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  • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
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  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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  • 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 Fibroblast Growth Factor (FGF)-Fibroblast Growth Factor Receptor (FGFR) signaling pathway is widely implicated in the development and maintenance of many different cancers.
• This signaling pathway comprises 4 different FGF receptors (FGFR1 , FGFR2, FGFR3, and FGFR4), some of which are also alternatively spliced, and 22 different FGF ligands.
• FGF receptor and splice form has different patterns of expression and different specificities for the various FGF ligands.
• FGFR1 is the best characterized of the four FGFRs. FGFR1 and its ligands have causal connections to cancer in animal models and strong correlative connections to human disease. Specific induction of FGFR 1 signaling in mouse prostate results in prostate hyperplasia and carcinoma (Freeman et al, Cancer Res. 2003;63:8256-63), demonstrating that abnormal hyperactivation of FGFR1 is sufficient to initiate tumorigenesis. Inhibition of FGFR1 activity inhibits tumor growth in xenograft models from multiple tissue types (Ogawa et al, Cancer Gene Ther. 2002;9:633-40).
• chromosomal amplification of the FGFR1 gene in a subset of breast cancer patients is associated with poor outcome (Gelsi-Boyer et al, Mol. Cancer Res. 2005;3:655-67) and overexpression or high systemic levels of FGF ligands correlate with tumorigenesis and poor patient outcome (Nguyen et al, J. Natl. Cancer Inst. 1994;86:356-61).
• FGFR1 has multiple mechanisms in the promotion of tumor cell growth and survival.
• FGFR1 signaling increases the mitotic rate of tumor cells, promotes tumor angiogenesis, and helps maintain the tumorigenicity of tumor stem cells (TSCs).
• TSCs tumor stem cells
• Many tumor cell lines are responsive to and dependent on FGFR1 signaling for growth in vitro, and tumor cell lines become resistant to cytotoxic agents when stimulated with FGF-2 (Song et al, PNAS
• FGF-FGFR1 vascular endothelial growth factor receptor
• TSCs malignant cells
• Soluble FGFR1 fusion proteins are able to bind to FGF ligands of the FGFR1 receptor, "trapping" the ligands and prevent them from activating FGFR1 receptors as well as other receptors for which the ligands have affinity. See, e.g., US Patent No. 7,678,890. Without being bound to a particular theory, it is believed that soluble FGFRl fusion proteins can inhibit tumorigenic activity through multiple mechanisms of action, including but not limited to, direct anti-tumor activity in cancers dependent on the FGF-FGFR pathway, inhibition of tumor angiogenesis, and/or inhibition of cancer stem cell maintenance.
• a soluble FGFRl /Fc fusion protein FP-1039
• concentrations of about 2 mg/kg body weight or higher (i.e., up to at least about 16 mg/kg) and that such concentrations are well- tolerated.
• treatment of humans with FP-1039 yields pharmacokinetic and pharmacodynamic profiles that indicate weekly or less frequent administration of doses above 2 mg/kg, 4 mg/kg, 8 mg/kg or 10 mg/kg is sufficient for sustained sequestration of target FGF ligands such as FGF-2.
• the present invention provides methods of treating a human having a cancer.
• the method comprises administering to the human a
• FGFRl soluble Fibroblast Growth Factor Receptor 1
• FGFRl fusion protein is administered at a dose of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28 or 30 mg/kg body weight, or within a range from one to another of the above dose values (the above doses being calculated using an extinction coefficient of 1.42 mL/mg*cm).
• the FGFRl fusion protein is administered at a dose of about 10 mg/kg body weight as calculated using an extinction coefficient of 1.1 1 mL/mg*cm.
• the FGFRl fusion protein is administered at a dose of about 20 mg/kg body weight as calculated using an extinction coefficient of 1.1 1 mL/mg*cm, or at a range of about 10 to about 20 mg/kg body weight as calculated using an extinction coefficient of 1.1 1 mIJmg*cm.
• the human has a fibroblast growth factor-2 (FGF-2) plasma concentration of at least 6 pg/ml.
• FGF-2 fibroblast growth factor-2
• the cancer is characterized by a ligand-dependent activating mutation in FGFR2.
• the ligand-dependent activating mutation in FGFR2 is S252W or P253R.
• the soluble FGFRl fusion protein is administered in combination with a chemotherapeutic agent or a VEGF antagonist.
• the FGFRl polypeptide is human FGFRl isoform IIIc.
• the fusion partner is an Fc polypeptide, which is the Fc region of human immunoglobulin Gl (IgGl).
• the FGFRl extracellular domain has the amino acid sequence of SEQ ID NO:5.
• the soluble FGFRl fusion protein has the amino acid sequence of SEQ ID NO:8.
• the soluble FGFRl fusion protein is administered at a dose of about 2 mg/kg body weight to about 30 mg/kg body weight. In some embodiments, the soluble FGFRl fusion protein is administered at a dose of about 8 mg/kg body weight to about 16 mg/kg body weight (or about 10 mg/kg body weight to about 20 mg/kg body weight when calculated using an extinction coefficient of 1.1 1 mIJmg*cm).
• the soluble FGFRl fusion protein is administered at a dose of about 8 mg/kg body weight, while in some embodiments, the soluble FGFRl fusion protein is administered at a dose of about 16 mg/kg body weight (or at about 10 mg/kg body weight or about 20 mg/kg body weight, respectively, when calculated using an extinction coefficient of 1.1 1 mL/mg*cm).
• the method comprises administering FP-1039 to a human patient having cancer, wherein the human has a fibroblast growth factor-2 (FGF-2) plasma concentration of at least 6 pg/ml and wherein FP-1039 is administered at a dose of about 2 mg/kg to about 30 mg/kg.
• FGF-2 fibroblast growth factor-2
• the soluble FGFRl fusion protein is administered at a dose of about 8 mg/kg body weight.
• the FP-1039 is administered at about 16 mg/kg.
• the soluble FGFRl fusion protein is administered twice a week, weekly, every other week, at a frequency between weekly and every other week, every three weeks, every four weeks, or every month.
• the soluble FGFRl fusion protein is administered intravenously or subcutaneously.
• the cancer is prostate cancer, breast cancer, colorectal cancer, lung cancer, endometrial cancer, head and neck cancer, laryngeal cancer, liver cancer, renal cancer, glioblastoma, or pancreatic cancer.
• the human has an FGF-2 plasma concentration of at least 10 pg/ml prior to the administration of the soluble FGFRl fusion protein.
• the soluble FGFRl fusion protein is administered at a dose such that at seven days after administration, the human has an FGF-2 plasma concentration of less than 4 pg/ml.
• the soluble FGFRl fusion protein is administered in
• the soluble FGFRl fusion protein is administered in combination with a chemotherapeutic agent.
• the chemotherapeutic agent is sorafenib.
• the soluble FGFRl fusion protein is administered in combination with a VEGF antagonist.
• the VEGF antagonist is a VEGF antibody, such as bevacizumab, or the VEGF antagonist is a VEGF trap, such as aflibercept.
• the soluble FGFRl fusion protein is administered in combination with an anti-angiogenic agent.
• the present invention also provides for methods of treating a human having a cancer, wherein the cancer is characterized by an Fibroblast Growth Factor Receptor 2 (FGFR2) having a ligand-dependent activating mutation, the method comprising: administering to the human a soluble Fibroblast Growth Factor Receptor 1 (FGFRl) fusion protein at a dose of about 2 mg/kg body weight to about 30 mg/kg body weight, wherein the fusion protein comprises an extracellular domain of an FGFRl polypeptide linked to a Fc polypeptide.
• FGFR2 Fibroblast Growth Factor Receptor 2
• FGFRl Fibroblast Growth Factor Receptor 1
• the soluble FGFRl fusion protein is administered at a dose of about 8 mg/kg body weight, ,while in some embodiments, the FP-1039 is administered at about 16 mg/kg body weight (or about 10 mg/kg body weight or about 20 mg/kg body weight, respectively when calculated using an extinction coefficient of 1.1 1 mL/mg*cm).
• the FGFRl polypeptide is human FGFRl isoform IIIc.
• the Fc polypeptide is an Fc region of human immunoglobulin Gl (IgGl ).
• the FGFR1 extracellular domain comprises the amino acid sequence of SEQ ID NO:5.
• the soluble FGFR1 fusion protein comprises the amino acid sequence of SEQ ID NO: 8.
• the soluble FGFR1 fusion protein is administered at a dose of about 2 mg/kg body weight to about 30 mg/kg body weight. In some embodiments for treating cancer characterized by an FGFR2 having a ligand-dependent activating mutation, the soluble FGFR1 fusion protein is administered at a dose of about 8 mg/kg body weight to about 16 mg/kg body weight (or about 10 mg/kg body weight to about 20 mg/kg body weight when calculated using an extinction coefficient of 1.1 1 mL/mg*cm).
• the soluble FGFR1 fusion protein is administered at a dose of about 8 mg/kg body weight, while in some embodiments for treating cancer characterized by an FGFR2 having a ligand-dependent activating mutation, is administered at a dose of about 16 mg/kg body weight (or about 10 mg/kg body weight or about 20 mg/kg body weight, respectively when calculated using an extinction coefficient of 1.1 1 mL/mg*cm).
• the FGFR1 fusion protein is administered at a dose of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28 or 30 mg/kg body weight, or within a range from one to another of the above dose values.
• dosages may be administered weekly, every other week, at a frequency between weekly and every other week, every three weeks, every four weeks, or every month.
• the method comprises administering FP-1039 to a human patient having cancer, wherein the cancer is characterized by an FGFR2 having a ligand-dependent activating mutation, and wherein FP- 1039 is administered at a dose of about 2 mg/kg to about 30 mg/kg.
• the FP-1039 is administered at about 8 mg/kg, while in some embodiments, the FP-1039 is administered at about 16 mg/kg (or about 10 mg/kg body weight to about 20 mg/kg body weight when calculated using an extinction coefficient of 1.1 1
• the soluble FGFRl fusion protein is administered weekly or every other week or a frequency between weekly and every other week.
• the soluble FGFRl fusion protein is administered intravenously or subcutaneously.
• the cancer is prostate cancer, breast cancer, colorectal cancer, lung cancer, endometrial cancer, head and neck cancer, laryngeal cancer, liver cancer, renal cancer, glioblastoma, or pancreatic cancer.
• the human has an FGF-2 plasma concentration of at least 10 pg/ml prior to the administration of the soluble FGFRl fusion protein.
• the soluble FGFRl fusion protein is administered at a dose such that at seven days after
• the human has an FGF-2 plasma concentration of less than 4 pg/ml.
• the soluble FGFRl fusion protein is administered in combination with a chemotherapeutic agent.
• the chemotherapeutic agent is sorafenib.
• the soluble FGFRl fusion protein is administered in combination with a chemotherapeutic agent, VEGF antagonist or anti-angiogenic agent.
• the VEGF antagonist is a VEGF antibody, such as bevacizumab, or the VEGF antagonist is a VEGF trap, such as aflibercept.
• the soluble FGFRl fusion protein is administered in combination with an anti-angiogenic agent.
• the ligand-dependent activating mutation in FGFR2 is S252W or P253R.
• the present invention also provides a composition comprising a soluble FGFRl fusion protein for use in the treatment of cancer, wherein the composition is administered at a dose of at least about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28 or 30 mg/kg body weight, or within a range from one to another of the above dose values.
• dosages may be administered twice a week, weekly, every other week, at a frequency between weekly and every other week, every three weeks, every four weeks, or every month.
• the human has a fibroblast growth factor-2 (FGF-2) plasma concentration of at least 6 pg/ml.
• FGF-2 fibroblast growth factor-2
• the cancer is characterized by a ligand-dependent activating mutation in FGFR2.
• the ligand-dependent activating mutation in FGFR2 is S252W or P253R.
• the soluble FGFRl fusion protein is administered in combination with a chemotherapeutic agent or a VEGF antagonist.
• a "fibroblast growth factor receptor 1" or "FGFRl” polypeptide refers to a polypeptide having the amino acid sequence of any one of the known FGFRl polypeptides, such as FGFRl -Illb and FGFRl -IIIc, and any variant, precursor, or fragment thereof, including those described in U.S. Patent Nos. 7,678,890; 6,656,728; 6,384, 191 ;
• An FGFRl polypeptide sequence is typically from, or derived from, a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow; pig; sheep; horse; or any other mammal.
• extracellular domain refers to the portion of a polypeptide that extends beyond the transmembrane domain of the polypeptide into the extracellular space.
• the ECD is an ECD of an FGFRl polypeptide, such as FGFRl -Illb and FGFRl -IIIc, or a variant thereof.
• FGFRl extracellular domain (“FGFRl ECD”) includes full-length FGFRl ECDs, FGFRl ECD fragments, and FGFRl ECD variants.
• FGFRl ECD refers to an FGFRl polypeptide that lacks the intracellular and transmembrane domains, with or without a signal peptide.
• the FGFRl ECD comprises an amino acid sequence that is substantially identical to an ECD having the amino acid sequence of SEQ ID NO: l .
• the FGFRl ECD has the amino acid sequence of any of SEQ ID NOs: 1 -6.
• a "soluble FGFR fusion protein” or an "FGFRl fusion protein” or “FGFRl ECD fusion molecule” refers to a protein comprising an FGFRl ECD linked to one or more fusion partners, wherein the soluble FGFR fusion protein lacks a transmembrane domain (e.g., an FGFRl transmembrane domain) and is not bound to the cellular membrane.
• a "fusion partner” is a molecule that is linked to the FGFRl ECD that imparts favorable pharmacokinetics and/or pharmacodynamics on the FGFRl ECD protein.
• a fusion partner may comprise a polypeptide, such as a fragment of an immunoglobulin molecule or albumin, or it may comprise a non-polypeptide moiety, for example, polyethylene glycol.
• the fusion partner is an Fc domain of an antibody.
• signal peptide refers to a sequence of amino acid residues located at the N terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell.
• a signal peptide may be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein.
• Signal peptides may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached. Exemplary signal peptides also include signal peptides from heterologous proteins.
• a "signal sequence” refers to a polynucleotide sequence that encodes a signal peptide.
• an FGFRl ECD lacks a signal peptide.
• an FGFRl ECD includes at least one signal peptide, which may be a native FGFRl signal peptide or a heterologous signal peptide.
• a "ligand-dependent activating mutation" of FGFR2 refers to a mutation that increases the biological activity of FGFR2, for example, a mutation causing FGFR2 to become activated more readily in comparison to the wildtype FGFR2 in response to certain stimuli, wherein the biological effects of the mutation depend on the binding of FGFR2 to one or more of its ligands.
• An example is a mutation that causes alterations in the ligand binding properties of FGFR2.
• the terms "native FGFRl ECD” and "wildtype FGFRl ECD” are used interchangeably to refer to an FGFRl ECD with a naturally occurring amino acid sequence.
• Native FGFRl ECDs and wildtype FGFRl ECDs also include FGFRl ECD splice variants or isoforms.
• FGFRl ECD splice variants or “splice isoforms” are used interchangeably to refer to alternative splice forms of FGFRl ECD, such as FGFRl -Illb and FGFRl -IIIc ECD.
• FGFRl ECD variants refers to FGFRl ECDs containing amino acid additions, deletions, and/or substitutions in comparison to the native FGFRl ECDs. FGFRl ECD variants retain the ability to bind FGF2. Such variants may be at least 90%, 92%, 95%, 97%, 98%, or 99% identical to the parent FGFRl ECD.
• full-length FGFRl ECD refers to an FGFRl ECD that extends to the last amino acid of the extracellular domain, and may or may not include an N- terminal signal peptide.
• FGFRl ECD fragment refers to an FGFRl ECD having an amino acid sequence modified in that amino acid residues have been deleted from the amino-terminus and/or from the carboxy-terminus of the polypeptide, wherein the fragment retains the ability to bind FGF2.
• native FGFRl ECD fragment refers to an FGFRl ECD fragment in which the retained portions of the FGFRl ECD sequence are naturally occurring, wherein the fragment retains the ability to bind FGF2.
• FGFRl ECD fragment variant and “variant of FGFRl ECD fragment” are used interchangeably to refer to FGFR l ECDs containing, not only amino acid deletions from the amino- and/or carboxy-terminus of native FGFRl ECD, but also amino acid additions, deletions, and/or substitutions within the retained portion of the FGFRl ECD. FGFRl ECD fragment variants also retain the ability to bind FGF2.
• nucleic acid or “polynucleotide” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
• degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 ( 1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 ( 1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 ( 1994)).
• nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
• peptide refers to a polymer of amino acid residues.
• the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
• the terms also include post-translational modifications of the polypeptide, including, for example, glycosylation, sialylation, acetylation, and phosphorylation.
• a polypeptide consists of a particular amino acid sequence, it may still contain post- translational modifications, such as glycosylation and sialylation.
• amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
• Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
• Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a ⁇ -carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
• Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
• Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical
• Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
• nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
• TGG which is ordinarily the only codon for tryptophan
• amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
• Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences.
• the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
• nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
• the invention provides polypeptides that are substantially identical to the polypeptides exemplified herein (e.g., the polypeptides exemplified in SEQ ID NOs: l -8).
• the identity exists over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence.
• identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence.
• shorter amino acid sequences e.g., amino acid sequences of 20 or fewer amino acids
• substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.
• sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
• test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
• sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
• a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman ( 1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
• BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. ( 1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. ( 1990) J. Mol. Biol. 215:403-410, respectively.
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra).
• initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
• the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001 .
• polypeptides are substantially identical is that the first polypeptide is immunologically cross reactive with the antibodies raised against the second polypeptide.
• a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
• cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
• cancer is a human cancer.
• examples of cancer include but are not limited to carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, solid and lymphoid cancers, etc.
• cancers examples include, but are not limited to, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, oral cancer, ovarian cancer, thyroid cancer, lung cancer (non- small cell lung cancer, small cell lung cancer, squamous cell lung cancer), (non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer), prostate cancer, uterine cancer, endometrial cancer, testicular cancer, neuroblastoma, squamous cell carcinoma of the head, neck, cervix and vagina, multiple myeloma, soft tissue and osteogenic sarcoma, colorectal cancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), mesothelioma, cervical cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), esophageal cancer, laryngeal cancer, gall bladder cancer, small intestine cancer
• treating refers to inhibiting a disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or relieving a disease, for example, by causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process.
• treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
• Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
• subject and “patient” are used interchangeably herein to refer to mammals, including, but not limited to, rodents, simians, humans, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets.
• the subject is a human.
• a "pharmaceutically acceptable carrier” refers to a solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent for administration to a subject.
• a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
• Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.
• FP-1039 refers to a protein having the amino acid sequence set forth in SEQ ID NO:8. FP-1039 can be produced, for example, as is described generally for FGFR fusion molecules in US Patent No. 7,678,890, the entire disclosure of which is expressly incorporated herein by reference.
• anti-neoplastic composition refers to a composition useful in treating cancer comprising at least one active therapeutic agent, e.g., "anti-cancer agent.”
• therapeutic agents include, but are not limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti- angiogenic agents, apoptotic agents, anti-tubulin agents, and other-agents to treat cancer, such as anti-VEGF antibodies (e.g., bevacizumab, AVASTIN ® ), anti-HER-2 antibodies (e.g., trastuzumab, HERCEPTIN ® ), anti-CD20 antibodies (e.g., rituximab, RITUXAN ® ), an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., er
• GLEEVEC ® (Imatinib Mesylate)
• a COX-2 inhibitor e.g., celecoxib
• interferons e.g., cytokines
• antagonists e.g., neutralizing antibodies
• a "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
• chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone;
• alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®)
• lapachol lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT- 1 1 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1 ); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chloropho
• calicheamicin especially calicheamicin gamma II and calicheamicin omegall (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
• ADRIAMYCIN® morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin, doxorubicin HC1 liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C,
• mycophenolic acid nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), pemetrexed (ALIMTA®); tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxif
• diaziquone diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate;
• hydroxyurea lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins;
• mitoguazone mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
• triaziquone 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;
• thiotepa taxoid, e.g. , paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine;
• mercaptopurine mercaptopurine
• methotrexate platinum agents such as cisplatin, oxaliplatin (e.g.,
• ELOXATIN® eLOXATIN®
• carboplatin eLOXATIN®
• vincas which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine
• ELDISINE®, FILDESIN®, and vinorelbine ELDISINE®
• etoposide VP- 16
• ifosfamide mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate;
• topoisomerase inhibitor RFS 2000 difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog);
• antisense oligonucleotides particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor ⁇ e.g., LURTOTECAN®); rmRH ⁇ e.g., ABARELIX®); BAY439006 (sorafenib, NEXAVAR ® ; Bayer); SU- 1 1248 (sunitinib,
• SUTENT®, Pfizer perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine- threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclopho
• Chemofherapeutic agents as defined herein include “anti-hormonal agents” or
• hormones which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen
• SERM3 selective estrogen receptor modulators
• SERM3 pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole
• FEMARA® aminoglutethimide
• other aromatase inhibitors include vorozole
• RIVISOR® megestrol acetate
• MEGASE® megestrol acetate
• fadrozole 4(5)-imidazoles
• lutenizing hormone-releasing hormone agonists including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin
• sex steroids including progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretinoic acid and fenretinide; onapristone; anti-progesterones; estrogen receptor down-regulators (ERDs); anti-androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.
• angiogenic factor or agent is a growth factor which stimulates the development of blood vessels, e.g., promote angiogenesis, endothelial cell growth, stability of blood vessels, and/or vasculogenesis, etc.
• angiogenic factors include, but are not limited to, e.g., VEGF and members of the VEGF family (VEGF-B, VEGF-C and VEGF-D), P1GF, PDGF family, fibroblast growth factor family (FGFs), TIE ligands (Angiopoietins), ephrins, delta-like ligand 4 (DLL4), del- 1 , fibroblast growth factors: acidic (aFGF) and basic (bFGF), follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF) /scatter factor (SF), interleukin-8 (IL-8), leptin, mid
• IGF-I insulin-like growth factor-I
• VIGF insulin-like growth factor
• EGF epidermal growth factor
• CTGF tumor necrosis factor
• TGF-alpha and TGF-beta TGF-beta.
• Klagsbrun and D'Amore (1991) Ann . Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172- 3179; Ferrara & Alitalo (1999) Nature Medicine 5(12): 1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 ⁇ e.g., Table 1 listing known angiogenic factors); and, Sato (2003) Int. J. Clin. Oncol. 8:200-206.
• an "anti-angiogenic agent” or “angiogenesis inhibitor” refers to a small molecular weight substance, a polynucleotide (including, e.g., an inhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly.
• RNAi or siRNA inhibitory RNA
• the anti-angiogenic agent includes those agents that bind and block the angiogenic activity of the angiogenic factor or its receptor.
• an anti-angiogenic agent is an antibody or other antagonist to an angiogenic agent as defined above, e.g., fusion proteins that binds to VEGF-A such as ZALTRAPTM (Aflibercept), antibodies to VEGF-A such as AVASTIN ® (bevacizumab) or to the VEGF-A receptor ⁇ e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as GLEEVEC ® (Imatinib
• Anti-angiogenic agents also include native angiogenesis inhibitors , e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D'Amore ⁇ 99 ⁇ ) Annu. Rev. Physiol.
• VEGF refers to the 165-amino acid human vascular endothelial cell growth factor and related 121-, 189-, and 206- amino acid human vascular endothelial cell growth factors, as described by Leung et al. (1989) Science 246: 1306, and Houck et al. (1991) Mol. Endocrin, 5: 1806, together with the naturally occurring allelic and processed forms thereof.
• VEGF also refers to VEGFs from non-human species such as mouse, rat or primate.
• VEGF vascular endothelial growth factor
• Reference to any such forms of VEGF may be identified in the present application, e.g., by "VEGF (8-109),” “VEGF (1 -109),” “VEGF-A109” or “VEGF 165.”
• the amino acid positions for a "truncated" native VEGF are numbered as indicated in the native VEGF sequence.
• amino acid position 17 (methionine) in truncated native VEGF is also position 17 (methionine) in native VEGF.
• the truncated native VEGF has binding affinity for the KDR and Fit- 1 receptors comparable to native VEGF.
• a "VEGF antagonist” refers to a molecule capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with VEGF activities including, but not limited to, its binding to one or more VEGF receptors.
• VEGF antagonists include, without limitation, anti- VEGF antibodies and antigen-binding fragments thereof, receptor molecules and derivatives which bind specifically to VEGF thereby sequestering its binding to one or more receptors, anti- VEGF receptor antibodies, VEGF receptor antagonists such as small molecule inhibitors of the VEGFR tyrosine kinases, and immunoadhesins that bind to VEGF such as VEGF traps (e.g., aflibercept).
• VEGF traps e.g., aflibercept
• VEGF antagonist specifically includes molecules, including antibodies, antibody fragments, other binding polypeptides, peptides, and non-peptide small molecules, that bind to VEGF and are capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with VEGF activities.
• VEGF activities specifically includes VEGF mediated biological activities of VEGF.
• VEGF trap means a protein, such as a fusion molecule, that binds to VEGF and is capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with VEGF activities.
• An example of a VEGF trap is aflibercept.
• anti-VEGF antibody or “an antibody that binds to VEGF” refers to an antibody that is capable of binding to VEGF with sufficient affinity and specificity that the antibody is useful as a diagnostic and/or therapeutic agent in targeting VEGF.
• Anti-VEGF antibodies suppress the growth of a variety of human tumor cell lines in nude mice (Kim et al, Nature 362:841 -844 (1993); Warren et al., J. Clin. Invest. 95: 1789- 1797 (1995); Borgstrom et al, Cancer Res. 56:4032-4039 (1996); Melnyk et al, Cancer Res. 56:921 -924 ( 1996)) and also inhibit intraocular angiogenesis in models of ischemic retinal disorders. Adamis et al, Arch. Ophthalmol 1 14:66-71 (1996).
• the anti-VEGF antibody can be used as a therapeutic agent in targeting and interfering with diseases or conditions wherein the VEGF activity is involved.
• the antibody selected will normally have a sufficiently strong binding affinity for VEGF.
• the antibody may bind hVEGF with a 3 ⁇ 4 value of between 100 nM-1 pM.
• Antibody affinities may be determined by a surface plasmon resonance based assay (such as the BIAcore assay as described in PCT Application Publication No. WO2005/012359); enzyme-linked
• ELISA immunoabsorbent assay
• competition assays e.g. RIA's
• the antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic.
• assays are known in the art and depend on the target antigen and intended use for the antibody. Examples include the HUVEC inhibition assay; tumor cell growth inhibition assays (as described in WO 89/06692, for example); antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (US Patent 5,500,362); and agonistic activity or hematopoiesis assays (see WO 95/27062).
• An anti-VEGF antibody will usually not bind to other VEGF homologues such as VEGF-B, VEGF-C, VEGF-D or VEGF-E, nor other growth factors such as P1GF, PDGF or bFGF.
• anti-VEGF antibodies include a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanized anti-VEGF monoclonal antibody (see Presta et al (1997) Cancer Res. 57:4593-4599), including but not limited to the antibody known as "bevacizumab” also known as “rhuMAb VEGF” or "AVASTIN ® .” AVASTIN ® is presently commercially available.
• Nonlimiting exemplary cancers that may be treated with bevacizumab include non- small cell lung cancer, colorectal cancer, breast cancer, renal cancer, ovarian cancer, glioblastoma multiforme, pediatric osteosarcoma, gastric cancer and pancreatic cancer.
• Bevacizumab comprises mutated human IgGi framework regions and antigen-binding complementarity-determining regions from the murine antibody A.4.6.1 that blocks binding of human VEGF to its receptors.
• Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. Pat. Nos. 6,884,879, and 7, 169,901. Additional anti-VEGF antibodies are described in PCT Application Publication Nos.
• a "effective amount” or “therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a subject. In certain embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
• a therapeutically effective amount of FGFR1 fusion protein of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the FGFR1 fusion proteins are outweighed by the therapeutically beneficial effects.
• the effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit ⁇ i.e., slow to some extent and typically stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and typically stop) tumor metastasis; inhibit, to some extent, tumor growth; allow for treatment of the tumor, and/or relieve to some extent one or more of the symptoms associated with the disorder.
• the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
• a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
• concurrent dosing refers to the administration of two therapeutic molecules within an eight hour time period.
• two therapeutic molecules are administered at the same time.
• Two therapeutic molecules are considered to be administered at the same time (i.e. simultaneously) if at least a portion of a dose of each therapeutic molecule is administered within 1 hour.
• Two therapeutic molecules are administered concurrently if at least one dose is administered concurrently, even if one or more other doses are not administered concurrently.
• concurrent administration includes a dosing regimen when the administration of one or more therapeutic molecule(s) continues after discontinuing the administration of one or more other therapeutic molecules(s).
• Administration "in combination with” one or more further therapeutic agents includes concurrent (including simultaneous) and consecutive (i.e., sequential) administration in any order.
• FIG. 1 Pharmacokinetics of FP- 1039 at doses from 0.5- 16.0 mg/kg. Arrows indicate days of FP- 1039 administration. PK samples on Days 8 and 15 are pre-dose (trough), while detailed PK sampling is performed following Dose 1 (Day 1 ) and after 4 weekly doses (Day 22).
• FIG. 1 Summary of plasma free FGF-2 levels at different timepoints across all dosing cohorts (0.5- 16 mg/kg). Free FGF-2 was measured pre-dose Day 1 , 24-hours post the 1 st dose, pre-dose Day 8, Day 15, and Day 36. The Day 36 samples are two weeks following FP- 1039 dosing.
• FIG. 3 Free FGF-2 plasma levels in normal subjects and patients before and after a single dose of FP- 1039. All patients had elevated FGF-2 plasma levels compared to plasma from normal subjects. Plasma free-FGF-2 levels in cancer patients treated with FP-1039 all decreased relative to pre-dose levels (overall average decrease of 76%).
• a soluble Fibroblast Growth Factor Receptor 1 (FGFRl )/Fc fusion protein FP- 1039
• FGFRl Fibroblast Growth Factor Receptor 1
• the present invention provides for administration of FP-1039 (i.e., SEQ ID NO:8) to human individuals, e.g., cancer patients, at concentrations from a dose of about 2 mg/kg body weight to at least about 30 mg/kg.
• FP-1039 i.e., SEQ ID NO:8
• other soluble FGFRl fusion proteins can also be safely administered to humans at these same elevated concentrations, for example, to treat cancer.
• the present invention provides methods of treating a human having a cancer, the method comprising administering to the human a soluble FGFRl fusion protein (e.g., FP-1039) as described herein at a dose of about 2 mg/kg body weight to about 30 mg/kg body weight.
• a soluble FGFRl fusion protein e.g., FP-1039
• the human having a cancer has an FGF-2 plasma concentration above the average FGF-2 plasma concentration of the human without cancer. In some embodiments, the human having a cancer has an FGF-2 plasma concentration of at least 6 pg/ml or at least 10 pg/ml prior to the administration of the soluble FGFRl fusion protein. In some embodiments, as used herein, FGF-2 levels are determined as measured by an
• ECL electrochemiluminescence
• MSD SI2400 reader Meso Scale Discovery, Sector Image 2400, Model # 1250.
• Assay Diluent GF1 is replaced with Calibrator Diluent GF1.
• the method of treating the human having the cancer comprises administering soluble FGFRl fusion protein (e.g., FP- 1039) in a dose that results in sustained target engagement in the plasma even after seven days post-administration or longer.
• the soluble FGFRl fusion protein is administered in a dose that results in sustained engagement of the target FGF-2 in the plasma even after seven days post-administration or longer.
• the soluble FGFRl fusion protein is administered at a dose such that, at seven days after administration, the human has a free FGF-2 plasma concentration of less than 4 pg/ml.
• the soluble FGFRl fusion protein is administered at a dose such that, at seven days after administration, the human has a free FGF-2 plasma concentration of less than 3 pg/ml.
• the soluble FGFRl fusion protein consists of or comprises SEQ ID NO:8.
• the soluble FGFRl fusion proteins of the present invention find use in treating both metastatic and non-metastatic forms of cancer, including but not limited to, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, oral cancer, ovarian cancer, thyroid cancer, lung cancer, prostate cancer, uterine cancer, endometrial cancer, testicular cancer, neuroblastoma, squamous cell carcinoma of the head, neck, cervix and vagina, multiple myeloma, soft tissue and osteogenic sarcoma, colorectal cancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), mesothelioma, cervical cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), esophageal cancer, laryngeal cancer, gall bladder cancer, small intestine cancer, cancer of the central nervous system
• pancreatic cancer breast cancer, gastric cancer, bladder
• the cancer to be treated is prostate cancer, breast cancer, colorectal cancer, lung cancer, endometrial cancer, head and neck cancer, laryngeal cancer, liver cancer, renal cancer, glioblastoma or pancreatic cancer.
• the humans treated with a soluble FGFR1 fusion protein have a cancer that is characterized by a Fibroblast Growth Factor Receptor 2 (FGFR2) with a ligand- dependent activating mutation.
• FGFR2 Fibroblast Growth Factor Receptor 2
• a ligand-dependent activating mutant is a FGFR2 variant whose biological effects depend on the binding of FGFR2 to one or more of its ligands, such as a mutation that causes alterations in the ligand binding properties of FGFR2.
• An observed FGFR2 mutation in endometrial tumor cells is S252W, found in about 7% of cases.
• a P253R mutation occurs in about 2% of endometrial cancer cases.
• expression of an FGFR2 having a ligand-dependent activating mutation is detected by biopsying the cancer and analyzing the nucleic acid of the tumor cells of the cancer. Whether the ligand-dependent activating mutation is detectably expressed in the nucleic acid can be analyzed using any method known in the art, including but not limited to polymerase chain reaction (PCR), quantitative PCR, RT-PCR, or sequencing analysis.
• PCR polymerase chain reaction
• quantitative PCR quantitative PCR
• RT-PCR RT-PCR
• compositions of the present invention will be formulated, dosed, and administered in a fashion consistent with good medical practice.
• Factors for consideration in this context include, but are not limited to, the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
• an FGFR1 fusion protein of the invention when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the severity and course of the disease, whether the FGFR1 fusion protein is administered for preventive or therapeutic purposes, the intended aggressiveness of the treatment regime, previous therapy, the patient's clinical history and response to the FGFR1 fusion protein, and the discretion of the attending physician.
• compositions for administration will commonly comprise a soluble FGFRl fusion protein ⁇ i.e., an extracellular domain of an FGFRl polypeptide linked to a Fc polypeptide, such as but not limited to FP-1039) dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier.
• a pharmaceutically acceptable carrier e.g., an aqueous carrier.
• aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
• These compositions may be sterilized by conventional, well known sterilization techniques.
• compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
• concentration of soluble FGFRl fusion protein in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
• a typical pharmaceutical composition for administration will vary according to the agent and method of administration (e.g. intravenous or subcutaneous). Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).
• Pharmaceutical formulations for use with the present invention can be prepared by mixing a soluble FGFR1 fusion protein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers. Such formulations can be lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used.
• Acceptable carriers, excipients or stabilizers can be acetate, phosphate, citrate, and other organic acids; antioxidant (e.g., ascorbic acid); preservatives; low molecular weight polypeptides; proteins, such as serum albumin or gelatin, or hydrophilic polymers such as polyvinylpyllolidone; and amino acids,
• monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents; and ionic and non-ionic surfactants (e.g., polysorbate); salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants.
• the soluble FGFR1 fusion protein (including but not limited to FP-1039) is administered at a dose of about 2 mg/kg body weight to about 30 mg/kg body weight. In some embodiments, the soluble FGFR1 fusion protein (including but not limited to FP-1039) is administered at a dose of about 8 mg/kg body weight to about 20 mg/kg body weight.
• the soluble FGFR1 fusion protein (including but not limited to FP-1039) is administered at a dose of about 8 mg/kg body weight, about 10 mg/kg body weight, about 1 1 mg/kg body weight, about 12 mg/kg body weight, about 13 mg/kg body weight, about 14 mg/kg body weight, about 15 mg/kg body weight, about 16 mg/kg body weight, about 17 mg/kg body weight, about 18 mg/kg body weight, about 19 mg/kg body weight, about 20 mg/kg body weight, about 24 mg/kg body weight, or about 30 mg/kg body weight, or in a dose ranging from one to another of the above values.
• dosages may be administered twice a week, weekly, every other week, at a frequency between weekly and every other week, every three weeks, every four weeks, or every month.
• dosages of the soluble FGFR1 fusion protein can be calculated in two ways depending on the extinction coefficient (EC) used.
• the extinction coefficient differs depending on whether the glycosylation of the protein is taken into account.
• the extinction coefficient based on the amino acid composition of FP- 1039 for example, is 1.42 mL/mg*cm.
• the extinction coefficient is 1.1 1 mIJmg*cm. Calculation of the FP-1039 dose using an EC of 1.1 1 mL/mg*cm increases the calculated dose by 28%, as shown in Table 1.
• the doses calculated using the two extinction coefficients are different, the molar concentrations, or the actual amounts of drug administered, are identical. Unless otherwise noted, the doses disclosed herein are each calculated using the extinction coefficient that does not take account of glycosylation. For FP-1039, this extinction coefficient is 1.42 mL/mg*cm. How these dosages compare to those calculated using the extinction coefficient that takes account of glycosylation is shown in Table 1. As can be seen from Table 1 , a dosage of 8 mg/kg (e.g., 7.8 and 8.0) using an EC of 1.42 mL/mg*cm herein corresponds to a dosage of 10 mg/kg (e.g.
• a dosage of 16 mg/kg (e.g. 15.6 and 16.0 mg/kg) herein using an EC of 1.42 mL/mg*cm corresponds to a dosage of about 20 mg/kg (e.g. 20.0 and 20.5) when calculated using an EC of 1.1 1 mL/mg*cm.
• measured numbers provided herein are approximate and encompass values having additional significant digits that are rounded off. For instance, 8 mg/kg encompasses values with two significant digits such as 7.6, 7.8, 8.0, 8.2, 8.4, and 8.45, each of which round to 8. Likewise, a value such as 16 mg/kg encompasses values with three significant digits that round to 16, such as, for example 15.6 and 16.0.
• the patient is treated with a combination of the FGFR1 fusion protein (e.g., FP-1039) and one or more other therapeutic agents(s).
• FGFR1 fusion protein e.g., FP-1039
• other therapeutic agents(s) e.g., FP-1039
• administration includes coadministration or concurrent administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein optionally there is a time period while both (or all) active agents simultaneously exert their biological activities.
• the effective amounts of therapeutic agents administered in combination with an FGFR1 fusion protein will be at the physician's or veterinarian's discretion. Dosage administration and adjustment is done to achieve maximal management of the conditions to be treated. The dose will additionally depend on such factors as the type of therapeutic agent to be used and the specific patient being treated.
• the patient is treated with a combination of the FGFR1 fusion protein (e.g., FP-1039) and a VEGF antagonist.
• the VEGF antagonist is a VEGF trap, such as aflibercept.
• the VEGF antagonist is a VEGF antibody.
• the VEGF antibody is bevacizumab.
• One exemplary dosage of bevacizumab would be in the range from about 0.05 mg/kg to about 20 mg/kg.
• one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 7.5 mg/kg, 10 mg/kg or 15 mg/kg (or any combination thereof) may be administered to the patient.
• Such doses may be administered intermittently, e.g., every week, every two, or every three weeks.
• the FGFR1 fusion protein (e.g., FP-1039) is administered in combination with another therapeutic agent, such as chemotherapeutic agent or anti-angiogenic agent, at the recommended or prescribed dosage and/or frequency of the therapeutic agent.
• another therapeutic agent such as chemotherapeutic agent or anti-angiogenic agent
• the soluble FGFR1 fusion proteins of the present invention are administered to a human patient in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal,
• the soluble FGFR1 fusion proteins are administered intravenously.
• the methods described herein are based on administering a soluble FGFRl fusion protein to a human patient as a continuous infusion over a period of 30 minutes.
• the present invention also contemplates administering the soluble FGFRl fusion protein over a shorter or longer period of time, e.g., over a period of one hour.
• compositions comprising the FGFRl fusion proteins of the invention can be administered as needed to subjects.
• an effective dose of the FGFRl fusion proteins of the invention is administered to a subject one or more times.
• an effective dose of the FGFRl fusion proteins of the invention is administered to the subject at least once a month, at least twice a month, once a week, twice a week, or three times a week.
• an effective dose of the FGFRl fusion proteins of the invention is administered to the subject for at least a week, at least a month, at least three months, at least six months, or at least a year.
• FGFRl fusion proteins of the present invention may be administered alone or with other modes of treatment. They may be provided before, substantially contemporaneous with, or after other modes of treatment, for example, surgery, chemotherapy, radiation therapy, or the administration of other therapeutics, including for example, small molecules and other biologies, such as a therapeutic antibody. [0099] In some embodiments, an FGFRl fusion protein of the present invention is the only therapeutically active agent administered to a patient.
• the FGFRl fusion protein is administered in combination with one or more other therapeutic agents, including but not limited to anti-angiogenic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, cytotoxic agents, cardioprotectants, or other therapeutic agents.
• the FGFRl fusion protein may be administered concurrently with one or more other therapeutic regimens.
• the FGFRl fusion protein may be administered in combination with one or more antibodies.
• Anti-angiogenic therapy in relationship to cancer is a cancer treatment strategy aimed at inhibiting the development of tumor blood vessels required for providing nutrients to support tumor growth.
• the anti-angiogenic treatment provided by the invention is capable of inhibiting the neoplastic growth of tumor at the primary site as well as preventing metastasis of tumors at the secondary sites, therefore allowing attack of the tumors by other therapeutics.
• anti-cancer agent or therapeutic is an anti-angiogenic agent.
• anti-cancer agent is a chemotherapeutic agent.
• angiogenic agents have been identified and are known in the arts, including those listed herein, e.g., listed under Definitions, and by, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et al., Nature Reviews:Drug Discovery, 3:391-400 (2004); and Sato Int. J. Clin. Oncol, 8:200-206 (2003). See also, US Patent Application US20030055006.
• two or more angiogenic inhibitors may optionally be co-administered to the patient in addition to an FGFRl fusion protein of the invention.
• VEGF antagonists or VEGF receptor antagonists.
• other therapeutic agents useful for combination tumor therapy with the FGFRl fusion protein include antagonists of other factors that are involved in tumor growth, such as EGFR, ErbB2 (also known as Her2) ErbB3, ErbB4, or TNF.
• the FGFRl fusion protein can be used in combination with small molecule receptor tyrosine kinase inhibitors (RTKIs) that target one or more tyrosine kinase receptors such as VEGF receptors, FGF receptors, EGF receptors and PDGF receptors.
• RTKIs small molecule receptor tyrosine kinase inhibitors
• Many therapeutic small molecule RTKIs are known in the art, including, but are not limited to, vatalanib (PTK787), erlotinib (TARCEVA ® ), OSI-7904, ZD6474
• ZACTIMA ® ZD6126 (ANG453), ZD1839, sunitinib (SUTENT ® ), semaxanib (SU5416), AMG706, AG013736, Imatinib (GLEEVEC ), MLN-518, CEP-701 , PKC- 412, Lapatinib (GSK572016), VELCADE ® , AZD2171 , sorafenib (NEXAVAR ® ), XL880, and CHIR-265.
• the FGFRl fusion protein and the one or more other therapeutic agents can be administered concurrently, simultaneously, or sequentially in an amount and for a time sufficient to reduce or eliminate the occurrence or recurrence of a tumor, a dormant tumor, or a micrometastases.
• the FGFRl fusion protein and the one or more other therapeutic agents can be administered as maintenance therapy to prevent or reduce the likelihood of recurrence of the tumor.
• Soluble FGFRl fusion proteins or FGFRl ECD fusion molecules refer to proteins comprising an FGFRl ECD polypeptide linked to at least one fusion partner, wherein the soluble FGFRl fusion protein lacks a transmembrane domain (e.g., an FGFRl transmembrane domain) and is not bound to the cellular membrane.
• the fusion partner may be joined to either the N- terminus or the C-terminus of the FGFRl ECD polypeptide.
• the FGFRl ECD may be joined to either the N-terminus or the C-terminus of the fusion partner.
• FGFRl ECD molecules are provided.
• FGFRl ECDs consist of native FGFRl ECDs, FGFRl ECD variants, FGFRl ECDs comprising an Ig domain III chosen from Illb and IIIc, native FGFRl -Illb ECD, native FGFRl -IIIc ECD, FGFRl -Illb ECD variants, FGFRl-IIIc ECD variants, FGFRl ECD fragments, native FGFRl ECD fragments, variants of FGFRl ECD fragments, FGFRl ECD glycosylation mutants, and FGFRl ECD fusion molecules, as well as non-human FGFRl ECDs.
• FGFRl ECDs are able to bind FGF-2.
• the FGFRl ECD includes a signal peptide, either from FGFRl, or from another FGFR, or from another protein. In other embodiments, no signal peptide is included.
• the FGFRl ECD proteins of the invention can comprise an entire FGFRl ECD, including that of wildtype FGFRl -Illb or wildtype FGFRl -IIIc ECD, or for example a variant or fragment of the FGFRl ECD (e.g., a variant or fragment having at least 95% amino acid sequence identity to a wildtype FGFRl ECD) that retains the ability to bind FGF-2.
• a variant of the native FGFRl ECD for example, lacking the first immunoglobulin domain is provided. See, e.g., US Patent No. 6,384, 191.
• FGFRl ECD having a deletion of one or more and up to 22 amino acid residues counting from the C-terminus of the native FGFRl ECD of SEQ ID NO: l .
• the FGFRl ECD has the final 22 amino acids of the C-terminus deleted, while in others, the FGFRl ECD has the final 19, 14, 9, 8, or 4 C-terminal amino acids deleted in comparison to SEQ ID NO: 1. See, e.g., SEQ ID NOs:2-6.
• the FGFRl ECD of the present invention has at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher amino acid sequence identity to a wildtype FGFRl-IIIc ECD (SEQ ID NO: l ). In some embodiments, the FGFRl ECD of the present invention has at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher amino acid sequence identity to a wildtype FGFRl -IIIc ECD (SEQ ID NO: l ) and has the ability to bind FGF-2.
• Non-limiting exemplary FGFRl ECD fragments include human FGFRl ECD ending at amino acid 339 (counting from the first amino acid of the mature form, without the signal peptide). In some embodiments, an FGFRl ECD fragment ends at an amino acid between amino acid 339 and amino acid 360 (counting from the first amino acid of the mature form, without the signal peptide).
• Examples of FGFRl ECD fragments include those having the C-terminal amino acid residues LYLE (SEQ ID NO:9) or MTSPLYLE (SEQ ID NO: 10) or VMTSPLYLE (SEQ ID NO: l 1) or A VMTSPLYLE (SEQ ID NO: 12) or EERP A VMTSPLYLE (SEQ ID NO: 13) or LEERPAVMTSPLYLE (SEQ ID NO: 14) or ALEERPAVMTSPLYLE (SEQ ID NO: 15) deleted as compared to either a native FGFRl -Illb or FGFRl -IIIc.
• Further examples include those having the C-terminal amino acid residues KALEERPAVMTSPLYLE (SEQ ID NO: 16) or RPVAKALEERPAVMTSPLYLE (SEQ ID NO: 17) deleted as compared to a native FGFRl-IIIb or EALEERPAVMTSPLYLE (SEQ ID NO: 18) deleted as compared to a native FGFRl -IIIc.
• Point mutations, truncations, or internal deletions or insertions within the ECD amino acid sequence may be made within the FGFRl ECD so long as FGF-2 binding activity is retained.
• a fusion partner is selected that imparts favorable
• a fusion partner is selected that increases the half-life of the FGFRl ECD fusion molecule relative to the corresponding FGFRl ECD without the fusion partner.
• a lower dose and/or less-frequent dosing regimen may be required in therapeutic treatment.
• the resulting decreased fluctuation in FGFR1 ECD serum levels may improve the safety and tolerability of the FGFR1 ECD-based therapeutics.
• fusion partners Many different types are known in the art. One skilled in the art can select a suitable fusion partner according to the intended use.
• Non-limiting exemplary fusion partners include polymers, polypeptides, lipophilic moieties, and succinyl groups.
• Exemplary polypeptide fusion partners include serum albumin (e.g. human serum albumin or HSA) and an antibody Fc domain.
• Exemplary polymer fusion partners include, but are not limited to, polyethylene glycol, including polyethylene glycols having branched and/or linear chains.
• oligomerization offers certain functional advantages to a fusion protein, including, but not limited to, multivalency, increased binding strength, and the combined function of different domains.
• a fusion partner comprises an oligomerization domain, for example, a dimerization domain.
• oligomerization domains include, but are not limited to, coiled-coil domains, including alpha- helical coiled-coil domains; collagen domains; collagen-like domains, and certain
• Certain exemplary coiled-coil polypeptide fusion partners include the tetranectin coiled-coil domain; the coiled-coil domain of cartilage oligomeric matrix protein; angiopoietin coiled-coil domains; and leucine zipper domains.
• Certain exemplary collagen or collagen-like oligomerization domains include, but are not limited to, those found in collagens, mannose binding lectin, lung surfactant proteins A and D, adiponectin, ficolin, conglutinin, macrophage scavenger receptor, and emilin.
• fusion partners are known in the art. One skilled in the art can select an appropriate Fc domain fusion partner according to the intended use.
• a fusion partner is an Fc immunoglobulin domain.
• An Fc fusion partner may be a wildtype Fc found in a naturally occurring antibody, a variant thereof, or a fragment thereof.
• Non-limiting exemplary Fc fusion partners include Fes comprising a hinge and the CH2 and CH3 constant domains of a human IgG, for example, human IgGl, IgG2, IgG3, or IgG4. In some embodiments, the Fc fusion partner does not include an Fc hinge region.
• Certain additional Fc fusion partners include, but are not limited to, human IgA and IgM.
• an Fc fusion partner comprises a C237S mutation.
• an Fc fusion partner comprises a hinge, CH2, and CH3 domains of human IgG2 with a P33 IS mutation, as described in U.S. Patent No. 6,900,292.
• a fusion partner is an albumin.
• Certain exemplary albumins include, but are not limited to, human serum albumin (HSA) and fragments of HSA that are capable of increasing the serum half-life and/or bioavailability of the polypeptide to which they are fused.
• a fusion partner is an albumin-binding molecule, such as, for example, a peptide that binds albumin or a molecule that conjugates with a lipid or other molecule that binds albumin.
• a fusion molecule comprising HSA is prepared as described, e.g., in U.S. Patent No. 6,686, 179.
• a fusion partner is a polymer, for example, polyethylene glycol (PEG).
• PEG may comprise branched and/or linear chains.
• a fusion partner comprises a chemically-derivatized polypeptide having at least one PEG moiety attached.
• Pegylation of a polypeptide may be carried out by any method known in the art. One skilled in the art can select an appropriate method of pegylating a particular polypeptide, taking into consideration the intended use of the polypeptide.
• Certain exemplary PEG attachment methods include, for example, EP 0 401 384; Malik et al., Exp.
• pegylation may be performed via an acylation reaction or an alkylation reaction, resulting in attachment of one or more PEG moieties via acyl or alkyl groups.
• PEG moieties are attached to a polypeptide through the a- or ⁇ - amino group of one or more amino acids, although any other points of attachment known in the art are also contemplated.
• Pegylation by acylation typically involves reacting an activated ester derivative of a PEG moiety with a polypeptide.
• a non-limiting exemplary activated PEG ester is PEG esterified to N-hydroxysuccinimide (NHS).
• NHS N-hydroxysuccinimide
• acylation is contemplated to include, without limitation, the following types of linkages between a polypeptide and PEG: amide, carbamate, and urethane. See, e.g., Chamow, Bioconjugate Chem., 5: 133-140 (1994).
• Pegylation by alkylation typically involves reacting a terminal aldehyde derivative of a PEG moiety with a polypeptide in the presence of a reducing agent.
• Non-limiting exemplary reactive PEG aldehydes include PEG propionaldehyde, which is water stable, and mono CI -CIO alkoxy or aryloxy derivatives thereof. See, e.g., U.S. Patent No. 5,252,714.
• a pegylation reaction results in poly-pegylated polypeptides. In certain embodiments, a pegylation reaction results in mono-, di-, and/or tri-pegylated polypeptides. Further, desired pegylated species may be separated from a mixture containing other pegylated species and/or unreacted starting materials using various purification techniques known in the art, including among others, dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gel filtration chromatography, and electrophoresis.
• the fusion partner may be attached, either covalently or non-covalently, to the amino- terminus or the carboxy-terminus of the FGFRl ECD.
• the attachment may also occur at a location within the FGFRl ECD other than the amino-terminus or the carboxy-terminus, for example, through an amino acid side chain (such as, for example, the side chain of cysteine, lysine, histidine, serine, or threonine).
• a linker may be included between the fusion partner and the FGFRl ECD.
• Such linkers may be comprised of amino acids and/or chemical moieties.
• Exemplary methods of covalently attaching a fusion partner to an FGFRl ECD include, but are not limited to, translation of the fusion partner and the FGFRl ECD as a single amino acid sequence and chemical attachment of the fusion partner to the FGFRl ECD.
• additional amino acids may be included between the fusion partner and the FGFRl ECD as a linker.
• the linker is selected based on the polynucleotide sequence that encodes it, to facilitate cloning the fusion partner and/or FGFRl ECD into a single expression construct (for example, a polynucleotide containing a particular restriction site may be placed between the polynucleotide encoding the fusion partner and the polynucleotide encoding the FGFRl ECD, wherein the polynucleotide containing the restriction site encodes a short amino acid linker sequence).
• linkers of various sizes can typically be included during the coupling reaction.
• linkers of various sizes can typically be included during the coupling reaction.
• Several methods of covalent coupling of a polypeptide to another molecule ⁇ i.e. fusion partner) are known.
• the polypeptide and fusion partner can also be non-covalently coupled.
• Exemplary methods of non-covalently attaching a fusion partner to an FGFRl ECD include, but are not limited to, attachment through a binding pair.
• Exemplary binding pairs include, but are not limited to, biotin and avidin or streptavidin, an antibody and its antigen, etc.
• Nucleic acid molecules encoding FGFRl ECDs and/or soluble FGFRl fusion proteins can be synthesized by chemical methods or prepared by techniques well known in the art. See, for example, Sambrook, et al, Molecular Cloning, A Laboratory Manual, Vols. 1 -3, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989).
• a polynucleotide encoding a polypeptide of the invention comprises a nucleotide sequence that encodes a signal peptide, which, when translated, will be fused to the amino-terminus of the FGFRl polypeptide.
• the nucleotide sequence does not include a sequence encoding a signal peptide.
• the signal peptide may be the native signal peptide, the signal peptide of FGFRl , FGFR2, FGFR3, or FGFR4, or may be another heterologous signal peptide.
• Exemplary signal peptides are known in the art, and are described, e.g., in PCT Publication No. WO 2006/081430.
• the nucleic acid molecule comprising the polynucleotide encoding the soluble FGFRl fusion protein is an expression vector that is suitable for expression in a selected host cell.
• the polynucleotide encoding the FGFRl ECD polypeptide ⁇ e.g., the polynucleotide encoding the polypeptide of any of SEQ ID NOs: l-6) is inserted into the expression vector at a linker site, and the polynucleotide encoding the fusion partner polypeptide ⁇ e.g., the polynucleotide encoding the Fc polypeptide of human IgGl) is inserted at a site following the FGFRl ECD such that the FGFRl ECD and Fc components are in-frame when the nucleic acid molecule is transcribed and translated.
• the FGFRl fusion proteins of the present invention may be expressed from a vector in a host cell.
• a vector is selected that is optimized for expression of polypeptides in CHO-S or CHO-S-derived cells. Exemplary such vectors are described, e.g., in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).
• a vector is chosen for in vivo expression of the polypeptides of the invention in animals, including humans.
• expression of the polypeptide is under the control of a promoter that functions in a tissue-specific manner.
• Suitable host cells for expression of the FGFR1 fusion proteins of the present invention include, for example, prokaryotic cells, such as bacterial cells; or eukaryotic cells, such as fungal cells, plant cells, insect cells, and mammalian cells.
• prokaryotic cells such as bacterial cells
• eukaryotic cells such as fungal cells, plant cells, insect cells, and mammalian cells.
• exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, Cos cells, including Cos 7 cells; 293 cells, including 293-6E and 293-T cells; CHO cells, including CHO-S and DG44 cells; and NS0 cells.
• nucleic acid vector into a desired host cell can be accomplished by any method known in the art, including, but not limited to, calcium phosphate transfection, DEAE- dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Certain exemplary methods are described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids may be transiently or stably transfected in the desired host cells, according to methods known in the art.
• a polypeptide can be produced in vivo in an animal that has been engineered or transfected with a nucleic acid molecule encoding the polypeptide, according to methods known in the art.
• the FGFR1 fusion proteins of the present invention can be purified by various methods known in the art. Such methods include, but are not limited to, the use of affinity matrices, ion exchange chromatography, and/or hydrophobic interaction chromatography. Suitable affinity ligands include any ligands of the FGFR1 ECD or of the fusion partner, or antibodies thereto. For example, in the case of a fusion protein, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind to an Fc fusion partner to purify a polypeptide of the invention. Antibodies to the polypeptides of the invention may also be used to purify the polypeptides of the invention. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying certain polypeptides. Many methods of purifying polypeptides are known in the art
• Example 1 Dose-Finding Study to Evaluate the Safety and Tolerability of the FGFRl-Fc Fusion Protein FP-1039 in Patients with Advanced Malignancies
• FP-1039 (SEQ ID NO:8) is a highly glycosylated, dimerized, soluble fusion protein consisting of a truncated extracellular domain of human FGFRl linked to the Fc region of human IgGi .
• FP-1039 demonstrated significant anti-tumor activity in a variety of different xenograft models; enhanced anti-tumor activity when combined with cytotoxic or targeted anti-cancer drugs; and inhibited both FGF- and VEGF-mediated angiogenesis.
• a Phase I clinical trial with FP-1039 had dosing cohorts at 0.5 mg/kg body weight through 16 mg/kg body weight.
• Inclusion criteria included subjects having histologically or cytologically proven metastatic or locally advanced, unresectable solid tumors for which standard curative or palliative measures do not exist or are no longer effective; Eastern Cooperative Oncology Group (ECOG) Performance Status grade 0-2 (see Oken et ai, Am. J. Clin. Oncol. 5:649-655 (1982), incorporated by reference herein in its entirety); and washout of prior anticancer therapy.
• ECOG Eastern Cooperative Oncology Group
• FP-1039 was administered to subjects intravenously over 30 minutes once a week for 4 infusions, followed by a two-week observation period. Based on the observation of two dose- limiting toxicities (DLTs) in the three subjects dosed at 1 mg/kg (one episode of bowel perforation and sepsis and one episode of grade 3 neutropenia), doses of 0.5 mg/kg and 0.75 mg/kg were explored. Six subjects were dosed at 0.5 mg/kg and six subjects were dosed at 0.75 mg/kg. As shown in Table 3 below, adverse effects included one adverse effect was observed at 0.5 mg/kg (grade 1 erythema) and one adverse effect was observed at 0.75 mg/kg (grade 2 urticaria). Other adverse events were observed but were not responsible for removal of subjects from treatment due to the adverse effects.
• DLTs dose- limiting toxicities
• FP- 1039 was administered intravenously over 30 minutes once a week for 4 infusions, followed by a two-week observation period. Safety of the dosing level was evaluated by assessing for adverse events at each visit. Adverse Events were graded by the Common Terminology Criteria for Adverse Events v3.0 (CTCAE). DLT was defined as any FP- 1039-associated Adverse Event of CTCAE grade 3 or higher. After completion of the initial treatment and observation period, subjects with no evidence of disease progression or DLT after 4 infusions were eligible to receive additional weekly infusions of FP-1039. The cohorts of subjects and dispositions or best responses for each cohort are shown in Table 3.
• AE Adverse Event
• PD Progressive Disease
• SAE Serious Adverse Event
• SD Stable Disease a Column indicates best response or if subjects withdrew from treatment due to AEs
• FP-1039 was well-tolerated without observations of drug-related weight loss, hypertension, or soft tissue calcification at doses up through 16 mg/kg. No DLTs were observed in the 15 subjects dosed from 2 mg/kg to 16 mg/kg.
• FP-1039 concentration was measured in plasma from the subjects using a quantitative enzyme immunoassay methodology that measures free, active FP-1039 in K 2 EDTA plasma. Briefly, samples and controls were diluted in assay diluent containing excess heparin and loaded onto plates pre-coated with recombinant human FGF-2 and pre-blocked. After a 1-hour incubation, the plate was washed and an anti-human IgG-Fc horseradish peroxidase (HRP) antibody added to detect bound FP-1039 using standard colorimetric ELISA detection. The method was validated in accordance with bioanalytical method validation guidelines and following Good Laboratory Practices (GLP) prior to clinical sample testing at Prevalere Life Sciences.
• GLP Good Laboratory Practices
• Plasma clearance (CL) was determined from the following equation:
• the plasma concentration of FP-1039 remains above 10 ⁇ g/ml even at one week post dosing. Furthermore, there was accumulation of FP-1039 in the plasma when comparing the C max , C min , and AUC data between the Day 1 and Day 22 doses (first vs. fourth dose). In summary, the pharmacokinetic profile of FP- 1039 supports twice weekly, weekly, or less frequent dosing.
• Plasma free FGF-2 levels were measured using a modified commercial immunoassay kit (Meso Scale Discovery, MSD) which utilizes an electrochemiluminescent (ECL) technology based on paired antibodies for detection of FGF-2.
• the detection system employs a ruthenium metal chelate (Sulfo-Tag) antibody as the ECL label.
• the relative mass values for natural FGF-2 in the plasma or serum samples is determined using the recombinant protein standards provided in the kit. Briefly, samples and controls were diluted in assay diluent and loaded onto plates pre- coated with antibodies against FGF-2 and pre-blocked.
• FGF-2 levels Prior to FP-1039 treatment, clinical subjects had elevated mean FGF-2 plasma concentrations relative to normal donors. As shown in Figures 2-3, treatment with FP- 1039 results in a decrease in free plasma FGF-2, suggesting that FP- 1039 sequesters FGF-2 present in the blood.
• Figure 2 shows that within 48 hours post dosing with FP-1039, there is a significant decrease in plasma FGF-2 levels. Among all of the dosing levels shown in Figures 2- 3, plasma free FGF-2 levels in cancer patients decreased relative to pre-dose levels (overall average decrease of 76%). This data demonstrates that FP-1039 exhibits a high degree of target engagement in plasma. Moreover, target engagement is maintained throughout the dosing schedule of weekly dosing. The data further suggest that target engagement may be maintained even after 2 weeks, supporting less frequent dosing.
• EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

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