Neutralizing human anti-IGFR antibody

专利摘要:

公开号:AU2007200876A1
申请号:U2007200876
申请日:2007-02-28
公开日:2007-03-22
发明作者:Peter Brams；Mary Diane Feingersh；Robert Greenberg；Judith Hailey；Jonathan A. Pachter；Leonard Presta；Mohan Srinivasan；Yan Wang；Denise Williams
申请人:Schering Corp；
IPC主号:C12N15-09

专利说明:
AUSTRALIA
Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: SCHERING CORPORATION Invention Title: NEUTRALIZING HUMAN ANTI-IGFR ANTIBODY The following statement is a full description of this invention, including the best method of performing it known to us: NEUTRALIZING HUMAN ANTI-IGFR ANTIBODY 00 This application claims the benefit of U.S. Provisional Patent Application No.
60/383,459, filed May 24, 2002; U.S. Provisional Patent Application No. 60/393,214, Q filed July 2, 2002 and U.S. Provisional Patent Application No. 60/436,254, filed r- December 23, 2002 each of which is herein incorporated by reference in its entirety.
O
(N FIELD OF THE INVENTION The present invention relates to fully human, monoclonal anti-Insulin-like Growth Factor Receptor-I (IGFR1) antibodies as well as methods of using the antibodies and methods of producing the antibodies.
BACKGROUND OF THE INVENTION The insulin-like growth factors, also known as somatomedins, include insulinlike growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II) (Klapper, et al., (1983) Endocrinol. 112:2215 and Rinderknecht, et al., (1978) Febs.Lett. 89:283).
These growth factors exert mitogenic activity on various cell types, including tumor cells (Macaulay, (1992) Br. J. Cancer 65:311), by binding to a common receptor named the insulin-like growth factor receptor-1 (IGFR1) (Sepp-Lorenzino, (1998) Breast Cancer Research and Treatment 47:235). Interaction of IGFs with IGFR1 activates the receptor by triggering autophosphorylation of the receptor on tyrosine residues (Butler, et al., (1998) Comparative Biochemistry and Physiology 121:19).
Once activated, IGFR1, in turn, phosphorylates intracellular targets to activate cellular signaling pathways. This receptor activation is critical for stimulation of tumor cell growth and survival. Therefore, inhibition of IGFR1 activity represents a valuable potential method to treat or prevent growth of human cancers and other proliferative diseases.
Several lines of evidence indicate that IGF-I, IGF-II and their receptor IGFR1 are important mediators of the malignant phenotype. Plasma levels of IGF-I have been found to be the strongest predictor of prostate cancer risk (Chan, et (1998) Science 279:563) and similar epidemiological studies strongly link plasma IGF-I levels with breast, colon and lung cancer risk.
S2 CN Overexpression of Insulin-like Growth Factor Receptor-I has also been demonstrated in several cancer cell lines and tumor tissues. IGFR1 is overexpressed in 40% of all breast cancer cell lines (Pandini, et al., (1999) Cancer Res. 5:1935) and 00 CN in 15% of lung cancer cell lines. In breast cancer tumor tissue, IGFR1 is overexpressed 6-14 fold and IGFR1 exhibits 2-4 fold higher kinase activity as NO compared to normal tissue (Webster, et aL, (1996) Cancer Res. 56:2781 and oO Pekonen, et al., (1998) Cancer Res. 48:1343). Ninety percent of colorectal cancer 0 tissue biopsies exhibit elevated IGFR1 levels wherein the extent of IGFR1 expression Sis correlated with the severity of the disease. Analysis of primary cervical cancer cell 0 10 cultures and cervical cancer cell lines revealed 3- and 5-fold overexpression of IGFR1, respectively, as compared to normal ectocervical cells (Steller, et al., (1996) Cancer Res. 56:1762). Expression of IGFR1 in synovial sarcoma cells also correlated with an aggressive phenotype metastasis and high rate of proliferation; Xie, et al., (1999) Cancer Res. 59:3588).
Acromegaly, a slowly developing disease, is caused by hypersecretion of growth hormone and IGF-I (Ben-Schlomo, etal., (2001) Endocrin. Metab.Clin. North.
Am. 30:565-583). Antagonism of IGFR1 function may be helpful in treating the disease.
There are several antibodies, which are known in the art, which inhibit the activity of (GFR1. However, these are of relatively low therapeutic value. For example, a-IR3 (Kull, et al., (1983) J. Biol. Chem. 258:6561), 1H7 (Li et al., (1993) Biochem. Biophys. Res. Comm. 196.92-98 and Xiong et al., (1992) Proc. Natl. Acad.
Sci., U.S.A. 89:5356-5360; Santa Cruz biotechnology, Inc.; Santa Cruz, CA) and MAB391 (R&D Systems; Minneapolis, MN) are mouse monoclonal antibodies which interact with IGFR1 and inhibit its activity. Since these are mouse antibodies, their therapeutic utility in humans is limited. When immunocompetent human subjects are administered a dose of mouse antibodies, the subjects produce antibodies against the mouse immunoglobulin sequences. These human anti-mouse antibodies (HAMA) neutralize the therapeutic antibodies and may induce acute toxicity a HAMA response).
One method by which to avert a HAMA response is through the use of fullyhuman antibodies which lack any foreign mouse) amino acid sequences.
Although the use of fully-human antibodies is an effective method by which to reduce or prevent human host immune rejection of the therapeutic antibody, rejection of the 3 c fully-human antibody can occur. Human rejection of human antibodies may be referred to as a human anti-human antibody response (HAHA response). HAHA T response can be mediated by factors such as the presence of rare, low occurrence 00 C amino acid sequences in the fully-human antibodies. For this reason, therapeutic antibodies may also be optimized by the inclusion of non-immunogenic or only weakly IDimmunogenic human antibody framework sequences. Preferably, the sequences oO occur frequently in other human antibodies.
SUMMARY OF THE INVENTION S 10 The present invention provides fully human anti-human IGFR1 monoclonal antibodies which, preferably, will not induce a HAMA response or will not induce a HAHA response when administered to human subjects and which are useful for treating or preventing diseases which are mediated by IGFR1 malignancy).
The present invention provides a binding composition an antibody or antigen-binding fragment thereof) comprising a light chain, wherein the chain comprises the amino acid sequence of the light chain CDR-L1 defined by SEQ ID NO: 8 or 31, the amino acid sequence of the light chain CDR-L2 defined by SEQ ID NO: 9 or 32 and the amino acid sequence of the light chain CDR-L3 defined by SEQ ID NO: 10 or 33. Also provided is a binding composition an antibody or antigenbinding fragment thereof) including a heavy chain, wherein the chain includes the amino acid sequence of the heavy chain CDR-H1 defined by SEQ ID NO: 14 or 37, the amino acid sequence of the heavy chain CDR-H2 defined by SEQ ID NO: 15 or 38 and the amino acid sequence of the heavy chain CDR-H3 defined by SEQ ID NO: 16 or 39.
Preferably, the binding composition an antibody or antigen-binding fragment thereof) of the invention comprises a light chain variable region, preferably a mature light chain variable region, which includes amino acids 20-128 of SEQ ID NO: 2, amino acids 21-130 of SEQ ID NO: 25, amino acids 20-128 of SEQ ID NO: 41 or 43 or amino acids 20-128 of SEQ ID NO: 41,43, 72, 74, 76 or 78 and/or a heavy chain variable region, preferably a mature heavy chain variable region, which includes amino acids 20-137 of SEQ ID NO: 4, amino acids 20-140 of SEQ ID NO: 27, amino acids 20-137 of SEQ ID NO: 45 or amino acids 20-137 of SEQ ID NO: 112.
Pharmaceutical compositions comprising a binding composition of the present invention and a pharmaceutically acceptable carrier are also provided by the present S4 N invention. The binding composition of the invention may also be conjugated to a a substance such as polyethylene glycol.
The present invention also includes a binding composition a human 00 C1 antibody or antigen binding fragment thereof) which specifically binds to human IGFR1 comprising a property selected from the group consisting of: IN binds to IGFR1 human IGFR1) with a Kd of about 86 X 10-1' or less; 0 0 Has an off rate (Kon) for IGFR1 human IGFR1) of about 6.50 X 10 5 or Ssmaller, S(c) Has an on rate (Kon) for IGFR1 human IGFR1) of about 0.7 X 10 5 or greater; S 10 Competes with IGF1 for binding to IGFR1 human IGFR1); Inhibits autophosphorylation with an ICso of 0.10 nM) of IGFR1 human IGFR1); and Inhibits anchorage-independent growth of a cell expressing IGFR1 human IGFR1).
Preferably, the binding composition comprises all of said properties More preferably, the binding composition a human antibody or antigen binding fragment thereof) comprises a member selected from: a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 8, CDR-L2 defined by SEQ ID NO: 9 and CDR-L3 defined by SEQ ID NO: a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 31, CDR-L2 defined by SEQ ID NO: 32 and CDR-L3 defined by SEQ ID NO: 33; a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 14 or SEQ ID NO: 17, CDR-H2 defined by SEQ ID NO: 15 and CDR-H3 defined by SEQ ID NO: 16; and a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 37 or SEQ ID NO: 70, CDR-H2 defined by SEQ ID NO: 38 and CDR-H3 defined by SEQ ID NO: 39.
The present invention also includes an isolated nucleic acid encoding a peptide selected from: amino acids 20-128 of SEQ ID NO: 2; amino acids 21-130 of SEQ ID NO: amino acids 20-128 of SEQ ID NO: 72; amino acids 20-128 of SEQ ID NO: 74; C amino acids 20-137 of SEQ ID NO: 4; S(b) amino acids 20-140 of SEQ ID NO: 27; 0 amino acids 20-137 of SEQ ID NO: CN amino acids 20-137 of SEQ ID NO: 112; amino acids 20-128 of SEQ ID NO: 76; and IN amino acids 20-128 of SEQ ID NO: 78.
r0 0 O Preferably, the nucleic acid is selected from: 0 nucleotides 58-384 of SEQ ID NO: 1; nucleotides 61-390 of SEQ ID NO: 24; nucleotides 58-384 of SEQ ID NO: 71; nucleotides 58-384 of SEQ ID NO: 73.
nucleotides 58-411 of SEQ ID NO: 3; nucleotides 58-420 of SEQ ID NO: 26; nucleotides 58-411 of SEQ ID NO: 44; nucleotides 58-411 of SEQ ID NO: 111; nucleotides 58-384 of SEQ ID NO: 75; and nucleotides 58-384 of SEQ ID NO: 77.
The present invention also provides a recombinant vector comprising any of the foregoing polynucleotides along with a host cell comprising the vector.
The present invention also comprises a polypeptide selected from: amino acids 20-128 of SEQ ID NO: 2; amino acids 21-130 of SEQ ID NO: amino acids 20-128 of SEQ ID NO: 72; amino acids 20-128 of SEQ ID NO: 74; amino acids 20-137 of SEQ ID NO: 4; amino acids 20-140 of SEQ ID NO: 27; amino acids 20-137 of SEQ ID NO: amino acids 20-137 of SEQ ID NO: 112; amino acids 20-128 of SEQ ID NO: 76; and amino acids 20-128 of SEQ ID NO: 78.
Preferably, the binding composition of the present invention is a human antibody comprising at least one 1 or 2) light chain/heavy chain combination selected from: 6 Sa) a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 2 and d) a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 4; S[15H12/19D12 mature LC 15H12/19D12 mature HC] Sb) a light chain variable region comprising amino acids 21-130 of SEQ ID NO: and a heavy chain variable region comprising amino acids 20-140 of SEQ ID NO: N 27; [1H3 mature LC 1H3 mature HC] 0 0 c) a light variable region comprising amino acids 20-128 of SEQ ID NO: 72 and a Oheavy chain variable region comprising amino acids 20-137 of SEQ ID NO: S[mature LCC mature HCA] O 10 d) a light variable region comprising amino acids 20-128 of SEQ ID NO: 74 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: [mature LCD mature HCA] e) a light variable region comprising amino acids 20-128 of SEQ ID NO: 76 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: [mature LCE mature HCA] f) a light variable region comprising amino acids 20-128 of SEQ ID NO: 78 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: [mature LCF mature HCA] g) a light variable region comprising amino acids 20-128 of SEQ ID NO: 72 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112; [mature LCC mature HCB] h) a light variable region comprising amino acids 20-128 of SEQ ID NO: 74 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112; [mature LCD mature HCB] i) a light variable region comprising amino acids 20-128 of SEQ ID NO: 76 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112; [mature LCE mature HCB] and j) a light variable region comprising amino acids 20-128 of SEQ ID NO: 78 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112.
[mature LCF mature HCB].
More preferably, the human antibody is a tetramer comprising two of the foregoing light/heavy chain pairs. Preferably, the human antibody includes mature LCF paired with mature HCA or mature HCB.
Also provided is a method for making a polypeptide comprising amino Sacids 20-128 of SEQ ID NO: 2, amino acids 20-137 of SEQ ID NO: 4 amino acids 21-130 of SEQ ID NO: 25, amino acids 20-140 of SEQ ID NO: 27, amino Sacids 20-128 of SEQ ID NO: 41, 43, 72, 74, 76 or 78, amino acids 20-137 of SEQ 00 5 ID NO: 45 or amino acids 20-137 of SEQ ID NO: 112 comprising culturing the host cell under conditions in which the polypeptide is produced. Preferably, the polypeptide is also isolated from the host cell.
SThe invention also provides a method for treating or preventing a medical 00 O condition in a subject which is mediated by elevated expression or activity of C 10 Insulin-like Growth Factor Receptor-I or by elevated expression of one or more of Sits ligands IGF-I or IGF-II) comprising administering a binding composition CN of the invention antibody or antigen-binding fragment of the invention) to the subject. Preferably, the binding composition comprises a member selected from: a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 8, CDR-L2 defined by SEQ ID NO: 9 and CDR-L3 defined by SEQ ID NO: a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 31, CDR-L2 defined by SEQ ID NO: 32 and CDR-L3 defined by SEQ ID NO: 33; a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 14 or SEQ ID NO: 17, CDR-H2 defined by SEQ ID NO: 15 and CDR-H3 defined by SEQ ID NO: 16; and a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 37 or SEQ ID NO: 70, CDR-H2 defined by SEQ ID NO: 38 and CDR-H3 defined by SEQ ID NO: 39.
The present invention includes any plasmid selected from the group consisting of CMV promoter-15H12/19D12 HCA (y4)- Deposit name: "15H12/19D12 HCA ATCC accession No.: PTA-5214; (ii) CMV promoter-15H12/19D12 HCB (y4)- Deposit name: "15H12/19D12 HCB ATCC accession No.: PTA-5215; (iii) CMV promoter-15H12/19D12 HCA (yl)- Deposit name: "15H12/19D12 HCA ATCC accession No.: PTA-5216; (iv) CMV promoter-15H12/19D12 LCC Deposit name: "15H12/19D12 LCC SATCC accession No.: PTA-5217; 00 5 CMV promoter-15H12/19D12 LCD Deposit name: "15H12/19D12 LCD O ATCC accession No.: PTA-5218; 00 (vi) CMV promoter-15H12/19D12 LCE 0 Deposit name: "15H12/19D12 LCE S 10 ATCC accession No.: PTA-5219; and S(vii) CMV promoter-15H12/19D12 LCF Deposit name: "15H12/19D12 LCF ATCC accession No.: PTA-5220; as well as the nucleic acid inserts of any of the foregoing plasmids. Also included are the nucleic acid portions of the inserts encoding the immunoglobulin variable regions included in the plasmid inserts optionally including the immunoglobulin constant region excluding the signal sequence). Also included are any polypeptides encoded by the nucleic acids of any of the foregoing plasmid inserts as well as polypeptides encoding the immunoglobulin variable regions included in any insert optionally including the immunoglobulin constant region excluding the signal sequence).
The above-identified plasmids were deposited, under the Budapest Treaty, on May 21, 2003 with the American Type Culture Collection (ATCC); 10801 University Boulevard; Manassas, Virginia 20110-2209. All restrictions on access to the plasmids deposited in ATCC will be removed upon grant of a patent.
Preferably, the binding composition is combined with a pharmaceutically acceptable carrier in a pharmaceutical composition. Such medical conditions, as contemplated by the present invention, include acromegaly, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumors, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation.
9 C1 The binding compositions may be administered to a subject, for example, by a parenteral route. Combination therapies comprising administration of a binding composition of the present invention in association with an anti-cancer therapy agent 00 oO Cor in association with an anti-cancer therapeutic procedure are also provided.
A method for producing a fully-human anti-IGFR1 antibody which comprises Sthe steps of immunizing a transgenic non-human animal having a genome comprising 00 a human heavy chain transgene and a human light chain transgene with IGFR1 0 antigenic polypeptide, preferably amino acids 30-902 of SEQ ID NO: 19 and/or a cell HEK293) which expresses IGFR1 on its surface, such that antibodies are 0 10 produced by B cells of the animal; isolating B cells of the animal; fusing the B cells with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies specific for IGFR1; and isolating the human monoclonal antibodies specific for IGFR1 is also provided.
DETAILED DESCRIPTION Preferred embodiments of the present invention include a fully human, monoclonal antibody or antigen-binding fragment thereof which specifically recognizes and binds to Insulin-like Growth Factor Receptor-1, preferably amino acids 30-902 of SEQ ID NO: 19. Preferably, the antibody or antigen-binding fragment thereof is 1H3, 15H12, 19D12, 15H12/19D12 LCA, 15H12/19D12 LCB, 15H12/19D12 LCC, 15H12/19D12 LCD, 15H12/19D12 LCE, 15H12/19D12 LCF, 15H12/19D12 HCA or 15H12/19D12 HCB.
A binding composition or agent refers to a molecule that binds with specificity to IGFR1, in a ligand-receptor type fashion or an antibody-antigen interaction, proteins which specifically associate with IGFR1, in a natural physiologically relevant protein-protein interaction, either covalent or non-covalent. The term "binding composition" is preferably a polypeptide, such as a full antibody or antigenbinding fragment thereof of the present invention 15H12/19D12 LCA, 15H12/19D12 LCB, 15H12/19D12 LCC, 15H12/19D12 LCD, 15H12/19D12 LCE, 15H12/19D12 LCF, 15H12/19D12 HCA OR 15H12/19D12 HCB or any peptide set forth, below, in Table 1).
The antibodies and antigen-binding fragments of the invention may be used to inhibit growth of cells, preferably malignant cells, both in vitro and in vivo. Without being bound by a single theory, the antibodies and antigen-binding fragments of the c invention may inhibit cellular growth by inhibiting the interaction between IGFR1 and a Sligand for the receptor, such as Insulin-like Growth Factor-I (IGF-I) or Insulin-like 0 Growth Factor-II (IGF-II). The antibodies and antigen-binding fragments may also C inhibit IGFR1 autophosphorylation, inhibit anchorage-independent growth of cells cancer cells) expressing IGFR1 and inhibit activation of AKT kinase by inducing r degradation of IGFR1. Preferably, the antibodies and antigen-binding fragments O neutralize the activity of IGFR1 and/or down-regulate IGFR1. The antibodies and antigen-binding fragments may be used to treat or prevent diseases which are mediated by IGFR1. The present invention also provides methods for making the antibodies and antigen-binding fragments of the invention.
The term "antibody molecule" refers to whole antibodies IgG, preferably, IgG1 or IgG4) and fragments, preferably antigen-binding fragments, thereof.
Antibody fragments include Fab antibody fragments, F(ab) 2 antibody fragments, Fv antibody fragments, single chain Fv antibody fragments and dsFv antibody fragments.
The terms "IGFR1" "Insulin-like Growth Factor Receptor-I" and "Insulin-like Growth Factor Receptor, type I" are well known in the art. Although IGFR1 may be from any organism, it is preferably from an animal, more preferably from a mammal mouse, rat, rabbit, sheep or dog) and most preferably from a human. The nucleotide and amino acid sequence of a typical human IGFR1 precursor has the Genbank Accession No. X04434 or NM_000875 (SEQ ID NO: 19). Cleavage of the precursor between amino acids 710 and 711) produces an a-subunit and a 3subunit which associate to form a mature receptor. In preferred embodiments of the invention, amino acids 30-902, from the full length IGFR1 polypeptide are used as an antigen for generation of anti-IGFR1 antibodies.
The terms "IGF-I" "Insulin-like Growth Factor-I" and "Insulin-like Growth Factor, type I" are also well known in the art. The terms "IGF-II" "Insulin-like Growth Factor-Il" and "Insulin-like Growth Factor, type II" are also well known in the art.
Although IGF-I or IGF-II may be from any organism, they are preferably from an animal, more preferably from a mammal mouse, rat, rabbit, sheep or dog) and most preferably from a human. The nucleic acid and amino acid sequence of typical, human IGF-I and IGF-II have the Genbank Accession No. XM_052648 (SEQ ID NO: and NM_000612 (SEQ ID NO: 21), respectively. The term "slGFRI" or "soluble IGFR1" includes any soluble fragment of IGFR1 human IGFR1), preferably a 11 fragment from which the receptor trans-membrane region has been deleted, more preferably amino acids 30-902 of SEQ ID NO: 19.
The amino acid sequence of the variable region of preferred, fully human, monoclonal anti-IGFR1 antibody molecules of the invention 1H3, 15H12 and 19D12) along with the nucleotide sequences of nucleic acids which encode the regions are summarized in Table 1. The present invention includes any nucleic acid or polypeptide antibody) which comprises one or more 1, 2, 3, 4, 5, 6, 7 or 8) of any of the nucleic acids or polypeptides (including mature fragments thereof) set forth, below, in Table 1. Table 1 also includes a summary of the amino acid and nucleotide sequences which correspond to the CDR regions of the antibodies. The amino acid and nucleotide sequences corresponding to the variable region of 15H12 and 19D12 are identical; for this reason, only a single sequence for each variable region or CDR is shown.
Table 1. Summary of amino acid and nucleotide sequences of the invention.
SEQUENCE SEQUENCE IDENTIFIER Nucleotide sequence encoding SEQ ID NO: 1 the 15H12 and 19D12 light chain variable region-including signal peptide (15H12/19D12 LC) Amino acid sequence of SEQ ID NO: 2 the15H12 and 19D12 light chain variable region-including signal peptide Nucleotide sequence encoding SEQ ID NO: 3 thel5H12 and 19D12 heavy chain variable region including signal peptide (15H12/19D12
HC)
Amino acid sequence of the SEQ ID NO: 4 15H12 and 19D12 heavy chain variable region including signal peptide Nucleotide sequence encoding SEQ ID NO: the 15H12 and 19D12 CDR-L1 Nucleotide sequence encoding SEQ ID NO: 6 the 15H12 and 19D12 CDR-L2 Nucleotide sequence encoding SEQ ID NO: 7 the 15H12 and 19D12 CDR-L3 Amino acid sequence of the SEQ ID NO: 8 15H12 and 19D12 CDR-L1 SEQUENCE SEQUENCE IDENTIFIER Amino acid sequence of the SEQ ID NO: 9 15H12 and 19D12 CDR-L2 Amino acid sequence of the SEQ ID NO: 15H12 and 19D12 CDR-L3 Nucleotide sequence encoding SEQ ID NO: 11 the 15H12 and 19D12 CDR-H1 Nucleotide sequence encoding SEQ ID NO: 12 the 15H12 and 19D12 CDR-H2 Nucleotide sequence encoding SEQ ID NO: 13 the 15H12 and 19D12 CDR-H3 Amino acid sequence of the SEQ ID NO: 14 15H12 and 19D12 CDR-H1 Amino acid sequence of the SEQ ID NO: 15H12 and 19D12 CDR-H2 Amino acid sequence of the SEQ ID NO: 16 15H12 and 19D12 CDR-H3 Amino acid sequence of an SEQ ID NO: 17 alternative 15H12 and 19D12 CDR-H1 Nucleotide sequence encoding SEQ ID NO: 18 an alternative 15H12 and 19D12 CDR-H1 Amino acid sequence of Insulin- SEQ ID NO: 19 like Growth Factor Receptor-I (IGFR1) Amino acid sequence of Insulin- SEQ ID NO: like Growth Factor-I (IGF1) Amino acid sequence of Insulin- SEQ ID NO: 21 like Growth Factor-ll (IGF2)_ Nucleotide sequence of PCR SEQ ID NO: 22 primer Nucleotide sequence of PCR SEQ ID NO: 23 primer Nucleotide sequence encoding SEQ ID NO: 24 the 1H3 light chain variable region-including signal peptide (1H3 LC) Amino acid sequence of the 1 H3 SEQ ID NO: light chain variable regionincluding signal peptide Nucleotide sequence encoding SEQ ID NO: 26 the 1H3 heavy chain variable region including signal peptide (1 H3 HC) Amino acid sequence of the 1 H3 SEQ ID NO: 27 heavy chain variable region including signal peptide SEQUENCE SEQUENCE IDENTIFIER Nucleotide sequence encoding SEQ ID NO: 28 the 1H3 CDR-L1 Nucleotide sequence encoding SEQ ID NO: 29 the 1H3 CDR-L2 Nucleotide sequence encoding SEQ ID NO: the 1H3 CDR-L3 Amino acid sequence of the 1H3 SEQ ID NO: 31 CDR-L1 Amino acid sequence of the 1H3 SEQ ID NO: 32 CDR-L2 Amino acid sequence of the 1H3 SEQ ID NO: 33 CDR-L3 Nucleotide sequence encoding SEQ ID NO: 34 the 1H3 CDR-H1 Nucleotide sequence encoding SEQ ID NO: the 1H3 CDR-H2 Nucleotide sequence encoding SEQ ID NO: 36 the 1H3 CDR-H3 Amino acid sequence of the 1 H3 SEQ ID NO: 37 CDR-H1 Amino acid sequence of the 1H3 SEQ ID NO: 38 CDR-H2 Amino acid sequence of the 1H3 SEQ ID NO: 39 CDR-H3 Nucleotide sequence encoding SEQ ID the 15H12/19D12 light chain A
(LCA)
Amino acid sequence of the SEQ ID NO:41 15H12/19D12 light chain A Nucleotide sequence encoding SEQ ID NO:42 the 15H12/19D12 light chain B
(LCB)
Amino acid sequence of the SEQ ID NO:43 15H12/19D12 light chain B Nucleotide sequence encoding SEQ ID NO:44 the 15H12/19D12 heavy chain A
(HCA)
Amino acid sequence of the SEQ ID 15H12/19D12 heavy chain A Nucleotide sequence encoding SEQ ID NO:46 the 15H12/19D12 light chain A framework region 1 Amino acid sequence of the SEQ ID NO:47 15H12/19D12 light chain A framework region 1 Nucleotide sequence encoding SEQ ID NO:48 the 15H12/19D12 light chain A framework region 2 SEQUENCE SEQUENCE IDENTIFIER Amino acid sequence of the SEQ ID NO:49 15H12/19D12 light chain A framework region 2 Nucleotide sequence encoding SEQ ID the 15H12/19D12 light chain A framework region 3 Amino acid sequence of the SEQ ID NO:51 15H12/19D12 light chain A framework region 3 Nucleotide sequence encoding SEQ ID NO:52 the 15H12/19D12 light chain A framework region 4 Amino acid sequence of the SEQ ID NO:53 15H12/19D12 light chain A framework region 4 Nucleotide sequence encoding SEQ ID NO:54 the 15H12/19D12 light chain B framework region 1 Amino acid sequence of the SEQ ID 15H12/19D12 light chain B framework region 1 Nucleotide sequence encoding SEQ ID NO:56 the 15H12/19D12 light chain B framework region 2 Amino acid sequence of the SEQ ID NO:57 15H12/19D12 light chain B framework region 2 Nucleotide sequence encoding SEQ ID NO:58 the 15H12/19D12 light chain B framework region 3 Amino acid sequence of the SEQ ID NO:59 15H12/19D12 light chain B framework region 3 Nucleotide sequence encoding SEQ ID the 15H12/19D12 light chain B framework region 4 Amino acid sequence of the SEQ ID NO:61 15H12/19D12 light chain B framework region 4 Nucleotide sequence encoding SEQ ID NO:62 the 15H12/19D12 heavy chain A framework region 1 Amino acid sequence of the SEQ ID NO:63 15H12/19D12 heavy chain A framework region 1 Nucleotide sequence encoding SEQ ID NO:64 the 15H12/19D12 heavy chain A framework region 2 SEQUENCE SEQUENCE IDENTIFIER Amino acid sequence of the SEQ ID 15H12/19D12 heavy chain A framework region 2 Nucleotide sequence encoding SEQ ID NO:66 the 15H12/19D12 heavy chain A framework region 3 Amino acid sequence of the SEQ ID NO:67 15H12/19D12 heavy chain A framework region 3 Nucleotide sequence encoding SEQ ID NO:68 the 15H12/19D12 heavy chain A framework region 4 Amino acid sequence of the SEQ ID NO:69 15H12/19D12 heavy chain A framework region 4 Amino acid sequence of the SEQ ID NO: alternative 1H3 CDR-H1 Nucleotide sequence encoding SEQ ID NO: 71 the 15H12/19D12 light chain C
(LCC)
Amino acid sequence of the SEQ ID NO: 72 15H12/19D12 light chain C Nucleotide sequence encoding SEQ ID NO: 73 the 15H12/19D12 light chain D
(LCD)
Amino acid sequence of the SEQ ID NO: 74 15H12/19D12 light chain D Nucleotide sequence encoding SEQ ID NO: the 15H12/19D12 light chain E
(LCE)
Amino acid sequence of the SEQ ID NO: 76 15H12/19D12 light chain E Nucleotide sequence encoding SEQ ID NO: 77 the 15H12/19D12 light chain F
(LCF)
Amino acid sequence of the SEQ ID NO: 78 15H12/19D12 light chain F Nucleotide sequence encoding SEQ ID NO: 79 the 15H12/19D12 light chain C framework region 1 Amino acid sequence of the SEQ ID NO: 15H12/19D12 light chain C framework region 1 Nucleotide sequence encoding SEQ ID NO: 81 the 15H12/19D12 light chain C framework region 2 SEQUENCE SEQUENCE IDENTIFIER Amino acid sequence of the SEQ ID NO: 82 15H12/19D12 light chain C framework region 2 Nucleotide sequence encoding SEQ ID NO: 83 the 15H12/19D12 light chain C framework region 3 Amino acid sequence of the SEQ ID NO: 84 15H12/19D12 light chain C framework region 3 Nucleotide sequence encoding SEQ ID NO: the 15H12/19D12 light chain C framework region 4 Amino acid sequence of the SEQ ID NO: 86 15H12/19D12 light chain C framework region 4 Nucleotide sequence encoding SEQ ID NO: 87 the 15H12/19D12 light chain D framework region 1 Amino acid sequence of the SEQ ID NO: 88 15H12/19D12 light chain D framework region 1 Nucleotide sequence encoding SEQ ID NO: 89 the 15H12/19D12 light chain D framework region 2 Amino acid sequence of the SEQ ID NO: 15H12/19D12 light chain D framework region 2 Nucleotide sequence encoding SEQ ID NO: 91 the 15H12/19D12 light chain D framework region 3 Amino acid sequence of the SEQ ID NO: 92 15H12/19D12 light chain D framework region 3 Nucleotide sequence encoding SEQ ID NO: 93 the 15H12/19D12 light chain D framework region 4 Amino acid sequence of the SEQ ID NO: 94 15H12/19D12 light chain D framework region 4 Nucleotide sequence encoding SEQ ID NO: the 15H12/19D12 light chain E framework region 1 Amino acid sequence of the SEQ ID NO: 96 15H12/19D12 light chain E framework region 1 Nucleotide sequence encoding SEQ ID NO: 97 the 15H12/19D12 light chain E framework region 2 SEQUENCE SEQUENCE IDENTIFIER Amino acid sequence of the SEQ ID NO: 98 15H12/19D12 light chain E framework region 2 Nucleotide sequence encoding SEQ ID NO: 99 the 15H12/19D12 light chain E framework region 3 Amino acid sequence of the SEQ ID NO: 100 15H12/19D12 light chain E framework region 3 Nucleotide sequence encoding SEQ ID NO: 101 the 15H12/19D12 light chain E framework region 4 Amino acid sequence of the SEQ ID NO: 102 15H12/19D12 light chain E framework region 4 Nucleotide sequence encoding SEQ ID NO: 103 the 15H12/19D12 light chain F framework region 1 Amino acid sequence of the SEQ ID NO: 104 15H12/19D12 light chain F framework region 1 Nucleotide sequence encoding SEQ ID NO: 105 the 15H12/19D12 light chain F framework region 2 Amino acid sequence of the SEQ ID NO: 106 15H12/19D12 light chain F framework region 2 Nucleotide sequence encoding SEQ ID NO: 107 the 15H12/19D12 light chain F framework region 3 Amino acid sequence of the SEQ ID NO: 108 15H12/19D12 light chain F framework region 3 Nucleotide sequence encoding SEQ ID NO: 109 the 15H12/19D12 light chain F framework region 4 Amino acid sequence of the SEQ ID NO: 110 15H12/19D12 light chain F framework region 4 Nucleotide sequence encoding SEQ ID NO: 111 the 15H12/19D12 heavy chain B
(HCB)
Amino acid sequence of the SEQ ID NO: 112 15H12/19D12 heavy chain B Nucleotide sequence encoding SEQ ID NO: 113 the 15H12/19D12 heavy chain B framework region 1 SEQUENCE SEQUENCE IDENTIFIER Amino acid sequence of the SEQ ID NO: 114 15H12/19D12 heavy chain B framework region 1 Nucleotide sequence encoding SEQ ID NO: 115 the 15H12/19D12 heavy chain B F framework region 2 Amino acid sequence of the SEQ ID NO: 116 15H12/19D12 heavy chain B framework region 2 Nucleotide sequence encoding SEQ ID NO: 117 the 15H12/19D12 heavy chain B framework region 3 Amino acid sequence of the SEQ ID NO: 118 15H.12/19D12 heavy chain B framework region 3 Nucleotide sequence encoding SEQ ID NO: 119 the 15H12/19D12 heavy chain B framework region 4 Amino acid sequence of the SEQ ID NO: 120 15H12/19D12 heavy chain B framework region 4 CDR-L1 is the first complementarity determining region (CDR) which occurs in the light chain, CDR-L2 is the second CDR which occurs on the light chain and CDR- L3 is the third CDR which occurs on the light chain.
Similarly, CDR-H1 is the first CDR which occurs on the heavy chain, CDR-H2 is the second CDR which occurs on the heavy chain and CDR-H3 is the third CDR which occurs on the heavy chain.
FR-L1 is the first framework region of the light chain, FR-L2 is the second framework region of the light chain, FR-L3 is the third framework region of the light chain, FR-L4 is the fourth framework region on the light chain, FR-H1 is the first framework region of the heavy chain, FR-H2 is the second framework region of the heavy chain, FR-H3 is the third framework region of the heavy chain and FR-H4 is the fourth framework region of the heavy chain. These terms and the arrangement of CDRs and FRs on an immunoglobulin chain are well known in the art.
A mature light chain variable region of the invention, which lacks the signal peptide first 19 or 20 residues), is amino acids 20-128 of SEQ ID NO: 2, 41, 43, 72, 74, 76 or 78 which is encoded by nucleotides 58-384 of SEQ ID NO: 1, 40, 42, 71, 73, 75, or 77 or amino acids 21-130 of SEQ ID NO: 25 which is encoded by nucleotides 61-390 of SEQ ID NO: 24.
C 19 c A mature heavy chain variable region, which lacks the signal peptide first d§ 19 residues), is amino acids 20-137 of SEQ ID NO: 4, 45 or 112 which is encoded by oO nucleotides 58-411 of SEQ ID NO: 3, 44 or 111 or amino acids 20-140 of SEQ ID NO:
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27 which is encoded by nucleotides 58-420 of SEQ ID NO: 26.
In some embodiments the 15H12 and 19D12 CDR-H1 is GFTFSSFAMH (SEQ ID NO: 17) which is encoded by the nucleotide sequence of SEQ ID NO: 18. In 0 some embodiments the 1H3 CDR-H1 is NYAMH (SEQ ID NO: 0 The present invention also includes antibodies and antigen-binding fragments which include the framework regions of the antibodies and antigen-binding fragments of the invention. Preferably, FR-L1 is amino acids 20-42 of SEQ ID NO: 2 or amino acids 21-43 of SEQ ID NO: 25; FR-L2 is amino acids 54-68 of SEQ ID NO: 2 or amino acids 55-69 of SEQ ID NO: 25; FR-L3 is amino acids 76-107 of SEQ ID NO: 2 or amino acids 77-108 of SEQ ID NO: 25; FR-L4 is amino acids 117-128 of SEQ ID NO: 2 or amino acids 128-130 of SEQ ID NO: 25; FR-H1 is amino acids 20-44 or 49 of SEQ ID NO: 4 or amino acids 20-44 or 20-49 of SEQ ID NO: 27; FR-H2 is amino acids 55-68 of SEQ ID NO: 4 or amino acids 55-68 of SEQ ID NO: 27; FR-H3 is amino acids 85-116 of SEQ ID NO: 4 or amino acids 85-116 of SEQ ID NO: 27 and FR-H4 is amino acids 127-137 of SEQ ID NO: 4 or amino acids 130-140 of SEQ ID NO: 27.
In preferred embodiments, the antibody molecules of the present invention include FR-L1 defined by amino acids 20-42 of SEQ ID NO: 41 or 43; FR-L2 defined by amino acids 54-68 of SEQ ID NO: 41 or 43; FR-L3 defined by amino acids 76-107 of SEQ ID NO: 41 or 43; and FR-L4 defined by amino acids 117-128 of SEQ ID NO: 41 or 43. Furthermore, preferred embodiments include antibody molecules including FR-H1 defined by amino acids 20-44 of SEQ ID NO: 45; FR-H2 defined by amino acids 55-68 of SEQ ID NO: 45; FR-H3 defined by amino acids 85-116 of SEQ ID NO: and FR-H4 defined by amino acids 127-137 of SEQ ID NO: Molecular Biology In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, Sambrook, Fritsch Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein 0 2 "Sambrook, et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II Glover ed. 1985); Oliqonucleotide Synthesis Gait ed. 1984); Nucleic Acid oO Hybridization Hames S.J. Higgins eds. (1985)); Transcription And Translation C Hames S.J. Higgins, eds. (1984)); Animal Cell Culture Freshney, ed.
(1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical SGuide To Molecular Cloning (1984); F.M. Ausubel, et al. Current Protocols in oO 0 Molecular Biology, John Wiley Sons, Inc. (1994).
SA "polynucleotide", "nucleic acid or "nucleic acid molecule" may refer to the Sphosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in single stranded form, double-stranded form or otherwise.
A "polynucleotide sequence", "nucleic acid sequence" or "nucleotide sequence" is a series of nucleotide bases (also called "nucleotides") in a nucleic acid, such as DNA or RNA, and means any chain of two or more nucleotides.
A "coding sequence" or a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in production of the product.
The term "gene" means a DNA sequence that codes for or corresponds to a particular sequence of ribonucleotides or amino acids which comprise all or part of one or more RNA molecules, proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine, for example, the conditions under which the gene is expressed. Genes may be transcribed from DNA to RNA which may or may not be translated into an amino acid sequence.
"Amplification" of DNA as used herein may denote the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of PCR see Saiki, et al., Science (1988) 239: 487. In a specific embodiment, the present invention includes a nucleic acid, which encodes an anti-IGFR1 antibody, an anti-IGFR1 antibody heavy or light chain, an anti-IGFR1 antibody heavy or light chain variable region, an anti- IGFR1 antibody heavy or light chain constant region or anti-IGFR1 antibody CDR O 21
O
CDR- L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 or CDR-H3) which can be amplified by PCR.
oO As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 10 10, 11, 12, 13 or 14), preferably at least 15 15, 16, 17, 18 or 19), and more preferably at least 20 nucleotides 20, 21, 22, 23, 24, 25, 26, 27, r28, 29 or 30), preferably no more than 100 nucleotides 40, 50, 60, 70, 80 or
OO
0 that may be hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA Smolecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest.
SOligonucleotides can be labeled, by incorporation of 32 P-nucleotides, 3
H-
nucleotides, 14 C-nucleotides, 3S-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of the gene, or to detect the presence of nucleic acids. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer.
The sequence of any nucleic acid a nucleic acid encoding an IGFR1 gene or a nucleic acid encoding an anti-IGFR1 antibody or a fragment or portion thereof) may be sequenced by any method known in the art chemical sequencing or enzymatic sequencing). "Chemical sequencing" of DNA may denote methods such as that of Maxam and Gilbert (1977) (Proc. Natl. Acad. Sci. USA 74:560), in which DNA is randomly cleaved using individual base-specific reactions.
"Enzymatic sequencing" of DNA may denote methods such as that of Sanger (Sanger, et al., (1977) Proc. Natl. Acad. Sci. USA 74:5463).
The nucleic acids herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, and non-coding regions, and the like.
A "promoter" or "promoter sequence" is a DNA regulatory region capable of binding an RNA polymerase in a cell directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bounded at its 3' terminus by the transcription initiation site and extends upstream direction) to include the minimum number of bases or O 22 c elements necessary to initiate transcription at any level. Within the promoter Ssequence may be found a transcription initiation site (conveniently defined, for 00 example, by mapping with nuclease S1), as well as protein binding domains
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(consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with other expression control sequences, rincluding enhancer and repressor sequences or with a nucleic acid of the invention SEQ ID NO: 1, 3, 5-7, 11-13,18, 22-24, 26, 28-30 or 34-36). Promoters which may be used to control gene expression include, but are not limited to, Scytomegalovirus (CMV) promoter Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, et al., (1981) Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, etal., (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner, et a., (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster, et al., (1982) Nature 296:39-42); prokaryotic expression vectors such as the P-lactamase promoter (Villa-Komaroff, et al., (1978) Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer, et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American (1980) 242:74-94; and promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter or the alkaline phosphatase promoter.
A coding sequence is "under the control of", "functionally associated with" or "operably associated with" transcriptional and translational control sequences in a cell when the sequences direct RNA polymerase mediated transcription of the coding sequence into RNA, preferably mRNA, which then may be trans-RNA spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence.
The terms "express" and "expression" mean allowing or causing the information in a gene, RNA or DNA sequence to become manifest; for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene. A DNA sequence is expressed in or by a cell to form an "expression product" such as an RNA mRNA) or a protein antibody 1H3, 15H12 or 19D12 or a fragment thereof). The expression product itself may also be said to be "expressed" by the cell.
0 23 The terms "vector", "cloning vector" and "expression vector" mean the vehicle d a plasmid) by which a DNA or RNA sequence can be introduced into a host cell, oO so as to transform the host and, optionally, promote expression and/or replication of c the introduced sequence.
The term "transfection" or "transformation" means the introduction of a nucleic Sacid into a cell. These terms may refer to the introduction of a nucleic acid encoding 00 0 an anti-IGFR1 antibody or fragment thereof into a cell. The introduced gene or 0 sequence may be called a "clone". A host cell that receives the introduced DNA or RNA has been "transformed" and is a "transformant" or a "clone". The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.
The term "host cell" means any cell of any organism that is selected, modified, transfected, transformed, grown, or used or manipulated In any way, for the production of a substance by the cell, for example the expression or replication, by the cell, of a gene, a DNA or RNA sequence, a protein or an enzyme.
The term "expression system" means a host cell and compatible vector which, under suitable conditions, can express a protein or nucleic acid which is carried by the vector and introduced to the host cell. Common expression systems include E.
coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. In a specific embodiment, IGFR1 or an antibody and antigen-binding fragment of the invention may be expressed in human embryonic kidney cells (HEK293). Other suitable cells include CHO (chinese hamster ovary) cells, HeLa cells and NIH 3T3 cells and NSO cells (non-lg-producing murine myeloma cell line). Nucleic acids encoding an antibody or antigen-binding fragment of the invention, slGFR1 or IGFR1 may be expressed at high levels in an E.coli/T7 expression system as disclosed in U.S. Patent Nos. 4,952,496, 5,693,489 and 5,869,320 and in Davanloo, et al., (1984) Proc. Natl. Acad. Sci. USA 81,2035- 2039; Studier, F. et al., (1986) J. Mol. Biol. 189: 113-130; Rosenberg, A. et a., (1987) Gene 56: 125-135; and Dunn, J. et al., (1988) Gene 68: 259 which are herein incorporated by reference.
The present invention contemplates any superficial or slight modification to the amino acid or nucleotide sequences which correspond to the antibodies or antigenbinding fragments of the invention. In particular, the present invention contemplates sequence conservative variants of the nucleic acids which encode the antibodies or O 24 c antigen-binding fragments of the invention. "Sequence-conservative variants" of a L polynucleotide sequence are those in which a change of one or more nucleotides in a 00 given codon results in no alteration in the amino acid encoded at that position.
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Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. "Function-conservative variants" are those in Swhich one or more amino acid residues in a protein or enzyme have been changed 0 without altering the overall conformation and function of the polypeptide, including, N but, by no means, limited to, replacement of an amino acid with one having similar Sproperties. Amino acids with similar properties are well known in the art. For c 10 example, polar/hydrophilic amino acids which may be interchangeable include asparagine, glutamine, serine, cysteine, threonine, lysine, arginine, histidine, aspartic acid and glutamic acid; nonpolar/hydrophobic amino acids which may be interchangeable include glycine, alanine, valine, leucine, isoleucine, proline, tyrosine, phenylalanine, tryptophan and methionine; acidic amino acids which may be interchangeable include aspartic acid and glutamic acid and basic amino acids which may be interchangeable include histidine, lysine and arginine.
The present invention includes anti-IGFR1 antibodies and fragments thereof which are encoded by nucleic acids as described in Table 1 as well as nucleic acids which hybridize thereto. Preferably, the nucleic acids hybridize under low stringency conditions, more preferably under moderate stringency conditions and most preferably under high stringency conditions and, preferably, exhibit IGFR1 binding activity. A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook, et al., supra).
The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Typical low stringency hybridization conditions may be 550C, 5X SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5X SSC, 0.5% SDS.
Typical, moderate stringency hybridization conditions are similar to the low stringency conditions except the hybridization is carried out in 40% formamide, with 5X or 6X SSC. High stringency hybridization conditions are similar to low stringency conditions except the hybridization conditions are carried out in 50% formamide, 5X or 6X SSC and, optionally, at a higher temperature 57 59 OC, 60 62 63 OC, or 68 In general, SSC is 0.15M NaC1 and 0.015M Na-citrate. Hybridization requires that the two nucleic acids contain complementary sequences, although, a depending on the stringency of the hybridization, mismatches between bases are 00oO possible. The appropriate stringency for hybridizing nucleic acids depends on the Nlength of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide rsequences, the higher the stringency under which the nucleic acids may hybridize.
00 For hybrids of greater than 100 nucleotides in length, equations for calculating the C melting temperature have been derived (see Sambrook, et al., supra, 9.50-9.51). For hybridization with shorter nucleic acids, oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook, et al., supra, 11.7-11.8).
Also included in the present invention are nucleic acids comprising nucleotide sequences and polypeptides comprising amino acid sequences which are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical 96%, 97%, 98%, 99%, 100%) to the reference nucleotide and amino acid sequences of Table 1 when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences.
Polypeptides comprising amino acid sequences which are at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar and most preferably at least about 95% similar 95%, 96%, 97%, 98%, 99%, 100%) to the reference amino acid sequences of Table 1 SEQ ID NOs. 2 amino acids 20-128), 4 amino acids 20-137), 8-10, 14-16, 17, 25 amino acids 21- 130), 27 amino acids 20-140), 31-33 or 37-39) when the comparison is performed with a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences, are also included in the present invention.
Sequence identity refers to exact matches between the nucleotides or amino acids of two sequences which are being compared. Sequence similarity refers to both exact matches between the amino acids of two polypeptides which are being compared in addition to matches between nonidentical, biochemically related amino O 26
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acids. Biochemically related amino acids which share similar properties and may be 0 interchangeable are discussed above.
OO The following references regarding the BLAST algorithm are herein N incorporated by reference: BLAST ALGORITHMS: Altschul, et al., (1990) J.
Mol. Biol. 215:403-410; Gish, et al., (1993) Nature Genet. 3:266-272; Madden, r et al., (1996) Meth. Enzymol. 266:131-141; Altschul, et al., (1997) Nucleic 00 0 Acids Res. 25:3389-3402; Zhang, et al., (1997) Genome Res. 7:649-656; Wootton, SJ.C., et al., (1993) Comput. Chem. 17:149-163; Hancock, J.M. et al., (1994) Comput.
Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, et al., "A model of evolutionary change in proteins." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff pp. 345-352, Natl. Biomed. Res.
Found., Washington, DC; Schwartz, et al., "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3." M.O. Dayhoff pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, (1991) J. Mol. Biol. 219:555-565; States, et al., (1991) Methods 3:66-70; Henikoff, etal., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, etal., (1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, et a., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, etal., (1994) Ann.
Prob. 22:2022-2039; and Altschul, S.F. "Evaluating the statistical significance of multiple distinct local alignments." in Theoretical and Computational Methods in Genome Research Suhai, (1997) pp. 1-14, Plenum, New York.
Antibody Structure In general, the basic antibody structural unit is known to comprise a tetramer.
Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain may include a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant 0 27 regions are joined by a region of about 12 or more amino acids, with the heavy Schain also including a region of about 10 more amino acids. See generally, oo Fundamental Immunology Ch. 7 (Paul, ed., 2nd ed. Raven Press, N.Y. (1989))
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(incorporated by reference in its entirety for all purposes).
The variable regions of each light/heavy chain pair may form the antibody r-binding site. Thus, in general, an intact IgG antibody has two binding sites. Except in 0 bifunctional or bispecific antibodies, the two binding sites are, in general, the same.
SNormally, the chains all exhibit the same general structure of relatively Sconserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md. 5 t ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, etal., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. The present invention provides antibodies or antigen-binding fragments of the invention comprising CDRs and FRs from the light and heavy chains of 1H3, 15H12 and 19D12 15H12/19D12 LCA, 15H12/19D12 LCB, 15H12/19D12 HCA, SEQ ID NOs: 2, 4, 25, 27, 41, 43 and 45) as defined by Kabat and Chothia (see above references).
Antibody Molecules The term "antibody molecule" includes, but is not limited to, antibodies and fragments, preferably antigen-binding fragments, thereof. The term includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab) 2 antibody fragments, Fv antibody fragments VH or VL), single chain Fv antibody fragments and dsFv antibody fragments. Furthermore, the antibody molecules of the invention may be fully human antibodies or chimeric antibodies. Preferably, the antibody molecules are monoclonal, fully human antibodies; more preferably, the antibody molecules are 1H3, 15H12 or 19D12.
8 28 Preferably, the antibody molecules include one or more of the variable regions and 0 CDRs whose amino acid and nucleotide sequences are set forth in Table 1.
00 The present invention includes any antibody molecule comprising a CDR selected from: RASQSIGSSLH (SEQ ID NO: 8); O YASQSLS (SEQ ID NO: 9); r- HQSSRLPHT (SEQ ID NO: 00 SFAMH (SEQ ID NO: 14) 0 GFTFSSFAMH (SEQ ID NO:17); 0 10 VIDTRGATYYADSVKG (SEQ ID NO: LGNFYYGMDV (SEQ ID NO: 16); RASQSVSSFLA (SEQ ID NO: 31); DASNRAP (SEQ ID NO: 32); QQRSNWPRWT (SEQ ID NO: 33); GFTFSNYAMH (SEQ ID NO: 37); AIGAGGDTYYADSVKG (SEQ ID NO:38); and GRHRNWYYYNKDY (SEQ ID NO: 39); NYAMH (SEQ ID NO: The scope of the present invention includes antibody variable regions of the present invention any variable region, mature or unprocessed, indicated in Table 1) linked to any immunoglobulin constant region. If a light chain variable region is linked to a constant region, preferably it is a K chain. If a heavy chain variable region is linked to a constant region, preferably it is a yl, y2, y3 or y4 constant region, more preferably, yl, y2 or y4 and even more preferably yl or y4.
The anti-IGFR1 antibody molecules of the invention preferably recognize human IGFR1, preferably slGFR1; however, the present invention includes antibody molecules which recognize IGFR1 from different species, preferably mammals mouse, rat, rabbit, sheep or dog). The present invention also includes anti-IGFR1 antibodies or fragments thereof which are complexed with IGFR1 or any fragment thereof amino acids 30-902 of SEQ ID NO: 19) or with any cell which is expressing IGFR1 or any portion or fragment thereof on the cell surface HEK293 cells stably transformed with human IGFR1 or MCF7 ATCC Cell Line No. HTB-22)). Such complexes may be made by contacting the antibody or antibody fragment with IGFR1 or the IGFR1 fragment.
In a preferred embodiment, fully-human monoclonal antibodies directed against IGFR1 are generated using transgenic mice carrying parts of the human immune system rather than the mouse system. These transgenic mice, which may be referred to, herein, as "HuMAb" mice, contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (4 and y) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the 0 29
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endogenous p and K chain loci (Lonberg, et al., (1994) Nature 368(6474): 856- 859). Accordingly, the mice exhibit reduced expression of mouse IgM or K, and in o0 response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies (Lonberg, et al., (1994), supra; reviewed in Lonberg, N.
(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, et al., oO O (1995) Intern. Rev. Immunol. 13:65-93, and Harding, etal., (1995) Ann. N. Y Acad.
N Sci 764:536-546). The preparation of HuMab mice is commonly known in the art and 0is described, for example, in Taylor, et al., (1992) Nucleic Acids Research C 10 20:6287-6295; Chen, et al., (1993) International Immunology 5: 647-656; Tuaillon, et al., (1993) Proc. Natl. Acad. Sci USA 90:3720-3724; Choi, et al., (1993) Nature Genetics 4:117-123; Chen, et al., (1993)EMBO J. 12: 821- 830; Tuaillon, et al., (1994) J Immunol. 152:2912-2920; Lonberg, et al., (1994) Nature 368(6474): 856- 859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Taylor, et al., (1994) Intemational Immunology 6: 579-591; Lonberg, et al., (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, etal., (1995) Ann. N.Y Acad. Sci 764:536-546; Fishwild, et al., (1996) Nature Biotechnology 14: 845-851 and Harding, et al., (1995) Annals NY Acad. Sci. 764:536-546; the contents of all of which are hereby incorporated by reference in their entirety. See further, U.S. Patent Nos. 5,545,806; 5, 569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874, 299; 5,770,429 and 5,545,807; and International Patent Application Publication Nos. WO 98/24884; WO 94/25585; WO 93/12227; WO 92/22645 and WO 92/03918 the disclosures of all of which are hereby incorporated by reference in their entity.
To generate fully human, monoclonal antibodies to IGFR1, HuMab mice can be immunized with an antigenic IGFR1 polypeptide, preferably amino acids 30-902 of SEQ ID NO: 19, as described by Lonberg, et al., (1994) Nature 368(6474): 856- 859; Fishwild, et al., (1996) Nature Biotechnology 14: 845-851 and WO 98/24884.
Preferably, the mice will be 6-16 weeks of age upon the first immunization. For example, a purified preparation of IGFR1 or slGFR1 can be used to immunize the HuMab mice intraperitoneally. The mice can also be immunized with whole HEK293 cells which are stably transformed or transfected with an IGFR1 gene. An "antigenic IGFR1 polypeptide" may refer to an IGFR1 polypeptide of any fragment thereof, c preferably amino acids 30-902 of SEQ ID NO: 19, which elicits an anti-IGFR1 immune response, preferably in HuMab mice.
In general, HuMAb transgenic mice respond well when initially immunized 00 C1 intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by every other week IP immunizations (usually, up to a total of 6) with antigen in incomplete N Freund's adjuvant. Mice can be immunized, first, with cells expressing IGFR1 oO stably transformed HEK293 cells), then with a soluble fragment of IGFR1 amino Sacids 30-902 of SEQ ID NO: 19) and continually receive alternating immunizations with the two antigens. The immune response can be monitored over the course of O 10 the immunization protocol with plasma samples being obtained by retroorbital bleeds.
The plasma can be screened for the presence of anti-IGFR1 antibodies, for example by ELISA, and mice with sufficient titers of immunoglobulin can be used for fusions.
Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each antigen may need to be performed. Several mice can be immunized for each antigen. For example, a total of twelve HuMAb mice of the HC07 and HC012 strains can be immunized.
Hybridoma cells which produce the monoclonal, fully human anti-IGFR1 antibodies may be produced by methods which are commonly known in the art.
These methods include, but are not limited to, the hybridoma technique originally developed by Kohler, et al., (1975) (Nature 256:495-497), as well as the trioma technique (Hering, et al., (1988) Biomed. Biochim. Acta. 47:211-216 and Hagiwara, et al., (1993) Hum. Antibod. Hybridomas 4:15), the human B-cell hybridoma technique (Kozbor, et al., (1983) Immunology Today 4:72 and Cote, et al., (1983) Proc. Natl.
Acad. Sci. U.S.A 80:2026-2030), and the EBV-hybridoma technique (Cole, et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985).
Preferably, mouse splenocytes are isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting hybridomas may then be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice may by fused to one-sixth the number of P3X63- Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells may be plated at approximately 2 x 10 5 cells/mL in a flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5mM HEPES, 0.055 31 C mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml C gentamycin and 1X HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells may be cultured in medium in which the HAT is replaced with HT.
00 C1 Individual wells may then be screened by ELISA for human anti-IGFR1 monoclonal IgG antibodies. Once extensive hybridoma growth occurs, medium can be observed N usually after 10-14 days. The antibody secreting hybridomas may be replated, 0 0 screened again, and if still positive for human IgG, anti-IGFR1 monoclonal antibodies, Scan be subcloned at least twice by limiting dilution. The stable subclones may then be cultured in vitro to generate small amounts of antibody in tissue culture medium for 0 10 characterization.
The anti-IGFR antibody molecules of the present invention may also be produced recombinantly in an E.coliT7 expression system as discussed above). In this embodiment, nucleic acids encoding the antibody molecules of the invention VH or VL) may be inserted into a pET-based plasmid and expressed in the E.coliT7 system. There are several methods by which to produce recombinant antibodies which are known in the art. One example of a method for recombinant production of antibodies is disclosed in U.S. Patent No. 4,816,567 which is herein incorporated by reference. Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for Introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextranmediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.
Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells Hep G2), A549 cells, 3T3 cells, and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression 32 Slevels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion thereof, the 00 Clight chain and/or antigen-binding portion thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time Ssufficient to allow for expression of the antibody in the host cells or, more preferably, 00 5 secretion of the antibody into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.
It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the instant invention, regardless of the glycosylation of the antibodies.
refers to the off-rate constant for dissociation of the antibody from an antibody/antigen complex.
refers to the rate at which the antibody associates with the antigen.
"Kd" refers to the dissociation constant of a particular antibody/antigen interaction. Kd= Koff/Kon.
The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
Monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies, and is not to be construed as C 33 c- requiring production of the antibody by any particular method. As mentioned above, Cd the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler, et al, (1975) Nature 00 CN 256: 495.
A polyclonal antibody is an antibody which was produced among or in the N presence of one or more other, non-identical antibodies. In general, polyclonal 00 0 antibodies are produced from a B-lymphocyte in the presence of several other B- Slymphocytes which produced non-identical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.
O 10 A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321, Kostelny, etal., (1992) J Immunol. 148:1547- 1553. In addition, bispecific antibodies may be formed as "diabodies" (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as "Janusins" (Traunecker, et al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52).
The term "fully human antibody" refers to an antibody which comprises human immunoglobulin protein sequences only. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell. Similarly, "mouse antibody" refers to an antibody which comprises mouse immunoglobulin sequences only.
The present invention includes "chimeric antibodies"- an antibody which comprises a variable region of the present invention fused or chimerized with an antibody region constant region) from another, non-human species mouse, horse, rabbit, dog, cow, chicken). These antibodies may be used to modulate the expression or activity of IGFR1 in the non-human species.
"Single-chain Fv" or "sFv" antibody fragments have the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
Generally, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. Techniques described for the production of single chain antibodies (U.S.
Patent Nos. 5,476,786; 5,132,405 and 4,946,778) can be adapted to produce anti- IGFR1-specific single chain antibodies. For a review of sFv see Pluckthun in The 34 C- Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.
Springer-Verlag, pp. 269-315 (1994).
"Disulfide stabilized Fv fragments" and "dsFv" refer to antibody molecules 00 00 comprising a variable heavy chain (VH) and a variable light chain (VL which are linked by a disulfide bridge.
O Antibody fragments within the scope of the present invention also include 00 F(ab) 2 fragments which may be produced by enzymatic cleavage of an IgG by, for Sexample, pepsin. Fab fragments may be produced by, for example, reduction of F(ab) 2 with dithiothreitol or mercaptoethylamine. A Fab fragment is a VL-CL chain appended to a VH-CH1 chain by a disulfide bridge. A F(ab) 2 fragment is two Fab fragments which, in turn, are appended by two disulfide bridges. The Fab portion of an F(ab) 2 molecule includes a portion of the Fc region between which disulfide bridges are located.
An Fv fragment is a VL or VH region.
Depending on the amino acid sequences of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG-1, lgG-2, lgG-3 and lgG-4; IgA-1 and lgA-2.
The anti-IGFR1 antibody molecules of the invention may also be conjugated to a chemical moiety. The chemical moiety may be, inter alia, a polymer, a radionuclide or a cytotoxic factor. Preferably the chemical moiety is a polymer which increases the half-life of the antibody molecule in the body of a subject. Suitable polymers include, but are not limited to, polyethylene glycol (PEG) PEG with a molecular weight of 2kDa, 5 kDa, 10 kDa, 12kDa, 20 kDa, 30kDa or 40kDa), dextran and monomethoxypolyethylene glycol (mPEG). Lee, etal., (1999) (Bioconj. Chem.
10:973-981) discloses PEG conjugated single-chain antibodies. Wen, etal., (2001) (Bioconj. Chem. 12:545-553) disclose conjugating antibodies with PEG which is attached to a radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).
The antibodies and antibody fragments of the invention may also be conjugated with labels such as 99 Tc, 9 Y, 111In 3 2 P, 14C, 1251, 3 H, 1311 11C, 15s, 13 N, 18
F,
3S, 1 Cr, 5To, 226 Ra, 60Co, 59 Fe, "Se, 15 2 Eu, 67 CU, 217 Ci, 2 11 At, 2 12 Pb, 47 Sc, 109 Pd, 234Th, and 40 K, 57 Gd, 55 Mn, 52 Tr and 6Fe.
The antibodies and antibody fragments of the invention may also be a conjugated with fluorescent or chemilluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, 00 N isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine, 152 Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, I an aromatic acridinium ester label, an imidazole label, an acridimium salt label, an 0- 0 o oxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones, biotin/avidin, Sspin labels and stable free radicals.
The antibody molecules may also be conjugated to a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP- S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.
Any method known in the art for conjugating the antibody molecules of the invention to the various moieties may be employed, including those methods described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, (1982) Histochem. and Cytochem. 30:407. Methods for conjugating antibodies are conventional and very well known in the art.
Modified Antibody Molecules The present invention includes antibodies and antigen-binding fragments fully-human antibodies, SFv, dsFv, Fv, chimeric antibodies) comprising a light chain of SEQ ID NOs: 41, 43, 72, 74, 76 or 78 (15H12/19D12 LCA, LCB, LCC, LCD, LCE or LCF); preferably amino acids 20-128 of SEQ ID NOs: 41, 43, 72, 74, 76 or 78 (mature 15H12/19D12 LCA, LCB, LCC, LCD, LCE or LCF). The present invention also includes antibody molecules including the heavy chain of SEQ ID NO: 45 or 112 (15H12/19D12 HCA, HCB); preferably amino acids 20-137 of SEQ ID NO: 45 or 112 (mature 15H12/19D12 HCA, HCB).
The 15H12/19D12 LCA, LCB, LCC, LCD, LCE and LCF may be dimerized with any other immunoglobulin heavy chain, preferably an immunoglobulin heavy chain of the present invention. Likewise, 15H12/19D12 HCA or HCB may be dimerized with any light chain, preferably a light chain of the present invention. For example, 36 15H12/19D12 HCA or HCB may be dimerized with 15H12/19D12 LCC, LCD, LCE or
LCF.
Antibodies and antigen-binding fragments comprising 15H12/19D12 LCA, 15H12/19D12 LCB, 15H12/19D12 LCC, 15H12/19D12 LCD, 15H12/19D12 LCE, 15H12/19D12 LCF, 15H12/19D12 HCA or 15H12/19D12 HCB or any fragment thereof exhibit minimal immunogenicity in a human subject; thereby, leading to a low incidence of HAHA response when administered to a human subject.
Preferred antibody chains are shown below. Dotted underscored type encodes the signal peptide. Solid underscored type encodes the CDRs. Plain type encodes the framework regions. Most preferably, the antibody chains are mature fragments which lack the signal peptide.
Modified 19D12/15H12 Light Chain-C (SEQ ID NO: 71) ATG TCG CCA TCA CAA CTC ATT GGG TTT CTG CTG CTC TGG GTT CCA GCC TCC AGG GGT GAA ATT GTG CTG ACT CAG AGC CCA GAC GGC GAG AGA GTC ACC ATC ACC TGC CGG GCC AGT TTA CAC TGG TAC CAG CAG AAA CCA GGT CAG TCT TAT GCA TCC CAG TCC CTC TCA GGG GTC CCC TCG TCT GGG ACA GAT TTC ACC CTC ACC ATC AGT AGC GCA GCG TAT TAC TGT CAT CAG AGT AGT CGT TTA GGG ACC AAG GTG GAG ATC AAA CGT ACG TCT CT TCT GTG ACT CCA CAG ACC ATT GGT AGT AGC CCA AAG CTT AGG TTC AGT CTC GAG GCT CCT CAC ACT CTC ATC AAG GGC AGT GGA GAA GAT GCT TTC GGC CAA (SEQ ID NO: 72) S L I F
RGEIVLTQSP
GERVTITCRA
C E I V L T Q S P C E R V T I T C R A L W V P S
DSLSVTP
D S L S V T P S Q S I G S S LHWYQQKPGQSPK LLK Y Q Q K P Q S P K L L I K
YASQSLSGVP
SGTDFTLTIS
AAYYCHQSSR
Y A S Q S L S V P S C T D F T L T I S A A Y Y C H Q S S R G T K V E I K R T S R F S G S G S L E A E D A L P H T F G Q 37 Modified 19D1 2/1 5H12 Light Chain-D (SEQ ID NO: 73) ATG TCG CCA TCA CAA CTC ATT GGG TTT CTG CTG CTC TGG GTT OCA GCC TCC 00 C1 5 AGG GGT GAA ATT GTG CTG ACT CAG AGC CCA GAC TCT CTG TCT GTG ACT CCA GGC GAG AGA GTC ACC ATC ACC TGC CGG GCC AGT CAG AGC ATT GGT AGT AGO TT~A CAC TGG TAC CAG CAG AAA CCA GGT CAG TOT CCA AAG CTT CTC ATC AAG 00 TAT GCA TCC CAG TCC CIC TCA GGO GTC CCC TCG AGG 1TC AGT GGC AGT GGA TCT 000 ACA GAT TTC ACC CIC ACC ATC AGT AGC CTC GAG GOT GAA GAT TTC GCA GTG TAT TAC TOT CAT CAG AGT AGT CGT TTA CCT CAC ACT TTC GGC CAA GGG ACC AAG GTG GAG ATC AAA CGT ACG (SEQ ID NO: 74) M S P S q L I G F L L L W V P A S R G E I V L T Q S P D S L S V T P G E R V T I T C R A S Q S I G S S L H W Y Q Q K P G Q S P K L L I K Y A S Q S L S G V P S R F S G S G S G T D F T L T I S S L E A E D F A V Y Y C H Q S S R L P H T F 0 Q G T K V E I K R T Modified 19D12/15H12 Light Chaln-E (SEQ ID NO: A TO TCG OqCA TCA CAA CTC ATT GGG TTT CTG CTO CTC TGG GTT CCA GOC TCC AGG GOT GAA ATT GTG CTG ACT CAG AGC CCA GGT ACC CTG TOT GTG TOT CCA GGC GAG AGA GOC ACC OTO TCC TGO CGG GCC AGT CAG AGO ATT GOT AGT AGO TTA CAC TOG TAC CAG CAG AAA CCA GOT CAG GOT CCA AGO CTT CTC ATC AAG TAT OCA TCC CAG TCC CTC TCA 000 ATC CCC OAT AGO TTC AGT GGC AGT GGA TOT GOG ACA OAT TTC ACC CTC ACC ATC AGT AGA CTG GAG CCT GAA OAT OCT OCA GCG TAT TAO TOT CAT CAG AGT AGT COT TTA CCT CAC ACT TTC GGC CAA 000 ACC AAG GTG GAG ATC AAA CGT ACA (SEQ ID NO: 76) M S S Q L I G F L L L W V P A S R0 E I V L T Q S P G T L S V S P C1G E R A T L S C R A S Q s I G S S L H W Y Q Q K P G Q A P R L L I K r- 10 Y A S Q S L S G I P D R F S G S G 00 S G T D F T L T I S R L E P E D A Y Q S S R L P H T F G Q G T K V E I K R T Modified 19D12/151-12 Light Chain-F (SEQ ID NO: 77) ATG TCG CCA TCA CAA CTC ATT GGG TTT CTG CTG CTC TGG G'1' CCA GCC TCC AGG GGT GAA ATT GTG CTG ACT CAG AGC CCA GGT ACC CTG TCT GTG TCT CCA GGC GAG AGA GCC ACC CTC TCC TGC CGG GCC AGT CAG AGC ATT GGT AGT AGC TTA CAC TGG TAC CAG CAG AAA CCA GGT CAG GCT CCA AGG CTT CTC ATC AAG TAT GCA TCC CAG TCC CTC TCA GGG ATC CCC GAT AGG TTC AGT GGC AGT GGA TCT GOG ACA GAT TTC ACC CTC ACC ATC AGT AGA CTG GAG CCT GAA GAT TTC GCA GTG TAT TAC TGT CAT CAG AGT AGT CGT TTA CCT CAC ACT TTC GGC CAA GGG ACC AAG GTG GAG ATC AAA CGT ACA (SEQ ID NO: 78) ~M S P S Q I F L W V P A S R E I V L T Q S P G T L S V S P G E R A T L S C R A S Q S I G S S L H W Y Q Q K P G Q A P R L L I K Y A S Q S L S G I P D R F S G S G S G T D F T L T I S R L E P E D F A V Y Y C H Q S S R L P H T F G Q G T K V E I K R T Modified 19D1 2/151112 heavy chain-A (SEQ ID NO: 44) 39 c-I ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GOT ATA TTA AAA GGT GTC CAG TGT GAG GTT CAG CTG GTG CAG TCT GGG GGA GGC TTIG GTA AAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GOAGCC TCTGGA TTC ACC TTC AGT AGC TTT 00 cIGOT ATG CAC TGG GT'r CGC CAG GOT CCA GGA AAA GGT CTG GAG TGG ATA TCA GTT ATT GAT ACT CGT GGT GCC ACA TAC TAT GCA GAC TCC GTG AAG GGC CGA TTC ACC ATC TCC AGA GAC AAT GCC AAG AAC TCC TTG TAT CTT CAA ATG AAC 00 AGC CTG AGA GCC GAG GAC ACT GCT GTG TAT TAC TGT GCA AGA CTG GGG AAC TITC TAC TAC GGT A1T3 GAC GTC TGG GGC CAA GGG ACC ACG GTC ACC GTC TCC
TCA
(SEQ ID NO: Met Glu Phe Gly Leu Ser !.Trp Val Phe Leu Val Ala Ile Leu .Lys Gly Val G CsGlu Val Gin Leu Val Gin Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Giu Trp Ile Ser Val. Ile Asp Thr Arg Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Asn Phe Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val. Thr Val Ser Ser Modified 19D1 2/15H1 2 heavy chain-B (SEQ ID NO: 111) ATG GAG TTT GGG CTG AGO TGG GTT TTC CTT GTT GCT ATA TTA AAA GGT GTC CAG TGT GAG GTT CAG CTG GTG CAG TCT GGG GGA GGO TTG GTA CAG CCC GGG GGG TCC CTG AGA CTC TCC TGT GOA GCC TOT GGA TC ACC TTC AGT AGC TTT GOT ATG CAC TGG GTT CGC CAG GOT CCA GGA AAA GGT CTG GAG TGG ATA TOA GTT ATT GAT ACT CGT GGT GOC ACA TAC TAT GCA GAC TCC GTG AAG GGC CGA TTC ACC ATC TCC AGA GAC AAT GCC AAG AAC TCC TTG TAT CTT CAA ATG AAC AGC CTG AGA GOC GAG GAO ACT GOT GTG TAT TAC TGT GOA AGA CTG GGG AAC TTC TAC TAC GGT ATG GAC GTC TGG GGC CAA GGG ACC ACG GTC ACC GTC TCC
TCA
(SEQ ID NO: 112) O 4 SMet Glu Phe Gy Leu Ser Trp Val Phe Leu Val Ala Ile Leu LYS Gly Val e Gin..y Cys Glu Val Gin Leu Val Gin Ser Gly Gly Gly Leu Val Gin Pro Gly 00 5 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Ile Ser C 10 Val Ile Asp Thr Arg Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 00 Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Asn CN 15 Phe Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Gene Therapy The anti-IGFR1 antibodies of the invention may also be administered to a subject in a gene therapy approach. In a gene therapy approach, the cells of a subject are transformed with nucleic acids which encode the antibodies of the invention. Subjects comprising the nucleic acids will then produce the antibody molecules endogenously. Previously, Alvarez, et al., (2000) (Clinical Cancer Research 6:3081-3087) introduced single-chain anti-ErbB2 antibodies to subjects using a gene therapy approach. The methods disclosed by Alvarez, et aL, may be easily adapted for the introduction of nucleic acids encoding an anti-IGFR1 antibody molecule of the invention to a subject.
Although nucleic acids encoding any polypeptide or antibody molecule of the invention may be introduced to a subject, in preferred embodiments, the antibody molecule is a fully human, single-chain antibody.
The nucleic acids may be introduced to the cells of a subject by any means known in the art. In preferred embodiments, the nucleic acids are introduced as part of a viral vector. Examples of preferred viruses from which the vectors may be derived include lentiviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, alphavirus, influenza virus, and other recombinant viruses with desirable cellular tropism.
Various companies produce viral vectors commercially, including, but by no means limited to, Avigen, Inc. (Alameda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, PA; adenoviral and AAV vectors), Genvec (adenoviral vectors), IntroGene (Leiden, Netherlands; 41 C adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, 00 1 retroviral, and lentiviral vectors).
Methods for constructing and using viral vectors are known in the art (see, Sg Miller, et al., (1992) BioTechniques 7:980-990). Preferably, the viral vectors are 0 0 replication defective, that is, they are unable to replicate autonomously, and thus are O not infectious, in the target cell. Preferably, the replication defective virus is a minimal Svirus, it retains only the sequences of its genome which are necessary for 0 10 encapsidating the genome to produce viral particles. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted.
Examples of vectors comprising attenuated or defective DNA virus sequences include, but are not limited to, a defective herpes virus vector (Kanno, et al., (1999) Cancer Gen. Ther. 6:147-154; Kaplitt, et al., (1997) J. Neurosci. Meth. 71:125-132 and Kaplitt, etal., (1994) J. Neuro Onc. 19:137-147).
Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Attenuated adenovirus vectors, such as the vector described by Stratford-Perricaudet, et al., (1992) Clin.
Invest. 90:626-630) are desirable in some instances. Various replication defective adenovirus and minimum adenovirus vectors have been described (PCT Publication Nos. W094/26914, W094/28938, W094/28152, W094/12649, W095/02697 and W096/22378). The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero, et al., (1991) Gene 101:195; EP 185573; Graham, (1984) EMBO J. 3:2917; Graham, et al., (1977) J. Gen. Virol. 36:59).
The adeno-associated viruses (AAV) are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see Daly, et al., (2001) Gene Ther. 8:1343-1346, 1245-1315; Larson, et al., (2001) Adv. Exp. Med. Bio.
42 S489:45-57; PCT Publication Nos. W091/18088 and W093/09239; U.S. Patent Nos.
"d 4,797,368 and 5,139,941 and EP 488528B1).
In another embodiment, the gene can be introduced in a retroviral vector, e.g., 00 oO N as described in U.S. Patent Nos. 5,399,346, 4,650,764, 4,980,289, and 5,124,263; Mann, etal., (1983) Cell 33:153; Markowitz, etal., (1988) J. Virol., 62:1120; EP IN 453242 and EP178220. The retroviruses are integrating viruses which infect dividing 00 0 0 cells.
O Lentiviral vectors can be used as agents for the direct delivery and sustained expression of nucleic acids encoding an antibody molecule of the invention in several tissue types, including brain, retina, muscle, liver and blood. The vectors can efficiently transduce dividing and nondividing cells in these tissues, and maintain long-term expression of the antibody molecule. For a review, see Zufferey, et al., (1998) J. Virol. 72:9873-80 and Kafri, et al., (2001) Curr. Opin. Mol. Ther. 3:316-326.
Lentiviral packaging cell lines are available and known generally in the art. They facilitate the production of high-titer lentivirus vectors for gene therapy. An example is a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 106 IU/ml for at least 3 to 4 days; see Kafri, et al., (1999) Virol. 73: 576-584). The vector produced by the inducible cell line can be concentrated as needed for efficiently transducing nondividing cells in vitro and in vivo.
Sindbis virus is a member of the alphavirus genus and has been studied extensively since its discovery in various parts of the world beginning in 1953. Gene transduction based on alphavirus, particularly Sindbis virus, has been well-studied in vitro (see Straus, et al., (1994) Microbiol. Rev., 58:491-562; Bredenbeek, et al., (1993) J. Virol., 67; 6439-6446 lijima, et al., (1999) Int. J. Cancer 80:110-118 and Sawai, et al., (1998) Biochim. Biophyr. Res. Comm. 248:315-323). Many properties of alphavirus vectors make them a desirable alternative to other virus-derived vector systems being developed, including rapid engineering of expression constructs, production of high-titered stocks of infectious particles, infection of nondividing cells, and high levels of expression (Strauss, etal., (1994) Microbiol. Rev. 58:491-562).
Use of Sindbis virus for gene therapy has been described. (Wahlfors, et al., (2000) Gene. Ther. 7:472-480 and Lundstrom (1999) J. Recep. Sig. Transduct. Res. 19(1- 4):673-686).
43 C1 In another embodiment, a vector can be introduced to cells by lipofection or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic llpids can be used to prepare liposomes for in vivo and In vitro transfection of a gene oO CN encoding a marker (Feigner, et al., (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417 and Wang, et (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855). Useful lipid O compounds and compositions for transfer of nucleic acids are described in PCT 00 Publication Nos. WO 95/18863 and W096/17823, and in U.S. Patent No. 5,459,127.
O It is also possible to introduce the vector in vivo as a naked DNA plasmid.
Naked DNA vectors for gene therapy can be introduced into the desired host cells by 0 10 methods known in the art, electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, Wilson, etal., (1992) J. Biol. Chem. 267:963-967; Williams, et al., (1991) Proc. Natl. Acad. Sci. USA 88:2726-2730). Receptor-mediated DNA delivery approaches can also be used (Wu, et al., (1988) J. Biol. Chem. 263:14621- 14624). U.S. Patent Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Vilquin, et al., (2001) Gene Ther. 8:1097; Payen, et al., (2001) Exp. Hematol. 29:295-300; Mir (2001) Bioelectrochemistry 53:1-10; PCT Publication Nos. W099/01157, W099/01158 and W099/01175).
Pharmaceutical Compositions An antibody or antigen-binding fragment of the invention can be incorporated into a pharmaceutical composition, along with a pharmaceutically acceptable carrier, suitable for administration to a subject in vivo. Although the scope of the present invention includes pharmaceutical compositions which may be administered to a subject by any route oral, ocular, topical or pulmonary (inhalation)), administration by a parenteral route such as intratumoral injection, intravenous injection, subcutaneous injection or intramuscular injection is preferred. In a preferred embodiment, the pharmaceutical compositions of the invention comprise 1H3, 15H12, 19D12, 15H12/19D12 LCA, 15H12/19D12 LCB, 15H12/19D12 LCC, 15H12/19D12 LCD, 15H12/19D12 LCE, 15H12/19D12 LCF, 15H12/19D12 HCA or 15H12/19D12 HCB and a pharmaceutically acceptable carrier.
44 c For general information concerning formulations, see, Gilman, et al., (O (eds.) (1990), The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; A. Gennaro Remington's Pharmaceutical Sciences, 18th Edition, (1990), Mack oO r Publishing Co., Easton, Pennsylvania.; Avis, et al., (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York; Lieberman, et al., (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, New York; and Lieberman, et Sal., (eds.) (1990), Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York, Kenneth A. Walters (2002) Dermatoloqical and Transdermal Formulations (Drugs and the Pharmaceutical Sciences), Vol 119, Marcel Dekker.
O 10 Pharmaceutically acceptable carriers are conventional and very well known in the art. Examples include aqueous and nonaqueous carriers, stabilizers, antioxidants, solvents, dispersion media, coatings, antimicrobial agents, buffers, serum proteins, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for injection into a subject's body.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
Stabilizers, such as a, a-trehalose dihydrate may be included for stabilizing the antibody molecules of the invention from degrading effects of dessication or freezedrying.
Examples of pharmaceutically-acceptable antioxidants include: water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; and oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
SPrevention of the presence of microorganisms may be ensured both by a sterilization procedures, and by the inclusion of various antimicrobial agents such as EDTA, EGTA, paraben, chlorobutanol, phenol sorbic acid, and the like.
oO C Suitable buffers which may be included in the pharmaceutical compositions of the invention include L-histidine based buffers, phosphate based buffers NOphosphate buffered saline, pH sorbate based buffers or glycine-based buffers.
0 Serum proteins which may be included in the pharmaceutical compositions of Sthe invention may include human serum albumin.
Isotonic agents, such as sugars, ethanol, polyalcohols glycerol, S 10 propylene glycol, liquid polyethylene glycol, mannitol or sorbitol), sodium citrate or sodium chloride buffered saline) may also be included in the pharmaceutical compositions of the invention.
Prolonged absorption of an injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and/or gelatin.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art.
Sterile injectable solutions can be prepared by incorporating the antibody or antigen-binding fragment of the invention in the required amount in an appropriate solvent, optionally with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the antibody molecule into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional, desired ingredient from a previously sterile-filtered solution thereof.
The antibody or antigen-binding fragment of the invention may also be orally administered. Pharmaceutical compositions for oral administration may contain, in addition to the binding composition, additives such as starch potato, maize or 46 Swheat starch or cellulose), starch derivatives microcrystalline cellulose or silica), sugars lactose), talc, stearate, magnesium carbonate or calcium phosphate. In 0 order to ensure that oral compositions comprising an antibody or antigen-binding Sfragment of the invention are well tolerated by the patient's digestive system, mucus formers or resins may be included. It may also be desirable to improve tolerance by IND formulating the antibody or antigen-binding fragment in a capsule which is insoluble in 0 the gastric juices. An exemplary pharmaceutical composition of this invention in the Sform of a capsule is prepared by filling a standard two-piece hard gelatin capsule with the antibody or antigen-binding fragment of the invention in powdered form, lactose, talc and magnesium stearate. Oral administration of immunoglobulins has been described (Foster, et al., (2001) Cochrane Database System rev. 3:CD001816) An antibody or antigen-binding fragment of the invention may also be included in a pharmaceutical composition for topical administration. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the antibody or antigenbinding fragment in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified by filtration.
Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile, aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the antibody or antigen-binding fragment of the invention in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a 47 Smetallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, d castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogels. The formulation oO Smay incorporate any suitable surface active agent such as an anionic, cationic or nonionic surface active such as sorbitan esters or polyoxyethylene derivatives thereof.
IDSuspending agents such as natural gums, cellulose derivatives or inorganic materials rO 0such as silicaceous silicas, and other ingredients such as lanolin, may also be Sincluded.
The antibodies and antigen-binding fragments of the invention may also be administered by Inhalation. A suitable pharmaceutical composition for inhalation may be an aerosol. An exemplary pharmaceutical composition for inhalation of an antibody or antigen-binding fragment of the invention may include: an aerosol container with a capacity of 15-20 ml comprising the antibody or antigen-binding fragment of the invention, a lubricating agent, such as polysorbate 85 or oleic acid, dispersed in a propellant, such as freon, preferably in a combination of 1,2dichlorotetrafluoroethane and difl uorochloromethane. Preferably, the composition is in an appropriate aerosol container adapted for either intranasal or oral inhalation administration.
In yet another embodiment of the present invention, the pharmaceutical composition can be administered by combination therapy. For example, the combination therapy can include a pharmaceutical composition of the present invention in association with one or more anti-cancer therapeutic agents alkylating agents, antimetabolites, anti-tumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenesis agents, anti-estrogens, anti-androgens, antibody therapies or immunomodulators). An "anti-cancer therapeutic agent' is a substance which, when administered to a subject, treats or prevents the development of cancer in the subject's body. The compositions of the invention may be administered in association with one or more anti-cancer therapeutic procedures radiation therapy or surgical tumorectomy). An "anti-cancer therapeutic procedure" is a process which is performed on a subject which treats or reduces the incidence of cancer in the subject. When a combination therapy is used, the antibodies or antigen-binding fragments of the invention, or pharmaceutical compositions thereof, may be formulated into a single composition for simultaneous delivery or formulated separately into two or more compositions a kit). Furthermore, the antibody or 48 c antigen-binding fragment may be administered to a subject at a different time than a when the other therapeutic agent or therapeutic procedure is administered; for example, each administration may be given non-simultaneously at several intervals CI over a given period of time.
"Alkylating agent" refers to any substance which can cross-link or alkylate any N molecule, preferably nucleic acid DNA), within a cell. Examples of alkylating 0 0 agents include mechlorethamine, cyclophosphamide, ifosfamide, phenylalanine Smustard, melphalen, chlorambucol, uracil mustard, estramustine, thiotepa, busulfan, lomustine, carmustine, streptozocin, dacarbazine, cis-platinum, carboplatin and 0 10 altretamine.
"Antimetabolites" refer to substances that block cell growth and/or metabolism by interfering with certain activities, usually DNA synthesis. Examples of antimetabolites include methotrexate, 5-fluoruracil, floxuridine, capecitabine, fludarabine, cytosine arabinoside, 6-mercaptopurine, 6-thioguanine, gemcitabine, cladribine, deoxycoformycin and pentostatin.
"Anti-tumor antibiotics" refer to compounds which may prevent or inhibit DNA, RNA and/or protein synthesis. Examples of anti-tumor antibiotics include doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone, dactinomycin, mithramycin, plicamycin, mitomycin C, bleomycin, and procarbazine.
"Mitotic inhibitors" prevent normal progression of the cell cycle and mitosis. In general, microtubule inhibitors such as paclitaxel and docetaxel are capable of inhibiting mitosis. Vinca alkaloids such as vinblastine, vincristine and vinorelbine are also capable of inhibiting mitosis.
"Chromatin function inhibitors" refer to substances which inhibit the normal function of chromatin modeling proteins such as topoisomerase I or topoisomerase II.
Examples of chromatin function inhibitors include topotecan, irinotecan, etoposide and teniposide.
"Anti-anglogenesis agent" refers to any drug, compound, substance or agent which inhibits growth of blood vessels. Exemplary anti-angiogenesis agents include, but are by no means limited to, razoxin, marimastat, COL-3, neovastat, BMS-275291, thalidomide, squalamine, endostatin, SU5416, SU6668, interferon-alpha, EMD121974, intereukin-12, IM862, angiostatin and vitaxin.
"Anti-estrogen" or "anti-estrogenic agent" refer to any substance which reduces, antagonizes or inhibits the action of estrogen. Examples of anti-estrogen S49 Sagents are tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, anastrozole, a letrozole, and exemestane.
"Anti-androgens" or "anti-androgen agents" refer to any substance which 00 C1 reduces, antagonizes or inhibits the action of an androgen. Examples of antiandrogens are flutamide, nilutamide, bicalutamide, sprironolactone, cyproterone I acetate, finasteride and cimitidine.
0- 0 Antibody therapies which may be administered in conjunction with the Santibodies or antigen-binding fragments of the invention include trastuzumab herceptin) (see, for example, Sliwkowski, et al., (1999) Semin. Oncol. 26(4 Suppl O 10 12):60-70), vitaxin and rituximab.
"Immunomodulators" are substances which stimulate the immune system.
Examples of immunomodulators include denileukin diftitox, levamisole in conjunction with 5-fluorouracil, interferon and interleukin-2.
"Radiotherapy" or "radiation therapy" refers to treating a disease, such as cancer, by administration of ionizing radiation (preferably to a tumor site). Examples of ionizing radiation which may be administered include X-rays, gamma rays emitted by radium, uranium or cobalt 60), and particle beam radiation protons, neutrons, pions or heavy ions).
Dosage Preferably, an antibody or antigen-binding fragment of the invention is administered to a subject at a "therapeutically effective dosage" which preferably inhibits a disease or condition which is mediated by IGFR1 tumor growth) to any extent-preferably by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%-100% relative to untreated subjects. The ability of an antibody or antigenbinding fragment of the invention to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of an antibody or antigenbinding fragment of the invention to inhibit tumor cell growth in vitro by assays (see below) well-known to the skilled practitioner. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
C Dosage regimens may be adjusted to provide the optimum desired response a therapeutic response). For example, a single bolus may be administered, T several divided doses may be administered over time or the dose may be 00 0proportionally reduced or increased as indicated by exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in O dosage unit form for ease of administration and uniformity of dosage.
00 A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the antibody or antigenbinding fragment of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention may be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be by injection, preferably proximal to the site of the target tumor). If desired, the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day.
Therapeutic Methods and Administration The antibodies or antigen-binding fragments of the invention and pharmaceutical compositions comprising the antibodies or antigen-binding fragments of the invention may be used for treating or preventing any disease or condition in a subject which is mediated by elevated expression or activity of IGFR1 or by elevated expression of its ligand IGF-I or IGF-II) and which may be treated or prevented by modulation of IGFR1 ligand binding, activity or expression. Preferably, the disease or condition is mediated by an increased level of IGFR1, IGF-I or IGF-II and is treated or prevented by decreasing IGFR1 ligand binding, activity autophosphorylation activity) or expression. Preferably, the disease or condition is malignancy, more preferably a malignancy characterized by a tumor which expresses IGFR1, such as, but not limited to, bladder cancer, Wilm's cancer, bone cancer, prostate cancer, lung cancer, colorectal cancer, breast cancer, cervical cancer, synovial sarcoma, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia (BPH), diarrhea associated 51 Swith metastatic carcinoid and vasoactive intestinal peptide secreting tumors VIPoma or Werner-Morrison syndrome). Acromegaly may also be treated with the C4antibody molecules of the invention. Antagonism of IGF-I has been reported for 00 00treatment of acromegaly (Drake, et al., (2001) Trends Endocrin. Metab. 12: 408-413).
Other non-malignant medical conditions which may also be treated, in a subject, by O administering an anti-IGFR1 antibody of the invention include gigantism, psoriasis, 00 atherosclerosis, smooth muscle restenosis of blood vessels or inappropriate microvascular proliferation, such as that found as a complication of diabetes, especially of the eye.
The term "subject" may refer to any organism, preferably an animal, more preferably a mammal rat, mouse, dog, cat, rabbit) and most preferably a human.
In preferred embodiments, the antibodies and antigen-binding fragments of the invention and pharmaceutical compositions thereof are administered by an invasive route such as by injection (see above). Administration by a non-invasive route (see above) is also within the scope of the present invention.
Compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle.
The pharmaceutical compositions of the invention may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S.
Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.
Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments.
Many other such implants, delivery systems, and modules are well known to those skilled in the art.
52
O
Cl Assays The anti-IGFR1 antibodies may be used to detect IGFR1 in a biological T sample in vitro or in vivo (see, for example, Zola, Monoclonal Antibodies: A Manual of 00 CTechniques, pp. 147-158 (CRC Press, Inc., 1987)). The anti-IGFR1 antibodies may be used in a conventional immunoassay, including, without limitation, an ELISA, an SRIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation. The 00 anti-IGFR1 antibodies of the invention may be used to detect IGFR1 from humans.
SThe invention provides a method for detecting IGFR1 in the biological sample comprising contacting the biological sample with an anti-IGFR1 antibody of the S 10 invention and detecting the anti-IGFR1 antibody bound to IGFR1, thereby indicating the presence of the IGFR1 in the biological sample. In one embodiment, the anti- IGFR1 antibody is directly labeled with a detectable label and may be detected directly. In another embodiment, the anti-IGFR1 antibody (the first antibody) is unlabeled and a secondary antibody or other molecule that can bind the anti-IGFR1 antibody is labeled. As is well known to one of skill in the art, a secondary antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the anti-IGFR1 antibody is a human IgG, then the secondary antibody may be an anti-human-lgG. The presence of an anti- IGFR1/IGFR1 complex in the biological sample can be detected by detecting the presence of the labeled secondary antibody. Other molecules that can bind to antibodies anti-IGFR1 antibodies) include, without limitation, Protein A and Protein G, both of which are available commercially, from Pierce Chemical Co.
(Rockford, IL) Suitable labels for the anti-IGFR1 antibody or secondary antibody have been disclosed supra, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, 1-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avldin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; an example of a magnetic agent includes gadolinium; and examples of suitable radioactive material include 1251, 1311, S or 3
H.
53
O
C1 In an alternative embodiment, IGFR1 can be assayed in a biological sample by a competition immunoassay utilizing IGFR1 standards labeled with a detectable 00 substance and an unlabeled anti-IGFR1 antibody. In this assay, the biological 00 CN sample, the labeled IGFR1 standards and the anti-IGFR1 antibody are combined and the amount of labeled IGFR1 standard bound to the unlabeled antibody is s o 0 determined. The amount of IGFR1 in the biological sample is inversely proportional 00 to the amount of labeled IGFR1 standard bound to the anti-IGFR1 antibody. One
O
O may use the Immunoassays disclosed above for a number of purposes. In one Sembodiment, the anti-IGFR1 antibodies may be used to detect IGFR1 in cells in cell 0 10 culture. In a preferred embodiment, the anti-IGFR1 antibodies may be used to determine the level of tyrosine phosphorylation, tyrosine autophosphorylation of IGFR1, and/or the amount of IGFR1 on the cell surface after treatment of the cells with various compounds. This method can be used to test compounds that may be used to activate or inhibit IGFR1. In this method, one sample of cells is treated with a test compound for a period of time while another sample is left untreated. If tyrosine autophosphorylation is to be measured, the cells are lysed and tyrosine phosphorylation of the IGFR1 is measured using an immunoassay, for example, as described above. If the total level of IGFR1 is to be measured, the cells are lysed and the total IGFR1 level is measured using one of the immunoassays described above.
A preferred immunoassay for determining IGFR1 tyrosine phosphorylation or for measuring total IGFR1 levels is an ELISA or Westem blot. If only the cell surface level of IGFR1 is to be measured, the cells are not lysed, and the cell surface levels of IGFR1 are measured using one of the immunoassays described above. A preferred immunoassay for determining cell surface levels of IGFR1 includes the steps of labeling the cell surface proteins with a detectable label, such as biotin or 1 2 1, immunoprecipitating the IGFR1 with an anti-IGFR1 antibody and then detecting the labeled IGFR1. Another preferred immunoassay for determining the localization of IGFR1, cell surface levels, is by using immunohistochemistry. Methods such as ELISA, RIA, Westem blot, immunohistochemistry, cell surface labeling of integral membrane proteins and immunoprecipitation are well known in the art. In addition, the immunoassays may be scaled up for high throughput screening in order to test a large number of compounds for either activation or inhibition of IGFR1.
54 C The anti-IGFR1 antibodies of the invention may also be used to determine the levels of IGFR1 in a tissue or in cells derived from the tissue. In a preferred l embodiment, the tissue is a diseased tissue. In a more preferred embodiment, the 00 0 tissue is a tumor or a biopsy thereof. In a preferred embodiment of the method, a tissue or a biopsy thereof is excised from a patient. The tissue or biopsy is then used IN in an immunoassay to determine, IGFR1 levels, cell surface levels of IGFR1, 00 levels of tyrosine phosphorylation of IGFR1, or localization of IGFR1 by the methods 0discussed above. The method can be used to determine if a tumor expresses IGFR1 Sat a high level.
The above-described diagnostic method can be used to determine whether a tumor expresses high levels of IGFR1, which may he indicative that the tumor will respond well to treatment with anti-IGFR1 antibody. The diagnostic method may also be used to determine whether a tumor is potentially cancerous, if it expresses high levels of IGFR1, or benign, if it expresses low levels of IGFR1. Further, the diagnostic method may also be used to determine whether treatment with anti-IGFR1 antibody is causing a tumor to express lower levels of IGFR1 and/or to exhibit lower levels of tyrosine autophosphorylation, and thus can be used to determine whether the treatment is successful. In general, a method to determine whether an anti- IGFR1 antibody decreases tyrosine phosphorylation comprises the steps of measuring the level of tyrosine phosphorylation in a cell or tissue of interest, incubating the cell or tissue with an antl-IGFR1 antibody or antigen-binding portion thereof, then re-measuring the level of tyrosine phosphorylation in the cell or tissue.
The tyrosine phosphorylation of IGFR1 or of another protein(s) may be measured.
The diagnostic method may also be used to determine whether a tissue or cell is not expressing high enough levels of IGFR1 or high enough levels of activated IGFR1, which may be the case for individuals with dwarfism, osteoporosis or diabetes. A diagnosis that levels of IGFR1 or active IGFR1 are too low could be used for treatment with activating anti-IGFR1 antibodies, IGF-1, IGF-2 or other therapeutic agents for increasing IGFR1 levels or activity.
The antibodies of the present invention may also be used in vivo to localize tissues and organs that express IGFR1. In a preferred embodiment, the anti-IGFR1 antibodies can be used to localize IGFR1-expressing tumors. The advantage of the anti-IGFR1 antibodies of the present invention is that they will not generate an immune response upon administration. The method comprises the steps of C1 administering an anti-IGFR1 antibody or a pharmaceutical composition thereof to a Spatient in need of such a diagnostic test and subjecting the patient to imaging analysis to determine the location of the IGFR1 -expressing tissues. Imaging analysis 00 CN is well known in the medical art, and includes, without limitation, x-ray analysis, magnetic resonance imaging (MRI) or computed tomography In another embodiment of the method, a biopsy is obtained from the patient to determine 00 whether the tissue of interest expresses IGFR1 rather than subjecting the patient to 0imaging analysis. In a preferred embodiment, the anti-IGFR1 antibodies may be labeled with a detectable agent that can be imaged in a patient. For example, the 0 10 antibody may be labeled with a contrast agent, such as barium, which can be used for x-ray analysis, or a magnetic contrast agent, such as a gadolinium chelate, which can be used for MRI or CE. Other labeling agents include, without limitation, radioisotopes, such as 9 Tc. In another embodiment, the anti-IGFR1 antibody will be unlabeled and will be imaged by administering a secondary antibody or other molecule that is detectable and that can bind the anti-IGFR1 antibody.
EXAMPLES
The following examples are provided to further describe the present invention and should not be construed to limit the scope of the invention in any way.
EXAMPLE 1: Construction of Fully Human Anti-IGFR1 Antibodies.
Introduction.
Fully human monoclonal antibodies specific for human insulin-like growth factor receptor 1 (IGFR1) were generated from HuMab mice of the Hco7 genotype (see below), immunized with recombinant slGFR1 and IGFR1 transfected HEK293 cells. A detailed description of Hco7 mice is provided in U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,874,299 and 5,877,397 and in Harding, et al., (1995) Ann. NY Acad. Sci. 764:536- 546. Antibodies 1H3, 15H12 and 19D12 were isolated from a HuMab mouse (referred to herein as #23716) which was selected for fusion based on the presence of antigen specific serum IgG titers of 25,600 to the immunizing antigen. The 1 H3, 15H12 and 19D121 antibodies were found to bind IGFR1.
56 Materials and Methods and Results.
00 C 2.1. Antigen.
O 2.1.1. Mice were immunized with two forms of antigen: live cells r0 (IGFR1 transfected HEK293 cells) and purified protein (slGFR1; an NSO-expressed recombinant protein encompassing the a-subunit and the extracellular domain of the 13-subunit of IGFR1). The biologically active version of this protein is in glycosylated form.
c 2.1.2. Three immunizations with soluble IGFR1 antigen and final tail vein boosts were performed with a purified IGFR1 preparation at a concentration of 2.67 mg/ml. Soluble IGFR1 was mixed with either complete or incomplete Freund's adjuvant (CFA and IFA) and mice were injected with 0.2 cc (cubic centimeters) prepared antigen into the intraperitoneal cavity. Final tail vein immunizations were performed with soluble IGFR1 in sterile PBS (phosphate buffer saline).
2.1.3. Immunizations were also performed with HEK293 cells transfected with IGFR1 DNA. Specifically, each mouse was immunized, by injection into the intraperitoneal cavity, with 0.2 cc of sterile saline containing 1.0-2.0 x 10' HEK293 cells expressing IGFR1.
2.2. Transgenic Mice.
2.2.1. Mice were housed in filter cages and were evaluated to be in good physical condition at the time of immunization, at the time of the bleeds and on the day fusions were produced.
2.2.2. The mouse that produced the selected hybridomas was a male (mouse ID #23716) of the (Hco7) 11952+; (JKD) 9272+ genotype. Individual transgene designations are in parentheses, followed by line numbers for randomly integrated transgenes. The symbols and indicate homozygous or hemizygous; however, because the mice are routinely screened using a PCR-based assay that does not allow us to distinguish between heterozygosity and homozygosity for the randomly integrated human Ig transgenes, a 57 designation may be given to mice that are actually homozygous for these elements.
2.3. Immunization Procedure and Schedule.
2.3.1. The immunization schedule is shown in the following table.
Table 2. Mouse immunization schedule.
Day Immunization: Bleed and Titer' adjuvant, antigen Dayl1 1.0 X 10'live IGFR1 transfected HEK293 cells in saline Day 15 CFA adjuvant, s1GERi Day 29 1.010 ive lGT transfected HEK293 cells in saline Day 37 antibody titer measured Day 43 lEA adjuvant, sIGERi gg)_ Day 54 antibody titer measured Day 57 1.0 X 107 live IGERi transfected HEK293 cells in saline Day 96 1.0 X 10' live IGFR1 transfected HEK293 cells in saline_ Day 103 antibody titer measured Day 112 CFA adjuvant, sGFR1 (25 lg) Day 126 antibody titer measured Days 128 and 129 Final tail vein intravenousboosts with sIGERi 2 1Titer information is shown below.
2 Fusions were performed on day 131.
Table 3. Titers of IGFR1 specific antibody during the immunization period of mouse 23716 described In Table 2 (see above).
Day Titer 37 100 58 C- 54 800 103 6400 126 25600 00 2.4. Hybridoma Preparation and Testing.
r- 0 2.4.1. The SP2/0-AG14 myeloma cell line (ATCC CRL 1581) was used for the fusions. The original ATCC vial was thawed and expanded in culture. A seed stock of frozen vials was prepared from this expansion.
SCells were maintained in culture for 6-8 weeks and passed twice a week.
2.4.2. High Glucose DMEM containing 10% FBS, Antibiotic-antimycotic (1OOX), and 0.1% L-glutamine was used to culture myeloma cells.
Additional media supplements were added to the hybridoma growth media which included: 5% Origen Hybridoma Cloning Factor (Fischer Scientific; Suwanee, GA), 4.5 x10" 4 M sodium Pyruvate, HAT 1.0 X 10 4 M Hypoxanthine, 4.0 x 10" M Aminopterin, 1.6 xl 05 M Thymidine, or HT 1.0 x 10 4 M Hypoxanthine, 1.6 x10, 5 M Thymidine; and characterized fetal bovine serum.
2.4.3. The spleen from mouse number #23716 was normal in size and yielded 5.73 x 108 viable cells.
2.4.4. The splenocytes were fused according to the following procedure: 1. Place approximately 10 ml of DMEM 10%FBS into a 50 mL tube.
2. Sacrifice the Intravenously boosted mouse.
3. Transfer the mouse into a hood onto a paper towel.
4. Soak the mouse with alcohol, and place onto its right side left side up.
Make a small cut into the skin above the spleen area.
6. Pull skin away from the mouse using both hands.
7. Soak with alcohol again.
8. Use sterile Instruments to open the peritoneum.
59 C 9. Insert the scissor points under the spleen and open the Sscissors so as to allow room to grasp the spleen with the forceps.
10. Remove the spleen and place into the tube containing 00 oO C DMEM 10%FBS. Transfer to a sterile tissue culture room.
11. Inside a sterile hood, add approximately 7 mL of DMEM IND without serum to each of 2 sterile 60 mm culture dishes.
00 12. Transfer the spleen to the first dish.
S13. Remove any adhesions from the spleen using sterile instruments.
0 10 14. Place a sterile homogenizer base into a test tube rack (for support).
Add the cleaned spleen into the homogenizer.
16. Add approximately 5 mL of DMEM and homogenize 4 passes. Pour off into a sterile 50 mL centrifuge tube.
17. Add another 5-6 mL of DMEM into the homogenizer and make another 3-4 additional passes.
18. Pour off into the same tube as described above.
19. Spin the cells at 1000 rpm for 10 minutes in a centrifuge.
Remove the supernatant. Pour off and resuspend pellets in
DMEM.
21. Count the spleen cells.
22. Transfer appropriate volume of SP2/0 cells (6 spleen cells per 1 cell of SP2/0) to a 50 mL centrifuge tube. Record volume.
23. Adjust volume of spleen cells with DMEM for more convenient balancing for centrifugation.
24. Spin cells for 10 minutes at 1000 rpm in a centrifuge.
Remove supematants pour off and resuspend pellets in 30-40 mL of DMEM wash medium (serum free). Combine all cells in one tube.
26. Spin again as above.
27. Pour off supematant and resuspend pellet.
28. Add approximately 1.2 mL of PEG (polyethylene glycol) at about 1 minute while gently swirling the tube in a beaker containing 370C water.
C 29. Let the tube sit for 90 seconds, then add 15 mL of DMEM Swash medium at 3 minutes.
L 30. Spin the tube as described above.
0 0 31. Remove the supematant and resuspend the pellet gently.
32. Add approximately 10 mL of Hat medium to the tube.
O 33. Pipette cells into the full volume of HAT medium. Allow the 00 cells to sit for 30-60 minutes in an incubator before plating.
034. Plate cells into 96 well culture plates, 200 PL/well (about cells per 96-well plate).
S 10 35. Feed cells on day 7 with HT media, 250gpl/well. (HT media, same as HAT media, with Aminopterin removed) 2.4.5. An initial ELISA screen for human IgGK antibodies was performed 7-10 days post fusion according to the following procedure: 1. Coat plate overnight with anti-hu-K, I pg/mL or anti-hu-y, 1 pg/mL in 1X PBS, 50 pL/well. Store in refrigerator.
2. Empty plate and block plate in 1X PBST (PBS with Tween) chicken serum for 1 hour at room temperature (100 pL/well).
3. Empty plate and wash manually with wash bottle (3X) or plate washer (3X) using 1X PBST. If wash bottle used, drain plates on paper towels.
4. Standards are used for testing production level of the clones.
Make dilutions with unknowns (1:10 in first well and dilute 2 fold across plate). Hu-IgG standards start at 1000 ng/mL and dilute 2 fold across plate. Leave a few wells for blanks: 1X PBST 5% chicken serum which is used for dilutions, 100 gL/well. Incubate at room temperature for 1 hour. Fusion screens and subclones are generally tested diluted 1:2 in blocking buffer. A positive control may also be used when screening fusions and subclones.
Repeat wash step #3.
6. Dilute secondary antibody HRP(horse radish peroxidase)anti-hu IgG-Fc reagent 1:5000 or HRP-anti-hu-K in 1XPBST chicken serum, add 100 IL/well. Incubate 1 hour at room temperature.
7. Repeat wash step#3. (2X) 61 Ci 8. Develop plate using 10 ml citrate phosphate buffer pH4.0, ap PIL ABTS, 8 LL H 2 0 2 per plate.
9. Incubate 30 minutes to 1 hour at room temperature. Read 00 oO CN plate at OD415 nm-490 nm.
Solutions: 1X PBST= 1xPBS 0.05% 00 Citrate phosphate buffer 21 gm/L citric acid, 14.2 gm/L 0disodium hydrogen phosphate (anhydrous); ABTS= 27.8 mg/mL 2,2'-azino-bis (3-ethylbenz- 0 10 thiazoline-6-sulfonic acid) diammonium salt in citrate buffer, freeze 1 mL aliquots.
Plate 96 well assay plate.
A positive ELISA signal was detected in the wells corresponding to hybridomas 1H3, 15H12 and 19D12, demonstrating that these hybridomas produced human IgG antibodies.
2.4.6. Hybridoma supernatants corresponding to human IgGK positive wells were then screened on soluble IGFR1 coated ELISA plates according to the following procedure: 1. Coat plate overnight with IGFR1 (1.0 pg/mL) in 1X PBS, pL/well. Store in refrigerator. Five milliliters needed for coating plate.
2. Empty plate and block plate in 1X PBST 5% chicken serum for 1 hour at room temperature (100 g.L/well).
3. Empty plate and wash manually with wash bottle (3X) or plate washer (3X) using 1X PBST. If wash bottle used, drain plates on paper towels.
4. Use blocking buffer as diluent. Test sera, beginning at 1:50 dilution in the top row of the plate and dilute 2 fold/row down the plate Incubate at room temperature 1 hour. For subclone screening, a 1:1 dilution of culture supernatant in blocking buffer is used as starting material.
Repeat wash step #3.
6. Dilute secondary HRP-anti-hu IgG-Fc specific and/or HRPanti-hu-K reagent 1:2500-5000 in 1X PBST chicken serum, add 100 pL/well. Incubate 1 hour at room temperature.
62 Ci 7. Repeat wash step#3. (2X) 8. Develop plate using 10 mL citrate-phosphate buffer T 80 I.L ABTS, 8 IL H 2 0 2 per plate.
00 C9. Incubate 30 minutes to 1 hour at room temperature. Read plate at OD 415 nm-490 nm. Consider twice above background titer limit.
N In these assays, hybridomas 15H12 and 19D12 produced a positive ELISA 00 signal. These data demonstrate that the hybridomas produced antibodies which can Sbind to soluble IGFR1.
SAntigen positive hybridomas were then transferred to 24 well plates, and 0 10 eventually to tissue culture flasks. IGFR1 specific hybridomas were subcloned by limiting dilution to assure monoclonality. Antigen positive hybridomas were preserved at several stages in the development process by freezing cells in Origen DMSO freeze medium (Fischer Scientific; Suwanee, GA).
2.4.7. Antibody isotypes were determined according to the following procedure: 1. Coat plate overnight in refrigerator at lug/ml soluble IGFR1 in 1X PBS, 50gL/well. Empty plate.
2. Add 1X PBST 5% chicken serum for 1 hour at room temperature. (100 PL/well). Empty plate.
3. Use blocking buffer as a diluent, add supematant or purified material to be tested in 1 well per secondary antibody to be tested- Incubate for 90 minutes at room temperature. Empty plate.
4. Empty plate and wash manually with wash bottle (3X) or plate washer (3X) using 1X PBST. If wash bottle used, drain plates on paper towels.
Using blocking buffer as a diluent, add secondary antibodies: HRP-anti-hu-gamma; HRP-anti-hu kappa; HRP-anti-human IgGI; or HRP-anti-human IgG3 diluted 1:1000. Incubate for 45 minutes at room temperature. Empty plate.
63 C 6. Repeat wash step #4 (3X).
7. Develop plate using 10 mL citrate-phosphate buffer L ABTS, 8 gL H 2 0 2 per plate.
00 cO 8. Incubate 30 minutes to 1 hour at room temperature. Read plate at OD415nm-490nm.
O The data from these assays is shown, below, in Table 4.
r- 00 cO 0 Table 4. Isotype ELISA results'.
pY K y1 y3 C- 1 2 3 4 5 6 7 I I I I I I II clone 1.903 1.003 0.064 0.813 115H12 *Each number represents the magnitude of the ELISA signal observed for each secondary antibody.
These data demonstrate that antibody 15H12 is an IgG3K antibody.
2.4.8. Hybridoma supernatants (1H3, 15H12 and 19D12) and MAB391 were also tested, in a fixed cell ELISA assay, for the ability to directly bind cells expressing IGFR1. In the assay, MCF-7 cells or HEK293 cells transfected with IGFR1 DNA were used. The assays were performed as follows: 1. Add 50 pIg/well of a 20 Lg/mL solution of Poly-L-lysine in 1 X PBS to each well of a 96 well plate and incubate for 30 minutes at room temperature or overnight at 4 0 C. Empty plate to remove Poly-L-lysine from the wells and allow to dry at room temperature until use.
2. Wash live cells three times with 1X PBS by centrifugation (1000 RPM/5 minutes). Adjust final cell concentration to 2 X 106 cells per well in 1X PBS. Add 50pL per well of this cell suspension.
3. Spin cells 5 minutes at 2000 RPM. Empty buffer.
4. Add 50pL/well of 0.5% ice cold glutaraldehyde in 1X PBS.
Let sit for 15 minutes at room temperature. Empty plate.
Add 1X PBST 5% chicken serum and incubate for 1 hour at room temperature (100 iL/well). Empty plate.
64 C6. Wash plate gently using 1X PBST To avoid cell loss, this step should be done manually in a container avoiding any plate T washers.
00 0 7. Using blocking buffer as a diluent, test culture supernatant by adding 100pg of a 1:1 dilution. Incubate 1 hour at room temperature.
O 8. Repeat step 6 (3X).
00 9. Dilute secondary HRP anti-hu IgG-Fc specific and/or HRP O anti-hu-, reagent 1:2500-5000 in 1X PBST 5% chicken serum, add 17- 100 pL/well. Incubate 1 hour at room temperature.
0 10 10. Repeat step 6 (3X).
11. Develop plate using 10 ml citrate-phosphate buffer L, ABTS, 8 pL- H 2 0 2 per plate.
12. Incubate 15-20 minutes at room temperature. Read plate at OD 415nm-490nm.
The results from these assays demonstrated that hybridomas 1H3, 15H12 and 19D12 produced an immunoglobulin which binds to HEK293 cells expressing IGFR1 and that hybridomas 1H3, 15H12 and 19D12 produced an immunoglobulin which binds to MCF-7 cells which express endogenous IGFR1. Additionally, the results demonstrated that MAB391 bound to IGFR1 expressing HEK293 cells and to MCF-7 cells.
2.4.9. The ability of hybridoma supernatants (1H3, 15H12 and 19D12) to block binding of IGF1 to IGFR1 was evaluated by measuring 1) staining intensity of the supematant on IGFR1 expressing HEK293 cells and on MCF7 cells and 2) the ability of the supernatants to block binding of IGF1-biotin to IGFR1 expressing cells. Initially, biotinylated IGF1 was titrated on IGFR1 expressing HEK293 cells in order to establish the proper concentration to evaluate blocking of IGF1 binding to its receptor by the antibodies of the present invention. This was done by the following procedure: 1. IGFR1 expressing HEK293 cells are harvested from a flask by slapping the flask to loosen the cells which were pipeted in to a conical tube. The cells are then centrifuged at 300 X g for 5 minutes to pellet the cells. The medium is then aspirated.
C 2. The cells are washed in 10-20 mL PBS containing 0.02% a[ sodium azide and resuspended in the same buffer at approximately X 108 cells/mL (106 cells). The cells are aliquoted, 200pL/well into a 00 C 96 well microtiter plate in the same buffer at 4 0 C. The cells are pelleted and the supernatant is aspirated.
I 3. The cells are stained by adding 50L/well serially diluted r-jr 0O IGFl-biotin in the same buffer, starting at a 1:5 dilution followed by 4- Sfold serial dilutions. The plate is tapped or gently vortexed to ensure an even suspension of cells are suspended. The cells are then incubated for 30 minutes at 4. The cells are washed 3X by adding 150pL buffer for the first wash and then pelleted. The supernatant is aspirated and 200pL buffer is added. Again, the cells are pelleted and the supernatant is aspirated; this wash step is repeated once more. Streptavidin-PE (streptavidin-R-Phycoerythrin) is added and the cells are incubated for minutes at 4 0
C.
The cells are washed once in PBS containing 2% FBS and 0.02% azide and resuspended in the same buffer except containing also 50 pg/mL propidium iodide to exclude dead cells.
6. The cells are analyzed by FACS.
The blocking assays were performed as follows: 1. Harvest MCF7 cells or HEK293/IGFR1 cells from a tissue culture flask by slapping the flask sides to loosen the cells. Pipet the cells into a conical tube. Centrifuge the tube for 5 minutes at 300 X g to pellet the cells. Aspirate the medium.
2. Wash the cells in 10-20 mL PBS containing 2% FBS and 0.02% sodium azide (PFA), and resuspend in the same buffer at approximately 2.5 X 106 1 X 106). Aliquot 200 uL/well into a 96 well microtiter plate in the same buffer at 40C. Pellet the cells and aspirate the buffer.
3. Stain the cells with each IGFR1 hybridoma supernatant by adding 100 UL/well, including a medium (negative) control, and MAB391 as a positive control. Tap the plate to ensure even suspension of the cells. Incubate 30-60 minutes at 4 0
C.
66 CI 4. Wash the cells 3 times in PFA by adding 100p~L buffer for the d) first wash, pellet, aspirate, resuspend in 2001L buffer, pellet, aspirate, resuspend again in 200pL buffer, divide each sample into two wells C and pellet.
5. To one set of wells, add anti-human IgG-FITC diluted 1:100 Sin PFA (para-formaldehyde) to the supernatant stained samples and oO Sthe medium control, and anti-mouse IgG-FITC at 1:200 to the MAB391 stained samples, again ensuring even dispersal of the cells (staining assay). Incubate for 30 minutes at 6. To the second set of wells, add IGF1-biotin diluted 1:500 in PBS containing 0.02% azide (no FBS) and incubate for 30 minutes at Wash the cells 3 times as described in step 4 (but without dividing the sample). Stain these cells by adding streptavidin-PE (streptavidin-R-Phycoerythrin) in PFA (blocking assay). Incubate for minutes at 4 0
C.
7. Wash all the samples once in PFA, and resuspend in the same buffer except containing also 50pg/mL propidium iodide to exclude dead cells.
8. Analyze by FACS analysis.
The results from these blocking assays demonstrated that the supematants from hybridomas 1H3, 15H12 and 19D12 block binding of biotinylated IGF1 to IGFR1, stain MCF7 cells which express endogenous IGFR1 and stain HEK293 cells expressing IGFR1.
2.4.10. The ability of purified antibodies 1 H3 and 15H12 to block binding of biotinylated IGF1 to IGFR1 in an ELISA assay and of antibodies 1H3, 15H12 and 19D12 to block binding of biotinylated MAB391 to IGFR1 in an ELISA assay was also evaluated according to the following procedure: 1. Coat plate overnight in a refrigerator with 1 gg/mL soluble IGFR1 in 1 X 2. Add 1 X PBST 5% chicken serum for 1 hour at room temperature- 100JL/well. Empty plate.
3. Wash plate 3X with wash buffer (1X PBS 0.05% tween- Slap plate dry.
C 67 S4. 2 gg/mL 1H3, 15H12 or 19D12 or positive or negative control Dantibodies are diluted in blocking buffer across the plate. The plates are incubated at room temperature for 1 hour.
O
C 5. Wash plates 3X in wash buffer.
6. Biotin-IGF1 or Biotin-MAB391 is added-50pL/well- and
D
incubated for 30 minutes at room temperature.
oO 0 7. Wash plate 3X 8. Add 100pL/well of streptavidin labeled alkaline phosphatase or horse radish peroxidase, incubate for 30 minutes at room temperature.
9. Wash plate 3X. Develop with appropriate reagent depending on the label used.
Read after 10-15 minutes.
MAB391 was biotinylated according to the following procedure: 1. Prepare MAB391 in PBS buffer (dialyze or use desalting column to remove unwanted buffers such as Tris or glycine).
2. Prepare a fresh stock solution of Sulfo-NHS-LC-biotin solution just before use. Add 2.0 mg of Sulfo-NHS-LC-biotin to 200 OL distilled water. Add this reagent to MAB391 at a 12-fold molar excess if working with a 10 mg/mL solution of MAB391, or a 20-fold molar excess when working with a dilute preparation of MAB391 (2 mg/mL).
3. Calculation: mmoles MAB391 mg protein/150,000 mmoles X 12 or 20 mmoles biotin reagent to add mmoles biotin to add X 556 mg biotin reagent to add For 1 mg/mL: 1/150000 6.6 X X 6.6 X 10.6 mmoles 1/32 X 10 4 NHS-LC-biotin 1.32 X 10- 4 X 556 0.073 mg sulfo NHS-LC-biotin From the stock NHS-LC-biotin solution, use 10 i.L (100 Rg) of solution per mg IgG for 1 or 2 mg.
4. Incubate for 2 hours on ice or for 30 minutes at room temperature. Dialyze against PBS or use desalting column to remove unreacted biotin reagent. Store at 40C in PBS 0.1% sodium azide.
In general, 3-5 biotins should be added to each IgG molecule labeled.
68
O
O The results from these blocking assays demonstrated that antibodies 1 H3 and .D 15H12 blocks binding of biotinylated IGF1 to slGFR1 and that antibodies 1H3, 15H12 and 19D12 block biotinylated MAB391 binding to slGFRI.
00 2.4.11. Binding between IGFR1 and the 1H3, 15H12 and 19D12 antibodies was evaluated in a BIAcore/surface plasmon resonance NO assay according to the following procedure: 0 1. IGFR1 is immobilized on a CM-5 chip by amine coupling, to a level of 350.4 response units on flow cells. The concentration of IGFR1 which is used to immobilize is 2.5 pg/mL in sodium acetate buffer and the protein is immobilized at pH c 2. Antibodies 1H3, 15H12 and 19D12 are purified from hybridoma supematants over a Protein-A or Protein-G column and tested for purity by SDS-PAGE analysis Tris-Glycine).
3. The antibodies are made to flow over the IGFR1 surface prepared above.
4. The concentration range of antibodies used is 4, 2, 1, and 0.25 pg/mL. A blank is also used for background substitution.
Samples are prepared in HBS buffer.
Injection time (association phase) is 10 minutes, at a flow rate of 20pL/minute, dissociation time (dissociation phase) is 1 hour at the same flow rate.
6. The assays are run at both 25 0 C and 370C. All experiments are done in duplicate.
7. Data analysis is carried out using Bia-Evaluation software v.3.0.2 (Biacore, Inc; Piscataway, NJ).
8. All experiments are carried out using a Biacore 3000 surface plasmon resonance instrument (Biacore, Inc; Piscataway, NJ).
The results for these assays demonstrated that antibodies 15H12 and 19D12 associate with IGFR1 at 250C and at 37°C and that antibody 1H3 associates with IGFR1 at 250C. The data from these experiments were also used to calculate the affinity and rate constants of 1H3, 15H12 and 19D12 binding to IGFR1 (see Table below).
0
N
oo 00 O
(N
O.
69 Table 5. Affinity and rate constants of antibodies 1H3, 15H12 and 19D12 with IGFR1.
Temp. Antibody Sample Assoc. Dissoc. k, kon Ko Half-life size time (min.) time (mln.) (min.) 250C 15H12 2 10 60 5.0 X 2.24 X10" 4.48 X 515.73 5 _10 1 1 250C 19D12 2 10 60 4.0 X 2.65 X 10-" 5.92 X 435.94 ___10 5 10 11 250C 1 H3 2 10 60 0.7 X 6.50 X 10-" 86 X 177.73 s 10- 1 1 37°C 15H12 2 10 60 7.2 X 4.01 X 10-' 5.57 X 288.09 10 5 10' 1 1 37°C 19D12 2 10 60 6.8 X 4.93 X 10-" 7.22 X 234.33
S
10 11 Calculated as Half life In(2/kof) EXAMPLE 2: Cell Based Receptor Binding Assay.
A cell based receptor binding assay was used to determine if antibodies 1 H3, 15H12 and 19D12 competed with IGF1 for binding to IGFR1.
In the assays, 96 well filter plates (1.2 uim pore) were pre-wet with 0.5% bovine serum albumin (BSA)/PBS for 2 hours at 40C. The buffer was then removed with a vacuum manifold. Various concentrations of 6X control or test antibody (1H3, 15H12 or 19D12) were added to the wells (25 IL). The 1 2 5 1]-IGF-1 ligand was then added to the wells at a final concentration of 0.375 nM in BSA/PBS. Cells were harvested with cell dissociation solution, counted with trypan blue, and resuspended in BSA/PBS to a cell number of 1-3 X 10 5 /ml. One hundred jl1 of cells (10,000-30,000) were added to each well. The plate was shaken at 4°C for 1 hour. The plate was then aspirated and washed three times with ice cold PBS using a vacuum manifold.
The filters were punched out and counted on a gamma counter. Data were analyzed for competitive binding.
The results of these experiments indicated that 1H3, 15H12 and 19D12 were capable of competing with IGF-I for binding to IGFR1.
EXAMPLE 3: IGFR1 Autophosphorylation Assay.
The ability of 1H3, 15H12 and 19D12 to inhibit IGFR1 autophosphorylation was also determined.
Antibodies (1H3, 15H12 or 19D12) were added to cells bearing IGFR1 for various lengths of times. Cells were then stimulated with 10 ng/ml IGF-I for 5 min at
C
370C. Cells were washed twice with cold PBS containing 0.1mM sodium vanadate d§ and lysed in lysis buffer (50 mM HEPES, pH7.4, 150 mM NaCI, 10% glycerol, 1% Triton X-100, 1.5 mM MgCI 2 protease inhibitors and 2 mM sodium vanadate).
oO C' Lysates were incubated on ice for 30 min and then centrifuged at 13,000 RPM for min at 40C. Protein concentrations of the lysates were measured by a Coomassie colorimetric assay, and subjected to immunoprecipitation and Western blot analysis.
0 The results of these assays indicated that antibodies 1H3, 15H12 and 19D12 Sinhibited IGFR1 autophosphorylation with an ICso of 0.10 nM.
0 10 EXAMPLE 4: Anchorage-Independent Growth (Soft Agar) Assay.
The ability of an anti-IGFR1 antibodies 1H3, 15H12, 19D12 and MAB391 to inhibit anchorage-independent growth of various cells, including human breast cancer cell line MCF7, human colorectal cancer cell HT29 and human prostatic cancer cell DU145, was evaluated.
In these experiments, three milliliters of 0.6% agarose in complete MEM medium were added to each well of 6 well tissue culture plates and allowed to solidify (bottom layer). One hundred microliters of antibody 1H3, 15H12, 19D12 or MAB391 (discussed above), at various concentrations, was added to culture tubes. Cells were harvested. Aliquots of the cells (15,000 cells) were added to the culture tubes containing the antibody and incubated at room temperature for 10-15 minutes. Three milliliters of a 0.35% agarose/complete minimal essential media (MEM) layer (top layer) were added to the antibody/cell mixture and then plated onto the solidified bottom layer. The top layer was allowed to solidify. The plates were then incubated for three weeks. MTT (3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl-2H-Tetrazolium Bromide) was added to the wells and incubated for 1-2 hours. The plates were scanned and the colonies counted and analyzed using a customized colony counter application program.
The results of these experiments demonstrated that an anti-IGFR1 antibody can inhibit anchorage-independent growth of all three malignant cell lines tested.
EXAMPLE 5: Cloning of the Variable Regions of an Antibody from Hybridomas.
Nucleic acids encoding the 1H3, 15H12 and 19D12 variable regions were obtained from hybridomas according to the following procedure.
71 NMessenger RNA (mRNA) from 2x10 6 hybridoma cells was prepared by using a Micro-Fast Track kit (Invitrogen; Carlsbad, CA) Cellular DNA (cDNA) encoding the variable region was prepared according the procedure described in "cDNA Cycle" kit 00 0 (Invitrogen; Carlsbad, CA).
The antibody variable regions were PCR amplified using the cDNA as a 0 template using 5'RACE (Clotech; Palo Alto, CA) technology. The following 3'primer 00 sequence was used to amplify the heavy chain: 03' (SEQ ID NO: 22) and following 3'primer sequence was used to amplify the light chain: 5'-CGGGAAGATGAAGACAGATG-3' (SEQ ID NO:23). Additionally, 0 10 PCR primers (Clotech; Palo Alto, CA) were used in each amplification.
The PCR reaction mixture included 2.5 units of Pfu I polymerase in its appropriate buffer (Stratagene; La Joola, CA), 0.2 mM of each dNTP, 750 nM of each and 3' primer and cDNA template. Total reaction volume was 50 u1. The following PCR cycling program was performed using a thermocycler: 1X 94 0 C, 2 min.
94 0 C, 45 sec.
0 C, 45 sec. Minus 1°C per cycle 72 0 C, 1 min.
25X 94 0 C, 45 sec.
0 C, 45 sec.
72 0 C, 1 min.
1X 72 0 C, 15 min.
The resulting PCR amplification product was inserted into the Zero Blunt TOPO PCR cloning vector (Invitrogen; Carlsbad, CA). The identity of the insert was verified by restriction enzyme analysis and then the nucleotide sequence of the insert was obtained by sequencing.
Example 6: Recombinant Expression of Antibody Chains In this example, nucleic acids encoding various anti-IGFR1 antibody chains of the present invention were used to transfect a dhfr-mammalian cell line (CHO- DXB11) wherein the chains were expressed. Transient transfections were carried out by cotransfection of the cell line with various combinations of one heavy (yl or y4) and one light chain plasmid, selected from plasmids 1-11, listed below. Construction of stable cell lines was performed by transfection by a single plasmid, either 12 or O 13, listed below, as follows: The nucleic acids were located in a single plasmid N and were operably linked to cytomegalovirus (CMV) promoters. The plasmids also contained DHFR cDNA operably linked to a mouse mammary tumor virus long terminal repeat (MMTV-LTR) which was used for plasmid amplification. The 00 C plasmid further included the hygromycin B gene operably linked to the TK promoter for selection in mammalian cells.
Below is a description of the promoter-expression cassette in the13 Splasmids which were constructed. The indicated plasmids (2-4 and 8-11) were N. 10 deposited, under the Budapest Treaty, on May 21, 2003 with the American Type SCulture Collection (ATCC); 10801 University Boulevard; Manassas, Virginia S 20110-2209 under the indicated name and accession number: CMV promoter-15H12/19D12 HC (y4) Insert Sequence: SEQ ID NO: 3; CMV promoter-15H12/19D12 HCA (y4)- Deposit name: "15H12/19D12 HCA (y4)" ATCC accession No.: PTA-5214 Insert Sequence: SEQ ID NO: 44; CMV promoter-15H12/19D12 HCB (y4)- Deposit name: "15H12/19D12 HCB (y4)" ATCC accession No.: PTA-5215 Insert Sequence: SEQ ID NO: I11; CMV promoter-15H12/19D12 HCA (yl)- Deposit name: "15H12/19D12 HCA ATCC accession No.:_PTA-5216 Insert Sequence: SEQ ID NO: 44; CMV promoter-15H12/19D12 LC (K) Insert Sequence: SEQ ID NO: 1; CMV promoter-15H12/19D12 LCA (K) Insert Sequence: SEQ ID NO: CMV promoter-15H12/19D12 LCB (K) Insert Sequence: SEQ ID NO: 42; CMV promoter-15H12/19D12 LCC SDeposit name: "15H12/19D12 LCC ATCC accession No.: PTA-5217 Insert Sequence: SEQ ID NO: 71; 9 CMV promoter-15H12/19D12 LCD 00 5 Deposit name: "15H12/19D12 LCD ATCC accession No.: PTA-5218 SInsert Sequence: SEQ ID NO: 73; 0 (10) CMV promoter-15H12/19D12 LCE
O
Deposit name: "15H12/19D12 LCE ATCC accession No.: PTA-5219 Insert Sequence: SEQ ID NO: (11) CMV promoter-15H12/19D12 LCF Deposit name: "15H12/19D12 LCF ATCC accession No.: PTA-5220 Insert Sequence: SEQ ID NO: 77; (12) CMV promoter-15HI2/19D12 HC (y4) and CMV promoter-15H12/19D12 LC (13) CMV promoter-15H12/19D12 HCA (yl) and CMV promoter-15H12/19D12 LC (K) All restrictions on access to the plasmids deposited in ATCC will be removed upon grant of a patent.
The 3' end of each cassette was linked to a beta-globin poly A signal. The variable chains which were expressed were linked to the constant region indicated in parentheses yl, y4 or Analysis of the transfected cell lines containing each plasmid indicated that the corresponding antibody chain polypeptides were expressed (amino acid sequences of the expression products not confirmed).
Each of the above-referenced plasmids constitutes part of the present invention. Further, the nucleic acid located within each expression cassette, along with the immunoglobulin variable region therein, along with the mature, processed version thereof lacking the signal sequence), particularly, SEQ ID NO: 44, mature HCA (nucleotides 58-411 of SEQ ID NO: 44), SEQ ID NO: 111, mature HCB (nucleotides 58-411 of SEQ ID NO: 111), SEQ ID NO: 71, mature LCC (nucleotides 58-384 of SEQ ID NO: 71), SEQ ID NO: 73, mature LCD (nucleotides 58-384 of SEQ ID NO: 73), SEQ ID NO: 75, mature LCE (nucleotides 58-384 of SEQ ID NO: 75), SEQ ID NO: 77 or mature LCF (nucleotides 58-384 of SEQ ID NO: 77), optionally including an immunoglobulin constant region, along with any polypeptide encoded by O any of the foregoing nucleic acids, including mature or unprocessed chains, optionally N including an immunoglobulin constant region, is a part of the present invention.
a Moreover, any antibody or antigen-binding fragment thereof comprising one of the encoded polypeptides is part of the present invention.
00 (3O C IThe present invention is not to be limited in scope by the specific embodiments oO 00described herein. Indeed, various modifications of the invention in addition to those Sdescribed herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall Swithin the scope of the appended claims.
Patents, patent applications, Genbank Accession Numbers and publications are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties.
In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia or any other country.

权利要求:
Claims (14)
[1] 1. A binding composition that specifically binds to IGFR1 comprising a member 00 C selected from the group consisting of: a) a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID ^g NO: 8, CDR-L2 defined by SEQ ID NO: 9 and CDR-L3 defined by SEQ ID NO: 00 b) a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID O NO: 31, CDR-L2 defined by SEQ ID NO: 32 and CDR-L3 defined by SEQ ID NO: 33; c) a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 14 or SEQ ID NO: 17, CDR-H2 defined by SEQ ID NO: 15 and CDR-H3 defined by SEQ ID NO: 16; and d) a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 37 or SEQ ID NO: 70, CDR-H2 defined by SEQ ID NO: 38 and CDR-H3 defined by SEQ ID NO: 39.
[2] 2. A binding composition of claim 1 that specifically binds to IGFR1 comprising a variable region selected from the group consisting of: amino acids 20-128 of SEQ ID NO: 2; amino acids 21-130 of SEQ ID NO: amino acids 20-128 of SEQ ID NO: 72; amino acids 20-128 of SEQ ID NO: 74. amino acids 20-137 of SEQ ID NO: 4; amino acids 20-140 of SEQ ID NO: 27; amino acids 20-137 of SEQ ID NO: amino acids 20-137 of SEQ ID NO: 112; amino acids 20-128 of SEQ ID NO: 76; and amino acids 20-128 of SEQ ID NO: 78.
[3] 3. A binding composition that specifically binds to IGFR1 comprising a member selected from the group consisting of: a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 2 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 4; 76 C a light chain variable region comprising amino acids 21-130 of SEQ ID NO: d and a heavy chain variable region comprising amino acids 20-140 of SEQ ID NO: 27; a light variable region comprising amino acids 20-128 of SEQ ID NO: 72 and a oO N heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: a light variable region comprising amino acids 20-128 of SEQ ID NO: 74 and a I heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 0 0 a light variable region comprising amino acids 20-128 of SEQ ID NO: 76 and a Oheavy chain variable region comprising amino acids 20-137 of SEQ ID NO: S(f) a light variable region comprising amino acids 20-128 of SEQ ID NO: 78 and a 0 10 heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: a light variable region comprising amino acids 20-128 of SEQ ID NO: 72 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112; a light variable region comprising amino acids 20-128 of SEQ ID NO: 74 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112; a light variable region comprising amino acids 20-128 of SEQ ID NO: 76 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112; and a light variable region comprising amino acids 20-128 of SEQ ID NO: 78 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112.
[4] 4. A pharmaceutical composition comprising a composition of claim 1 and a pharmaceutically acceptable carrier. An isolated nucleic acid encoding a polypeptide selected from the group consisting of: amino acids 20-128 of SEQ ID NO: 2; amino acids 21-130 of SEQ ID NO: amino acids 20-128 of SEQ ID NO: 72; amino acids 20-128 of SEQ ID NO: 74; amino acids 20-137 of SEQ ID NO: 4; amino acids 20-140 of SEQ ID NO: 27; amino acids 20-137 of SEQ ID NO: amino acids 20-137 of SEQ ID NO: 112; amino acids 20-128 of SEQ ID NO: 76; and amino acids 20-128 of SEQ ID NO: 78. 77 S6. A nucleic acid of claim 5 selected from the group consisting of: S(a) nucleotides 58-384 of SEQ ID NO: 1; 00 N nucleotides 61-390 of SEQ ID NO: 24; nucleotides 58-384 of SEQ ID NO: 71; IN nucleotides 58-384 of SEQ ID NO: 73. 0 0 nucleotides 58-411 of SEQ ID NO: 3; O(f) nucleotides 58-420 of SEQ ID NO: 26; nucleotides 58-411 of SEQ ID NO: 44; nucleotides 58-411 of SEQ ID NO: 111; nucleotides 58-384 of SEQ ID NO: 75; and nucleotides 58-384 of SEQ ID NO: 77.
[5] 7. A recombinant vector comprising a nucleic acid of claim
[6] 8. A host cell comprising a vector of claim 7.
[7] 9. A method for producing a polypeptide comprising culturing the host cell of claim 8 under conditions in which the polypeptide is produced. A method for treating or preventing a medical condition in a subject, which medical condition is mediated by elevated expression or activity of Insulin-like Growth Factor Receptor-I, comprising administering a binding composition of claim 1 to the subject.
[8] 11. The method of claim 10 wherein the medical condition is selected from the group consisting of acromegaly, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumors, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation. 78 O S12. The method of claim 10 wherein the binding composition is administered to the 1C subject by a parenteral route. 00
[9] 13. The method of claim 10 wherein the binding composition is administered to the subject in association with an additional, anti-cancer, therapeutic agent or anti-cancer, o therapeutic procedure. oO 00 S14. A method for treating or preventing a medical condition in a subject, which medical condition is mediated by elevated expression or activity of Insulin-like Growth S 10 Factor Receptor-I, comprising administering a binding composition that specifically binds to IGFR1 comprising a member selected from the group consisting of: a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 8, CDR-L2 defined by SEQ ID NO: 9 and CDR-L3 defined by SEQ ID NO: a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 31, CDR-L2 defined by SEQ ID NO: 32 and CDR-L3 defined by SEQ ID NO: 33; a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 14 or SEQ ID NO: 17, CDR-H2 defined by SEQ ID NO: 15 and CDR-H3 defined by SEQ ID NO: 16; and a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 37 or SEQ ID NO: 70, CDR-H2 defined by SEQ ID NO: 38 and CDR-H3 defined by SEQ ID NO: 39; to the subject.
[10] 15. The method of claim 14 wherein the medical condition is selected from the group consisting of acromegaly, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumors, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation.
[11] 16. A method for producing a fully human, monoclonal antibody which specifically binds to IGFR1 comprising the steps of: 79 immunizing a transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene with an LC IGFR1 antigenic polypeptide, such that the antibody is produced by a B cell of 00 the animal; (ii) isolating said B cell of the animal; I (iii) fusing the B cell with a myeloma cell to form an immortal hybridoma cell that 00 secretes said antibody; and (iv) isolating the antibody from the hybridoma cell.
[12] 17. The method of claim 16 wherein the antigenic polypeptide is amino acids 30-902 Sof SEQ ID NO: 19.
[13] 18. A binding composition which specifically binds to human IGFR1 comprising a property selected from the group consisting of: Binds to IGFR1 with a Kd of about 86 X 10 11 or less; Has an off rate (Kof) for IGFR1 of about 6.50 X 10- 5 or smaller; Has an on rate (Kon) for IGFR1 of about 0.7 X 10 5 or greater; Competes with IGF1 for binding to IGFR1; Inhibits autophosphorylation of IGFR1; and Inhibits anchorage-independent growth of a cell expressing IGFR1.
[14] 19. A binding composition of claim 18 comprising all of said properties. A binding composition of claim 18 comprising a member selected from the group consisting of: a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 8, CDR-L2 defined by SEQ ID NO: 9 and CDR-L3 defined by SEQ ID NO: a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 31, CDR-L2 defined by SEQ ID NO: 32 and CDR-L3 defined by SEQ ID NO: 33; a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 14 or SEQ ID NO: 17, CDR-H2 defined by SEQ ID NO: 15 and CDR-H3 defined by SEQ ID NO: 16; and CN a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 37 or SEQ ID NO: 70, CDR-H2 defined by SEQ ID NO: 38 and CDR-H3 defined by SEQ ID NO: 39. 00 00 (Nq l'- 0~ SEQUENCE LISTING <110> Schering Corp. Wang, Yan Pachter, Jonathan A Hailey, Judith Greenberg, Robert Leonard, Presta Brams, Peter Feingersh, Diane Williams, Denise Srinivasan, Mohan <120> NEUTRALIZING HUMAN ANTI-IGFR ANTIBODY <130> OC01533-K-WI <140> <141> <150> 60/383,459 <151> May 24, 2002 <150> 60/393,214 <151> July 2, 2002 <150> 60/436,254 <151> December 23, 2002 <160> 120 <170> Patentln version 3.1 <210> 1 00 <211> 384 <212> DNA <213> Homo sapiens r- 00 <220> <221> CDS <222> 1..384 <223> <220> <221> sigpeptide <222> 1..57 <223> <400> 1 atg tcg cca tca caa ctc att ggg ttt ctg ctg ctc tgg gtt cca gcc 48 Met Ser Pro Ser Gin Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala 1 5 10 tcc agg ggt gaa att gtg ctg act cag gtt cca gac ttt cag tct gtg 96 Ser Arg Gly Giu Ile Val Leu Thr Gin Val Pro Asp Phe Gin Ser Val 25 act cca aag gag aaa gtc acc atc acc tgc cgg gcc agt cag agc att 144 Thr Pro Lys Giu Lys Val Thr Ile Thr Cys Arg Ala Ser Gin Ser Ile 40 ggt agt agc tta cac tgg tac cag cag aaa cca gat cag tct cca aag 192 Gly Ser Ser Leu His Trp Tyr Gin Gln*Lys Pro Asp Gin Ser Pro Lys 55 ctc ctc atc aag tat gct tcc cag tcc ctc tca ggg gtc ccc tcg agg 240 Leu Leu Ile Lys Tyr Ala Ser Gin Ser Leu Ser Gly Vai Pro Ser Arg 70 75 ttc aqt ggc agt gga tct ggg aca gat ttc acc ctc acc atc aat agc 288 Phe Ser Giy Ser Gly Ser Giy Thr Asp Phe Thr Leu Thr Ile Asn Ser 90 ctg gaa gct gaa gat gct gca gcg tat tac tqt cat cag agt agt cgt 336 Leu Giu Ala Giu Asp Ala Ala Ala Tyr Tyr Cys His Gin Ser Ser Arg 2/62 100 105 tta cct cac Leu Pro His 115 <210> 2 <211> 128 act ttc ggc gga ggg Thr Phe Gly Gly Giy 120 acc aag gtg gag atc aaa cga act Thr Lys Vai Glu Ile Lys Arg Thr 125 <212> PRT <213> Homo sapiens <400> 2 Ser Pro Ser Leu Ile Gly *Phe Leu Leu Trp Val Pro Ala Ser Arg Gly Thr Pro Lys Ile Vai Leu Thr Val Pro Asp Phe Gin Ser Val Gin Ser Ile Giu Lys Val Thr Thr Cys Arg Ala Giy Ser Ser Leu His Trp Gin Gin Lys Pro Asp Gin Ser Pro Lys Giy Val Pro Ser Arg Leu Ile Lys Tyr Ser Gin Ser Leu Phe Ser Gly Ser Ser Giy Thr Asp Thr Leu Thr Ile Asn Ser Leu Giu Ala Leu Pro His 115 Asp Ala Ala Ala Tyr Cys His Gin Ser Ser Arq 110 Thr Phe Gly Gly Thr Lys Val Glu Ile Lys Arg Thr <210> 3 <211> 411 <212> DNA <213> Homoa sapiens <220> <221> <222> <223> <220> <221> <222> <223> C DS 1. .411 si gpeptide 1. .57 <400> 3 atg gag ttt Met Glu Phe ggg ctg agc tgg gtt ttc ctt gtt gct ata tta aaa Gly Leu Ser Trp Vai Phe Leu Val Ala Ile Leu Lys 10 gt c Val cct Pro agt Ser gag Gi u t cc S er ttg Leu tac Tyr cag Gin ggg Gly agc Ser tgg Trp gtg Val1 tat Tyr tgt Cys t gt Cys ggg Giy t tt Phe ata Ile a ag Lys ctt Leu gca Aia 115 gag Giu t cc S er gct Aila tca Ser ggc Giy caa Gin 100 aga Ar g gt t Vali ct g Leu atg Met gtt Val1 cga Arg at g Met ct g Leu ca g Gin aga Arg ca c His att Ile 70 ttc Phe aac As n ggg Gly ctg Leu ct C Leu t gg Trp 55 ga t Asp a cc Th r agc S er aac Asn gt g Val1 tcc Ser 40 gtt Val act Thr at c Ile ctg Leu ttc Phe 120 cag Gin 25 tgt Cys cgc Arg cgt Arg tcc Ser aga Arg 105 tac Tyr tct Ser gca Al a cag Gin ggt Gi y aga Arg 90 gcc Ala tac Tyr ggg Gly gcc Ala gct Ala gcc Ala 75 gac Asp gag Glu gga Gly t ct S er cca Pro aca Thr aat Asn gac Asp ggc Giy gga Gly gga Gly tac Tyr gcc Ala atg Met ttg Leu ttc Phe aaa Lys tat Tyr aag Lys gct Ala 110 gta Val a cc Thr ggt Gi y gca Ala aac Asn gtg Val ggt Gi y cat His ttc Phe ctg Leu ga c Asp t cc Ser tat Tyr ggt atg gac gtc tgg ggc Gly Met Asp Vai Trp Gly 125 caa ggg acc acg Gin Giy Thr Thr 130 acc gtc tcc tca Thr Vai Ser Ser 135 <210> 4 <211> 137 <212> PRT <213> Homo sapiens <400> 4 Met Giu Phe Gly Ser Trp Val. Phe Leu Val Ala Ile Leu Lys Gly Val Gin Cys Pro Gly Gly Glu Val Gin Leu Val Ser Leu Arg Leu Ser Gin 25 Ser Giy Giy Gly Leu Val His Phe Thr Phe Cys Ala Ala Ser Ser Ser Phe Ala Met His Trp Val Arg Gin Ala Gly Lys Gly Leu Trp Ile Ser Val Asp Thr Arq Gly Ala Thr Tyr Tyr Ala Ser Val Lys Gly Phe Thr Ile Ser Asp Asn Ala Lys Asn Ser Leu Tyr Leu Tyr Cys Ala 115 Gin 100 Met Asn Ser Leu Ala Giu Asp Met Ala Val Tyr 110 Vai Trp Gly Arg Leu Gly Asn Tyr Tyr Gly Met Gin Gly 130 Thr Thr Val Thr Val Ser Ser 135 <210> <211> 33 <212> DNA <213> Homo sapiens <400> cgggccagtc agagcattgq taqtagctta cac 33 <210> <211> <212> <213> 6 21 DNA Homo sapiens <400> 6 tatgcttccc agtccctctc a <210> <211> <212> <213> 7 27 DNA Homo sapiens <400> 7 catcagagta 24 gtcgtttacc tcacact <210> <211> <212> <213> 8 11 PRT Homo sapiens <400> 8 Arg Ala Ser 1 Gin Ser Ile Gly Ser Ser Leu His 5 <210> <211> <212> <213> 9 7 PRT Homo sapiens <400> 9 Tyr Ala Ser 1 Gin Ser Leu Ser <210> (:1<211> 9 <212> PRT 00) <213> Homo sapiens INO <400> 00 His Gin Ser Ser Arg Leu Pro His Thr 1 <210> 11 <211> <212> DNA <213> Homo sapiens <400> 11 agctttgcta tgcac <210> 12 <211> 48 <212> DNA <213> Homo sapiens <400> 12 gttattgata ctcgtgqtgc cacatactat gcagactccg tqaagggc 48 <210> 13 <211> <212> DNA <213> Homo sapiens <400> 13 ctggggaact tctactacgg tatggacgtc <210> 14 <211> <212> PRT <213> Homno sapiens (400> 14 Ser Phe Ala Met His 2. <210> <211> <212> <213> 16 PRT Homo sapiens <400> Val Ile Asp 1 Thr Arg Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly 5 10 <210> <211> <212> <213> 16 PRT Homno sapiens <400> 16 Leu Gly Asn 1 Phe Tyr Tyr Gly Met Asp Val 5 <210> 17 <211> <212> PRT <213> Homno sapiens <400> 17 Gly Phe Thr 1 Phe Ser Ser Phe Ala Met His 5 <210> 18 <211> <212> DNA <213> Homo sapiens <400> 18 ggattcacct tcagtagctt tgctatgcac <210> 19 <211> 1367 <212> PRT <213> Homo sapiens <400> 19 Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu Leu Phe Leu Ser Ala Ala Leu Ser Leu 25 Trp Pro Thr Ser Gly Glu Ile Leu Lys Arg Cys Gly Pro Gly Ile Asp Ile Arq Asn Asp Tyr Gin Gin Leu Glu Asn Cys Thr Val Ile Glu Gly Tyr Leu His Ile Leu Leu Ile S er Lys Ala Giu Asp Arg Ser Tyr Arg Phe Pro Lys Leu Ala Gly Leu Giu Thr Ile Thr Giu Tyr Leu Leu Phe Arg Val Ser Leu Gly Asp Leu Pro Asn Leu Thr Vai Ile Arg Gly Trp Lys Leu Phe Tyr Asn Tyr Ala Leu Val Ilie Phe Glu Met Thr Asn Leu Lys Asp Ile Gly Leu 130 Tyr Asn Leu Arg Gl Leule Thr Arg Gly Ala Ile Arg Ile Glu 130 140 Lys 145 Asn Ala Asp Leu Cys 150 Tyr Leu Ser Thr Asp Trp Ser Leu Leu Asp Ala Val. Asn Asn Tyr Ile Giy Asn Lys Pro Pro Lys 175 Glu Cys Gly Glu Lys Thr 195 Asp 180 Leu Cys Pro Gly Thr 185 Met Glu Glu Lys Pro Met Cys 190 Trp Thr Thr Thr Ile Asn Asn Gi u 200 Tyr Asn Tyr Arg Asn Arg 210 Cys Gin Lys Met Cys 215 Pro Ser Thr Cys Lys Arg Ala Cys Thr 225 Glu Asn Asn Giu Cys 230 Cys His Pro Glu Leu Gly Ser Cys Ala Pro Asp Asn Thr Ala Cys Val Cys Arg His Tyr Tyr Tyr 255 Ala Gly Val Gly Trp Arg 275 Val Pro Ala Cys Pro Asn Thr Tyr Arg Phe Glu 270 Leu Ser Ala Cys Val Asp Arg Phe Cys Ala Asn Glu Ser 290 Ser Asp Ser Glu Phe Val Ile His Gly Glu Cys Met Gin Glu Cys Pro Ser 305 Cys Ilie Pro Cys Glu 325 Phe Ile Arg Asn Ser Gin Ser Met Gly Pro Cys Pro Vai Cys Glu Glu Giu Lys 335 Lys Thr Lys Cys Thr Ile 355 Ile Asp Ser Val Ser Ala Gin Met Leu Gin Gly 350 Arg Gly Asn Phe Lys Giy Asn Leu Ile Asn Ile Asn Ile 370 Ala Ser Giu Leu Asn Phe Met Gly Ile Glu Val Val 10/62 Thr Gly Tyr Vai Lys 385 Phe Leu Lys Asn Leu 405 Arg His Ser His Leu Val Ser Leu Arg Leu Ile Leu Giu Giu Gin Leu Giu Giy 415 Asn Tyr Ser Asp Trp Asp 435 Phe 420 Tyr Vai Leu Asp Gin Asn Leu Gin Gin Leu Trp 430 His Arg Asn Leu Ile Lys Ala Gly Lys Met Tyr Phe Ala Phe 450 Asn Pro Lys Leu Val Ser Giu Ile Arg Met Giu Giu Thr Giy Thr Lys Gly Arg Gin Ser Lys Giy Asp Ile Asn Thr Arg 470 475 480 Asn Asn Gly Giu Ala Ser Cys Giu Asp Val Leu His Phe Thr 495 Ser Thr Thr Ser Lys Asn Arg Ile Ile Thr Trp His Arq Tyr 510 Arg Pro Pro 515 Asp Tyr Arg Asp Leu Ile Ser Phe Thr Val Tyr Tyr Lys 520 525 Thr Giu Tyr Asp Gly Gin Asp Ala Cys 540 Glu Ala 530 Pro Phe Lys Asn Gly 545 Ser Asn Ser Trp, Met Val Asp Val Asp 555 Leu Pro Pro Asn Asp Vai Giu Pro Ile Leu Leu His Gi y 570 Leu Lys Pro Trp Thr Gin 575 Tyr Ala Val His Ile Arg 595 Val Lys Ala Val Thr Leu Thr Met Val 585 Giu Asn Asp 590 Thr Asn Ala Gly Ala Lys Ser Ile Leu Tyr Ile Arg 605 Ser Val 610 Pro Ser Ile Pro Asp Vai Leu Ser Al a 620 Ser Asn Ser Ser 11/62 Ser Gin Leu Ile Val 625 Leu Ser Tyr Tyr Ile 645 Lys 630 Trp Asn Pro Pro Leu Pro Asn Gly Val Arg Trp Gin Gin Pro Gin Asp Giy Tyr 655 Leu Tyr Arg Tyr Aia Asp 675 His Asn 660 Tyr Cys Ser Asp Lys Ile Pro Ile Arg Lys 670 Asn Pro Lys Giy Thr Ile Asp Giu Giu Vai Thr Thr Giu 690 Val Cys Gly Gly Giu 695 Lys Gly Pro Cys Ala Cys Pro Lys Giu Ala Giu Lys Gin 710 Aia Giu Lys Giu Ala Giu Tyr Arg Vai Phe Giu Asn Leu His Asn Ser Phe Val Pro Arg Pro Giu 735 Arg Lys Arg Arq Ser Arg 755 Arg 740 Asp Vai Met Gin Ala Asn Thr Thr Met Ser Ser 750 Thr Asp Pro Asn Thr Thr Ala Asp Thr Tyr Asn Giu Giu 770 Leu Giu Thr Giu Pro Phe Phe Giu Arg Val Asp Asn Lys 785 Giu Arg Thr Vai Ser Asn Leu Arq Pro 795 Phe Thr Leu Tyr Ile Asp Ile His Cys Asn His Giu Giu Lys Leu Gly Cys Ser 815 Ala Ser Asn Asp Ile Pro 835 Phe 820 Val Phe Ala Arg Met Pro Ala Giu Gly Ala Asp 830 Gly Pro Val Thr Giu Pro Arg Pro Giu Asn Ser Ile 845 Phe Leu 850 Lys Trp Pro Giu Giu Asn Pro Asn Leu Ile Leu Met Tyr Giu Ile Lys Tyr Gly Ser Gin Vai Giu Asp Gin Arg Giu Cys Vai 12/62 870 875 Ser Arq Gin Glu Tyr 885 Arg Lys Tyr Gly Gi y 890 Ala Lys Leu Asn Arg Leu 895 Asn Pro Gly Asn Gly Ser 915 As n 900 Tyr Thr Ala Arg Gin Ala Thr Ser Leu Ser Gly 910 Ala Lys Thr Trp Thr Asp Pro Phe Phe Tyr Val Gly Tyr 930 Giu Asn Phe Ilie Leu Ile Ile Ala Leu Pro Val Ala Val 940 Leu Ile Val Gly Gly 950 Leu Val Ile Met Tyr Val Phe His Lys Arg Asn Asn Ser 965 Arg Leu Gly Asn Val Leu Tyr Ala Ser Val 975 Asn Pro Giu Giu Val Ala 995 Tyr 980 Phe Ser Ala Ala Val Tyr Val Pro Asp Giu Trp 990 Arg Giu Lys Ile Thr Met Ser Arg Giu Leu Gly Gin Gly 1000 1005 Ser Phe 1010 Asp Giu 1025 Ala Ser 1040 Met Lys 1055 Val Ser 1070 Arg Gly 1085 Giu Asn 1100 Giy Met Val Tyr Pro Giu Thr Arg Gi u 1015 Val 1030 Gly Val Ala Lys Ala Ile Lys Thr Met Arg Giu Arg Ile Giu Phe Leu Asn 1045 Giu Phe Asn Cys Gin Gly Gin Pro Asp Leu Lys Ser Asn Pro Val Leu His 1060 Thr 1075 Tyr 1090 Al a 1105 His Val Val Arq Leu Vai Ile Met Leu Arg Ser Leu Pro Pro Ser Leu Gi y 1020 Val1 1035 Gi u 1050 Leu 1065 Gi u 1080 Arg 1095 Ser 1110 Ala Ser Val Vai Val Lys Asn Giu Ala Leu Gly Val Leu Met Thr Pro Giu Met Lys Met Ile 13/62 Gin Met Ala Gly Glu Ile Ala Asp Gly Met Ala Tyr Leu Asn Ala 00 00 1115 Asn Lys 1130 Ala Glu 1145 Asp Ile 1160 Leu Pro 1175 Phe Thr 1190 Glu Ile 1205 Glu Gin 1220 Pro Asp 1235 Trp Gin 1250 Ser Ser 1265 Phe Tyr 1280 Asp Leu 1295 Ala Ser 1310 Lys Ala 1325 Phe Val His Arg Asp Phe Thr Val Tyr Giu Thr Asp Vai Arg Trp Met Thr Tyr Ser Asp Ala Thr Leu Ala Val Leu Arg Phe Asn Cys Pro Asp Tyr Asn Pro Lys Ile Lys Giu Giu Tyr Ser Giu Glu Giu Pro Giu Asn Ser Ser Ser Leu Giu Asn Gly Pro 1120 Asp 1135 Lys 1150 Tyr 1165 S er 1180 Val1 1195 Glu 1210 Val1 1225 Met 1240 Met 1255 Met 1270 Asn 1285 Met 1300 Pro 1315 G 1 y 1330 Leu Ala Ala Ile Giy Asp Tyr Arg Lys Pro Giu Ser Trp Ser Phe Gin Pro Tyr Met Glu Gly Leu Phe Glu Arg Pro Ser Giu Pro Gly Lys Leu Pro Glu Ser Val Leu Pro Asp I Pro Gly Vai L 1125 Arq Asn Cys Met Val 1140 Phe Giy Met Thr Arg 1155 Gly Gly Lys Giy Leu 1170 Leu Lys Asp Giy Vai 1185 Gly Val Val Leu Trp 1200 Gin Gly Leu Ser Asn 1215 Sly Leu Leu Asp Lys 1230 Leu Met Arg Met Cys 1245 Phe Leu Glu Ile Ile 1260 Phe Arg Giu Val Ser 1275 3lu Pro Giu Giu Leu 1290 ro Leu Asp Pro Ser 1305 ~rg His Ser Gly His 1320 ~eu Val Leu Arg Ala 1335 14/62 Ser Phe Asp Glu Arq Gin Pro 1340 1345 Tyr Ala His Met Asn Gly Gly Arg 1350 Lys Asn Glu Arg Ala Leu Pro Leu Pro Gin Ser Ser Thr Cys 1355 1360 1365 <210> <211> 195 <212> PRT <213> Homo sapiens <400> Met Gly Lys Ile Ser Ser Leu Pro Thr 1 5 Cys Asp Phe Leu Lys Val Lys Met His Leu Phe Lys Cys Cys Phe Thr Met Ser Ser Ser His Leu Ala Thr Ala Phe Tyr Leu Ala Leu Cys Leu Thr Phe Thr Ser Gly Pro Glu Thr Leu Cys Gly 55 Ala Glu Leu Val Asp Ala Leu Gin Phe Val1Cys Gly Asp Arg Phe Tyr Phe Asn Pro Thr Gly Tyr Ser Ser Ser Arg Ala Pro Gin Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala Arq Ser Val Arg Ala Gin His Thr Asp Met Pro 130 Lys Thr Gin Lys Gin Pro Pro Ser Asn Lys Asn Thr Ser Gin Arg Arg Gly Trp Pro Lys Thr His Pro Gly Gly 155 15/62 Gin Lys Giu Giu Gin Arg Gly Thr 165 Arg Giu 180 Glu Ala Ser Leu Ile Arg Gly Lys Lys Lys 175 Ile Gly Ser Asn Ala Giu Cys Arg Gly Lys 190 Lys Gly Lys 195 <210> 21 <211> 180 <212> PRT <213> Homo sapiens <400> 21 Giy Ile Pro Met Gly Lys Ser Met Val Leu Leu Thr Phe Leu Aia Phe Ala Cys Gly Gly Ser Cys Cys Ile Ala Tyr Arg Pro Ser Giu Thr Leu Gly Asp Arg Giu Leu Val Asp Leu Gin Phe Val Gly Phe Tyr Phe Ser Arg Ala Ser Arg Val Arg Arg Ser Arq Ile Val Giu Giu Cys Phe Arg Ser Asp Leu Ala Leu Glu Thr Tyr Cys Thr Pro Ala Lys Glu Arg Asp Val Ser Thr Pro Pro Thr Phe Phe Gin 115 Leu Pro Asp Asn Pro Arg Tyr Pro Val Gly Lys 110 Leu Arg Arg Tyr Asp Thr Trp Gin Ser Thr Gin Gly Leu 130 Pro Ala Leu Leu Ala Arg Arg Gly Val Leu Ala Lys Giu Leu Giu Ala Phe Arg Glu Ala Lys Arg His Arg Pro Leu Ile Ala 16/62 r- 145 150 155 160 SLeu Pro Thr Gin Asp Pro Ala His Gly Gly Ala Pro Pro Glu Met Ala 165 170 175 00 r N Ser Asn Arg Lys 180 O <210> 22 r- 00 <211> 21 S<212> DNA <213> Artificial sequence <220> <223> PCR primer <400> 22 tgccaggggg aagaccgatg g 21 <210> 23 <211> <212> DNA <213> Artificial sequence <220> <223> PCR primer <400> 23 cgggaagatg aagacagatg <210> 24 <211> 390 <212> DNA <213> Homo sapiens <220> <221> CDS 17/62 <222> CK1 <223> 00 <220> <221> sigypeptide IND <222> 00 <223> <400> 24 atg gaa gcc cca get cag ctt ctc ttc etc ctg eta. ctc tgg ctc eca 48 Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 gat aec acc gga gaa att qtq ttg aca. cag tct cca gee ace etg tct 96 Asp Thr Thr Gly Glu Ile Val Leu Thr Gin Ser Pro Ala Thr Leu Ser 25 ttg tct eca ggg gaa aga gcc ace ctc tcc tgc agg gcc agt cag agt 144 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser 40 gtt age agt ttc tta gee tgg tac caa. cag aaa. ect qgc cag gct ccc 192 Val Ser Ser Phe Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro 55 agg etc etc atc tat gat gca tcc aac aqg qcc cct ggc atc cca gcc 240 Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Pro Gly Ile Pro Ala 70 75 agg ttc agt ggc agt ggg tct qgg aca. gac ttc act etc ace ate age 288 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 90 age eta gag ect gaa gat ttt gca gtt tat tac tgt cag eag cgt age 336 Ser Leu Giu Pro Giu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser 100 105 110 aac tgg ect egg tgg aeg ttc gge caa ggg ace aag gtg gaa ate aaa 384 Asn Trp Pro Arg Trp Thr Phe Gly Gin Gly Thr Lys Val Giu Ile Lys 115 120 125 ega act 390 Arg Thr 130 <210> <211> 130 <212> PRT 18/62 (213> Homo sapiens <400> Met Giu Ala Pro Gin Leu Leu Phe Leu Leu Leu Trp Leu Pro Asp Thr Thr Glu Ile Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Tyr Gin Gin Lys Pro Gly Gin Ala Pro Val Ser Ser Phe Leu Ala Leu Leu Ile Tyr Ala Ser Asn Arg Pro Gly Ile Pro Arg Phe Ser Gly Gly Ser Gly Thr Phe Thr Leu Thr Ile Ser Ser Leu Giu Asn Trp Pro 115 Giu Asp Phe Ala Tyr Tyr Cys Gin Gin Arg Ser 110 Giu Ile Lys Arg Trp Thr Phe Gin Gly Thr Lys Arq Thr 130 <210> 26 <211> 420 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (420) <223> 19/62 CK1 <220> 4) <221> sigpeptide 00 <222> <223> 00 <400> 26 atg gag ttt ggg ctg agc tgg gtt ttc ctt qtq gct ata tta aaa qgt 48 Met Giu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly C11 5 10 gtc cag tgt gag gtt cag ctg gtg cag tct ggg gga ggc ttg gta ctt 96 Val Gin Cys Giu Val Gin Leu Val Gin Ser Gly Gly Gly Leu Val Leu 25 cct ggg ggg tcc ctg aga ctc tcc tgt gca ggc tct gga ttc acc ttc 144 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe 40 agt aac tat gct atg cac tgg att cgc cag gct cca gqa aaa gqt ctq 192 Ser Asn Tyr Ala Met His Trp Ile Arg Gin Aia Pro Gly Lys Gly Leu 55 gag tgg gtg tca gct att ggt gct ggt ggt gac acg tac tat gca gac 240 Giu Trp Vai Ser Aia Ile Gly Ala Gly Gly Asp Thr Tyr Tyr Ala Asp 70 75 tcc gtg aag ggc cga ttc acc atc tcc aga gac aac gcc aag gac tcc 288 Ser Vai Lys Giy Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Ser 90 ttg tat ctt caa atg aac agc ctg aga gcc gag gac atg gct gtt tat 336 Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Giu Asp Met Ala Val Tyr 100 105 110 tac tgt gca aga ggc cgg cat agg aac tgg tac tac tac aat aag gac 384 Tyr Cys Ala Arg Gly Arg His Arg Asn Trp Tyr Tyr Tyr Ash Lys Asp 115 120 125 tac tgg ggc cag gga acc ctg gtc acc gtc tcc tca 420 Tyr Trp, Gly Gin Gly Thr Leu Val Thr Val Ser Ser 130 135 140 <210> 27 <211> 140 <212> PRT <213> Homo sapiens 20/62 <400> 27 Met 1 Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Giy Val Gin Cys Pro Gly Gly Val Gin Leu Val Gin Ser Gly Gly Gly Leu Val Leu Phe Thr Phe Ser Leu Arq Leu Cys Ala Gly Ser Ser Asn Glu Trp Tyr Ala Met His Val Ser Ala Ie 70 Ile Arg Gin Ala Gly Lys Gly Leu Gly Ala Gly Gly Asp Thr Tyr Tyr Ala Ser Vai Lys Gly Arg Phe Thr Ile Ser Asp Asn Ala Lys Asp Ser Leu Tyr Leu Tyr Cys Ala 115 Met Asn Ser Leu Ala Giu Asp Met Ala Val Tyr 110 Asn Lys Asp Arg Gly Arq His Asn Trp Tyr Tyr Tyr Trp 130 Gly Gin Gly Thr Val Thr Val Ser <210> 28 <211> 33 <212> DNA <213> Homo sapiens <400> 28 agggccagtc agagtgttaq cagtttctta gcc <210> 29 <211> 21 <212> DNA <213> Homo sapiens 21/62 <400> 29 gatgcatcca acagggcccc t <210> <211> <212> DNA <213> Homo sapiens <400> cagcagcgta gcaactggcc tcggtggacg <210> <211> <212> <213> 31 11 PRT Homo sapiens <400> 31 Arg Ala Ser 1 Gin Ser Val Ser Ser Phe Leu Ala 5 <210> <211> <212> <213> 32 7 PRT Homo sapiens <400> 32 Asp Ala Ser 1 Asn Arg Ala Pro <210> <211> <212> <213> 33 PRT Homo sapiens 22/62 <400> 33 Gin Gin Arg Ser Asn Trp Pro Arg Trp Thr 1. 5 <210> 34 00 ~KI<211> <212> DNA r- <213> Homo sapiens 00 C1<400> 34 ggattcacct tcagtaacta tgctatgcac <210> <211> 48 <212> DNA <213> Homo sapiens <400> gctattggtg ctgqtgqtga cacgtactat gcagactccq tqaaqggc 48 <210> 36 <211> 39 <212> DNA <213> Homo sapiens <400> 36 qqccgqcata qgaactggta ctactacaat aaggactac 39 <210> 37 <211> <212> PRT <213> Homo sapiens <400> 37 23/62 Gly Phe Thr Phe Ser Asn Tyr Ala Met His 1 5 <210> <211> <212> <213> 38 16 PRT Homo sapiens <400> 38 Ala Ile Gly 1 Ala Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys Gly 5 10 <210> <211> <212> <213> 39 13 PRT Homo sapiens <400> 39 Gly Arg His 1 Arg Asn Trp Tyr Tyr Tyr Asn Lys Asp Tyr 5 <210> <211> <212> <213> 384 DNA Homo sapiens <220> <221> <222> <223> C DS (384) <400> atg tcg cca tca caa ctc att qg Met Ser Pro Ser Gin Leu Ile Gly 1 5 ttt ctg ctg ctc tgg gtt cca gcc Phe Leu Leu Leu Trp Val Pro Ala 10 24/62 t cc Ser act Thr ggt Gly ct t Leu ttc Phe ct C Leu tta Leu [ggt gaa att fGly Gin Ile ggc gag aga Gly Gin Arg agc tta cac Ser Len His atc tac tat t le Tyr Tyr qgc agt gga Gly Ser Gly fgct gaa gat iAla Gin Asp 100 cac act ttc His Thr Phe 115 41 128 PRT Homo sapiens cca Pro cgg Arg cca Pro tca Ser 75 acc Thr t gt Cy s gtq Val1 ga c Asp gcc Al a ggt Gly ggg Mly ctc Len cat His gag GJ.u t ct Ser agt Se r cag Gin gt c Val1 acc Thr cag Gin at c Ilie 125 Ct g Leu cag Gin tct Ser ccc Pro at c Ile agt Ser 110 aaa Lys tct Ser agc Ser cca Pro tcg Ser aqt Se r agt Ser cgt Arg gt g Val1 att Ile aag Lys agg Ar g agc Se r cgt Arg acg Thr 96 144 192 240 288 336 384 <210> <211> <212> <213> <400> 41 Met Ser Pro 1 Ser Arg Giv Ser Gin 5 Gi Ile Len Ile Gly Phe Len Len Trp Vai Pro Ala Val Len Thr Pro Asp Ser Thr Pro Gly Gin Giv Ser Ser Leu Arg Val Thr His Trp Tyr 55 Tyr Aia Ser Cys Arg Aia Len Ser Val Gin Ser Ile Ser Pro Lys Gin Lys Pro Leu Leu Gly Giy Ile Tyr Gin Ser Leu Val Pro Ser 25/62 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser C185 90 Leu Giu Ala Giu Asp Phe Ala Val Tyr Tyr Cys His Gin Ser Ser Arg 00 100 105 110 Leu Pro His Thr Phe Giy Gin Gly Thr Lys Val Giu Ile Lys Arg Thr NO115 120 125 00 <210> 42 CK1 <211> 384 <212> DNA r~l<213> Homno sapiens <220> <221> CDS <222> <223> <400> 42 atg tog cca. tca. caa. cto att ggg ttt otg otg ctc tgg gtt cca. goc 48 Met Ser Pro Ser Gin Leu Ie Gly Phe Leu Leu Leu Trp Val Pro Ala 1 5 10 too agg ggt gaa att gtg ctg act cag agc cca ggt acc ctg tct gtg 96 Ser Arg Qly Giu Ile Val Leu Thr Gin Ser Pro Giy Thr Leu Ser Val 25 tct cca. ggc gag aga. gcc acc cto tcc tgo cgg gcc agt cag agc att 144 Ser Pro Gly Giu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Ile 40 ggt agt agc tta. cac tgg tao cag cag aaa coa ggt cag got oca agg 192 Gly Ser Ser Leu His Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg 55 ott ctc ato tac tat got too cag too otc tca ggg atc ccc gat agg 240 Leu Leu Ile Tyr Tyr Aia Ser Gin Ser Leu Ser Gly Ile Pro Asp Arg 70 75 tto agt ggo agt gga tot ggg aoa gat tto acc cto acc atc agt aga 288 Phe Ser Gly Ser Gly Ser Giy Thr Asp Phe Thr Leu Thr Ile Ser Arg 90 ctg gag oct gaa gat ttc gca gtg tat tao tgt oat cag agt agt ogt 336 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gin Ser Ser Arg 100 105 110 26/62 tta cct cac Leu Pro His 115 act ttc qgc caa Thr Phe Gly Gin ggg acc aag gtg gag Gly Thr Lys Val Giu 120 atc aaa cgt aca Ile Lys Arg Thr 125 <210> 43 <211> 128 <212> PRT <213> Homo sapiens <400> 43 Met Ser Pro Ser Gin 1 5 Leu Ile Gly Phe Leu Leu Trp Val Pro Ala Ser Arg Giy Ser Pro Gly Giu Ile Val Leu Thr Ser Pro Gly Thr Leu Ser Val Gin Ser Ile Giu Arg Ala Thr Ser Cys Arg Ala Gly Ser Ser Leu His Trp Tyr Gin Gin Lys Pro Gin Ala Pro Arg Leu Leu Ile Tyr Tyr Ser Gin Ser Leu Ser Gly Ile Pro Asp Phe Ser Gly Ser Ser Gly Thr Asp Thr Leu Thr Ile Ser Arg Leu Giu Pro Asp Phe Aia Val Tyr Cys His Gin Ser Ser Arg 110 Lys Arg Thr Leu Pro His Thr Phe Giy Gin Thr Lys Val Giu <210> 44 <21i> 411 <212> DNA <213> Homo sapiens 27/62 <220> <221> CDS <222> (411) 00 <223> <400> 44 atg gag ttt ggg ctg ago tgg gtt ttc ctt gtt got ata tta aaa ggt 48 00 Met Giu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 gtc cag tgt gag gtt cag ctg gtg cag tot ggg gga ggc ttg gta aag 96 Val Gin Cys Giu Val Gin Leu Val Gin Ser Giy Gly Gly Leu Val Lys 25 cct ggg ggg too ctg aga otc tc tgt gca gc tot gga tto aco tto 144 Pro Gly Giy Ser Leu Arg Leu Ser Cys Ala Ala Ser Giy Phe Thr Phe 40 agt agc ttt got atg cac tgg gtt cgc cag got cca gga aaa ggt otg 192 Ser Ser Phe Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Giy Leu 55 gag tgg ata toa gtt att gat aot ogt ggt goo aoa tao tat goa gao 240 Glu Trp Ile Ser Val Ile Asp Thr Arg Giy Ala Thr Tyr Tyr Ala Asp 70 75 too gtg aag ggo oga tto aoo ato too aga gao aat gc aag aao too 288 Ser Vai Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 90 ttg tat ott oaa atg aao ago ctg aga goo gag gao aot got gtg tat 336 Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Giu Asp Thr Ala Vai Tyr 100 105 110 tao tgt goa aga otg ggg aao tto tao tao ggt atg gao gto tgg ggo 384 Tyr Cys Ala Arg Leu Gly Asn Phe Tyr Tyr Gly Met Asp Val Trp Gly 115 120 125 oaa ggg aoo aog gto aoo gto too toa 411 Gin Gly Thr Thr Val Thr Val Ser Ser 130 135 <210> <211> 137 <212> PRT <213> Homo sapiens <400> Met Giu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly 28/62 Val Gin Cys Pro Gly Gly Giu Val Gin Leu Val Gin 25 Ser Gly Gly Gly Leu Val Lys Phe Thr Phe Ser Leu Arq Leu Se r Cys Ala Ala Ser Ser Ser Phe Ala Met His Trp Val Arg Gin Ala Gly Lys Gly Leu Giu Trp Ile Ser Val Asp Thr Arg Gly Thr Tyr Tyr Ala Ser Val Lys Gly Phe Thr Ile Ser Asp Asn Ala Lys Asn Ser Leu Tyr Leu Tyr Cys Ala 115 Gin Gly Thr 130 Met Asn Ser Leu Ala Giu Asp Thr Ala Val Tyr 110 Val Trp Gly Arg Leu Giy Asn Tyr Tyr Gly Met Thr Val Thr Vai Ser Ser 135 <210> 46 <211> 69 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 46 gaa att gtg ctg act cag agc cca gac Giu Ile Val Leu Thr Gin Ser Pro Asp tct ctg tct gtg act cca ggc Ser Leu Ser Val Thr Pro Giy gag aga gtc acc atc acc tgc 29/62 Giu Arg Val Thr Ile Thr Cys <210> 47 <211> 23 <212> PRT <213> Homo sapiens <400> 47 Glu Ile Val Leu Thr Gin Ser Pro Asp Ser Leu Ser Val Thr Pro Gly Giu Arg Val Thr Ile Thr Cys <210> 48 <211> <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 48 tqg tac cag Trp Tyr Gin 1 cag aaa cca ggt cag tct cca Gin Lys Pro Giy Gin Ser Pro 5 aag ctt ctc atc Lys Leu Leu Ile <210> 49 <211> <212> PRT <213> Homo sapiens <400> 49 30/62 Trp Tyr Gin Gin Lys Pro Gly Gin Ser 1 5 Pro Lys Leu Leu Ile Tyr <210> <211> 96 <212> DNA <213> Homno sapiens <220> <221> COS <222> (96) <223> <400> ggg gtc ccc Giy Vai Pro 1 ctc acc atc Leu Thr Ile tcg agg Ser Arg 5 ttc agt ggc aqt Phe Ser Gly Ser gga Gly 10 tot ggg aca gat Ser Gly Thr Asp ttc acc Phe Thr agt Se r ago oto gag gct gaa gat ttc gca gtg Ser Leu Giu Ala Giu Asp Phe Aia Vai tat tac tgt Tyr Tyr Cys <210> 51 <211> 32 <212> PRT <213> Homo sapiens <400> 51 Gly Val Pro Ser 1 Phe Ser Gly Ser Ser Giy Thr Asp Phe Thr Leu Thr Ile <210> 52 <211> 36 <212> DNA Ser Leu Giu Ala Asp Phe Aia Val Tyr Tyr Cys 31/62 <213> Homo sapiens <220> <221> CDS <222> <223> <400> 52 ttc ggc caa Phe Gly Gin 1 ggg ace aag gtg gag ate aaa cgt acg Gly Thr Lys Val Glu Ile Lys Arg Thr 5 <210> 53 <211> 12 <212> PRT <213> Homo sapiens <400> 53 Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr 1 5 <210> 54 <211> 69 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 54 gaa att gtg ctg act cag age cca Glu Ile Val Leu Thr Gin Ser Pro 1 5 ggt ace ctg tct gtg tct cca ggc 48 Gly Thr Leu Ser Val Ser Pro Gly 10 32/62 gag aga gcc Glu Arg Ala <210> <211> 23 <212> PRT acc ctc tcc tgc Thr Leu Ser Cys <213> Homo sapiens <400> Glu Ile Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Val Ser Pro Gly Giu Arg Ala <210> 56 <211> <212> DNA Thr Leu Ser Cys <213> Homno sapiens <220> <221> CDS <222> <223> <400> 56 tgg tac cag cag aaa cca ggt cag gct cca agg ctt ctc atc tac Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile Tyr 1 5 10 <210> 57 <211> <212> PRT <213> Homo sapiens 33/62 <400> 57 Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile Tyr <210> 58 <211> 96 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 58 ggg atc ccc gat agg ttc agt ggc agt Gly Ile Pro Asp Arg Phe Ser Gly Ser tot ggg aca gat Ser Gly Thr Asp ttc ac Phe Thr cto acc atc Leu Thr Ile agt aga Ser Arg ctg gag cct Leu Glu Pro gat ttc gca gtg Asp Phe Ala Val tat tao tgt Tyr Tyr Cys <210> 59 <211> 32 <212> PRT <213> Homo sapiens <400> 59 Gly Ile Pro 1 Asp Arg 5 Phe Ser Gly Ser Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Asp Phe Ala Val Tyr Tyr Cys <210> <211> 36 34/62 I <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> ftc ggc caa Phe Giy Gin ggg acc aag gtg gag atc aaa cgt aca Gly Thr Lys Val Glu Ile Lys Arg Thr <210> 61 <211> 12 <212> PRT <213> Homo sapiens <400> 61 Phe Gly Gin Giy Thr Lys Val Giu Ile Lys Arg Thr 1 5 <210> 62 <211> <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 62 gag gtt cag ctg gtg cag tct ggg gga ggc ttg gta aag cct ggg ggq Giu Val Gin Leu Vai Gin Ser Gly Giy Giy Leu Val Lys Pro Gly Gly 35/62 1 tcc ctg aga Ser Leu Arg 5 ctc tcc tqt gca gcc tct gga ttc acc ttc agt Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 25 <210> <211> <212> <213> 63 PRT Homo sapiens <400> 63 Glu Val Gin 1 Ser Leu Arg Leu Val Gin Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 5 10 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 25 <210> <211> <212> <213> <220> <221> <222> <223> 64 42 DNA Homo sapiens CDS <400> 64 tgg gtt cgc cag gct cca gqa aaa ggt ctq gag tgg ata tca Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Giu Trp Ile Ser 1 5 <210> <211> 14 <212> PRT 36/62 <213> Homo sapiens <400> Trp Vai Arg Gin Ala Pro Giy Lys Giy Leu Giu Trp Ile Ser 1 5 <210> 66 <211> 96 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 66 cga ttc acc Arg Phe Thr 1 atg aac agc Met Asn Ser atc tcc Ile Ser 5 aga gac aat gcc Arg Asp Asn Ala aac tcc ttg tat Asn Ser Leu Tyr ctt caa Leu Gin aga gac gag gac Arg Aia Glu Asp gat gtg tat tac Ala Val Tyr Tyr tgt gaa aga Cys Ala Arg <210> 67 <211> 32 <212> PRT <213> Holmo sapiens <400> 67 Arg Phe Thr 1 Met Asn Ser Ile Ser 5 Leu Arg Arg Asp Asn Ala Asn Ser Leu Tyr Leu Gin Ala Giu Asp Ala Vai Tyr Tyr Cys Ala Arg 37/62 <210> <211> <212> <213> <220> <221> <222> <223> 68 33 DNA Homo sapiens CDS (33) <400> 68 tgg ggc caa Trp Gly Gin qgg acc acq gtc acc qtc tcc tca 33 Gly Thr Thr Val Thr Val Ser Ser <210> <211> <212> <213> 69 11 PRT Homo sapiens <400> 69 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 1 5 <210> <211> <212> PRT <213> Homo sapiens <400> Asn*Tyr Ala Met His 1 <210> 71 38/62 <211> 384 <212> DNA <213> Home sapiens 00 <220> <221> COS 00 <222> <223> <400> 71 atg tog cca tca caa ctc att ggg ttt ctg ctg otc tqg qtt coa goc 48 Met Ser Pro Ser Gin Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala 1 5 10 tc agg ggt gaa att gtg otg act cag agc cca gac tct otg tct gtg 96 Ser Arg Gly Glu Ile Val Leu Thr Gin Ser Pro Asp Ser Leu Ser Val 25 act oca ggo gag aga gto acc ato acc tgc cgg gcc agt cag ago att 144 Thr Pro Gly Giu Arg Val Thr lie Thr Cys Arg Ala Ser Gin Ser Ile 40 ggt agt ago tta cac tgg tao cag cag aaa cca ggt cag tct coa aag 192 Gly Ser Ser Leu His Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys 55 ott otc atc aag tat goa tc cag too otc tca ggg gto ccc tcg agg 240 Leu Leu Ile Lys Tyr Ala Ser Gin Ser Leu Ser Gly Val Pro Ser Arg 70 75 ttc agt ggo aqt gga tot ggg aca gat tto aoc otc aco ato agt ago 288 Phe Ser Gly Ser Giy Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 90 otc gag got gaa gat got goa gog tat tao tgt oat oag agt agt ogt 336 Leu Giu Ala Giu Asp Ala Ala Ala Tyr Tyr Cys His Gin Ser Ser Arg 100 105 110 tta cot cac act ttc ggo caa ggg aoo aag gtg gag ato aaa cgt aog 384 Leu Pro His Thr Phe Gly Gin Giy Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 <210> 72 <211> 128 <212> PRT <213> Homo sapiens 39/62 00 <400> 72 met Ser Pro Ser Gin Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala Ser Arq Gly Giu Ile Val Leu Thr Gin Ser Pro Asp Ser Leu Ser Val Gin Ser Ile Thr Pro Gly Glu Arg Val Thr Thr Cys Arg Ala Gly Ser Ser Leu His Trp Gin Gin Lys Pro Gin Ser Pro Lys Leu Ile Lys Tyr Ser Gin Ser Leu Gly Val Pro Ser Phe Ser Gly Ser Gly Ser Gly Thr Asp Thr Leu Thr Ile Ser Ser Leu Giu Ala Leu Pro His 115 Asp Ala Ala Ala Tyr Cys His Gin Ser Ser Arg 110 Lys Arg Thr Thr Phe Gly Gin Gly 120 Thr Lys Val Giu <210> 73 <211> 384 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (384) <223> <400> 73 atg tcg cca tca caa ctc att ggg ttt Met Ser Pro Ser Gin Leu Ile Gly Phe ctg ctq ctc tgg gtt cca gcc 48 Leu Leu Leu Trp Val Pro Ala 40/62 a gg Arg cca Pro agt Ser ct C Leu a gt Ser gag Glu cet Pro gtg Val1 gt c Val1 t gg Trp gca Ala t ct Ser ttc Phe ggc Gi y act Thr atc Ile ca g Gin ca g Gin aca Thr gt g Val1 ggg Gly 120 cca Pro cgg Arg o ca Pro t ca Ser ac Thr t gt Cys gtg Val1 ga c Asp gcc Al a ggt Gly ggg Gly ct C Leu cat His gag Glu tct Ser a gt Ser cag Gin gte Val1 acc Thr cag Gin atc Ile 125 ctg Leu eag Gln t ct Ser ccc Pro ate Ile agt Se r 110 aa a Lys t ct Ser age Ser o ca Pro t eg Ser agt Ser agt Se r cgt Arg 96 144 192 240 288 336 384 <210> 74 <211> 128 <212> PRT <213> Homo sapiens <400> 74 Met Ser Pro 1 Ser Arg Gly Thr Pro Gly Gly Ser Ser Leu Leu Ilie Ser Gin 5 Giu Ile Leu Ile Giy Phe Leu Leu Trp Pro Asp Ser Val Leu Thr Val Pro Ala Leu Ser Val Gin Ser Ile Ser Pro Lys Arg Val Thr Cys Arg Ala Leu His Trp Lys Tyr Ala T yr 55 Ser Gin Lys Pro Gly Gly Gin Ser Leu Val Pro Ser 41/62 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 90 Leu Giu Ala Giu Asp Phe Ala Val Tyr Tyr Cys His Gin Ser Ser Arg 100 105 110 00 Leu Pro His Thr Phe Gly Gin Gly Thr Lys Val Giu Ile Lys Arg Thr 115 120 125 00 <210> <211> 384 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> atg teg cca tca caa ctc att ggg ttt etg ctg ctc tgg gtt eca gcc 48 Met Ser Pro Ser Gin Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala 1 5 10 tcc agg gqt gaa att gtg ctg act cag agc cca ggt acc ctg tct gtg 96 Ser Arg Gly Giu Ile Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Vai 25 tct eca ggc gag aga gcc acc etc tcc tgc cgg gcc agt cag agc att 144 Ser Pro Gly Giu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Ile 40 ggt agt age tta eac tgg tac cag eag aaa eca ggt cag gct eca agg 192 Gly Ser Ser Leu His Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg 55 ctt ctc atc aag tat gca tee cag tcc ctc tca ggg ate ecc gat agg 240 Leu Leu Ile Lys Tyr Ala Ser Gin Ser Leu Ser Gly Ile Pro Asp Arg 70 75 ttc agt ggc agt gga tct ggg aca gat ttc ace etc acc atc agt aga 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 90 ctg gag oct gaa gat get gca gcg tat tac tgt eat cag agt agt cgt 336 Leu Glu Pro Giu Asp Ala Ala Ala Tyr Tyr Cys His Gin Ser Ser Arg 100 105 110 42/62 tta cct cac Leu Pro His 115 act ttc qgc caa ggg acc aag gtg gag atc aaa cgt aca Thr Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr 120 125 <210> 76 <211> 128 <212> PRT <213> Homo sapiens <400> 76 Met Ser Pro Ser Gin Leu Ile Gly The Leu Leu Trp Val Pro Ala Ser Arg Gly Ser Pro Gly Glu Ile Val Leu Thr Ser Pro Gly Thr Leu Ser Val Gin Ser Ile Giu Arg Ala Thr Ser Cys Arq Ala Gly Ser Ser Leu His Trp Gin Gin Lys Pro Gin Aia Pro Arg Leu Ile Lys Tyr Ser Gin Ser Leu Gly Ile Pro Asp Phe Ser Gly Ser Ser Gly Thr Asp Thr Leu Thr Ile Ser Arq Leu Giu Pro Leu Pro His 115 Glu 100 Asp Ala Ala Ala Tyr Cys His Gin Ser Ser Arg 110 Lys Arg Thr Thr Phe Gly Gin Thr Lys Val Giu <210> 77 <211> 384 <212> DNA <213> Homo sapiens <220> 43/62 <221> CDS <222> (384) <223> <400> 77 atg Met I toc Ser t ct S er ggt Gly ctt Leu ttc Phe ctg Le u tt a Leu tcg cca tca oaa ctc att ggg ttt ctg ctg ctc tgg gtt cca gc Ser Pro Ser Gin Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala a gq Arg cca Pro agt Ser ct 0 Leu a gt Ser gag Giu cct Pro gqt Giy Giy agc Ser ato Ile Gly cot Pro ca 0 His 115 gaa Giu gag Giu tta Leu aag Lys agt Ser gaa Giu 100 act Thr 5 att Ile aga Arg cac His tat Tyr gga Gi y ga t Asp ttc Phe gt g Val1 gc Aila t gg Trp gca Aila 70 tot Se r ttc Phe ggc Gi y ot g Leu aoo Thr tao Tyr 55 too Ser ggg Gly goa Ala caa Gin act Thr ot 0 Le u 40 ca g Gin oa g Gin aoa Thr gt g Val1 ggg Gi y 120 cag Gin 25 too Ser oag Gin too Ser gat Asp tat Tyr 105 a cc Thr ago Se r tgc Cys aaa Lys ot 0 Leu tto Phe 90 tao Tyr aag Lys o ca Pro cqg Arg oca Pro t ca Ser 75 ac Thr tgt Cys gtg Val1 ggt Gly gc Ala gqt Gly ggg Gly ot 0 Leu cat His gag Glu aco Thr a gt Ser ca g Gin atc Ile aco Thr cag Gin ato Ile 125 ctg Leu oag Gin got Ala ccc Pro ato Ile agt Ser 110 aa a Lys tot S er ago Ser oca Pro gat Asp agt Ser a gt Ser cgt Arg gtg Val a tt Ile a gg Arg a gg Arg a ga Arg cgt Arg aca T hr 96 144 192 240 288 336 384 <210> <211> <212> <213> 78 128 PRT Homo sapiens <400> 78 Met Ser Pro 1 Ser Gin Leu Ile Gly Phe Leu Leu Leu Trp Vai Pro Ala Ser Arg Gly Giu Ile Vai Leu Thr Gin Ser Pro Gly Thr Leu Ser Val 25 44/62 Ser Pro Gly Giu Arq Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Ile Gly Ser Ser Leu His Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arq Le u Leu Ile Lys Tyr Ser Gin Ser Leu Gly Ile Pro Asp Phe Ser Gly Ser Ser Gly Thr Asp Thr Leu Thr Ile Ser Arg Leu Glu Pro Leu Pro His 115 <210> 79 <211> 69 <212> DNA Asp Phe Ala Val Tyr Cys His Gin Lys Arg Thr Thr Phe Gly Gin Thr Lys Val Glu <213> Homo sapiens <220> <221> CDS <222> (69) <223> <400> 79 gaa att qtg Giu Ile Vai 1 gag aqa qtc Giu Arg Val <210> <211> 23 <212> PRT ctg act Leu Thr 5 cag agc cca gac tct ctg tct gtg act cca qgc Gin Ser Pro Asp Ser Leu Ser Vai Thr Pro Giy 10 atc acc tgc Ile Thr Cys 45/62 <213> Homo sapiens <400> Giu Ile Val Leu Thr Gin Ser Pro Asp Ser Leu Ser Vai Thr Pro Gly Giu Arg Val Thr Ile Thr Cys <210> 81 <211> <2i2> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 81 tqq tac cag Trp Tyr Gin 1 <210> 82 <211> <212> PRT cag aaa cca ggt cag tct cca aag ctt ctc atc aag Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Lys 5 10 <213> Homo sapiens <400> 82 Trp Tyr Gin 1 <210> 83 <211> 96 Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Lys 5 10 46/62 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 83 ggg gtc ccc Gly Val Pro 1 etc ace ate Leu Thr Ile tcg agg ttc agt ggc agt Ser Arg Phe Ser Gly Ser 5 tct ggg aca gat Ser Gly Thr Asp ttc acc Phe Thr agt Ser age etc gag get Ser Leu Glu Ala gaa gat gct gca gcg tat tac tgt 96 Glu Asp Ala Ala Ala Tyr Tyr Cys 25 <210> 84 <211> 32 <212> PRT <213> Homo sapiens <400> 84 Gly Val Pro Ser 1 Phe Ser Gly Ser Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Glu Ala Asp Ala Ala Ala Tyr Tyr Cys <210> <211> 36 <212> DNA <213> Homo sapiens <220> <221> CDS 47/62 <222> (36) <223> <400> ttc ggo caa Phe Giy Gin 1 ggg aco aag gtg gag atc aaa ogt aog Gly Thr Lys Vai Giu Ile Lys Arg Thr 5 <210> 86 <211> 12 <212> PRT <213> Homo sapiens <400> 86 Phe Gly Gin Gly Thr Lys Val Giu Ile Lys Arg Thr 1 5 <210> 87 <211> 69 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 87 gaa att gtg ctq act Glu Ile Vai Leu Thr cag aqc cca gao tct ctg tot gtg act cca ggc Gin Ser Pro Asp Ser Leu Ser Val Thr Pro Gly gag aga gtc acc atc acc tgo Giu Arg Val Thr Ile Thr Cys <210> 88 48/62 <211> 23 <212> PRT <213> Homo sapiens <400> 88 Glu Ile Val Leu Thr Gin Ser Pro Asp Ser Leu Ser Val Thr Pro Gly 10 1 Glu Arg Val Thr Ile Thr Cys <210> 89 <211> <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 89 tgg tac cag Trp Tyr Gin 1 cag aaa cca ggt cag tct cca aag ctt ctc atc aag Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Lys 5 10 <210> <211> <212> PRT <213> Homo sapiens <400> Trp Tyr Gin 1 Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Lys 5 10 49/62 <210> 91 <211> 96 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 91 ggq gtc ccc Gly Val Pro 1 ctc acc atc Leu Thr Ile tcg agg ttc agt ggc agt gga tct ggg aca gat ttc acc 48 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr agc ctc gag gat Ser Leu Glu Ala gaa Glu gat ttc gca gtg Asp Phe Ala Val tat tac tgt Tyr Tyr Cys <210> 92 <211> 32 <212> PRT <213> Homo sapiens <400> 92 Gly Val Pro 1 Leu Thr Ile Ser Arg Phe Ser Gly 5 Ser Gly 10 Ser Gly Thr Asp Phe Thr Tyr Tyr Cys Ser Ser Leu Glu Ala Glu 25 Asp Phe Ala Val <210> 93 <211> 36 <212> DNA <213> Homo sapiens 50/62 <220> <221> CDS <222> (36) <223> <400> 93 ttc ggc caa Phe Gly Gin ggg acc aag gtg gag atc aaa cgt acg Gly Thr Lys Val Glu Ile Lys Arg Thr <210> 94 <211> 12 <212> PRT <213> Homo sapiens <400> 94 Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr <210> <211> 69 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (69) <223> <400> gaa att gtg ctg act Giu Ile Val Leu Thr 1 cag agc cca ggt acc ctg tct gtg tct cca ggc Gin Ser Pro Gly Thr Leu Ser Val Ser Pro Gly gag aga gcc acc ctc tcc tgc Glu Arg Ala Thr Leu Ser Cys 1/62 <210> <211> <212> <213> 96 23 PRT Homno sapiens <400> Giu Ile 1 Glu Arg <210> <211> <212> <213> <220> <221> <222> <223> 96 Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Val Ser Pro Gly 5 10 Ala Thr Leu Ser Cys 97 DNA Homo sapiens CDS <400> 97 tgg tac cag cag aaa cca ggt cag gct cca agg ctt ctc atc aag Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arq Leu Leu Ile Lys 1 5 10 <210> <211> <212> <213> 98 PRT Homo sapiens <400> 98 Trp Tyr Gin Gin Lys Pro Giy Gin Ala Pro Arg Leu Leu Ile Lys 1 5 10 52/62 <210> 99 <211> 96 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 99 ggg atc ccc Gly Ile Pro 1 ctc acc atc Leu Thr Ile gat agg ttc agt ggc agt gga tct ggg aca gat ttc acc Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr agt aga Ser Arg ctg gag cct Leu Glu Pro gat gct gca gcg Asp Ala Ala Ala tat tao tgt Tyr Tyr Cys <210> 100 <211> 32 <212> PRT <213> Homo sapiens <400> 100 Gly Ile Pro Asp Arq Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Ala Ala Ala 25 Tyr Tyr Cys <210> 101 <211> 36 <212> DNA <213> Homno sapiens 53/62 <220> <221> cDs <222> <223> <400> 101 ttc ggc caa Phe Gly Gin 1 ggg acc aag gtg gag atc aaa cqt aca Gly Thr Lys Val Glu Ile Lys Arg Thr 5 <210> 1.02 <211> 12 <212> PRT <213> Homo sapiens <400> 102 Phe Giy Gin Giy Thr Lys Val Giu Ile Lys Arg Thr 1 5 <210> 103 <211> 69 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 103 gaa att gtg ctg act Giu Ile Val Leu Thr 1 5 cag agc aca ggt acc ctg tct gtg tct cca gqc Gin Ser Pro Gly Thr Leu Ser Val Ser Pro Gly 10 gag aga gcc acc etc tcc tgc Giu Arg Ala Thr Leu Ser Cys 54/62 <210> <211> <212> <213> 104 23 PRT Homo sapiens <400> 104 Glu Ile Val 1 Glu Arg Ala Leu Thr Gin Ser Pro Gly Thr Leu Ser Val Ser Pro Gly 5 10 Thr Leu Ser Cys <210> 105 <211> <212> DNA <213> Homno sapiens <220> <221> CDS <222> <223> <400> 105 tgg tac cag cag aaa eca ggt cag get eca agg ctt ctc atc aag Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile Lys 1 5 10 <210> <211> <212> <213> 106 PRT Homo sapiens <400> 106 55/62 Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile Lys 1. 5 10 <210> 107 <211> 96 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 107 ggg ate ccc gat aqq Gly Ile Pro Asp Arg 1 5 ttc agt ggc agt Phe Ser Gly Ser tct ggg aca gat Ser Gly Thr Asp ttc acc Phe Thr etc ace atc Leu Thr Ile aqa ctg gag cet Arg Leu Glu Pro gat ttc qea gtg Asp Phe Ala Val tat tac tgt 96 Tyr Tyr Cys <210> 108 <211> 32 <212> PRT <213> Homo sapiens <400> 108 Giy Ile Pro 1 Leu Thr Ile <210> 109 <211> 36 <212> DNA Asp Arg Phe Ser Gly 5 Ser Gly Ser Gly Thr Asp 10 Phe Thr Ser Arg Leu Giu Pro Giu Asp Phe Aia Vai Tyr Tyr Cys 25 56/62 <213> Homo sapiens <220> <221> CDS <222> <223> <400> 109 ttc ggc caa Phe Gly Gin 1 gqg acc aag qtq gag atc aaa cgt aca Gly Thr Lys Val Glu Ile Lys Arg Thr 5 <210> 110 <211> 12 <212> PRT <213> Homo sapiens <400> 110 Phe Gly Gin Gly Thr Lys Val Glu Ilie Lys Arq Thr <210> 111 <211> 411 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (411) <223> <400> 111 atg gag ttt ggg ctg Met Glu Phe Gly Leu agc tgg gtt ttc ctt gtt gct ata tta aaa ggt Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly gtc cag tgt gag qtt cag ctg gtg cag tct ggg gga ggc ttg gta cag 57/62 Val Gin Cys Glu Val ccc ggg ggg tcc ctg Pro Gly Gly Ser Leu agt agc ttt gct atg Ser Ser Phe Ala Met gag tgg ata tca gtt Glu Trp Ile Ser Val tcc gtg aag ggc cga Ser Val Lys Gly Arg ttg tat ott caa atg Leu Tyr Leu Gin Met 100 tac tgt gca aga ctg Tyr Cys Ala Arg Leu 115 caa gqg acc acg gtc Gin Gly Thr Thr Vai 130 <210> 112 <211> 137 <212> PRT <213> Homno sapiens Gin 25 t gt Cys cgc Arg cgt Arg t cc Ser a ga Arg 105 tac Tyr t ca Ser Gly tct Ser cca Pro aca Thr aat As n ga c Asp atg Met Leu ttc Phe aaa Lys tat T yr aag Lys gct Al a 110 gt c Val1 Val1 acc Thr ggt Gi y gca Ala aac As n gtg Val tgg Trp Gin ttc Phe ctq Leu ga c Asp tcc Ser tat Tyr ggc Gly 144 192 240 288 336 384 <400> 112 Met Giu Phe 1 Val Gin Cys Pro Gly Gly Ser Ser Phe Gly Leu 5 Glu Val Ser Trp Val Phe Leu 10 Ser Val Ala Ile Leu Lys Gly Gin Leu Val Gly Gly Gly Ser Leu Arg Leu Ala Ala Ser Gly Gly Leu Val Gin Phe Thr Phe Lys Gly Leu Ala Met His Arg Glu Trp Ile Ser Val Ile Gin Ala Pro Gly Ala Thr Thr Arg 58/62 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Leu Tyr Leu Gin Met Asn Ser Leu Arg Asp Asn Ala Lys Asn Ser Ala Giu Asp Thr Ala Val Tyr 110 Val Trp Gly Tyr Cys Ala Arg Leu Gly Asn Tyr Tyr Gly Met Asp 125 Gin Gly 130 Thr Thr Val Thr Val Ser Ser 135 <210> 113 <211> <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 113 gag gtt cag Giu Val Gin 1 tcc ctq aqa Ser Leu Arg ctq gtg cag tct ggg gga Leu Val Gin Ser Giy Giy 5 ggc ttg gta cag cct qqq ggg Gly Leu Vai Gin Pro Giy Giy 10 ct c Leu tcc tqt gca gc Ser Cys Ala Ala t ct Se r <210> 114 <211> <212> PRT <213> Homo sapiens <400> 114 59/62 Glu Val Gin 1 Ser Leu Arg Leu Val 5 Leu Ser Gin Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 10 Cys Ala Ala 00 N~ <210> 115 <211> 42 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <223> <400> 115 tgg gtt cgc Trp Val Arg 1 <210> 116 <211> 14 <212> PRT cag gct cca gga aaa ggt ctg gag tgg ata tca Gin Ala Pro Gly Lys Gly Leu Glu Trp Ile Ser 5 <213> Homo sapiens <400> 116 Trp Val Arg 1 Gin Ala Pro Gly Lys Gly Leu Glu Trp Ile Ser <210> 117 <211> 96 <212> DNA <213> Homo sapiens 60/62 <220> <221> CDS <222> (96) <223> <400> 117 aga ttc acc atc Arg Phe Thr Ile tc Ser aga gac aat cc Arg Asp Asn Ala aac tcc ttg tat Asn Ser Leu Tyr ctt caa Leu Gin atg aac aqc Met Asn Ser 118 <2ii> 32 <212> PRT aga gcc gag gac Arg Ala Giu Asp act T hr gct gtg tat tac Ala Val Tyr Tyr tgt gca aga Cys Ala Arg <213> Homo sapiens <400> 118 Arg Phe Thr Ile I Arq Asp Asn Ala Asn Ser Leu Tyr Leu Gin Met Asn Ser Arq Ala Giu Asp Aia Val Tyr Tyr Cys Ala Arg <210> 119 <211> 33 <212> DNA <213> Homo sapiens <220> <221> CES <222> <223> <400> 119 61/62 tgg ggc caa Trp Gly Gin ggg acc acg gtc acc gtc Gly Thr Thr Val Thr Val tcc tca 33 Ser Ser <210> 120 <211>. 11 <212> PRT <213> Homo sapiens <400>t 120 Trp, Gly Gin 1 Gly Thr Thr Val Thr Val Ser Ser 5 62/62

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2011-03-03| FGA| Letters patent sealed or granted (standard patent)|

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2012-10-11| DA2| Applications for amendment section 104|Free format text: THE NATURE OF THE AMENDMENT IS: AMEND THE PATENTEE TO READ MERCK SHARP & DOHME CORP. . |

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2015-12-17| MK14| Patent ceased section 143(a) (annual fees not paid) or expired|

优先权:

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申请号 | 申请日 | 专利标题

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US38345902P| true| 2002-05-24|2002-05-24||

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US60/383,459||2002-05-24||

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US39321402P| true| 2002-07-02|2002-07-02||

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US60/393,214||2002-07-02||

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US43625402P| true| 2002-12-23|2002-12-23||

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US60/436,254||2002-12-23||

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AU2003241590A|AU2003241590B2|2002-05-24|2003-05-22|Neutralizing human anti-IGFR antibody|

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AU2007200876A|AU2007200876B2|2002-05-24|2007-02-28|Neutralizing human anti-IGFR antibody|AU2007200876A| AU2007200876B2|2002-05-24|2007-02-28|Neutralizing human anti-IGFR antibody|

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AU2010227116A| AU2010227116A1|2002-05-24|2010-10-08|Neutralizing human anti-IGFR antibody|