGACTT LYQQLNDLE CCGTCACGACT
    PCAWEVVRAEIMR CGGATTCCCTC ACVIQGVGV TCGGATTCCCT
    SFSLSTNLQESLRS AGGAAGAGTTT TETPLMKEDS CAGGAAGAGT
    KETVAAPSDIQMT GGAAACCAATT ILAVRKYFQR TTGGAAACCA
    QSPSSLSASVGDRV CCAAAAAGCA ITLYLKEKKY ATTCCAAAAA
    TITCRASQWIGSQL GAAACTATTCC SPCAWEVVR GCAGAAACTA
    SWYQQKPGKAPKL TGTCTTGCACG AEIMRSFSLS TTCCTGTCTTG
    LIMWRSSLQSGVPS AAATGATCCAG TNLQESLRSK CACGAAATGA
    RFSGSGSGTDFTLT CAAATATTCAA ETVAAPSDIQ TCCAGCAAATA
    ISSLQPEDFATYYC TTTGTTTTCTA MTQSPSSLSA TTCAATTTGTT
    AQGLMKPMTFGQ CAAAGGACTC SVGDRVTITC TTCTACAAAGG
    GTKVEIKRAAAEQ ATCAGCCGCTT RASQWIGSQL ACTCATCAGCC
    KLISEEDLN* (SEQ GGGATGAAAC SWYQQKPGK GCTTGGGATGA
    ID NO: 392) TCTGTTAGATA APKLLIMWR AACTCTGTTAG
    AATTCTACACT SSLQSGVPSR ATAAATTCTAC
    GAACTATATCA FSGSGSGTDF ACTGAACTATA
    ACAACTGAAC TLTISSLQPED TCAACAACTGA
    GATCTAGAGGC FATYYCAQG ACGATCTAGA
    TTGCGTTATTC LMKPMTFGQ GGCTTGCGTTA
    AGGGTGTAGG GTKVEIKR TTCAGGGTGTA
    AGTTACTGAAA (SEQ ID GGAGTTACTGA
    CTCCCCTAATG NO: 394) AACTCCCCTAA
    AAAGAAGATT TGAAAGAAGA
    CAATTCTAGCC TTCAATTCTAG
    GTTAGAAAATA CCGTTAGAAA
    CTTTCAGCGTA ATACTTTCAGC
    TCACATTGTAT GTATCACATTG
    TTAAAGGAAA TATTTAAAGGA
    AGAAATACTCC AAAGAAATAC
    CCATGTGCATG TCCCCATGTGC
    GGAGGTGGTTA ATGGGAGGTG
    GAGCAGAAAT GTTAGAGCAG
    TATGAGGTCCT AAATTATGAG
    TCTCTCTTTCT GTCCTTCTCTC
    ACGAATTTGCA TTTCTACGAAT
    AGAATCTTTGA TTGCAAGAATC
    GATCTAAGGA TTTGAGATCTA
    AACCGTCGCTG AGGAAACCGT
    CTCCATCTGAC CGCTGCTCCAT
    ATCCAGATGAC CTGACATCCAG
    CCAGTCTCCAT ATGACCCAGTC
    CCTCCCTGTCT TCCATCCTCCC
    GCATCTGTAGG TGTCTGCATCT
    AGACCGTGTCA GTAGGAGACC
    CCATCACTTGC GTGTCACCATC
    CGGGCAAGTC ACTTGCCGGGC
    AGTGGATTGGG AAGTCAGTGG
    TCTCAGTTATC ATTGGGTCTCA
    TTGGTACCAGC GTTATCTTGGT
    AGAAACCAGG ACCAGCAGAA
    GAAAGCCCCTA ACCAGGGAAA
    AGCTCCTGATC GCCCCTAAGCT
    ATGTGGCGTTC CCTGATCATGT
    CTCGTTGCAAA GGCGTTCCTCG
    GTGGGGTCCCA TTGCAAAGTGG
    TCACGTTTCAG GGTCCCATCAC
    TGGCAGTGGAT GTTTCAGTGGC
    CTGGGACAGAT AGTGGATCTGG
    TTCACTCTCAC GACAGATTTCA
    CATCAGCAGTC CTCTCACCATC
    TGCAACCTGAA AGCAGTCTGCA
    GATTTTGCTAC ACCTGAAGATT
    GTACTACTGTG TTGCTACGTAC
    CTCAGGGTCTT TACTGTGCTCA
    ATGAAGCCTAT GGGTCTTATGA
    GACGTTCGGCC AGCCTATGACG
    AAGGGACCAA TTCGGCCAAGG
    GGTGGAAATC GACCAAGGTG
    AAACGGGCGG GAAATCAAAC
    CCGCAGAACA GG (SEQ ID
    AAAACTCATC NO: 395)
    TCAGAAGAGG
    ATCTGAATTA
    A (SEQ ID
    NO: 393)
    DMS7324 CDLPQTHSLGSRRT TGCGACTTGCC CDLPQTHSLG TGCGACTTGCC
    (IFNα2b- LMLLAQMRRISLF SACAGACACAT SRRTLMLLA ACAGACACAT
    DOM7h- CLKDRHDFGFPQE AGTTTGGGATC QMRRISLFSC AGTTTGGGATC
    14-19) EFGNQFQKAETIPV AAGAAGAACA LKDRHDFGFP AAGAAGAACA
    LHEMIQQIFNLF STTTGATGTTATT QEEFGNQFQ TTGATGTTATT
    KDSSAAWDETLLD AGCACAAATG KAETIPVLHE AGCACAAATG
    KFYTELYQQLNDL CGTAGAATTTC MIQQIFNLFS CGTAGAATTTC
    EACVIQGVGVTETP TTTGTTCTCTT TKDSSAAWD TTTGTTCTCTT
    LMKEDSILAVRKY GTCTAAAGGAC ETLLDKFYTE GTCTAAAGGA
    FQRITLYLKEKKYS CGTCACGACTT LYQQLNDLE CCGTCACGACT
    PCAWEVVRAEIMR CGGATTCCCTC ACVIQGVGV TCGGATTCCCT
    SFSLSTNLQESLRS AGGAAGAGTTT TETPLMKEDS CAGGAAGAGT
    KETVAAPSDIQMT GGAAACCAATT ILAVRKYFQR TTGGAAACCA
    QSPSSLSASVGDRV CCAAAAAGCA ITLYLKEKKY ATTCCAAAAA
    TISCRASQWIGSQL GAAACTATTCC SPCAWEVVR GCAGAAACTA
    SWYQQKPGEAPKL TGTCTTGCACG AEIMRSFSLS TTCCTGTCTTG
    LIMWRSSLQSGVPS AAATGATCCAG TNLQESLRSK CACGAAATGA
    RFSGSGSGTDFTLT CAAATATTCAA ETVAAPSDIQ TCCAGCAAATA
    ISSLQPEDFATYYC TTTGTTTTCTA MTQSPSSLSA TTCAATTTGTT
    AQGAALPRTFGQG CAAAGGACTC SVGDRVTISC TTCTACAAAGG
    TKVEIKR ATCAGCCGCTT RASQWIGSQL ACTCATCAGCC
    AAAEQKLISEEDL GGGATGAAAC SWYQQKPGE GCTTGGGATGA
    N* (SEQ ID NO: 396) TCTGTTAGATA APKLLIMWR AACTCTGTTAG
    AATTCTACACT SSLQSGVPSR ATAAATTCTAC
    GAACTATATCA FSGSGSGTDF ACTGAACTATA
    ACAACTGAAC TLTISSLQPED TCAACAACTGA
    GATCTAGAGGC FATYYCAQG ACGATCTAGA
    TTGCGTTATTC AALPRTFGQ GGCTTGCGTTA
    AGGGTGTAGG GTKVEIKR TTCAGGGTGTA
    AGTTACTGAAA (SEQ ID GGAGTTACTGA
    CTCCCCTAATG NO: 398) AACTCCCCTAA
    AAAGAAGATT TGAAAGAAGA
    CAATTCTAGCC TTCAATTCTAG
    GTTAGAAAATA CCGTTAGAAA
    CTTTCAGCGTA ATACTTTCAGC
    TCACATTGTAT GTATCACATTG
    TTAAAGGAAA TATTTAAAGGA
    AGAAATACTCC AAAGAAATAC
    CCATGTGCATG TCCCCATGTGC
    GGAGGTGGTTA ATGGGAGGTG
    GAGCAGAAAT GTTAGAGCAG
    TATGAGGTCCT AAATTATGAG
    TCTCTCTTTCT GTCCTTCTCTC
    ACGAATTTGCA TTTCTACGAAT
    AGAATCTTTGA TTGCAAGAATC
    GATCTAAGGA TTTGAGATCTA
    AACCGTCGCTG AGGAAACCGT
    CTCCATCTGAC CGCTGCTCCAT
    ATCCAGATGAC CTGACATCCAG
    CCAGTcTCCAT ATGACCCAGTc
    CCTCCCTGTCT TCCATCCTCCC
    GCATCTGTAGG TGTCTGCATCT
    AGACCGTGTCA GTAGGAGACC
    CCATCTCTTGC GTGTCACCATC
    CGGGCAAGTC TCTTGCCGGGC
    AGTGGATTGGG AAGTCAGTGG
    TCTCAGTTATC ATTGGGTCTCA
    TTGGTACCAGC GTTATCTTGGT
    AGAAACCAGG ACCAGCAGAA
    GGAAGCCCCTA ACCAGGGGAA
    AGCTCCTGATC GCCCCTAAGCT
    ATGTGGCGTTC CCTGATCATGT
    CTCGTTGCAAA GGCGTTCCTCG
    GTGGGGTCCCA TTGCAAAGTGG
    TCACGTTTCAG GGTCCCATCAC
    TGGCAGTGGAT GTTTCAGTGGC
    CTGGGACAGAT AGTGGATCTGG
    TTCACTCTCAC GACAGATTTCA
    CATCAGCAGTC CTCTCACCATC
    TGCAACCTGAA AGCAGTCTGCA
    GATTTTGCTAC ACCTGAAGATT
    GTACTACTGTG TTGCTACGTAC
    CTCAGGGTGCG TACTGTGCTCA
    GCGTTGCCTAG GGGTGCGGCG
    GACGTTCGGCC TTGCCTAGGAC
    AAGGGACCAA GTTCGGCCAAG
    GGTGGAAATC GGACCAAGGT
    AAACGGGCGG GGAAATCAAA
    CCGCAGAACA CGG (SEQ ID
    AAAACTCATC NO: 399)
    TCAGAAGAGG
    ATCTGAATTA
    A (SEQ ID
    NO: 397)
    DMS7325 CDLPQTHSLGSRRT TGCGACTTGCC CDLPQTHSLG TGCGACTTGCC
    (IFNα2b- LMLLAQMRRISLFS ACAGACACAT SRRTLMLLA ACAGACACAT
    DOM7h- CLKDRHDFGFPQE AGTTTGGGATC QMRRISLFSC AGTTTGGGATC
    11) EFGNQFQKAETIPV AAGAAGAACA LKDRHDFGFP AAGAAGAACA
    LHEMIQQIFNLFST TTGATGTTATT QEEFGNQFQ TTGATGTTATT
    KDSSAAWDETLLD AGCACAAATG KAETIPVLHE AGCACAAATG
    KFYTELYQQLNDL CGTAGAATTTC MIQQIFNLFS CGTAGAATTTC
    EACVIQGVGVTETP TTTGTTCTCTT TKDSSAAWD TTTGTTCTCTT
    LMKEDSILAVRKY GTCTAAAGGAC ETLLDKFYTE GTCTAAAGGA
    FQRITLYLKEKKYS CGTCACGACTT LYQQLNDLE CCGTCACGACT
    PCAWEVVRAEIMR CGGATTCCCTC ACVIQGVGV TCGGATTCCCT
    SFSLSTNLQESLRS AGGAAGAGTTT TETPLMKEDS CAGGAAGAGT
    KETVAAPSDIQMT GGAAACCAATT ILAVRKYFQR TTGGAAACCA
    QSPSSLSASVGDRV CCAAAAAGCA ITLYLKEKKY ATTCCAAAAA
    TITCRASRPIGTTLS GAAACTATTCC SPCAWEVVR GCAGAAACTA
    WYQQKPGKAPKLL TGTCTTGCACG AEIMRSFSLS TTCCTGTCTTG
    IWFGSRLQSGVPSR AAATGATCCAG TNLQESLRSK CACGAAATGA
    FSGSGSGTDFTLTIS CAAATATTCAA ETVAAPSDIQ TCCAGCAAATA
    SLQPEDFATYYCA TTTGTTTTCTA MTQSPSSLSA TTCAATTTGTT
    QAGTHPTTFGQGT CAAAGGACTC SVGDRVTITC TTCTACAAAGG
    KVEIKR ATCAGCCGCTT RASRPIGTTL ACTCATCAGCC
    AAAEQKLISEEDL GGGATGAAAC SWYQQKPGK GCTTGGGATGA
    N* (SEQ ID NO: 400) TCTGTTAGATA APKLLIWFGS AACTCTGTTAG
    AATTCTACACT RLQSGVPSRF ATAAATTCTAC
    GAACTATATCA SGSGSGTDFT ACTGAACTATA
    ACAACTGAAC LTISSLQPEDF TCAACAACTGA
    GATCTAGAGGC ATYYCAQAG ACGATCTAGA
    TTGCGTTATTC THPTTFGQGT GGCTTGCGTTA
    AGGGTGTAGG KVEIKR (SEQ TTCAGGGTGTA
    AGTTACTGAAA ID NO: 402) GGAGTTACTGA
    CTCCCCTAATG AACTCCCCTAA
    AAAGAAGATT TGAAAGAAGA
    CAATTCTAGCC TTCAATTCTAG
    GTTAGAAAATA CCGTTAGAAA
    CTTTCAGCGTA ATACTTTCAGC
    TCACATTGTAT GTATCACATTG
    TTAAAGGAAA TATTTAAAGGA
    AGAAATACTCC AAAGAAATAC
    CCATGTGCATG TCCCCATGTGC
    GGAGGTGGTTA ATGGGAGGTG
    GAGCAGAAAT GTTAGAGCAG
    TATGAGGTCCT AAATTATGAG
    TCTCTCTTTCT GTCCTTCTCTC
    ACGAATTTGCA TTTCTACGAAT
    AGAATCTTTGA TTGCAAGAATC
    GATCTAAGGA TTTGAGATCTA
    AACCGTCGCTG AGGAAACCGT
    CTCCATCTGAC CGCTGCTCCAT
    ATCCAGATGAC CTGACATCCAG
    CCAGTCTCCAT ATGACCCAGTC
    CCTCCCTGTCT TCCATCCTCCC
    GCATCTGTAGG TGTCTGCATCT
    AGACCGTGTCA GTAGGAGACC
    CCATCACTTGC GTGTCACCATC
    CGGGCAAGTC ACTTGCCGGGC
    GTCCGATTGGG AAGTCGTCCGA
    ACGACGTTAAG TTGGGACGAC
    TTGGTACCAGC GTTAAGTTGGT
    AGAAACCAGG ACCAGCAGAA
    GAAAGCCCCTA ACCAGGGAAA
    AGCTCCTGATC GCCCCTAAGCT
    TGGTTTGGTTC CCTGATCTGGT
    CCGGTTGCAAA TTGGTTCCCGG
    GTGGGGTCCCA TTGCAAAGTGG
    TCACGTTTCAG GGTCCCATCAC
    TGGCAGTGGAT GTTTCAGTGGC
    CTGGGACAGAT AGTGGATCTGG
    TTCACTCTCAC GACAGATTTCA
    CATCAGCAGTC CTCTCACCATC
    TGCAACCTGAA AGCAGTCTGCA
    GATTTTGCTAC ACCTGAAGATT
    GTACTACTGTG TTGCTACGTAC
    CGCAGGCTGG TACTGTGCGCA
    GACGCATCCTA GGCTGGGACG
    CGACGTTCGGC CATCCTACGAC
    CAAGGGACCA GTTCGGCCAAG
    AGGTGGAAAT GGACCAAGGT
    CAAACGGGCG GGAAATCAAA
    GCCGCAGAAC CGG (SEQ ID
    AAAAACTCAT NO: 403)
    CTCAGAAGAG
    GATCTGAATT
    AA (SEQ ID
    NO: 401)
    DMS7326 CDLPQTHSLGSRRT TGCGACTTGCC CDLPQTHSLG TGCGACTTGCC
    (IFNα2b- LMLLAQMRRISLFS ACAGACACAT SRRTLMLLA ACAGACACAT
    DOM7h- CLKDRHDFGFPQE AGTTTGGGATC QMRRISLFSC AGTTTGGGATC
    11-12) EFGNQFQKAETIPV AAGAAGAACA LKDRHDFGFP AAGAAGAACA
    LHEMIQQIFNLF STTTGATGTTATT QEEFGNQFQ TTGATGTTATT
    KDSSAAWDETLLD AGCACAAATG KAETIPVLHE AGCACAAATG
    KFYTELYQQLNDL CGTAGAATTTC MIQQIFNLFS CGTAGAATTTC
    EACVIQGVGVTETP TTTGTTCTCTT TKDSSAAWD TTTGTTCTCTT
    LMKEDSILAVRKY GTCTAAAGGAC ETLLDKFYTE GTCTAAAGGA
    FQRITLYLKEKKYS CGTCACGACTT LYQQLNDLE CCGTCACGACT
    PCAWEVVRAEIMR CGGATTCCCTC ACVIQGVGV TCGGATTCCCT
    SFSLSTNLQESLRS AGGAAGAGTTT TETPLMKEDS CAGGAAGAGT
    KETVAAPSDIQMT GGAAACCAATT ILAVRKYFQR TTGGAAACCA
    QSPSSLSASVGDRV CCAAAAAGCA ITLYLKEKKY ATTCCAAAAA
    TITCRASRPIGTML GAAACTATTCC SPCAWEVVR GCAGAAACTA
    SWYQQKPGKAPKL TGTCTTGCACG AEIMRSFSLS TTCCTGTCTTG
    LILFGSRLQSGVPS AAATGATCCAG TNLQESLRSK CACGAAATGA
    RFSGSGSGTDFTLT CAAATATTCAA ETVAAPSDIQ TCCAGCAAATA
    ISSLQPEDFATYYC TTTGTTTTCTA MTQSPSSLSA TTCAATTTGTT
    AQAGTHPTTFGQG CAAAGGACTC SVGDRVTITC TTCTACAAAGG
    TKVEIKR ATCAGCCGCTT RASRPIGTML ACTCATCAGCC
    AAAEQKLISEEDL GGGATGAAAC SWYQQKPGK GCTTGGGATGA
    N* (SEQ ID NO: 404) TCTGTTAGATA APKLLILFGS AACTCTGTTAG
    AATTCTACACT RLQSGVPSRF ATAAATTCTAC
    GAACTATATCA SGSGSGTDFT ACTGAACTATA
    ACAACTGAAC LTISSLQPEDF TCAACAACTGA
    GATCTAGAGGC ATYYCAQAG ACGATCTAGA
    TTGCGTTATTC THPTTFGQGT GGCTTGCGTTA
    AGGGTGTAGG KVEIKR (SEQ TTCAGGGTGTA
    AGTTACTGAAA ID NO: 406) GGAGTTACTGA
    CTCCCCTAATG AACTCCCCTAA
    AAAGAAGATT TGAAAGAAGA
    CAATTCTAGCC TTCAATTCTAG
    GTTAGAAAATA CCGTTAGAAA
    CTTTCAGCGTA ATACTTTCAGC
    TCACATTGTAT GTATCACATTG
    TTAAAGGAAA TATTTAAAGGA
    AGAAATACTCC AAAGAAATAC
    CCATGTGCATG TCCCCATGTGC
    GGAGGTGGTTA ATGGGAGGTG
    GAGCAGAAAT GTTAGAGCAG
    TATGAGGTCCT AAATTATGAG
    TCTCTCTTTCT GTCCTTCTCTC
    ACGAATTTGCA TTTCTACGAAT
    AGAATCTTTGA TTGCAAGAATC
    GATCTAAGGA TTTGAGATCTA
    AACCGTCGCTG AGGAAACCGT
    CTCCATCTGAC CGCTGCTCCAT
    ATCCAGATGAC CTGACATCCAG
    CCAGTCTCCAT ATGACCCAGTC
    CCTCCCTGTCT TCCATCCTCCC
    GCATCTGTAGG TGTCTGCATCT
    AGACCGTGTCA GTAGGAGACC
    CCATCACTTGC GTGTCACCATC
    CGGGCAAGTC ACTTGCCGGGC
    GTCCGATTGGG AAGTCGTCCGA
    ACGATGTTAAG TTGGGACGATG
    TTGGTACCAGC TTAAGTTGGTA
    AGAAACCAGG CCAGCAGAAA
    GAAAGCCCCTA CCAGGGAAAG
    AGCTCCTGATC CCCCTAAGCTC
    TTGTTTGGTTC CTGATCTTGTT
    CCGGTTGCAAA TGGTTCCCGGT
    GTGGGGTCCCA TGCAAAGTGG
    TCACGTTTCAG GGTCCCATCAC
    TGGCAGTGGAT GTTTCAGTGGC
    CTGGGACAGAT AGTGGATCTGG
    TTCACTCTCAC GACAGATTTCA
    CATCAGCAGTC CTCTCACCATC
    TGCAACCTGAA AGCAGTCTGCA
    GATTTTGCTAC ACCTGAAGATT
    GTACTACTGTG TTGCTACGTAC
    CGCAGGCTGG TACTGTGCGCA
    GACGCATCCTA GGCTGGGACG
    CGACGTTCGGC CATCCTACGAC
    CAAGGGACCA GTTCGGCCAAG
    AGGTGGAAAT GGACCAAGGT
    CAAACGGGCG GGAAATCAAA
    GCCGCAGAAC CGG (SEQ ID
    AAAAACTCAT NO: 407)
    CTCAGAAGAG
    GATCTGAATT
    AA (SEQ ID
    NO: 405
    DMS7327 CDLPQTHSLGSRRT TGCGACTTGCC CDLPQTHSLG TGCGACTTGCC
    (IFNα2b- LMLLAQMRRISLFS ACAGACACAT SRRTLMLLA ACAGACACAT
    DOM7h- CLKDRHDFGFPQE AGTTTGGGATC QMRRISLFSC AGTTTGGGATC
    11-15) EFGNQFQKAETIPV AAGAAGAACA LKDRHDFGFP AAGAAGAACA
    LHEMIQQIFNLF STTTGATGTTATT QEEFGNQFQ TTGATGTTATT
    KDSSAAWDETLLD AGCACAAATG KAETIPVLHE AGCACAAATG
    KFYTELYQQLNDL CGTAGAATTTC MIQQIFNLFS CGTAGAATTTC
    EACVIQGVGVTETP TTTGTTCTCTT TKDSSAAWD TTTGTTCTCTT
    LMKEDSILAVRKY GTCTAAAGGAC ETLLDKFYTE GTCTAAAGGA
    FQRITLYLKEKKYS CGTCACGACTT LYQQLNDLE CCGTCACGACT
    PCAWEVVRAEIMR CGGATTCCCTC ACVIQGVGV TCGGATTCCCT
    SFSLSTNLQESLRS AGGAAGAGTTT TETPLMKEDS CAGGAAGAGT
    KETVAAPSDIQMT GGAAACCAATT ILAVRKYFQR TTGGAAACCA
    QSPSSLSASVGDRV CCAAAAAGCA ITLYLKEKKY ATTCCAAAAA
    TITCRASRPIGTML GAAACTATTCC SPCAWEVVR GCAGAAACTA
    SWYQQKPGKAPKL TGTCTTGCACG AEIMRSFSLS TTCCTGTCTTG
    LILAFSRLQSGVPS AAATGATCCAG TNLQESLRSK CACGAAATGA
    RFSGSGSGTDFTLT CAAATATTCAA ETVAAPSDIQ TCCAGCAAATA
    ISSLQPEDFATYYC TTTGTTTTCTA MTQSPSSLSA TTCAATTTGTT
    AQAGTHPTTFGQG CAAAGGACTC SVGDRVTITC TTCTACAAAGG
    TKVEIKR ATCAGCCGCTT RASRPIGTML ACTCATCAGCC
    AAAEQKLISEEDL GGGATGAAAC SWYQQKPGK GCTTGGGATGA
    N* (SEQ ID NO: 408) TCTGTTAGATA APKLLILAFS AACTCTGTTAG
    AATTCTACACT RLQSGVPSRF ATAAATTCTAC
    GAACTATATCA SGSGSGTDFT ACTGAACTATA
    ACAACTGAAC LTISSLQPEDF TCAACAACTGA
    GATCTAGAGGC ATYYCAQAG ACGATCTAGA
    TTGCGTTATTC THPTTFGQGT GGCTTGCGTTA
    AGGGTGTAGG KVEIKR (SEQ TTCAGGGTGTA
    AGTTACTGAAA ID NO: 410) GGAGTTACTGA
    CTCCCCTAATG AACTCCCCTAA
    AAAGAAGATT TGAAAGAAGA
    CAATTCTAGCC TTCAATTCTAG
    GTTAGAAAATA CCGTTAGAAA
    CTTTCAGCGTA ATACTTTCAGC
    TCACATTGTAT GTATCACATTG
    TTAAAGGAAA TATTTAAAGGA
    AGAAATACTCC AAAGAAATAC
    CCATGTGCATG TCCCCATGTGC
    GGAGGTGGTTA ATGGGAGGTG
    GAGCAGAAAT GTTAGAGCAG
    TATGAGGTCCT AAATTATGAG
    TCTCTCTTTCT GTCCTTCTCTC
    ACGAATTTGCA TTTCTACGAAT
    AGAATCTTTGA TTGCAAGAATC
    GATCTAAGGA TTTGAGATCTA
    AACCGTCGCTG AGGAAACCGT
    CTCCATCTGAC CGCTGCTCCAT
    ATCCAGATGAC CTGACATCCAG
    CCAGTCTCCAT ATGACCCAGTC
    CCTCCCTGTCT TCCATCCTCCC
    GCATCTGTAGG TGTCTGCATCT
    AGACCGTGTCA GTAGGAGACC
    CCATCACTTGC GTGTCACCATC
    CGGGCAAGTC ACTTGCCGGGC
    GTCCGATTGGG AAGTCGTCCGA
    ACGATGTTAAG TTGGGACGATG
    TTGGTACCAGC TTAAGTTGGTA
    AGAAACCAGG CCAGCAGAAA
    GAAAGCCCCTA CCAGGGAAAG
    AGCTCCTGATC CCCCTAAGCTC
    CTTGCTTTTTC CTGATCCTTGC
    CCGTTTGCAAA TTTTTCCCGTT
    GTGGGGTCCCA TGCAAAGTGG
    TCACGTTTCAG GGTCCCATCAC
    TGGCAGTGGAT GTTTCAGTGGC
    CTGGGACAGAT AGTGGATCTGG
    TTCACTCTCAC GACAGATTTCA
    CATCAGCAGTC CTCTCACCATC
    TGCAACCTGAA AGCAGTCTGCA
    GATTTTGCTAC ACCTGAAGATT
    GTACTACTGCG TTGCTACGTAC
    CGCAGGCTGG TACTGCGCGCA
    GACGCATCCTA GGCTGGGACG
    CGACGTTCGGC CATCCTACGAC
    CAAGGGACCA GTTCGGCCAAG
    AGGTGGAAAT GGACCAAGGT
    CAAACGGGCG GGAAATCAAA
    GCCGCAGAAC CGG (SEQ ID
    AAAAACTCAT NO: 411)
    CTCAGAAGAG
    GATCTGAATT
    AA (SEQ ID
    NO: 409)
    The amino acid and nucleotide sequences highlighted in bold represents the cloning site and MYC tag. * represents the stop codon at the end of the gene.
    • Affinity Determination and Biophysical Characterisation:
  • To determine the binding affinity (KD) of the ALBUDAB™-IFNα2b fusion proteins to each serum albumin; purified fusion proteins were analysed by BiaCore™ over albumin (immobilised by primary-amine coupling onto CM5 chips; BiaCore™) using fusion protein concentrations from 5000 nM to 39 nM (5000 nM, 2500 nM, 1250 nM, 625 nM, 312 nM, 156 nM, 78 nM, 39 nM) in HBS-EP BiaCore™ buffer.
  • TABLE 12
    Affinity to SA
    Affinity to
    ALBUDAB ™ Fusion SA (nM) Kd Ka
    Rat
    DOM7h-14 IFNα2b 350 4.500E−02  1.28E+05
    DOM7h-14-10 IFNα2b 16 4.970E−03  5.90E+05
    DOM 7h-14-18 IFNα2b 780 2.127E−01  5.80E+05
    DOM 7h-14-19 IFNα2b 1900 1.206E−01  7.96E+04
    DOM 7h-11 IFNα2b 6000 7.500E−01  nd
    DOM 7h-11-12 IFNα2b 1700 3.100E−01  1.30E+05
    DOM 7h-11-15 IFNα2b 200 1.660E−02  1.50E+05
    Cyno
    DOM 7h-14 IFNα2b 60 1.32E−02  5.0E+05
    DOM 7h-14-10 IFNα2b 19 7.05E−03 4.50E+05
    DOM 7h-14-18 IFNα2b no binding no binding no binding
    DOM 7h-14-19 IFNα2b 520 8.47E−02 2.73E+05
    DOM 7h-11 IFNα2b 3300 3.59E−01 1.20E+05
    DOM 7h-11-12 IFNα2b 630 3.45E−01 7.00E+05
    DOM 7h-11-15 IFNα2b 15 4.86E−03 3.60E+05
    Mouse
    DOM 7h-14 IFNα2b 240 3.21E−02 1.50E+06
    DOM 7h-14-10 IFNα2b 60 3.45E−02 6.86E+05
    DOM 7h-14-18 IFNα2b 180 1.50E−01 9.84E+05
    DOM 7h-14-19 IFNα2b 490 4.03E−02 1.19E+05
    DOM 7h-11 IFNα2b 6000 1.55E−01 nd
    DOM 7h-11-12 IFNα2b 150 9.49E−02 6.30E+05
    DOM 7h-11-15 IFNα2b 28 6.69E−03 2.80E+05
    Human
    DOM 7h-14 IFNα2b 244 2.21E−02 9.89E+04
    DOM 7h-14-10 IFNα2b 32 6.58E−03 3.48E+05
    DOM 7h-14-18 IFNα2b 470 2.75E−01 6.15E+05
    DOM 7h-14-19 IFNα2b 350 4.19E−02 1.55E+05
    DOM 7h-11 IFNα2b 670 2.02E−01 7.00E+05
    DOM 7h-11-12 IFNα2b 500 1.66E−01 3.90E+05
    DOM 7h-11-15 IFNα2b 10 1.87E−03 3.50E+05
    When IFNα2b is linked to the ALBUDAB ™ variants, in all cases the affinity of ALBUDAB ™ binding to serum albumin is reduced. DOM7h-14-10 and DOM7-11-15 retain improved binding affinity to serum albumin across species compared to parent. DOM7h-11-12 also shows improved binding affinity to serum albumin across species compared to parent.
  • TABLE 13
    Biophysical Characterisation
    Biophysical Characterisation was carried out by SEC
    MALLS and DSC as described above for the single ALBUDABs ™
    Biophysical parameters
    DMS SEC DSC
    ALBUDAB ™ Fusion number MALLS Tm(° C.)
    DOM 7h-14 IFNα2b DMS7321 M/D 58-65
    DOM 7h-14-10 IFNα2b DMS7322 M/D 55-65
    DOM 7h-14-18 IFNα2b DMS7323 M/D 55-65
    DOM 7h-14-19 IFNα2b DMS7324 M/D 59-66
    DOM 7h-11 IFNα2b DMS7325 M/D 65.8-66.2
    DOM 7h-11-12 IFNα2b DMS7326 M/D  67-67.3
    DOM 7h-11-15 IFNα2b DMS7327 M/D 56.3-66.2
    M/D indicates a monomer/dimer equilibrium as detected by SEC MALLS
    We observed expression for all clones in Tabale 13 in the range of 17.5 to 54 mg/L in HEK293.
    For IFNα2b-DOM7h-14 and IFNα2b-DOM7h-11 variants, favorable biophysical parameters and expression levels were maintained during affinity maturation.
    • PK Determination for ALBUDAB™-IFNα2b Fusions
  • ALBUDABs™ IFNα2b fusions DMS7321 (IFNα2b-DOM7h-14) DMS7322 (IFNα2b-DOM7h-14-10) DMS7323 (IFNα2b-DOM7h-14-18), DMS7324 (IFNα2b-DOM7h-14-19), DMS7325 (IFNα2b-DOM7h-11), DMS7326 (IFNα2b-DOM7h-11-12), DMS7327 (IFNα2b-DOM7h-11-15) were expressed with the myc tag at 20-50 mg quantities in HEK293 cells and purified from culture supernatant using protein L affinity resin and eluted with 100 mM glycine pH2. The proteins were concentrated to greater than 1 mg/ml, buffer exchanged into Dulbecco's PBS and endotoxin depleted using Q spin columns (Vivascience).
  • For Rat PK, IFN-ALBUDABs™ were dosed as single i.v injections at 2.0 mg/kg using 3 rats per compound. Serum samples were taken at 0.16, 1, 4, 8, 24, 48, 72, 120, 168 hrs. Analysis of serum levels was by EASY ELISA according to manufacturers instructions (GE Healthcare, catalogue number RPN5960).
  • For Mouse PK, DMS7322 (IFN2b-DOM7h-14-10) DMS7325 (IFN2b-DOM7h-11), DMS7326 (IFN2b-DOM7h-11-12), DMS7327 (IFN2b-DOM7h-11-15) all with myc tags were dosed as single i.v injections at 2.0 mg/kg per dose group of 3 subjects and serum samples taken at 10 mins; 1 h; 8h; 24h; 48h; 72h; 96h. Analysis of serum levels was by EASY ELISA according to manufacturers instructions (GE Healthcare, catalogue number RPN5960).
  • TABLE 14
    PK parameters
    Albumin AUC CL t½ Vz
    Species ALBUDAB ™ Fusion KD (nM) h × ug/ml ml/h/kg h ml/kg
    Rat 7h-14 IFNα2b 350 832.1 2.4 27 94.5
    7h-14-10 IFNα2b 16 1380.7 1.5 35.8 75.2
    7h-14-18 IFNα2b 780 691.2 2.9 22.4 93.7
    7h-14-19 IFNα2b 1900 969.4 2.2 25 78.7
    7h-11 IFNα2b 6000 327.9 6.5 11 101.9
    7h-11-12 IFNα2b 1700 747.1 2.8 25.8 104.7
    7h-11-15 IFNα2b 200 1118.7 1.8 39.5 103.6
    mouse 7h-14 IFNα2b 240 761.2 2.6 30.4 115.3
    7h-14-10 IFNα2b 60 750.5 2.7 30.9 118.6
    7h-11 IFNα2b 6000 493.9 4.0 8.8 51.2
    7h-11-12 IFNα2b 150 439.6 4.5 21.5 140.9
    7h-11-15 IFNα2b 28 971.8 2.1 33.6 99.6
    Pharmacokinetic parameters derived from rat and mouse studies were fitted using a non-compartmental model. Key: AUC: Area under the curve from dosing time extrapolated to infinity; CL: clearance; t½: is the time during which the blood concentration is halved; Vz: volume of distribution based on the terminal phase.
    IFNα2b -ALBUDABs ™ were tested in rat and mouse. For all IFNα2b-DOM7h-11 variant fusion proteins in both rat and mouse, t½ is improved compared to parent. The improvement in t½ correlates with the improved in vitro KD to serum albumin. For IFNα2b-DOM7h-14-10 variants, the improvement in in vitro KD to serum albumin also correlated to an improvement in t½ in rat.
    All IFNα2b -ALBUDAB ™ fusion proteins exhibit a 5 to 10-fold decrease in the binding to RSA compared to the single ALBUDAB ™. This effect is more pronounced (i.e. 10-fold) for the DOM7h-14 series than the DOM7h-11 series (only 5-fold decrease).
    • Example 8: Further ALBUDAB™ Fusions with Proteins, Peptides and NCEs
  • Various ALBUDABs™ fused to other chemical entities namely domain antibodies (dAbs), peptides and NCEs were tested. The results are shown in table 15.
  • TABLE 15
    PK parameters
    Albumin AUC CL t½ Vz
    Species ALBUDAB ™ Fusion KD (nM) h × ug/ml ml/h/kg h ml/kg
    Rat DOM7h-14 Exendin-4 2400 18 57.1 11 901.9
    DOM7h-14-10 Exendin-4 19 43.6 23.1 22.1 740.3
    DOM7h-14-18 Exendin-4 16000 16.9 75.7 9.4 1002.5
    DOM7h-14-19 Exendin-4 17000 31.4 32.5 11.9 556.7
    DOM7h-11 Exendin-4 24000 6.1 168 7.1 1684.1
    DOM7h-11-12 Exendin-4 1400 24.2 59.9 13 1068.7
    DOM7h-11-15 Exendin-4 130 36.3 27.6 19.3 765.7
    DOM7h14-10 NCE-GGGGSC 62
    DOM7h14-10 NCE-TVAAPSC 35
    Human DOM7h-14 NCE 204
    mouse DOM7h-11 DOM1m-21-23 234 10.7 4.7 72.5
    DOM7h-11-12 DOM1m-21-23 755 3.3 18 86.2
    DOM7h-11-15 DOM1m-21-23 1008 2.5 17.4 62.4
    Key: DOM1m-21-23 is an anti-TNFR1 dAb, Exendin-4 is a peptide (a GLP-1 agonist) of 39 amino acids length. NCE, NCE-GGGGSC and NCE-TVAAPSC are described below.
    Previously we have described the use of genetic fusions with an albumin-binding dAb (ALBUDAB ™) to extend the PK half-life of anti-TNFR1 dAbs in vivo (see, eg, WO04003019, WO2006038027, WO2008149148). Reference is made to the protocols in these PCT applications. In the table above, DOM1m-21-23 is an anti-mouse TNFR1 dAb.
  • To produce genetic fusions of exendin-4 or with DOM7h-14 (or other ALBUDAB′) which binds serum albumin, the exendin-4-linker-ALBUDAB′ sequence was cloned into the pTT-5 vector (obtainable from CNRC, Canada). In each case the exendin-4 was at the 5′ end of the construct and the dAb at the 3′ end. The linker was a (G4S)3 linker. Endotoxin-free DNA was prepared in E. coli using alkaline lysis (using the endotoxin-free plasmid Giga kit, obtainable from Qiagen CA) and used to transfect HEK293E cells (obtainable from CNRC, Canada). Transfection was into 250 ml/flask of HEK293E cells at 1.75×106 cells/ml using 333 ul of 293 fectin (Invotrogen) and 250 ug of DNA per flask and expression was at 30° C. for 5 days. The supernatant was harvested by centrifugation and purification was by affinity purification on protein L. Protein was batch bound to the resin, packed on a column and washed with 10 column volumes of PBS. Protein was eluted with 50 ml of 0.1M glycine pH2 and neatralised with Tris pH8. Protein of the expected size was identified on an SDS-PAGE gel.
    • NCE ALBUDAB™ Fusions:
  • A new chemical entity (NCE) ALBUDAB™ fusion was tested. The NCE, a small molecule ADAMTS-4 inhibitor was synthesised with a PEG linker (PEG 4 linker (ie 4 PEG molecules before the maleimide) and a maleimide group for conjugation to the ALBUDAB™. Conjugation of the NCE to the ALBUDAB™ is via an engineered cystine residue at amino acid position R108C, or following a 5 amino acid (GGGGSC) or 6 amino acid (TVAAPSC (SEQ ID NO: 419)) spacer engineered at the end of the ALBUDAB™. Briefly, the AALBUDAB™ was reduced with TCEP (Pierce, Catalogue Number 77720), desalted using a PD10 column (GE healthcare) into 25 mM Bis-Tris, 5 mM EDTA, 10% (v/v) glycerol pH6.5. A 5 fold molar excess of maleimide activated NCE was added in DMSO not to exceed 10% (V/V) final concentration. The reaction was incubated over night at room temperature and dialysed extensively into 20 mM Tris pH7.4
    • PEG Linker:
  • Figure US20200283512A1-20200910-C00001
    • Sequences:
  • DOM7h-14 R108C:
    (SEQ ID NO: 412)
    DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
    RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ
    GTKVEIKC
    Nucleotide:
    (SEQ ID NO: 413)
    GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
    CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
    CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
    CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
    TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
    CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA
    GGGACCAAGGTGGAAATCAAATGC
  • See table 5 for the sequences of DOM7h-14-10/TVAAPSC and DOM7h-14-10/GGGGSC (ie, DOM7h-14-10/G4 SC).
  • NCE-ALBUDABs™ DOM7h-14-10 GGGGSC(SEQ ID NO: 62) and DOM7h14-10 TVAAPSC, exhibit a 5 to 10 fold decrease in in vitro affinity (KD) to RSA as determined by BiaCore™ when fused to the chemical entity. PK data are not available for these molecules yet.
  • dAb-ALBUDAB™ fusion: the 2 DOM7h-11 ALBUDABs™ with the highest affinity to RSA experience a 2-fold decrease in affinity to RSA as on BiaCore™ when fused to a therapeutic domain antibody (DOM1m-21-23) compared to the unfused ALBUDAB™. The DOM7h-11 clone shows a micromolar KD when fused (2.8 uM) as well as when unfused (˜5 uM).
  • Exendin 4-ALBUDAB™ fusion: the effect of fusing the ALBUDABs™ to a peptide on the binding ability to RSA is about 10-fold, apart from DOM7h-14-10, which only shows a 4-fold decrease in binding. The effect, however, is more pronounced for the DOM7h-14 series (except DOM7h-14-10) than it appears to be for the DOM7h-11 series.
  • For all the above data, the T1/2 of the fusion increased with improved affinity to the species' SA.
  • We generally classify ALBUDAB™-therapeutics as being therapeutically amenable (for treatment and/or prophylaxis of diseases, conditions or indications) when the ALBUDAB™-drug fusions show an affinity range (KD) of from 0.1 nM to 10 mM for serum albumin binding.
  • We define the therapeutic ranges of ALBUDABs™ and ALBUDAB™ fusions (Protein-ALBUDABs™ for example IFNα2b-DOM7h-14-10; Peptide-ALBUDABs™ for example Exendin-4-DOM7h-14-10; dAb-ALBUDABs™ for example DOM1m21-23-DOM7h11-15; NCE-ALBUDAB™ for example ADAMTS-4-DOM7h-14-10) as follows: Affinity (KD) ranges that are useful for therapy of chronic or acute conditions, diseases or indictions are shown. Also shown are affinity ranges marked as “intermediate”. ALBUDABs™ and fusions in this range have utility for chronic or acute diseases, conditions or indications. In this way, the affinity of the ALBUDAB™ or fusion for serum albumin can be tailored or chosen according to the disease, condition or indication to be addressed. As described above, the invention provides ALBUDABs™ with affinities that allow for each ALBUDAB™ to be categorised as “high affinity”, “medium affinity” or “low affinity”, thus enabling the skilled person to select the appropriate ALBUDAB™ of the invention according to the therapy at hand. See FIG. 2.
    • Example 9: DOM7h-11-15S12P Sequences Amino Acid Sequence of DOM7h-11-15S12P
  • (SEQ ID NO: 414)
    DIQMTQSPSSLPASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLILA
    FSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQ
    GTKVEIKR
  • An aspect of the invention provides a nucleic acid comprising the nucleotide sequence of DOM7h-11-15S12P or a nucleotide sequence that is at least 80% identical to said selected sequence. DOM7h-11-15S12P was produced using the following nucleic acid sequence (the underlined C denotes the change (versus the nucleic acid encoding DOM7h-11-15) leading to a proline at position 12):—
  • (SEQ ID NO: 415)
    GACATCCAGATGACCCAGTCTCCATCCTCCCTGCCTGCATCTGTAGGAGA
    CCGTGTCACCATCACTTGCCGGGCAAGTCGTCCGATTGGGACGATGTTAA
    GTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCTTGCT
    TTTTCCCGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
    TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
    CTACGTACTACTGCGCGCAGGCTGGGACGCATCCTACGACGTTCGGCCAA
    GGGACCAAGGTGGAAATCAAACGG
  • DOM7h-11-15S12P was constructed by using DOM7h-11-15 as a template in a PCR where a primer was used to introduce the S12P mutation. The primer sequence is:—
  • (SEQ ID NO: 416)
    GCAACAGCGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGCC
    TGCATCTGTAGG
  • An alternative aspect of the invention provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 415 or a nucleotide sequence that is at least 80% identical to said selected sequence. In one embodiment, DOM7h-11-15S12P is encoded by, and expressed from, a vector that contains a linker region and a C-terminal sequence encoding a protein or peptide drug or a single variable domain or other antibody fragment to make the in-line protein fusion product. The linker, in one embodiment, comprises the amino acid sequence TVA, eg, TVAAPS (SEQ ID NO: 422). Other aspects of the invention are a vector comprising the nucleic acid; and an isolated host cell comprising the vector. The invention also provides a method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of DOM7h-11-15S12P to said patient.

Claims (28)

1. An anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11 (DOM7h-11 as shown in FIG. 1), wherein the variant comprises at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) compared to DOM7h-11, and wherein the variant has from 2 to 8 changes compared to the amino acid sequence of DOM7h-11.
2. The variant of claim 1, wherein position 49 (according to Kabat) is Leu.
3. The variant of claim 1, wherein position 50 (according to Kabat) is Ala or Trp.
4. The variant of claim 1, wherein position 51 (according to Kabat) is Phe or Asn.
5. The variant of claim 1, wherein the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-3 (SEQ ID NO: 5), DOM7h-11-15 (SEQ ID NO: 2), DOM7h-11-12 (SEQ ID NO: 1) and DOM7h-11-19 (SEQ ID NO: 4) or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has at least one mutation in the FW2/CDR2 junction.
6. The variant of claim 1, wherein the variant comprises an amino acid sequence that is identical to the amino acid sequence of DOM7h-11-15S12P (SEQ ID NO: 414) or has up to 4 changes compared to the amino acid sequence of DOM7h-11-15S12P, provided that the amino acid sequence of the variant has at least one mutation in the FW2/CDR2 junction.
7. An anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11, wherein the variant comprises a Met at position 32 (numbering according to Kabat) compared to DOM7h-11 (as shown in FIG. 1), and wherein the variant has from 0 to 4 further changes compared to the amino acid sequence of DOM7h-11.
8. The variant of claim 7, wherein the variant comprises at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) compared to DOM7h-11 (as shown in FIG. 1).
9. The variant of claim 7, wherein the variant comprises at least one mutation compared to DOM7h-11 (as shown in FIG. 1) selected from the following
Position 49=L,
Position 50=A or W,
Position 51=F or N,
Position 87=H, and
Position 91=T.
10. The variant of claim 7, wherein the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-12 (SEQ ID NO: 1), DOM7h-11-15 (SEQ ID NO: 2), DOM7h-11-18 (SEQ ID NO: 3) and DOM7h-11-19 (SEQ ID NO: 4) or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has Met at position 32.
11. The variant of claim 7, wherein the variant comprises an amino acid sequence that is identical to the amino acid sequence of DOM7h-11-15S12P (SEQ ID NO: 414) or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has Met at position 32.
12. The variant of claim 1, wherein the variant comprises a binding site that specifically binds human SA with a dissociation constant (KD) of from about 0.1 to about 10000 nM, optionally from about 1 to about 6000 nM, as determined by surface plasmon resonance.
13. The variant of claim 1, wherein the variant comprises a binding site that specifically binds human SA with an off-rate constant (Kd) of from about 1.5×10−4 to about 0.1 sec−1, optionally from about 3×10−4 to about 0.1 sec−1 as determined by surface plasmon resonance.
14. The variant of claim 1, wherein the variant comprises a binding site that specifically binds human SA with an on-rate constant (Ka) of from about 2×106 to about 1×104M−1 sec−1, optionally from about 1×106 to about 2×104M−1 sec−1 as determined by surface plasmon resonance.
15. The variant of claim 1, wherein the variant comprises a binding site that specifically binds Cynomolgus monkey SA with a dissociation constant (KD) of from about 0.1 to about 10000 nM, optionally from about 1 to about 6000 nM, as determined by surface plasmon resonance.
16. The variant of claim 1, wherein the variant comprises a binding site that specifically binds Cynomolgus monkey SA with an off-rate constant (Kd) of from about 1.5×10−4 to about 0.1 sec−1, optionally from about 3×10−4 to about 0.1 sec−1 as determined by surface plasmon resonance.
17. The variant of claim 1, wherein the variant comprises a binding site that specifically binds Cynomolgus monkey SA with an on-rate constant (Ka) of from about 2×106 to about 1×104M−1 sec−1, optionally from about 1×106 to about 5×103M−1 sec−1 as determined by surface plasmon resonance.
18. A multispecific ligand comprising an anti-SA variant of claim 1 and a binding moiety that specifically binds a target antigen other than SA.
19. An anti-SA variant single variable domain of claim 1, wherein the variable domain is conjugated to a drug (optionally an NCE drug), optionally wherein the selected variant is DOM7h-11-15 (SEQ ID NO: 2), DOM7h-11-15S12P (SEQ ID NO: 414) or DOM7h-11-12 (SEQ ID NO: 1).
20. A fusion protein comprising a polypeptide or peptide drug fused to a variant according to claim 1, optionally wherein the selected variant is DOM7h-11-15 (SEQ ID NO: 2), DOM7h-11-15S12P (SEQ ID NO: 414) or DOM7h-11-12 (SEQ ID NO: 1).
21. A fusion protein according to claim 20, wherein the fusion protein comprises a linker (eg, a linker comprising the amino acid sequence TVA, optionally TVAAPS) between the variant and the drug.
22. A composition comprising a variant, fusion protein or ligand of claim 1 and a pharmaceutically acceptable diluent, carrier, excipient or vehicle.
23. A nucleic acid comprising a nucleotide sequence encoding a variant according to claim 1.
24. A nucleic acid comprising the nucleotide sequence of a DOM7h-11 variant selected from the nucleotide sequence of DOM7h-11-3 (SEQ ID NO: 5), DOM7h-11-15 (SEQ ID NO: 2), DOM7h-11-12 (SEQ ID NO: 1), DOM7h-11-18 (SEQ ID NO: 3) and DOM7h-11-19 (SEQ ID NO: 4) or a nucleotide sequence that is at least 80% identical to said selected sequence.
25. A nucleic acid comprising the nucleotide sequence of DOM7h-11-15S12P (SEQ ID NO: 414) or a nucleotide sequence that is at least 80% identical to said selected sequence.
26. A vector comprising the nucleic acid of claim 23.
27. An isolated host cell comprising the vector of claim 26.
28. A method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of a variant according to claim 1 to said patient.
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