WO2008133873A2 - Fgf-binding fusion proteins - Google Patents

Fgf-binding fusion proteins Download PDF

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Publication number
WO2008133873A2
WO2008133873A2 PCT/US2008/005172 US2008005172W WO2008133873A2 WO 2008133873 A2 WO2008133873 A2 WO 2008133873A2 US 2008005172 W US2008005172 W US 2008005172W WO 2008133873 A2 WO2008133873 A2 WO 2008133873A2
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Prior art keywords
loop
vai
domain
consists essentially
fusion protein
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PCT/US2008/005172
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French (fr)
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WO2008133873A3 (en
WO2008133873A9 (en
Inventor
William M. Winston
Lynn Breault
Zhigang Weng
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Aveo Pharmaceuticals, Inc.
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Publication of WO2008133873A2 publication Critical patent/WO2008133873A2/en
Publication of WO2008133873A9 publication Critical patent/WO2008133873A9/en
Publication of WO2008133873A3 publication Critical patent/WO2008133873A3/en

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the field of the invention is molecular biology, cell biology, growth factors, cancer and medicine.
  • Fibroblast growth factors and fibroblast growth factor receptors (FGFRs) display various biological activities, including promotion of cell proliferation and angiogenesis .
  • FGFs act as mitogens, inducing cell migration and/or differentiation.
  • FGF signaling plays a fundamental role in animal development. See, e.g., De Moerlooze et al . , 2000, Development 127:483-492; Petiot et al . , 2003, Development 130:5493-5501; Revest et al . , 2001, J. Immunol. 167:1954-1961.
  • FGF/FGFR signaling appears to be involved in both normal angiogenesis and tumor angiogenesis.
  • FGF/FGFR signaling in angiogenesis see, e.g., Presta et al., 2005, Cytokine & Growth Factor Reviews, 16:159-178.
  • FGF/FGFR-mediated signaling in various types of cancer, including breast cancer, skin cancer, prostate cancer, and urothelial cancer.
  • FGF signaling in tumorigenesis see, e.g., Grose et al., 2005, Cytokine & Growth Factor Reviews, 16:179-186.
  • 22 mammalian FGFs are known.
  • FGFs are secreted glycoproteins.
  • FGF-FGFR binding results in formation of a complex that contains at least two FGFs and at least two FGFRs. Formation of such a complex results in signal transduction mediated by the FGFR intracellular kinase domain.
  • FGFRl, 2, 3, and 4 are type I transmembrane tyrosine kinases that belong to the immunoglobulin (Ig) superfamily
  • Native FGFRs include an amino-terminal signal sequence
  • Ig-like domains that arise by alternative splicing, an acid box that precedes Ig- like domain 2, a transmembrane domain, and a tyrosine kinase domain
  • FIG. 1 The extracellular domain, which includes the FGF binding site, contains two or three Ig-like domains.
  • FGFR- 1 and -2 alternative splicing produces a protein containing three Ig-like domains or two Ig-like domain (domains 2 and 3), which are referred to as the alpha and beta forms, respectively. Only the alpha form has been described for FGFR- 3 and -4.
  • alternative splicing produces variants within the carboxy terminal half of the Ig-like domain 3, known as the IHb or IIIc form, depending on the exon used (FIG. 1) . Only the IIIc form as been described for FGFR-4.
  • FGF binds FGFR Ig-like domains 2 and 3. Such binding leads to receptor dimerization and autophosphorylation (Mohammadi, supra).
  • the invention provides a variety of soluble fusion proteins engineered to bind fibroblast growth factor (FGF) molecules (ligands) efficiently in vitro and in vivo, thereby competing with endogenous FGF for binding sites on membrane- bound FGFRs. This inhibition of FGF/FGFR binding reduces biological signal transduction mediated by FGF/FGFR binding.
  • FGF fibroblast growth factor
  • the domain structure of soluble fusion proteins according to the invention comprises:
  • D2 consists essentially of the Ig-like domain 2 from FGFR2 (R2D2; SEQ ID NO: 49), FGFR3 (R3D2 ; SEQ ID NO: 50) or FGFR4 (R4D2; SEQ ID NO: 51) ;
  • L is DVLERxyHR (Leu loop; SEQ ID NO: 59) or DWERxyHR (VaI loop; SEQ ID NO: 60) , wherein x is S or W, and y is P or
  • R; D3 consists essentially of the Ig-like domain 3 of FGFR2- IHb (R2D3b; SEQ ID NO: 54), FGFR2-IIIC (R2D3C; SEQ ID NO: 55), FGFR3-IIIb (R3D3b; SEQ ID NO: 56), FGFR3-IIIC (R3D3C; SEQ ID NO: 57) , or FGFR4-IIIC (R4D3C; SEQ ID NO: 58) ; and
  • Fc consists essentially of the Fc domain of a human immunoglobulin.
  • D2 consists essentially of the Ig-like domain 2 from FGFR2. In some embodiments of the invention, D2 consists essentially of the Ig-like domain 2 from FGFR3. In some embodiments of the invention, D2 consists essentially of the Ig-like domain 2 from FGFR4.
  • D3 consists essentially of the Ig-like domain 3 from FGFR2-IIIb. In some embodiments of the invention, D3 consists essentially of the Ig-like domain 3 from FGFR2-IIIC. In some embodiments of the invention, D3 consists essentially of the Ig-like domain 3 from FGFR3-IIIb. In some embodiments of the invention, D3 consists essentially of the Ig-like domain 3 from FGFR3-IIIc. In some embodiments - A -
  • D3 consists essentially of the Ig-like domain 3 from FGFR4-IIIC.
  • L consists essentially of DWERSPHR (wild-type VaI loop; SEQ ID NO: 53) .
  • L consists essentially of DVLERSPHR (wild-type Leu loop,- SEQ ID NO: 52) .
  • the immunoglobulin is IgGl.
  • the invention also provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR2 , L consists essentially of DWERWPHR (the VaI loop comprising mutation S106W; SEQ ID NO: 61) and D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIC.
  • the invention also provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR2 , L consists essentially of DWERSRHR (the VaI loop comprising mutation P107R; SEQ ID NO: 62) and D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIc.
  • the invention further provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR2 , L consists essentially of DWERWRHR (the VaI loop comprising mutations S106W and P107R; SEQ ID NO: 63) and D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIc.
  • the invention also provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR2 , L consists essentially of DWERSRHR (the VaI loop comprising mutation P107R; SEQ ID NO: 62) and D3 consists essentially of the Ig-like domain 3 of FGFR3-IIIC.
  • the invention also provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR4 , L consists essentially of DWERSRHR (the VaI loop comprising mutation P106R; SEQ ID NO: 62) and D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIc.
  • the invention also provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR4 , L consists essentially of DVLERSRHR (the Leu loop comprising mutation P106R; SEQ ID NO: 64) and D3 consists essentially of the Ig- like domain 3 of FGFR2-IIIc.
  • the invention also provides fusion proteins wherein the domain structure is: R2D2 Leu Loop R3D3b Fc,-
  • R2D2 Leu Loop R3D3c Fc R2D2 Leu Loop R4D3C Fc; R2D2 VaI Loop R2D3b Fc; R2D2 VaI Loop R2D3c Fc; R2D2 VaI Loop R3D3b Fc;
  • the fusion proteins have the domain structure : R2D2 VaI Loop R2D3C Fc; R2D2 VaI Loop R3D3C Fc; R4D2 VaI Loop R2D3C Fc ; R2D2 VaI P107R Loop R2D3c Fc; R4D2 Leu Loop R2D3C Fc ;
  • R2D2 VaI P107R Loop R3D3C Fc R4D2 VaI P106R Loop R2D3C Fc; or R4D2 Leu P106R Loop R2D3c Fc.
  • the invention also provides a nucleic acid comprising a nucleotide sequence that encodes the FGF-binding fusion protein comprising the domain structure D2-L-D3-Fc as described above.
  • the invention also provides an expression vector comprising the nucleic acid and a host cell comprising the expression vector.
  • the invention further provides a method of making the FGF-binding fusion protein comprising the domain structure D2- L-D3-Fc, the method comprising the steps of (a) culturing the host cell containing the expression vector comprising the nucleic acid comprising the nucleotide sequence that encodes the FGF-binding fusion protein under conditions so that the host cell expresses the FGF-binding fusion protein, and (b) harvesting the fusion protein.
  • the invention also provides a method of inhibiting binding of an FGF molecule to an FGF receptor, comprising contacting the FGF-binding fusion protein of the invention with the FGF molecule.
  • the FGF molecule, FGF receptor and FGF-binding fusion protein are in vivo.
  • the invention also provides a method of inhibiting proliferation of a tumor cell in vitro, comprising contacting the tumor cell with the FGF-binding fusion protein.
  • the invention also provides a method of inhibiting proliferation of a tumor cell in a mammal, comprising administering to the mammal an effective amount of the FGF-binding fusion protein.
  • the invention further provides a method of treating a tumor in a mammal, comprising administering to the mammal an effective amount of the FGF-binding fusion protein.
  • the invention also provides for pharmaceutical compositions comprising the fusion proteins described above.
  • FIG. 1 is a schematic drawing of the domain structure of native FGFR alpha IHb and FGFR alpha IIIc (prior art) .
  • FIG. 2 is a sequence alignment of the FGFR domain 2 sequences, D2-D3 loop sequences, and FGFR domain 3 sequences that are used in different combinations to make the FGF-binding fusion proteins of the invention.
  • FIG. 3 is a graph summarizing data showing the effect of the R2D2 VaI Loop R2D3 Fc fusion protein in an FGF-dependent proliferation assay. An Fc protein was also tested as a control .
  • administering means delivering in a manner which is effected or performed using any of the various methods and delivery systems known to those skilled in the art.
  • Administering can be performed, for example, topically, intravenously, pericardially, orally, via implant, transmucosally, transdermally, intramuscularly, subcutaneously, intraperitoneally, intrathecally, intralymphatically, intralesionally, or epidurally. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • Expression vector means a nucleic acid encoding a nucleic acid of interest and/or a protein of interest, which nucleic acid, when placed in a cell, permits the expression of the nucleic acid or protein of interest.
  • Expression vectors are well known in the art. - S -
  • Fc means the immunoglobulin constant region.
  • a native sequence Fc or constant region comprises an amino acid sequence identical to the amino acid sequence of an Fc or constant region found in nature.
  • a variant or altered Fc or constant region comprises an amino acid sequence which differs from that of a native sequence heavy chain region by virtue of at least one amino acid modification, insertion, or deletion, for example. Additionally, the variant constant region may contain one or more amino acid substitutions, deletions, or insertions that results in altered post-translational modifications, including, for example, an altered glycosylation pattern.
  • Host cell means a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing a selected gene of interest. Host cells are well known in the art.
  • “Inhibiting" the onset of a disorder means lessening the likelihood of the disorder's onset, or preventing the onset of the disorder entirely.
  • Nucleic acid means any nucleic acid molecule, including DNA, RNA and hybrids thereof.
  • the nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR
  • Protein means a polymer of amino acid residues.
  • the amino acid residues can be naturally occurring or chemical analogues thereof.
  • Proteins also can include modifications such as glycosylation, lipid attachment, sulfation, hydroxylation, and ADP-ribosylation .
  • Subject means a mammal, including a human.
  • “Therapeutically effective amount” means an amount of an agent which, when administered to a subject afflicted with a disease or disorder against which the agent is effective, produces a beneficial effect with respect to the disease or disorder.
  • Vector means a molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell .
  • the invention provides soluble fusion proteins comprising D2 , L and D3 , which together form the FGF-binding region of the fusion proteins.
  • the fusion protein lacks the transmembrane region and the tyrosine kinase domain found in native FGFRs. Consequently, the fusion protein is soluble in physiological fluids.
  • FGF molecules ligands
  • fusion proteins of the invention can be classified as "decoy receptors" or "ligand traps" because the ligand binds to them in vivo, thereby competing with the functional endogenous receptors.
  • the ligands are diverted or sequestered, with the result being less FGF/FGFR-mediated signaling than would occur in the absence of the soluble fusion proteins.
  • the invention provides a variety of soluble FGF- binding fusion proteins.
  • the general structure of the FGF-binding fusion proteins according to the invention comprises D2-L-D3-Fc.
  • D2 may consist essentially of the Ig- like domain 2 from FGFR2 (R2D2; SEQ ID NO: 49), FGFR3 (R3D2 ; SEQ ID NO: 50) or FGFR4 (R4D2; SEQ ID NO: 51) .
  • L may consist essentially of DVLERSPHR (Leu loop,- SEQ ID NO: 52) or DWERSPHR (VaI loop; SEQ ID NO : 53) .
  • the L region may also comprise mutated loop sequences such as DWERWPHR (VaI loop comprising mutation S106W; SEQ ID NO: 61) , DWERSRHR (VaI loop comprising mutation P107R; SEQ ID NO: 62), DWERWRHR (VaI loop comprising mutations S106W and P107R; SEQ ID NO: 63) , and DVLERSRHR (Leu loop comprising mutation P106R; SEQ ID NO: 64) .
  • the invention provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR2 and L consists essentially of DWERSRHR (VaI loop comprising mutation P107R, SEQ ID NO: 62) .
  • the invention provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR4 and L consists essentially of DWERSRHR (VaI loop comprising mutation P106R, SEQ ID NO: 62) .
  • D3 may consist essentially of the Ig-like domain 3 of FGFR2-IIIb (R2D3b; SEQ ID NO: 54), FGFR2-IIIC (R2D3C; SEQ ID NO: 55), FGFR3-IIIb (R3D3b; SEQ ID NO: 56), FGFR3-IIIC (R3D3C; SEQ ID NO: 57) , or FGFR4-IIIC (R4D3C; SEQ ID NO : 58) .
  • Fc may consist essentially of the Fc domain of a human immunoglobulin.
  • each FGF-binding fusion protein The nucleotide and amino acid sequences of each FGF-binding fusion protein are provided in Table 1.
  • gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
  • gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
  • the human IgGl Fc domain was fused to each of the twenty-eight fusion protein constructs.
  • the nucleotide and amino acid sequences of the human IgGl Fc that were fused to each of the different fusion protein constructs are provided in Table 2.
  • Table 2. Nucleotide and Amino Acid Sequences of human IgGl Fc used for generating FGF-binding Fusion Proteins
  • IgGl Fc sequence used for R3D3b, R3D3c, and R4D3c
  • the present invention provides methods for making the FGF-binding fusion proteins of the invention using nucleic acid molecules herein described.
  • the production of a recombinant form of a protein typically involves the following steps.
  • a nucleic acid molecule is first obtained that encodes a FGF-binding fusion protein of the invention.
  • the nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame.
  • the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein.
  • the recombinant protein is isolated from the medium or from the cells. Purification of the protein may not be necessary in some instances where some impurities can be tolerated.
  • Each of the foregoing steps can be accomplished in a variety of ways.
  • the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier and are otherwise known to persons skilled in the art.
  • suitable restriction sites can be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
  • a skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce a desired recombinant protein.
  • cloned DNA sequences comprising FGF- binding fusion proteins of the invention can be introduced into cultured mammalian cells by, for example, calcium phosphate- mediated transfection (Wigler et al . , Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973) .
  • Other techniques for introducing cloned DNA sequences into mammalian cells such as electroporation (Neumann et al . , EMBO J. 1: 841-845, 1982), or lipofection also can be used.
  • a selectable marker is generally introduced into the cells along with the gene or cDNA of interest.
  • Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate.
  • the selectable marker can be an amplifiable selectable marker, such as the DHFR gene. Selectable markers are reviewed by Thilly ⁇ Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass. (1986) and the choice of selectable markers is within ordinary skill in the art.
  • Any expression system may be used, including yeast, bacterial, animal, plant, eukaryotic and prokaryotic systems.
  • yeast, mammalian cell culture and transgenic animal or plant production systems are preferred.
  • mammalian cell culture is used.
  • FGF-binding fusion proteins can be isolated from the medium of host cells grown under conditions that allow the expression and secretion of the fusion proteins.
  • the cell material is removed from the culture medium, and the fusion proteins are isolated using any suitable isolation techniques. Suitable isolation techniques include precipitation and fractionation by a variety of chromatographic methods, including gel filtration, ion exchange chromatography and affinity chromatography. 72
  • FGF-Binding Fusion Protein Pharmaceutical Compositions
  • Such FGF-binding fusion protein pharmaceutical compositions can comprise a therapeutically effective amount of a FGF-binding fusion protein of the invention in admixture with pharmaceutically or physiologically acceptable formulation carriers selected for suitability with the mode of administration.
  • Pharmaceutically acceptable carriers are well known in the art and include 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline.
  • Pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, or emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Porphyrin or Lipofectin also can be used as a delivery agent.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.
  • the optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage.
  • the appropriate dosage levels for treatment will vary depending, in part, upon the molecule delivered, the indication for which the FGF-binding fusion protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • the frequency of dosing will depend upon the pharmacokinetic parameters of the FGF-binding fusion protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition can be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art. Appropriate dosages can be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical composition can vary. For example, it can be oral, intravenous, intraperitoneal, intracerebral (intraparenchymal) , intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional . Administration also can be by sustained release systems; or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • the composition can be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be by diffusion, timed- release bolus, or continuous administration.
  • An FGF-binding fusion protein can be delivered by- implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the FGF-binding fusion protein.
  • Such cells can be animal or human cells. They can be autologous, heterologous, or xenogeneic. Optionally, the cells are immortalized.
  • the cells may be encapsulated to avoid infiltration of surrounding tissues.
  • the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product (s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
  • the fusion proteins of the invention are useful as therapeutic agents against diseases or disorders that depend, at least in part, on FGF/FGFR-mediated signaling.
  • diseases and disorders include various types of cancer, for example, prostate carcinogenesis, skin tumorigenesis, urothelial cancer and hematological malignancies.
  • Other types of cancer treatable by the fusion proteins of the invention include breast cancer, cervical cancer, ovarian cancer, gastric cancer, colorectal cancer, pancreatic cancer and lung cancer.
  • the fusion proteins of the invention can be used to inhibit FGF binding to FGFRs in any in vivo or in vitro cellular process involving FGF signaling.
  • FGF/FGFR- mediated signaling has been implicated in many cellular processes, including angiogenesis and cell proliferation. As described in Example 4, the fusion proteins of the invention can inhibit cell proliferation.
  • Twenty-two FGF Traps were constructed by fusing Ig domains 2 and 3 from human FGFR2 , 3, and 4.
  • Ig-like domain 2 (D2) regions from FGFR2, 3, and 4
  • two domain 2 - domain 3 loop (VaI Loop or Leu Loop) sequences one from FGFR2 and the other representing FGFR3 and 4, respectively
  • Ig-like domain 3 (D3) regions from FGFR2 IHb, FGFR2 IHc, FGFR3 IHb, FGFR3 IHc, and FGFR4 IIIc
  • PCR (Young et al . , 2004, Nucleic Acids Res. Apr 15; 32(7): e59) was conducted using the Expand High Fidelity PCR System (Roche Applied Science, Germany) according to the manufacturer's instructions and as outlined by PCR #1 in Table 5. Second, overlapping extension PCR (Young et al., 2004, Nucleic Acids
  • Mouse FGFR3 Ig domain 2 and Ig domain Ilia contain minor amino acid differences in comparison to human FGFR3.
  • PCRs #2, 3, 5-8 were conducted as outlined in Table 6 using KOD Hot Start DNA Polymerase (EMD Biosciences, Novagen, San Diego, CA) according to the manufacturer's instructions.
  • KOD Hot Start DNA Polymerase EMD Biosciences, Novagen, San Diego, CA
  • PCRs #9, 10, and 11 were conducted with KOD polymerase as outlined in Table 6.
  • FGFR2 IHc, FGFR3 IHb, FGFR3 IHc, and FGFR4 IHc with either D2 - D3 Loop option PCRs #20-29 were conducted with KOD polymerase as outlined in Table 6.
  • Human IgGl Fc (SEQ ID NO: 93 for R2D3b and R2D3c; SEQ ID NO: 94 for R3D3b, R3D3c and R4D3c) region was amplified in PCR #30 with KOD polymerase. Twenty-two combinations of Ig domain 2, D2 - D3 Loop, and Ig domain 3 were fused with human IgGl Fc by PCRs #31-52 as outlined in Table 6.
  • the resulting 22 PCR fragments contained the following in order: the attBl recombination site at the 5 1 end followed by a HindIII restriction site, a consensus Kozak translation initiation sequence, a secretion signal sequence, an FGFR D2 - Loop - D3 Fc combination, an EcoRI restriction site, and finally an attB2 recombination site at the 3' end.
  • One or two amino acid changes were also made in the Loop region of several traps. These mutations are analogous to Ser 252 to Trp and Pro 253 to Arg mutations described for FGFR2 (HIb or IIIc forms) .
  • FGF Trap recombinant proteins are designated as follows:
  • the point mutations were introduced by PCR.
  • Domain 2 + Loop was amplified to include the mutation (s) in the Loop (mutation introduced by the primer) .
  • Loop + Domain 3 + Fc was also amplified to include the mutation (s) in the Loop.
  • Corresponding Domain 2 + Loop and Loop + Domain 3 + Fc PCR fragments were then fused by PCR.
  • the construction of these FGF Traps is outlined by PCRs #53-70 in Table 6.
  • the resulting six full length FGF Trap PCRs also include the additional sequences described above for the first 22 traps (attBl, Hindlll, Kozak, signal sequence, EcoRI, and attB2) .
  • FGF Traps # 5, 7, 17, 22, 24, and 26-28 were also subcloned from pDONR221 into pEE14.4 (Lonza Biologies, Berkshire, UK) using unique Hindlll and EcoRI restriction sites.
  • the template used was a plasmid containing R4D2 Leu R2D3c Fc.
  • the preferred template would have been a plasmid containing R4D2 VaI R2D3c Fc, but the one used was sufficient to allow the creation of the desired product.
  • Similary, for PCRs #66 and 67 the preferred template would have been a plasmid containing R4D2 Leu R2D3c Fc, but a plasmid containing R4D2 VaI R2D3c Fc was actually used, also yielding the desired 0 product .
  • FGF Traps #1-25 were expressed in 293T cells by transiently transfecting the pcDNA3.2 based vectors with 5 GeneJuice® Transfection Reagent (EMD Biosciences, Novagen, San Diego, CA) according to the manufacturer's instructions.
  • Cells were cultured in DMEM media (Invitrogen) supplemented with 10% Ultra Low IgG Fetal Bovine Serum (Invitrogen) or 10% Fetal Bovine Serum (Invitrogen) .
  • Cell supernatant containing the 0 secreted FGF Trap was collected 72 hours after transfection. Media was replenished and supernatant was collected after an additional 72 hours.
  • FGF Trap-containing cell supernatant was used for surface plasmon resonance (BIAcore) characterization of FGF Trap binding to various FGFs (see below) .
  • Stable cell 5 lines producing FGF Traps # 5, 7, 17, 22, 24, and 26-28 were also produced by transfecting the pEE14.4 based vectors into CHOKlSV cells (Lonza Biologies) by electroporation.
  • Stable cell clones were selected with 25 or 50 uM methionine sulphoximine (MSX) in CD-CHO media (Invitrogen) .
  • FGF Traps unpurified and undiluted in cell supernatant were captured on individual flow cell at a flow rate of lO ⁇ l/min for 25 to 90 seconds depending on the experiment. Wild-type FGFRs (FGFRlalpha (HIb) ,
  • FGFRlbeta(IIIb) , FGFRlbeta (IIIc) , FGFR3 (HIb) and FGFR3 (IIIc) ) were purchased from R&D Systems and used as positive controls.
  • Recombinant human FGF-I, -3, -4, -6, -9, and -10 were purchased from R&D Systems (respective catalog number: 232-FA, 1206-F3, 235-FA, 238-F6, 273-F9, 345-FG) .
  • Two different forms of recombinant human FGF-2 were purchased from R&D Systems (233 -FB and 234 -FB) .
  • Recombinant mouse FGF- 8b and FGF- 8c were also purchased from R&D Systems (423-8b, 424-Fc) .
  • Mouse FGF-8b is 100% identical to human FGF- 8b.
  • a human equivalent of the FGF- 8c isoform does not appear to be expressed (Gemel et al . , 1996, Genomics 35:253-7).
  • Recombinant human FGF-7 was purchased from Cell Sciences (CRK300B) . All different recombinant FGFs were diluted individually in running buffer at a single concentration of 75nM and injected over the captured traps for 240 sec at 60 ⁇ l/min. The dissociation phase was monitored for 10 min before the surface was regenerated with 1OmM Glycine- HCl, pH 2.0 (BIAcore, #BR-1003-55) injected for 3 min at a flow rate of 60 ⁇ l/min.
  • Equilibrium constant (KD) can be used as an approximate guide to assess effectiveness of trap binding to a given ligand.
  • Equilibrium constants for the traps tested are shown in Tables 7-18.
  • Kinetic parameters for FGF binding of Traps 5, 23, 24 and 25 were compared (Table 18) .
  • the mutated Traps (23, 24 and 25) exhibited better binding to FGF7 and FGFlO as compared to the wild-type trap (5) .
  • curve fits calculated by the BIAevaluation software are non- ideal with respect to on-rate (ka) or off-rate (kd) . See Comments column of Tables 7-18. Confidence in these kinetic values might be increased, e.g., by an increased number of replicates and varied range of ligand concentration, or other assay optimization.
  • Table 7. FGFl Binding
  • FDCP-I cells mouse bone marrow cells obtained from German Collection of Microorganisms and Cell Cultures DSMZ
  • FDCP-FGFR3 cDNA expressing human FGFR3 IIIc
  • G418 600 ⁇ g/ml
  • Single clones were isolated and tested for their FGFl-dependent proliferation in the absence of IL-3-containing WEHI- conditioned medium.
  • FDCP-FGFR3#109 exhibited FGF-I induced proliferation in the absence of IL3 (WEHI-conditioned medium) .
  • FGF Trap R2D2 VaI R2D3c Fc
  • ILlRLl-Fc Recombinant Human IL-I R4/ST2/Fc Chimera, R&D Systems, 523-ST- 100
  • the mixtures were then added to FDCP-FGFR3 #109 cells seeded in basic growth medium [70% ISCOVE ' s Modified Dulbecco's Medium (Invitrogen, 12440-053) , 20% horse serum (Invitrogen 26050-088)) and 10% WEHI-culture medium (90% Iscove's MDM + 10% FBS (Invitrogen 10438-026) + 2 mM L-glutamine (Invitrogen, 25030-081) + 0.0025 mM 2-Mercaptoethanol (Invitrogen, 21985- 023)] in a 96-well plate (30,000 cells/ well).
  • the final concentration of FGFl and heparin used in the assay is 8 ng/ml and 5 ⁇ g/ml respectively.
  • a standard MTT assay was conducted three days post FGFl stimulation to assess relative proliferation.

Abstract

Soluble human fibroblast growth factor (FGF) -binding fusion proteins and a method of making these proteins are disclosed. Methods of using such fusion proteins to inhibit FGF binding to FGF receptors, to inhibit tumor cell proliferation in vitro and in vivo and to treat tumors in mammals are also disclosed.

Description

FGF-BINDING FUSION PROTEINS
Field of the Invention
[0001] The field of the invention is molecular biology, cell biology, growth factors, cancer and medicine.
Background of the Invention
[0002] Fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) display various biological activities, including promotion of cell proliferation and angiogenesis . FGFs act as mitogens, inducing cell migration and/or differentiation. FGF signaling plays a fundamental role in animal development. See, e.g., De Moerlooze et al . , 2000, Development 127:483-492; Petiot et al . , 2003, Development 130:5493-5501; Revest et al . , 2001, J. Immunol. 167:1954-1961. FGF/FGFR signaling appears to be involved in both normal angiogenesis and tumor angiogenesis. For a review of the role of FGF/FGFR signaling in angiogenesis, see, e.g., Presta et al., 2005, Cytokine & Growth Factor Reviews, 16:159-178. [0003] While the details of FGF involvement in tumorigenesis and/or tumor maintenance remain to be elucidated, numerous published studies indicate a role for FGF/FGFR-mediated signaling in various types of cancer, including breast cancer, skin cancer, prostate cancer, and urothelial cancer. For a review of FGF signaling in tumorigenesis, see, e.g., Grose et al., 2005, Cytokine & Growth Factor Reviews, 16:179-186. [0004] Currently, 22 mammalian FGFs are known. Most mammalian FGFs are secreted glycoproteins. FGF-FGFR binding results in formation of a complex that contains at least two FGFs and at least two FGFRs. Formation of such a complex results in signal transduction mediated by the FGFR intracellular kinase domain.
[0005] FGFRl, 2, 3, and 4 are type I transmembrane tyrosine kinases that belong to the immunoglobulin (Ig) superfamily
(Mohammadi, et al . , 2005, Cytokine Growth Factor Rev. 16:107- 37) . Native FGFRs include an amino-terminal signal sequence
(which gets cleaved) , two or three Ig-like domains that arise by alternative splicing, an acid box that precedes Ig- like domain 2, a transmembrane domain, and a tyrosine kinase domain
(FIG. 1) . The extracellular domain, which includes the FGF binding site, contains two or three Ig-like domains. For FGFR- 1 and -2, alternative splicing produces a protein containing three Ig-like domains or two Ig-like domain (domains 2 and 3), which are referred to as the alpha and beta forms, respectively. Only the alpha form has been described for FGFR- 3 and -4. Additionally, with respect to FGFR-I, -2, and -3, alternative splicing produces variants within the carboxy terminal half of the Ig-like domain 3, known as the IHb or IIIc form, depending on the exon used (FIG. 1) . Only the IIIc form as been described for FGFR-4. FGF binds FGFR Ig-like domains 2 and 3. Such binding leads to receptor dimerization and autophosphorylation (Mohammadi, supra).
[0006] Because various diseases and disorders depend, at least in part, on FGF/FGFR-mediated signaling, there is a medical need for improved therapeutic agents that inhibit FGF/FGFR-mediated signaling.
Summary of the Invention
[0007] The invention provides a variety of soluble fusion proteins engineered to bind fibroblast growth factor (FGF) molecules (ligands) efficiently in vitro and in vivo, thereby competing with endogenous FGF for binding sites on membrane- bound FGFRs. This inhibition of FGF/FGFR binding reduces biological signal transduction mediated by FGF/FGFR binding. [0008] The domain structure of soluble fusion proteins according to the invention comprises:
D2-L-D3-FC, wherein :
D2 consists essentially of the Ig-like domain 2 from FGFR2 (R2D2; SEQ ID NO: 49), FGFR3 (R3D2 ; SEQ ID NO: 50) or FGFR4 (R4D2; SEQ ID NO: 51) ;
L is DVLERxyHR (Leu loop; SEQ ID NO: 59) or DWERxyHR (VaI loop; SEQ ID NO: 60) , wherein x is S or W, and y is P or
R; D3 consists essentially of the Ig-like domain 3 of FGFR2- IHb (R2D3b; SEQ ID NO: 54), FGFR2-IIIC (R2D3C; SEQ ID NO: 55), FGFR3-IIIb (R3D3b; SEQ ID NO: 56), FGFR3-IIIC (R3D3C; SEQ ID NO: 57) , or FGFR4-IIIC (R4D3C; SEQ ID NO: 58) ; and
Fc consists essentially of the Fc domain of a human immunoglobulin.
[0009] In some embodiments of the invention, D2 consists essentially of the Ig-like domain 2 from FGFR2. In some embodiments of the invention, D2 consists essentially of the Ig-like domain 2 from FGFR3. In some embodiments of the invention, D2 consists essentially of the Ig-like domain 2 from FGFR4.
In some embodiments of the invention, D3 consists essentially of the Ig-like domain 3 from FGFR2-IIIb. In some embodiments of the invention, D3 consists essentially of the Ig-like domain 3 from FGFR2-IIIC. In some embodiments of the invention, D3 consists essentially of the Ig-like domain 3 from FGFR3-IIIb. In some embodiments of the invention, D3 consists essentially of the Ig-like domain 3 from FGFR3-IIIc. In some embodiments - A -
of the invention, D3 consists essentially of the Ig-like domain 3 from FGFR4-IIIC.
[0010] In some embodiments of the invention, L consists essentially of DWERSPHR (wild-type VaI loop; SEQ ID NO: 53) . In some embodiments of the invention, L consists essentially of DVLERSPHR (wild-type Leu loop,- SEQ ID NO: 52) . [0011] In some embodiments of the invention, the immunoglobulin is IgGl. [0012] The invention also provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR2 , L consists essentially of DWERWPHR (the VaI loop comprising mutation S106W; SEQ ID NO: 61) and D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIC. The invention also provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR2 , L consists essentially of DWERSRHR (the VaI loop comprising mutation P107R; SEQ ID NO: 62) and D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIc. The invention further provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR2 , L consists essentially of DWERWRHR (the VaI loop comprising mutations S106W and P107R; SEQ ID NO: 63) and D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIc. [0013] The invention also provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR2 , L consists essentially of DWERSRHR (the VaI loop comprising mutation P107R; SEQ ID NO: 62) and D3 consists essentially of the Ig-like domain 3 of FGFR3-IIIC. The invention also provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR4 , L consists essentially of DWERSRHR (the VaI loop comprising mutation P106R; SEQ ID NO: 62) and D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIc. The invention also provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR4 , L consists essentially of DVLERSRHR (the Leu loop comprising mutation P106R; SEQ ID NO: 64) and D3 consists essentially of the Ig- like domain 3 of FGFR2-IIIc.
[0014] The invention also provides fusion proteins wherein the domain structure is: R2D2 Leu Loop R3D3b Fc,-
R2D2 Leu Loop R3D3c Fc; R2D2 Leu Loop R4D3C Fc; R2D2 VaI Loop R2D3b Fc; R2D2 VaI Loop R2D3c Fc; R2D2 VaI Loop R3D3b Fc;
R2D2 VaI Loop R3D3C Fc; R2D2 VaI Loop R4D3C Fc; R3D2 Leu Loop R2D3b Fc ; R3D2 Leu Loop R2D3c Fc; R3D2 Leu Loop R3D3b Fc;
R3D2 Leu Loop R3D3c Fc; R3D2 Leu Loop R4D3c Fc; R3D2 VaI Loop R2D3b Fc; R3D2 VaI Loop R2D3c Fc; R4D2 Leu Loop R2D3b Fc;
R4D2 Leu Loop R2D3C Fc ; R4D2 Leu Loop R3D3b Fc ; R4D2 Leu Loop R3D3c Fc ; R4D2 Leu Loop R4D3C Fc ; R4D2 VaI Loop R2D3b Fc ;
R4D2 VaI Loop R2D3C Fc; R2D2 VaI S106W R2D3C Fc; R2D2 VaI P107R R2D3C Fc; R2D2 VaI S106W P107R R2D3C Fc ; R2D2 VaI P107R R3D3C Fc;
R4D2 VaI P106R R2D3C Fc; or R4D2 Leu P106R R2D3C Fc.
[0015] In preferred embodiments, the fusion proteins have the domain structure : R2D2 VaI Loop R2D3C Fc; R2D2 VaI Loop R3D3C Fc; R4D2 VaI Loop R2D3C Fc ; R2D2 VaI P107R Loop R2D3c Fc; R4D2 Leu Loop R2D3C Fc ;
R2D2 VaI P107R Loop R3D3C Fc; R4D2 VaI P106R Loop R2D3C Fc; or R4D2 Leu P106R Loop R2D3c Fc.
[0016] The invention also provides a nucleic acid comprising a nucleotide sequence that encodes the FGF-binding fusion protein comprising the domain structure D2-L-D3-Fc as described above. The invention also provides an expression vector comprising the nucleic acid and a host cell comprising the expression vector. [0017] The invention further provides a method of making the FGF-binding fusion protein comprising the domain structure D2- L-D3-Fc, the method comprising the steps of (a) culturing the host cell containing the expression vector comprising the nucleic acid comprising the nucleotide sequence that encodes the FGF-binding fusion protein under conditions so that the host cell expresses the FGF-binding fusion protein, and (b) harvesting the fusion protein.
[0018] The invention also provides a method of inhibiting binding of an FGF molecule to an FGF receptor, comprising contacting the FGF-binding fusion protein of the invention with the FGF molecule. In some embodiments of the invention, the FGF molecule, FGF receptor and FGF-binding fusion protein are in vivo.
[0019] The invention also provides a method of inhibiting proliferation of a tumor cell in vitro, comprising contacting the tumor cell with the FGF-binding fusion protein. The invention also provides a method of inhibiting proliferation of a tumor cell in a mammal, comprising administering to the mammal an effective amount of the FGF-binding fusion protein. The invention further provides a method of treating a tumor in a mammal, comprising administering to the mammal an effective amount of the FGF-binding fusion protein. [0020] The invention also provides for pharmaceutical compositions comprising the fusion proteins described above.
Brief Description of the Drawings
[0021] FIG. 1 is a schematic drawing of the domain structure of native FGFR alpha IHb and FGFR alpha IIIc (prior art) .
[0022] FIG. 2 is a sequence alignment of the FGFR domain 2 sequences, D2-D3 loop sequences, and FGFR domain 3 sequences that are used in different combinations to make the FGF-binding fusion proteins of the invention.
[0023] FIG. 3 is a graph summarizing data showing the effect of the R2D2 VaI Loop R2D3 Fc fusion protein in an FGF-dependent proliferation assay. An Fc protein was also tested as a control .
Detailed Description of the Invention
[0024] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. See, e.g., Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989) ;
Ausubel et al . , Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1990) ; Principles th of Neural Science, 4 ed. , Eric R. Kandel, James H. Schwart, Thomas M. Jessell editors. McGraw-Hill/Appleton & Lange : New York, NY (2000) . The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular neurobiology and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art . Conventional techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients .
[0025] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers .
Definitions
[0026] "Administering" means delivering in a manner which is effected or performed using any of the various methods and delivery systems known to those skilled in the art.
Administering can be performed, for example, topically, intravenously, pericardially, orally, via implant, transmucosally, transdermally, intramuscularly, subcutaneously, intraperitoneally, intrathecally, intralymphatically, intralesionally, or epidurally. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
[0027] "Expression vector" means a nucleic acid encoding a nucleic acid of interest and/or a protein of interest, which nucleic acid, when placed in a cell, permits the expression of the nucleic acid or protein of interest. Expression vectors are well known in the art. - S -
[0028] "Fc" means the immunoglobulin constant region. A native sequence Fc or constant region comprises an amino acid sequence identical to the amino acid sequence of an Fc or constant region found in nature. A variant or altered Fc or constant region comprises an amino acid sequence which differs from that of a native sequence heavy chain region by virtue of at least one amino acid modification, insertion, or deletion, for example. Additionally, the variant constant region may contain one or more amino acid substitutions, deletions, or insertions that results in altered post-translational modifications, including, for example, an altered glycosylation pattern.
[0029] "Host cell" means a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing a selected gene of interest. Host cells are well known in the art.
[0030] "Including" means including without limitation.
[0031] "Inhibiting" the onset of a disorder means lessening the likelihood of the disorder's onset, or preventing the onset of the disorder entirely.
[0032] "Nucleic acid" means any nucleic acid molecule, including DNA, RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR
Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996- 1997, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA) .
[0033] "Protein" means a polymer of amino acid residues. The amino acid residues can be naturally occurring or chemical analogues thereof. Proteins also can include modifications such as glycosylation, lipid attachment, sulfation, hydroxylation, and ADP-ribosylation .
[0034] "Subject" means a mammal, including a human. [0035] "Therapeutically effective amount" means an amount of an agent which, when administered to a subject afflicted with a disease or disorder against which the agent is effective, produces a beneficial effect with respect to the disease or disorder.
[0036] "Vector" means a molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell .
General Description
[0037] The invention provides soluble fusion proteins comprising D2 , L and D3 , which together form the FGF-binding region of the fusion proteins. The fusion protein lacks the transmembrane region and the tyrosine kinase domain found in native FGFRs. Consequently, the fusion protein is soluble in physiological fluids. When the fusion protein binds FGF molecules (ligands) , there is no FGF/FGFR-mediated signal transduction. Therefore, fusion proteins of the invention can be classified as "decoy receptors" or "ligand traps" because the ligand binds to them in vivo, thereby competing with the functional endogenous receptors. To the extent that the endogenous ligands bind to the soluble fusion proteins of the invention, the ligands are diverted or sequestered, with the result being less FGF/FGFR-mediated signaling than would occur in the absence of the soluble fusion proteins.
FGF-Binding Fusion Proteins
[0038] The invention provides a variety of soluble FGF- binding fusion proteins. [0039] The general structure of the FGF-binding fusion proteins according to the invention comprises D2-L-D3-Fc. D2 may consist essentially of the Ig- like domain 2 from FGFR2 (R2D2; SEQ ID NO: 49), FGFR3 (R3D2 ; SEQ ID NO: 50) or FGFR4 (R4D2; SEQ ID NO: 51) . [0040] L may consist essentially of DVLERSPHR (Leu loop,- SEQ ID NO: 52) or DWERSPHR (VaI loop; SEQ ID NO : 53) . The L region may also comprise mutated loop sequences such as DWERWPHR (VaI loop comprising mutation S106W; SEQ ID NO: 61) , DWERSRHR (VaI loop comprising mutation P107R; SEQ ID NO: 62), DWERWRHR (VaI loop comprising mutations S106W and P107R; SEQ ID NO: 63) , and DVLERSRHR (Leu loop comprising mutation P106R; SEQ ID NO: 64) . In certain embodiments, the invention provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR2 and L consists essentially of DWERSRHR (VaI loop comprising mutation P107R, SEQ ID NO: 62) . In other embodiments, the invention provides fusion proteins wherein D2 consists essentially of the Ig-like domain 2 of FGFR4 and L consists essentially of DWERSRHR (VaI loop comprising mutation P106R, SEQ ID NO: 62) .
[0041] D3 may consist essentially of the Ig-like domain 3 of FGFR2-IIIb (R2D3b; SEQ ID NO: 54), FGFR2-IIIC (R2D3C; SEQ ID NO: 55), FGFR3-IIIb (R3D3b; SEQ ID NO: 56), FGFR3-IIIC (R3D3C; SEQ ID NO: 57) , or FGFR4-IIIC (R4D3C; SEQ ID NO : 58) . [0042] Fc may consist essentially of the Fc domain of a human immunoglobulin.
[0043] Twenty-eight specific FGF-binding fusion proteins were constructed as described in Example 1 and expressed in 293T cells as described in Example 2. The fusion proteins are designated as follows:
1. R2D2 Leu R3D3b Fc
2. R2D2 Leu R3D3c Fc
3. R2D2 Leu R4D3C Fc
4. R2D2 VaI R2D3b Fc 5. R2D2 VaI R2D3c Fc
6. R2D2 VaI R3D3b Fc
7. R2D2 VaI R3D3c Fc
8. R2D2 VaI R4D3C Fc
9. R3D2 Leu R2D3b Fc 10. R3D2 Leu R2D3c Fc
11. R3D2 Leu R3D3b Fc
12. R3D2 Leu R3D3c Fc
13. R3D2 Leu R4D3c Fc
14. R3D2 VaI R2D3b Fc 15. R3D2 VaI R2D3c Fc
16. R4D2 Leu R2D3b Fc
17. R4D2 Leu R2D3c Fc
18. R4D2 Leu R3D3b Fc 19. R4D2 Leu R3D3c Fc
20. R4D2 Leu R4D3c Fc
21. R4D2 VaI R2D3b Fc
22. R4D2 VaI R2D3C Fc
23. R2D2 VaI S106W R2D3c Fc 24. R2D2 VaI P107R R2D3c Fc
25. R2D2 VaI S106W P107R R2D3C Fc
26. R2D2 VaI P107R R3D3c Fc
27. R4D2 VaI P106R R2D3c Fc
28. R4D2 Leu P106R R2D3c Fc
The nucleotide and amino acid sequences of each FGF-binding fusion protein are provided in Table 1.
Table 1. Nucleotide and Amino Acid Sequences of FGF-binding
Fusion
Proteins (FGF Traps)
Trap No.
Trap Description R2D2 Leu R3D3b Fc
DNA SEQ ID NO. 65
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccaac
61 agtaacaaca agagagcacc atactggacc aacacagaaa agatggaaaa gcggctccat
121 gctgtgcctg cggccaacac tgtcaagttt cgctgcccag ccggggggaa cccaatgcca
181 accatgcggt ggctgaaaaa cgggaaggag tttaagcagg agcatcgcat tggaggctac
241 aaggtacgaa accagcactg gagcctcatt atggaaagtg tggtcccatc tgacaaggga
301 aattatacct gtgtggtgga gaatgaatac gggtccatca atcacacgta ccacctggat
361 gtgctggagc ggtccccgca ccggcccatc ctgcaggctg ggctgccggc caaccagaca
421 gccgttctag gcagtgacgt ggagttccac tgcaaggtgt acagcgatgc acagccacac
481 atccagtggc tgaagcacgt ggaagtgaac ggcagcaagg tgggccctga cggcacgccc
541 tacgtcactg tactcaagtc ctggatcagt gagagtgtgg aggccgacgt gcgcctccgc
601 ctggccaatg tgtcggagcg ggacgggggc gagtacctct gtcgagccac caatttcata
661 ggcgtggccg agaaggcctt ttggctgagc gttcacgggc cccgagcagc cgaggaggag
721 ctggtggagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa
781 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc
841 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc
901 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag
961 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
1021 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag
1081 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca
1141 tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat
1201 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
1261 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
1321 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac
1381 aaccactaca cgcagaagag cctctccctg tctccgggta aatga
Protein SEQ ID NO. 95
1 nsnnkrapyw tntekmekrl havpaantvk frcpaggnpm ptmrwlkngk efkqeh 61 ykvrnqhws1 imeswpsdk gnytcwene ygsinhtyhl dvlersphrp ilqagl 121 tavlgsdvef hckvysdaqp hiqwlkhvev ngskvgpdgt pyvtvlkswi sesvea 181 rlanvserdg geylcratnf igvaekafwl svhgpraaee elvepkscdk thtcpp 241 ellggpsvfl fppkpkdtlm isrtpevtcv wdvshedpe vkfnwyvdgv evhnak 301 eeqynstyrv vsvltvlhqd wlngkeykck vsnkalpapi ektiskakgq prepqv
361 psreemtknq vsltclvkgf ypsdiavewe sngqpennyk ttppvldsdg sfflys
421 dksrwqqgnv fscsvmheal hnhytqksls lspgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 2
Trap Description R2D2 Leu R3D3C Fc
DNA SEQ ID NO. 66
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccaac
61 agtaacaaca agagagcacc atactggacc aacacagaaa agatggaaaa gcggctccat
121 gctgtgcctg cggccaacac tgtcaagttt cgctgcccag ccggggggaa cccaatgcca
181 accatgcggt ggctgaaaaa cgggaaggag tttaagcagg agcatcgcat tggaggctac
241 aaggtacgaa accagcactg gagcctcatt atggaaagtg tggtcccatc tgacaaggga
301 aattatacct gtgtggtgga gaatgaatac gggtccatca atcacacgta ccacctggat
361 gtgctggagc ggtccccgca ccggcccatc ctgcaggctg ggctgccggc caaccagaca
421 gccgttctag gcagtgacgt ggagttccac tgcaaggtgt acagcgatgc acagccacac
481 atccagtggc tgaagcacgt ggaagtgaac ggcagcaagg tgggccctga cggcacgccc
541 tacgtcactg tactcaagac tgcaggcgct aacaccaccg acaaggagct agaggttctg
601 tccttgcaca atgtcacctt tgaggacgcg ggggagtaca cctgcctggc gggcaattct
661 attgggtttt cccatcactc tgcgtggctg gtggtgctgc cagctgagga ggagctggtg
721 gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg
781 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg
841 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc
901 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
961 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat
1021 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc
1081 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg
1141 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc
1201 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
1261 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc
1321 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac
1381 tacacgcaga agagcctctc cctgtctccg ggtaaatga
Protein SEQ ID NO. 96
1 nsnnkrapyw tntekmekrl havpaantvk frcpaggnpm ptmrwlkngk efkqehrigg
61 ykvrnqhwsl imeswpsdk gnytcwene ygsinhtyhl dvlersphrp ilqaglpanq
121 tavlgsdvef hckvysdaqp hiqwlkhvev ngskvgpdgt pyvtvlktag anttdkelev
181 lslhnvtfed ageytclagn sigfshhsaw lwlpaeeel vepkscdkth tcppcpapel
241 lggpsvflfp pkpkdtlmis rtpevtcvw dvshedpevk fnwyvdgvev hnaktkpree
301 qynstyrws vltvlhqdwl ngkeykckvs nkalpapiek tiskakgqpr epqvytlpps
361 reemtknqvs ltclvkgfyp sdiavewesn gqpennyktt ppvldsdgsf flyskltvdk
421 srwqqgnvfs csvmhealhn hytqkslsls pgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence
Table 1 continued:
Figure imgf000016_0001
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 4
Trap Description R2D2 VaI R2D3b Fc
DNA SEQ ID NO. 70
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccaac
61 agtaacaaca agagagcacc atactggacc aacacagaaa agatggaaaa gcggctccat
121 gctgtgcctg cggccaacac tgtcaagttt cgctgcccag ccggggggaa cccaatgcca
181 accatgcggt ggctgaaaaa cgggaaggag tttaagcagg agcatcgcat tggaggctac
241 aaggtacgaa accagcactg gagcctcatt atggaaagtg tggtcccatc tgacaaggga
301 aattatacct gtgtggtgga gaatgaatac gggtccatca atcacacgta ccacctggat
361 gttgtggagc gatcgcctca ccggcccatc ctccaagccg gactgccggc aaatgcctcc
421 acagtggtcg gaggagacgt agagtttgtc tgcaaggttt acagtgatgc ccagccccac
481 atccagtgga tcaagcacgt ggaaaagaac ggcagtaaat acgggcccga cgggctgccc
541 tacctcaagg ttctcaagca ctcggggata aatagttcca atgcagaagt gctggctctg
601 ttcaatgtga ccgaggcgga tgctggggaa tatatatgta aggtctccaa ttatataggg
661 caggccaacc agtctgcctg gctcactgtc ctgccaaaac agcaagcgcc tggaagagaa
721 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg
781 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc
841 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac
901 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac
961 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc
1021 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc
1081 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag
1141 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac
1201 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
1261 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
1321 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
1381 acgcagaaga gcctctccct gtctccgggt aaatga
Protein SEQ ID 100 NO.
1 nsnnkrapyw tntekmekrl havpaantvk frcpaggnpm ptmrwlkngk efkqehrigg
61 ykvrnqhwsl imeswpsdk gnytcwene ygsinhtyhl dwersphrp ilqaglpana
121 stwggdvef vckvysdaqp hiqwikhvek ngskygpdgl pylkvlkhsg inssnaevla
181 lfnvteadag eyickvsnyi gqanqsawlt vlpkqqapgr epkscdktht cppcpapell
241 ggpsvfIfpp kpkdtlmisr tpevtcvwd vshedpevkf nwyvdgvevh naktkpreeq
301 ynstyrwsv ltvlhqdwln gkeykckvsn kalpapiekt iskakgqpre pqvytlppsr
361 eemtknqvsl tclvkgfyps diavewesng qpennykttp pvldsdgsff lyskltvdks
421 rwqqgnvfsc svmhealhnh ytqkslslsp gk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 5
Trap R2D2 VaI R2D3C Fc
Description
DNA SEQ ID 71 NO.
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccaac
61 agtaacaaca agagagcacc atactggacc aacacagaaa agatggaaaa gcggctccat
121 gctgtgcctg cggccaacac tgtcaagttt cgctgcccag ccggggggaa cccaatgcca
181 accatgcggt ggctgaaaaa cgggaaggag tttaagcagg agcatcgcat tggaggctac
241 aaggtacgaa accagcactg gagcctcatt atggaaagtg tggtcccatc tgacaaggga
301 aattatacct gtgtggtgga gaatgaatac gggtccatca atcacacgta ccacctggat
361 gttgtggagc gatcgcctca ccggcccatc ctccaagccg gactgccggc aaatgcctcc
421 acagtggtcg gaggagacgt agagtttgtc tgcaaggttt acagtgatgc ccagccccac
481 atccagtgga tcaagcacgt ggaaaagaac ggcagtaaat acgggcccga cgggctgccc
541 tacctcaagg ttctcaaggc cgccggtgtt aacaccacgg acaaagagat tgaggttctc
601 tatattcgga atgtaacttt tgaggacgct ggggaatata cgtgcttggc gggtaattct
661 attgggatat cctttcactc tgcatggttg acagttctgc cagcgcctgg aagagaaccc
721 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
781 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
841 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
901 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
961 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
1021 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
1081 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
1141 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
1201 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1261 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
1321 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1381 cagaagagcc tctccctgtc tccgggtaaa tga
Protein SEQ 101
ID NO.
1 nsnnkrapyw tntekmekrl havpaantvk frcpaggnpm ptmrwlkngk efkqehrigg
61 ykvrnqhwsl imeswpsdk gnytcwene ygsinhtyhl dwersphrp ilqaglpana
121 stwggdvef vckvysdaqp hiqwikhvek ngskygpdgl pylkvlkaag vnttdkeiev
181 lyirnvtfed ageytclagn sigisfhsaw ltvlpapgre pkscdkthtc ppcpapellg
241 gpsvflfppk pkdtlmisrt pevtcvwdv shedpevkfn wyvdgvevhn aktkpreeqy
301 nstyrwsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsre
361 emtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsffl yskltvdksr
421 wqqgnvfscs vmhealhnhy tqkslslspg k
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 6
Trap R2D2 VaI R3D3b Fc
Description
DNA SEQ ID NO. 7;
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccaac
61 agtaacaaca agagagcacc atactggacc aacacagaaa agatggaaaa gcggctccat
121 gctgtgcctg cggccaacac tgtcaagttt cgctgcccag ccggggggaa cccaatgcca
181 accatgcggt ggctgaaaaa cgggaaggag tttaagcagg agcatcgcat tggaggctac
241 aaggtacgaa accagcactg gagcctcatt atggaaagtg tggtcccatc tgacaaggga
301 aattatacct gtgtggtgga gaatgaatac gggtccatca atcacacgta ccacctggat
361 gttgtggagc gatcgcctca ccggcccatc ctgcaggctg ggctgccggc caaccagaca
421 gccgttctag gcagtgacgt ggagttccac tgcaaggtgt acagcgatgc acagccacac
481 atccagtggc tgaagcacgt ggaagtgaac ggcagcaagg tgggccctga cggcacgccc
541 tacgtcactg tactcaagtc ctggatcagt gagagtgtgg aggccgacgt gcgcctccgc
601 ctggccaatg tgtcggagcg ggacgggggc gagtacctct gtcgagccac caatttcata
661 ggcgtggccg agaaggcctt ttggctgagc gttcacgggc cccgagcagc cgaggaggag
721 ctggtggagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa
781 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc
841 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc
901 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag
961 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
1021 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag
1081 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca
1141 tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat
1201 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
1261 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
1321 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac
1381 aaccactaca cgcagaagag cctctccctg tctccgggta aatga
Protein SEQ ID 102 NO.
1 nsnnkrapyw tntekmekrl havpaantvk frcpaggnpm ptmrwlkngk efkqehrigg
61 ykvrnqhws1 imeswpsdk gnytcwene ygsinhtyhl dwersphrp ilqaglpanq
121 tavlgsdvef hckvysdaqp hiqwlkhvev ngskvgpdgt pyvtvlkswi sesveadvrl
181 rlanvserdg geylcratnf igvaekafwl svhgpraaee elvepkscdk thtcppcpap
241 ellggpsvf1 fppkpkdtlm isrtpevtcv wdvshedpe vkfnwyvdgv evhnaktkpr
301 eeqynstyrv vsvltvlhqd wlngkeykck vsnkalpapi ektiskakgq prepqvytlp
361 psreemtknq vsltclvkgf ypsdiavewe sngqpennyk ttppvldsdg sfflyskltv
421 dksrwqqgnv fscsvmheal hnhytqksls lspgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 7
Trap Description R2D2 VaI R3D3C FC
DNA SEQ ID NO. 73
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccaac
61 agtaacaaca agagagcacc atactggacc aacacagaaa agatggaaaa gcggctccat
121 gctgtgcctg cggccaacac tgtcaagttt cgctgcccag ccggggggaa cccaatgcca
181 accatgcggt ggctgaaaaa cgggaaggag tttaagcagg agcatcgcat tggaggctac
241 aaggtacgaa accagcactg gagcctcatt atggaaagtg tggtcccatc tgacaaggga
301 aattatacct gtgtggtgga gaatgaatac gggtccatca atcacacgta ccacctggat
361 gttgtggagc gatcgcctca ccggcccatc ctgcaggctg ggctgccggc caaccagaca
421 gccgttctag gcagtgacgt ggagttccac tgcaaggtgt acagcgatgc acagccacac
481 atccagtggc tgaagcacgt ggaagtgaac ggcagcaagg tgggccctga cggcacgccc
541 tacgtcactg tactcaagac tgcaggcgct aacaccaccg acaaggagct agaggttctg
601 tccttgcaca atgtcacctt tgaggacgcg ggggagtaca cctgcctggc gggcaattct
661 attgggtttt cccatcactc tgcgtggctg gtggtgctgc cagctgagga ggagctggtg
721 gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg
781 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg
841 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc
901 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
961 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat
1021 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc
1081 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg
1141 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc
1201 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
1261 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc
1321 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac
1381 tacacgcaga agagcctctc cctgtctccg ggtaaatga
Protein SEQ ID NO. 103
1 nsnnkrapyw tntekmekrl havpaantvk frcpaggnpm ptmrwlkngk efkqehrigg
61 ykvrnqhwsl imeswpsdk gnytcwene ygsinhtyhl dwersphrp ilqaglpanq
121 tavlgsdvef hckvysdaqp hiqwlkhvev ngskvgpdgt pyvtvlktag anttdkelev
181 lslhnvtfed ageytclagn sigfshhsaw lwlpaeeel vepkscdkth tcppcpapel
241 lggpsvflfp pkpkdtlmis rtpevtcvw dvshedpevk fnwyvdgvev hnaktkpree
301 qynstyrws vltvlhqdwl ngkeykckvs nkalpapiek tiskakgqpr epqvytlpps
361 reemtknqvs ltclvkgfyp sdiavewesn gqpennyktt ppvldsdgsf flyskltvdk
421 srwqqgnvfs csvmhealhn hytqkslsls pgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 8
Trap Description R2D2 VaI R4D3C FC
DNA SEQ ID NO. 74
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccaac
61 agtaacaaca agagagcacc atactggacc aacacagaaa agatggaaaa gcggctccat
121 gctgtgcctg cggccaacac tgtcaagttt cgctgcccag ccggggggaa cccaatgcca
181 accatgcggt ggctgaaaaa cgggaaggag tttaagcagg agcatcgcat tggaggctac
241 aaggtacgaa accagcactg gagcctcatt atggaaagtg tggtcccatc tgacaaggga
301 aattatacct gtgtggtgga gaatgaatac gggtccatca atcacacgta ccacctggat
361 gttgtggagc gatcgcctca ccggcccatc ctgcaggccg ggctcccggc caacaccaca
421 gccgtggtgg gcagcgacgt ggagctgctg tgcaaggtgt acagcgatgc ccagccccac
481 atccagtggc tgaagcacat cgtcatcaac ggcagcagct tcggagccga cggtttcccc
541 tatgtgcaag tcctaaagac tgcagacatc aatagctcag aggtggaggt cctgtacctg
601 cggaacgtgt cagccgagga cgcaggcgag tacacctgcc tcgcaggcaa ttccatcggc
661 ctctcctacc agtctgcctg gctcacggtg ctgccagagg aggaccccac atggaccgca
721 gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg
781 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg
841 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc
901 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
961 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat
1021 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc
1081 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg
1141 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc
1201 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
1261 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc
1321 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac
1381 tacacgcaga agagcctctc cctgtctccg ggtaaatga
Protein SEQ ID NO. 104
1 nsnnkrapyw tntekmekrl havpaantvk frcpaggnpm ptmrwlkngk efkqehrigg
61 ykvrnqhwsl imeswpsdk gnytcwene ygsinhtyhl dwersphrp ilqaglpant
121 tawgsdvel lckvysdaqp hiqwlkhivi ngssfgadgf pyvqvlktad inssevevly
181 lrnvsaedag eytclagnsi glsyqsawlt vlpeedptwt aepkscdkth tcppcpapel
241 lggpsvflfp pkpkdtlmis rtpevtcvw dvshedpevk fnwyvdgvev hnaktkpree
301 qynstyrws vltvlhqdwl ngkeykckvs nkalpapiek tiskakgqpr epqvytlpps
361 reemtknqvs ltclvkgfyp sdiavewesn gqpennyktt ppvldsdgsf flyskltvdk
421 srwqqgnvfs csvmhealhn hytqkslsls pgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No . 9
Trap Description R3D2 Leu R2D3b FC
DNA SEQ ID NO. 77
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgac
61 acaggtgtgg acacaggggc tccttattgg actcgcccgg agcgaatgga taagaaactg
121 ctggctgtgc cagccgcaaa cactgtccgc ttccgctgcc cagctgctgg caaccctacc
181 ccctccatct cctggctgaa gaatggcaga gagttccgag gggagcatcg cattgggggc
241 atcaagctcc ggcaccagca gtggagcttg gtcatggaaa gtgtggtacc ctccgatcgt
301 ggcaactata cctgtgtagt tgagaacaag tttggcagca tccggcagac atacacactg
361 gatgtgctgg agcggtcccc gcaccggccc atcctccaag ccggactgcc ggcaaatgcc
421 tccacagtgg tcggaggaga cgtagagttt gtctgcaagg tttacagtga tgcccagccc
481 cacatccagt ggatcaagca cgtggaaaag aacggcagta aatacgggcc cgacgggctg
541 ccctacctca aggttctcaa gcactcgggg ataaatagtt ccaatgcaga agtgctggct
601 ctgttcaatg tgaccgaggc ggatgctggg gaatatatat gtaaggtctc caattatata
661 gggcaggcca accagtctgc ctggctcact gtcctgccaa aacagcaagc gcctggaaga
721 gaacccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg
781 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg
841 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc
901 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
961 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat
1021 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc
1081 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg
1141 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc
1201 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
1261 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc
1321 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac
1381 tacacgcaga agagcctctc cctgtctccg ggtaaatga
Protein SEQ ID NO. 107
1 dtgvdtgapy wtrpermdkk llavpaantv rfrcpaagnp tpsiswlkng refrgehrig
61 giklrhqqws lvmeswpsd rgnytcwen kfgsirqtyt ldvlersphr pilqaglpan
121 astwggdve fvckvysdaq phiqwikhve kngskygpdg lpylkvlkhs ginssnaevl
181 alfnvteada geyickvsny igqanqsawl tvlpkqqapg repkscdkth tcppcpapel
241 lggpsvflfp pkpkdtlmis rtpevtcvw dvshedpevk fnwyvdgvev hnaktkpree
301 qynstyrws vltvlhqdwl ngkeykckvs nkalpapiek tiskakgqpr epqvytlpps
361 reemtknqvs ltclvkgfyp sdiavewesn gqpennyktt ppvldsdgsf flyskltvdk
421 srwqqgnvfs csvmhealhn hytqkslsls pgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Figure imgf000023_0001
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 11
Trap Description R3D2 Leu R3D3b Fc
DNA SEQ ID NO. 79
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgac
61 acaggtgtgg acacaggggc tccttattgg actcgcccgg agcgaatgga taagaaactg
121 ctggctgtgc cagccgcaaa cactgtccgc ttccgctgcc cagctgctgg caaccctacc
181 ccctccatct cctggctgaa gaatggcaga gagttccgag gggagcatcg cattgggggc
241 atcaagctcc ggcaccagca gtggagcttg gtcatggaaa gtgtggtacc ctccgatcgt
301 ggcaactata cctgtgtagt tgagaacaag tttggcagca tccggcagac atacacactg
361 gatgtgctgg agcggtcccc gcaccggccc atcctgcagg ctgggctgcc ggccaaccag
421 acagccgttc taggcagtga cgtggagttc cactgcaagg tgtacagcga tgcacagcca
481 cacatccagt ggctgaagca cgtggaagtg aacggcagca aggtgggccc tgacggcacg
541 ccctacgtca ctgtactcaa gtcctggatc agtgagagtg tggaggccga cgtgcgcctc
601 cgcctggcca atgtgtcgga gcgggacggg ggcgagtacc tctgtcgagc caccaatttc
661 ataggcgtgg ccgagaaggc cttttggctg agcgttcacg ggccccgagc agccgaggag
721 gagctggtgg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct
781 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg
841 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag
901 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg
961 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac
1021 tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc
1081 gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc
1141 ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc
1201 tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag
1261 accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg
1321 gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg
1381 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatga
Protein SEQ ID NO. 109
1 dtgvdtgapy wtrpermdkk llavpaantv rfrcpaagnp tpsiswlkng refrgehrig
61 giklrhqqws lvmeswpsd rgnytcwen kfgsirqtyt ldvlersphr pilqaglpan
121 qtavlgsdve fhckvysdaq phiqwlkhve vngskvgpdg tpyvtvlksw isesveadvr
181 lrlanvserd ggeylcratn figvaekafw lsvhgpraae eelvepkscd kthtcppcpa
241 pellggpsvf lfppkpkdtl misrtpevtc vwdvshedp evkfnwyvdg vevhnaktkp
301 reeqynstyr wsvltvlhq dwlngkeykc kvsnkalpap iektiskakg qprepqvytl
361 ppsreemtkn qvsltclvkg fypsdiavew esngqpenny kttppvldsd gsfflysklt
421 vdksrwqqgn vfscsvmhea lhnhytqksl slspgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No . 12
Trap Description R3D2 Leu R3D3C Fc
DNA SEQ ID NO. 80
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgac
61 acaggtgtgg acacaggggc tccttattgg actcgcccgg agcgaatgga taagaaactg
121 ctggctgtgc cagccgcaaa cactgtccgc ttccgctgcc cagctgctgg caaccctacc
181 ccctccatct cctggctgaa gaatggcaga gagttccgag gggagcatcg cattgggggc
241 atcaagctcc ggcaccagca gtggagcttg gtcatggaaa gtgtggtacc ctccgatcgt
301 ggcaactata cctgtgtagt tgagaacaag tttggcagca tccggcagac atacacactg
361 gatgtgctgg agcggtcccc gcaccggccc atcctgcagg ctgggctgcc ggccaaccag
421 acagccgttc taggcagtga cgtggagttc cactgcaagg tgtacagcga tgcacagcca
481 cacatccagt ggctgaagca cgtggaagtg aacggcagca aggtgggccc tgacggcacg
541 ccctacgtca ctgtactcaa gactgcaggc gctaacacca ccgacaagga gctagaggtt
601 ctgtccttgc acaatgtcac ctttgaggac gcgggggagt acacctgcct ggcgggcaat
661 tctattgggt tttcccatca ctctgcgtgg ctggtggtgc tgccagctga ggaggagctg
721 gtggagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc
781 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc
841 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag
901 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag
961 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg
1021 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa
1081 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc
1141 cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc
1201 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg
1261 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag
1321 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac
1381 cactacacgc agaagagcct ctccctgtct ccgggtaaat ga
Protein SEQ ID NO. 110
1 dtgvdtgapy wtrpermdkk llavpaantv rfrcpaagnp tpsiswlkng refrgehrig
61 giklrhqqws lvmeswpsd rgnytcwen kfgsirqtyt ldvlersphr pilqaglpan
121 qtavlgsdve fhckvysdaq phiqwlkhve vngskvgpdg tpyvtvlkta ganttdkele
181 vlslhnvtfe dageytclag nsigfshhsa wlwlpaeee lvepkscdkt htcppcpape
241 llggpsvfIf ppkpkdtlmi srtpevtcw vdvshedpev kfnwyvdgve vhnaktkpre
301 eqynstyrw svltvlhqdw lngkeykckv snkalpapie ktiskakgqp repqvytlpp
361 sreemtknqv sltclvkgfy psdiavewes ngqpennykt tppvldsdgs fflyskltvd
421 ksrwqqgnvf scsvmhealh nhytqkslsl spgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 13
Trap Description R3D2 Leu R4D3C FC
DNA SEQ ID NO. 81
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgac
61 acaggtgtgg acacaggggc tccttattgg actcgcccgg agcgaatgga taagaaactg
121 ctggctgtgc cagccgcaaa cactgtccgc ttccgctgcc cagctgctgg caaccctacc
181 ccctccatct cctggctgaa gaatggcaga gagttccgag gggagcatcg cattgggggc
241 atcaagctcc ggcaccagca gtggagcttg gtcatggaaa gtgtggtacc ctccgatcgt
301 ggcaactata cctgtgtagt tgagaacaag tttggcagca tccggcagac atacacactg
361 gatgtgctgg agcggtcccc gcaccggccc atcctgcagg ccgggctccc ggccaacacc
421 acagccgtgg tgggcagcga cgtggagctg ctgtgcaagg tgtacagcga tgcccagccc
481 cacatccagt ggctgaagca catcgtcatc aacggcagca gcttcggagc cgacggtttc
541 ccctatgtgc aagtcctaaa gactgcagac atcaatagct cagaggtgga ggtcctgtac
601 ctgcggaacg tgtcagccga ggacgcaggc gagtacacct gcctcgcagg caattccatc
661 ggcctctcct accagtctgc ctggctcacg gtgctgccag aggaggaccc cacatggacc
721 gcagagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc
781 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc
841 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag
901 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag
961 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg
1021 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa
1081 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc
1141 cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc
1201 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg
1261 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag
1321 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac
1381 cactacacgc agaagagcct ctccctgtct ccgggtaaat ga
Protein SEQ ID NO. 111
1 dtgvdtgapy wtrpermdkk llavpaantv rfrcpaagnp tpsiswlkng refrgehrig
61 giklrhqqws lvmeswpsd rgnytcwen kfgsirqtyt ldvlersphr pilqaglpan
121 ttawgsdve llckvysdaq phiqwlkhiv ingssfgadg fpyvqvlkta dinssevevl
181 ylrnvsaeda geytclagns iglsyqsawl tvlpeedptw taepkscdkt htcppcpape
241 llggpsvfIf ppkpkdtlmi srtpevtcw vdvshedpev kfnwyvdgve vhnaktkpre
301 eqynstyrw svltvlhqdw lngkeykckv snkalpapie ktiskakgqp repqvytlpp
361 sreemtknqv sltclvkgfy psdiavewes ngqpennykt tppvldsdgs fflyskltvd
421 ksrwqqgnvf scsvmhealh nhytqkslsl spgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 14
Trap Description R3D2 VaI R2D3b Fc
DNA SEQ ID NO. 82
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgac
61 acaggtgtgg acacaggggc tccttattgg actcgcccgg agcgaatgga taagaaactg
121 ctggctgtgc cagccgcaaa cactgtccgc ttccgctgcc cagctgctgg caaccctacc
181 ccctccatct cctggctgaa gaatggcaga gagttccgag gggagcatcg cattgggggc
241 atcaagctcc ggcaccagca gtggagcttg gtcatggaaa gtgtggtacc ctccgatcgt
301 ggcaactata cctgtgtagt tgagaacaag tttggcagca tccggcagac atacacactg
361 gatgttgtgg agcgatcgcc tcaccggccc atcctccaag ccggactgcc ggcaaatgcc
421 tccacagtgg tcggaggaga cgtagagttt gtctgcaagg tttacagtga tgcccagccc
481 cacatccagt ggatcaagca cgtggaaaag aacggcagta aatacgggcc cgacgggctg
541 ccctacctca aggttctcaa gcactcgggg ataaatagtt ccaatgcaga agtgctggct
601 ctgttcaatg tgaccgaggc ggatgctggg gaatatatat gtaaggtctc caattatata
661 gggcaggcca accagtctgc ctggctcact gtcctgccaa aacagcaagc gcctggaaga
721 gaacccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg
781 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg
841 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc
901 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
961 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat
1021 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc
1081 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg
1141 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc
1201 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
1261 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc
1321 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac
1381 tacacgcaga agagcctctc cctgtctccg ggtaaatga
Protein SEQ ID NO. 112
1 dtgvdtgapy wtrpermdkk llavpaantv rfrcpaagnp tpsiswlkng refrgehrig
61 giklrhqqws lvmeswpsd rgnytcwen kfgsirqtyt ldwersphr pilqaglpan
121 astwggdve fvckvysdaq phiqwikhve kngskygpdg lpylkvlkhs ginssnaevl
181 alfnvteada geyickvsny igqanqsawl tvlpkqqapg repkscdkth tcppcpapel
241 lggpsvflfp pkpkdtlmis rtpevtcvw dvshedpevk fnwyvdgvev hnaktkpree
301 qynstyrws vltvlhqdwl ngkeykckvs nkalpapiek tiskakgqpr epqvytlpps
361 reemtknqvs ltclvkgfyp sdiavewesn gqpennyktt ppvldsdgsf flyskltvdk
421 srwqqgnvfs csvmhealhn hytqkslsls pgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 15
Trap Description R3D2 VaI R2D3c FC
DNA SEQ ID NO. 83
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgac
61 acaggtgtgg acacaggggc tccttattgg actcgcccgg agcgaatgga taagaaactg
121 ctggctgtgc cagccgcaaa cactgtccgc ttccgctgcc cagctgctgg caaccctacc
181 ccctccatct cctggctgaa gaatggcaga gagttccgag gggagcatcg cattgggggc
241 atcaagctcc ggcaccagca gtggagcttg gtcatggaaa gtgtggtacc ctccgatcgt
301 ggcaactata cctgtgtagt tgagaacaag tttggcagca tccggcagac atacacactg
361 gatgttgtgg agcgatcgcc tcaccggccc atcctccaag ccggactgcc ggcaaatgcc
421 tccacagtgg tcggaggaga cgtagagttt gtctgcaagg tttacagtga tgcccagccc
481 cacatccagt ggatcaagca cgtggaaaag aacggcagta aatacgggcc cgacgggctg
541 ccctacctca aggttctcaa ggccgccggt gttaacacca cggacaaaga gattgaggtt
601 ctctatattc ggaatgtaac ttttgaggac gctggggaat atacgtgctt ggcgggtaat
661 tctattggga tatcctttca ctctgcatgg ttgacagttc tgccagcgcc tggaagagaa
721 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg
781 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc
841 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac
901 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac
961 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc
1021 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc
1081 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag
1141 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac
1201 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
1261 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
1321 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
1381 acgcagaaga gcctctccct gtctccgggt aaatga
Protein SEQ ID NO. 113
1 dtgvdtgapy wtrpermdkk llavpaantv rfrcpaagnp tpsiswlkng refrgehrig
61 giklrhqqws lvmeswpsd rgnytcwen kfgsirqtyt ldwersphr pilqaglpan
121 astwggdve fvckvysdaq phiqwikhve kngskygpdg lpylkvlkaa gvnttdkeie
181 vlyirnvtfe dageytclag nsigisfhsa wltvlpapgr epkscdktht cppcpapell
241 ggpsvfIfpp kpkdtlmisr tpevtcvwd vshedpevkf nwyvdgvevh naktkpreeq
301 ynstyrwsv ltvlhqdwln gkeykckvsn kalpapiekt iskakgqpre pqvytlppsr
361 eemtknqvsl tclvkgfyps diavewesng qpennykttp pvldsdgsff lyskltvdks
421 rwqqgnvfsc svmhealhnh ytqkslslsp gk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence 5172
- 28 -
Table 1 continued:
Trap No. 16
Trap Description R4D2 Leu R2D3b FC
DNA SEQ ID NO. 85
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccagt
61 tacccccagc aagcacccta ctggacacac ccccagcgca tggagaagaa actgcatgca
121 gtacctgcgg ggaacaccgt caagttccgc tgtccagctg caggcaaccc cacgcccacc
181 atccgctggc ttaaggatgg acaggccttt catggggaga accgcattgg aggcattcgg
241 ctgcgccatc agcactggag tctcgtgatg gagagcgtgg tgccctcgga ccgcggcaca
301 tacacctgcc tggtagagaa cgctgtgggc agcatccgct ataactacct gctagatgtg
361 ctggagcggt ccccgcaccg gcccatcctc caagccggac tgccggcaaa tgcctccaca
421 gtggtcggag gagacgtaga gtttgtctgc aaggtttaca gtgatgccca gccccacatc
481 cagtggatca agcacgtgga aaagaacggc agtaaatacg ggcccgacgg gctgccctac
541 ctcaaggttc tcaagcactc ggggataaat agttccaatg cagaagtgct ggctctgttc
601 aatgtgaccg aggcggatgc tggggaatat atatgtaagg tctccaatta tatagggcag
661 gccaaccagt ctgcctggct cactgtcctg ccaaaacagc aagcgcctgg aagagaaccc
721 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
781 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
841 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
901 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
961 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
1021 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
1081 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
1141 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
1201 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1261 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
1321 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1381 cagaagagcc tctccctgtc tccgggtaaa tga
Protein SEQ ID NO. 115
1 sypqqapywt hpqrmekklh avpagntvkf rcpaagnptp tirwlkdgqa fhgenriggi
61 rlrhqhwslv meswpsdrg tytclvenav gsirynylld vlersphrpi lqaglpanas
121 twggdvefv ckvysdaqph iqwikhvekn gskygpdglp ylkvlkhsgi nssnaevlal
181 fnvteadage yickvsnyig qanqsawltv lpkqqapgre pkscdkthtc ppcpapellg
241 gpsvflfppk pkdtlmisrt pevtcwvdv shedpevkfn wyvdgvevhn aktkpreeqy
301 nstyrwsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsre
361 emtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsff1 yskltvdksr
421 wqqgnvfscs vmhealhnhy tqkslslspg k
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Figure imgf000030_0001
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 18
Trap Description R4D2 Leu R3D3b Fc
DNA SEQ ID NO. 87
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccagt
61 tacccccagc aagcacccta ctggacacac ccccagcgca tggagaagaa actgcatgca
121 gtacctgcgg ggaacaccgt caagttccgc tgtccagctg caggcaaccc cacgcccacc
181 atccgctggc ttaaggatgg acaggccttt catggggaga accgcattgg aggcattcgg
241 ctgcgccatc agcactggag tctcgtgatg gagagcgtgg tgccctcgga ccgcggcaca
301 tacacctgcc tggtagagaa cgctgtgggc agcatccgct ataactacct gctagatgtg
361 ctggagcggt ccccgcaccg gcccatcctg caggctgggc tgccggccaa ccagacagcc
421 gttctaggca gtgacgtgga gttccactgc aaggtgtaca gcgatgcaca gccacacatc
481 cagtggctga agcacgtgga agtgaacggc agcaaggtgg gccctgacgg cacgccctac
541 gtcactgtac tcaagtcctg gatcagtgag agtgtggagg ccgacgtgcg cctccgcctg
601 gccaatgtgt cggagcggga cgggggcgag tacctctgtc gagccaccaa tttcataggc
661 gtggccgaga aggccttttg gctgagcgtt cacgggcccc gagcagccga ggaggagctg
721 gtggagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc
781 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc
841 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag
901 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag
961 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg
1021 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa
1081 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc
1141 cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc
1201 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg
1261 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag
1321 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac
1381 cactacacgc agaagagcct ctccctgtct ccgggtaaat ga
Protein SEQ ID NO. 117
1 sypqqapywt hpqrmekklh avpagntvkf rcpaagnptp tirwlkdgqa fhgenriggi
61 rlrhqhwslv meswpsdrg tytclvenav gsirynylld vlersphrpi lqaglpanqt
121 avlgsdvefh ckvysdaqph iqwlkhvevn gskvgpdgtp yvtvlkswis esveadvrlr
181 lanvserdgg eylcratnfi gvaekafwls vhgpraaeee lvepkscdkt htcppcpape
241 llggpsvfIf ppkpkdtlmi srtpevtcw vdvshedpev kfnwyvdgve vhnaktkpre
301 eqynstyrw svltvlhqdw lngkeykckv snkalpapie ktiskakgqp repqvytlpp
361 sreemtknqv sltclvkgfy psdiavewes ngqpennykt tppvldsdgs fflyskltvd
421 ksrwqqgnvf scsvmhealh nhytqkslsl spgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Figure imgf000032_0001
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Figure imgf000033_0001
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 21
Trap Description R4D2 VaI R2D3b FC
DNA SEQ ID NO. 91
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccagt
61 tacccccagc aagcacccta ctggacacac ccccagcgca tggagaagaa actgcatgca
121 gtacctgcgg ggaacaccgt caagttccgc tgtccagctg caggcaaccc cacgcccacc
181 atccgctggc ttaaggatgg acaggccttt catggggaga accgcattgg aggcattcgg
241 ctgcgccatc agcactggag tctcgtgatg gagagcgtgg tgccctcgga ccgcggcaca
301 tacacctgcc tggtagagaa cgctgtgggc agcatccgct ataactacct gctagatgtt
361 gtggagcgat cgcctcaccg gcccatcctc caagccggac tgccggcaaa tgcctccaca
421 gtggtcggag gagacgtaga gtttgtctgc aaggtttaca gtgatgccca gccccacatc
481 cagtggatca agcacgtgga aaagaacggc agtaaatacg ggcccgacgg gctgccctac
541 ctcaaggttc tcaagcactc ggggataaat agttccaatg cagaagtgct ggctctgttc
601 aatgtgaccg aggcggatgc tggggaatat atatgtaagg tctccaatta tatagggcag
661 gccaaccagt ctgcctggct cactgtcctg ccaaaacagc aagcgcctgg aagagaaccc
721 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
781 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
841 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
901 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
961 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
1021 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
1081 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
1141 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
1201 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1261 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
1321 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1381 cagaagagcc tctccctgtc tccgggtaaa tga
Protein SEQ ID NO. 121
1 sypqqapywt hpqrmekklh avpagntvkf rcpaagnptp tirwlkdgqa fhgenriggi
61 rlrhqhwslv meswpsdrg tytclvenav gsirynylld wersphrpi lqaglpanas
121 twggdvefv ckvysdaqph iqwikhvekn gskygpdglp ylkvlkhsgi nssnaevlal
181 fnvteadage yickvsnyig qanqsawltv lpkqqapgre pkscdkthtc ppcpapellg
241 gpsvflfppk pkdtlmisrt pevtcwvdv shedpevkfn wyvdgvevhn aktkpreeqy
301 nstyrwsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsre
361 emtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsff1 yskltvdksr
421 wqqgnvfscs vmhealhnhy tqkslslspg k
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Figure imgf000035_0001
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Figure imgf000036_0001
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 24
Trap Description R2D2 VaI P107R R2D3C Fc
DNA SEQ ID NO. 68
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccaac
61 agtaacaaca agagagcacc atactggacc aacacagaaa agatggaaaa gcggctccat
121 gctgtgcctg cggccaacac tgtcaagttt cgctgcccag ccggggggaa cccaatgcca
181 accatgcggt ggctgaaaaa cgggaaggag tttaagcagg agcatcgcat tggaggctac
241 aaggtacgaa accagcactg gagcctcatt atggaaagtg tggtcccatc tgacaaggga
301 aattatacct gtgtggtgga gaatgaatac gggtccatca atcacacgta ccacctggat
361 gttgtggagc gatcgcggca ccggcccatc ctccaagccg gactgccggc aaatgcctcc
421 acagtggtcg gaggagacgt agagtttgtc tgcaaggttt acagtgatgc ccagccccac
481 atccagtgga tcaagcacgt ggaaaagaac ggcagtaaat acgggcccga cgggctgccc
541 tacctcaagg ttctcaaggc cgccggtgtt aacaccacgg acaaagagat tgaggttctc
601 tatattcgga atgtaacttt tgaggacgct ggggaatata cgtgcttggc gggtaattct
661 attgggatat cctttcactc tgcatggttg acagttctgc cagcgcctgg aagagaaccc
721 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
781 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
841 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
901 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
961 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
1021 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa gaccatctcc
1081 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
1141 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
1201 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1261 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
1321 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1381 cagaagagcc tctccctgtc tccgggtaaa tga
Protein SEQ ID NO. 98
1 nsnnkrapyw tntekmekrl havpaantvk frcpaggnpm ptmrwlkngk efkqehrigg
61 ykvrnqhwsl imeswpsdk gnytcwene ygsinhtyhl dwersrhrp ilqaglpana
121 stwggdvef vckvysdaqp hiqwikhvek ngskygpdgl pylkvlkaag vnttdkeiev
181 lyirnvtfed ageytclagn sigisfhsaw ltvlpapgre pkscdkthtc ppcpapellg
241 gpsvflfppk pkdtlmisrt pevtcvwdv shedpevkfn wyvdgvevhn aktkpreeqy
301 nstyrwsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsre
361 emtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsff1 yskltvdksr
421 wqqgnvfscs vmhealhnhy tqkslslspg k
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No. 25
Trap Description R2D2 VaI S106W P107R R2D3C Fc
DNA SEQ ID NO. 75
1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccaac
61 agtaacaaca agagagcacc atactggacc aacacagaaa agatggaaaa gcggctccat
121 gctgtgcctg cggccaacac tgtcaagttt cgctgcccag ccggggggaa cccaatgcca
181 accatgcggt ggctgaaaaa cgggaaggag tttaagcagg agcatcgcat tggaggctac
241 aaggtacgaa accagcactg gagcctcatt atggaaagtg tggtcccatc tgacaaggga
301 aattatacct gtgtggtgga gaatgaatac gggtccatca atcacacgta ccacctggat
361 gttgtggagc gatggcggca ccggcccatc ctccaagccg gactgccggc aaatgcctcc
421 acagtggtcg gaggagacgt agagtttgtc tgcaaggttt acagtgatgc ccagccccac
481 atccagtgga tcaagcacgt ggaaaagaac ggcagtaaat acgggcccga cgggctgccc
541 tacctcaagg ttctcaaggc cgccggtgtt aacaccacgg acaaagagat tgaggttctc
601 tatattcgga atgtaacttt tgaggacgct ggggaatata cgtgcttggc gggtaattct
661 attgggatat cctttcactc tgcatggttg acagttctgc cagcgcctgg aagagaaccc
721 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
781 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
841 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
901 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
961 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
1021 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa gaccatctcc
1081 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
1141 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
1201 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1261 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
1321 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1381 cagaagagcc tctccctgtc tccgggtaaa tga
Protein SEQ ID NO. 105
1 nsnnkrapyw tntekmekrl havpaantvk frcpaggnpm ptmrwlkngk efkqehrigg
61 ykvrnqhwsl imeswpsdk gnytcwene ygsinhtyhl dwerwrhrp ilqaglpana
121 stwggdvef vckvysdaqp hiqwikhvek ngskygpdgl pylkvlkaag vnttdkeiev
181 lyirnvtfed ageytclagn sigisfhsaw ltvlpapgre pkscdkthtc ppcpapellg
241 gpsvflfppk pkdtlmisrt pevtcwvdv shedpevkfn wyvdgvevhn aktkpreeqy
301 nstyrwsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsre
361 emtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsffl yskltvdksr
421 wqqgnvfscs vmhealhnhy tqkslslspg k
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Figure imgf000039_0001
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Figure imgf000040_0001
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence Table 1 continued:
Trap No . 28
Trap Description R4D2 Leu P106R R2D3C Fc
DNA SEQ ID NO. 84
1 atgqggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccagt
61 tacccccagc aagcacccta ctggacacac ccccagcgca tggagaagaa actgcatgca
121 gtacctgcgg ggaacaccgt caagttccgc tgtccagctg caggcaaccc cacgcccacc
181 atccgctggc ttaaggatgg acaggccttt catggggaga accgcattgg aggcattcgg
241 ctgcgccatc agcactggag tctcgtgatg gagagcgtgg tgccctcgga ccgcggcaca
301 tacacctgcc tggtagagaa cgctgtgggc agcatccgct ataactacct gctagatgtg
361 ctggagcggt cccggcaccg gcccatcctc caagccggac tgccggcaaa tgcctccaca
421 gtggtcggag gagacgtaga gtttgtctgc aaggtttaca gtgatgccca gccccacatc
481 cagtggatca agcacgtgga aaagaacggc agtaaatacg ggcccgacgg gctgccctac
541 ctcaaggttc tcaaggccgc cggtgttaac accacggaca aagagattga ggttctctat
601 attcggaatg taacttttga ggacgctggg gaatatacgt gcttggcggg taattctatt
661 gggatatcct ttcactctgc atggttgaca gttctgccag cgcctggaag agaacccaaa
721 tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg
781 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag
841 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac
901 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc
961 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag
1021 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaagac catctccaaa
1081 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg
1141 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc
1201 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg
1261 gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag
1321 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
1381 aagagcctct ccctgtctcc gggtaaatga
Protein SEQ ID NO. 114
1 sypqqapywt hpqrmekklh avpagntvkf rcpaagnptp tirwlkdgqa fhgenriggi
61 rlrhqhwslv meswpsdrg tytclvenav gsirynylld vlersrhrpi lqaglpanas
121 twggdvefv ckvysdaqph iqwikhvekn gskygpdglp ylkvlkaagv nttdkeievl
181 yirnvtfeda geytclagns igisfhsawl tvlpapgrep kscdkthtcp pcpapellgg
241 psvflfppkp kdtlmisrtp evtcwvdvs hedpevkfnw yvdgvevhna ktkpreeqyn
301 styrwsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree
361 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffIy skltvdksrw
421 qqgnvfscsv mhealhnhyt qkslslspgk
Signal sequence is underlined in the nucleic acid sequence and is not present in the protein sequence
[0044] The human IgGl Fc domain was fused to each of the twenty-eight fusion protein constructs. The nucleotide and amino acid sequences of the human IgGl Fc that were fused to each of the different fusion protein constructs are provided in Table 2. Table 2. Nucleotide and Amino Acid Sequences of human IgGl Fc used for generating FGF-binding Fusion Proteins
DNA SEQ ID NO: 93
Description: IgGl Fc sequence used for R2D3b and R2D3c
1 gaacccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 61 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 121 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 181 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 241 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 301 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 361 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 421 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 481 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 541 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 601 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 661 tacacgcaga agagcctctc cctgtctccg ggtaaatga
DNA SEQ ID NO. 94
Description: IgGl Fc sequence used for R3D3b, R3D3c, and R4D3c
1 gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 61 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 121 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 181 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 241 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 301 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 361 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 421 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 481 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 541 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 601 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 661 tacacgcaga agagcctctc cctgtctccg ggtaaatga
Protein SEQ ID NO. 123
Description: IgGl Fc sequence used for all proteins
1 epkscdktht cppcpapell ggpsvflfpp kpkdtlmisr tpevtcvwd vshedpevkf 61 nwyvdgvevh naktkpreeq ynstyrwsv ltvlhqdwln gkeykckvsn kalpapiekt 121 iskakgqpre pqvytlppsr eemtknqvsl tclvkgfyps diavewesng qpennykttp 181 pvldsdgsff lyskltvdks rwqqgnvfsc svπvhealhnh ytqkslslsp gk
008/005172
- 42 -
Production and Expression of FGF-Binding Fusion Proteins [0045] The present invention provides methods for making the FGF-binding fusion proteins of the invention using nucleic acid molecules herein described. In general terms, the production of a recombinant form of a protein typically involves the following steps.
[0046] A nucleic acid molecule is first obtained that encodes a FGF-binding fusion protein of the invention. The nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame. The expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein. Optionally the recombinant protein is isolated from the medium or from the cells. Purification of the protein may not be necessary in some instances where some impurities can be tolerated. [0047] Each of the foregoing steps can be accomplished in a variety of ways. For example, the construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier and are otherwise known to persons skilled in the art. If necessary, suitable restriction sites can be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce a desired recombinant protein.
[0048] Alternatively, cloned DNA sequences comprising FGF- binding fusion proteins of the invention can be introduced into cultured mammalian cells by, for example, calcium phosphate- mediated transfection (Wigler et al . , Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973) . Other techniques for introducing cloned DNA sequences into mammalian cells, such as electroporation (Neumann et al . , EMBO J. 1: 841-845, 1982), or lipofection also can be used. In order to identify cells that have integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker can be an amplifiable selectable marker, such as the DHFR gene. Selectable markers are reviewed by Thilly {Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass. (1986) and the choice of selectable markers is within ordinary skill in the art.
[0049] Any expression system may be used, including yeast, bacterial, animal, plant, eukaryotic and prokaryotic systems. In some embodiments, yeast, mammalian cell culture and transgenic animal or plant production systems are preferred. In preferred embodiments, mammalian cell culture is used.
Isolation and Purification of FGF-Binding Fusion Proteins [0050] FGF-binding fusion proteins can be isolated from the medium of host cells grown under conditions that allow the expression and secretion of the fusion proteins. The cell material is removed from the culture medium, and the fusion proteins are isolated using any suitable isolation techniques. Suitable isolation techniques include precipitation and fractionation by a variety of chromatographic methods, including gel filtration, ion exchange chromatography and affinity chromatography. 72
- 44 -
FGF-Binding Fusion Protein Pharmaceutical Compositions [0051] Therapeutic compositions are within the scope of the present invention. Such FGF-binding fusion protein pharmaceutical compositions can comprise a therapeutically effective amount of a FGF-binding fusion protein of the invention in admixture with pharmaceutically or physiologically acceptable formulation carriers selected for suitability with the mode of administration. [0052] Pharmaceutically acceptable carriers are well known in the art and include 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Porphyrin or Lipofectin also can be used as a delivery agent. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.
[0053] The optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage.
[0054] One skilled in the art will appreciate that the appropriate dosage levels for treatment will vary depending, in part, upon the molecule delivered, the indication for which the FGF-binding fusion protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
[0055] The frequency of dosing will depend upon the pharmacokinetic parameters of the FGF-binding fusion protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art. Appropriate dosages can be ascertained through use of appropriate dose-response data.
[0056] The route of administration of the pharmaceutical composition can vary. For example, it can be oral, intravenous, intraperitoneal, intracerebral (intraparenchymal) , intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional . Administration also can be by sustained release systems; or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
[0057] The composition can be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be by diffusion, timed- release bolus, or continuous administration. [0058] An FGF-binding fusion protein can be delivered by- implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the FGF-binding fusion protein. Such cells can be animal or human cells. They can be autologous, heterologous, or xenogeneic. Optionally, the cells are immortalized. In order to decrease the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues. The encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product (s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
Use of Fusion Proteins
[0059] The fusion proteins of the invention are useful as therapeutic agents against diseases or disorders that depend, at least in part, on FGF/FGFR-mediated signaling. Such diseases and disorders include various types of cancer, for example, prostate carcinogenesis, skin tumorigenesis, urothelial cancer and hematological malignancies. Other types of cancer treatable by the fusion proteins of the invention include breast cancer, cervical cancer, ovarian cancer, gastric cancer, colorectal cancer, pancreatic cancer and lung cancer. [0060] In addition, the fusion proteins of the invention can be used to inhibit FGF binding to FGFRs in any in vivo or in vitro cellular process involving FGF signaling. FGF/FGFR- mediated signaling has been implicated in many cellular processes, including angiogenesis and cell proliferation. As described in Example 4, the fusion proteins of the invention can inhibit cell proliferation. EXAMPLES
Example 1: Construction of FGF-Binding Fusion Proteins (FGF Traps)
[0061] Twenty-two FGF Traps were constructed by fusing Ig domains 2 and 3 from human FGFR2 , 3, and 4. Three Ig-like domain 2 (D2) regions (from FGFR2, 3, and 4), two domain 2 - domain 3 loop (VaI Loop or Leu Loop) sequences (one from FGFR2 and the other representing FGFR3 and 4, respectively), and five Ig-like domain 3 (D3) regions (from FGFR2 IHb, FGFR2 IHc, FGFR3 IHb, FGFR3 IHc, and FGFR4 IIIc) were used to create a D2 - Loop - D3 fusion. If a domain 2 - domain 3 combination was between two domains from receptors containing different loops, then both loops were used for making combinations. The amino acid sequence of the FGFR domains used is outlined in FIG 2. A secretion signal sequence was fused to the amino terminus of each D2 - Loop - D3 version. The Fc portion of human IgGl (Table 2; SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 123) was fused to the carboxy terminus of each D2 - Loop - D3 version, thus creating a homodimeric secreted protein. The resulting recombinant proteins are designated as follows:
1. R2D2 Leu Loop R3D3b Fc
2. R2D2 Leu Loop R3D3c Fc 3. R2D2 Leu Loop R4D3c Fc
4. R2D2 VaI Loop R2D3b Fc
5. R2D2 VaI Loop R2D3c Fc
6. R2D2 VaI Loop R3D3b Fc
7. R2D2 VaI Loop R3D3C Fc 8. R2D2 VaI Loop R4D3c Fc
9. R3D2 Leu Loop R2D3b Fc
10. R3D2 Leu Loop R2D3c Fc
11. R3D2 Leu Loop R3D3b Fc
12. R3D2 Leu Loop R3D3c Fc 13. R3D2 Leu Loop R4D3c Fc
14. R3D2 VaI Loop R2D3b Fc
15. R3D2 VaI Loop R2D3c Fc
16. R4D2 Leu Loop R2D3b Fc
17. R4D2 Leu Loop R2D3c Fc 18. R4D2 Leu Loop R3D3b Fc 19. R4D2 Leu Loop R3D3c Fc
20. R4D2 Leu Loop R4D3c Fc
21. R4D2 VaI Loop R2D3b Fc
22. R4D2 VaI Loop R2D3c Fc
[0062] The DNA sequences for the above FGF Traps were constructed using Polymerase Chain Reaction (PCR) . DNA segments of each domain were amplified by PCR and contained overlapping sequence, thus allowing them to be fused together by additional PCR reactions. Oligonucleotide primers and DNA templates used for PCR reactions are listed in Tables 3 and 4, respectively. Combinations of oligonucleotide primers and DNA templates used for each PCR reaction are described in Tables 5 and 6. [0063] To create the IHb domain of FGFR3 , dual asymmetric
PCR (Young et al . , 2004, Nucleic Acids Res. Apr 15; 32(7): e59) was conducted using the Expand High Fidelity PCR System (Roche Applied Science, Germany) according to the manufacturer's instructions and as outlined by PCR #1 in Table 5. Second, overlapping extension PCR (Young et al., 2004, Nucleic Acids
Res. Apr 15; 32(7) : e59) was conducted using Expand polymerase as outlined by PCR #4 in Table 6.
[0064] Mouse FGFR3 Ig domain 2 and Ig domain Ilia contain minor amino acid differences in comparison to human FGFR3. To alter mouse FGFR3 cDNA sequences to correspond to the fully human amino acid sequence, PCRs #2, 3, 5-8 were conducted as outlined in Table 6 using KOD Hot Start DNA Polymerase (EMD Biosciences, Novagen, San Diego, CA) according to the manufacturer's instructions. [0065] To amplify the contiguous Ig domains 2 and 3 of FGFR2 IHb, FGFR2 IHc, and FGFR4 HIc, PCRs #9, 10, and 11 were conducted with KOD polymerase as outlined in Table 6. To amplify Ig domain 2 of FGFR2 , 3, and 4 with either D2 - D3 Loop option, PCRs #12-19 were conducted with KOD polymerase as outlined in Table 6. To amplify Ig domain 3 of FGFR2 IHb,
FGFR2 IHc, FGFR3 IHb, FGFR3 IHc, and FGFR4 IHc with either D2 - D3 Loop option, PCRs #20-29 were conducted with KOD polymerase as outlined in Table 6. Human IgGl Fc (SEQ ID NO: 93 for R2D3b and R2D3c; SEQ ID NO: 94 for R3D3b, R3D3c and R4D3c) region was amplified in PCR #30 with KOD polymerase. Twenty-two combinations of Ig domain 2, D2 - D3 Loop, and Ig domain 3 were fused with human IgGl Fc by PCRs #31-52 as outlined in Table 6. The resulting 22 PCR fragments contained the following in order: the attBl recombination site at the 51 end followed by a HindIII restriction site, a consensus Kozak translation initiation sequence, a secretion signal sequence, an FGFR D2 - Loop - D3 Fc combination, an EcoRI restriction site, and finally an attB2 recombination site at the 3' end. [0066] One or two amino acid changes were also made in the Loop region of several traps. These mutations are analogous to Ser 252 to Trp and Pro 253 to Arg mutations described for FGFR2 (HIb or IIIc forms) . These single mutations have been shown to increase affinity and broaden specificity of FGFR2 to FGFs when compared to wild-type FGFR2 (Ibrahimi, et al . , 2004, Hum MoI Genet. 13: 2313-24). The resulting FGF Trap recombinant proteins are designated as follows:
23. R2D2 VaI S106W R2D3C Fc
24. R2D2 VaI P107R R2D3C Fc
25. R2D2 VaI S106W P107R R2D3C Fc 26. R2D2 VaI P107R R3D3C Fc
27. R4D2 VaI P106R R2D3C Fc
28. R4D2 Leu P106R R2D3C Fc
[0067] The point mutations were introduced by PCR. Using KOD polymerase, Domain 2 + Loop was amplified to include the mutation (s) in the Loop (mutation introduced by the primer) . Loop + Domain 3 + Fc was also amplified to include the mutation (s) in the Loop. Corresponding Domain 2 + Loop and Loop + Domain 3 + Fc PCR fragments were then fused by PCR. The construction of these FGF Traps is outlined by PCRs #53-70 in Table 6. The resulting six full length FGF Trap PCRs also include the additional sequences described above for the first 22 traps (attBl, Hindlll, Kozak, signal sequence, EcoRI, and attB2) .
[0068] All 28 FGF Trap PCR fragments were cloned into pD0NR221 (Invitrogen, Carlsbad, CA) using the Gateway® BP Clonase™ II Enzyme Mix (Invitrogen) according to the manufacturer's instructions and using standard molecular biology techniques. FGF Traps #1-25 were subcloned into pcDNA3.2 V5-DEST using the Gateway® LR Clonase™ II Enzyme Mix
(Invitrogen) according to the manufacturer's instructions. FGF Traps # 5, 7, 17, 22, 24, and 26-28 were also subcloned from pDONR221 into pEE14.4 (Lonza Biologies, Berkshire, UK) using unique Hindlll and EcoRI restriction sites.
Table 3. DNA Oligonucleotides for PCRs
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Table 4. Source of DNA templates for PCR
Figure imgf000055_0001
Table 5. PCR Reactions
Figure imgf000055_0002
Table 6. PCR Reactions Continued
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
*For PCRs #64 and 65, the template used was a plasmid containing R4D2 Leu R2D3c Fc. The preferred template would have been a plasmid containing R4D2 VaI R2D3c Fc, but the one used was sufficient to allow the creation of the desired product. Similary, for PCRs #66 and 67 the preferred template would have been a plasmid containing R4D2 Leu R2D3c Fc, but a plasmid containing R4D2 VaI R2D3c Fc was actually used, also yielding the desired 0 product .
Example 2 : Expression of FGF Traps
[0069] FGF Traps #1-25 were expressed in 293T cells by transiently transfecting the pcDNA3.2 based vectors with 5 GeneJuice® Transfection Reagent (EMD Biosciences, Novagen, San Diego, CA) according to the manufacturer's instructions. Cells were cultured in DMEM media (Invitrogen) supplemented with 10% Ultra Low IgG Fetal Bovine Serum (Invitrogen) or 10% Fetal Bovine Serum (Invitrogen) . Cell supernatant containing the 0 secreted FGF Trap was collected 72 hours after transfection. Media was replenished and supernatant was collected after an additional 72 hours. FGF Trap-containing cell supernatant was used for surface plasmon resonance (BIAcore) characterization of FGF Trap binding to various FGFs (see below) . Stable cell 5 lines producing FGF Traps # 5, 7, 17, 22, 24, and 26-28 were also produced by transfecting the pEE14.4 based vectors into CHOKlSV cells (Lonza Biologies) by electroporation. Stable cell clones were selected with 25 or 50 uM methionine sulphoximine (MSX) in CD-CHO media (Invitrogen) .
Example 3 : Screening of FGF Traps for Binding to FGFs
[0070] The ability of the FGF Traps to bind to recombinant FGF molecules was assessed by surface plasmon resonance technology using a BlAcore TlOO instrument. Mouse anti-human immunoglobulins (Jackson ImmunoResearch Labs, 209-005-098) were immobilized on carboxymethylated dextran CM4 sensor chips (BlAcore, #BR-1005-34) by amine coupling (BlAcore, #BR-1000-50) using a standard coupling protocol according to manufacturer's recommendations. The analyses were performed at 25° C using PBS (GIBCO, #14040-133) containing 0.05% surfactant P20 (BlAcore, #BR-1000-54) , 1 mg/ml BSA (EMD, #2930) and 5μg/ml heparin sodium salt (Sigma H4784) as running buffer. [0071] FGF Traps (unpurified and undiluted in cell supernatant) were captured on individual flow cell at a flow rate of lOμl/min for 25 to 90 seconds depending on the experiment. Wild-type FGFRs (FGFRlalpha (HIb) ,
FGFRlbeta(IIIb) , FGFRlbeta (IIIc) , FGFR3 (HIb) and FGFR3 (IIIc) ) were purchased from R&D Systems and used as positive controls. Recombinant human FGF-I, -3, -4, -6, -9, and -10 were purchased from R&D Systems (respective catalog number: 232-FA, 1206-F3, 235-FA, 238-F6, 273-F9, 345-FG) . Two different forms of recombinant human FGF-2 were purchased from R&D Systems (233 -FB and 234 -FB) . Recombinant mouse FGF- 8b and FGF- 8c were also purchased from R&D Systems (423-8b, 424-Fc) . Mouse FGF-8b is 100% identical to human FGF- 8b. A human equivalent of the FGF- 8c isoform does not appear to be expressed (Gemel et al . , 1996, Genomics 35:253-7). Recombinant human FGF-7 was purchased from Cell Sciences (CRK300B) . All different recombinant FGFs were diluted individually in running buffer at a single concentration of 75nM and injected over the captured traps for 240 sec at 60μl/min. The dissociation phase was monitored for 10 min before the surface was regenerated with 1OmM Glycine- HCl, pH 2.0 (BIAcore, #BR-1003-55) injected for 3 min at a flow rate of 60μl/min.
[0072] Kinetic parameters ka (association rate constant) , kd (dissociation rate constant) and KD (equilibrium dissociation constant) for FGF/FGF trap interactions were determined using the kinetic function of the BIAevalutation software (BIAcore) with reference subtraction and a 1-to-l binding model. Additionally, report points were taken for each trap screened at selected times (5 sec before the end of the sample injection phase for the late binding report point and 10 sec before the end of the dissociation phase for the late stability report point) . These values were then normalized to the amount captured for each trap and used to evaluate the relative kinetic parameters of each trap towards each FGF tested (Tables 7-17) . Higher late binding values represent faster on rates and higher late stability values represent slower off rates. [0073] Equilibrium constant (KD) can be used as an approximate guide to assess effectiveness of trap binding to a given ligand. Equilibrium constants for the traps tested are shown in Tables 7-18. Kinetic parameters for FGF binding of Traps 5, 23, 24 and 25 were compared (Table 18) . The mutated Traps (23, 24 and 25) exhibited better binding to FGF7 and FGFlO as compared to the wild-type trap (5) . In some cases, curve fits calculated by the BIAevaluation software are non- ideal with respect to on-rate (ka) or off-rate (kd) . See Comments column of Tables 7-18. Confidence in these kinetic values might be increased, e.g., by an increased number of replicates and varied range of ligand concentration, or other assay optimization. Table 7. FGFl Binding
Figure imgf000061_0001
Table 8. FGF2 Long Form (234-FB) Binding
Figure imgf000062_0001
NB = no or very little binding
Table 9. FGF2 Short Form (233-FB) Binding
Figure imgf000063_0001
NB = no or very little binding
Table 10. FGF3 Binding
Figure imgf000064_0001
NB = no or very little binding
Table 11. FGF4 Binding
Figure imgf000065_0001
Table 12. FGF6 Binding
Figure imgf000066_0001
Table 13 . FGF7 Binding
Figure imgf000067_0001
NB = no or very little binding
Table 14. FGF8b Binding
Figure imgf000068_0001
NB = no or very little binding
Table 15 . FGF8c Binding
Figure imgf000069_0001
NB = no or very little binding
Table 16. FGF9 Binding
Figure imgf000070_0001
NB = no or very little binding
Table 17. FGFlO Binding
Figure imgf000071_0001
NB = no or very little binding
Table 18. FGF1-4, 6-10: Comparison of Point Mutant Traps to Wild- type
Figure imgf000072_0001
Figure imgf000073_0001
Example 4: FGF-Dependent Proliferation Assay
[0074] FDCP-I cells (mouse bone marrow cells obtained from German Collection of Microorganisms and Cell Cultures DSMZ) were transfected with a cDNA expressing human FGFR3 IIIc (referred to as FDCP-FGFR3) and selected with G418 (600 μg/ml) . Single clones were isolated and tested for their FGFl-dependent proliferation in the absence of IL-3-containing WEHI- conditioned medium. FDCP-FGFR3#109 exhibited FGF-I induced proliferation in the absence of IL3 (WEHI-conditioned medium) . [0075] To test the effect of FGF trap on the proliferation of FDCP-FGFR3#109 stimulated by FGF-I, varying amounts of FGF Trap (R2D2 VaI R2D3c Fc) or a control protein (ILlRLl-Fc (Recombinant Human IL-I R4/ST2/Fc Chimera, R&D Systems, 523-ST- 100)) were preincubated with FGFl and heparin (Sigma H4784) for an hour. The mixtures were then added to FDCP-FGFR3 #109 cells seeded in basic growth medium [70% ISCOVE ' s Modified Dulbecco's Medium (Invitrogen, 12440-053) , 20% horse serum (Invitrogen 26050-088)) and 10% WEHI-culture medium (90% Iscove's MDM + 10% FBS (Invitrogen 10438-026) + 2 mM L-glutamine (Invitrogen, 25030-081) + 0.0025 mM 2-Mercaptoethanol (Invitrogen, 21985- 023)] in a 96-well plate (30,000 cells/ well). The final concentration of FGFl and heparin used in the assay is 8 ng/ml and 5 μg/ml respectively. A standard MTT assay was conducted three days post FGFl stimulation to assess relative proliferation.
[0076] As illustrated in Figure 3, the R2D2 VaI R2D3c Fc Trap inhibited proliferation of FGF-dependent FDCP-FGFR3, whereas the control ILlRLl-Fc did not.
Other embodiments are within the following claims.

Claims

What is Claimed is:
1. A soluble human fibroblast growth factor (FGF)- binding fusion protein, comprising the domain structure D2-L- D3-Fc, wherein the Fc domain is at the carboxy terminus of the protein, and
D2 consists essentially of the Ig-like domain 2 from FGFR2 (R2D2), FGFR3 (R3D2) or FGFR4 (R4D2);
L is DVLERSPHR (Leu loop) or DWERSPHR (VaI loop) ;
D3 consists essentially of the Ig-like domain 3
Of FGFR2-IIIb (R2D3b) , FGFR2-IIIC (R2D3C) , FGFR3-IIIb (R3D3b) , FGFR3-IIIC (R3D3c) , or FGFR4-IIIC (R4D3c) ; and
Fc consists essentially of the Fc domain of a human immunoglobulin.
2. The fusion protein of claim 1, wherein the immunoglobulin is IgGl.
3. The fusion protein of claim 1, wherein D2 consists essentially of the Ig-like domain 2 from FGFR2.
4. The fusion protein of claim 1, wherein D2 consists essentially of the Ig-like domain 2 from FGFR3.
5. The fusion protein of claim 1, wherein D2 consists essentially of the Ig-like domain 2 from FGFR4.
6. The fusion protein of claim 1, wherein D3 consists essentially of the Ig-like domain 3 from FGFR2-IIIb.
7. The fusion protein of claim 1, wherein D3 consists essentially of the Ig-like domain 3 from FGFR2-IIIc.
8. The fusion protein of claim 1, wherein D3 consists essentially of the Ig-like domain 3 from FGFR3-IIIb.
9. The fusion protein of claim 1, wherein D3 consists essentially of the Ig-like domain 3 from FGFR3-IIIC.
10. The fusion protein of claim 1, wherein D3 consists essentially of the Ig-like domain 3 from FGFR4-IIIc.
11. The fusion protein of claim 1, wherein L consists essentially of DWERSPHR (wt VaI loop) .
12. The fusion protein of claim 1, wherein L consists essentially of DVLERSPHR (wt Leu loop) .
13. The fusion protein of claim 1, wherein
D2 consists essentially of the Ig-like domain 2 Of FGFR2;
L consists essentially of DWERWPHR (VaI loop comprising mutation S106W) ; and
D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIC.
14. The fusion protein of claim 1, wherein
D2 consists essentially of the Ig-like domain 2 of FGFR2;
L consists essentially of DWERSRHR (VaI loop comprising mutation P107R) ; and
D3 consists essentially of the Ig-like domain 3 Of FGFR2-IIIC.
15. The fusion protein of claim 1, wherein
D2 consists essentially of the Ig-like domain 2 Of FGFR2;
L consists essentially of DWERWRHR (VaI loop comprising mutations S106W and P107R) ; and
D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIC.
16. The fusion protein of claim 1, wherein D2 consists essentially of the Ig-like domain 2 Of FGFR2;
L consists essentially of DWERSRHR (VaI loop comprising mutation P107R) ; and
D3 consists essentially of the Ig-like domain 3 of FGFR3-IIIC.
17. The fusion protein of claim 1, wherein
D2 consists essentially of the Ig-like domain 2 Of FGFR4;
L consists essentially of DWERSRHR (VaI loop comprising mutation P106R) ; and
D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIC.
18. The fusion protein of claim 1, wherein
D2 consists essentially of the Ig-like domain 2 Of FGFR4;
L consists essentially of the DVLERSRHR (Leu loop comprising mutation P106R) ; and
D3 consists essentially of the Ig-like domain 3 of FGFR2-IIIC.
19. The fusion protein of claim 1, wherein the domain structure is selected from the group consisting of:
R2D2 Leu Loop R3D3b Fc;
R2D2 Leu Loop R3D3C Fc;
R2D2 Leu Loop R4D3C Fc ;
R2D2 VaI Loop R2D3b Fc;
R2D2 VaI Loop R2D3C Fc;
R2D2 VaI Loop R3D3b Fc ;
R2D2 VaI Loop R3D3C Fc;
R2D2 VaI Loop R4D3C Fc;
R3D2 Leu Loop R2D3b Fc ;
R3D2 Leu Loop R2D3C Fc; R3D2 Leu Loop R3D3b Fc ;
R3D2 Leu Loop R3D3c Fc;
R3D2 Leu Loop R4D3C Fc;
R3D2 VaI Loop R2D3b Fc;
R3D2 VaI Loop R2D3c Fc;
R4D2 Leu Loop R2D3b Fc ;
R4D2 Leu Loop R2D3C Fc ;
R4D2 Leu Loop R3D3b Fc;
R4D2 Leu Loop R3D3C Fc;
R4D2 Leu Loop R4D3C Fc ;
R4D2 VaI Loop R2D3b Fc ;
R4D2 VaI Loop R2D3C Fc;
R2D2 VaI S106W R2D3c Fc;
R2D2 VaI P107R R2D3c Fc;
R2D2 VaI S106W P107R R2D3C Fc ;
R2D2 VaI P107R R3D3c Fc;
R4D2 VaI P106R R2D3c Fc; and
R4D2 Leu P106R R2D3c Fc.
20. The fusion protein of claim 19, wherein the domain structure is selected from the group consisting of:
R2D2 VaI Loop R2D3C Fc ;
R2D2 VaI Loop R3D3C Fc ;
R4D2 VaI Loop R2D3C Fc ;
R2D2 VaI P107R Loop R2D3c Fc ;
R4D2 Leu Loop R2D3C Fc ;
R2D2 VaI P107R Loop R3D3c Fc ;
R4D2 VaI P106R Loop R2D3c Fc ; and
R4D2 Leu P106R Loop R2D3c Fc.
21. A nucleic acid comprising a nucleotide sequence that encodes the fusion protein of claim 1.
22. An expression vector comprising the nucleic acid of claim 21.
23. A host cell comprising the expression vector of claim 22.
24. A method of making the fusion protein of claim 1, comprising (a) culturing the host cell of claim 23 under conditions so that the host cell expresses the fusion protein of claim 1, and (b) harvesting the fusion protein.
25. A method of inhibiting binding of an FGF molecule to an FGF receptor, comprising contacting the fusion protein of claim 1 with the FGF molecule.
26. The method of claim 25, wherein the FGF molecule, FGF receptor and fusion protein are in vivo.
27. A method of inhibiting proliferation of a tumor cell in vitro, comprising contacting the tumor cell with the fusion protein of claim 1.
28. A method of inhibiting proliferation of a tumor cell in a mammal, comprising administering to the mammal an effective amount of the fusion protein of claim 1.
29. A method of treating a tumor in a mammal, comprising administering to the mammal an effective amount of the fusion protein of claim 1.
30. A pharmaceutical composition comprising the fusion protein of claim 1.
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102219860A (en) * 2011-05-20 2011-10-19 烟台荣昌生物工程有限公司 FGFR-Fc fusion protein and applications thereof
EP2478003A1 (en) * 2009-09-15 2012-07-25 Five Prime Therapeutics, Inc. Hair growth methods using fgfr4 extracellular domains
US8614183B2 (en) 2009-11-13 2013-12-24 Five Prime Therapeutics, Inc. Use of FGFR1 extra cellular domain proteins to treat cancers characterized by ligand-dependent activating mutations in FGFR2
WO2014111744A1 (en) * 2013-01-16 2014-07-24 INSERM (Institut National de la Santé et de la Recherche Médicale) A soluble fibroblast growth factor receptor 3 (fgr3) polypeptide for use in the prevention or treatment of skeletal growth retardation disorders
US8920800B2 (en) 2010-11-15 2014-12-30 Five Prime Therapeutics, Inc. Treatment of cancer with elevated dosages of soluble FGFR1 fusion proteins
US9173957B2 (en) 2005-07-22 2015-11-03 Five Prime Therapeutics, Inc. FGFR2 fusion proteins comprising C-terminal deletion mutants of FGFR2-IIIC
US9175065B2 (en) 2008-08-04 2015-11-03 Five Prime Therapeutics, Inc. FGFR extracellular domain acidic region muteins
WO2016110786A1 (en) * 2015-01-07 2016-07-14 Therachon Solube fgfr3 decoys for treating skeletal growth disorders
US9957313B2 (en) 2013-03-15 2018-05-01 Remegen, Ltd. FGFR-FC fusion proteins and the use thereof
US10016484B2 (en) 2011-11-14 2018-07-10 Five Prime Therapeutics, Inc. Methods of treating lung cancer
US10294289B2 (en) 2016-07-07 2019-05-21 Therachon Sas Soluble fibroblast growth factor receptor 3 (SFGFR3) polypeptides and uses thereof
JP2020015733A (en) * 2019-08-19 2020-01-30 アンセルムＩｎｓｅｒｍ Soluble fibroblast growth factor receptor 3 (fgr3) polypeptide for use in prevention or treatment of skeletal growth retardation disorders

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021258A1 (en) * 1994-02-01 1995-08-10 United States Of America, Represented By The Secretary, Department Of Health And Human Services Fusion proteins that include antibody and nonantibody portions
WO2000046380A2 (en) * 1999-02-08 2000-08-10 Chiron Corporation Fibroblast growth factor receptor-immunoglobulin fusion
WO2005066211A2 (en) * 2003-12-19 2005-07-21 Five Prime Therapeutics, Inc. Fibroblast growth factor receptors 1, 2, 3, and 4 as targets for therapeutic intervention
WO2007014123A2 (en) * 2005-07-22 2007-02-01 Five Prime Therapeutics, Inc. Compositions and methods of treating disease with fgfr fusion proteins

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021258A1 (en) * 1994-02-01 1995-08-10 United States Of America, Represented By The Secretary, Department Of Health And Human Services Fusion proteins that include antibody and nonantibody portions
WO2000046380A2 (en) * 1999-02-08 2000-08-10 Chiron Corporation Fibroblast growth factor receptor-immunoglobulin fusion
US6656728B1 (en) * 1999-02-08 2003-12-02 Chiron Corporation Fibroblast growth factor receptor-immunoglobulin fusion
WO2005066211A2 (en) * 2003-12-19 2005-07-21 Five Prime Therapeutics, Inc. Fibroblast growth factor receptors 1, 2, 3, and 4 as targets for therapeutic intervention
WO2007014123A2 (en) * 2005-07-22 2007-02-01 Five Prime Therapeutics, Inc. Compositions and methods of treating disease with fgfr fusion proteins

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US9192683B2 (en) 2005-07-22 2015-11-24 Five Prime Therapeutics, Inc. Methods of treating disease with FGFR1 fusion proteins
US9175065B2 (en) 2008-08-04 2015-11-03 Five Prime Therapeutics, Inc. FGFR extracellular domain acidic region muteins
EP2478003A1 (en) * 2009-09-15 2012-07-25 Five Prime Therapeutics, Inc. Hair growth methods using fgfr4 extracellular domains
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US9169313B2 (en) 2009-11-13 2015-10-27 Five Prime Therapeutics, Inc. Method of treatment of cancers characterized by ligand-dependent activating mutations in FGFR2 with FGFR1 ECD proteins
US8614183B2 (en) 2009-11-13 2013-12-24 Five Prime Therapeutics, Inc. Use of FGFR1 extra cellular domain proteins to treat cancers characterized by ligand-dependent activating mutations in FGFR2
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US9475871B2 (en) 2010-11-15 2016-10-25 Five Prime Therapeutics, Inc. Treatment of cancer with elevated dosages of soluble FGFR1 fusion proteins
CN102219860A (en) * 2011-05-20 2011-10-19 烟台荣昌生物工程有限公司 FGFR-Fc fusion protein and applications thereof
EP2711376A4 (en) * 2011-05-20 2015-06-03 Yantai Rongchang Biotechnologies Co Ltd Fgfr-fc fusion protein and use thereof
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US10537611B2 (en) 2011-11-14 2020-01-21 Five Prime Therapeutics, Inc. Methods of treating lung cancer
US10016484B2 (en) 2011-11-14 2018-07-10 Five Prime Therapeutics, Inc. Methods of treating lung cancer
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