Bispecific EGFR / c-met antibodies

Abstract

To provide bispecific EGFR / c-Met antibodies, and methods of making and using the molecules.SOLUTION: An isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor (c-Met) comprising a first heavy chain (HC1) comprising a HC1CH3 constant domain 3 (HC1) and a HC1 variable region 1 (VH1), a second heavy chain (HC2) comprising a HC2CH3 constant domain 3 (HC2) and a variable region 2 (VH2), a first light chain () comprising a light chain variable region (), and a second light chain () comprising a light chain variable region (). HC2 LC2 VL1 VL2 LC1, bispecific antibodies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to bispecific EGFR / c-Met antibodies, as well as methods for producing and using such molecules. Regarding the method. [Background technology]

[0002] The epidermal growth factor receptor (EGFR, ErbB1 or HER1) is a c-erbB1 oncogenic gene. EGFR is a 170 kDa type I transmembrane glycoprotein encoded by the gene These include HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). The human epidermal growth factor receptor (HER) family of receptor tyrosine kinases (RTKs) Signaling by EGFR is mediated by other Erb receptors following ligand binding. Conformational changes in receptors by members of the B family, homodimerization, or heterodimerization Helodimerization and trans-autophosphorylation of the receptor (Ferguson et al., Annu Rev Biophys, 37:353-73, 2008) This initiates a signal transduction cascade that ultimately leads to cell proliferation and Affects various cell functions, including survival. Increased expression of EGFR has been associated with a wide range of human cancers, making it attractive for therapeutic intervention. It has become a popular target (Mendelsohn et al., Oncogene 19 :6550~6565,2000;Grunwald et al.,J Natl C ancer Inst 95:851~67,2003;Mendelsohn et. al., Semin Oncol 33:369-85, 2006). Both copy number and protein expression gains are associated with EGFR transcriptional regulation in non-small cell lung cancer. Favorable response to the steroid kinase inhibitor IRESSA™ (gefitinib) (Hirsch et al., Ann Oncol 18:7 52-60,2007).

[0003] EGFR therapy is used in colorectal cancer, pancreatic cancer, head and neck cancer, and non-small cell lung cancer (NSCLC). This includes both small molecule compounds and anti-EGFR antibodies approved for the treatment of ga and Arteaga, J Clin Oncol 23:2445~2459 (20005;Gill et al.,J Biol Chem,259:7755~ 7760,1984;Goldstein et al., Clin Cancer R es,1:131 1~1318;1995;Prewett et al.,Clin Cancer Res, 4:2957~2966, 1998).

[0004] The efficacy of anti-EGFR therapy varies depending on tumor type and EGFR mutation / amplification status of the tumor. Side effects of current therapies can include skin toxicity (De Roock et al. al.,Lancet Oncol 11:753~762,2010;Linard ou et al., Nat Rev Clin Oncol, 6:352~366,2 009;Li and Perez-Soler,Targ Oncol 4:107~ 119, 2009). EGFR tyrosine kinase inhibitors (TKIs) are effective in treating non-small cell lung cancer (NSCLC) It is widely used as a second-line therapy for SCLC, but due to resistance pathways, The effect often wears off within 2 months (Riely et al., Clin Can cer Res 12:839~44,2006).

[0005] c-Met encodes a transmembrane tyrosine kinase receptor, which is involved in the regulation of certain oncogenic compounds. Treatment with TPR-MET results in a fusion protein that exhibits constitutive activity. It was first identified as a proto-oncogene in 1984 after it was discovered that (Nature 311:29-33, 1984). Ligand for c-Met Activation of c-Met by hepatocyte growth factor (HGF), a marker of cell proliferation, motility, invasion, and migration, is essential for the development of cell proliferation. A plethora of cellular processes, including migration, epithelial-mesenchymal transition, angiogenesis / wound healing, and tissue regeneration. stimulates the immune system (Christensen et al., Cancer Lett 22 5:1~26,2005;Peters and Adjei,Nat Rev Cli n Oncol 9:314-26, 2012). c-Met is bound by disulfide bonds. The protein is composed of a 50 kDa α subunit and a 140 kDa β subunit bound together by It is synthesized as a single-chain protein that is cleaved by proteolysis (Ma et al., C ancer and Metastasis Reviews,22:309~325, 2003). c-Met is structurally similar to other membrane receptors such as RON and Sea. Although the exact stoichiometry of HGF:c-Met binding has not been elucidated, it is generally believed that two Binding of an HGF molecule to two c-Met molecules results in receptor dimerization and tyrosine 12 binding. This is thought to lead to autophosphorylation at 30, 1234, and 1235 (St amos et al.,The EMBO Journal 23:2325~233 5, 2004). Gene amplification, mutation, or receptor overexpression can result in ligand-independent Autophosphorylation of c-Met can also occur.

[0006] c-Met is a cancer that affects the stomach, lung, colon, breast, bladder, head and neck, ovarian, prostate, and thyroid cancers. Amplified in many types of cancer, including adenocarcinoma, pancreatic cancer, and cancer of the central nervous system (CNS), Mutations or overexpression are common. Missense mutations are usually confined to the kinase domain. in hereditary papillary renal cell carcinoma (PRCC) and in 13% of sporadic PRCC commonly seen (Schmidt et al., Oncogene 18:2343 ~2350, 1999). It is localized to the semaphorin or juxtamembrane domain of c-Met. Mutations in c-Met are frequently found in gastric, head and neck, liver, ovarian, NSCLC, and thyroid cancers. This is often seen (Ma et al., Cancer and Metastasis Research eviews,22:309~325,2003;Sakakura et al.,C Hromosomes and Cancer, 1999.24:299~305). c Amplification of -Met has been detected in brain, colorectal, gastric, and lung cancers, These often correlate with disease progression (Ma et al., Cancer and Metastasis Reviews, 22:309~325, 2003). non-small Up to 4% and 20% of non-small cell lung cancers (NSCLC) and gastric cancers, respectively, show c-Met amplification. (Sakakura et al., Chromosomes and Can cer,1999.24:299~305:Sierra and Tsao,Ther Apeutic Advances in Medical Oncology,3:S 21-35, 2011). In lung cancer, even if gene amplification is not observed, Overexpression is often observed (Ichimura et al., Jpn J Can Cer Res, 87:1063-9, 1996). Furthermore, in clinical samples, almost all lung adenocarcinomas Half of the patients had high levels of leukemia, which have been shown to correlate with increased tumor growth rate, metastasis, and poor prognosis. c-Met and HGF were shown (Sierra and Tsao, Therapeutics utic Advances in Medical Oncology,3:S21~ 35,2011;Siegfried et al.,Ann Thorac Surg 66:1915~8,1998).

[0007] Approximately 60% of all tumors that become resistant to EGFR tyrosine kinase receptor inhibitors, c Increased expression of c-Met, amplification of c-Met, or the only known ligand of c-Met Increased HGF expression due to steroid hormone (Turke et al., Cancer Cell, 17:77-8) 8, 2010), suggesting the existence of a compensatory pathway of EGFR via c-Met. Amplification of c-Met is associated with resistance to gefitinib, an EGFR kinase inhibitor. This was first confirmed in cultured cells, where it was found to be highly viable via the Her3 pathway. (Engelman et al.,Science,316:1039~43,200 7) This indicates that only 2 of 62 untreated patients had c-Met amplification. 43 patients developed resistance to erlotinib or gefitinib, compared with 16 patients Nine of these patients were further confirmed with clinical samples showing c-Met amplification. Four of the patients also had an activating EGFR mutation, T790M, which was present at the same time. (Beat et al., Proc Natl Acad Sc i USA,104:20932~7,2007).

[0008] The individual roles of EGFR and c-Met in cancer are well established. Both receptors mediate the same survival pathways, making these targets attractive for combination therapy. signaling through pro- and anti-apoptotic pathways (ERK and AKT) Therefore, inhibiting this pairing may limit the activation of compensatory pathways. This can limit the number of EGFR and c-Met receptors, thereby improving overall efficacy. Targeted combination therapy with Tarceva® (erlotinib) in NSCLC in combination with an anti-c-Met monovalent antibody (Spigel et al., 2011 ASCO Annual Meeting Proceedings 2011,Jou clinical oncology:Chicago,IL.p.7 505), and Tarceva (erlotinib) and AR, a small molecule inhibitor of c-Met. Combination with Q-197 (Adjei et al., Oncologist, 16: Clinical trials are being conducted using combination or bispecific antibodies. For the EGFR / c-Met molecule, see, for example, International Patent Publication No. WO2008 / 1277 10, WO2009 / 111691, WO2009 / 126834, WO2010 / 039248, WO2010 / 115551, and U.S. patent applications Disclosed in Publication No. US2009 / 0042906.

[0009] Current therapeutic approaches that antagonize the EGFR and / or c-Met signaling pathways Small and large molecule therapeutic approaches have demonstrated specificity that may be seen with small molecule inhibitors. Suboptimal due to potential lack of activity, potential off-target activity, and dose-limiting toxicity. Common monospecific bivalent antibodies inhibit membrane-bound receptor clustering and downstream signaling pathways Monovalent antibodies with full-length heavy chains (half arms) can lead to unwanted activation of the antibody. This makes the manufacturing process significantly more complex and costly. Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, further monospecific and bispecific EGFs for both therapeutic and diagnostic purposes are available. R and / or c-Met inhibitors are needed. [Means for solving the problem]

[0011] One embodiment of the present invention is an isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte a cell growth factor receptor (c-Met) antibody, The first gene includes HC1 constant domain 3 (HC1 CH3) and HC1 variable domain 1 (VH1). and the heavy chain (HC1) of a second domain comprising HC2 constant domain 3 (HC2 CH3) and HC2 variable domain 2 (VH2); and the heavy chain (HC2) of a first light chain (LC1) comprising a light chain variable region (VL1); a second light chain (LC2) comprising a light chain variable region (VL2), wherein the VH1 and the V L1 pairs with VH2 to form a first antigen-binding site that specifically binds to EGFR, and VL2 pair to form a second antigen-binding site that specifically binds to c-Met, HC1 contains at least one substitution in said HC1 CH3, and said HC2 contains at least one substitution in said HC2 at least one substitution in CH3, If the substitution in CH3 is: A bispecific antibody is one in which the two antibodies are generated at different amino acid residue positions.

[0012] In another embodiment, the present invention provides a bispecific EGFR / c-Met antibody, In this case, the antibody was detected in NCI-H292, NCI-H1975 or SKMES-1 cell lines. inhibits the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 / 2) in the C 50 Values ​​are for a control monovalent EGFR antibody containing heavy chain 3 (HC3) and light chain 3 (LC3). and a control monovalent c-Met antibody containing heavy chain 4 (HC4) and light chain 4 (LC4). Effect of the compound on NCI-H292, NCI-H1975, or SKMES-1 cell lines IC for inhibition of RK1 / 2 phosphorylation 50 At least about 10 times lower, or at least about 20 times lower, at least about 30 times lower, at least about 40 times lower, at least about 50-fold lower, or at least about 60-fold lower IC 50 value, where HC3 and HC1, L C3 and LC1, HC4 and HC2, and LC4 and LC2 each have the same amino acid sequence. The phosphorylation of ERK1 / 2 was measured by electrochemical analysis using anti-phospho-ERK1 / 2 antibody as a capture antibody. The detection antibody was an antibody that binds to non-phosphorylated and phosphorylated ERK1 / 2 conjugated with a photochemical compound. It is measured in whole cell lysates using a sandwich immunoassay.

[0013] In another embodiment, the present invention provides a bispecific EGFR / c-Met antibody, In the case of the antibody, the antibody inhibits the expression of protein kinase B (AKT) in the NCI-H1975 cell line. Inhibits phosphorylation at Ser473 and its IC 50 The values ​​are for components containing HC3 and LC3. Control monovalent EGFR antibodies and control monovalent c-Met antibodies including HC4 and LC4 Phosphorylation of Ser473 of AKT in NCI-H1975 cells by a mixture of IC of inhibition of cytotoxicity 50 At least approximately 70-fold lower IC compared to the 50 value, where , HC3 and HC1, LC3 and LC1, HC4 and HC2, LC4 and LC2 are the same The phosphorylation of Ser473 in AKT is The antibody that binds to phospho-AKT was used as the capture antibody, and anti-phospho-AKT conjugated with an electrochemiluminescent compound was used. A sandwich immunoassay using T Ser473 antibody as the detection antibody was performed. Measured in cell lysates.

[0014] In another embodiment, the present invention relates to the EGFR of SEQ ID NO: 73 and EGFR residue K489, It binds to c-Met at I491, K467, ​​and S492 and to residues PEFRDSYPIKYV HAF (SEQ ID NO: 238) and FAQSKPDSAEPMDRSA (SEQ ID NO: 239) A bispecific EGFR / c-Met antibody that binds is provided.

[0015] In another embodiment, the present invention relates to the proliferation of NCI-H292 or NCI-H1975 cells. The growth of NCI-H292 or NCI-H1975 cells was measured by the cell culture method. IC of inhibition of proliferation of NCI-H292 or NCI-H1975 cells by tuximab 50 value at least about 300 times lower, at least about 400 times lower, at least about 500 times lower, at least about 600 times lower, at least about 700 times lower, at least about 800 times lower, at least about 900 times lower, at least about 1000 times lower, at least about 1100 times lower, at least about 1200 times lower, at least about 1300 times lower, at least about 1400 times lower, at least about 500 times lower, at least about 600 times lower, at least about 700 times lower, or at least Approximately 800 times lower IC 50 The present invention provides a bispecific EGFR / c-Met antibody that inhibits EGFR at a low level.

[0016] In another embodiment, the present invention provides a method for administering a bispecific antibody and cetuximab at a concentration of 20 mg / kg. HGF-expressing SKMES in SCID Beige mice when administered at a dose of -1 cell tumor growth at least 500-fold lower T / T at day 36 compared to cetuximab The present invention provides a bispecific EGFR / c-Met antibody that inhibits C values ​​(%).

[0017] In another embodiment, the present invention provides a bispecific EGFR / c-Met antibody, where HC1CH3 contains a substitution of K409R or F405L and HC2CH3 contains a substitution of K4 and the substitutions F409R or F405L, where residue numbering is according to the EU index. It is something that happens.

[0018] In other embodiments, the present invention provides specific heavy and light chain CDRs, VH1, VL1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, Bispecific EGFR / c- containing H2, VL2, HC1, LC1, HC2 and LC2 sequences Met antibodies are provided.

[0019] Another embodiment of the invention is a nucleic acid encoding the HC1, HC2, LC1, or LC2 of the invention. It is an isolated synthetic polynucleotide.

[0020] Another embodiment of the present invention is a vector comprising a polynucleotide of the present invention.

[0021] Another embodiment of the present invention is a host cell comprising a vector of the present invention.

[0022] Another embodiment of the present invention produces isolated bispecific EGFR / c-Met antibodies. 1. A method comprising: An isolated single-stranded antibody comprising two heavy chains of SEQ ID NO: 199 and two light chains of SEQ ID NO: 200. An isomeric bivalent anti-EGFR antibody and two heavy chains of SEQ ID NO: 201 and two light chains of SEQ ID NO: 202. and an isolated monospecific bivalent anti-c-Met antibody comprising the α- and β-chains, in a molar ratio of about 1:1. and adding them together, introducing a reducing agent into the mixture; incubating the mixture for about 90 minutes to about 6 hours; removing the reducing agent; A first heavy chain of SEQ ID NO: 199 and a second heavy chain of SEQ ID NO: 201, and a second heavy chain of SEQ ID NO: 200 and a second light chain of SEQ ID NO: 202. and purifying said first heavy chain of SEQ ID NO: 199 with said second heavy chain of SEQ ID NO: 200. a first light chain that pairs with the first binding domain to specifically bind to EGFR, The second heavy chain of SEQ ID NO: 201 pairs with the second light chain of SEQ ID NO: 202 to specifically bind c-Met. forming a second binding domain that binds heterologously to the target protein.

[0023] Another embodiment of the invention comprises a bispecific antibody of the invention and a pharmaceutically acceptable carrier. It is a pharmaceutical composition.

[0024] Another embodiment of the present invention is a method of treating a subject having cancer, comprising administering a therapeutically effective amount of The bispecific EGFR / c-Met antibodies of the invention can be administered to patients in need thereof to treat cancer. The method comprises administering the compound for a sufficient period of time.

[0025] Another embodiment of the present invention is directed to the growth or proliferation of cells expressing EGFR and / or c-Met. A method for inhibiting proliferation of a cell, the method comprising contacting the cell with a bispecific antibody of the invention. is.

[0026] Another embodiment of the present invention is a method for the detection of EGFR and / or c-Met expressing tumor or cancer cells in a subject. A method for inhibiting proliferation or metastasis, comprising administering to said subject an effective amount of a bispecific antibody of the invention. to inhibit the growth or metastasis of EGFR- and / or c-Met-expressing tumor or cancer cells. The method includes: [Brief explanation of the drawings]

[0027] [Figure 1A] Amino acid alignment of the EGFR-binding FN3 domain. The BC and FG loops are shown boxed at residues 22-28 and 75-86 of SEQ ID NO: 18. Some mutants contain L17A, N46K, and E86I substitutions that increase thermostability (residue numbering is based on Tencon (SEQ ID NO: 1)). [Figure 1B] Amino acid alignment of the EGFR-binding FN3 domain. The BC and FG loops are shown boxed at residues 22-28 and 75-86 of SEQ ID NO: 18. Some mutants contain L17A, N46K, and E86I substitutions that increase thermostability (residue numbering is based on Tencon (SEQ ID NO: 1)). [Figure 2] Sequence alignment of the Tencon27 backbone (SEQ ID NO: 99) and the TCL14 library (SEQ ID NO: 100) with a randomized C-CD-F-FG alternative surface. Loop residues are boxed. Loops and strands are indicated above the sequences. [Figure 3]Sequence alignment of the c-Met-binding FN3 domain. The C-loop and CD strand, and the F-loop and FG strand are boxed and span residues 29-43 and 65-81. [Figure 4] Figure 1 shows inhibition of c-Met phosphorylation in NCI-H292 cells pretreated with monospecific or bispecific FN3 domain-containing molecules and stimulated with HGF. A significant increase in the potency of the bispecific EGFR / c-Met molecule (ECB1) was observed when compared with the monospecific c-Met-binding FN3 domain (P114AR5P74-A5, designated A5 in the figure) alone or in combination with the EGFR-binding FN3 domain (P54AR4-83v2, designated 83v2 in the figure). [Figure 5] Inhibition of EGFR and c-Met phosphorylation in cells pretreated with monospecific or bispecific FN3 domain-containing molecules. In cell lines expressing high levels of EGFR, NCI-H292 (Figure 5A) and H596 (Figure 5B), anti-EGFR monospecific and bispecific FN3 domain-containing molecules were similarly effective in reducing EGFR phosphorylation. In cell lines expressing low levels of EGFR relative to c-Met, NCI-H441 (Figure 5C), the bispecific EGFR / c-Met molecule improved the potency of inhibiting EGFR phosphorylation compared with the monospecific EGFR-binding FN3 domain alone. In cell lines expressing low levels of c-Met relative to EGFR, NCI-H292 (Figure 5D) and H596 (Figure 5E), the bispecific EGFR / c-Met molecule significantly increased the inhibitory effect of c-Met phosphorylation compared with the monospecific c-Met-binding FN3 domain alone. The molecules used in this study were the bispecific ECB5 (shown as 17-A3 in the figure), the monospecific EGFR-binding FN3 domain P53A1R5-17 (shown as 17 in the figure), the bispecific EGFR / c-Met molecule ECB3 (shown as 83-H9 in the figure), and the monospecific c-Met-binding FN3 domain P114AR7P93-H9 (shown as H9 in the figure). [Figure 6]Pharmacodynamic signaling in tumors isolated from mice treated with bispecific EGFR / c-Met molecules for 6 or 72 hours. Both molecules significantly reduced phosphorylation of c-Met, EGFR, and ERK after 6 and 72 hours, with the extent of inhibition dependent on the affinity of the FN3 domain for EGFR and / or c-Met. Bispecific molecules were generated by linking a high-affinity (83 in the figure is p54AR4-83v2) or intermediate-affinity (17v2 in the figure is P53A1R5-17v2) EGFR-binding FN3 domain with a high-affinity (A3 in the figure is P114AR7P94-A3) or intermediate-affinity (A5 in the figure is P114AR5P74-A5) c-Met-binding FN3 domain. [Figure 7] Plasma (top panel) and tumor (bottom panel) accumulation of bispecific EGFR / c-Met molecules with different affinity linked to albumin-binding domains (ABDs) is shown 6 hours (left panel) and 72 hours (right panel) after IP administration. At 6 hours, tumor accumulation was greatest in mice treated with bispecific molecules bearing either a medium-affinity EGFR-binding FN3 domain (17v2) or a high-affinity EGFR-binding domain (83v2). Bispecific molecules incorporated high- or medium-affinity EGFR- or c-Met-binding FN3 domains: 83v2-A5-ABD (ECB18; high / medium for EGFR / c-Met), 83v2-A3-ABD (ECB38; high / high), 17v2-A5 (ECB28; medium / medium), and 17v2-A3-ABD (ECB39; medium / high). In the figure, 83v2 refers to p54AR4-83v2, 17v2 refers to p53A1R5-17v2, A3 refers to p114AR7P94-A3, and A5 refers to p114AR5P74-A5. [Figure 8]H292-HGF tumor xenografts were implanted into SCID Beige mice. When tumors reached an average volume of approximately 80 mm, the mice were treated with the bispecific EGFR / c-Met molecule (25 mg / kg) or PBS vehicle three times a week. Both bispecific molecules reduced tumor growth, with the tumor growth inhibition (TGI) dependent on the molecule's affinity for c-Met and EGFR (high EGFR-high cMet refers to p54AR4-83v2-p114AR7P94-A3 (ECB38); high EGFR-med cMet refers to p54AR4-83v2-p114AR5P74-A5 (ECB18); medium EGFR-high cMet refers to p53A1R5-17v2-p114AR7P94-A3 (ECB39); and medium EGFR-med cMet refers to p53A1R5-17-p114AR5P74-A5 (ECB28)). [Figure 9] H292-HGF tumor xenografts were implanted into SCID Beige mice, and the mice were treated with different therapeutic agents. The antitumor activity of the therapeutic agents was demonstrated. (The bispecific EGFR / c-Met molecule refers to p54AR4-83v2-p114AR7P94-A3-ABD (ECB38). Other therapeutic agents included crizotinib, erlotinib, cetuximab, and a combination of crizotinib and erlotinib.) [Figure 10] SKMES-HGF tumor xenografts were implanted into SCID Beige mice, and the mice were treated with different therapeutic agents. The antitumor activity of the therapeutic agents is shown as the change in tumor size (mm3) over time. The bispecific EGFR / c-Met antibody EM1-mAb was administered intraperitoneally (ip) at 20 mg / kg, 5 mg / kg, or 1 mg / kg twice weekly, and cetuximab was administered intraperitoneally at 20 mg / kg twice weekly. Arrows in the figure indicate the days of administration. The numbers after the antibodies indicate the administered dose. [Figure 11] HCC827 tumor xenografts were implanted into nude mice and treated with the indicated doses of erlotinib or EM1-mAb. EM1-mAb was administered twice weekly and erlotinib once daily for 4 weeks. Arrows indicate the days of administration. Antitumor activity of the therapeutic agents is shown as the change in tumor size (mm3) over time. [Figure 12]SNU-5 tumor xenografts were implanted into CB17 / SCID mice, which were then treated with 10 mg / kg cetuximab or 10 mg / kg or 1 mg / kg EM1-mAb. The antibodies were administered twice weekly for four weeks. Arrows indicate the days of administration. The antitumor activity of the therapeutic agents is shown as the change in tumor size (mm3) over time. [Figure 13] H1975-HGF tumor xenografts were implanted into nude mice and treated with 10 mg / kg cetuximab, 10 mg / kg EM1-mAb, 50 mg / kg erlotinib, 15 mg / kg afatinib, or a combination of 10 mg / kg EM1-mAb and 15 mg / kg afatinib. The antibodies were administered twice weekly, and the small molecules were administered once daily for three weeks. Arrows indicate the days of administration. The antitumor activity of the therapeutic agents is shown as the change in tumor size (mm3) over time. [Figure 14] HCC827-ER1 tumor xenografts were implanted into nude mice, which were then treated with 10 mg / kg of EM1-mAb, 25 mg / kg of erlotinib, or a combination of these. EM1-mAb was administered twice weekly and erlotinib once daily for 19 days. Arrows indicate the days of administration. The antitumor activity of the therapeutic agents is shown as the change in tumor size (mm3) over time. [Figure 15] Mean EGFR and c-Met levels in tumor lysates isolated from H1975HGF tumor xenografts implanted in SCID Beige mice after a single dose of 20 mg / kg EM1-mAb. Receptor levels are shown as % of PBS controls at the indicated times after treatment. [Figure 16] H1975-HGF tumor xenografts were implanted into nude mice and treated with 10 mg / kg of EM1-mAb or 10 mg / kg of the EM1-mAb mutant IgG2V234A / G237A / P238S / H268A / V309L / A330S / P331S, which lacks Fc receptor binding and effector function. The antibody was administered twice weekly on the indicated days. The antitumor activity of the therapeutic agents is shown as the change in tumor size (mm) over time. DETAILED DESCRIPTION OF THE INVENTION

[0028] As used herein, "fibronectin type III (FNIII) domain (F The term "N3 domain" refers to proteins such as fibronectin, tenascin, and intracellular cytoskeletal proteins. frequently found in proteins, including proteins, cytokine receptors, and prokaryotic enzymes. Maine (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990~8994,1992;Meinke et al., J Bacteriol 175:1910~1918,1993;Watanabe et al., J Biol Chem 265:15659~15665, 1990) Representative FN3 domains are the 15 different FN3 domains present in human tenascin-C. 15 different FN3 domains present in human fibronectin (FN), and e.g. For example, the non-natural synthetic FN3 domains described in U.S. Patent Application Publication No. 2010 / 0216708. Individual FN3 domains are identified by domain number and protein name, e.g., The third FN3 domain of tenascin (TN3) or the tenth FN3 domain of fibronectin It is called Inn (FN10).

[0029] As used herein, "substituting" or "substituted" or "mutating" means Alternatively, the term "mutated" refers to one or more mutations in a polypeptide or polynucleotide sequence. By changing, deleting or inserting amino acids or nucleotides in the It refers to achieving something.

[0030] As used herein, "randomize" or "randomized" or " The term "diversified" or "diversifying" refers to a polynucleotide or polypeptide It refers to making at least one substitution, insertion, or deletion in a sequence.

[0031] As used herein, the term "mutant" refers to a variant that includes, for example, a substitution, insertion, or deletion. and differ from a reference polypeptide or polynucleotide by one or more modifications of It refers to a polypeptide or polynucleotide that

[0032] As used herein, "specifically bind" or "specifically bound" means The term refers to a given antigen and approximately 1 x 10 -6 M or less, for example, about 1 × 10 -7 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, approximately 1×10 -12 M or less, or about 1 x 10 -13 The dissociation constant (K D ) FN3 domain of the present invention Main, a bispecific agent that specifically binds to EGFR and c-Met, or a bispecific E Generally, the FN3 domains of the present invention, EGFR and c-Met antibodies, and c-Met, or bispecific EGFR / c-Met Antibodies can be analyzed by surface plasmon resonance using, for example, a Proteon instrument (BioRad). Its K for non-specific antigens (e.g., BSA or casein) measured by D Less than At least 10 times smaller K D binds to a given antibody (i.e., EGFR or c-Met) Therefore, the bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention, E Bispecific agents that specifically bind to GFR and c-Met, or bispecific EGFR / c -Met antibody has at least about 1 x 10 -6 M or less, for example Approximately 1×10 -7 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less , about 1×10 -11 M or less, approximately 1×10 -12 M or less, or about 1 x 10 -13 Binding affinity below M (K D However, the bispecific antibodies of the present invention that bind to a given antigen EGFR / c-Met FN3 domain-containing molecule, specifically binds to EGFR and c-Met Bispecific drugs combining EGFR and c-Met or bispecific EGFR / c-Met antibodies may be used to treat other related antigens. , for example, may have cross-reactivity with the same given antigen from other species (homologues).

[0033] The term "library" refers to a collection of variants. The nucleic acid sequence may consist of a nucleic acid variant.

[0034] As used herein, the term "stability" refers to, for example, the stability of EGFR or c-Met so as to maintain at least one of its normal functional activities, such as binding to a predetermined antigen. Refers to the ability of a molecule to remain folded under physiological conditions.

[0035] As used herein, "epidermal growth factor receptor" or "EGFR" refers to the gene encoding the epidermal growth factor receptor (EGFR) of SEQ ID NO: 7 3 and the amino acid sequence shown in GenBank accession number NP_005219. Human EGFR (also known as HER1 or ErbB1) and its naturally occurring variants It is known (Ullrich et al., Nature 309:418-425, 1 984). Such variants include the well-known EGFRvIII and other Alternatively spliced ​​variants of (e.g., SwissProt accession no. P00533-1 (wild type; same as SEQ ID NO: 73 and NP_005219), P0053 3-2(F404L / L405S), P00533-3(628~705:CTGPGL EGCP...GEAPNQALLR→PGNESLKAML...SVIITASSC H, and deletions 706-1210), P00533-4 (C628S and 629-121 0), mutants GlnQ98, R266, K521, I674, G962, and P9 88(Livingston et al.,NIEHS-SNPs,environm ental genome project, NIEHS ES15478), T790 M, L858R / T790M, and del (E746, A750) can be done.

[0036] As used herein, "EGFR ligand" includes EGF, TGFα, heparin, Bound EGF (HB-EGF), amphiregulin (AR), and epiregulin (EPI) These include all (eg, physiological) ligands of EGFR, such as:

[0037] As used herein, "epidermal growth factor (EGF)" refers to the EGF protein shown in SEQ ID NO: 74. It refers to the well-known human EGF, which consists of 53 amino acids and has the amino acid sequence shown below.

[0038] As used herein, "hepatocyte growth factor receptor" or "c-Met" refers to the receptor having the sequence No. 101 or Genbank accession number NP_001120972 It refers to human c-Met and its naturally occurring variants having the amino acid sequence

[0039] As used herein, "hepatocyte growth factor" (HGF) refers to a factor that is cleaved to form hepatocyte growth factor (HGF). SEQ ID NO: 10, which forms a dimer of α and β chains linked by disulfide bonds. 2.

[0040] As used herein, "blocking binding" or "inhibiting binding" means EGFR ligands such as EGF for GFR and / or HGF for c-Met The FN3 domain of the present invention, a bispecific EGFR / c- Met FN3 domain-containing molecule, a bispecific molecule that specifically binds EGFR and c-Met This refers interchangeably to the ability of a specific drug, or a bispecific EGFR / c-Met antibody, to The FN3 domains of the present invention include partial and complete blocking / inhibition. EGFR / c-Met FN3 domain-containing molecule, specifically binds to EGFR and c-Met Anti-EGFR therapy with a bispecific drug combining EGFR with a bispecific EGFR / c-Met antibody Blockade of EGFR ligands, such as EGF for c-Met and / or HGF for c-Met. Blocking / inhibition refers to the binding of EGFR ligands to EGFR without blocking or inhibiting them. and / or HGF binding to c-Met compared to EGFR signaling and / or cM The FN3 domain of the present invention partially or completely reduces the normal level of et signaling. EGFR / c-Met FN3 domain-containing bispecific molecule Bispecific agents that specifically bind to EGFR or bispecific c-Met antibodies can inhibit Harm rates of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, and 65% , 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or 100%. Blocks the binding of EGFR ligands, such as HGF and / or c-Met. Inhibition of binding can be determined, for example, using FACS and the methods described herein. FN3 domains of the present invention, bispecific EGFR / c-Met FN3 domain-containing molecules, Bispecific agents that specifically bind to EGFR and c-Met, or bispecific EGFR / Inhibition of biotinylated EGF binding in EGFR-expressing A431 cells exposed to c-Met antibodies c-Met assays are performed to measure the toxicity or to detect c-Met activity using known methods and those described herein. Using well-known methods, such as measuring inhibition of binding of biotinylated HGF to the extracellular domain, It can be measured.

[0041] The term "EGFR signaling" refers to the autotransfer of at least one tyrosine residue of EGFR. Signaling induced by binding of EGFR ligands to EGFR, leading to autophosphorylation One of the major EGFR ligands is EGF.

[0042] As used herein, "neutralizing EGFR signaling" refers to neutralizing EGFR signaling. at least 30% of EGFR signaling induced by EGFR ligands such as F , 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% inhibition of the FN3 domain of the present invention, bispecific EGFR / cM etFN3 domain-containing molecule, a bispecific drug that specifically binds to EGFR and c-Met This refers to the ability of a therapeutic agent, or a bispecific EGFR / c-Met antibody, to

[0043] The term "c-Met signaling" refers to the activation of at least one tyrosine residue of c-Met. Signal transduction induced by HGF binding to c-Met, leading to autophosphorylation of c-Met. Usually, HGF binding induces 1230, 1234, and 12 At least one tyrosine residue at position 35 or 1349 is autophosphorylated.

[0044] As used herein, "neutralizing c-Met signaling" refers to GF-induced c-Met signaling by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 The FN3 domains of the present invention, including the bispecific EGFR / c-Met FN3 domain, inhibit a bispecific agent that specifically binds to EGFR and c-Met, or a bispecific This refers to the ability of specific EGFR / c-Met antibodies.

[0045] As used herein, "overexpress," "overexpressed," and "overexpressed" "has" the expression of EGFR and / or c-Met on the surface compared to normal cells of the same tissue type. These terms interchangeably refer to cancer or malignant cells in which the level of leukemia is measurably higher. Such overexpression can be caused by gene amplification or by increased transcription or translation. Expression and overexpression of EGFR and / or c-Met can be measured, for example, in living or lysed cells. ELISA, immunofluorescence, flow cytometry, or radioimmunoassay on isolated cells Alternatively, or alternatively, the amount of ATP can be measured using well-known assays using the In addition, levels of nucleic acids encoding EGFR and / or c-Met can be measured, for example, by fluorescence using in situ hybridization, Southern blotting, or PCR It can also be measured intracellularly. EGFR and / or c-Met bind to EGFR on the surface of cells. If the levels of FR and / or c-Met are at least 1.5 times higher than in normal cells, It is overexpressed when

[0046] As used herein, "Tencon" refers to a strain of rice having the sequence shown in SEQ ID NO: 1. Synthetic fibronectin, as described in US Patent Application Publication No. US2010 / 0216708. Refers to the type III (FN3) domain.

[0047] As used herein, a "cancer cell" or "tumor cell" refers to a cell that has undergone the uptake of new genetic material. In vivo, ex vivo, spontaneous or induced phenotypic changes that do not necessarily involve Transformation refers to the transformation of cancerous, precancerous, or transformed cells in vivo and in tissue culture. Infection with transforming viruses and integration of new genomic nucleic acid or uptake of exogenous nucleic acid It can occur due to carcinogenicity, but can also occur naturally or after exposure to carcinogens, Transformation / cancer can be caused by, for example, in vitro or in vivo mutations in endogenous genes. Morphological changes in vivo and ex vivo, cell immobilization, abnormal growth control, foci formation, proliferation, Malignant lesions, tumor-specific marker levels, invasiveness, and tumor size in appropriate animal hosts such as nude mice This is demonstrated by tumor growth or inhibition in the culture of mice (Freshney, Culture o f Animal Cells:A Manual of Basic Techniq ue(3rd ed.1994)).

[0048] The term "vector" refers to a molecule that can be replicated within a biological system or transferred between such systems. Vector polynucleotides generally refer to the transfer of these vectors to living systems. Origins of replication, polyadenylation sequences, and sequences that function to facilitate the replication or maintenance of polynucleotides. Examples of such biological systems include cells, Reconstruction using biological elements capable of replicating viruses, animals, plants, and vectors. The polynucleotides comprising the vectors can be DNA or The nucleic acid molecule may be a nucleic acid molecule, a nucleic acid molecule, or a hybrid thereof.

[0049] The term "expression vector" refers to a vector that is used in a biological system or a reconstituted biological system. directing the translation of a polypeptide encoded by a polynucleotide sequence present in the target The term "vector" refers to a vector that can be used for the purpose of

[0050] The term "polynucleotide" refers to a polynucleotide that is composed of a sugar-phosphate backbone or other equivalent covalent chemical structure. It refers to a molecule containing a chain of nucleotides covalently linked by a double-stranded or single-stranded Strands of DNA and RNA are typical examples of polynucleotides.

[0051] "Complementary DNA" or "cDNA" refers to a DNA fragment containing intervening introns present in genomic DNA. Organization of sequence elements found in naturally occurring mature mRNA species with exon deletions and consecutive exons The codon encoding the start methionine is cDNA may or may not be present in the cDNA. It can be synthesized by gene assembly.

[0052] As used herein, "synthetic" or "non-natural" or "artificial" refers to a polynucleotide that is It refers to a nucleotide or polypeptide molecule that is not naturally occurring.

[0053] The term "polypeptide" or "protein" refers to a polynucleotide that is linked by peptide bonds. It means a molecule containing at least two amino acid residues that form a peptide. Less than about 50 Small polypeptides consisting of amino acids are sometimes called "peptides."

[0054] As used herein, a "bispecific EGFR / c-Met molecule" or "bispecific The term "specific EGFR / c-Met FN3 domain-containing molecule" refers to a molecule that is linked to a target molecule, either directly or via a linker. The EGFR-binding FN3 domain and the distinct c-Met A typical bispecific EGFR / cM molecule contains the FN3 binding domain. The et-binding molecule contains a first FN3 domain that specifically binds to EGFR and a second FN3 domain that specifically binds to c-Met. and a second FN3 domain that heterologously binds to the FN3 domain.

[0055] As used herein, "valency" refers to the presence within a molecule of a specific antigen. refers to the number of binding sites present. Hence, "monovalent," "divalent," and "tetravalent" and the term "hexavalent" refers to the presence of one, two, four, or more specific antigens in a molecule. and six binding sites, respectively.

[0056] As used herein, a "mixture" refers to a group of molecules that are not covalently linked to each other. A mixture refers to a sample or preparation of two or more FN3 domains of the same type. It may consist of three domains or may consist of different FN3 domains. The mixture used herein is monovalent for EGFR and / or monovalent for c-Met. It also refers to a sample or preparation of two or more monovalent antibodies that are monovalent in nature.

[0057] As used herein, "bispecific binding specifically to EGFR and c-Met" refers to a The term "agent" refers to a compound having a first domain that specifically binds to EGFR and a second domain that specifically binds to c-Met. and a second domain that specifically binds to EGFR and c-Met. One of the representative drugs that combines these is a bispecific antibody. Another exemplary bispecific agent that binds to an EGFR-binding FN3 domain and a separate c- It is a molecule containing the Met-binding FN3 domain. It specifically binds to EGFR and c-Met. The combined bispecific agent may be loaded with one polynucleotide or with more than one. It may be composed of a polynucleotide.

[0058] As used herein, a "bispecific anti-EGFR / c-Met antibody" or "bispecific The term "antibody" refers to a first domain that specifically binds to EGFR. refers to a bispecific antibody having a first domain that specifically binds to c-Met and a second domain that specifically binds to c-Met. The domains that specifically bind to EGFR and c-Met are usually VH / VL pairs. The bispecific anti-EGFR / c-Met antibody has the following properties regarding binding to EGFR and c-Met: is monovalent.

[0059] As used herein, the term "antibody" is used in a broad sense and includes polyclonal antibodies. monoclonal antibodies, including murine, human, human-adapted, humanized and chimeric monoclonal antibodies Clonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimers, tetramers The term "antibody" includes immunoglobulin molecules including monomeric or multimeric antibodies, as well as single chain antibodies.

[0060] Immunoglobulins are classified into IgA, IgD, and IgI based on the amino acid sequence of the constant domain of the heavy chain. They can be divided into five major classes: IgE, IgG, and IgM. G is the isotype of IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The light chains of antibodies from any vertebrate species are further classified based on the amino acid sequence of the constant domain. Based on this, they are classified into one of two distinct types: kappa (κ) and lambda (λ). It is possible.

[0061] The term "antibody fragment" refers to a fragment of an antibody comprising heavy chain complementarity determining regions (HCDRs) 1, 2 and 3, a light chain complementarity determining region (HCDR) 1, 2 and 3, and a light chain complementarity determining region (HCDR) 2, 3 and 3. Chain complementarity determining regions (LCDRs) 1, 2, and 3, heavy chain variable region (VH), or light chain variable region The portion of an immunoglobulin molecule that maintains the antigen-binding site of the heavy and / or light chains, such as (VL) Antibody fragments include Fab fragments; VL, VH, CL, and Monovalent fragments consisting of the CHI domain; F(ab)2 fragments, hinge region, A bivalent fragment containing two Fab fragments linked by a disulfide bridge in fragment; Fd fragment consisting of VH and CHI domains; V of one arm of an antibody Fv fragment consisting of L and VH domains; domain antibody (d Ab) fragment (Ward et al (1989) Nature 341:544 VH and VL domains can be engineered to form synthetic phosphorylases. By linking them together via a carrier, various types of single-chain antibody designs can be formed. In this case, the VH / VL domains are arranged intramolecularly or the VH domain and the VL domain are arranged intramolecularly. When expressed as separate single-chain antibody constructs, they are inter-molecularly assemblable. , those that form a monovalent antigen-binding site, such as single-chain Fv (scFv) or diabodies This is described, for example, in International Patent Publication Nos. WO1998 / 44001 and WO 1988 / 01649, WO1994 / 13804, and WO1992 / 0 1047. These antibody fragments can be obtained using techniques well known to those skilled in the art. Such fragments may be used in the same manner as full-length antibodies for practical purposes. are screened.

[0062] The phrase "isolated antibody" refers to an antibody or antibody that is substantially free of other antibodies having different antigen specificities. refers to an antibody fragment (e.g., that specifically binds EGFR and c-Met) The isolated bispecific antibody specifically binds to an antigen other than human EGFR and c-Met. However, it does not contain antibodies that specifically bind to EGFR and c-Met. The isolated antibody may be human EGFR and / or c-Met, e.g., Macaca fascicularis. orthologues of EGFR and / or c-Met in Macaca fascicularis (cynomolgus monkey) Furthermore, an isolated antibody may have cross-reactivity with other cellular material and / or chemicals. In some cases, it may not actually be included.

[0063] The antibody variable region consists of a "framework" region that contains three "antigen binding sites." The antigen-binding site is defined by different terms: (i) three ( HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, L The complementarity determining regions (CDRs) of the CDR3 are based on sequence variability (Wu and Kabat(1970)J Exp Med 132:211~50,1970 ;Kabat et al Sequences of Proteins of Im munological Interest,5th Ed.Public Healt h Service, National Institutes of Health, (Bethesda, Md., 1991), (ii) 3 in VH (H1, H2, H3) and and the three "hypervariable regions," "HVRs," or "HVs" within VL (L1, L2, L3), Chothia and Lesk(Chothia and Lesk Mol Biol 196:901-17, 1987) are highly variable in structure. This refers to the region of the variable domain of an antibody. Other terms include "IMGT-CDR" (Left CDR). Anc et al., Dev Comparat Immunol 27:55~77 , 2003) and "Specificity Determining Residue Usage" (SDRU) (Almagro Mol R International Journal of Cognition 17:132-43, 2004). ImMunoGeneTics (IMGT) database (http: / / www_i The CD4000 antigen binding site numbering and definition is provided by the CD4000 antigen binding site numbering and definition. For correspondence between the notations R, HV, and IMGT, see Lefranc et al., De v Comparat Immunol 27:55-77, 2003 .

[0064] As used herein, "Chothia residue" refers to the residue of Al-Lazikani ( Al-Lazikani et al., J Mol Biol 273:927~48 The antibody VL and VH residues are numbered according to (Ibid., 1997).

[0065] "Framework" or "framework sequence" is defined as the antigen binding site. The antigen-binding site is the remaining sequence of the variable region other than the sequence that is being The exact amino acid sequence of the framework can be determined by the antigen binding site. It depends on how it is defined.

[0066] A "humanized antibody" is an antibody in which the antigen-binding site is derived from a species other than human and the variable region framework is Humanized antibodies refer to antibodies in which the framework regions are derived from human immunoglobulin sequences. The framework may contain substitutions within the It is possible that the gene may not be an exact copy of the native or germline gene sequence.

[0067] "Human-adapted" antibodies or "human framework adapted (HFA)" antibodies are defined in the U.S. Pat. A human adapted according to the method described in Application Publication No. US2009 / 0118127. Human-adapted antibodies refer to antibodies that have the greatest CDR and FR similarity, CDR1 and CDR2. Based on the length compatibility and sequence similarity of the R2 loop and a portion of the light chain CDR3 loop The protein is humanized by selecting a human framework to serve as an acceptor.

[0068] A "human antibody" is an antibody in which both the framework and antigen-binding sites are derived from sequences of human origin. When an antibody contains a constant region, the constant region is also derived from a sequence of human origin.

[0069] Human antibodies are antibodies whose variable regions are derived from human germline immunoglobulins or rearranged immunoglobulins. When derived from a system that uses human globulin genes, the " containing heavy or light chain variable regions. Such systems include those displayed on phage. Human immunoglobulin gene libraries and human immunoglobulins as described herein. Examples of such transgenic non-human animals include mice carrying the phospho-phosphorylation locus. An "antibody" is a molecule that, when compared to human germline or rearranged immunoglobulin sequences, e.g., , naturally occurring somatic mutations or the introduction of deliberate substitutions in the framework or antigen-binding site. Generally, a "human antibody" is an antibody in which the amino acid sequence is identical to that of a human antibody. and the amino acid sequence encoded by the germline or rearranged immunoglobulin gene. At least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% identical. In some cases, a "human antibody" may be, for example, a human antibody. Nappik et al., J Mol Biol 296:57~86,2000 The consensus framework derived by analysis of the human framework sequences as described The target sequence, or Shi et al., J Mol Biol 397:385-96, 2010 and International Patent Publication No. WO2009 / 085462, Synthetic HCs incorporated into a human immunoglobulin gene library displayed on a PDMS chip Antibodies whose antigen-binding site is derived from a species other than human fall within the definition of a "human antibody." is not included.

[0070] Isolated humanized antibodies may be synthetic. Human antibodies are antibodies derived from human immunoglobulin sequences. derived from the sequence but incorporating synthetic CDRs and / or synthetic frameworks. Antibodies may be generated using systems such as image display or may be used to improve antibody properties. In order to achieve this, in vivo human antibody germline mutations may be generated. This results in antibodies that do not naturally exist in the human repertoire.

[0071] As used herein, the term "recombinant antibody" includes antibodies derived from human immunoglobulin genes. Transgenic or transchromosomal animals (e.g., mice) or animals prepared therefrom Antibodies isolated from hybridomas (described further below), transformed to express the antibodies Antibodies isolated from recombinant host cells, isolated from recombinant combinatorial antibody libraries antibodies and splicing human immunoglobulin gene sequences into other DNA sequences or Fab, prepared, expressed, produced, or isolated by any other means involving Prepared and expressed by recombinant means, such as antibodies generated in vitro using arm exchange The term "antibody" includes any antibody that is produced, generated, or isolated.

[0072] As used herein, the term "monoclonal antibody" refers to an antibody of single molecular composition. A monoclonal antibody composition refers to a preparation of antibody molecules that are directed against a single antibody against a particular epitope. exhibit binding specificity and affinity, or in the case of bispecific monoclonal antibodies, two It exhibits dual binding specificity for different epitopes.

[0073] As used herein, the term "substantially identical" refers to the degree to which the two antibodies being compared are substantially identical. This means that the amino acid sequences of the variant regions are identical or have only "slight differences." The minor differences are those that do not adversely affect the antibody properties, such as 1, 2, 3, or 4 changes in the sequence of the antibody variable region. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions Amino acid sequences substantially identical to the variable region sequences disclosed herein are included in the present invention. In some embodiments, the sequence identity is in the range of about 90%, 91%, 92%, It may be 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. The identity (%) was calculated using, for example, Vector NTI v.9.0.0 (Invitrogen The AlignX module from the University of California, San Jose, CA (USA) was used with the default settings. The protein sequence of the present invention can be determined by aligning the sequence with the Use the sequence as a starting point, e.g., to search public databases to identify related sequences. or to carry out a search in a patent database. Typical programs used are XBLAST or B LASTP program (http_ / / www_ncbi_nlm / nih_gov), or GenomeQuest™ (GenomeQuest, Massachusetts, USA) Westborough Suite.

[0074] As used herein, the term "epitope" refers to an antigen to which an antibody specifically binds. An epitope is usually a chemically active portion of a molecule, such as an amino acid or polysaccharide side chain. They consist of various (polar, non-polar or hydrophobic) surface groups, with specific three-dimensional structural characteristics and specific Epitopes may have continuous and / or discontinuous structures that form structural spatial units. A discontinuous epitope can consist of different parts of the linear sequence of an antigen. The amino acids of a protein are brought closer together in three-dimensional space by the folding of the protein molecule.

[0075] As used herein, the term "in combination" refers to the administration of two or more therapeutic agents to a subject in combination. together in a combination, simultaneously as single agents, or sequentially in any order as single agents. It means you can.

[0076] Throughout this specification, the numbering of amino acid residues in antibody constant regions is the same as above unless otherwise specified. Unless otherwise specified, see Kabat et al., Sequences of Proteins f Immunological Interest,5th Ed.Public H health service,National Institutes of Hea According to the EU Index as stated in Ith, Bethesda, MD. (1991) went.

[0077] Material composition The present invention provides bispecific agents that specifically bind to EGFR and c-Met. The invention also provides polypeptides and polynucleotides encoding the bispecific agents of the invention, or Complementary nucleic acids, vectors, host cells, and methods for making and using them are also provided. do.

[0078] Monospecific and bispecific EGFR and / or c-MetFN3 domain-containing binding molecules child Monospecific EGFR FN3 domain-containing binding molecules The present invention relates to a method for the production of epidermal growth factor (EGF) receptor-specific EGFR-binding proteins. Provides a fibronectin type III (FN3) domain that blocks binding of EGFR to The present invention can therefore be widely used in therapeutic and diagnostic applications. The present invention relates to a polynucleotide encoding the FN3 domain of the present invention, a nucleic acid complementary thereto, a vector The present invention provides vectors, host cells, and methods for making and using the same.

[0079] The FN3 domain of the present invention binds to EGFR with high affinity and inhibits EGFR signaling. and reduced off-target toxicity compared to small molecule EGFR inhibitors in terms of off-target toxicity and improved tissue response compared to conventional antibody therapies This may provide advantages in terms of penetration.

[0080] One embodiment of the present invention is a method for producing an antibody that specifically binds to epidermal growth factor receptor (EGFR) and inhibits epidermal growth factor receptor (EGFR). Isolated fibronectin type III blocks EGF binding to EGFR (FN3) domain.

[0081] The FN3 domain of the present invention was used in A431 cells in the presence or absence of the FN3 domain of the present invention. A431 cells incubated in the absence of 600 nM streptavidin-fibrinogen were The amount of fluorescence from bound biotinylated EGF was measured using a coerythrin conjugate. In a competitive assay, approximately 1 × 10 -7 Less than M, approximately 1 x 10 -8 Less than M, approximately 1 x 10 -9 Less than M, approximately 1 x 10 -10 Less than M, approximately 1 x 10 -11 Less than M or about 1 x 10 -12 IC5 less than M A value of 0 can block EGF binding to EGFR. EGFR-binding FN3 domains having the amino acid sequence of amino acids 9, 107-110, or 122-137. A typical FN3 domain, such as the main domain, has a total of approximately 1 × 10 -9 M ~ approx. 1×10 -7 M IC 50 By value The FN3 domain of the present invention can block EGF binding to EGFR. was compared to EGFR in the absence of the FN3 domain of the present invention using the same assay conditions. inhibits EGF binding to EGFR by at least 30% compared to EGF binding to EGFR alone , 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% blocking.

[0082] The FN3 domains of the present invention were assayed using the same assay conditions as in the absence of the FN3 domains of the present invention. EGFR signaling at a lower level compared to the level of signaling in the presence of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100% inhibition may be achieved.

[0083] Binding of ligands such as EGF to EGFR induces receptor dimerization, autophosphorylation, and Activation of the receptor's internal cytoplasmic tyrosine kinase domain and regulation of DNA synthesis (genetic Multiple signal transducers involved in cell cycle progression or cell division Inhibition of EGFR signaling stimulates the initiation of EGFR-mediated transcription and transactivation pathways. Disabling EGFR can lead to inhibition of signaling pathways downstream of EGFR. These include the inhibition of cell proliferation and differentiation, angiogenesis, cell motility, and metastasis. It may have various effects.

[0084] EGFR signaling is mediated by, for example, tyrosines Y1068, Y1148, and Y1173. Receptor autophosphorylation in either the cytosol or the cytosol (Downward et al., Nature re 311:483-5, 1984), and / or phosphorylation of natural or synthetic substrates Phosphorylation can be measured using a variety of well-known methods, such as measuring ELI. Well-known methods such as SA assay or Western blot using phosphotyrosine-specific antibodies A representative assay can be used to detect the presence of β-glucan in the chromatin. Pharmacol Exp Thera 283:1433-44, 1997 and B Atley et al., Life Sci 62:143-50, 1998, and This can be seen in the assays described herein.

[0085] In one embodiment, the FN3 domain of the present invention is amplified using 50 ng / mL human EGF. EGF induction of tyrosine residue at position 1173 of EGFR as measured in 431 cells EGFR phosphorylation was measured at approximately 2.5 × 10 -6 Less than M, e.g., about 1 x 10 -6 Less than M, approx. 1 x 1 0 -7 Less than M, approximately 1 x 10 -8 Less than M, approximately 1 x 10 -9 Less than M, approximately 1 x 10 -10 Less than M, approx. 1 x 10 -11 M, or approximately 1 x 10 -12 IC less than M 50 Inhibit by value.

[0086] In one embodiment, the FN3 domain of the present invention is amplified using 50 ng / mL human EGF. EGF induction of tyrosine residue at position 1173 of EGFR as measured in 431 cells EGFR phosphorylation was measured at approximately 1.8 × 10 -8 M ~ approx. 2.5×10 -6 M IC 50 Inhibit by value. Such representative FN3 domains include those set forth in SEQ ID NOs: 18-29, 107-110, or 1 Some have amino acid sequences of 22 to 137.

[0087] In one embodiment, the FN3 domains of the present invention are surface proteins as practiced by those skilled in the art. Dissociation constant (K) measured by raspberry resonance or Kinexa method D ) is about 1 × 10 -8 M Less than, for example, about 1 x 10 -9 Less than M, approximately 1 x 10 -10 Less than M, approximately 1 x 10 -11 Less than M, approx. 1 x 10 -12 M, or approximately 1 x 10 -13 In certain embodiments, the antibody binds to human EGFR with a binding affinity of less than M. The FN3 domain of the present invention is about 2×10 -10 ~Approx. 1×10 -8 K of M D Binds to human EGFR The affinity of the FN3 domain for EGFR can be experimentally determined using any suitable method. (See, for example, Berzofsky, et al., "Antibo dy-Antigen Interactions,” In Fundamental Immunology,Paul,WE,Ed.,Raven Press:New York, NY (1984); Kuby, Janis Immunology, W.H. .Freeman and Company: New York, NY (1992); and methods described herein.) The specific FN3 domain-antigen being measured The affinity of the interaction can be measured under different conditions (e.g., osmolarity, pH) Therefore, affinity and other antigen binding parameters (e.g., K D , K. on , K off ) is preferably measured using standardized solutions of protein scaffolds and antigens, and the methods described herein. This is done using standardized buffers such as those described in

[0088] Representative FN3 domains of the present invention that bind to EGFR include those represented by SEQ ID NOs: 18 to 29, Examples of FN3 domains include FN3 domains 107 to 110 and 122 to 137.

[0089] In one embodiment, the FN3 domain that specifically binds to EGFR is the amino acid sequence of SEQ ID NO:27. It comprises an amino acid sequence that has at least 87% identity with the amino acid sequence of the present invention.

[0090] In one embodiment, the FN3 domain that specifically binds to EGFR is Sequence HNVYKDTNX9RGL (SEQ ID NO: 179) or sequence LGSYVFEHDVM L (SEQ ID NO: 180) (wherein X9 is M or I); and A BC loop comprising the sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 181), with the proviso that X1 is A, T, G or D; X2 is A, D, Y or W; X3 is P, D or N; X4 is L or absent, X5 is D, H, R, G, Y or W; X6 is G, D or A; X7 is A, F, G, H or D; X8 is Y, F or L).

[0091] The FN3 domain of the present invention specifically binds to EGFR and inhibits autophosphorylation of EGFR. The sequence HNVYKDTNX9RGL (SEQ ID NO: 179) or the sequence A FG loop containing the sequence LGSYVFEHDVML (SEQ ID NO: 180) (wherein X9 is Such FN3 domains may be 8 or 9 amino acids long. and a BC rule defined by the sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 181). The antibody may further contain a peptide, and when measured using A431 cells with 50 ng / mL human EGF, Approximately 2.5 x 10 -6 IC less than M 50 value, or approximately 1.8 x 10 -8 M ~ approx. 2.5×10 -6 M IC 50 It can inhibit the autophosphorylation of EGFR at low levels.

[0092] The FN3 domain of the present invention specifically binds to EGFR and inhibits autophosphorylation of EGFR. The sequence LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8DSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV HNVYKDTNX9RGLPLSAEFTT (SEQ ID NO: 182), or the sequence LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8DSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV LGSYVFEHDVMLPLSAEFTT (SEQ ID NO: 183), (however, X1 is A, T, G or D; X2 is A, D, Y or W; X3 is P, D or N; X4 is L or absent, X5 is D, H, R, G, Y or W; X6 is G, D or A; X7 is A, F, G, H or D; X8 is Y, F or L; X9 is M or I).

[0093] EGFR-binding FN3 domains were generated using well-known methods and those described herein. and can be tested for their ability to inhibit autophosphorylation of EGFR.

[0094] Another embodiment of the present invention is an isolated FN3 domain that specifically binds to EGFR. Therefore, the sequences shown in SEQ ID NOs: 18 to 29, 107 to 110, or 122 to 137 The FN3 domain contains the sequence shown in FIG.

[0095] In some embodiments, the EGFR-binding FN3 domain is a cytotoxic molecule, e.g., ... Initiator molecules linked to the N-terminus of specific FN3 domains to facilitate expression and / or conjugation It contains a thionine (Met) or a cysteine ​​(Cys) linked to the C-terminus.

[0096] Another embodiment of the present invention is a method for detecting EGF binding to EGFR by specifically binding to EGFR. An isolated fibronectin type III (FN3) domain that blocks FN3 domain isolated from a library designed based on Tencon sequence number 1 be.

[0097] Monospecific c-Met FN3 domain-containing binding molecule The present invention relates to a method for treating hepatocyte growth factor receptor (c-Met) by specifically binding to the c-Met receptor. Fibronectin type III (FN3) blocks the binding of hepatocyte growth factor (HGF) to ) domain and can therefore be widely used in therapeutic and diagnostic applications. The present invention relates to a polynucleotide encoding the FN3 domain of the present invention, or a complementary nucleic acid thereof. Acids, vectors, host cells, and methods of making and using them are provided.

[0098] The FN3 domain of the present invention binds to c-Met with high affinity and mediates c-Met signaling. inhibits c-Met receptors with specificity and reduced off-target activity compared to small molecule c-Met inhibitors Improved in terms of off-target toxicity and when compared to conventional antibody therapies This may provide advantages in terms of tissue penetration. The FN3 domains of the present invention are monovalent and Therefore, unwanted receptor clustering and activation that can occur with other bivalent molecules is prevented. It is prevented.

[0099] One embodiment of the present invention is a method for treating hepatocyte growth factor receptor (c-Met)-associated leukemia. Isolated fibronectin blocks hepatocyte growth factor (HGF) binding to Met. It is a cutin type III (FN3) domain.

[0100] The FN3 domain of the present invention may be used to synthesize a c-Met-Fc fusion protein in the presence of the FN3 domain of the present invention. In an assay that detects inhibition of binding of biotinylated HGF to proteins, approximately 1× 10 -7 Less than M, approximately 1 x 10 -8 Less than M, approximately 1 x 10 -9 Less than M, approximately 1 x 10 -10 Less than M, about 1 x10 -11 Less than M or about 1 x 10 -12 IC less than M 50 The effect of HGF on c-Met was A typical FN3 domain can block approximately 2 × 10 -10 M ~ approx. 6× 10 -8 M IC 50 This antibody can block HGF binding to c-Met at a low level. The FN3 domain of the invention was assayed using the same assay conditions as in the absence of the FN3 domain of the invention. HGF binding to c-Met compared with HGF binding to c-Met in of bonding at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% blocking.

[0101] The FN3 domains of the present invention were assayed using the same assay conditions as in the absence of the FN3 domains of the present invention. c-Met signaling at a lower level compared to the level of signaling in the absence of At least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 7 5%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 It can be inhibited by 7%, 98%, 99%, or 100%.

[0102] Binding of HGF to c-Met is mediated by receptor dimerization, autophosphorylation, and internalization of the receptor. Activation of the cytoplasmic tyrosine kinase domain of β-glucan and regulation of DNA synthesis (gene activation) ) and multiple signal transduction and transcription factors involved in cell cycle progression or cell division. Inhibition of c-Met signaling stimulates the initiation of the transactivation pathway. Disabling c-Met may lead to inhibition of signaling pathways downstream of et It has various functions, including inhibiting cell proliferation and differentiation, angiogenesis, cell motility, and metastasis. It may have a beneficial effect.

[0103] c-Met signaling is mediated by, for example, tyrosine residues Y1230, Y1234, and Y123 autophosphorylation of the receptor at at least one of Y1349, Y1359, or Y1369, and / or Y1370. or by measuring the phosphorylation of natural or synthetic substrates. Phosphorylation can be measured, for example, by ELISA assay or Western blot. It can be detected using antibodies specific for phosphotyrosine. Assays for enzyme activity are described, for example, in Panek et al., J Pharmacol E xp Thera 283:1433~44,1997 and Batley et al. , Life Sci 62:143-50, 1998, and also as described herein. The assay is described in

[0104] In one embodiment, the FN3 domain of the present invention binds to 100 ng / mL of recombinant human HGF. The cleavage of c-Met at residue 1349 was measured in NCI-H441 cells using HGF-induced c-Met phosphorylation was measured using approximately 1 × 10 -6 Less than M, approximately 1 x 10 -7 Less than M, approx. 1 x 1 0 -8 Less than M, approximately 1 x 10 -9 Less than M, approximately 1 x 10 -10 Less than M, approximately 1 x 10 -11 M, or approximately 1 x 10 -12 IC less than M 50 Inhibit by value.

[0105] In one embodiment, the FN3 domain of the present invention binds to 100 ng / mL of recombinant human HGF. at c-Met tyrosine Y1349 when measured in NCI-H441 cells using HGF-induced c-Met phosphorylation was measured using approximately 4 × 10 -9 M ~ approx. 1×10 -6 M IC 50 Inhibit by value do.

[0106] In one embodiment, the FN3 domains of the present invention are surface proteins as practiced by those skilled in the art. Dissociation constant (K) measured by raspberry resonance or Kinexa method D ) is about 1 × 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 x 10 -12 M, 1 x10 -13 M, 1 x 10 -14 M, or 1 x 10 -15 It binds to human c-Met at a molecular weight of less than M. In some embodiments, the FN3 domain of the invention is about 3×10 -10 M ~ approx. 5×10 -8 M K DThe affinity of the FN3 domain for c-Met is It can be experimentally determined using any suitable method (e.g., Berzofsky ,et al.,“Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology,WH Freeman and Company:New New York, NY (1992); and methods described therein. The affinity of a particular FN3 domain-antigen interaction varies under different conditions (e.g., osmolarity). Therefore, the affinity and other antigen binding properties may differ when measured under different conditions (temperature, pH). parameters (e.g., K D , K. on , K. off ) is preferably measured by measuring the protein backbone and antigen Standardization solutions of the above, as well as standardization buffers such as those described herein, can be.

[0107] Representative FN3 domains of the present invention that bind to c-Met include those of SEQ ID NOs: 32 to 49 Alternatively, an FN3 domain having an amino acid sequence of 111 to 114 may be mentioned.

[0108] In one embodiment, the FN3 domain that specifically binds to c-Met is the amino acid sequence of SEQ ID NO: 41. It contains an amino acid sequence that has at least 83% identity with the amino acid sequence.

[0109] In one embodiment, the FN3 domain that specifically binds c-Met is Array DSFX 10 IRYX 11 EX 12 X13 X 14 X 15 GX 16 C-strand containing (SEQ ID NO: 184) and CD loops (but X 10 is W, F or V, X 11 is D, F or L, X 12 is V, F or L, X 13 is V, L or T, X 14 is V, R, G, L, T, or S; X 15 is G, S, A, T or K, X 16 is E or D) and Array TEYX 17 VX 18 IX 19 X 20 VKGGX 21 X 22 SX 23 (SEQ ID NO: 185) chain and FG loop (where X 17 is Y, W, I, V, G or A, X 18 is N, T, Q or G, X 19 is L, M, N or I, X 20 is G or S, X 21 is S, L, G, Y, T, R, H or K, X 22 is I, V or L, X 23 is V, T, H, I, P, Y or L).

[0110] The FN3 domain of the present invention specifically binds to c-Met and inhibits the autophosphorylation of c-Met. In is an array LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 10IRYX 11 E X 12 X 13 X 14 X 15 GX 16 AIVLTVPGSERSYDLTGLKPGTEYX 17 VX 18 IX 19 X 20 VKGGX 21 X 22 SX 23 PLSAEFTT (SEQ ID NO: 186) (however, X 10 is W, F or V, X 11 is D, F or L, X 12 is V, F or L, X 13 is V, L or T, X 14 is V, R, G, L, T or S, X 15 is G, S, A, T or K, X 16 is E or D, X 17 is Y, W, I, V, G or A, X 18 is N, T, Q or G, X 19 is L, M, N or I, X 20 is G or S, X 21 is S, L, G, Y, T, R, H or K, X 22 is I, V or L, X 23 is V, T, H, I, P, Y or L).

[0111] Another embodiment of the present invention is an isolated FN3 domain that specifically binds c-Met. FN3 comprising the sequences shown in SEQ ID NOs: 32 to 49 or 111 to 114 It is a domain.

[0112] Another embodiment of the present invention is a method for inhibiting the binding of HGF to c-Met by specifically binding to c-Met. An isolated fibronectin type III (FN3) domain that blocks binding, FN3 domains isolated from a library designed based on Tencon sequences in column number 1 is.

[0113] Single EGFR or c-MetFN3 domains from Tencon sequence-based libraries away Tencon (SEQ ID NO: 1) is the consensus sequence of the 15 FN3 domains of human tenascin-C. A non-naturally occurring fibronectin type III (FN3) domain designed from the FN3 sequence (Jacobs et al., Protein Engineering, D esign,and Selection,25:107~117,2012;U.S. Patent The crystal structure of Tencon is characterized by the FN3 domain. It exhibits six surface-exposed loops connecting seven characteristic β-strands, which are These loops are called B, C, D, E, F, and G, and are arranged as follows: AB, BC, CD, DE, EF , and FG loops (Bork and Doolittle, Proc Natl Acad Sci USA 89:8990-8992, 1992; U.S. Patent (No. 6,673,901). These loops, or selected residues within each loop, are randomly selected. These compounds can be used to select novel molecules that bind to EGFR or c-Met by synthesizing them. To construct a library of fibronectin type III (FN3) domains that can be used in Table 1 shows the positions and sequences of each loop and β-strand of Tencon (SEQ ID NO: 1). show.

[0114] Therefore, the library designed based on the Tencon sequence is Randomized FG loops, such as TCL1 or TCL2, or randomized It may have a BC and FG loop. The BC loop of Tencon is 7 amino acids long, Therefore, the library designed based on the Tencon sequence diversified in the BC loop In a sequence, 1, 2, 3, 4, 5, 6, or 7 amino acids can be randomized. The FG loop of Con is 7 amino acids long, and therefore, there is diversity in the FG loop. The libraries designed based on the Tencon sequence are 1, 2, 3, 4, 5, 6, or 7. The amino acids in the loop can be randomized. This allows for greater diversity in the loops in the Tencon library. For example, The FG and / or BC loops can be extended by 1 to 22 amino acids, or by 1 to 3 amino acids. The FG loop in Tencon is 7 amino acids long, but In contrast, the corresponding loops in antibody heavy chains range from 4 to 28 residues. To achieve this, the FG loop was designed to correspond to the length range of antibody CDR3 from 4 to 28 residues. The sequence and length can be varied. For example, the length of the FG loop can be increased by an additional 1 amino acid. Further diversification can be achieved by extending the loop by 2, 3, 4 or 5 positions.

[0115] The library designed based on the Tencon sequence was designed to identify the FN3 domain and its flanking regions. It may have a randomized surface containing two or more beta strands and at least one loop. One such surface is located between amino acids in the C and F β strands and the CD and FG loops. (C-CD-F-FG surface) Another C-CD-F-FG surface of Tencon The design of the library based on this is shown in Figure 1, and the detailed generation of such a library The method is described in U.S. Patent Application Publication No. US2013 / 0226834.

[0116] The libraries designed based on the Tencon sequence include 11, 14, 17, 37, 46, having a substitution at residue 73 or 86 (residue numbering corresponds to SEQ ID NO: 1); Liver engineered based on Tencon mutants, such as Tencon mutants that exhibit high thermostability Representative Tencon mutants are described in U.S. Patent Application Publication No. 2011 / 027462. 3, and the substitutions E11R, L17A when compared with Tencon of SEQ ID NO: 1. , Tencon27 (SEQ ID NO: 99) with N46V and E86I.

[0117] [Table 1]

[0118] Tencon and other FN3 sequence-based libraries are available with random or defined amino acids. A set of residues can be used to randomize at selected residue positions. The variants in the library with dam substitutions represent all 20 naturally occurring amino acids. In other diversification schemes, DV The K codon is used to identify the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, It can encode Ser, Arg, Asp, Glu, Gly, and Cys. Alternatively, NNS codons can be used to generate all 20 amino acid residues, with a stop codon at the same time. The frequency of mutations can be reduced by using a method that has a biased amino acid distribution at the position to be diversified. Libraries of FN3 domains are available, for example, from Slonomics® Technology. -(http:_ / / www_sloning_com) can be used to synthesize This technology allows for the creation of universal building blocks sufficient for thousands of gene synthesis processes. A library of pre-prepared double-stranded triplets is used. The library contains all the possible sequence combinations needed to construct any desired DNA molecule. Codon designations are in accordance with the well-known IUB code.

[0119] The FN3 domain of the present invention that specifically binds to EGFR or c-Met is A tag formed after in vitro translation that is stably associated with the DNA encoding it DNA encoding scaffold proteins to generate a pool of protein-DNA complexes c for ligating the fragment to the DNA fragment encoding RepA Using the is display to generate FN3 libraries such as the Tencon library Therefore (U.S. Patent No. 7,842,476, Odegrip et al., Proc Natl Acad Sci USA 101,2806~2810,2004), EGF can also be obtained by any method known in the art and described in the Examples. By assaying the library for specific binding to R and / or c-Met Representative well-known methods that can be used include ELISA, SDS-PAGE, and the like. These include sandwich immunoassays, as well as competitive and non-competitive assays (e.g., Ausub el et al., eds, 1994, Current Protocols in Molecular Biology,Vol.1,John Wiley & Son (See, for example, Sigma-Aldrich, Inc., New York). Identified FN3 domains that bind to EGFR can be identified using the methods described herein. Blocking EGFR ligands, such as EGF binding to c-Met or HGF binding to c-Met. for its ability to lock in and inhibit EGFR and / or c-Met signaling The capabilities are further characterized.

[0120] The FN3 domain of the present invention that specifically binds to EGFR or c-Met can be any FN3 The domains are used as templates to generate libraries and the methods provided herein This library was screened for molecules that specifically bind to EGFR or c-Met using Typical FN3 domains that can be used are The third FN3 domain of tenascin-C (TN3) (SEQ ID NO: 75), fibcon ( SEQ ID NO: 76), and the 10th FN3 domain of fibronectin (SEQ ID NO: 77) To express or translate the library in vitro, standard cloning and Expression methods are used to clone the library into a vector or to generate a double stranded version of the library. Synthesize a double strand cDNA cassette, e.g., using ribosome display (Hanes and Pluckthun,Proc Natl Acad Sci USA,94,493 7-4942, 1997), mRNA display (Roberts and Szos tak,Proc Natl Acad Sci USA,94,12297~1230 2,1997) or other cell-free systems (U.S. Patent No. 5,643,768). The library of FN3 domain variants can be prepared, for example, from any suitable bacteriophage. They can be expressed as fusion proteins that are found on the surface of phages. Methods for displaying fusion polypeptides on the surface of a page are well known (U.S. Pat. No. 6,423,199). Patent Publication No. 2011 / 0118144, International Patent Publication No. WO2009 / 085462, U.S. Patent No. 6,969,108, U.S. Patent No. 6,172,197, U.S. Patent No. 5,223 ,409, U.S. Patent No. 6,582,915, U.S. Patent No. 6,472,147).

[0121] By modifying the FN3 domain of the present invention that specifically binds to EGFR or c-Met, Therefore, the properties can be improved, such as thermal stability and reversibility of thermal folding and unfolding. It can increase the apparent thermal stability of proteins and enzymes, resulting in extremely Rational design based on comparison with similar thermostable sequences, and design of stabilizing disulfide bridges , mutations that increase the propensity for α-helixes, manipulation of salt bridges, alteration of the surface charge of proteins, and directed orientation Several methods have been applied, including evolution and consensus sequence composition (Lehm Ann and Wyss,Curr Opin Biotechnol,12,371 ~375, 2001). High thermostability increases the yield of expressed proteins and improves their solubility or enhances activity, reduces immunogenicity, and minimizes cold chain requirements in manufacturing Substitutions can be made to increase the thermal stability of Tencon (SEQ ID NO: 1). Possible residues include residues at positions 11, 14, 17, 37, 46, 73, or 86. These residues are described in U.S. Patent Application Publication No. 2011 / 0274623. The corresponding substitutions are incorporated into the FN3 domains or bispecific FN3 domain-containing molecules of the invention. You can see it in.

[0122] Another embodiment of the present invention is a method for producing the nucleic acids shown in SEQ ID NOs: 18-29, 107-110, and 122-137. 11, 14, 17, 37, 46, 73, and 54 of Tencon (SEQ ID NO: 1). and 86, and further comprising substitutions at one or more residue positions corresponding to positions 87 and 88. It is an isolated FN3 domain that blocks EGF binding to the FR.

[0123] Another embodiment of the present invention is a method for producing a nucleic acid comprising the sequence shown in SEQ ID NO: 32 to 49 or 111 to 114. and corresponding to positions 11, 14, 17, 37, 46, 73, and 86 of Tencon (SEQ ID NO: 1). c-Met specifically binds to c-Met and contains substitutions at one or more residue positions corresponding to the c-Met Isolated FN3 domain that blocks HGF binding.

[0124] Representative substitutions include E11N, E14P, L17A, E37P, N46V, and G 73Y and E86I (numbering based on SEQ ID NO: 1).

[0125] In certain embodiments, the FN3 domain of the present invention comprises the substitution L1 of Tencon (SEQ ID NO: 1). Contains substitutions corresponding to 7A, N46V, and E86I.

[0126] The FN3 domain (Figure 1) that specifically binds to EGFR is compared with Tencon (SEQ ID NO: 1). It has a longer FG loop compared to the rest of Tencon (SEQ ID NO: 1). Residues corresponding to 11, 14, 17, 37, 46, 73, and 86 are FN of SEQ ID NO:24. Residues 11, 1 of the EGFR FN3 domain are shown in Figures 1A and 1B, excluding the 3 domain. 4, 17, 37, 46, 73, and 91, and the insertion of a single amino acid into the BC loop Thus, the corresponding residues are residues 11, 14, 17, 38, 74, and 92.

[0127] Another embodiment of the present invention is a sequence number 18-29, 107-110, or 122-137. and the amino acid sequence shown in Figure 1, which is a sequence of the amino acid sequence of Tencon (SEQ ID NO: 1) with the substitutions L17A, N46V, and and optionally having substitutions corresponding to E86I and E86I, Isolated FN3 domain that blocks EGF binding.

[0128] Another embodiment of the present invention is a method for producing a medicament comprising administering to a subject the amino acid sequence set forth in SEQ ID NOs: 34-49 or 111-114. The amino acid sequence corresponds to the substitutions L17A, N46V, and E86I in Tencon (SEQ ID NO: 1). Binding of HGF to c-Met, optionally having a substitution that specifically binds to c-Met It is an isolated FN3 domain that blocks

[0129] Are measurements of protein stability and protein instability the same as protein integrity? Proteins can be degraded by heat, ultraviolet light, or ionizing radiation, In solutions, changes in ambient osmolarity and pH, micropore filtration, ultraviolet radiation, and γ Ionizing radiation such as irradiation, chemical or thermal dehydration, or destruction of protein structure susceptible to denaturation caused by mechanical shear forces due to any other action or force that may affect the The stability of a molecule can be determined using standard methods. For example, the stability of a molecule can be determined using standard methods by measuring the temperature in degrees Celsius at which half of the molecule is unfolded. The temperature can be determined by measuring the thermal melting (Tm) temperature, which is in degrees Celsius. Generally, the higher the TM, the more stable the molecule. Besides heat, the chemical environment also affects the protein. It alters the ability of proteins to maintain their specific three-dimensional structure.

[0130] In one embodiment, the EGFR or c-Met binding FN3 domain of the present invention is TM at least 5%, 10%, or 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, Shows 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater stability.

[0131] Chemical modification can also be measured by a variety of methods. Chemical modifiers include: Guanidine hydrochloride, guanidinium thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride), reducing agents (e.g., dithiothreitol Benzene, β-mercaptoethanol, dinitrothiobenzene, and hydrides such as borohydride sodium chloride), non-ionic and ionic detergents, acids (e.g., hydrochloric acid (HCl), acetic acid (CH 3COOH), halogenated acetic acids), hydrophobic molecules (e.g., phospholipids), and target denaturants. Quantification of the degree of denaturation can be achieved by determining the loss of functional properties, such as the ability to bind to a target molecule, or Tendency to aggregate, exposure of previously solvent inaccessible residues, or disulfide bonds The effect of the hydroxyl group on the hydroxyl group may depend on the physicochemical properties of the hydroxyl group, such as the disintegration or formation of hydroxyl groups.

[0132] In one embodiment, the FN3 domains of the invention that bind to EGFR or c-Met are The denaturing activity was measured using guanidine hydrochloride as a reactive denaturant and compared to the same backbone before manipulation. Compared to at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more greater stability. Increased stability can be measured by increasing the concentration using well-known methods. Measured as a function of tryptophan fluorescence reduction upon treatment with guanidine hydrochloride. It is possible.

[0133] The FN3 domains of the present invention can be used as monomers, dimers, or multimers to bind to, for example, target molecules. as a means to increase the valence, and hence avidity, of a bond, or to combine two or more different as a means to generate bi- or multispecific scaffolds that simultaneously bind multiple target molecules. Dimers and multimers can be formed, for example, by using amino acid linkers, e.g., polyglycerides. by including a linker containing amine, glycine and serine, or alanine and proline , generated by linking monospecific, bispecific or multispecific protein scaffolds. Representative linkers include (GS)2, (SEQ ID NO: 78), (GGG (GS)5 (SEQ ID NO: 79), (AP)2 (SEQ ID NO: 80), (AP)5 (SEQ ID NO: 81), (AP) 10 (SEQ ID NO: 82), (AP) 20 (SEQ ID NO: 83) and A(EAAAK)5AA A linker A (SEQ ID NO: 84) is an example. Dimers and multimers are linked to each other in the N-C direction. The natural polypeptides can be linked to the novel ligated fusion polypeptides. The use of synthetic peptide linkers is well known in the literature (Hallewelyn et al., 2004). l et al., J Biol Chem 264,5260~5268,1989; Alfthan et al.,Protein Eng.8,725~731,199 5;Robinson & Sauer,Biochemistry 35,109~1 16, 1996; U.S. Patent No. 5,856,456).

[0134] Bispecific drug that specifically binds to EGFR and c-Met The bispecific agents of the present invention that specifically bind EGFR and c-Met are small molecule EGFR inhibitors. Specificity and reduced off-target toxicity compared to FR and / or c-Met inhibitors The present invention provides an antibody that specifically binds to EGFR and c-Met. The bispecific drugs are a combination of a monospecific drug that binds to EGFR and a monospecific drug that binds to c-Met. It provides significantly improved synergistic inhibition when compared to mixtures of isomeric drugs. This work is based, at least in part, on the surprising discovery that these molecules inhibit EGFR and c-Met, thereby improving tumor penetration and Bispecific binding to EGFR and c-Met can be achieved. The active agent is more effective at inhibiting EGFR and / or EGFR-specific markers than cetuximab (Erbitux®). or inhibiting the c-Met signaling pathway, thereby more efficiently inhibiting tumor growth. That is why.

[0135] Bispecific agents that specifically bind to EGFR and c-Met include the EGFR-binding domain and by any polypeptide or multimeric polypeptide containing a c-Met binding domain. The EGFR and c-Met binding domains can be formed in the antigen-binding site of the antibody. , antibody VH / VL pairs, or fibronectin type III (FN3) domains, other types of binding molecules, such as domains based on the fibrinogen IFN9 (FN9) domain; Or it may be any combination of these.

[0136] EGFR and c-Met binding polypeptides are available from existing monospecific EGFR and c-Met The polypeptide may be derived from a t-binding polypeptide or may be newly isolated.

[0137] Bispecific EGFR / c-Met FN3 domain-containing molecule One embodiment of the present invention is a fibronectin type III (FN3) domain comprising a first fibronectin type III (FN3) domain and a second fibronectin type III (FN3) domain. an isolated bispecific FN3 domain-containing molecule comprising an FN3 domain of The FN3 domain of 1 specifically binds to the epidermal growth factor receptor (EGFR) and induces EGFR-specific responses. The second FN3 domain blocks the binding of epidermal growth factor (EGF), which inhibits hepatocyte proliferation. It specifically binds to the hepatocyte growth factor (HGF) receptor (c-Met) and inhibits the hepatocyte growth factor (HGF) ) is an isolated bispecific FN3 domain-containing molecule that blocks binding of FN3.

[0138] The bispecific EGFR / c-MetFN3 domain-containing molecules of the invention can be prepared using any of the methods of the invention. directly between the EGFR-binding FN3 domain and any c-Met-binding FN3 domain, or It can be produced by covalently linking via a linker. Thus, the first FN3 domain of the bispecific molecule is an EGFR-binding FN3 domain. and the second FN3 domain of the bispecific molecule may have the properties described above, -Met-binding FN3 domains may have the properties described above for the -Met-binding FN3 domains.

[0139] In one embodiment, the first of the bispecific EGFR / c-Met FN3 domain-containing molecules The FN3 domain mediates EGF-induced EGFR phosphorylation at EGFR residue tyrosine 1173. was approximately 2.5 x 1 when measured in A431 cells using 50 ng / mL human EGF. 0 -6 IC less than M 50 Inhibition at low levels, containing the bispecific EGFR / c-Met FN3 domain The second FN3 domain of the molecule mediates HGF-induced c-Met cleavage at c-Met residue tyrosine 1349. -Met phosphorylation was measured in NCI-H441 cells using 100 ng / mL human HGF. When measured, it is approximately 1.5 x 10 -6 IC less than M 50 Inhibit by value.

[0140] In another embodiment, the first of the bispecific EGFR / c-Met FN3 domain-containing molecules The FN3 domain of EGFR binds to EGF-induced EGFR phosphorylation at EGFR residue tyrosine 1173. The activation was approximately 1.8x when measured in A431 cells using 50 ng / mL human EGF. 10 -8 M ~ approx. 2.5×10 -6 M IC 50 inhibited at low levels and bispecific EGFR / c-Met The second FN3 domain of the FN3 domain-containing molecule binds to c-Met residue tyrosine 1349. HGF-induced c-Met phosphorylation in NCs was assessed using 100 ng / mL human HGF. When measured with I-H441 cells, approximately 4 × 10 -9 M~approx. 1.5×10 -6 M IC 50 Value To harm.

[0141] In another embodiment, the first of the bispecific EGFR / c-Met FN3 domain-containing molecules The FN3 domain of approximately 1 × 10 -8 A dissociation constant (K D ) binds human EGFR, The second FN3 domain of the bispecific EGFR / c-Met FN3 domain-containing molecule comprises: Approximately 5×10 -8 K less than M D binds human c-Met.

[0142] In a bispecific molecule that binds both EGFR and c-Met, the first FN3 domain is approximately 2 x 10 -10 ~Approx. 1×10 -8 K of M D It binds human EGFR and binds the second FN3 domain. is approximately 3 x 10 -10 ~Approx. 5×10 -8 K of M D binds human c-Met.

[0143] The affinity of the bispecific EGFR / c-Met molecule for EGFR and c-Met is The polyspecific molecules can be determined as described above.

[0144] The first FN3 domain of the bispecific EGFR / c-Met molecule of the present invention is The cells were used to measure the activity of A431 cells incubated with or without the first FN3 domain. 600 nM streptavidin-phycoerythrin conjugate was used for the cells. and detect the amount of fluorescence from bound biotinylated EGF in an assay that detects the amount of fluorescence from bound biotinylated EGF. The binding of EGF to the -9 M~approx. 1.5×10 -7 M IC 50 Blocking by value The first FN3 domain of the bispecific EGFR / c-Met molecule of the invention can be E to EGFR in the absence of the first FN3 domain using the same assay conditions 3. Increase EGF binding to EGFR by at least 30%, 35% or more compared to the binding of EGFR. %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, or 100% can be blocked.

[0145] The second FN3 domain of the bispecific EGFR / c-Met molecule of the present invention comprises a second FN Binding of biotinylated HGF to c-Met-Fc fusion protein in the presence of the 3 domain In an assay that detects inhibition of approximately 2 x 10 -10 M ~ approx. 6×10 -8 M IC 50 Value c -Met. The second FN3 domain of the Met molecule was assayed using the same assay conditions. HGF binding to c-Met was significantly increased compared to that in the absence of HGF binding was increased by at least 30%, 35%, 40%, 45%, 50%, 55%, and 60%. %, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94 Can block 95%, 96%, 97%, 98%, 99%, or 100% .

[0146] The bispecific EGFR / c-Met molecules of the present invention were assayed using the same assay conditions as in the present invention. Compared to the level of signaling in the absence of the bispecific EGFR / c-Met molecule and inhibiting EGFR and / or c-Met signaling by at least 30%, 35%, or 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, Or it can be inhibited 100%.

[0147] EGFR and c-Met signaling are mediated by various monospecific molecules as described above. It can be measured using well-known methods.

[0148] The first FN3 domain specifically binds to EGFR and c-Met The bispecific EGFR / c-Met molecules of the invention comprising a second FN3 domain may comprise a first and Significantly higher EGF than synergistic inhibition observed with a mixture of the second FN3 domain resulting in synergistic inhibition of R and c-Met signaling and tumor cell proliferation. For example, ERK activation by bispecific EGFR / c-Met FN3 domain-containing molecules and two monospecific EGFR-binding proteins, one of which binds to EGFR and the other to c-Met. The inhibition of ERK phosphorylation by a mixture of active molecules can be assessed by measuring The bispecific EGFR / c-Met molecules of the present invention can be synthesized by combining two monospecific FN3 domains. Main mixture IC 50 at least about 100 times smaller than the value, e.g., less ICs that are 500, 1000, 5000 or 10,000 times smaller 50 ERK phosphorylation at and the bispecific FN3 domains can inhibit Exhibits at least 100-fold higher binding avidity than EGFR / c-Met FN3 domain-containing molecules Representative bispecific EGFR-c-Met FN3 domain-containing molecules are 5×10 -9The following IC 50 ERK phosphorylation can be inhibited at low levels. , can be measured using standard methods and those described herein.

[0149] The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention were administered at a dose of 7.5 ng / NCI-H292 induced by medium containing 10% FBS supplemented with mL of HGF IC for inhibition of cell proliferation by a mixture of the first FN3 domain and the second FN3 50 Value and At least 30-fold lower IC 50 Inhibits proliferation of NCI-H292 cells at The bispecific molecules of the present invention can be composed of a first FN3 domain and a second FN3 domain. IC of tumor cell growth inhibition by its mixture with acetaminophen 50 When compared with the values ​​of approximately 30, 40, and 5 0, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 60 0, 700, 800, or approximately 1000 times lower IC 50 It is possible to inhibit tumor cell proliferation at Inhibition of tumor cell growth can be measured using standard methods and those described herein. It is possible.

[0150] Another embodiment of the present invention is a method for producing a fibronectin type III (FN3) domain comprising: two FN3 domains, wherein the first FN3 domain binds to the epidermal growth factor receptor (EGFR) ) specifically binds to EGFR and blocks the binding of epidermal growth factor (EGF) to the EGFR, The second FN3 domain specifically binds to the hepatocyte growth factor receptor (c-Met) and A bispecific FN3 domain-containing antibody that blocks hepatocyte growth factor (HGF) binding to t It is a molecular the first FN3 domain comprises: Sequence HNVYKDTNX9RGL (SEQ ID NO: 179) or sequence LGSYVFEHDVM L (SEQ ID NO: 180) (wherein X9 is M or I); and A BC loop comprising the sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 181), with the proviso that X1 is A, T, G or D; X2 is A, D, Y or W; X3 is P, D or N; X4 is L or absent, X5 is D, H, R, G, Y or W; X6 is G, D or A; X7 is A, F, G, H or D; X8 is Y, F or L; the second FN3 domain comprises: Array DSFX 10 IRYX 11 EX 12 X 13 X 14 X 15 GX 16 C-strand containing (SEQ ID NO: 184) and CD loops (but X 10 is W, F or V, X 11 is D, F or L, X 12 is V, F or L, X 13 is V, L or T, X 14 is V, R, G, L, T or S, X 15 is G, S, A, T or K, X 16 is E or D) and Array TEYX 17 VX 18 IX 19 X 20 VKGGX 21 X 22 SX 23(SEQ ID NO: 185) chain and FG loop (where X 17 is Y, W, I, V, G or A, X 18 is N, T, Q or G, X 19 is L, M, N or I, X 20 is G or S, X 21 is S, L, G, Y, T, R, H or K, X 22 is I, V or L, X 23 is V, T, H, I, P, Y, or L), and It is a fluorine-containing molecule.

[0151] In another embodiment, the bispecific molecule has the sequence: LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8DSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV HNVYKDTNX9RGLPLSAEFTT (SEQ ID NO: 182), or the sequence: LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8DSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV LGSYVFEHDVMLPLSAEFTT (SEQ ID NO: 183) (However, in SEQ ID NOs: 182 and 183, X1 is A, T, G or D; X2 is A, D, Y or W; X3 is P, D or N; X4 is L or absent, X5 is D, H, R, G, Y or W; X6 is G, D or A; X7 is A, F, G, H or D; X8 is Y, F or L; X9 is M or I).

[0152] In another embodiment, the bispecific molecule has the sequence: LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 10 IRYX 11 E X 12 X 13 X 14 X 15 GX 16 AIVLTVPGSERSYDLTGLKPGTEYX 17 VX 18 IX 19 X 20 VKGGX 21 X 22 SX 23 PLSAEFTT (SEQ ID NO: 186) (however, X 10 is W, F or V, X 11 is D, F or L, X 12 is V, F or L, X 13 is V, L or T, X 14 is V, R, G, L, T or S, X 15 is G, S, A, T or K, X 16 is E or D, X 17 is Y, W, I, V, G or A, X 18 is N, T, Q or G, X 19 is L, M, N or I, X 20 is G or S, X 21 is S, L, G, Y, T, R, H or K, X 22 is I, V or L, X 23 is V, T, H, I, P, Y, T, or L), It contains two FN3 domains.

[0153] An exemplary bispecific EGFR / c-Met FN3 domain-containing molecule is SEQ ID NO: 50 -72, 106, 118-121, or 138-167 of the amino acid sequence.

[0154] The bispecific EGFR / c-Met molecules of the present invention have functions such as inhibiting EGFR autophosphorylation. The sequence HNVYKDTNX9RGL (SEQ ID NO: 179) or is the sequence LGSYVFEHDVML (SEQ ID NO: 180) (wherein X9 is M or I) Certain structural features, such as the FG loop of the first FN3 domain, bind to EGFR, including Includes signs.

[0155] In one embodiment, the bispecific EGFR / c-MetFN3 domain-containing molecule of the invention is , EGF-induced EGFR phosphorylation at EGFR residue tyrosine 1173 was measured at 50 ng / ml Approximately 8 x 10 L of human EGF was measured in H292 cells. -7 IC less than M 50 By value To hinder or HGF-induced c-Met phosphorylation at c-Met residue tyrosine 1349 was measured using 100n When measured in NCI-H441 cells using 10 μg / mL human HGF, the -7 IC less than M 50 Inhibit by value, HGF-induced NCI-H29 induced by 10% FBS containing 7.5 ng of HGF 2 Cell proliferation was approximately 9.5 x 10 -6 IC less than M 50 Inhibit by value, Approximately 2.0×10 -8 K less than M D binds to EGFR at Approximately 2.0×10 -8 K less than M D It binds to c-Met.

[0156] In another embodiment, the bispecific EGFR / c-Met FN3 domain-containing molecule of the invention The child, EGF-induced EGFR phosphorylation at EGFR residue tyrosine 1173 was measured at 50 ng / ml When measured with H292 cells using L of human EGF, approximately 4.2 x 10 -9 M~8×10 -7 M IC 50 Inhibit by value, HGF-induced c-Met phosphorylation at c-Met residue tyrosine 1349 was measured using 100n Approximately 2.4 × 10 -8 M to approx. 8.4 x 10 -7 M IC 50 Inhibit by value, HGF-induced NCI-H29 induced by 10% FBS containing 7.5 ng of HGF 2. Cell proliferation was approximately 2.3 x 10 -8 M ~ approx. 9.5×10 -6 M IC 50 Inhibit by value, Approximately 2×10 -10 M to approximately 2 x 10 -8 K of M D binds EGFR with Approximately 1×10 -9 M ~ approx. 2.0×10 -8 K of M D binds c-Met.

[0157] In one embodiment, the bispecific EGFR / c-Met molecule has the sequence: LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8DSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV EGFR-binding F comprising HNVYKDTNX9RGLPLSAEFTT (SEQ ID NO: 182) N3 domain (except X1 is D, X2 is D, X3 is P, X4 does not exist, X5 is H or W; X6 is A, X7 is F X8 is Y, X9 is M or I) and array: LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 10 IRYX 11 E X 12 X 13 X 14 X 15 GX 16 AIVLTVPGSERSYDLTGLKPGTEYX 17 VX 18 IX 19 X 20 VKGGX 21 X 22 SX 23 c-Me containing PLSAEFTT (SEQ ID NO: 186) t-binding FN3 domain (however, X 10 is W, X 11 is F, X 12 is F, X 13 is V or L, X 14 is G or S, X 15 is S or K, X 16 is E or D, X 17 is V, X18 is N, X 19 is L or M, X 20 is G or S, X 21 is S or K, X 22 is I, X 23 is P).

[0158] Representative bispecific EGFR / c-Met molecules are set forth in SEQ ID NOs: 57, 61, 62, 63, 64, 65, 66, 67 and 68.

[0159] The bispecific molecules of the present invention comprise the 11th, 14th and 17th amino acids of Tencon (SEQ ID NO: 1) described above. , 37, 46, 73, and 86 of the first FN3 domain and / or the second FN3 It may further include substitutions at one or more residue positions in the domain, and a substitution at position 29. Representative Substitutions Replaces E11N, E14P, L17A, E37P, N46V, G73Y, E86 I, and D29E (numbering is based on SEQ ID NO: 1). Other amino acids, such as amino acids within a family of amino acids related in their side chains, may be used. It will be appreciated that amino acids may also be used for substitution. The stability and binding to EGFR and / or c-Met were determined using the methods described in the literature. Sex can be tested.

[0160] In one embodiment, the bispecific EGFR / c-MetFN3 domain-containing molecule is The first FN3 domain specifically binds to R, and the second FN3 domain specifically binds to c-Met. and a first FN3 domain, wherein the first FN3 domain has the sequence: LPAPKNLVVSX24 VTX 25 DSX 26 RLSWDDPX 27 AFYX 28 SFLIQ YQX 29 SEKVGEAIX 30 LTVPGSERSYDLTGLKPGTEYTVSIY X 31 VHNVYKDTNX 32 RGLPLSAX 33 FTT (SEQ ID NO: 187) (wherein X 24 is E, N or R, X 25 is E or P, X 26 is L or A, X 27 is H or W, X 28 is E or D, X 29 is E or P, X 30 is N or V, X 31 is G or Y, X 32 is M or I, X 33 is E or I, The second FN3 domain has the sequence: LPAPKNLVVSX 34 VTX 35 DSX 36 RLSWTAPDAAFDSFWIRYF X 37 FX 38 X 39 X 40 GX 41 AIX 42 LTVPGSERSYDLTGLKPGTEYVVN IX 43 X 44 VKGGX 45 ISPPLSAX 46 FTT (SEQ ID NO: 188) (wherein X 34is E, N or R, X 35 is E or P, X 36 is L or A, X 37 is E or P, X 38 is V or L, X 39 is G or S, X 40 is S or K, X 41 is E or D, X 42 is N or V, X 43 is L or M, X 44 is G or S, X 45 is S or K, X 46 is E or I).

[0161] In other embodiments, the bispecific EGFR / c-Met FN3 domain containing molecule comprises the sequence The amino acid sequence of sequence number 27 is at least 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity a first FN3 domain comprising the amino acid sequence of SEQ ID NO: 41 and at least 8 amino acids; 3%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9 Amino acids with 3%, 94%, 95%, 96%, 97%, 98%, or 99% identity and a second FN3 domain comprising the sequence

[0162] The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention bind to EGFR and Tumor accumulation is maximized by adjusting the specific affinity for c-Met. It is possible.

[0163] Another embodiment of the present invention is a method for producing a fibronectin type III (FN3) domain comprising: a molecule comprising two FN3 domains, the first FN3 domain being an epidermal growth factor receptor agonist; It specifically binds to epidermal growth factor (EGFR) and blocks the binding of epidermal growth factor (EGF) to EGFR. The second FN3 domain specifically binds to the hepatocyte growth factor receptor (c-Met). and blocking the binding of hepatocyte growth factor (HGF) to c-Met, The N3 domain and the second FN3 domain are designed based on the Tencon sequence of SEQ ID NO: 1. isolated from a designed library.

[0164] The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention can be prepared by well-known methods. The EGFR-binding FN3 domain and the c-Met-binding FN3 domain of the present invention are covalently linked using FN3 domains can be linked together by, for example, polymerase chain reaction (PCR). linkers such as linkers containing glycine, glycine and serine, or alanine and proline; Representative linkers include (GS)2, (SEQ ID NO: No. 78), (GGGGS) (SEQ ID NO: 79), (AP) (SEQ ID NO: 80), (AP) (SEQ ID NO: 81), (AP) 10 (SEQ ID NO: 82), (AP) 20 (SEQ ID NO: 83), A(E AAAK)5AAA (SEQ ID NO: 84) linker. The use of natural and artificial peptide linkers to join fusion polypeptides has been reported in the literature. It is well known that 64,5260~5268,1989;Alfthan et al.,Protein Eng.8,725~731,1995;Robinson & Sauer,Bio Chemistry 35,109-116,1996; U.S. Patent No. 5,856,456 The bispecific EGFR / c-Met molecules of the present invention comprise a C-terminal fragment of the first FN3 domain. to the N-terminus of the second FN3 domain, or from the C-terminus of the second FN3 domain to the first F Any EGFR-binding FN3 domain can be linked to the N-terminus of the N3 domain. The c-Met-binding FN3 domain can be covalently linked to the FN3 domain. EGFR-binding FN3 domains include those represented by SEQ ID NOs: 18 to 29, 107 to 110, and 12 There is a domain with the amino acid sequence shown in 2-137, which is a representative c-Met binding F The N3 domain includes amino acid sequences shown in SEQ ID NOs: 32 to 49 and 111 to 114. The EGFR-binding FN3 domains linked to the bispecific molecule are , and may further comprise an initiating methionine (Met) at their N-terminus.

[0165] Within the scope of the present invention are mutations in bispecific EGFR / c-Met FN3 domain-containing molecules. For example, the resulting mutants may be EGFR and cM when compared to the parent molecule. Bispecific EGFR / c-Met as long as it retains similar selectivity and efficacy to et Substitutions can be made in FN3 domain-containing molecules. Exemplary modifications include, for example: There are conservative substitutions that result in variants with similar properties to the parent molecule. Substitutions that occur within a family of related amino acids in a gene-encoded Amino acids are classified into (1) acidic amino acids (aspartate, glutamate), (2) basic amino acids Amino acids (lysine, arginine, histidine), (3) nonpolar amino acids (alanine, Leucine, Isoleucine, Proline, Phenylalanine, Methionine, Tryptophan (4) uncharged polar amino acids (glycine, asparagine, glutamine, cysteine) They can be classified into four families: phenyl, serine, threonine, and tyrosine. Amino acids such as thialanine, tryptophan, and tyrosine are collectively classified as aromatic amino acids. Alternatively, the amino acid repertoire may consist of (1) acidic amino acids (asparagine (2) basic amino acids (lysine, arginine, histidine), (3) Aliphatic amino acids (glycine, alanine, valine, leucine, isoleucine, serine) , threonine) (serine and threonine are arbitrarily classified separately as aliphatic hydroxyl (4) aromatic amino acids (phenylalanine, tyrosine, tryptophan), (5) amides (asparagine, glutamine), and (6) sulfur-containing amino acids (cysteine ​​and methyl thionin) (Stryer (ed.), Biochemis try, 2nd ed, WH Freeman and Co., 1981). non-conservative Bispecific EGFs involving substitutions of amino acid residues between different classes of amino acids. Improving the properties of bispecific molecules by using R / c-Met FN3 domain-containing molecules A change in the amino acid sequence of a polypeptide or fragment thereof may result in a functional homologue. The presence or absence of a logarithmic change can be determined by measuring the unmodified polynucleotide using the assays described herein. The modified polypeptide or fragment produces a response in the same manner as the peptide or fragment. This can be easily determined by assessing the ability of peptides with more than one substitution to Any peptide, polypeptide or protein can be readily tested in the same manner.

[0166] The bispecific EGFR / c-Met FN3 domain-containing molecules of the invention can be used to target, for example, As a means to increase the valence and therefore avidity of the bond, Multimers can be produced by incorporating amino acid linkers, for example, using well-known methods. By incorporating one or more EGFR-binding FN3 domains and one or more c-Met-binding FN The three domains are linked to form a molecule with at least dual specificity for either EGFR or c-Met. by forming a molecule containing at least three individual FN3 domains of different amino acids. It is possible.

[0167] Another embodiment of the present invention is a method for producing a fibronectin type III (FN3) domain comprising: a molecule comprising two FN3 domains, the first FN3 domain being an epidermal growth factor receptor agonist; It specifically binds to epidermal growth factor (EGFR) and blocks the binding of epidermal growth factor (EGF) to EGFR. The second FN3 domain specifically binds to the hepatocyte growth factor receptor (c-Met). SEQ ID NO: 5, which binds to and blocks the binding of hepatocyte growth factor (HGF) to c-Met A bispecific FN comprising an amino acid sequence shown in 0 to 72, 106, or 138 to 165. There are three domains.

[0168] Half-life extension moiety The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention or the monospecific EGFR The GFR or c-Met binding FN3 domains bind to the GFR or c-Met receptors via, for example, covalent interactions. In one aspect of the invention, the bispecific EGFR of the invention may incorporate other subunits. The FR / c-Met FN3 domain-containing molecule further comprises a half-life extending moiety. The phase extender moiety may comprise albumin, albumin variants, albumin binding proteins and / or domains, transferrin, and fragments and analogs thereof, and Fc regions One representative albumin binding domain is shown in SEQ ID NO:117.

[0169] Antibody-like properties, particularly C1q binding, complement-dependent cytotoxicity (CDC), and Fc receptor binding , antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, cell surface receptors (e.g., B cell receptors) Fc region, such as Fc effector functions, including downregulation of the BCR (body receptor) The whole or part of an antibody constant region is linked to the molecule of the invention to confer properties associated with These activities can be further improved by modifying the residues in the Fc that are responsible for these activities. (For an overview, see Strohl, Curr Opin Biote (See chnol.20,685~691,2009).

[0170] The bispecific molecules of the invention may be modified with PEG5000 or PEG2 to achieve the desired properties. Polyethylene glycol (PEG) molecules such as 0000, e.g., laurate, myristyl Phosphate, stearate, arachidate, behenate, oleate, arachidonate , octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, etc. Fatty acids and fatty acid esters, polylysine, octane, carbohydrates (dextran, cellulose Further moieties such as saccharides, oligosaccharides or polysaccharides can be incorporated. The fusion may be a direct fusion with the coding sequence of the protein scaffold, and may be carried out using standard cloning and Alternatively, the recombinant protein can be prepared by well-known chemical conjugation methods. Such moieties can also be attached to recombinantly produced molecules of the invention.

[0171] For example, a cysteine ​​residue can be incorporated into the C-terminus of the molecule using well-known methods, and this cysteine ​​can be used to By attaching a PEGyl group to the amino acid, the bispecific or monospecific molecules of the invention can be prepared. A typical bispecific nucleotide sequence with a C-terminal cysteine ​​can be added. The functional molecules include those having the amino acid sequences shown in SEQ ID NOs: 170 to 178.

[0172] Monospecific and bispecific molecules of the invention incorporating additional moieties can be synthesized using several well-known antigens. For example, the Fc domain and / or Altered characteristics of monospecific and / or bispecific molecules by incorporation of Fc domain variants The activity is mediated by soluble FcγRI, FcγRII, FcγRIII, or FcRn receptors. using receptors in the form of antibodies or cell-based antibodies that measure, for example, ADCC or CDC. The pharmacokinetic characteristics of the molecules of the invention can be characterized using assays based on the pharmacokinetics of the molecules of the invention or in in vivo models. can be assayed in an Fc receptor binding assay by assessing .

[0173] Polynucleotides, Vectors, and Host Cells The present invention relates to EGFR-binding or c-Met-binding FN3 domains or bispecific FN3 domains of the invention. The active EGFR / c-Met FN3 domain-containing molecule is provided as an isolated polynucleotide. a nucleic acid encoding the nucleic acid as part of an expression vector or as part of a linear DNA sequence The present invention provides linear DNA sequences, compositions or and the prokaryotic, eukaryotic, or filamentous phage expression and secretion of these directed mutagens. and / or display compatible vectors. The present disclosure discloses polynucleotides that are suitable for use in the present invention, but may be modified due to the degeneracy of the genetic code or the codons in a given expression system. In consideration of selectivity, the EGFR-binding or c-Met-binding FN3 domain of the present invention or Other polynucleotides encoding bispecific EGFR / c-Met FN3 domain-containing molecules Otides are also within the scope of the present invention.

[0174] One embodiment of the present invention is a method for producing a nucleic acid sequence of SEQ ID NO: 18 to 29, 107 to 110, or 122 to 137. An isolated nucleic acid encoding an FN3 domain that specifically binds to EGFR, having the amino acid sequence It is a polynucleotide.

[0175] One embodiment of the present invention is a method for producing a polynucleotide sequence of SEQ ID NO: 97-98 or 168-169. It is an isolated polynucleotide comprising a sequence.

[0176] One embodiment of the present invention is a method for producing a nucleic acid sequence comprising the sequence shown in SEQ ID NOs: 32 to 49 or 111 to 114. An isolated polypeptide encoding an FN3 domain that specifically binds to c-Met, having the amino acid sequence It is a polynucleotide.

[0177] One embodiment of the present invention is a method for producing a nucleotide sequence of SEQ ID NO: 50 to 72, 106, 118 to 121, or 138 to 140. Contains a bispecific EGFR / c-Met FN3 domain with a 165 amino acid sequence It is an isolated polynucleotide encoding the molecule.

[0178] One embodiment of the present invention is a polynucleotide sequence of SEQ ID NO: 115-116 or 166-167. It is an isolated polynucleotide comprising a nucleotide sequence.

[0179] The polynucleotides of the present invention can be synthesized by solid-phase polynucleotide synthesis in an automated polynucleotide synthesizer. They can be produced by chemical synthesis, such as by ligation synthesis, and can be assembled as complete single- or double-stranded molecules. Alternatively, the polynucleotides of the present invention can be prepared by PCR followed by conventional cloning. Polynucleotides of a specific known sequence can be generated by other methods, such as by Methods for producing or obtaining tides are well known in the art.

[0180] The polynucleotide of the present invention may contain promoter or enhancer sequences, introns, polynucleotides, At least one non-coding sequence, such as an adenylation signal or a cis sequence that promotes RepA binding, is required. The polynucleotide sequence may also facilitate purification or detection of the protein. marker or tag sequence, signal, such as a histidine tag or an HA tag, for sequences, RepA, Fc, or bacteriophage coat proteins such as pIX or pIII Further amino acids encoding the fusion protein partner, such as a fusion protein, may be added. It may also include a sequence

[0181] Another embodiment of the present invention is a vector comprising at least one polynucleotide of the present invention. Such vectors include plasmid vectors, viral vectors, baculovirus vectors, and vectors, transposon-based vectors, or vectors that are specifically engineered by any means suitable for introducing a polynucleotide of the present invention into a target organism or genetic background. The vector may be any other vector. A nucleic acid sequence capable of controlling, regulating, inducing, or allowing expression of a polypeptide of interest. The vector may contain a transcription enhancer binding site, R The RNA polymerase initiation site, ribosome binding site, and the encoded The expression system may contain other sites that facilitate expression of the desired polypeptide. Such expression systems are well known in the art. These may be well-known cell-based or cell-free systems.

[0182] Another embodiment of the present invention is a host cell comprising the vector of the present invention. Single-specific EGFR-binding or c-Met-binding FN3 domain or bispecific EGFR / cMet etFN3 domain-containing molecules can be expressed in various media, including cell lines, mixed cell lines, and the like, as is well known in the art. Optionally, the cells may be produced by a line, an immortalized cell, or a clonal population of immortalized cells. For example, see Ausubel, et al., ed., Current Protocols ls in Molecular Biology,John Wiley & Son s,Inc.,NY,NY(1987~2001);Sambrook,et al., Molecular Cloning:A Laboratory Manual,2 nd Edition,Cold Spring Harbor,NY(1989);Har low and Lane, Antibodies, a Laboratory Man ual,Cold Spring Harbor,NY(1989);Colligan ,et al.,eds.,Current Protocols in Immuno logy, John Wiley & Sons, Inc., NY (1994~2001 );Colligan et al.,Current Protocols in P rotein Science,John Wiley & Sons,NY,NY,( The host cells selected for expression are of mammalian origin. Alternatively, COS-1, COS-7, HEK293, BHK21, CHO, B SC-1, He G2, SP2 / 0, HeLa, myeloma, lymphoma, yeast, insect or The cell may be selected from a plant cell, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell may be derived from a species or organism that is unable to glycosylate polypeptides. , e.g., BL21, BL21(DE3), BL21-GOLD(DE3), XL1-B lue, JM109, HMS174, HMS174(DE3), and natural or engineered from prokaryotic cells or organisms such as strains of Escherichia coli, Klebsiella, or Pseudomonas. You can also select from:

[0183] Another embodiment of the present invention is a monoclonal antibody of the present invention that specifically binds to EGFR or c-Met. isolated FN3 domain or isolated bispecific EGFR / c-Met F domain of the invention A method for producing an N3 domain-containing molecule, comprising: An isolated FN3 domain that specifically binds to EGFR or c-Met, or an isolated The bispecific EGFR / c-Met FN3 domain-containing molecule was cultured under conditions that allowed its expression. and purifying said domain or molecule.

[0184] The FN3 domain of the present invention that specifically binds to EGFR or c-Met, or an isolated The bispecific EGFR / c-Met FN3 domain-containing molecule can be, for example, a protein Purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography Chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography affinity chromatography, hydroxyapatite chromatography, and and lectin chromatography, or high performance liquid chromatography (HPLC) It can be purified from recombinant cell culture by any well-known method.

[0185] Bispecific EGFR / c-Met antibody Bispecific EGFR / c-Met antibodies may be generated de novo or derived from existing monospecific antibodies. EGFR antibodies and anti-c-Met antibodies can be engineered.

[0186] Representative anti-EGFR antibodies that can be used to engineer bispecific molecules include Examples include panitumumab (ABX-EGF), nimotuzumab, necitumumab, and matuzumab. and, for example, U.S. Patent No. 7,595,378, U.S. Patent No. 7,247,301, U.S. Patent Application Publication No. 2011 / 0256142, U.S. Patent No. 5,891,996, U.S. Patent No. No. 5,212,290, U.S. Pat. No. 5,558,864, or U.S. Pat. No. 7, 589, 180. For example, those shown in SEQ ID NO: 189 or 191. an antibody VH domain having the amino acid sequence shown in SEQ ID NO: 190 or 192; An antibody VL domain having an amino acid sequence can be used.

[0187] Representative anti-c-Met antibodies that can be used to engineer bispecific molecules include For example, rilotumumab, onartuzumab, ficlatuzumab, and other compounds listed in the International Patent Publication No. WO2011 / 110642, U.S. Patent Application Publication No. 2004 / 0166544 , International Patent Publication No. WO2005 / 016382, or International Patent Publication No. WO2006 / 0 For example, the amino acid sequence shown in SEQ ID NO: 193 or 195 is described in SEQ ID NO: 15371. An antibody VH domain having the amino acid sequence shown in SEQ ID NO: 194 or 196 An antibody VL domain having the sequence: The heavy and light chain amino acid sequences of the identified antibodies are available at http: / / _www_ama-as Available from the American Medical Association at sn_org or by CAS Registry Number.

[0188] Monospecific EGFR and c-Met binding variable domains can be, for example, human immunoglobulin or Fab, single chain antibody (scFV) or unpaired or paired antibody variable fragments A phage display live cell that has been engineered to express a portion of the region, such as a A newly selected strain was selected from the library (Knappik et al., J Mol Biol 296 :57~86,2000;Krebs et al., J Immunol Meth 254:67~84,2001;Vaughan et al.,Nature Bio technology 14:309~314,1996;Sheets et al. , PITAS(USA)95:6157~6162,1998;Hoogenboom and Winter, J Mol Biol 227:381,1991;Marks et al., J Mol Biol 222:581, 1991), followed by manipulation This allows for a more bispecific format. The combined variable domains are described in Shi et al. (2010) J. Mol. Biol. 397:3 85-96 and International Patent Publication No. WO09 / 085462, for example, The heavy and light chain variable regions of the antibody are fused to the bacteriophage pIX coat protein. These antibodies can be isolated from phage display libraries expressed as proteins. The antibody library was screened for binding to the extracellular domain of human EGFR or c-Met. The resulting positive clones were further characterized and Fab was isolated from the clone lysates. Such phage display methods for isolating human antibodies are well known in the art. For example, U.S. Patent No. 5,223,409 and U.S. Patent No. 5,223,409. ,403,484, U.S. Patent No. 5,571,698, U.S. Patent No. 5,427,908 No., U.S. Patent No. 5,580,717, U.S. Patent No. 5,969,108, U.S. Patent No. No. 6,172,197, U.S. Pat. No. 5,885,793, U.S. Pat. No. 6,521,40 No. 4, U.S. Patent No. 6,544,731, U.S. Patent No. 6,555,313, U.S. Patent No. See U.S. Patent Nos. 6,582,915 and 6,593,081. New variable regions that bind to EGFR or c-Met can be generated using the methods described herein. and engineered into a bispecific format.

[0189] Bispecific antibody format The antibodies of the present invention have two or more antigen-binding sites and are bispecific. Antibodies include antibodies having a full-length antibody structure.

[0190] As used herein, a "full-length antibody" refers to an antibody that contains two full-length antibody heavy chains and two A full-length antibody heavy chain (HC) is an antibody having a full-length antibody light chain. Full-length antibody light chains consist of variable and constant domains VH, CH1, CH2, and CH3. The full-length antibody (LC) is composed of the well-known variable and constant domains of the light chain, VL and CL. The antibody may lack a C-terminal lysine (K) in one or both heavy chains.

[0191] The term "Fab arm" or "half molecule" refers to one of the heavy / light chains that specifically binds to an antigen. Refers to a pair of

[0192] The full-length bispecific antibodies herein can be expressed in vitro in a cell-free environment or by co-expression. conditions that favor the formation of heterodimers of two antibody half molecules with different specificities. To achieve this, two monospecific bivalent antibodies were generated by introducing substitutions at the interface of the heavy chain CH3 of each half molecule. It can be generated by Fab arm exchange (or half molecule exchange) between antibodies. The ab arm exchange reaction is a mechanism for the disulfide bond isomerization and dissociation / association of the CH3 domain. As a result, the heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine ​​of one parent monospecific antibody is transferred to the second parent monospecific antibody molecule. It forms an inter-heavy chain disulfide bond with cysteine ​​residues, and at the same time, the CH3 domain of the parent antibody is decomposed. The CH3 domain of the Fab arm is released and reassembled by dissociation / association. The resulting product can be engineered to favor heterodimerization over dimerization. Each epitope is a different epitope, i.e., an epitope on EGFR and an epitope on c-Met. It is a bispecific antibody that has two Fab arms or half molecules that bind a specific tope.

[0193] As used herein, "homodimerization" refers to the formation of a homodimer having identical CH3 amino acid sequences. As used herein, "homodimer" refers to the interaction of two heavy chains that are identical. It refers to an antibody having two heavy chains with the CH3 amino acid sequence:

[0194] As used herein, "heterodimerization" refers to the process of combining non-identical CH3 amino acid sequences. As used herein, "heterodimer" refers to the interaction of two heavy chains having , refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0195] The "knob-in-hole" method (see, e.g., International Patent Publication No. WO 2006 / 028936) ) can be used to generate full-length bispecific antibodies. Selected amino acids that form the interface of the CH3 domains of IgG were identified as Mutations can be made at positions that affect the activity and promote heterodimer formation. The amino acid with a small side chain (hole) is located at the heavy chain of the antibody that specifically binds to the first antigen. The amino acid with a large side chain (knob) is introduced into the chain to specifically bind to the second antigen. The co-expression of these two antibodies results in the formation of a "hole" The heavy chain with the "knob" selectively interacts with the heavy chain with the "knob" to form heterodimers. Representative pairs of CH3 substitutions that form "knobs" and "holes" include (first Modification position in the first CH3 domain of the heavy chain / modification in the second CH3 domain of the second heavy chain position), T366Y / F405A, T366W / F405W, F405 W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T3 94S, F405W / T394S and T366W / T366S_L368A_Y407V There is.

[0196] U.S. Patent Application Publication No. 2010 / 0015133, U.S. Patent Application Publication No. 2009 / 01 82127, U.S. Patent Application Publication No. 2010 / 028637, or U.S. Patent Application Publication No. As described in 2011 / 0123532, the positively charged residue on one of the CH3 surfaces Electrostatic interactions can be utilized by substituting the negatively charged residues on the CH3 surface and the second CH3 surface. Other approaches can be used, such as using ribonucleotides to promote heavy chain heterodimerization. In this method, heterodimerization can be promoted by the following substitutions (the first heavy chain first as modification positions within the CH3 domain of the first heavy chain / modification positions within the second CH3 domain of the second heavy chain (i.e., L351Y_F405A_Y407V / T394W, T366 I_K392M_T394W / F405A_Y407V, T366L_K392M_T3 94W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409 F, or T350V_L351Y_F405A_Y407V / T350V_T366L_ K392L_T394W (U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Patent (as described in application publication no. 2013 / 0195849).

[0197] In addition to the methods described above, the bispecific antibodies of the present invention can also be produced by the methods described in International Patent Publication No. WO2011 / 013166. C of two monospecific homodimeric antibodies according to the method described in US Pat. No. 131746. Asymmetric mutations were introduced into the H3 region, and the resulting product was purified under reducing conditions to induce disulfide bond isomerization. to form a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies These methods can be performed in vitro in a cell-free environment. a monospecific bivalent antibody (e.g., an anti-c-Met antibody) and a second monospecific bivalent antibody (e.g., anti-EGFR antibody) with specific substitutions in the CH domain that enhance heterodimer stability In this case, these antibodies are engineered so that the cysteines in the hinge region are disulfide bonded. By incubating them together under reducing conditions sufficient to isomerize the Fab fragments. Bispecific antibodies are generated by arm exchange. Incubation conditions are non-reducing. The typical reducing agent that can be used is 2-methylpropanol. 2-Methoxyethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol Dimethicone (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCE P), L-cysteine ​​and β-mercaptoethanol, preferably 2-mercaptoethanol. triethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine For example, the presence of at least 25 mM 2-MEA. or in the presence of at least 0.5 mM dithiothreitol, at pH 5 to 8, e.g., pH Incubate at pH 7.0 or 7.4 for at least 90 minutes at a temperature of at least 20°C. A solution can be used.

[0198] Bispecific EGFR / c-Met antibody The bispecific EGFR / c-Met antibodies of the present invention are directed to small molecule EGFR and / or cMet receptors. Offering advantages in terms of specificity and reduced off-target toxicity compared to et inhibitors The present invention relates to a method for treating EGFR-associated c-Met cells, comprising administering to a subject a subject in need thereof a bispecific EGFR / c-Met antibody. A mixture of a bispecific antibody and a c-Met binding monospecific antibody or a dual antibody as described in the literature It also showed significantly improved synergistic inhibitory activity compared to specific EGFR / c-Met antibodies. This was based, at least in part, on the surprising discovery that Accordingly, the observed synergistic effect varied from about 14 to more than about 800 fold. EGFR / c-Met antibodies are more effective than cetuximab (Erbitux®) This results in effective inhibition of the EGFR and / or c-Met signaling pathways, thereby more efficiently inhibiting tumor growth. The bispecific EGFR / c-Met antibodies of the present invention inhibit EGF R-activating mutations and / or treatment with tyrosine kinase inhibitors such as gefitinib Tumors and / or tumor cell lines with EGFR mutations known to confer resistance inhibits EGFR signaling in NSCLC and inhibits EGFR tyrosine kinase activity in cancers such as NSCLC. It is upregulated upon treatment with enzyme inhibitors and provides compensatory signaling. The present invention inhibits the c-Met signaling pathway, a pathway that has been identified as being involved in the treatment of cancer. Bispecific EGFR / c-Met antibodies directly inhibit EGFR and c-Met signaling. In addition to inhibiting EGFR and c-Me, it also enhances antibody-dependent cellular cytotoxicity (ADCC) and Current EGFR treatments (cetuximab and pancreatic cancer) have shown antitumor activity by disrupting the EGFR receptor. Unlike EGFR / c-Met (anticoagulant), the bispecific EGFR / c-Met antibodies of the present invention are CC induces the death of tumor cells harboring KRAS mutations.

[0199] International Patent Publication No. WO2010 / 115551 is currently undergoing Phase III clinical trials. The EGFR-binding VH / VL pair of cetuximab and the antibody 5D5 (MetMab, IgG-scFv format using the c-Met binding VH / VL pair of nartuzumab We describe an engineered bispecific EGFR / c-Met antibody (BSAB01) BSAB01 showed approximately two-fold (additive) increased activity of A431 cells compared to the parent antibody. Inhibition of cell proliferation (Example 7 of International Patent Publication No. WO2010 / 115551, Figure 8b) ), which has a significant effect on the proliferation of Ovarc-8 cells when compared to the combination of the two parental antibodies. The inhibitor exhibits an additive inhibition rate of 15% compared to 10% (International Patent Publication No. WO2010 10a, Example 16 of US Patent No. 2011 / 0115551). Thus, surprisingly and unexpectedly, Furthermore, the present invention relates to the regulation of EGFR and c-Met signaling, cancer cell survival, and tumor growth. and a bispecific EGFR / c-Met antibody that exhibits significant synergistic activity in inhibiting Without wishing to be bound by any theory, the bispecific The remarkable synergistic effect of the antibody on both the EGFR-binding arm and the c-Met-binding arm was observed. This is due, at least in part, to the specificity of the target site, and the homodimerization of EGFR and c-Met. This leads to inhibition of signal transduction by not only the EGFR / HERx heterodimer but also the EGFR / HERx heterodimer. It is thought that this is possible.

[0200] One embodiment of the present invention is an isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte a cell growth factor receptor (c-Met) antibody, a) Contains HC1 constant domain 3 (HC1 CH3) and HC1 variable domain 1 (VH1) a first heavy chain (HC1); b) containing HC2 constant domain 3 (HC2 CH3) and HC2 variable domain 2 (VH2) a second heavy chain (HC2); and c) a first light chain (LC1) comprising a light chain variable region (VL1); a second light chain (LC2) comprising a light chain variable region (VL2), wherein VH1 and VL1 are VH2 and VL2 pair together to form a first antigen-binding site that specifically binds to EGFR. pair to form a second antigen-binding site that specifically binds to c-Met, and HC1 is 1 CH3 contains at least one substitution, HC2 contains at least one substitution in HC2 CH3 Substitutions within HC1 CH3 and substitutions within HC2 CH3 are included in the residue numbering. occurs at different amino acid residue positions when It is a bispecific EGFR / c-Met antibody.

[0201] In some embodiments described herein, the bispecific EGFR / c-Met antibody The extracellular signaling in NCI-H292, NCI-H1975, or SKMES-1 cell lines was inhibits the phosphorylation of signal-related kinases 1 and 2 (ERK1 / 2) and its IC 50 The value is A control monovalent EGFR antibody containing heavy chain 3 (HC3) and light chain 3 (LC3) and heavy chain 4 (H C4) and a control monovalent c-Met antibody containing light chain 4 (LC4) ERK1 / 2 expression in CI-H292, NCI-H1975, or SKMES-1 cell lines IC for inhibition of phosphorylation50 At least about 10 times lower, at least about 2 times lower, when compared to the 0 times lower, at least about 30 times lower, at least about 40 times lower, at least about 50 times lower , or at least about 60-fold lower IC 50 However, HC3 and HC1, LC3 and LC 1. HC4 and HC2, and LC4 and LC2 have identical amino acid sequences, respectively, and ERK1 / Phosphorylation of 2 was detected using anti-phospho-ERK1 / 2 antibody as a capture antibody and conjugated with an electrochemiluminescent compound. The antibody that binds to the non-phosphorylated and phosphorylated ERK1 / 2 was used as the detection antibody. The antibody is measured in whole cell lysates using a sandwich immunoassay. The inhibitory rate of EGFR / c-Met antibodies was assessed by inhibition of ERK1 / 2 phosphorylation. in EGFR patients compared with a monospecific EGFR antibody and a monospecific c-Met antibody This results in a synergistic and more pronounced inhibition of FR and c-Met signaling. A suitable bispecific EGFR / c-Met antibody is the antibody EM1-mAb of the present invention. .

[0202] As used herein, a "control monospecific EGFR antibody" refers to a monospecific EGFR antibody that is not tested. The Fab arm of the EGFR-binding bispecific EGFR / c-Met antibody has the same amino acid sequence as the Fab arm. The first Fab arm binds to EGFR and is "inactive." The first antibody binds to an unrelated / unrelated antigen, human immunodeficiency virus (HIV) gp120. This refers to an antibody with two Fab arms. The second Fab arm is the antibody with the dual specificity being tested. When the EGFR-binding Fab arm of an isomeric EGFR / c-Met antibody contains an F405L substitution , having a light chain having the sequence of SEQ ID NO: 209 and a heavy chain having the sequence of SEQ ID NO: 198. The second Fab arm controls the EGFR binding of the bispecific EGFR / c-Met antibody being tested. If the Fab arm contains a K409R substitution, then the light chain has the sequence of SEQ ID NO: 209 and the light chain has the sequence of SEQ ID NO: and a heavy chain having the sequence of SEQ ID NO:197.

[0203] As used herein, a "control monospecific c-Met antibody" refers to a monospecific c-Met antibody that is not tested. The c-Met-binding Fab arm of the bispecific EGFR / c-Met antibody is identical to The first Fab arm has a c-Met binding sequence and is an "inactive" " and a second Fab arm that binds to an unrelated / unrelated antigen, HIV gp120. The second Fab arm is the antibody that binds to the bispecific EGFR / c SEQ ID NO: 20, when the c-Met-binding Fab arm of the -Met antibody contains the F405L substitution 9 and a heavy chain having the sequence of SEQ ID NO: 198. The ab arm is the c-Met binding Fab of the bispecific EGFR / c-Met antibody being tested. If the arm contains a K409R substitution, the light chain has the sequence of SEQ ID NO: 209 and the arm has the sequence of SEQ ID NO: 19 and a heavy chain having the sequence of SEQ ID NO:7.

[0204] In some embodiments described herein, the bispecific EGFR / c-Met antibody increases ERK1 / 2 phosphorylation by approximately 2 × 10 -9 M or less, approximately 1×10 -9 M or less, or about 1 x 10 -10 IC below M 50 Inhibit by value.

[0205] In some embodiments described herein, ERK1 is a nucleotide sequence comprising residues Thr202 and T ERK2 is phosphorylated at residues Thr185 and Tyr197. It is phosphorylated.

[0206] In some embodiments described herein, the bispecific EGFR / c-Met antibody Ser473 of protein kinase B (AKT) in NCI-H1975 cell line inhibits phosphorylation in the IC 50 Values ​​are control monovalents containing HC3 and LC3 A mixture of EGFR antibodies and control monovalent c-Met antibodies containing HC4 and LC4 IC of AKT Ser473 phosphorylation inhibition in NCI-H1975 cell line 50 At least approximately 70-fold lower IC compared to the 50 value, but HC3 and HC 1. LC3 and LC1, HC4 and HC2, and LC4 and LC2 have the same amino acid sequence. The phosphorylation of AKT at Ser473 was compared with non-phosphorylated and phosphorylated AKT. The binding antibody was used as the capture antibody, and anti-phospho-AKT Ser conjugated with an electrochemiluminescent compound was used. Whole cell lysates were analyzed using a sandwich immunoassay with 473 antibody as the detection antibody. It is measured in the

[0207] In some embodiments described herein, the bispecific EGFR / c-Met antibody Thr308 of protein kinase B (AKT) in NCI-H1975 cell line inhibits phosphorylation in the IC 50 Values ​​are control monovalents containing HC3 and LC3 A mixture of EGFR antibodies and control monovalent c-Met antibodies containing HC4 and LC4 Inhibition of AKT phosphorylation at Thr308 in NCI-H1975 cell line C50 At least approximately 100-fold lower IC compared to the 50 value, where HC3 and HC1, LC3 and LC1, HC4 and HC2, and LC4 and LC2 each have the same amino acid sequence. The phosphorylation of AKT at Thr308 is a key step in determining whether the AKT is phosphorylated or phosphorylated. The antibody that binds to T was used as the capture antibody, and anti-phospho-AKT T conjugated with an electrochemiluminescent compound was used. Whole cell laminin was detected using a sandwich immunoassay with hr308 antibody as the detection antibody. It is measured in isate.

[0208] The bispecific EGFR / c-Met antibodies of the present invention increase the inhibition rate by inhibiting AKT phosphorylation. The combination of a monospecific EGFR antibody and a monospecific c-Met antibody was compared when evaluated by This results in a synergistic and more pronounced inhibition of EGFR and c-Met signaling compared to Representative bispecific EGFR / c-Met antibodies include the antibody EM1- There is mAb.

[0209] In some embodiments described herein, the bispecific EGFR / c-Met antibody phosphorylation of AKT at Ser473 or Thr308 was approximately 1 × 10 -9 I below M C 50 Inhibit by value.

[0210] In some embodiments described herein, the bispecific EGFR / c-Met antibody is EGFR of SEQ ID NO: 73 and EGFR residues K489, I491, K467 and S492 and c-Met and residues PEFRDSYPIKYVHAF (SEQ ID NO: 238) and FAQSKPDSAEPMDRSA (SEQ ID NO: 239). A representative bispecific antibody is EM1-mAb. As described above and in International Patent Publication No. WO2010 / 115551, the antibody BSAB Binds to different epitopes to EGFR and c-Met compared to 01. The parent EGFR binding arm of 01 (cetuximab) is a sequence similar to residues of full-length EGFR in SEQ ID NO: 73. R367, Q408, Q432, H433, F436, S442, S464, K467, EGFR amino acid sequence of mature EGFR corresponding to K489, I491, S492, and N497. Noic acid residues R353, Q384, Q408, H409, F412, S418, S440, K 443, K465, I467, S468, and N473 (Li et al. , Cancer Cell 7:301-311, 2005). Parent cM of BSAB01 The et-binding arm (mAb 5D5) binds to residues 325–340 of c-Met. EGFR-binding parent antibody of EM1-mAb. For epitope mapping of the antibody (2F8), see U.S. Patent Application Publication No. US2011 / 004999. Cetuximab and the parent 2F8 antibody are described in patent application Ser. No. 256142A1. However, they bind to distinct epitopes.

[0211] Epitope mapping can be performed using standard methods. For example, If the structures of both proteins are known, in silico protein-protein docking can be performed. This allows us to identify the sites of compatible interactions. By performing a hydrogen / dioxide exchange, it is possible to map the regions of an antigen to which an antibody can bind. By using segment and point mutagenesis of the antigen, it is possible to identify the regions important for antibody binding. The position of the amino acid can be identified.

[0212] In some embodiments described herein, the bispecific EGFR / c-Met antibody abrogates EGFR and c-Met signaling.

[0213] The bispecific EGFR / c-Met antibodies of the invention were assayed using the same assay conditions as those of the invention. Compared to the level of signaling in the absence of the bispecific EGFR / c-Met molecule When administered, EGFR and c-Met signaling is suppressed by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 It can be disabled to 0%.

[0214] Binding of ligands such as EGF to EGFR induces receptor dimerization, autophosphorylation, and Activation of the receptor's internal cytoplasmic tyrosine kinase domain and regulation of DNA synthesis (genetic Multiple signal transducers involved in cell cycle progression or cell division stimulates the initiation of transcription and transactivation pathways. This may lead to inhibition of one or more downstream signaling pathways of EGFR, Disabling this leads to inhibition of cell proliferation and differentiation, angiogenesis, cell motility, and metastasis, as well as These compounds may have a variety of effects, including the inhibition of downstream signal transduction pathways.

[0215] EGFR signaling and abrogation of EGFR signaling can be achieved, for example, by the bispecific antibody of the present invention. Receptor at any of tyrosines Y1068, Y1148, and Y1173 by a specific antibody Autophosphorylation of the body (Downward et al., Nature 311:483~ 5, 1984), and / or phosphorylation of natural or synthetic substrates, and natural or synthetic groups using various well-known methods, such as measuring inhibition of protein autophosphorylation and / or phosphorylation. Phosphorylation can be measured using an ELISA assay or a phosphotyrosine-specific antibody. This can be detected using well-known methods such as Western blotting using a representative The assay was performed as described by Panek et al., J Pharmacol Exp Thera 283:1433-44, 1997 and Batley et al., Life Sc i 62:143-50, 1998, and further described herein.

[0216] Binding of HGF to c-Met induces receptor dimerization, autophosphorylation, and cell proliferation of the receptor. Activation of the protein tyrosine kinase domain and regulation of DNA synthesis (gene activation) and Multiple signal transduction and transduction pathways involved in cell cycle progression or cell division Inhibition of c-Met signaling stimulates the initiation of one or more c-Met activation pathways. Disabling c-Met may improve cell proliferation, as it may lead to inhibition of downstream signaling pathways. Various actions, including inhibition of cell proliferation and differentiation, angiogenesis, cell motility, and metastasis may have:

[0217] c-Met signaling and c-Met signaling inactivation are mediated by, for example, tyrosine residues. in at least one of groups Y1230, Y1234, Y1235, or Y1349 Various methods are available, such as measuring receptor autophosphorylation and / or phosphorylation of natural or synthetic substrates. Phosphorylation can be measured using various well-known methods. For example, phosphorylation can be measured using an ELISA assay. or Western blot using antibodies specific to phosphotyrosine. A representative assay is described in Panek et al., J Ph armacol Exp Thera 283:1433~44,1997 and Batl ey et al., Life Sci 62:143-50, 1998, and As described herein.

[0218] EGFR and c-Met signaling inhibits ERK1 / 2 and AKT phosphorylation The amount of ATP can be measured using a variety of well-known methods, such as those described herein, such as measuring ERK1 phosphorylation at Thr202 and Tyr204 and Thr185 and inhibition of ERK2 phosphorylation at Tyr187, and Ser473 or Thr3 Inhibition of AKT in 08, for example, in lysates of NCI-H1975 cells, The antibody is coated onto a solid support and the detection antibody is attached to the Meso Scale Disco conjugated with electrochemiluminescent compounds such as ver(MSD) SULFO-TAG labels. by using a sandwich assay and then detecting the signal with a plate reader. It can be measured.

[0219] In some embodiments described herein, the bispecific EGFR / c-Met antibody The growth of NCI-H292 or NCI-H1975 cells was measured using the NCI-H29 with cetuximab when I-H1975 cells were grown under low-adherence conditions IC of inhibition of proliferation of 2 or NCI-H1975 cells 50 At least approximately 300 times lower, at least about 400 times lower, at least about 500 times lower, at least about 6 00-fold lower, at least about 700-fold lower, or at least about 800-fold lower IC 50 Inhibit by value do.

[0220] Inhibition of cell proliferation can be assessed by known methods, e.g., by preventing cell adhesion. or hydrogels or biomimetic polymers (e.g., Corning Coated plates (Ultra Low Attachment plates) The effect of the antibody on cell proliferation induced by 7.5 ng / mL HGF was examined. Cell viability (%) was measured after 72 hours of incubation using standard methods. It can be evaluated by:

[0221] The bispecific EGFR / c-Met antibodies of the present invention are monospecific EGFR antibodies and monospecific E compared with the combination of a specific c-Met antibody and the standard of care, cetuximab This results in a synergistic and more pronounced inhibition of GFR and c-Met expressing cancer cells. A representative bispecific EGFR / c-Met antibody is the antibody EM1-mAb of the present invention. The bispecific EGFR / c-Met antibodies of the present invention bind to wild-type EGFR and wild-type c-Met. -Met-expressing cancer cells and small molecule tyrosine kinase inhibitors such as gefitinib ( L858R in EGFR has been identified as contributing to resistance to treatment with TKIs Therefore, the bispecific EGFR-1 of the present invention inhibits cancer cells expressing the T790M / T790M mutant. FR / c-Met antibodies have been shown to be effective in a broader patient population compared with cetuximab and TKIs. This can bring benefits.

[0222] In some embodiments described herein, the bispecific EGFR / c-Met antibody showed that the bispecific antibody and cetuximab administered at a dose of 20 mg / kg were SC Growth of HGF-expressing SKMES-1 cell tumors in ID Beige mice was investigated using cerebrospinal fluid (CPE) assay. Inhibits at least 500-fold lower T / C values ​​(%) at day 36 compared to mab.

[0223] A tumor xenograft model using SCID Beige mice is well known. The cells can be engineered to express human HGF using standard methods. Typically, in SCID Beige mice, Culturex was administered subcutaneously in the dorsal flank of each animal. Inoculation of SKMES-1 cells expressing human HGF embedded in an extracellular matrix such as One week after the transplantation, the mice were divided into groups with the same tumor volume. the bispecific EGFR / c-Met antibody of the invention, a control or benchmark antibody, or The small molecule can be administered, for example, three times a week. Tumor volume is recorded twice a week, and tumor growth inhibition is assessed. The TGI can be observed by calculating the T / C value (%). The values ​​(%) are indicators of antitumor efficacy. T and C are the treatment and control groups, respectively, on a particular day. is the average volume of the trawl group.

[0224] The bispecific EGFR / c-Met antibody of the present invention has a This resulted in significantly improved efficacy in killing tumors in vivo compared to This may provide benefit in this patient population when compared to cetuximab.

[0225] In some embodiments described herein, the bispecific EGFR / c-Met antibody HC1 and HC2 are of the isotype IgG1, IgG2, IgG3, or IgG4. Includes:

[0226] In some embodiments described herein, the bispecific EGFR / c-Met antibody includes HC1 and HC2, which are of the IgG1 isotype.

[0227] In some embodiments described herein, residue numbering is according to the EU index. 350, 366, 368, 370, 399, 405, 407, if based on or residue 409, the HC1 CH3 of the bispecific EGFR / c-Met antibody containing at most 1, 2, 3, 4, 5, 6, 7, or 8 substitutions, H3 contains at least 1, 2, 3, 4, 5, 6, 7, or 8 substitutions.

[0228] In some embodiments described herein, residue numbering is according to the EU index. If the amino acid sequence is based on the nucleotide sequence of the present invention, the amino acid sequence may contain a bispecific amino acid sequence at residues 350, 370, 405, or 409. EGFR / c-Met antibody has at least 1, 2, 3, or 4 HC1 CH3s and HC2 CH3 contains at least 1, 2, 3, or 4 substitutions.

[0229] The domains and numbering of antibodies are well known. Two CH3 domains (or CH3 Regions) are non-identical if they differ from each other by at least one amino acid substitution. The CH3 region of IgG1 generally consists of residues 341 to 446 (residue numbering is E (based on the U index). One representative IgG1 constant region is shown in SEQ ID NO: 203. The CH3 domain spans residues 224-329 of SEQ ID NO: 203 and is represented by the EU index. This corresponds to residues 341 to 446 based on the

[0230] In some embodiments described herein, residue numbering is according to the EU index. If the antibody is based on the EGFR / cM antibody, the antibody may contain a bispecific EGFR / cM antibody at residues 405 or 409. The HC1 CH3 of the et antibody contains at least one substitution and the HC2 CH3 contains at least Contains one substitution.

[0231] In some embodiments described herein, residue numbering is according to the EU index. When based on the HC1 CH3 of the bispecific EGFR / c-Met antibody, HC2 CH3 contains K409R or F405L substitutions Includes exchange.

[0232] In some embodiments described herein, the bispecific EGFR / c-Met antibody HC1 CH3 contains a F405L substitution and HC2 CH3 contains a K409R substitution .

[0233] In some embodiments described herein, HC1 CH3 and HC2 CH3 The substitution is at position 366, 368, 370, 399, 405, 407 or 409. (Residue numbering is based on the EU index). These positions are Nos. 203 and 204, 248, 250, 252, 281, 287, and 288 of the heavy chain constant region; These correspond to residues 289 and 291.

[0234] In certain embodiments described herein, position 409 of HC1 CH3 is Lys, L and an amino acid substitution other than eu or Met, and position 405 of HC2 CH3 is other than Phe. It has amino acid substitutions.

[0235] In some embodiments described herein, position 405 of HC1 CH3 is Phe and position 409 of HC2 CH3 is not Lys, Leu or Met. It has other amino acid substitutions.

[0236] In some embodiments described herein, position 409 of HC1 CH3 is Lys , Leu or Met, and position 405 of HC2 CH3 is Phe, Contains amino acid substitutions other than Arg or Gly.

[0237] In some embodiments described herein, position 405 of HC1 CH3 is Phe , and an amino acid substitution other than Arg or Gly, and position 409 of HC2 CH3 is Lys, Contains an amino acid substitution other than Leu or Met.

[0238] In some embodiments described herein, HC1 CH3 contains Phe at position 405. and an amino acid other than Lys, Leu, or Met at position 409, and HC2 CH3 is 4. It has an amino acid other than Phe at position 05 and Lys at position 409.

[0239] In some embodiments described herein, HC1 CH3 has a Phe or greater at position 405. It has other amino acids, Lys at position 409, and HC2 CH3 has Phe at position 405. and has an amino acid other than Lys, Leu, or Met at position 409.

[0240] In some embodiments described herein, HC1 CH3 contains Phe at position 405. and an amino acid other than Lys, Leu, or Met at position 409, and HC2 CH3 is 4. It has a substitution other than Phe, Arg, or Gly at position 05 and Lys at position 409.

[0241] In some embodiments described herein, HC1 CH3 is Phe at position 405; A substitution other than Arg or Gly is present at position 409, and HC2 CH3 is present at position 405. and at position 409 an amino acid other than Lys, Leu, or Met.

[0242] In some embodiments described herein, HC1 CH3 contains Phe at position 405. and an amino acid other than Lys, Leu, or Met at position 409, and HC2 CH3 is 4. It has Leu at position 05 and Lys at position 409.

[0243] In some embodiments described herein, HC1 CH3 contains Leu at position 405. HC2 CH3 has Phe at position 405 and Lys at position 409 Contains an amino acid other than Lys, Leu, or Met.

[0244] In some embodiments described herein, HC1 CH3 contains Phe at position 405. and aArg at position 409, and HC2 CH3 at position 405 is Phe, Arg or GI. It has amino acids other than y and has Lys at position 409.

[0245] In some embodiments described herein, HC1 CH3 is Phe at position 405; It has an amino acid other than Arg or Gly, has Lys at position 409, and HC2 CH3 is 4 It has Phe at position 05 and Arg at position 409.

[0246] In some embodiments described herein, HC1 CH3 contains Phe at position 405. HC2 CH3 has Leu at position 405 and Arg at position 409 Contains Lys.

[0247] In some embodiments described herein, HC1 CH3 contains Leu at position 405. HC2 CH3 has Phe at position 405 and Lys at position 409 Contains Arg.

[0248] In some embodiments described herein, HC1 CH3 contains Phe at position 405. HC2 CH3 has Leu at position 405 and Lys at position 409 Has aArg.

[0249] In some embodiments described herein, HC1 CH3 contains Leu at position 405. HC2 CH3 has a Phe at position 405 and a Arg at position 409 It has Lys.

[0250] In some embodiments described herein, HC1 CH3 contains Lys at position 409, It has an amino acid other than Leu or Met, and HC2 CH3 has Lys at position 409 and It has Thr at position 405 and Leu at position 406.

[0251] In some embodiments described herein, HC1 CH3 contains Lys at position 409. , Thr at position 370, Leu at position 405, HC2 CH3 at position 409, Lys, Contains an amino acid other than Leu or Met.

[0252] In some embodiments described herein, HC1 CH3 contains Arg at position 409. HC2 CH3 has Lys at position 409, Thr at position 370, and Leu at position 405. Has.

[0253] In some embodiments described herein, HC1 CH3 contains Lys at position 409. , Thr at position 370, Leu at position 405, and HC2 CH3 Arg at position 409. Has.

[0254] In some embodiments described herein, HC1 CH3 contains Lys at position 370. , Phe at position 405, aArg at position 409, and HC2 CH3 at position 409. It has a Thr at position 370 and a Leu at position 405.

[0255] In some embodiments described herein, HC1 CH3 contains Lys at position 409. , Thr at position 370 and Leu at position 405, and HC2 CH3 has Lys at position 370. , with Phe at position 405 and Arg at position 409.

[0256] In some embodiments described herein, HC1 CH3 contains Lys at position 409, It has an amino acid other than Leu or Met, and HC2 CH3 has Tyr, Asp, It has an amino acid other than Glu, Phe, Lys, Gln, Arg, Ser or Thr.

[0257] In some embodiments described herein, HC1 CH3 contains Tyr at position 407, Amino acids other than Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr and HC2 CH3 has an amino acid at position 409 other than Lys, Leu, or Met.

[0258] In some embodiments described herein, HC1 CH3 contains Lys at position 409, HC2 CH3 has an amino acid other than Leu or Met at position 407, and Contains His, Ile, Leu, Met, Asn, Val, or Trp.

[0259] In some embodiments described herein, HC1 CH3 contains Ala at position 407, Gly, His, Ile, Leu, Met, Asn, Val or Trp, HC2 CH3 has an amino acid at position 409 other than Lys, Leu, or Met.

[0260] In some embodiments described herein, HC1 CH3 contains Lys at position 409, HC2 CH3 has an amino acid other than Leu or Met at position 407, and Contains Met, Asn, or Trp.

[0261] In some embodiments described herein, HC1 CH3 contains Gly at position 407, Leu, Met, Asn or Trp, and HC2 CH3 has Lys, Leu at position 409 or has an amino acid other than Met.

[0262] In some embodiments described herein, HC1 CH3 contains Tyr at position 407. , has an amino acid other than Lys, Leu or Met at position 409, and HC2 CH3 is at position other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr It has the amino acids:

[0263] In some embodiments described herein, HC1 CH3 contains Tyr at position 407, Amino acids other than Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr , has Lys at position 409, HC2 CH3 has Tyr at position 407, Lys at position 409, Contains an amino acid other than Leu or Met.

[0264] In some embodiments described herein, HC1 CH3 contains Tyr at position 407. , an amino acid other than Lys, Leu, or Met at position 409, and HC2 CH3 at position 407 Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at positions It has Lys at position 409.

[0265] In some embodiments described herein, HC1 CH3 contains Ala at position 407, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 409 HC2 CH3 has Tyr at position 407 and Lys, Leu or M at position 409 Contains amino acids other than et.

[0266] In some embodiments described herein, HC1 CH3 contains Tyr at position 407. , an amino acid other than Lys, Leu, or Met at position 409, and HC2 CH3 at position 407 It has Gly, Leu, Met, Asn, or Trp at position 409 and Lys at position 409.

[0267] In some embodiments described herein, HC1 CH3 contains Gly at position 407, Leu, Met, Asn or Trp, Lys at position 409, and HC2 CH3 at position 40 It has Tyr at position 7 and an amino acid other than Lys, Leu, or Met at position 409.

[0268] In some embodiments described herein, HC1 CH3 contains Tyr at position 407. , Arg at position 409, HC2 CH3 at position 407, Tyr, Asp, Glu, Ph an amino acid other than e, Lys, Gln, Arg, Ser, or Thr, and Lys at position 409 Has.

[0269] In some embodiments described herein, HC1 CH3 contains Tyr at position 407, Amino acids other than Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr , Lys at position 409, HC2 CH3 Tyr at position 407, Arg at position 409 Has.

[0270] In some embodiments described herein, HC1 CH3 contains Tyr at position 407. , Arg at position 409, and HC2 CH3 at position 407 with Ala, Gly, His, or Il e, Leu, Met, Asn, Val or Trp, and at position 409 Lys.

[0271] In some embodiments described herein, HC1 CH3 contains Ala at position 407, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 409 ys, and HC2 CH3 has Tyr at position 407 and Arg at position 409.

[0272] In some embodiments described herein, HC1 CH3 contains Tyr at position 407. , Arg at position 409, HC2 CH3 at position 407, Gly, Leu, Met, As n or Trp, and Lys at position 409.

[0273] In some embodiments described herein, HC1 CH3 contains Gly at position 407, Leu, Met, Asn or Trp, Lys at position 409, and HC2 CH3 at position 40 It has Tyr at position 7 and Arg at position 409.

[0274] In some embodiments described herein, HC1 CH3 contains Lys at position 409, and an amino acid other than Leu or Met, and HC2 CH3 is (i) Phe at position 368, L (ii) has an amino acid other than eu or Met, or (ii) has Trp at position 370 or (iii) an amino acid other than Asp, Cys, Pro, Glu, or Gln at position 399 It has amino acids.

[0275] In some embodiments described herein, HC1 CH3 has (i) P at position 368 or (ii) an amino acid other than He, Leu, or Met at position 370; or (iii) Asp, Cys, Pro, Glu, or Gln at position 399 and HC2 CH3 has an amino acid other than Lys, Leu or Met at position 409. It has the amino acids:

[0276] In some embodiments described herein, HC1 CH3 is Arg at position 409, HC2 CH3 has (i) Lys, GI at position 368; n, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Th (ii) has r, Val or Trp at position 370, or (iii) 399th place is Ala, Gly, Ile, Leu, Met, Asn, Ser, Th It has r, Trp, Phe, His, Lys, Arg, or Tyr.

[0277] In some embodiments described herein, HC1 CH3 is (i) L at position 368 ys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, S or (ii) has Trp at position 370. or (iii) Ala, Gly, Ile, Leu, Met, Asn, S at position 399 Er, Thr, Trp, Phe, His, Lys, Arg or Tyr, and HC2 CH3 has Arg, Ala, His, or Gly at position 409.

[0278] In some embodiments described herein, HC1 CH3 contains Arg at position 409. HC2 CH3 has (i) Asp, Glu, Gly, Asn, Arg, or S at position 368 or (ii) has Trp at position 370. or (iii) has Phe, His, Lys, Arg, or Tyr at position 399 do.

[0279] In some embodiments described herein, HC1 CH3 has (i) A at position 368 Contains sp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp or (ii) has Trp at position 370, or (iii) has Phe, H at position 399 HC2 CH3 has Arg at position 409 do.

[0280] In some embodiments described herein, residue numbering is according to the EU index. If the compound is based on the HC1 CH3, it may contain a K409R substitution or a F405L substitution. HC2CH3 contains a K409R substitution or a F405L substitution.

[0281] In some embodiments described herein, HC1 CH3 contains a substitution of F405L. and HC2 CH3 contains a substitution of K409R.

[0282] Substitutions are typically made using standard methods for molecules such as antibody constant domains. It is done at the level.

[0283] In some embodiments described herein, the bispecific EGFR / c-Met antibody comprises VH1 and VL1, The VH1 sequences are composed of heavy chain complementarity determining regions (HHs) of SEQ ID NOs: 210, 211, and 212, respectively. CDR) 1(HCDR1), HCDR 2(HCDR2) and HCDR 3(HCDR3 ) amino acid sequence, The VL1 sequences are composed of light chain complementarity determining regions (Ls) of SEQ ID NOs: 213, 214, and 215, respectively. CDR) 1(LCDR1), LCDR 2(LCDR2) and LCDR 3(LCDR3 ) amino acid sequence.

[0284] In some embodiments described herein, the bispecific EGFR / c-Met antibody comprises VH2 and VL2, The VH2 has HCDR1 and HCDR2 sequences of SEQ ID NOs: 216, 217, and 218, respectively. and the amino acid sequence of HCDR3, The VL2 has LCDR1 and LCDR2 sequences of SEQ ID NOs: 219, 220, and 221, respectively. and the amino acid sequence of LCDR3.

[0285] In some embodiments described herein, the bispecific EGFR / c-Met antibody are VH1, VL1, VH2 and VL3 of SEQ ID NOs: 189, 190, 193 and 194, respectively. Contains the amino acid sequence of VL2.

[0286] In some embodiments described herein, the bispecific EGFR / c-Met antibody are HC1, LC1, HC2 and LC3 of SEQ ID NOs: 199, 200, 201 and 202, respectively. Contains the amino acid sequence of LC2, and is C-terminally separated from HC1, HC2, or both HC1 and HC2 Optionally, the lysines may be removed.

[0287] In some embodiments described herein, the bispecific EGFR / c-Met antibody comprises VH1 and VL1, The VH1 has HCDR1 and HCDR2 of SEQ ID NOs: 222, 223, and 224, respectively. and the amino acid sequence of HCDR3, The VL1 has LCDR1 and LCDR2 sequences of SEQ ID NOs: 225, 226, and 227, respectively. and the amino acid sequence of LCDR3.

[0288] In some embodiments described herein, the bispecific EGFR / c-Met antibody comprises VH2 and VL2, The VH2 has HCDR1 and HCDR2 sequences of SEQ ID NOs: 228, 229, and 230, respectively. and the amino acid sequence of HCDR3, The VL2 has LCDR1 and LCDR2 sequences of SEQ ID NOs: 231, 232, and 233, respectively. and the amino acid sequence of LCDR3.

[0289] In some embodiments described herein, the bispecific EGFR / c-Met antibody are VH1, VL1, VH2 and VL3 of SEQ ID NOs: 191, 192, 195 and 196, respectively. Contains the amino acid sequence of VL2.

[0290] In some embodiments described herein, the bispecific EGFR / c-Met antibody are HC1, LC1, HC2 and LC3 of SEQ ID NOs: 234, 235, 236 and 237, respectively. Contains the amino acid sequence of LC2, and is C-terminally separated from HC1, HC2, or both HC1 and HC2 Optionally, the lysines may be removed.

[0291] In some embodiments described herein, the bispecific EGFR / c-Met antibody is coated on a plate and the bispecific EGFR / c-Met antibody of the present invention Recombinant human EGFR or recombinant human c-Me incubated in the presence or absence In a competition assay using the extracellular domain of t, EGF binding to EGFR and c The binding of HGF to Met was measured at approximately 1 × 10 -8 Less than M, approximately 1 x 10 -9 Less than M, approx. 1 x 1 0 -10 Less than M, approximately 1 x 10 -11 Less than M or about 1 x 10 -12 IC less than M 50 Block by value The bispecific EGFR / c-Met antibodies described herein can be used in combination with other antibodies of the same type. Bispecific EGFR / c-Met antibodies of the invention described herein using assay conditions Binding of EGF to EGFR and HGF to c-Met in the absence of Binding of EGF to EGFR and HGF to c-Met compared to At least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 7 0%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 9 It can block 6%, 97%, 98%, 99% or 100%.

[0292] In some embodiments described herein, the bispecific EGFR / c-Met antibody includes HC1, LC1, HC2 and LC2, and the HC1, the LC1, the HC2 and and the LC2 are synthetic nucleotides comprising the sequences of SEQ ID NOs: 205, 206, 207 and 208, respectively. It is encoded by a polynucleotide.

[0293] The bispecific EGFR / c-Met antibodies of the present invention were synthesized using CH3 engineering and in vitro Fa Generated using the methods described herein, such as generating antibodies using b-arm exchange A typical bispecific antibody can be prepared by combining two monospecific antibodies at 75 mM 2 in a buffer with a final concentration of 2-mercaptoethanolamine (2-MEA) at pH 7 Approximately 1-20 mg / mL of each antibody was added in a 1:1 molar ratio in 0.0-7.4% PBS. After incubation at 25-37°C for 2-6 hours, the solution was subjected to dialysis, diafiltration, Tangential flow filtration and spinned cell filtration Bispecific antibodies can be generated by removing the 2-MEA by cleavage. The antibody yield was about 80% or more, about 90% or more, about 91%, 92%, 93%, 94%, 95%, It may be greater than 96%, 97%, 98% or 99%.

[0294] Some embodiments described herein include an isolated bispecific EGFR / cM 1. A method for producing an et antibody, comprising: An isolated single-stranded antibody comprising two heavy chains of SEQ ID NO: 199 and two light chains of SEQ ID NO: 200. An isomeric bivalent anti-EGFR antibody and two heavy chains of SEQ ID NO: 201 and two light chains of SEQ ID NO: 202. and an isolated monospecific bivalent anti-c-Met antibody comprising the α- and β-chains, in a molar ratio of about 1:1. and adding them together, introducing a reducing agent into the mixture; incubating the mixture for about 90 minutes to about 6 hours; removing the reducing agent; A first heavy chain of SEQ ID NO: 199 and a second heavy chain of SEQ ID NO: 201, and a second heavy chain of SEQ ID NO: 200 and a second light chain of SEQ ID NO: 202. and purifying said first heavy chain of SEQ ID NO: 199 with said second heavy chain of SEQ ID NO: 200. a first light chain that pairs with the first binding domain to specifically bind to EGFR, The second heavy chain of SEQ ID NO: 201 pairs with the second light chain of SEQ ID NO: 202 to specifically bind c-Met. A method is provided for forming a heterologous binding second binding domain.

[0295] In some embodiments described herein, the reducing agent is 2-mercaptoethanol. The compound is 2-MEA.

[0296] In some embodiments described herein, 2-MEA is present in an amount of about 25 mM to about 75 mM. It is present at a concentration of M.

[0297] In some embodiments described herein, the incubating step is for about 2 It is carried out at a temperature of 5°C to about 37°C.

[0298] Some embodiments described herein include HC1, LC1, HC2, and LC2. 1. An isolated bispecific EGFR / c-Met antibody comprising: 9, wherein said LC1 comprises the sequence of SEQ ID NO: 200 and said HC2 comprises the sequence of SEQ ID NO: 20 1, wherein the LC2 comprises the sequence of SEQ ID NO: 202, and the HC1, the LC1, The HC2 and / or the LC2 are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 conservative amino acid substitutions. GFR / c-Met antibodies are provided.

[0299] Some embodiments described herein may be the same or different from the HC1, LC1, HC2, and and said LC2, wherein said HC 1 comprises the sequence of SEQ ID NO: 234, said LC1 comprises the sequence of SEQ ID NO: 235, and said HC 2 comprises the sequence of SEQ ID NO: 236, said LC2 comprises the sequence of SEQ ID NO: 237, and said HC 1. The LC1, the HC2 and / or the LC2 are 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions. The present invention provides a bispecific EGFR / c-Met antibody.

[0300] The HC1, LC1, HC2 and LC2 amino acid sequences are broadly similar to the antibodies disclosed herein. Bispecific EGFR / c-Met antibodies that do not differ are included within the scope of the present invention. , including antigen-binding sites or frameworks that do not adversely alter the properties of the antibody. One or more conservative amino acids with similar charge, hydrophobicity, and stereochemical properties in Conservative substitutions include amino acid substitutions. Conservative substitutions improve antibody properties, such as stability or affinity. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 2, 13, 14, or 15 amino acid substitutions can be made, for example, in VH1, VL1, VH2 and / or For example, a "conservative amino acid substitution" can be made in VL2. The natural amino acid residues are replaced with non-natural amino acid residues so as to have little or no effect on the polarity or charge of the acid residues. Furthermore, alanine scanning mutagenesis has been used to As described above, substitution of any naturally occurring residue in the polypeptide with alanine may be used. (MacLennan et al., Acta Physiol Scand Suppl 643:55~67,1998;Sasaki et al.,Adv Biophys 35:1-24, 1998). Those skilled in the art can easily identify the desired amino acid substitutions. It can be determined when such substitutions are desirable. For example, amino acid substitutions can be used. and identify important residues in the molecular sequence or to identify the parent molecules described herein. Representative conservative amino acid substitutions are listed above. As stated above.

[0301] Amino acid substitutions can be made, for example, by PCR mutagenesis (U.S. Patent No. 4,683,195). For example, random codons (NNK) or non-random codons, e.g., 11 types of amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Using DVK codons encoding Ser, Tyr, and Trp, mutants with desired properties can be generated. a library of mutants using well-known methods, such as screening a library for can be generated.

[0302] In some embodiments described herein, amino acid substitutions are made in the constant region of the antibody. For example, the well-known G1m17 allotype, G1m3 allotype, or G1 Different IgG1 allotypes, such as m1 allotypes, or combinations thereof, can be used in the methods of the present invention. It can be used for bispecific EGFR / c-Met antibodies.

[0303] In some embodiments described herein, the bispecific EGFR / c-Met antibody The pharmacokinetic properties of α-glucan are improved by substitutions in the Fc domain that modulate antibody half-life. In some embodiments described herein, the bispecific EGFR / c-Met antibody has substitutions M252Y / S254T / T256 in HC1 and / or HC2 E, where residue numbering is according to the EU index. The / S254T / T256E substitutions have been shown to extend antibody half-life (Dall's Acqua et al., J Biol Chem 281:23514~24,20 06).

[0304] Bispecific EGFR / c-Met with conservative and / or additional substitutions in the Fc region The antibodies are tested for their properties using the methods described herein.

[0305] In some embodiments described herein, the bispecific EGFR / c-Met antibody The immune effector properties of the antibody can be enhanced or abolished by modifying the Fc by methods known to those skilled in the art. For example, C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent Cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cell surface receptor Fc effectors, such as downregulation of the B cell receptor (BCR) -functions by modifying residues of Fc that are responsible for these activities and / or can be controlled.

[0306] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to the process of blocking Fc receptor (FcR)-expressing antibodies. Nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and Macrophages (cells) recognize antibodies bound to target cells and then cause lysis of the target cells. This refers to a cell-mediated reaction that

[0307] The ability of monoclonal antibodies to induce ADCC can be improved by manipulating their oligosaccharide components. Human IgG1 or IgG3 has an N-terminal amino acid sequence at Asn297. Glycosylated, with the majority of glycans being of the well-known biantennary types G0, G0F, G1, and G1F , G2 or G2F. Produced by unengineered CHO cells. Antibodies containing Fc usually have a glycan fucose content of approximately at least 85%. Removal of the core fucose from the biantennary complex oligosaccharides attached to the region results in the antigen Enhanced FcγRIIIa binding without altering binding or CDC activity This enhances the ADCC of the antibody. et al., Cytotechnology 64(:249~65,2012), Application of the mutant CHO cell line Lec13 as a host cell line (Shields et al., J Biol Chem 277:26733~26740 2002), as a host cell line and the application of mutant CHO strain EB66 (Olivier et al., MAbs; 2(4) , 2010; published online ahead of print; PMID: 20562582), host cells Application of rat hybridoma cell line YB2 / 0 as a cell line (Shinkawa et al. l., J Biol Chem 278:3466~3473, 2003), α1,6- Introduction of small interfering RNA specific for the fucosyltransferase (FUT8) gene (Mori et al.,Biotechnol Bioeng 88:901~90 8, 2004), or β1,4-N-acetylglucosaminyltransferase III and kifunen, a potent inhibitor of Golgi α-mannosidase II or α-mannosidase I. Co-expression with ribosomal kinase (Ferrara et al., J Biol Chem 281: 5032~5036,2006,Ferrara et al.,Biotechnol Bioeng 93:851~861,2006;Xhou et al.,Biot Echnol Bioeng 99:652-65, 2008), etc. This leads to the efficient expression of relatively highly defucosylated antibodies with branched complex structures. can be obtained using different methods that have been reported.

[0308] In some embodiments described herein, the bispecific EGFR / c-Met antibody ADCC elicited by antibodies can also be enhanced by specific substitutions within the antibody Fc. Representative substitutions include, for example, the amino acids described in U.S. Pat. No. 6,737,056. 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or there is a substitution at position 430 (residue numbering is according to the EU index).

[0309] In some embodiments described herein, the bispecific EGFR / cM The et antibody is about 1% to about 15%, for example, 15%, 14%, 13%, 12%, 11%, 10% , 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% fucose content. In some embodiments, the bispecific EGFR / cM ET antibodies are approximately 50%, 40%, 45%, 40%, 35%, 30%, 25%, or 20% It has a glycan structure with a fucose content of

[0310] "Fucose content" means the amount of fucose monosaccharide in the glycan at Asn297. The relative amount of fucose is the ratio (%) of fucose-containing structures to all glycan structures. These include, for example, 1) as described in International Patent Publication No. WO2008 / 077546 Samples (e.g., complexes, hybrids, and oligos) treated with N-glycosidase F and high mannose structures) using MALDI-TOF, 2) Asn297 glycan After enzymatic release, derivatization and fluorescence detection and / or HPLC-MS ( 3) detection / quantification by HPLC (UPLC) using UPLC-MS; and the second GlcNAc monosaccharide, leaving the fucose attached to the first GlcNAc. With or without processing of the Asn297 glycan by Endo S or other enzymes 3) intact protein analysis of native or reduced mAbs, 4) enzymatic digestion of mAbs (e.g., transfection) After digestion with lysin or endopeptidase Lys-C into constituent peptides, HPLC 5) PNGase F was separated, detected, and quantified by C-MS (UPLC-MS). Specific enzymatic deglycosylation of Asn297 by mAb olefins from mAb proteins This can be characterized and quantified by a number of methods, including the separation of oligosaccharides. The oligosaccharides thus released can be labeled with fluorophores and analyzed by a variety of complementary methods. Although it is possible to separate and identify these compounds, these methods are based on matrix-assisted laser desorption. Glycan structure by comparison of experimental mass with theoretical mass using magnetic resonance ionization (MALDI) mass spectrometry Detailed characterization of the structure, degree of sialylation by ion-exchange HPLC (GlycoSep C) Determination of oligosaccharides based on hydrophilicity criteria by standard-phase HPLC (GlycoSep N) Separation and quantification of body morphology and high performance capillary electrophoresis laser-induced fluorescence (HP-LCF) This allows for the separation and quantification of oligosaccharides by CE-LIF.

[0311] As used in this application, "low fucose" or "low fucose content" refers to a fucose content This refers to antibodies with a specificity of approximately 1% to 15%.

[0312] As used herein, "normal fucose" or "normal fucose content" refers to fucose The antibody has a serotonin content of greater than about 50%, typically greater than about 80% or greater than 85%. This refers to the following.

[0313] Some embodiments of the present invention are directed to the bispecific EGFR / EGFR-binding protein of the invention described herein. The heavy and light chains of the c-Met binding antibodies can be produced as isolated polynucleotides or as expression vectors. providing synthetic nucleic acids encoding the vectors as part of a vector or as part of a linear DNA sequence; and linear DNA sequences, compositions or their derivatives used in in vitro transcription / translation. Expression, secretion and characterization of directed mutagens in prokaryotic cells, eukaryotic cells or filamentous phages and / or display compatible vectors.

[0314] Some embodiments herein include the polynucleotides of SEQ ID NOs: 205, 206, 207, or 208. and providing an isolated polynucleotide comprising the polynucleotide sequence of the nucleotide sequence. .

[0315] The polynucleotides of the present invention can be synthesized by solid-phase polynucleotide synthesis in an automated polynucleotide synthesizer. Produced by chemical synthesis, such as by oxidase synthesis, and assembled as a complete single- or double-stranded molecule Alternatively, the polynucleotides of the present invention can be prepared by PCR followed by conventional cloning. Polynucleotides of specific known sequences can also be generated by other methods, such as by PCR. Techniques for producing or obtaining oxides are well known in the art.

[0316] The polynucleotide of the present invention may contain promoter or enhancer sequences, introns, polynucleotides, At least one non-coding sequence, such as an adenylation signal or a cis sequence that promotes RepA binding, is required. The polynucleotide sequence may also facilitate purification or detection of the protein. marker or tag sequence, signal, such as a histidine tag or an HA tag, for sequences, RepA, Fc, or bacteriophage coat proteins such as pIX or pIII Further amino acids encoding the fusion protein partner, such as a fusion protein, may be added. It may also include a sequence

[0317] Some embodiments described herein involve vectors comprising the polynucleotides of the invention. Such vectors include plasmid vectors, viral vectors, and vaccinia vectors. by a vector, a transposon-based vector, or by any other means Suitable for introducing polynucleotides of the invention into a particular organism or genetic background. For example, the heavy and light chains of the bispecific antibody of the present invention may be expressed as The polynucleotides encoding the light and heavy chains can be inserted into an expression vector. The immunoglobulin chains can be cloned into the same or different expression vectors. The DNA segment to be loaded is then inserted into an expression vector that ensures expression of an immunoglobulin polypeptide. Such a control sequence can be operably linked to a control sequence within the promoter. null sequence, promoter (e.g., naturally associated or heterologous promoter) , enhancer elements, and transcription termination sequences selected for antibody expression. The vector can be selected to be compatible with the host cell in which it is to be introduced. When incorporated, the host is able to synthesize the protein encoded by the integrated synthetic polynucleotide. The vector can be maintained under conditions suitable for high level expression of the vector.

[0318] Suitable expression vectors are generally expressed either episomes or as part of the host chromosomal DNA. Replicable in a host organism. Generally, an expression vector is a vector containing a desired DNA sequence. Ampicillin resistance, hygromycin resistance, and tetanus resistance were identified to allow detection of transformed cells. Contains a selection marker such as tetracycline resistance, kanamycin resistance, or neomycin resistance. I am doing this.

[0319] Some embodiments described herein provide host cells comprising the vectors of the invention. The term "host cell" refers to a cell into which a vector has been introduced. It is understood that the term refers not only to the particular subject cell but to the progeny of such a cell. Because certain modifications can occur in subsequent generations due to mutations or environmental influences, these Although the resulting progeny may not be identical to the parent cell, they are still referred to as "host cells" as that term is used herein. Such host cells include eukaryotic cells, prokaryotic cells, and the like. The cell may be a nuclear cell, a plant cell or an archaeal cell.

[0320] Exemplary eukaryotic cells include those of mammalian, insect, avian or other animal origin. As mammalian eukaryotic cells, SP2 / 0 (American Type Culture Collection) re Collection (ATCC), Manassa, Virginia, CRL-1581). ,NS0(ECACC(European Collection of Cell C Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. .85110503), and Ag653 (ATCC CRL-1580) mouse cell lines. Examples include immortalized cell lines such as hybridoma or myeloma cell lines. An example of a myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61), or DG 44 derived from Chinese hamster ovary (CHO) cells.

[0321] The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention, Use of EGFR / c-Met Antibodies and EGFR- or c-Met-Binding FN3 Domains The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention, N3 domain, c-Met binding FN3 domain, or bispecific EGFR / c-Met antibody The body is a system of cells, tissues, organs, body fluids, or the host in general that can be affected by a particular disease in humans. Diagnose, monitor, regulate, treat, or alleviate a physiological condition, aid in the prevention of its onset, or alleviate the symptoms of The method of the present invention can be used to alleviate the symptoms of any animal. It can be used to treat patients, such as humans, rodents, and the like. mammals such as animals, dogs, cats, and livestock.

[0322] One aspect of the present invention is a method for inhibiting the growth or proliferation of cells expressing EGFR and / or c-Met. The method comprises administering to a cell an isolated bispecific EGFR / c-Me receptor agonist of the present invention. t FN3 domain-containing molecules, EGFR-binding FN3 domains, c-Met-binding FN3 domains The method comprises contacting the patient with a bispecific EGFR / c-Met antibody.

[0323] Another aspect of the present invention is a method for detecting EGFR and / or c-Met expressing tumors or cancer cells in a subject. 10. A method for inhibiting the growth or metastasis of a plant comprising administering to a subject an effective amount of an isolated plant of the present invention. bispecific EGFR / c-Met FN3 domain-containing molecule, EGFR-binding FN3 domain administration of EGFR, c-Met-binding FN3 domain, or bispecific EGFR / c-Met antibody and inhibiting the growth or metastasis of EGFR- and / or c-Met-expressing tumor or cancer cells. The method includes:

[0324] Another aspect of the invention is a method of treating a subject having cancer, comprising administering to a subject a compound sufficient to treat the cancer. and administering a therapeutically effective amount of the isolated diamine hydroxybenzoate of the present invention to a patient in need of such treatment for a sufficient period of time. Bispecific EGFR / c-Met FN3 domain-containing molecules, EGFR-binding FN3 domains , a c-Met-binding FN3 domain, or a bispecific EGFR / c-Met antibody. The method includes:

[0325] The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention, N3 domain, c-Met binding FN3 domain, or bispecific EGFR / c-Met antibody The body may contain EGFR, c-Met, EGF, soluble EGFR, soluble c-Met or any of its Any disease characterized by abnormal activation or production of other EGFR ligands or HGF a disease or disorder, or EGFR, c-Met, EGF, or other EGFR ligands, or abnormal activation and / or production of HGF is a cause of or predisposition to said disease or disorder. It may also involve malignant tumors or cancers arising in the cells or tissues of a subject with It can be used to treat disorders associated with EGFR or c-Met expression, which may not be Cut.

[0326] The compound of the present invention specifically binds to c-Met and blocks HGF binding to c-Met. The FN3 domains of the present invention can be used to treat tumors, including cancer and benign tumors. Cancers suitable for treatment with the c-Met-binding FN3 domain of the present invention include those that overexpress c-Met. Representative cancers suitable for treatment with the FN3 domain of the present invention include those that overexpress FN3. These include epithelial cell carcinoma, breast cancer, ovarian cancer, lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, and bladder cancer. Bladder cancer, head and neck cancer, stomach cancer, ovarian cancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen Cancer of the pancreas, testicles, and thymus.

[0327] The FN of the present invention specifically binds to EGFR and blocks EGF binding to EGFR. The FN3 domains of the present invention can be used to treat tumors, including cancer and benign tumors. Cancers suitable for treatment with the three domains include those that overexpress EGFR or its mutants. Representative cancers suitable for treatment with the FN3 domain of the present invention include epithelial cell carcinoma (EC2-1.2.1 ... Cellular cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer , ovarian cancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, and The bispecific EGFR / c-Met FN3 domain-containing Molecular or bispecific EGFR / c-Met antibodies are used to treat tumors, including cancer and benign tumors. The bispecific EGFR / c-Met FN3 domain-containing compounds of the present invention can be used. Representative cancers suitable for treatment with EGFR or bispecific c-Met antibodies include E Those that overexpress GFR and / or c-Met, EGFR activity and / or expression levels Increased expression of EGFR (e.g., due to activating mutations, EGFR gene amplification, or ligand-mediated EGFR activation by EGFR) and increased activity and / or expression levels of c-Met (e.g., For example, activating mutations in c-Met, amplification of the c-Met gene, or HGF-mediated c-Met activation may be involved. Examples include cancers associated with Met activation.

[0328] Representative EGFR activating mutations that can be associated with cancer include tyrosine kinase E, such as increased activity, receptor homodimer and heterodimer formation, and enhanced ligand binding. Point mutations, deletion mutations, insertion mutations that lead to an increase in at least one biological activity of GFR, Mutations include EGFR gene or gene amplification. It can be located in any part of the regulatory region surrounding exon 18, 19, 20, or 21. These include mutations in the EGFR kinase domain and mutations in the kinase domain. The amino acids are G719A, L861X (X is any amino acid), L858R, E746K, and L7 47S, E749Q, A750P, A755V, V765M, L858P, or T790 M substitution, E746-A750 deletion, R748-P753 deletion, M766 and A767 Ala insertion between S768 and V769, SVA (Ser, Val, Ala) between S768 and V769 and an NS (Asn, Ser) insertion between P772 and H773. Other examples of R-activating mutations are known in the art (see, e.g., U.S. Pat. Appln. KOKAI Publication No. 2004 / 0123994, U.S. Pat. No. 6,223,169). (See US2005 / 0272083). Receptor homo- and heterodimers, receptor linkers Gands, autophosphorylation sites, and signaling molecules involved in ErbB-mediated signal transduction Information regarding EGFR and other ErbB receptors, including those listed above, is known in the art. (e.g., Hynes and Lane, Nature Reviews, Cancer 5:341-354, 2005).

[0329] Typical c-Met activating mutations include increased tyrosine kinase activity and receptor homozygotes. The formation of dimers and heterodimers, enhanced ligand binding, and other minor functions of the c-Met protein point mutations, deletion mutations, insertion mutations, inversions, or mutations that result in an increase in at least one biological activity Mutations include c-Met, such as mutations in the kinase domain of c-Met. It may be located in any part of the regulatory region associated with the Met gene or the c-Met gene. Representative c-Met activating mutations include N375, V13, V923, and R175. , V136, L229, S323, R988, S1058 / T1010 and E168 There are mutations at residues in EGFR and c-Met. Methods for detecting EGFR and c-Met mutations are well known. be.

[0330] Treatment with bispecific molecules of the invention, such as bispecific EGFR / c-Met antibodies of the invention Representative cancers suitable for treatment include epithelial cell carcinoma, breast cancer, ovarian cancer, lung cancer, and non-small cell lung cancer (NSCLC). CLC), lung adenocarcinoma, small cell lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer Neck cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer Cancer of the breast, testicle cancer, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer (hepatocellular carcinoma (HCC)), or These include sporadic or hereditary papillary renal cell carcinoma (PRCC).

[0331] Another aspect of the invention is a method of treating a subject with cancer, comprising administering a therapeutically effective amount of the present invention to a subject. The isolated bispecific EGFR / c-Met antibody is administered to patients in need thereof to treat cancer. and administering to the subject a CD16 fragment at position 158 for a period sufficient to induce the growth of the tumor. isotypic for phenylalanine (genotype FcγRIIIa-158F / F), or are heterozygous for valine and phenylalanine at position 158 of CD16 (genotype Fc γRIIIa-158F / V) method. CD16 binds to Fcγ receptor IIIa (Fcγ RIIIa) or low-affinity immunoglobulin gamma Fc region receptor III-A isoform Also known as the valine / phenylalanine at residue 158 of the FcγRIIIa protein. Viral / F polymorphism affects the affinity of FcγRIIIa for human IgG It has been shown that FcγRIIIa-158F / F or FcγRIIIa-158F Receptors with the FcγRIIIa-158V / V polymorphism exhibit weaker Fc binding compared to FcγRIIIa-158V / V. The absence of fucose in human N-linked oligosaccharides indicates poor binding and therefore poor ADCC. or low amounts enhance the binding of the antibody to human FcγRIIIa (CD16). This increases the antibody's ability to induce ADCC (Shields et al., J Biol Chem 277:26733-40, 2002). In some embodiments, the fucose content is low, between 1% and about 10%. The specific EGFR / c-Met antibody has a fucose content of approximately 50%, 40%, 45%, and 40%. ,35%,30%,25%,20%,15%,10%,9%,8%,7%,6%,5%, 4%, 3%, 2% or 1% of the glycan structures. Thus, the antibodies of the present invention have Fc Patients with the genotypes FcγRIIIa-158F / F or FcγRIIIa-158F / V The efficacy of FcγRIIIa in treating patients with FcγRIIIa-associated leukemia may be higher. The patient can be analyzed for type.

[0332] In some methods described herein, the antibodies of the invention are administered in combination with one or more EGFR inhibitors. for use in treating a subject with cancer that is resistant or has acquired resistance to treatment with Representative EGFR inhibitors to which cancers can acquire resistance include anti-EGF R antibodies cetuximab (Erbitux®), panitumumab (Vecti bix®), matuzumab, nimotuzumab, and the small molecule EGFR inhibitor Tar ceva® (erlotinib), IRESSA (gefitinib), EKB-56 9 (pelitinib, irreversible EGFR TKI), pan-ErbB and other receptor tyrosine kinase inhibitors kinase inhibitors, lapatinib (EGFR and HER2 inhibitor), pelitinib (EGFR and HER2 inhibitors), vandetanib (ZD6474, ZACTIMA™), EGF R, VEGFR2 and RET TKI), PF00299804 (dacomitinib, irreversible CI-1033 (irreversible pan-erbB TKI) , afatinib (BIBW2992, irreversible pan-ErbB TKI), AV-41 2 (dual EGFR and ErbB2 inhibitor), EXEL-7647 (EGFR, ErbB2 , GEVGR and EphB4 inhibitor), CO-1686 (irreversible mutant selective EGFR TKI), AZD9291 (irreversible mutant-selective EGFR TKI), and HKI -272 (neratinib, an irreversible EGFR / ErbB2 inhibitor). The available treatments are gefitinib, erlotinib, afatinib, CO-1686, and AZD9 291 and / or cetuximab for the treatment of cancers resistant to treatment with cetuximab. One of the representative antibodies that can be used is EM1-mAb.

[0333] Another aspect of the invention is a method of treating a subject with cancer, comprising administering a therapeutically effective amount of the present invention to a subject. The present invention provides a novel bispecific EGFR / c-Met antibody sufficient to treat cancer in patients in need thereof. and administering to the subject over a period of time, the subject being administered erlotinib, gefitinib, alfalfa, or rifabutinib. Resistance to treatment with atinib, CO-1686, AZD9291, or cetuximab The method is characterized by the fact that the patient has acquired or has developed resistance to the drug.

[0334] A variety of qualitative and / or quantitative methods can be used to assess whether a subject responds to treatment with an EGFR inhibitor. It can be determined whether a patient has, has developed resistance to, or is susceptible to developing resistance to the drug. Conditions that can be associated with resistance to EGFR inhibitors include, for example, Worsening or stagnating health status, increase in tumor size, halt or slowing of tumor growth, and / or or the spread of cancerous cells from one location in the body to other organs, tissues, or cells. Loss of appetite, cognitive impairment, depression, shortness of breath, fatigue, hormone disturbances, neutropenia, pain The recurrence or worsening of various cancer-related symptoms, such as steroid use, peripheral neuropathy, and sexual dysfunction, may also occur. may be an indicator that a patient has developed or is susceptible to developing resistance to EGFR inhibitors. Symptoms associated with cancer may vary depending on the type of cancer. For example, symptoms associated with cervical cancer may Symptoms include abnormal bleeding, an abnormally heavy vaginal discharge, pelvic pain unrelated to the normal menstrual cycle, and urinary tract infections. Bladder pain or painful urination, and bleeding between regular menstrual periods after intercourse, douching, or pelvic examination may occur. Symptoms associated with lung cancer include persistent cough, coughing up blood, shortness of breath, and chest pain with wheezing. Symptoms of liver cancer include: loss of appetite, unintentional weight loss, and fatigue. Symptoms include loss of appetite and weight, abdominal pain (especially in the right upper abdomen, back, and shoulders), nausea and vomiting. Vomiting, general weakness and tiredness, enlarged liver, abdominal distension (ascites), and yellowing of the skin and whites of the eyes Those skilled in the art of oncology will recognize the symptoms associated with specific cancer types. Symptoms can be identified immediately.

[0335] Another means for determining whether a subject has developed resistance to an EGFR inhibitor is to measure the level of resistance to the cancer. EGFR phosphorylation, ERK1 / 2 phosphorylation, and / or AKT phosphorylation in cells were investigated. In such cases, increased phosphorylation may indicate that the subject has developed resistance to EGFR inhibitors. EGFR, ERK1 / 2 and / or Methods for measuring AKT phosphorylation are well known and are described herein. To identify a subject that has developed resistance to a toxic agent, for example, an increase in circulating levels of HGF can be detected. c-Met expression due to activating mutations in the c-Met gene or amplification of the c-Met gene This may involve detecting elevated current levels or elevated activity of c-Met.

[0336] Another embodiment of the present invention is directed to NS with EGFR activating mutations or EGFR gene amplification. 1. A method of treating NSCLC in a patient having a NSCLC tumor or tumor metastasis, comprising administering to said patient administering a therapeutically effective amount of a bispecific EGFR / c-Met antibody of the invention It is a method.

[0337] The bispecific EGFR / c-Met antibodies of the present invention are useful in treating squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. It can be used to treat cancer, including non-small cell lung cancer (NSCLC). In some embodiments, the cells of the NSCLC have an epithelial phenotype. , NSCLC has acquired resistance to treatment with one or more EGFR inhibitors.

[0338] In NSCLC, certain mutations in the EGFR gene are associated with the development of EGFR tyrosine kinase inhibitors ( EGFR-TKIs have a high response rate (70-80%). The amino acid deletion or EGFR point mutation L858R is associated with EGFR TKI sensitivity. Iru(Nakata and Gotoh,Expert Opin Ther Tar gets 16:771~781, 2012). These mutations inhibit EGFR kinase activity. EGFR activating mutations occur in 10–30% of NSCLC patients. 0% of cases, East Asian, female, never-smoker, and with histological findings of adenocarcinoma significantly more common in patients with glaucoma (Janne and Johnson Clin C ancer Res 12(14 Suppl):4416s~4420s,2006) EGFR gene amplification also strongly correlates with response after treatment with EGFR-TKIs. (Cappuzzo et al., J Natl Cancer Inst 97: 643~55,2005).

[0339] Most patients with EGFR-mutated NSCLC respond early to EGFR TKI treatment However, virtually all patients develop resistance, preventing a sustained response. 0% of patients have a point mutation (T790M) in the second site of the kinase domain of EGFR. Approximately 10% of all tumors that have acquired resistance to EGFR tyrosine kinase inhibitors 60% of cases show increased c-Met expression, amplification of the c-Met gene, or its only known ligand. Increased HGF, the target of IL-1, is observed (Turke et al., Cancer Cell l,17:77~88,2010).

[0340] Another embodiment of the present invention is a method of treating a patient with cancer, comprising administering a therapeutically effective amount of The bispecific EGFR / c-Met antibodies of the invention can be administered to patients in need thereof to treat cancer. and administering the compound for a sufficient period of time, wherein the cancer is characterized by an EGFR activating mutation, an EGFR gene Gene amplification, increased levels of circulating HGF, c-Met activating mutations, c-Met gene The method is associated with amplification of or mutant KRAS.

[0341] In certain embodiments, the EGFR activating mutation is G719A, G719X (where X is any amino acid). amino acid), L861X (X is any amino acid), L858R, E746K, L747S, Replaces E749Q, A750P, A755V, V765M, L858P or T790M, Deletion of E746 to A750, deletion of R748 to P753, and A between M766 and A767 insertion of Ser, Val, and Ala (SVA) between S768 and V769; insertion, and an Asn and Ser (NS) insertion between P772 and H773.

[0342] Another embodiment of the present invention is a method of treating a patient with cancer, comprising administering a therapeutically effective amount of The bispecific EGFR / c-Met antibodies of the invention can be administered to patients in need thereof to treat cancer. and administering the compound for a sufficient period of time, wherein the cancer is characterized by EGFR mutation L858R, T79 0M, or deletion of residues E746 to A750 (del(E746,A750)), EGFR amplification, or c-Met amplification.

[0343] In some embodiments, the cancer is associated with wild-type EGFR and wild-type c-Met .

[0344] In some embodiments, the cancer is associated with wild-type EGFR and amplification of c-Met. .

[0345] In some embodiments, the cancer is EGFR L858R and T790M mutated and wild type It is related to c-Met.

[0346] In some embodiments, the cancer is EGFR deletion del(E764,A750) and wild-type. It is related to type c-Met.

[0347] In some embodiments, the cancer is EGFR deletion del(E764,A750) and c- Associated with Met amplification.

[0348] In some embodiments, the cancer is EGFR deletion del(E764,A750), EGF It is associated with R amplification and c-Met amplification.

[0349] In some embodiments, the patient has EGFR L858R and T790M mutations, and have wild-type c-Met-associated NSCLC.

[0350] In some embodiments, the patient has NS associated with EGFR amplification and wild-type c-Met. Has CLC.

[0351] In some embodiments, the patient has NSCs associated with EGFR amplification and c-Met amplification. It has LC.

[0352] In some embodiments, the patient has an EGFR deletion del(E764,A750) and have live-type c-Met-associated NSCLC.

[0353] In some embodiments, the patient has an EGFR deletion del(E764,A750) and -Has NSCLC associated with Met amplification.

[0354] In some embodiments, treatment is with the EM1-mAb of the present invention. -mAb is used to treat tumors with L858R, T790M, del(E746,A750) EGFR , when associated with EGFR amplification, wild-type c-Met, and / or c-Met amplification, It has been shown to be effective in in vivo tumor animal models. Southern blotting, FISH, or comparative genomic hybridization (CGH) by standard methods such as determining the copy number of the EGFR or c-Met gene by It can be evaluated based on this.

[0355] Another embodiment of the present invention is a method of treating a patient with cancer, comprising administering a bispecific antibody of the present invention. and administering a therapeutically effective amount of a specific EGFR / c-Met antibody to a patient in need thereof to treat cancer. and administering the compound for a sufficient period of time, wherein the cancer is characterized by EGFR mutation L858R, T79 0M, or deletion of residues E746 to A750 (del(E746,A750)), EGFR The method is associated with c-Met amplification and mutant KRAS.

[0356] In some embodiments, the mutant KRAS has a G12V substitution. The family of RAS proto-oncogenes encoding nosine triphosphatases (GTPases) It belongs to the family of receptors that mediate EGFR signal transduction downstream of the receptor. Tumors with proto-oncogene KRAS mutations, such as activating G12V or G12C mutations, are more susceptible to EGF It is predicted that this disease will not be treatable by R antibodies. Clinical trials with panitumumab and rituximab have demonstrated efficacy in patients with KRAS-mutated colorectal tumors. Patients have been shown to be unresponsive to these drugs (Van Cutsem et al. l.,N Eng J Med 360:1408~1417,2009;Lievre et al., J Clin Oncol 26:374~379,2008;Ama do et al., J Clin Oncol 26:1626~1634m 200 8) The bispecific EGFR / c-Met antibody of the present invention inhibits KRA through effective ADCC. Unlike currently used anti-EGFR therapeutics, it mediates killing of S mutant cell lines. , which may be effective in treating patients with cancers associated with KRAS-activating mutations A representative example of such an antibody is EM1-mAb.

[0357] The terms "treat" or "treatment" refer to both therapeutic treatment or prophylactic or preventative measures. The purpose is to prevent undesirable physiological changes or diseases, such as the development or spread of cancer. For purposes of this invention, beneficial or desirable Clinical outcomes include, but are not limited to, detectable or undetectable Regardless of whether symptoms are alleviated, the extent of the disease is reduced, or the disease remains stable (i.e., not worsening) ) condition, delay or slowing of disease progression, alleviation or relief of symptoms, and remission (partial or complete) "Treatment" means any condition that prolongs survival compared to expected survival if not receiving treatment. It can also mean the length of time a patient has been treated. not only those who are at high risk of having a condition or disease, but also those who are at high risk of having a condition or disease This includes those who need to be protected.

[0358] A "therapeutically effective amount" means a therapeutically effective amount, at dosages and for periods of time necessary to achieve a desired therapeutic result. Therapeutically effective amount of the bispecific EGFR / c-Met antibody of the present invention refers to a therapeutically effective amount. The bispecific EGFR / c- The ability of Met antibodies to produce the desired response in that individual is a factor. Potential bispecific EGFR / EGFR-associated vasculitis may be reduced or attenuated with resistance. Typical outcomes of c-Met treatment include, for example, improvement in patient health and tumor size. reduction or shrinkage of tumors, stopping or slowing tumor growth, and / or spreading of cancer cells elsewhere in the body These include the absence of metastasis.

[0359] Administration / Pharmaceutical Compositions The present invention relates to a bispecific EGFR / c-Met antibody of the invention and a pharmaceutically acceptable carrier. In therapeutic applications, the bispecific EGFR / c-Met antibody of the present invention is provided. FN3 domain-containing molecules, EGFR-binding FN3 domains, c-Met-binding FN3 domains or a bispecific EGFR / c-Met antibody, comprising an effective amount of said domain, molecule, or antibody. as an active ingredient in a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, or excipient with which an active compound is administered. or solvents. Such solvents include peanut oil, soybean oil, mineral oil, sesame oil, etc. It may be a liquid such as water and oil, including those of petroleum, animal, vegetable, or synthetic origin. For example, 0.4% saline and 0.3% glycine can be used. These solutions should be kept sterile. and are generally free of particulate matter. These can be sterilized using conventional and well-known sterilization techniques (e.g., filtration). The composition may contain pH adjusting and buffering agents, stabilizing agents, thickening agents, lubricating agents, etc. Pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as lubricants and colorants. The concentration of the molecule or antibody of the invention in such pharmaceutical formulations may be less than about 0.5% by weight. It can vary widely, usually at least about 1% by weight, up to 15 or 20% by weight. and is based primarily on the required dose, fluid volume, viscosity, etc., according to the particular method of administration selected. Suitable solvents and formulations (including other human proteins, e.g., human serum albumin) are selected based on the For details on the science and technology of the 19th century, see, for example, Remington: The Science and d Practice of Pharmacy, 21 st Edition,Troy,D .B.ed., Lipincott Williams and Wilkins, Ph. iladelphia,PA 2006,Part 5,Pharmaceutical Manufacturing pp. 691-1092 (especially pp. 958-989) (see reference).

[0360] The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention, N3 domain, c-Met binding FN3 domain, or bispecific EGFR / c-Met antibody For therapeutic use in the body, the dosage is as a tablet, capsule, solution, powder, gel, or granule, or by syringe. , implantable devices, osmotic pumps, cartridges, micropumps, or other devices known in the art. Parenteral administration, e.g., using formulations packaged in other ways known or recognized by those skilled in the art. For example, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal) This may be any suitable route of delivery of the agent to the host. Site-specific administration may be, for example, , intra-articular, intra-bronchial, intra-abdominal, intra-articular capsule, intra-cartilaginous, intra-sinus, intra-cavity, intra-cerebellar, intra-ventricular, intra-colonic, Intracervical canal, intragastric, intrahepatic, intramyocardial, intrabone, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary , Intrarectum, Intrakidney, Intraretina, Intraspinal cord, Intrasynovial bursa, Intrathoracic, Intrauterine, Intravascular, Intrabladder, Lesion This can be achieved by intravenous, intravaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery. Cut.

[0361] Thus, a pharmaceutical composition of the present invention for intramuscular injection may contain 1 ml of sterile buffered water and approximately 1 ng to about 100 mg / kg, for example, about 50 ng to about 30 mg / kg, or more preferably about 5 25 mg / kg to about 25 mg / kg of the bispecific EGFR / c-MetFN3 domain of the present invention molecules, such as those containing an EGFR-binding FN3 domain or a c-Met-binding FN3 domain It can be prepared.

[0362] The bispecific EGFR / c-Met antibodies of the invention can be administered, for example, by intravenous (IV) infusion or by bolus injection. The administration may be parenteral, by any suitable route, such as by intramuscular or subcutaneous injection, or intraperitoneal administration. It can be administered to patients by a variety of routes. IV infusion can be administered over a minimum of 15 minutes, with many In some cases, the dose may be administered over 30, 60, 90 minutes, or even 2 or 3 hours. The bispecific EGFR / c-Met antibodies of the present invention can be administered to the site of disease (e.g., The dose administered to a patient with cancer can be determined by direct injection into the tumor itself. sufficient to alleviate or at least partially arrest the disease being treated (therapeutic efficacy) Efficacy), often 0.1 to 10 mg per kg of body weight, e.g., 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 mg / kg, but may be higher, e.g., 15, 20, It may be 30, 40, 50, 60, 70, 80, 90 or 100 mg / kg. For example, fixed unit doses of 50, 100, 200, 500, or 1000 mg may be administered. Alternatively, the dose may be increased to, for example, 400, 300, 250, 200 or 100 mg / m 2 and It may be based on the patient's body surface area, as in the case of cancer treatment. 10 doses (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 doses) can be administered, 12, 20 or more doses may be administered. c-Met antibody was administered on the following days: 1, 2, 3, 4, 5, 6, 1 week, 2 weeks, and 3 weeks. 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more Repeated treatment regimens are possible, as are chronic administration. The repeated doses can be the same dose or different doses.

[0363] Pharmaceutical compositions comprising the bispecific EGFR / c-Met antibodies of the invention, e.g., for intravenous infusion For administration to an 80 kg patient, approximately 200 ml of sterile Ringer's solution and approximately 8 mg to approximately 2400 mg, about 400 mg to about 1600 mg, or about 400 mg to about 800 mg double It can be formulated to contain specific EGFR / c-Met antibodies. Methods for preparing compositions are well known and are described, for example, in Remington's Pharmaceuticals. Maceutical Science,15th ed.,Mack Publish ing Company, Easton, PA.

[0364] The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention, N3 domain, c-Met binding FN3 domain, or bispecific EGFR / c-Met antibody The bodies can be lyophilized for storage and reconstituted in a suitable carrier prior to use. has been shown to be effective in conventional protein preparations and is a well-known method for lyophilization and A reconstitution method can be used.

[0365] The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention, N3 domain, c-Met binding FN3 domain, or bispecific EGFR / c-Met antibody The compound can be administered in combination with a second therapeutic agent either simultaneously, sequentially, or separately. The therapeutic agent can be a chemotherapeutic agent or a targeted anti-cancer therapeutic agent.

[0366] The bispecific EGFR / c-Met antibody may be administered in combination with one or more of any chemotherapeutic agent or drug known to those skilled in the art. Therapeutic agents may be administered with other known anti-cancer therapeutic agents. Chemotherapeutic agents are chemical agents useful in the treatment of cancer. compounds, including antiproliferative agents or other cytotoxic agents, including alkylating agents, antimetabolites, antimicrobials, Tubulin inhibitors, topoisomerase inhibitors, receptor tyrosine kinase inhibitors, angiogenesis inhibitors Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cycloheximide. Phosphamide (CYTOXAN®); alkyl sulfonates, e.g., Busulf aziridines, such as benzodopa, carbamazepine, benzophenone ... Bocon, meturedopa and uredopa; ethyleneimine and methylameramine (Altre thamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide including melamine and trimethylolmelamine); nitrogen mustards, such as chloramphen Bucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, Mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phene Sterin, prednimustine, trophosphamide, uracil mustard; nitrourea , such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and Ranimustine; antibiotics such as aclacinomycin, actinomycin, australamycin Cin, azaserine, bleomycin, cactinomycin, calicheamicin, calabicin , carminomycin, carzinophilin, chromomycin, dactinomycin, Daunol Bicine, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, Epirubicin, esorubicin, idambicine, marcelomycin, mitomycin, myco Phenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin , puromycin, queramycin, rhodolubicin, streptonigrin, strept Zosyn, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, e.g. methotrexate and 5-FU; folic acid analogues, e.g., denopterin, methotrexate , pteropterin and trimetrexate; purine analogues such as fludarabine, 6-methyl- thiamipurine and thioguanine; pyrimidine analogues such as ancitabine azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine , doxifluridine, enocitabine and floxuridine; androgens, e.g., callus Teron, dromostanolone propionate, epithiostanol, lunepiciostane and Testolactone; antiadrenal agents such as aminoglutethimide, mitotane and trilostane; leaves Acid supplements, such as furoic acid; aceglatone; aldophosphamide glycosides; aminole Pyruvulinic acid; Amsacrine; Bestravsil; Bisantrene; Edatraxate; Defof amine; demecolcine; diazicon; elfornithine; elliptinium acetate; etoglucan Cid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mito Xantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubi Syn; Podophyllic acid; 2-ethylhydrazide; Procarbazine; PSKR® ;Razoxane;Schizofuran;Spirogermanium;Tenuazonic acid;Triaziquone;2, 2',2"-Trichlorotriethylamine;Urethane;Vindesine;Dacarbazine;Mann Nomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabino Cid (Ara-C); Cyclophosphamide; Thiotepa; Taxoid or Taxane family members of the family, such as paclitaxel (TAXOL®) and docetaxel (T AXOTERE® and analogs thereof; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues, e.g., cisplatin vinblastine and carboplatin; vinblastine; platinum; etoposide (VP-16); Ifosf amide; mitomycin C; mitoxantrone; vincristine; vinorelbine; Navel Bin; Novantrone; Teniposide; Daunomycin; Aminopterin; Xeloda; Ivan Doronate; CPT-11; Topoisomerase inhibitor RFS2000; Difluoromethyl Ornithine (DMFO); Retinoic acid; Esperamicin; Capecitabine; Receptor tyrosine kinase and / or angiogenesis inhibitors (sorafenib (NEXAVAR®)) , sunitinib (SUTENT®), pazopanib (VOTRIENT™) , toceranib (PALLADIA™), vandetanib (ZACTIMA™) , cedrilanib (RECENTIN®), regorafenib (BAY73-45 06), axitinib (AG013736), lestaurtinib (CEP-701), Rulotinib (TARCEVA®), gefitinib (IRESSA™), BIBW2992 (TOVOK™), lapatinib (TYKERB®), neratinib (HKI-272), etc.); and any of the above pharmaceutically acceptable Also included in this definition are salts, acids, or derivatives of tamagoyaki. Xifene, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxybenzoate Oxitamoxifen, Trioxifen, Keoxifen, LY117018, Onapri antiestrogens, such as steroid and toremifene (FARESTON®); and anti-inflammatory drugs such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin. antitumor agents, such as antitumor agents; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. There are antihormonal drugs that act to regulate or block hormone action on tumors. Cytotoxic chemical compounds (Wiemann et al., 1985, Medical Oncology(Calabresi et aL, eds.),Chapter 10, McMillan Publishing) also include the methods of the present invention. is applicable to.

[0367] The bispecific EGFR / c-Met FN3 domain-containing molecules of the present invention, N3 domain, c-Met binding FN3 domain, or bispecific EGFR / c-Met antibody Representative drugs that can be used in combination with the body include tyrosine kinase inhibitors and targeted anti-cancer drugs. therapeutic agents such as Iressa® (gefitinib) and Tarceva® (Ero) tinib), as well as other antagonists of HER2, HER3, HER4, or VEGF. Representative HER2 antagonists include CP-724-714, HERC EPTIN™ (trastuzumab), OMNITARG™ (pertuzumab), TAK-165, lapatinib (EGFR and HER2 inhibitor), and GW-282974 Representative HER3 antagonists include anti-Her3 antibodies. (See, e.g., U.S. Patent Application Publication No. 2004 / 0197332). Antagonists include anti-HER4 siRNA (see, e.g., Maatta et al. al., Mol Biol Cell 17:67~79, 2006). representative A potent VEGF antagonist is bevacizumab (Avastin™).

[0368] When a small molecule is used in combination with a bispecific EGFR / c-Met antibody of the invention, the bispecific Isomeric EGFR / c-Met antibodies are usually administered more frequently, preferably once daily. However, it can be 2, 3, 4 or more times a day, every other day, every week, or other times. Small molecule drugs are often administered orally, but may also be administered by IV infusion or via a syringe. Parenteral administration by intravenous injection or subcutaneous or intramuscular administration is also possible. The dose is usually 10 to 1000 mg, or about 100, 150, 200, or 250 mg. That's fine.

[0369] When the bispecific EGFR / c-Met antibodies of the invention are administered in combination with a second therapeutic agent, In this case, the combination can be administered at any convenient time frame. For example, a bispecific EGFR / The c-Met antibody and the second therapeutic agent may be administered to the patient on the same day and even via the same intravenous infusion. However, the bispecific EGFR / c-Met antibody and the second therapeutic agent Alternate every day, or every week, or every two weeks, or every month In some methods, two drugs are administered simultaneously. The bispecific E is then co-present in the patient at detectable concentrations (e.g., in serum). The GFR / c-Met antibody and the second therapeutic agent are administered sufficiently close in time. The method involves administering a bispecific EGFR / c- A second treatment, also consisting of multiple doses, given after or before the full course of Met antibody. In some methods, a second course of treatment is administered. Treatment with c-Met antibodies may be delayed if patients develop resistance to the first second treatment. Patients will receive a bispecific EGFR / c-Met antibody A single course of treatment with one or both of the first and second therapeutic agents or multiple treatments. The bispecific EGFR / c-Met antibody and second therapeutic agent can be administered. A recovery period of one, two, or several days or weeks may be used between administrations of the second If an appropriate treatment regimen has already been established for a therapeutic drug, that regimen may be used in the present invention. These antibodies may be used in combination with other known bispecific EGFR / c-Met antibodies, for example: Tarceva® (erlotinib) comes as a 100 mg or 150 mg pill Taken once daily, Iressa® (gefitinib) comes in 250 mg tablets. It is taken once a day.

[0370] The bispecific EGFR / c-Met antibody, optionally in combination with a second therapeutic agent, is Irradiation, Intensity Modulated Radiation Therapy (IMRT), as well as Gamma Knife, CyberKnife, and Lin ac and interstitial radiation (e.g., implanted radioactive seeds, GliaSite balloons) administered in conjunction with any form of radiation therapy, including any form of radiosurgery, and / or surgery. Combination with radiation therapy may be particularly appropriate for head and neck cancers and brain tumors.

[0371] The present invention has been described in general terms above, but the embodiments of the present invention are limited to the scope of the claims. The present invention is further disclosed in the following examples, which should not be construed as constituting an indication of the effectiveness of the present invention. [Example]

[0372] Example 1: Construction of the Tencon library Tencon (SEQ ID NO: 1) is the consensus sequence of the 15 FN3 domains of human tenascin-C. Fibronectin type III (FN3), an immunoglobulin-like scaffold designed from the nucleotide sequence ) domain (Jacobs et al., Protein Engineeri ng,Design,and Selection,25:107~117,2012; (US Patent Application Publication No. 2010 / 0216708). The crystal structure of Tencon is composed of seven beta chains. The figure shows six exposed surface loops connecting the Fibronectin type III (FN3) was synthesized by randomizing selected residues in Construct a library of domains and use it to select novel molecules that bind to specific targets. It is possible.

[0373] Tencon: Lpapknlvvsevtedslrlswtapdaafdsfliqyqese kvgeainltvpgsersydltglkpgteytvsiygvkgghr snplsaeftt (SEQ ID NO: 1):

[0374] Building the TCL1 library cis display system (Jacobs et al., Protein Eng ineering,Design,and Selection,25:107~117 For use with the Tencon DNA fragment (SEQ ID NO: 1), only the FG loop of Tencon DNA fragment was randomized. We have constructed a library (TCL1) designed to dam the Ta c promoter, Tencon library coding sequence, RepA coding sequence, cis element A single-stranded DNA incorporating the sequence of the nucleotide and ori element is generated. When expressed in a transcription / translation system, the Tencon-RepA fusion protein A complex bound to the DNA in cis is generated. Then, as described below, it binds to the target molecule. The binding complexes are isolated and amplified by polymerase chain reaction (PCR).

[0375] Construction of a TCL1 library for use with cis-display involves PCR. Successive rounds of DNA synthesis are carried out to generate half of the final linear double-stranded DNA molecule. (The 5' fragment contains the promoter and Tencon sequences, whereas the The 3' fragment contains the RepA gene and cis and ori elements. Combine the two halves of the construct by restriction digestion to generate the entire construct. This TCL1 library contains the FG loop of Tencon KGGHRSN (SEQ ID NO: 86). The library was designed to incorporate random amino acids. Using the nucleotide sequence, all 20 amino acids and one stop codon were incorporated into the FG loop. This TCL1 library is made possible by the use of different Six distinct subgroups were prepared with different randomized FG loop lengths (7 to 12 residues). The design of the library based on Tencon is shown in Table 2.

[0376] [Table 2] * TAPDAAFD: residues 22-28 of SEQ ID NO: 1 ** KGGHRSN: SEQ ID NO: 86 X refers to a degenerate amino acid encoded by the NNS codon. # refers to the "designed amino acid distribution" described in the text.

[0377] To construct the TCL1 library, successive rounds of PCR were performed to generate the Tac promoter. Addition of a nucleotide sequence, incorporating degeneracy into the FG loop, and the necessary restriction sites for final assembly. First, a DNA sequence containing the promoter sequence and the Tencon sequence 5' of the FG loop was added. The sequence was generated by PCR in two steps. DNA corresponding to the complete Tencon gene sequence was As the R template, primers POP2220 (SEQ ID NO: 2) and TC5'toFG (SEQ ID NO: 3) was used together with the PCR product obtained in this reaction as a template. After PCR amplification, the primers were 130mer (SEQ ID NO: 4) and TC5'toFG. This was used in rounds 1 and 2 to complete the addition of the 5' and promoter sequences to Tencon. The DNA product generated in the first step was amplified using forward primer POP2222 (SEQ ID NO: 5) ) and reverse primers TCF7 (SEQ ID NO: 6), TCF8 (SEQ ID NO: 7) containing degenerate nucleotides. Sequence number 7), TCF9 (SEQ ID NO: 8), TCF10 (SEQ ID NO: 9), TCF11 (SEQ ID NO: No. 10), or TCF12 (SEQ ID NO: 11) to amplify the FG loop. The number of PCR cycles was kept to a minimum to maximize library diversity. At least eight 100 μL PCR reactions were performed for each sublibrary to At least 5 μg of this PCR product was gel purified and ligated with primer POP2222 (SEQ ID NO: 1). No. 5) and POP2234 (SEQ ID NO: 12) in the next PCR step to produce 6xH An is tag and a NotI restriction site were added to the 3' end of the Tencon sequence. , using only 15 PCR cycles and at least 500 ng of template DNA. The resulting PCR product was gel purified, digested with NotI restriction enzyme, and purified by Qiagen. It was purified by column chromatography.

[0378] The 3' fragment of the library contains the PspOMI restriction site, the coding region of the repA gene, Certain D regions, including display elements such as cis and ori elements The plasmid containing this DNA fragment (pCR4Blunt) (I (Invitrogen) was used with M13 forward and M13 reverse primers. The resulting PCR product was digested with PspOMI overnight and gel purified. The 5' portion of the library DNA was ligated to the 3' DNA containing the repA gene. To do this, 2 pmol of 5' DNA was mixed with an equimolar amount of 3' repA DNA and NotI and Ligation was carried out in the presence of PspOMI enzyme and T4 ligase. Ligation was carried out overnight at 37°C. After gating, a small amount of the ligated DNA is run on a gel to confirm the ligation. The efficiency of the PCR was confirmed. The ligated library product was divided into 12 PCR amplicons. The primers of POP2250 (SEQ ID NO: 13) and DidLigRev (SEQ ID NO: 14) PCR was performed for 12 cycles using mer pairs. The DNA yields from the lab ranged from 32 to 34 μg.

[0379] To assess the quality of the library, a small amount of the active library was analyzed using primer Tcon5new 2 (SEQ ID NO: 15) and Tcon6 (SEQ ID NO: 16) were amplified and ligase-independent cloning was performed. The plasmid DNA was cloned into a modified pET vector by cloning. 1-GOLD(DE3) competent cells (Stratagene) were transfected and run Ninety-six dam-selected colonies were sequenced using T7 promoter primers. No duplicated sequences were observed. Overall, approximately 70-85% of the clones contained frameshift mutations. The gene contained the complete promoter and Tencon coding sequence without any differences. The percentage of functional sequences, excluding clones with different sequences, was 59% to 80%.

[0380] Building the TCL2 library Both the BC and FG loops of Tencon were randomized, and the amino acid sequence at each position was We constructed a TCL2 library with tightly controlled structure. Table 3 shows the location of the TCL2 library. The amino acid distribution at the desired loop position is shown. The amino acid distribution by design is based on two criteria. First, based on the crystal structure analysis of Tencon and / or homology modeling, From the results, residues predicted to be structurally important for the folding and stability of Tencon were identified. The library was biased, for example, at position 29, as this residue is hydrophobic in the Tencon fold. Since the amino acids are embedded in the hydrophobic core, only a subset of hydrophobic amino acids was fixed. The second layer of the analysis aligns the distribution of amino acids to the distribution of residues preferentially found in the heavy chain HCDR3 of antibodies. By biasing the molecules closer together, we can efficiently generate binders with high affinity. (Birtalan et al., J Mol Biol 377:151 8~28,2008;Olson et al., Protein Sci 16:47 6-84, 2007). For this purpose, the "distribution by design" in Table 3 refers to the following: The distribution of 6% alanine, 6% arginine, 3.9% asparagine, 7.5 % aspartic acid, 2.5% glutamic acid, 1.5% glutamine, 15% glycine, 2. 3% histidine, 2.5% isoleucine, 5% leucine, 1.5% lysine, 2.5% phenyl 4% proline, 10% serine, 4.5% threonine, 4% tryptophan , 17.3% tyrosine, and 4% valine. This distribution also includes methionine, cysteine, and No stop codon is included.

[0381] [Table 3] * Residue numbering is based on the Tencon sequence of SEQ ID NO:1.

[0382] The 5' fragment of the TCL2 library was inserted into the promoter and library pool (S The coding region of Tencon was chemically synthesized as a part of Lonning Biotechnology The DNA pool contained at least 1 x 10 11 Different types At the end of the fragment, the fragment was ligated to RepA. A BsaI restriction site for the cleavage of the nucleotide sequence was incorporated into the design.

[0383] The 3' fragment of the library contained a 6xHis tag, the coding region of the repA gene, and It was a fixed DNA sequence containing elements for display, including cis elements. The DNA was prepared using a pre-existing DNA template (see above) and / or primer LS1008 (sequence no. Prepared by PCR using DidLigRev (sequence number 14) and DidLigRev (sequence number 17) To construct the complete TCL2 library, a total of 1 μg of BsaI-digested 5' 3.5 μg of 3' Tencon library DNA prepared by restriction digestion with the same enzymes After overnight ligation, the DNA was ligated to Quiag The DNA was purified using an en column and quantified by measuring the absorbance at 260 nm. The ligand library product was purified using the primer pair POP2250 (SEQ ID NO: 13) and D The fragment was amplified by a 12-cycle PCR reaction using idLigRev (SEQ ID NO: 14). In total, each contained 50 ng of the ligated DNA product as template. 72 reactions were performed. The overall yield of active library DNA for TCL2 was approximately 100 μg. A small portion of the active library was subcloned as described above for TCL1. Approximately 80% of these sequences were fragments. It contained the complete promoter and coding sequence of Tencon without any region-shift mutations.

[0384] Building the TCL14 library Upper (BC, DE, and FG) and lower (AB, CD, and EF) loops, e.g., F The reported binding surface within the N3 domain is dominated by the β-strands that form the core of the FN3 structure. The FN3 domain has a shape different from the surface formed by the loops alone. Another surface present on the two "sides" of the in is two antiparallel β-strands, the C and F β-strands, and The CD and FG loops form the FN3 domain on one side of the FN3 domain. -This is called the CD-F-FG surface.

[0385] Another surface-randomized library of Tencon was developed to randomize the surface exposure of selected C and F chains. By randomizing the residues and parts of the CD and FG loops as shown in Figure 1, The following substitutions were made when compared to Tencon (SEQ ID NO: 1): E11R Tencon27 (SEQ ID NO: 1), a Tencon mutant with L17A, N46V, and E86I The library was generated using the method used to construct this library. A detailed description of the method is set forth in U.S. Patent Application Publication No. US2013 / 0226834. do.

[0386] Example 2: Fibronectin type III domain binds to EGFR and inhibits EGF binding Inn Selection Library screening Using cis display, EGFR-binding domains were isolated from the TCL1 and TCL2 libraries. The main antibody selected was a recombinant human extracellular domain of EGFR fused to IgG1 Fc ( R&D Systems) was biotinylated using standard methods and used for panning. (residues 25-645 of full-length EGFR of SEQ ID NO: 73). In vitro transcription and translation (IT To perform the T), add 2-6 µg of library DNA to a 1x S buffer containing 0.1 mM total amino acids, 1x S 30 premix components and 30 μL of S30 extract (Promega) and total The total volume was 100 μL and the mixture was incubated at 30°C. After 1 hour, 450 μL of blocking solution was added. The solution (2% bovine serum albumin, 100 μg / mL herring sperm DNA, and 1 mg PBS (pH 7.4) supplemented with 1 / mL heparin was added and the reaction was incubated on ice for 15 min. The cells were incubated in blocking solution with recombinant human EGF (R&D Systems ) was mixed with biotinylated recombinant EGFR-Fc at room temperature for 1 hour to obtain EGF. R-Fc:EGF complexes were constructed at molar ratios of EGFR to EGF of 1:1 and 10:1. To perform the binding, 500 μL of the blocked ITT reaction was added to 100 μL of EGFR-F c: After mixing with the EGF complex and incubating at room temperature for 1 hour, the bound complex was detected by magnetic Precipitated by neutravidin or streptavidin beads (Seradyne) Unbound library members were removed by successive washes with PBST and PBS. After washing, the DNA was eluted from the bound complex by heating to 65°C for 10 minutes. The resulting fragments were then amplified by PCR and subjected to restriction digestion and ligation for further rounds of panning. The DNA fragment encoding RepA was added by gating. The target EGFR-Fc concentration in each round was changed from 200 nM to 50 nM. Isolation was performed by successively decreasing and increasing the stringency of the washes. 4 and 5 were incubated in PBS in the presence of a 10-fold molar excess of non-biotinylated EGFR-Fc. Unbound or weakly bound FN3 domains were removed by overnight washing.

[0387] After panning, selected FN3 domains were ligated with the oligonucleotide Tcon5new2 (SEQ ID NO: 15) and Tcon6 (SEQ ID NO: 16) were amplified by PCR and ligated. Subcloned into a pET vector modified to contain a DNA-independent cloning site. , using standard molecular biology techniques, BL21-GOLD(DE3) (Stratage ne) cells and soluble expression in E. coli to allow for purification and detection. A gene sequence encoding a C-terminal polyhistidine tag was added to each FC3 domain. The culture was incubated at 37°C in 1 mL of a 96-well block containing 100 μg / mL carbenicillin. After growth in 2YT medium to an optical density of 0.6–0.8, IPTG was added to 1 mM. At that point, the temperature was reduced to 30°C. After approximately 16 hours, the cells were harvested by centrifugation. Each pellet was diluted with 0.6 mL of BugBuster® ) HT lysis buffer (Novagen EMD Biosciences) at room temperature with shaking. Cell lysis was achieved by incubating with shaking for 45 minutes.

[0388] Selection of FN3 domains that bind to EGFR on cells To assess the ability of different FN3 domains to bind EGFR in a more physiological setting The ability of the FN3 domain to bind to A431 cells (Amer) was then measured. ican Type Culture Collection, Catalog No. CRL-1 555) expresses EGFR at approximately 2 × 10 6 receptors are overexpressed in cells. 5,000 cells / well were seeded in an opaque black 96-well plate and incubated in a humidified 5% CO2 atmosphere. The bacterial lysate expressing the FN3 domain was then incubated overnight at 37°C in an air atmosphere. Dilute 1,000-fold in S staining buffer (Becton Dickinson) and test in triplicate. The plate was incubated at room temperature for 1 hour. The lysate was removed and 150 μL of cells were added. The cells were washed three times with 50 μL / well of FACS staining buffer. Anti-PentaHis-Alexa488 antibody conjugate diluted 1:100 in color buffer The cells were incubated with 150 μL / well of PBS (Qiagen) at room temperature for 20 minutes. After washing three times with FACS staining buffer, the wells were filled with 100 μL of FACS staining buffer. The plate was filled and the fluorescence was read at 488 nm using an Acumen eX3 reader. Bacterial lysates containing the three domains were screened for their ability to bind to A431 cells. (1320 crude bacterial lysates for the TCL1 and TCL2 libraries) Sixteen positive clones were identified, with binding between 10-fold higher than background signal. 300 lysates from the TCL14 library were screened for binding. As a result, 58 unique FN3 domain sequences that bind to EGFR were obtained.

[0389] Selection of FN3 domains that inhibit EGF binding to EGFR on cells To further characterize the mechanism of EGFR binding, we investigated the binding mechanism of different EGFR-EGF competitively. The ability of the identified FN3 domain clones to bind to A431 cells was measured. A431 cells were plated in an opaque black 96-well plate. Plate 100 cells at 5,000 cells / well and incubate overnight at 37°C in a humidified 5% CO2 atmosphere. Cells were allowed to adhere. 50 μL / well of 1:1,000 diluted bacteria was added to triplicate plates. The lysate was incubated at room temperature for 1 hour. Biotinylated EGF (Invitrogen) en, Catalog No. E-3477) to each well to a final concentration of 30 ng / mL The cells were then incubated at room temperature for 10 minutes. The cells were then washed three times with PBS. The cells were then soaked in 50 μL / well of FACS staining buffer at a 1:100 dilution. Diluted streptavidin-phycoerythrin conjugate (Invitrogen ) for 20 minutes at room temperature. The cells were then transferred to 150 μL / well of FACS staining buffer. After washing three times with FACS, fill each well with 100 μL of FACS staining buffer and incubate for 1 minute. Fluorescence was read at 600 nm using a men eX3 reader.

[0390] Bacterial lysates containing the FN3 domain were screened using the EGF competition assay described above. 1320 crude bacterial lysates from the TCL1 and TCL2 libraries were screened. As a result of screening, 451 positive clones were obtained that inhibited EGF binding by 50% or more. .

[0391] Expression and purification of identified FN3 domains that bind to EGFR The His-tagged FN3 domain was then plated onto the His MultiTrap™ HP plate. The lysate was purified from clarified E. coli lysate by a 200-mL syringe pump (GE Healthcare). 500 mM sodium phosphate, 500 mM sodium chloride, and 250 mM imidazole The purified sample was eluted in a pH 7.4 buffer containing PBS (pH 7. 4) Replace the plate with a PD MultiTrap™ G-25 plate (GE Heal) The analysis was performed using the thcare.

[0392] Size exclusion chromatography analysis Aggregation state of FN3 domains bound to EGFR using size exclusion chromatography An aliquot (10 μL) of each purified FN3 domain was measured using Superdex Transfer the PBS (pH 7.4) to a 75 5 / 150 column (GE Healthcare). The mobile phase was injected at a flow rate of 0.3 mL / min. The eluate from the column was monitored by absorbance at 280 nm. FN3 domains that showed high levels of aggregation by SEC were excluded from further analysis. .

[0393] Dissociation rates of selected EGFR-binding FN3 domains from EGFR-Fc To facilitate the selection of high-affinity binders, a ProteOn XPR-36 instrument was used. (Bio-Rad) selected EGFR-binding FN3 domains were screened to identify EGFR-binding FN3 domains. Dissociation rate (k off ) was identified as slow. Goat anti-human Fc IgG (R&D systems) was diluted with 0.005% Tween Six lateral ligand channels on the chip were prepared at a flow rate of 30 μL / min in PBS containing α-20. All of the antibodies were directly immobilized on the cells by amine coupling (pH 5.0). The average was approximately 1500 response units (RU), with less than 5% variation between different channels. EGFR-Fc was bound to anti-human Fc-Ig in the longitudinal direction to a density of approximately 600 RU. All FN3 domains tested were normalized to a concentration of 1 μM and were then captured on the G surface. To maximize the throughput of the screening, we tested their binding in the horizontal direction. Therefore, all six analyte channels were used for the FN3 domain. The flow rate was 1 μL / min and the measurement was performed for 10 min. The binding signal between spots was used as a reference to monitor binding, and all binding reactions were analyzed. The processed binding data were fitted to a simple 1:1 Langmuir binding model. By locally fitting, each FN3 to the captured EGFR-Fc Domain Combination off was extracted.

[0394] Inhibition of EGF-stimulated EGFR phosphorylation Purified EGFR-binding FN3 domains were tested at a single concentration in A431 cells to inhibit EGF The ability of EGFR to inhibit phosphorylation stimulated by EGFR was tested. Oxidation was performed using the EGFR phospho (Tyr1173) kit (Meso Scale Disco Cells were plated in clear 96-well tissue culture-treated plates. (Nunc), GlutaMA with 10% fetal bovine serum (FBS) (Gibco). 20,000 in 100 μL / well of RPMI medium (Gibco) containing X™ Cells were seeded at 100 cells / well and allowed to attach overnight at 37°C in a humidified 5% CO2 atmosphere. After complete removal, the cells were incubated at 37°C in a humidified 5% CO atmosphere without FBS for 10 min. The cells were then starved overnight in 0 μL / well of medium. 100 μL of EGFR-binding FN3 domain at a concentration of 2 μM was incubated at 37°C for 1 hour in a 2 atmosphere. Treated with 1 / well of pre-warmed (37°C) starvation medium. Controls were treated with starvation medium alone. The cells were treated with 100 ng / mL recombinant human EGF (R&D Systems, Add 100 μl / well of pre-warmed (37°C) starvation medium containing 100 μl of PBS ... Stimulate by adding and gently mixing EGF to a final concentration of 50 ng / mL and 1 μM The EGFR-binding FN3 domain was incubated at 37°C in 5% CO2 for 15 minutes. One set of control wells was left unstimulated as a negative control. The medium was completely removed, and the cells were resuspended in 100 μL / well of Co according to the manufacturer's instructions. Complete Lysis Buffer (Meso Scale Discovery) The EGF was dissolved by shaking for 10 minutes at room temperature. Assay plate configured to measure R (Meso Scale Discov ery) at room temperature for 1.5 min in the provided blocking solution according to the manufacturer's instructions. Blocking was performed for 2 hours. The plate was then washed with 200 μL / well of 1× Tris W. The cells were washed four times with ash buffer (Meso Scale Discovery). Transfer an aliquot of the isate (30 µL / well) to the assay plate and plate it The plate was covered with sealing film (VWR) and incubated at room temperature with shaking for 1 hour. The assay plate was washed four times with 200 μL / 2 well of Tris Wash buffer. Then, add 25 μL of ice-cold Detection Antibody Solution (M eso Scale Discovery) into each section, being careful not to trap air bubbles. The plate was incubated at room temperature with shaking for 1 hour, and then 200 μL Wash four times with 150 μL / well of Tris Wash Buffer. Buffer (Meso Scale Discovery) was added and the solution was then washed with water as instructed by the manufacturer. SECTOR® Imager with pre-installed assay-specific default settings The results were obtained by reading on a 6000 instrument (Meso Scale Discovery). The signal was detected. The inhibition rate of the positive control signal stimulated by EGF (%) was calculated for each EGFR binding domain.

[0395] Inhibition of EGF-stimulated EGFR phosphorylation was measured using TCL1 and TCL2 libraries. The 232 identified clones obtained from the library were assayed. Two of them inhibited EGFR phosphorylation by 50% at a concentration of 1 μM. After removing clones determined to be multimeric by chromatography, nine clones were obtained. Further biological characterization was performed on the clones. The sequences of the BC and FG loops of these clones were The columns are shown in Table 4. Eight of the nine selected clones shared the common FG loop sequence (H NVYKDTNMRGL; SEQ ID NO: 95), and the BC loop was A region of significant sequence similarity was observed.

[0396] [Table 4]

[0397] Example 3: Characterization of EGFR-binding FN3 domains that inhibit EGF binding Large-scale expression, purification, and endotoxin removal To provide more material for detailed characterization, we mass-produced the FN3 domains shown in Table 4. Overnight cultures containing each EGFR-binding FN3 domain mutant were used to incubate at 100 μg / ml The overnight culture was diluted at a 1 / 80 dilution in 0.8 L of Ternary Buffer A (TBA) supplemented with 1 L of ampicillin. Fresh medium was inoculated with rific broth medium and incubated at 37°C with shaking. When the optical density at 600 nm reached approximately 1.2-1.5, add IPTG to a final concentration of 1 mM. The culture was induced by adding 100 mM NaCl and the temperature was reduced to 30°C. After 4 hours, the cells were Cells were harvested by centrifugation and the cell pellets were stored at -80°C until needed.

[0398] To lyse the cells, the thawed pellet was diluted with 25 U / mL Benzonase ( (Sigma-Aldrich) and 1 kU / mL of rLysozyme ( ) (Novagen EMD Biosciences) er® at a ratio of 5 mL of BugBuster® per 1 g of pellets Lysis was allowed to proceed for 1 hour at room temperature with gentle agitation, followed by incubation at 4°C for 50 minutes. The supernatant was collected and filtered through a 0.2 μm filter. AKTAexplorer 100s chromatography system (GE Heal) Use the appropriate care to prepare Buffer A (50 mM Tris-HCl, pH 7 5 mL of HisCl2 pre-equilibrated in 0.5 mM NaCl, 500 mM NaCl, 10 mM imidazole Load the TraFF column and wash with 20 column volumes of Buffer A. Then, add 6 columns' worth of 16% Buffer B (50 mM Tris-HCl , pH 7.5, 500 mM NaCl, 250 mM imidazole). The domain was eluted with 50% B over 10 column volumes and 6 column volumes. A gradient of 50-100% B was run. Fractions containing FN3 domain proteins were pooled. The solution was concentrated using a Millipore 10K MWCO concentrator, filtered, and then added to P HiLoad™ 16 / 60 Superdex™ 75 pre-equilibrated with BS The proteins were loaded onto a column (GE Healthcare). The peak of the protein monomer was confirmed.

[0399] ActiClean Etox resin (Sterogene Bioseparation Endotoxin was removed using a batch approach using ns. Prior to removal, the resin was pretreated with 1N NaOH for 2 hours at 37°C (or overnight at 4°C), The purified product was washed thoroughly with PBS until the pH stabilized at approximately 7 when measured with pH indicator paper. The protein was filtered through a 0.2 μm filter, and then 10 mL of PEG was added to 1 mL of resin. The binding of endotoxin to the resin was determined by: Allow to proceed for at least 2 hours at room temperature with gentle rotation. Centrifuge at 500g for 2 minutes. The resin was removed and the protein supernatant was collected. Endotoxin levels were measured using EndoSafe™ cartridges. The endotoxin levels were analyzed using a PEG-MCS reader (Charles River). If the level was higher than 5 EU / mg after the first Etox treatment, the endotoxin level was The above procedure was repeated until the endotoxin level was reduced to 5 × 5 EU / mg. If higher than EU / mg and stabilized after two successive treatments with Etox, The protein is then subjected to anion exchange or hydrophobic interaction chromatography to remove residual endotoxins. The topography conditions are now established.

[0400] Affinity of selected EGFR-binding FN3 domains for EGFR-Fc (EGFR- Fc affinity measurement Binding of selected EGFR-binding FN3 domains to recombinant EGFR extracellular domain For affinity, surface plasmon resonance was performed using a Proteon instrument (BioRad). Further characterization was performed by the following assay setup (chip preparation, EGFR-Fc The dissociation rate analysis was similar to that described above. Binding FN3 domains were tested in a 3-fold serial dilution series across the lateral direction at a concentration of 1 μM. Buffer samples were also injected to monitor the stability of the baseline. The dissociation phase at all concentrations of the three domains was performed for 30 min at a flow rate of 100 μL / min ( k from dissociation rate screening off is about 10 -2 s -1 1 hour (for items) or o ff is about 10 -3 s -1 or slower). The sets were subtracted from the response data: 1) EGFR-binding FN3 domain and fixed FN3 domain Spot-to-spot signal to correct for nonspecific interactions with the standardized IgG surface and 2) correct for baseline fluctuations due to dissociation of the captured EGFR-Fc surface over time. Buffer channel signal for each FN3 domain at all concentrations. The binding data were globally fitted to a simple 1:1 Langmuir binding model. By doing this, the dynamics (k on ,k off ) and affinity (K D ) constant estimates were extracted. Table 5 shows the kinetic constants for each of the constructs, with affinities of 2 It varies from 0.00pM to 9.6nM.

[0401] Binding of selected EGFR-binding FN3 domains to EGFR on cells (A431 cells) Binding Assay A431 cells were seeded at 5,000 cells / well in an opaque black 96-well plate and humidified. The cells were allowed to adhere overnight at 37°C in a 5% CO2 atmosphere. The cells were treated with 1.5 μM to 30 μM of ATP in 50 μL in triplicate plates for 1 hour at room temperature. The supernatant was removed and the cells were washed three times with 150 μL / well of FACS staining buffer. Cells were stained with 50 μL / well of anti-pentahydroxybenzoate (PentaH) diluted 1:100 in FACS staining buffer. Incubate with is-Alexa488 antibody conjugate (Qiagen) at room temperature for 20 minutes. The cells were washed three times with 150 μL / well of FACS staining buffer and then plated onto the wells. Fill the tube with 100 μL of FACS staining buffer and use an Acumen eX3 reader. The fluorescence was read at 488 nm. Data were plotted against the logarithm of the molar concentration of the FN3 domain. Plotted as raw fluorescence signals and analyzed using GraphPad Prism4 (GraphPad d Software) was used to fit a sigmoidal dose-response curve with a variable slope. EC 50Table 5 shows the concentrations of α- and β-actin in the control group, ranging from 2.2 nM to over 20 μM. EC of each tract 50 Shows.

[0402] Binding of EGF to EGFR on cells using selected EGFR-binding FN3 domains Inhibition of (A431 cell EGF competition assay) A431 cells were seeded at 5,000 cells / well in an opaque black 96-well plate and humidified. The cells were allowed to adhere overnight at 37°C in a 5% CO2 atmosphere. In triplicate plates, cells (50 μL / well) were treated with 1.5 μM to 30 μM of ATP for 1 hour. Biotinylated EGF (Invitrogen, Cat. No. E-3477) was added at room temperature. to each well to a final concentration of 30 ng / mL and incubate at room temperature for 10 minutes. The cells were washed three times with 150 μL / well of FACS staining buffer. 50 μL / well of streptavidin-fluorescence stain diluted 1:100 in ACS staining buffer Incubate with erythrin conjugate (Invitrogen) at room temperature for 20 minutes. The cells were washed three times with 150 μL / well of FACS staining buffer, and then the wells were Fill with 100 μL of FACS staining buffer and use an Acumen eX3 reader Fluorescence was read at 600 nm. Data were plotted against the logarithm of the molar concentration of the FN3 domain. The fluorescence signal was plotted and analyzed using GraphPad Prism 4 (GraphPad Fitting a sigmoidal dose-response curve with a variable slope was performed using the RT-MS software. EC 50 Table 5 shows the IC values ​​ranging from 1.8 nM to 121 nM. 50 value Shows.

[0403] Inhibition of EGF-stimulated EGFR phosphorylation (phospho-EGFR assay) Selected FN3 domains significantly inhibit EGF-stimulated EGFR phosphorylation , IC of inhibitors 50 This was more fully evaluated by measuring the EGF-stimulated Inhibition of EGFR phosphorylation stimulated by EGF is referred to as "inhibition of EGF-stimulated EGFR phosphorylation." As described above, the FN3 domain was evaluated at different concentrations (0.5 nM to 10 μM). Data are plotted as electrochemiluminescence signal against the logarithm of the molar concentration of FN3 domain. and GraphPad Prism 4 (GraphPad Software) and by fitting the data to a sigmoidal dose-response with a varying slope I C 50 Table 5 shows the IC values ​​ranging from 18 nM to over 2.5 nM. 50 Indicates the value.

[0404] Inhibition of human tumor cell proliferation (NCI-H292 proliferation and NCI-H322 proliferation assays) Inhibition of EGFR-dependent cell proliferation was confirmed by the use of NC, a human tumor cell line that overexpresses EGFR. I-H292 and NCI-H322 (American Type Culture Collection collection, catalog numbers CRL-1848 and CRL-5806, respectively) This was assessed by measuring survival after exposure to the EGFR-binding FN3 domain. The cells were grown in opaque white 96-well tissue culture treated plates (Nunc) using 10% heat inactivated Nitrated fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Gibco) GlutaMAX™ and 10 mM HEPES were added to 100 μL / well. 500 cells / well (NCI-H292) or 100 cells / well (NCI-H292) in RPMI medium (Gibco) ,000 cells / well (NCI-H322) in a humidified 5% CO2 atmosphere. Cells were allowed to attach overnight at 37°C. Cells were incubated with a range of EGFR-binding FN3 domains. Treatment was performed by adding 5 μL / well of phosphate-buffered saline (PBS). Rolls were plated with 5 μL / well of PBS alone or 25 mM ethylenediaminetetraacetic acid in PBS. The cells were treated with acid. The cells were incubated at 37°C, 5% CO2 for 120 hours. 75 μL / well CellTiter-Glo® Reagent (Promega) was added Mix on a plate shaker for 2 minutes and incubate in the dark at room temperature for an additional 10 minutes. Viable cells were detected by scanning the plate through an Sp and a ectraMax M5 plate reader (Molecular Devices). The data were read against a medium-only blank at a read time of 0.5 seconds per well. Proliferation of PBS-treated cells (%) versus the logarithm of the molar concentration of FN3 domains. GraphPad Prism 4 (GraphPad Software) Fitting the data to a sigmoidal dose-response equation with a variable slope using By this, IC 50 Table 5 shows the results for NCI-H292 and NCI-H322. When cells were used, the ranges were 5.9 nM to 1.15 μM and 9.2 nM to over 3.1 μM. Wataru IC 50 Table 5 shows the biomarkers of EGFR-binding FN3 domains in each assay. An overview of the biological properties is given below.

[0405] [Table 5]

[0406] Example 4: Engineering EGFR-binding FN3 domains A subset of EGFR-binding FN3 domains was isolated by increasing the conformational stability of each molecule. The mutation L17A, which has been shown to increase the stability of the FN3 domain, was engineered to , N46V, and E86I (described in U.S. Patent Application No. US2011 / 0274623) The clones P54AR4-83, P54CR4-31, and P54CR4-41 were synthesized by DNA synthesis. and P54AR4-37. New mutants P54AR5-83v2, P54C R431-v2 and P54AR4-37v2 were expressed and purified as described above. By assessing the stability of each mutant using differential scanning calorimetry in PBS, The stability of the T m The average increase in This indicates that the heterologous molecule was significantly stabilized.

[0407] [Table 6]

[0408] Example 5: Fibronectin type III (F) binds to c-Met and inhibits HGF binding N3) Selecting a domain Panning of human c-Met To identify the FN3 domain capable of specifically binding to c-Met, TCL The 14 libraries were constructed using biotinylated human c-Met extracellular domain (bt-c-Met) For selection, 3 μg of the TCL14 library was S30 Linear Extract (Promega, Madison, WI) The expressed library was subjected to in vitro transcription and translation (IVTT) in a cis-blocker. 2% BSA (Sigma-Aldrich, St. Louis, MO), 100 μg / ml herring sperm DNA (Promega), 1 mg / mL heparin (Sigma- Aldrich). 100 nM (Round 1), 200 nM (Rounds 2 and 3), and 100 nM (Rounds 4 and NeutrAvidin magnetic beads (Thermo Fisher Scientific) were added at concentrations of 0.5 and 5. , Rockford, IL) (rounds 1, 3, and 5) or streptavidin magnetic Beads (Promega) (rounds 2 and 4) were used to separate the members of the ligated library. Collect the bars and wash the beads 5 to 14 times with 500 μL of PBS-T, followed by 500 μL of Unbound library members were removed by washing twice with PBS. .

[0409] Further rounds of selection were performed to identify molecules with FN3 domains that exhibited improved affinity. In summary, the harvest from round 5 was prepared as above with the following modifications: The selection rounds were repeated, i.e., incubation with bt-c-Met was repeated for 1 The time required for bead capture was reduced from 20 minutes to 15 minutes, and the time required for bt-c-Met was reduced from 20 minutes to 15 minutes. The excess was reduced to 25 nM (rounds 6 and 7) or 2.5 nM (rounds 8 and 9). An additional 1-hour wash was performed in the presence of 100 μL of non-biotinylated c-Met. has a significantly lower K D potentially have faster binding rates and slower dissociation rates, leading to The key is to simultaneously select a binder that will work.

[0410] The collected fractions from rounds 5, 7, and 9 were used as TCON6 (SEQ ID NO: 30) and TCON5 E86 The I short (SEQ ID NO: 31) primer was used to clone the ligase-independent cloning site (p A modified pET15 vector (EMD Biosciences) containing ET154-LIC The clones were cloned by PCR at the National Atomic Energy Agency (NEURALSKA, Gibbstown, NJ) and analyzed using standard protocols. After transformation with PBS and induction with IPTG (final concentration 1 mM, 16 hours at 30°C), The protein was expressed as a C-terminal His6 tagged protein. After separating and harvesting the hearts, 0.2 mg / mL of Chicken Egg White Ly Bugbuster HT ( The bacterial lysate was clarified by centrifugation and the supernatant was transferred to a new 96 deep well plate.

[0411] Screening of FN3 domains that inhibit HGF binding to c-Met The FN3 domain present in E. coli lysate was purified in a biochemical format. These were screened for their ability to inhibit HGF binding to the c-Met extracellular domain. Recombinant human c-Met / Fc chimera (0.5 μg / mL in PBS, 100 μL / well) The plate was coated onto 96-hole White Maxisorp Plates (Nunc). The plates were washed with a Biotek plate washer and incubated overnight at 4°C. 300 μl / well of Tris-buffered saline containing 0.05% Tween 20 The assay plate was washed twice with TBS-T (Sigma-Aldrich). Starting Block T20 (200 μL / well, Thermo Fisher Scientific) The mixture was incubated at 4°C for 1 hour at room temperature (RT) with shaking. The plate was blocked with 0.5% COOH and washed twice with 300 μl of TBS-T. Lysates were transferred to the STARplus robotics system using the Hamilton STARplus robotics system. Diluted (1:10 to 1:100,000) in StartingBlock T20. Lysate (50 μL / well) was incubated on the assay plate for 1 hour at room temperature with shaking. Without washing the plate, bt-HGF (Starting Block 1 μg / mL in T20, 50 μL / well, biotinylated) onto the plate for 30 minutes. The control wells contained Tencon 27 lysate and were shaken for 1 minute at room temperature. Starting Block T20 or diluted bt-HGF was added to the 1000 cells. Wash the plate four times with 300 μl / well of TBS-T and then add 100 μl / well of Streptavidin-HRP (1:2000 in TBS-T, Jackson Immun oresearch, West Grove, PA) for 30-40 minutes at room temperature. The plate was again washed four times with TBS-T. Prepare POD according to the manufacturer's instructions to generate Chemiluminescence ence Substrate (50 μL / well, Roche Diagnostics , Indianapolis, IN) was added to the plate and the SoftMax Luminescence was read on a Molecular Devices M5 Pro. The inhibition rate (%) was calculated using the formula: 100-((RLU 試料 -Average RLUbt-HGFを加 えないコントロール ) / (average RLU bt-HGFコントロール -Average RLU bt-HGFを加えない コントロール ) × 100) Values ​​of 50% or higher inhibition rate (%) were considered hits.

[0412] High-throughput expression and purification of FN3 domains His-tagged FN3 domains were isolated from clarified E. coli lysates using His Mu Purification was performed using a ltiTrap™ HP plate (GE Healthcare) and 20 ml 500 mM sodium phosphate, 500 mM sodium chloride, and 250 mM imidazole The purified sample was eluted in a buffer containing PBS (pH 7.4). and replaced with a PD MultiTrap™ G-25 plate (GE Healthcare The analysis was performed using (are).

[0413] IC of inhibition of HGF binding to c-Met 50 Decision Selected FN3 domains were further characterized in an HGF competition assay. A dose-response curve of purified FN3 domains was generated using the assay (starting concentration of 5 μM). The percent inhibition values ​​were calculated. Data were plotted against the logarithm of the molar concentration of the FN3 domain. The changes were plotted as % inhibition and analyzed using GraphPad Prism 4. The IC was calculated by fitting the data to a sigmoidal dose-response with a slope of 50 Find the value Ta.

[0414] From round 5, activity was observed at a dilution of 1:10, with IC values ​​ranging from 0.5 to 1500 nM. 50 From round 7, 35 unique sequences with a 1:100 dilution were identified. with IC in the range of 0.16–2.9 nM 50 39 unique sequences with values ​​were obtained. From round 9 onwards, hits were defined as active at a dilution of 1:1000. Six unique sequences were identified. In round 9, IC was as low as 0.2 nM. 50 Value is recognized (Table 8).

[0415] Affinity of selected c-Met-binding FN3 domains for c-Met-Fc (EGF Determination of R-Fc affinity Selected EGFR-binding FNs were used except that c-Met-Fc was used in the assay. Selected c-Met binding domains were determined as described in Example 3 for the determination of affinity of the three domains. The affinity of the FN3 domain was determined.

[0416] Example 6: Characterization of FN3 domains that bind to c-Met and inhibit HGF binding The FN3 domain was expressed and purified as described above in Example 2. Size exclusion chromatography and kinetic analysis were performed as described above. The sequences of the chain, CD loop, F chain, and FG loop, as well as the complete amino acid sequence of each domain, are shown. Indicates the column number.

[0417] [Table 7]

[0418] The C-loop residues correspond to residues 28-37 of the SEQ ID NOs shown. The CD chain residues correspond to residues 38-43 of the SEQ ID NOs shown. The F loop residues correspond to residues 65-74 of the SEQ ID NOs shown. The FG chain residues correspond to residues 75-81 of the SEQ ID NOs shown.

[0419] Binding of selected c-Met-binding FN3 domains to c-Met on cells (H441 Cell Binding Assay NCI-H441 cells (Cat. No. HTB-174, American Type Culture Collection, Manassas, Virginia) to Lysine-coated black clear 96-well plates (BD Biosciences, Cells were seeded at 20,000 cells / well in a 1000-well plate (San Jose, California) and incubated at 37°C in 5% CO2. The purified FN3 domain (50 μL / well, 0-1000 nM) was added to the wells. Cells were added in duplicate plates for 1 hour at 4°C. The supernatant was removed and the cells were subjected to FACS staining. Buffer (150 μL / well, BD Biosciences, Cat. No. 55465 The cells were washed three times with biotinylated anti-HIS antibody (1:1 in FACS staining buffer). Dilute with 160, 50 μL / well, R&D Systems, Catalog No. BAM050 ) for 30 minutes at 4°C. The cells were then incubated with FACS staining buffer (150 μL / After washing three times with anti-mouse IgG1-Alexa 488 conjugated antibody ( Diluted 1:80 in FACS staining buffer, 50 μL / well, Life Techno The cells were incubated with 100µL of PBS (Prod. No. A21121) for 30 minutes at 4°C. Wash the cells three times with FACS staining buffer (150 μL / well) and then resuspend them in FACS staining buffer. (50 μL / well). Total fluorescence was measured using an Acumen eX3 reader. Data were plotted as raw fluorescence signal against the logarithm of the molar concentration of FN3 domain. GraphPad Prism 4 (GraphPad Software) EC by fitting a sigmoidal dose-response curve with a varying slope using 50 Calculate the value As shown in Table 8, the FN3 domain had an EC 50 It was found that the binding activity of the IgG1A-binding domains was broad and varied.

[0420] Inhibition of HGF-stimulated c-Met phosphorylation The purified FN3 domain was analyzed by Meso Scale Discovery (Maryland). c-Met phospho (Tyr1349) kit from the company Gaithersburg, MD, was used. The ability to inhibit HGF-stimulated c-Met phosphorylation in CI-H441 Cells were plated in clear 96-well tissue culture treated plates in 10% fetal bovine serum. 100 μL / well of RP with supernatant (FBS, Life Technologies) MI medium (Glutamax and HEPES (Life Technologies) The cells were seeded at 20,000 cells / well in a humidified 5% CO2 atmosphere at 37°C. The medium was completely removed, and the cells were placed in serum-free RPMI medium (100 μL / well). The cells were starved overnight in PBS at 37°C and 5% CO2. After this, the cells were transfected with the FN3 domain. Fresh serum-free RPMI medium (100 μL / well) containing acetaminophen at a concentration of 20 μM or less was added to the wells. The cells were incubated at 10°C with 5% CO₂ for 1 hour. The control was treated with medium alone. 0 ng / mL recombinant human HGF (100 μL / well, R&D Systems catalog) The cells were stimulated with 100 μg of erythrocytes containing 100 μg of erythrocyte stimulatory protein (antibody number 294-HGN) and incubated at 37°C, 5% CO2 for 15 minutes. One set of control wells was left unstimulated as a negative control. Afterwards, the medium was completely removed and the cells were incubated in Complete Lysis according to the manufacturer's instructions. s buffer (50 μL / well, Meso Scale Discovery) for 10 min. The solution was dissolved by shaking at room temperature for 1 minute. The assay plate was then blocked with the provided blocking solution according to the manufacturer's instructions. The plate was then washed with Tris Wash buffer (2 The cells were washed three times with 1000 μL / well of Meso Scale Discovery. Transfer the lysate (30 μL / well) to an assay plate and incubate at room temperature with shaking for 1 hour. The assay plate was then washed four times with TrisWash buffer. Then, add ice-cold Detection Antibody Solution (25 μL Add 100 ml of PBS (Meso Scale Discovery) to each well and shake for 1 hour at room temperature. The plate was again washed four times with TrisWash buffer. Read buffer (150 μL / well, Meso Scale Discovery ) and run SECT using the assay-specific default values ​​installed by the manufacturer. OR® Imager 6000 instrument (Meso Scale Discover The signal was detected by reading the data on a fluorometer (FN3). Plots were made as electrochemiluminescence signal against the logarithm of the degree of polarization and analyzed using GraphPad Prism. Fitting the data to a sigmoidal dose-response with a variable slope using m4 By IC 50 As shown in Table 8, the FN3 domain exhibited a serotonin concentration of 4.6 nM to 1 IC in the range of 415nM 50It was shown to inhibit phosphorylated c-Met at high levels.

[0421] Inhibition of human tumor cell proliferation or survival After exposure to the c-Met-binding FN3 domain, U87-MG cells (American T Viability of Type Culture Collection, Catalog No. HTB-14 Inhibition of c-Met-dependent cell proliferation was assessed by measuring the β-catenin-dependent cytotoxicity (c-Met) of the cells. In a standard white 96-well tissue culture treated plate (Nunc), 10% FBS was added to 100 ml of PBS. Seed 8000 cells / well in 100 μL / well of RPMI medium and culture at 37°C, 5% CO. 24 hours after seeding, the medium was aspirated and the cells were refilled with serum-free RPMI medium. After 24 hours of serum starvation, the c-Met-binding FN3 domain (30 μL / well) was added. The cells were treated by adding serum-free medium containing 100% ethanol (100% ethanol). The cells were incubated at 37°C, 5% CO₂ Incubated at 25°C for 72 hours. 100 μL / well of CellTiter-Glo After adding the Promega® reagent, mix on a plate shaker for 10 minutes. Viable cells were detected by scanning the plate through an Sp and a ectraMax M5 plate reader (Molecular Devices). The read time was 0.5 seconds per well. Data were collected as a function of FN3 domain molar concentration. Plotted as raw luminescence units (RLU) versus number. GraphPad P Fit the data to a sigmoidal dose-response equation with varying slope using Rism 4 By doing so, the IC 50 Table 8 shows the I values ​​in the range of 1 nM to over 1000 nM. C50 values ​​are shown. The properties of the c-Met binding FN3 domains are summarized in Table 8.

[0422] [Table 8]

[0423] Thermal stability of the c-Met-binding FN3 domain The stability of each FN3 domain was assessed using differential scanning calorimetry in PBS. The results are shown in Table 9.

[0424] [Table 9]

[0425] Example 7. Generation and characterization of bispecific anti-EGFR / c-Met molecules Generation of bispecific anti-EGFR / c-Met molecules The EGFR-binding FN3 domain and the c-Met-binding FN3 domain described in Examples 1 to 6 Multiple combinations of bispecific antibodies capable of binding both EGFR and c-Met Furthermore, E having the amino acid sequences shown in SEQ ID NOs: 107 to 110 was linked as a molecule. having a GFR-binding FN3 domain and the amino acid sequences shown in SEQ ID NOs: 111 to 114 c-Met-binding FN3 domains were prepared and linked as bispecific molecules. mat, i.e., an EGFR-binding FN3 domain followed by a peptide linker, Subsequently, the c-Met binding FN3 domain was maintained by using SEQ ID NOs: 50 to 72 and 1. A synthetic gene encoding the amino acid sequence set forth in Table 10 was prepared. In addition to the molecules shown in Table 10, The linker between the two FN3 domains was selected according to the length, sequence, and other parameters shown in Table 11. Many other linkers were used to link these FN3 domains, depending on the composition and structure of the sequence. It is envisioned that this method can be used to link the two. IMAC and Gel Filtration Chromatography Using a fusion process, monospecific EGFR or c-Met FN3 domains were identified as described. The bispecific EGFR / c-Met molecule was expressed and purified from E. coli.

[0426] [Table 10]

[0427] [Table 11]

[0428] The bispecific EGFR / c-Met molecule has enhanced efficacy and avidity compared to the monomolecule alone. It suggests ditty.

[0429] NCI-H292 cells were cultured in 96-well plates in RPMI medium containing 10% FBS. After 24 hours, the medium was replaced with serum-free RPMI. , cells were treated with different concentrations of FN3 domains, i.e., high affinity monospecific EGFR. FR FN3 domain (P54AR4-83v2), weak affinity monospecific c-Met FN3 domain (P114AR5P74-A5), two monospecific EGFR FN3 a mixture of the c-Met FN3 domain and the high-affinity EGFR FN3 domain Bispecific EGFR / c receptors consisting of in-linked low-affinity c-MetvFN3 domains The cells were incubated for 1 hour with either monospecific or bispecific α-Met molecules (ECB1). After treatment with the molecule, EGF, HGF, or a combination of EGF and HGF were used for 15 minutes, followed by 3 The cells were stimulated at 7°C and 5% CO2. The cells were lysed in MSD Lysis buffer and analyzed as described above. As previously described, cell signaling was performed using the appropriate MSD assay plate according to the manufacturer's instructions. Null transmission was assessed.

[0430] The low-affinity c-Met FN3 domain is IC 50 = 610 nM c-Met phosphate As expected, the EGFR FN3 domain inhibited the activation of c-Met. The mixture of monospecific molecules was unable to inhibit phosphorylation of the c-Met FN3 domain. However, the bispecific EGFR / c-Met molecule I C 50 = 1 nM inhibited c-Met phosphorylation (Fig. 4), and The efficacy was increased by more than 2 logs compared to the control group.

[0431] Dual-specificity enhances inhibition of c-Met and / or EGFR phosphorylation through avidity effects The density and ratio of c-Met and EGFR for the ability of isomeric EGFR / c-Met molecules The assay was performed in multiple cell types, varying the concentration of NCI-H292, NCI-H4, and NCI-H6 (Figure 5). 41 or NCI-H596 cells were cultured in RPMI medium containing 10% FBS in a 96-well plate. After 24 hours, the medium was replaced with serum-free RPMI. After incubation, cells were treated with different concentrations of the monospecific EGFR-binding FN3 domain, the monospecific c-Me t-binding FN3 domain, or bispecific EGFR / c-Met molecules (P53A1R5-1 The cells were treated with either ECB5 (consisting of 7v2 and P114AR7P94-A3). were treated with monospecific or bispecific molecules for 1 hour, followed by EGF, HGF, or EGF and The cells were stimulated with HGF in combination for 15 minutes at 37°C and 5% CO2. Lyse in lysis buffer and incubate in the appropriate MSD assay according to the manufacturer's instructions, as described above. Assay plates were used to assess cell signaling.

[0432] Figure 5 (A-C) shows the bispecific EGFR / c-Met fragment in three different cell lines. Inhibition of EGFR using monospecific EGFR-binding FN3 domains compared to To assess avidity in the EGFR phosphorylation assay, we used a medium affinity E The GFR-binding FN3 domain (1.9 nM) (P53A1R5-17v2) was A duplex containing the same EGFR-binding FN3 domain linked to a c-Met-binding FN3 domain Comparison with specific EGFR / c-Met molecule (0.4nM) (P114AR7P94-A3) In NCI-H292 and H596 cells, the inhibition of EGFR phosphorylation was observed with monospecific The results were comparable for the bispecific and bispecific molecules (Figures 5A and 5B), which is consistent with the results for these cell lines. This is thought to be due to the high ratio of EGFR receptors to c-Met receptors in this model. To verify this, we used NCI-H441 cells, which express more c-Met receptors than EGFR. Inhibition of EGFR phosphorylation was evaluated in the bispecific EGFR / c-Met molecule. When NCI-H441 cells were treated with α-glucan, the IC of EGFR phosphorylation was 50 is a single special This was 30-fold reduced compared to the isomeric EGFR-binding FN3 domain (Fig. 5C).

[0433] Potential for enhanced efficacy with bispecific EGFR / c-Met molecules: EGF It has high affinity for R (0.26 nM) and moderate affinity for c-Met. The results were evaluated in a c-Met phosphorylation assay using a molecule with a specific activity (10.1 nM). In both NCI-H292 and NCI-H596 cells, inhibition of c-Met phosphorylation was The bispecific molecule resulted in 13% more c-Met-binding FN3 domains than the monospecific c-Met-binding FN3 domains, respectively. 4-fold and 1012-fold higher (Figs. 3D and 3E).

[0434] Inhibition of EGFR and c-Met phosphorylation by bispecific EGFR / c-Met molecules It was determined that the increased potency was linked to increased inhibition of signal transduction and proliferation. In their experiments, a mixture of the FN3 EGFR-binding domain and the c-Met-binding FN3 domain was The compounds were compared to the bispecific EGFR / c-Met molecule. As shown in Tables 12 and 13 The IC50 values ​​for ERK phosphorylation (Table 12) and proliferation of NCI-H292 cells (Table 13) were also calculated. 50 Values ​​are based on the monospecific binding activity when cells were treated with the bispecific EGFR / c-Met molecule. The bispecific EGFR / c-Met molecule IC for inhibition of ERK phosphorylation 50 Two monospecific EGFR-binding FN3 domains The FN3 domain was 143-fold lower than the mixture of FN3 and the c-Met-binding domain. Table 1 shows the effect of avidity on the potency of molecules in this assay. 12, a monospecific EGFR-binding FN3 domain and a c-Met-binding FN3 domain does not completely inhibit activity, and therefore the IC 50 The value is considered a lower limit This proliferation assay combines different combinations of EGFR-binding FN3 domains with c -Met-binding FN3 domains linked as a mixture or in a bispecific format The bispecific EGFR / c-Met molecule inhibits proliferation. I C 50 The monospecific parent EGFR-binding FN3 domain or c-Met-binding FN3 domain This is consistent with the anti-inflammatory effect seen at the receptor level. The vidity effect (Figures 4 and 5) was observed in cell signaling (Table 12) and cell proliferation (Table 13) This confirmed that this leads to improved inhibition of .

[0435] [Table 12]

[0436] [Table 13]

[0437] In vivo tumor xenografts: PK / PD To investigate the efficacy of monospecific and bispecific FN3 domain molecules in vivo, Tumor cells were engineered to secrete mouse HGF (mouse HGF binds to human c-Met) Lentiviral infection (human HGF-expressing lentiviral DNA vector (A) Accession number X16322) and lentiviral package inserts from Genecopoeia Human HGF was stably expressed in NCI-H292 cells using a caging kit. After infection, the HGF-expressing cells were incubated with 4 μg / mL puromycin (Invitrogen). Human HGF protein was selected using an algorithm from MesoScale Discovery. The antibodies were detected in the conditioned medium of pooled cells using assay plates.

[0438] NCI-H292 cells (Cultrex, Trevigen) expressing human HGF ) Medium, 2.0×10 6 The cells (volume: 200 μL) were placed on the dorsal flank of each SCID Beige mouse. The tumor was injected subcutaneously into the abdomen. 3 Measure tumors twice weekly until tumors reach the range Mice were then injected with a bispecific EGFR / c-Met molecule (injected with EGFR-C1000 for increased half-life). The mice were given a single intraperitoneal administration of either the IgG1-linked antibody (linked to an albumin-binding domain) or PBS as a vehicle. After 3 or 72 hours, tumors were excised and immediately frozen in liquid nitrogen. Blood samples were collected in 8% citrate containing protease inhibitors. The tumor was centrifuged and the resulting plasma was transferred to a sample tube and stored at -80°C. , cut into small pieces, and soak in RIPA buffer and HALT protease / phosphatase inhibitors ( Pierce), 50 mM sodium fluoride (Sigma), 2 mM activated orthovanadate sodium phosphate (Sigma), and 1 mM PMSF (Meso Scale Di recovery) and in a Lysing Matrix A tube (LMA) containing Remove the lysate from the LMA matrix and centrifuge to remove insoluble proteins. Soluble tumor proteins were quantified by BCA protein assay, and tumor lysis buffer was determined. The proteins were diluted to the same protein concentration in the buffer. FR and ERK were measured using assay plates from MesoScale Discovery. (according to the manufacturer's protocol and as described above).

[0439] The experimental results are shown in Figure 6. Each bispecific EGFR / c-Met molecule exhibited phosphorylated Significantly increased the levels of c-Met, EGFR, and ERK at both 6 and 72 hours. The data shown in Figure 6 demonstrate that simultaneous inhibition of both c-Met and EGFR The importance of this and the affinity of the bispecific EGFR / c-Met molecule for each receptor This indicates that EGF plays a role in the downstream inhibition of ERK. R-binding FN3 domain (P54AR4-83v2, shown as "8" in the figure, K D =0 The molecule containing the intermediate affinity EGFR-binding FN3 domain (shown as "1.26 nM") P53A1R5-17v2, denoted as "7", K D = 1.9 nM) compared to a molecule containing and significantly inhibited EGFR phosphorylation at both 6 and 72 hours. All four bispecific molecules tested showed no significant effect on ERK phosphorylation at 6 hours, regardless of affinity. At 72 hours, the high-affinity c-Met-binding FN3 domain was In (P114AR7P94-A3, shown as "A3" in the figure, K D =0.4nM) The molecule containing the intermediate-affinity c-Met-binding FN3 domain (shown as "A5" in the figure) Shown is P114AR5P74-A5, K D = 10.1 nM, Figure 6) compared with ERK phosphorylation was significantly inhibited.

[0440] The concentration of each bispecific EGFR / c-Met molecule was measured in the blood and tumor 6 hours after administration. Interestingly, the intermediate affinity EGFR binding Conjugation domain (P53A1R5-17v2, K D = 1.9 nM) and high affinity c-Met binding The FN3 domain (P114AR7P94-A3, K D =0.4nM) The active molecule showed significantly more accumulation in the tumor after 6 hours than the other molecules. The difference diminished by 72 hours. Cells outside the tumor expressed both EGFR and c-Met. Due to their low surface expression levels, intermediate affinity EGFR molecules are more sensitive to the activity of higher affinity EGFRs. We hypothesize that it binds less strongly to normal tissues than the FR-binding FN3 domain. Therefore, in the tumor, there are many free intermediate affinity molecules available for binding. There are multiple EGFR-binding FN3 domains, so appropriate affinities for each receptor are available. By identifying the characteristics of the drug, it is possible to identify therapeutic drugs with low systemic toxicity and high tumor accumulation. It may be possible to do so.

[0441] Tumor efficacy trial with bispecific EGFR / c-Met molecules NCI-H292 cells (Cultrex, Trevigen) expressing human HGF ) Medium, 2.0×10 6 200 μL of cells were injected into the dorsal flank of each SCID Beige mouse. One week after implantation, mice were divided into groups with comparable tumor volumes (mean tumor volume). Tumor volume=77.9+ / -1.7mm 3 Mice were injected with the bispecific molecule three times a week to induce tumor growth. Tumor volumes were recorded twice weekly. Tumor growth inhibition (TGI) was measured with four different bispecific molecules. These showed different affinities for c-Met and EGFR. If the binding FN3 domain is intermediate affinity, then the high affinity EGFR-binding FN3 domain is In mice treated with a molecule containing the EGFR-binding FN3 domain, the EGFR-binding activity was significantly higher than that of the intermediate-affinity FN3 domain. The effect of inhibiting both c-Met and EGFR on tumor growth was observed. Shown (open triangle and filled triangle; P54AR4-83v2-P114 AR5P74-A5 vs. P53A1R5-17-P114AR5P74-A5). Furthermore, this data does not support the use of either high- or intermediate-affinity EGFR-binding FN3 domains. The importance of having a high-affinity c-Met-binding FN3 domain as a bispecific molecule containing Although the high-affinity c-Met binding FN3 domain showed the highest efficacy (Dashed gray and black lines; P54AR4-83v2-P114AR7P94-A3 and P 53A1R5-17v2-P114AR7P94-A3).

[0442] Efficacy of bispecific molecules and other EGFR and c-Met inhibitors Bispecific EGFR / c-Met molecule (ECB38) and each as a single agent , the small molecule inhibitors crizotinib (c-Met inhibitor) and erlotinib (EGFR inhibitor) anti-EGFR antibody), cetuximab (anti-EGFR antibody), and the combination of crizotinib and erlotinib The in vivo therapeutic efficacy of the combination was evaluated by subcutaneous administration of H29 in SCID / Beige mice. 2-HGF was evaluated in the treatment of a human lung cancer xenograft model.

[0443] H292-HGF cells were cultured in a medium supplemented with fetal bovine serum (10% v / v) and L-glutamine (2 m The cells were cultured in RPMI 1640 medium supplemented with HCl at 37°C in an atmosphere of 5% CO2 in air. Cells were maintained in vitro and routinely subcultured twice a week by trypsin-EDTA treatment. Exponentially growing cells were harvested and counted for tumor inoculation.

[0444] The right flank region of each mouse was treated with 0.1 ml of PBS supplemented with Cultrex (1:1). H292-HGF tumor cells (2 × 10 6 ) was subcutaneously inoculated to induce tumors. Size is 139mm 3 Treatment was initiated when the test substance administration and animal The numbers are shown in the experimental design table below. The day of tumor cell inoculation is shown as day 0. Table 14 shows the treatment Show the group.

[0445] [Table 14] N = number of animals, po = oral administration, ip = intraperitoneal injection 3 times / week, weeks 1, 3, and It was administered on the fifth day. QD = once daily, Q4d = once every four days, between crizotinib and erlotinib combination The interval was 0.5 hours, the administration volume was adjusted based on body weight (10 l / g), a = 1 after grouping No dosing was administered on the fourth day.

[0446] Prior to the start of treatment, all animals were weighed and tumor volumes were measured. Mice were randomized based on tumor volume, as this may affect the efficacy of any treatment administered. A block design was used to allocate patients to each group, allowing all groups to be compared at baseline. A randomized block design was used to allocate the experimental animals to each group. First, the experimental animals were divided into uniform blocks based on the initial tumor volume. Randomization of experimental animals to treatments was performed within ...

Claims

1. Isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor (c- Met) antibody, a) comprising HC1 constant domain 3 (HC1 CH3) and HC1 variable domain 1 (VH1); a first heavy chain (HC1); and b) HC2 constant domain 3 (HC2 CH3) and HC2 variable domain 2 (VH2) a second heavy chain (HC2); and c) a first light chain (LC1) comprising a light chain variable region (VL1); d) a second light chain (LC2) comprising a light chain variable region (VL2); and Including, a first antigen-binding site in which the VH1 and the VL1 are paired to specifically bind to EGFR; and a second antibody that specifically binds to c-Met by pairing the VH2 and the VL2. forming a primary binding site, said HC1 comprising at least one substitution in said HC1 CH3; said HC2 contains at least one substitution in said HC2 CH3, and and the substitutions in HC2 CH3 are Bispecific antibodies, which are based on the above, are generated at different amino acid residue positions.

2. The antibody inhibits the activity of NCI-H292, NCI-H1975, or SKMES-1 cell lines. inhibits the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 / 2) and its IC 50 Values ​​are compared with a control monovalent EGFR antibody containing heavy chain 3 (HC3) and light chain 3 (LC3). Mixed with a control monovalent c-Met antibody containing heavy chain 4 (HC4) and light chain 4 (LC4) ER in NCI-H292, NCI-H1975 or SKMES-1 cell lines by IC of K1 / 2 phosphorylation inhibition 50 At least about 10 times lower, or at least at least about 20 times lower, at least about 30 times lower, at least about 40 times lower, at least about 5 times lower 0-fold lower, or at least about 60-fold lower IC 50 is the value, The HC3 and the HC1, the LC3 and the LC1, the HC4 and the HC2, and the LC4 and the LC2 each have the same amino acid sequence; The phosphorylation of ERK1 / 2 was detected by electrophoresis using anti-phosphorylated ERK1 / 2 antibody as a capture antibody. A detection antibody that binds to non-phosphorylated and phosphorylated ERK1 / 2 conjugated with a chemiluminescent compound is used. Measured in whole cell lysates using a sandwich immunoassay The bispecific antibody of claim 1.

3. The antibody inhibits ERK1 / 2 phosphorylation by approximately 2×10 -9 M or less, approximately 1×10 -9 M and below, or about 1 x 10 -10 IC below M 50 The bispecific antibody of claim 1 or 2, body.

4. ERK1 is phosphorylated at residues Thr202 and Tyr204, and ERK2 is phosphorylated at residues The double antibody of claim 2 or 3, which is phosphorylated at Thr185 and Tyr197. Heterogeneous antibodies.

5. The antibody inhibits the activity of protein kinase B (AKT) in the NCI-H1975 cell line. Inhibits phosphorylation at er473 and its IC 50 The value includes the HC3 and the LC3. A control monovalent EGFR antibody containing the HC4 and the LC4. Ser473 of AKT in NCI-H1975 cell line by a mixture of AKT and Met antibodies IC for inhibition of phosphorylation in 50 At least about 70-fold lower IC 50 value and The HC3 and the HC1, the LC3 and the LC1, the HC4 and the HC2, and the LC4 and the LC2 each have the same amino acid sequence; Phosphorylation of AKT at Ser473 binds to unphosphorylated or phosphorylated AKT The antibody was used as a capture antibody, and anti-phosphorylated AKT Ser473 conjugated with an electrochemiluminescent compound was used. in whole cell lysates using a sandwich immunoassay with antibodies as detection antibodies. The bispecific antibody of any one of claims 1 to 4, wherein the antibody is measured by

6. The antibody inhibits phosphorylation of AKT at Thr308 in the NCI-H1975 cell line. inhibits the ionization and its IC 50 Values ​​are for the control monovalent EGF containing the HC3 and the LC3. A mixture of R antibody and a control monovalent c-Met antibody containing the HC4 and LC4 Inhibition of AKT phosphorylation at Thr308 in NCI-H1975 cell line by I C 50 At least about 100-fold lower IC 50 is the value, The HC3 and the HC1, the LC3 and the LC1, the HC4 and the HC2, and the LC4 and the LC2 each have the same amino acid sequence; The phosphorylation of AKT at Thr308 binds to non-phosphorylated or phosphorylated AKT. The capture antibody was an antibody against phosphorylated AKT Thr3 conjugated with an electrochemiluminescent compound. Whole cell lysates were analyzed using a sandwich immunoassay with antibody 08 as the detection antibody. The bispecific antibody of any one of claims 1 to 5, wherein the antibody is measured in a sera.

7. The antibody inhibits phosphorylation of AKT at Ser473 or Thr308 by about 1×10 -9 IC below M 50 The bispecific antibody of claim 5 or 6, which inhibits at a level

8. The HC1, the LC1, the HC2, and the LC2 are sequences selected from SEQ ID NOs: 199 and 200, respectively. HC1, HC2, or HC3 comprises the amino acid sequence of 00, 201, or 202, Optionally, the C-terminal lysine may be removed from both HC1 and HC2.

8. The bispecific antibody according to any one of claims 7 to 7.

9. The bispecific antibody is an EGFR-EGFR antibody having the amino acid sequence shown in SEQ ID NO:

73. It binds to c-Met at FR residues K489, I491, K467, ​​and S492. PEFRDSYPIKYVHAF (SEQ ID NO: 238) and FAQSKPDSAEPMDR 9. The double antibody of claim 1, wherein the double antibody binds to the nucleotide sequence of ... Heterogeneous antibodies.

10. The antibody inhibits the proliferation of NCI-H292 or NCI-H1975 cells and its IC 50 Values ​​are for NCI-H292 or NCI-H1975 cells grown under low-adhesion conditions. IC of growth inhibition of NCI-H292 or NCI-H1975 cells by cetuximab 50 value at least about 300 times lower, at least about 400 times lower, at least about 500 times lower, at least about 600 times lower, at least about 700 times lower, at least about 800 times lower, at least about 900 times lower, at least about 1000 times lower, at least about 1200 times lower, at least about 1400 times lower, at least about 1600 times lower, at least about 1800 times lower, at least about 500 times lower, at least about 600 times lower, at least about 700 times lower, or at least Approximately 800 times lower IC 50 The bispecific antibody according to any one of claims 1 to 9, 。

11. The antibody was administered at a dose of 20 mg / kg of the bispecific antibody and cetuximab. Growth of HGF-expressing SKMES-1 cell tumors in SCID Beige mice with at least 500-fold lower T / C values ​​(%) at day 36 compared to cetuximab The bispecific antibody according to any one of claims 1 to 10.

12. 12. The method of claim 1, wherein the antibody neutralizes EGFR and c-Met signaling. The bispecific antibody according to any one of claims 1 to 4.

13. The HC1 and the HC2 are IgG1, IgG2, IgG3, or IgG4 isotypes. The bispecific antibody of any one of claims 1 to 12.

14. The dual antibody of claim 13, wherein the HC1 and the HC2 are of the IgG1 isotype. Specific antibodies.

15. If the residue numbering is based on the EU index, At residues 68, 370, 399, 405, 407, or 409, the HC1 CH3 at least 1, 2, 3, 4, 5, 6, 7, or 8 substitutions; C2 CH3 has at least 1, 2, 3, 4, 5, 6, 7, or 8 substitutions 15. The bispecific antibody of claim 13 or 14, comprising:

16. At least one of the HC1 CH3 is present at residues 350, 370, 405, or 409. , 2, 3, or 4 substitutions, and said HC2 CH3 has at least 1, 2, 3, or 4 substitutions.

16. The bispecific antibody of claim 15, comprising one or four substitutions.

17. At residues 405 or 409, said HC1 CH3 contains at least one substitution, 17. The bispecific antibody of claim 16, wherein the HC2 CH3 comprises at least one substitution.

18. The HC1 CH3 contains a substitution of K409R or F405L, and the HC2 CH3 18. The bispecific antibody of claim 17, wherein said antibody comprises the substitution K409R or F405L.

19. The HC1 CH3 contains the F405L substitution and the HC2 CH3 contains the K40 19. The bispecific antibody of claim 18, comprising a 9R substitution.

20. a) said VH1 comprises heavy chain complementarity determining regions of SEQ ID NOs: 210, 211 and 212, respectively; comprising the amino acid sequences of (HCDR)1 (HCDR1), HCDR2 and HCDR3, b) said VL1 comprises the light chain complementarity determining regions of SEQ ID NOs: 213, 214 and 215, respectively; (LCDR)1 (LCDR1), LCDR2 and LCDR3 amino acid sequences. Item 20. The bispecific antibody according to any one of Items 1 to 19.

21. a) the VH2 comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 216, 217 and 218, respectively; comprising the amino acid sequences of HCDR2 and HCDR3, b) the VL2 comprises an LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 219, 220 and 221, respectively; 21. The bispecific antibody of claim 20, comprising the amino acid sequences of LCDR2 and LCDR3.

22. The VH1, the VL1, the VH2 and the VL2 are sequences of SEQ ID NOs: 189 and 190, respectively.

22. The bispecific antibody of claim 21, comprising the amino acid sequences 90, 193 and 194.

23. a) the VH1 comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 222, 223 and 224, respectively; comprising the amino acid sequences of HCDR2 and HCDR3, b) the VL1 is selected from the group consisting of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 225, 226, and 227, respectively; Any of claims 1 to 7 or claims 9 to 18, comprising the amino acid sequences of LCDR2 and LCDR3. The bispecific antibody of claim 1.

24. a) the VH2 comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 228, 229 and 230, respectively; comprising the amino acid sequences of HCDR2 and HCDR3, b) the VL2 comprises LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 231, 232 and 233, respectively; 24. The bispecific antibody of claim 23, comprising the amino acid sequences of LCDR2 and LCDR3.

25. The VH1, the VL1, the VH2 and the VL2 are sequences of SEQ ID NOs: 191 and 192, respectively.

25. The bispecific antibody of claim 24, comprising the amino acid sequences of 92, 195 and 196.

26. The HC1, the LC1, the HC2, and the LC2 are sequences selected from SEQ ID NOs: 234 and 235, respectively. the HC1, the HC2, or the HC, comprising the amino acid sequences of 35, 236, and 237; 25. The C-terminal lysine may optionally be removed from both HC1 and HC2. The bispecific antibody described in .

27. 1, 2, 3, 4, 5, 6, 7 to the HC1, the LC1, the HC2 and the LC2 , 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions; 27. A bispecific antibody according to claim 22 or 26.

28. said HC1 and / or said HC2 comprising substitutions M252Y / S254T / T256E; 28. The bispecific antibody of claim 27, wherein the residue numbering is according to the EU index. antibody.

29. The antibody has a biantennary glycan structure with a fucose content of about 1% to about 15%. Item 30. The bispecific antibody according to any one of Items 1 to 29.

30. The HC1, the LC1, the HC2 and the LC2 are sequences selected from SEQ ID NOs: 205 and 206, respectively. 206, 207 and 208. The bispecific antibody according to claim 2.

31. The HC1, the LC1, the HC2 and the LC2 according to claim 8 or 26 are coated. An isolated synthetic polynucleotide that encodes a nucleotide sequence ...

32. 3. The polynucleotide sequence of claim 3, comprising SEQ ID NO: 205, 206, 207 or 208.

2. The isolated polynucleotide according to claim 1.

33. A vector comprising the polynucleotide of claim 31 or 32.

34. 34. An isolated host cell comprising the vector of claim 33.

35. 9. A method for producing the isolated bispecific EGFR / c-Met antibody of claim 8. So, a) an isolated antibody comprising two heavy chains of SEQ ID NO: 199 and two light chains of SEQ ID NO: 200; A bispecific bivalent anti-EGFR antibody and two heavy chains of SEQ ID NO: 201 and two heavy chains of SEQ ID NO:

202. and an isolated monospecific bivalent anti-c-Met antibody comprising a light chain of Adding things together, b) introducing a reducing agent into the mixture; c) incubating the mixture for about 90 minutes to about 6 hours; d) removing the reducing agent; e) a first heavy chain of SEQ ID NO: 199 and a second heavy chain of SEQ ID NO: 201, and a 00 and a second light chain of SEQ ID NO:

202. purifying the antibody; Including, The first heavy chain of SEQ ID NO: 199 is paired with the first light chain of SEQ ID NO: 200 to bind EGF R, and said second heavy chain of SEQ ID NO: 201 forms a first binding domain that specifically binds to R a second binding domain that pairs with the second light chain of SEQ ID NO: 202 and specifically binds to c-Met; Form the main, method.

36. 36. The method of claim 35, wherein the reducing agent is 2-mercaptoethanolamine (2-MEA). The method described.

37. 37. The method of claim 36, wherein the 2-MEA is present at a concentration of about 25 mM to about 75 mM. Law.

38. 38. The method of claim 37, wherein the incubating step is carried out at a temperature of about 25°C to about 37°C. The method described.

39. A composition comprising the bispecific antibody of any one of claims 1 to 30 and a pharmaceutically acceptable carrier. Mmm, pharmaceutical composition.

40. 27. A method of treating a subject with cancer, comprising administering to a subject a bispecific E antibody of claim 2, 8 or 26. A therapeutically effective amount of the GFR / c-Met antibody is administered to a patient in need thereof, the amount being sufficient to treat cancer. The method comprises administering the compound over a period of time.

41. The cancer is characterized by an activating EGFR mutation, an amplification of the EGFR gene, or an increased level of circulating HGF. , associated with c-Met activating mutations, c-Met gene amplification, or mutant KRAS 41. The method of claim 40.

42. The EGFR activating mutation is G719A, G719X (X is any amino acid), L86 1X (X is any amino acid), L858R, E746K, L747S, E749Q, A7 Replaces 50P, A755V, V765M, L858P or T790M, E746 to A75 0 deletion, deletion of R748 to P753, insertion of Ala (A) between M766 and A767 , an insertion of Ser, Val, and Ala (SVA) between S768 and V769, and P7 42. The method of claim 41, wherein the insertion of Asn and Ser (NS) between H72 and H773. Law.

43. The EGFR activating mutation is L858R, del (E476, A750) and / or T 43. The method of claim 42, wherein the substitution is 790M.

44. 42. The method of claim 41, wherein the mutant KRAS has a G12V or G12C substitution. Law.

45. 45. The method of claim 44, wherein the mutant KRAS has a G12V substitution.

46. The subject is a patient receiving erlotinib, gefitinib, afatinib, CO-1686, AZD9 192 or cetuximab treatment or have acquired resistance.

42. The method of claim 41.

47. The cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, Small cell lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal Cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC)), or sporadic or inherited 42. The method of claim 41, wherein the cancer is propagating papillary renal cell carcinoma (PRCC).

48. the subject is homozygous for phenylalanine at position 158 of CD16, or 16 is heterologous for valine and phenylalanine at position 158 of claim 40 How to do it.

49. 48. The method of claim 47, further comprising administering a second therapeutic agent.

50. 50. The method of claim 49, wherein the second therapeutic agent is a chemotherapeutic agent or a targeted anti-cancer therapeutic agent. 。

51. 51. The method of claim 50, wherein the chemotherapeutic agent is cisplatin or vinblastine. 。

52. The chemotherapeutic agent or the targeted anti-cancer therapeutic agent is a compound that targets EGFR, c-Met, HER2, HER3, 3. The method of claim 50, which is a tyrosine kinase inhibitor of HER4 or VEGFR. 。

53. The tyrosine kinase inhibitor is erlotinib, gefitinib, or afatinib.

53. The method of claim 52,

54. 50. The method of claim 49, wherein the second therapeutic agent is administered simultaneously, sequentially, or separately.

55. A method for inhibiting the growth or proliferation of cells expressing EGFR and / or c-Met. contacting the cells with the bispecific antibody of claim 2, 8 or 26. Law.

56. Methods for inhibiting the growth or metastasis of EGFR- and / or c-Met-expressing tumor or cancer cells in a subject 27. A method of administering to the subject an effective amount of the bispecific antibody of claim 2, 8 or 26. and inhibiting the growth or metastasis of EGFR and / or c-Met expressing tumor or cancer cells. A method comprising:

57. The EGFR and / or c-Met expressing tumor is selected from the group consisting of epithelial cell carcinoma, breast cancer, ovarian cancer, lung cancer, non-small cell carcinoma, and the like. Small cell lung cancer (NSCLC), lung adenocarcinoma, small cell lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer Cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer , cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC)), or sporadic or hereditary papillary renal cell carcinoma (PRCC).

6. The method according to claim 6.

58. The EGFR and / or c-Met expressing tumor is characterized by an EGFR activating mutation, an EGFR gene amplification of HGF, increased circulating HGF levels, c-Met activating mutations, and c-Met gene amplification 58. The method of claim 57, wherein the gene is associated with a mutant KRAS.

59. The EGFR activating mutation is G719A, G719X (X is any amino acid), L86 1X (X is any amino acid), L858R, E746K, L747S, E749Q, A7 Replaces 50P, A755V, V765M, L858P or T790M, E746 to A75 0 deletion, deletion of R748 to P753, insertion of Ala (A) between M766 and A767 , an insertion of Ser, Val, and Ala (SVA) between S768 and V769, and P7 58. The method of claim 58, wherein the insertion of Asn and Ser (NS) between H72 and H773. Law.

60. The EGFR activating mutation is L858R, del (E476, A750) and / or T 60. The method of claim 59, wherein the substitution is 790M.

61. 59. The method of claim 58, wherein the mutant KRAS has a G12V or G12C substitution. 。

62. 62. The method of claim 61, wherein the mutant KRAS has a G12V substitution.

63. Use of a bispecific antibody according to any one of claims 1 to 30 for therapy.

64. A bispecific antibody according to any one of claims 1 to 30 for use in the treatment of cancer. antibody.

65. The cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, Small cell lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal Cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC)), or sporadic or inherited 65. The method according to claim 64, wherein the treatment is a prognostic papillary renal cell carcinoma (PRCC).

10. The bispecific antibody of any one of claims 1 to 0.

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