TW201642904A - Composition comprising anti-FGFR2 antibody and other agent - Google Patents

Abstract

A combination comprising an antibody capable of binding to human fibroblast growth factor receptor 2 (hFGFR2) and an agent that is different from the antibody.

Description

Composition containing anti-FGFR2 antibody and other agents

The present invention relates to a novel antibody or a functional fragment thereof, or a combination thereof, and other active ingredients, a pharmaceutical composition containing the antibody or a functional fragment thereof or a modified form thereof, and other active ingredients, and the like.

In the treatment of cancer, it is expected to cure or prolong life by killing cancerous parts by surgery, using anticancer agents or radiation to kill cancer cells. However, for most cancers, since the treatment with only a single anticancer agent fails to achieve sufficient effects, it has been attempted to be treated by treatment with a wide variety of agents. For anticancer agents, there are chemotherapeutic agents such as doxorubicin, cisplatin, taxol, or camptothecin; or crizotinib or dasatinib. Molecular standard drugs such as (dasatinib), lapatinib, etc.; cancer therapeutic antibody such as bevacizumab, cetuximab, trastuzumab, etc.; An anticancer agent or a combination therapy thereof is determined depending on the type, degree of progress, and treatment state of a hormone such as anastrozole, exemestane, and tamoxifen. It has been previously used to directly block the proliferation message by acting on the receptor that controls cell proliferation and survival, acting on the extracellular domain, or displaying antitumor activity by ADCC. A cancer-treating antibody" and a "agent for the same receptor or a drug that inhibits a different receptor, or a chemotherapeutic agent, etc." enhances the anticancer effect.

The combination of Lapinib (Her2 TKI) and Herceptin (anti-Her2 antibody) for breast cancer treatment is considered to have a combined effect, suggesting that Her2 accumulates on the cell surface by the action of lapatinib. Thereby, the possibility of the effect of Her2 is enhanced (Non-Patent Document 1). Further, it has been clearly recognized that the dual inhibition of EGFR which inhibits the afatinib of the ErbB family and the anti-EGFR monoclonal antibody cetuximab also shows a strong anticancer effect (Non-Patent Document 2) ). Thus, the combination of "identified antibody" and "low-molecular agent for inhibiting tyrosine kinase activity" of receptor-type tyrosine kinase causes strong inhibition of proliferation, and may be associated with a useful therapeutic method. Sex. In addition, in the treatment of colorectal cancer, the combination of chemotherapeutic camptothecin and EGFR inhibitor cetuximab, or in combination with avastin has been established as a standard treatment for colorectal cancer. Treatment shows great advantages.

However, the agent which is enhanced by the anticancer action by the use together with the anti-FGFR2 antibody is almost unknown (Patent Documents 1 and 2).

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] US Patent Application Publication No. US 2014/0322220 A1

[Patent Document 2] US Patent Application Publication No. US 2015/0050273 A1

[Non-patent literature]

[Non-Patent Document 1] Scaltriti et al. (Scaltriti, M. et al.), Oncogene, February 2009, Vol. 28 (No. 6), 803-814, electronic publication on December 8, 2008

[Non-Patent Document 2] Regales et al. (Regales, L., et al.), Journal of Clinical Research (J. Clin. Invest.), October 2009, vol. 119 (No. 10), 3000 to 3010, 2009 Electronic disclosure on September 14th

One object of the present invention is to provide a combination of an anti-FGFR2 antibody and other pharmaceutical agents, and a pharmaceutical composition containing the antibody and other pharmaceutical agents.

Further, another object of the present invention is to provide an anti-FGFR2 antibody and other agents which are used in combination for the treatment or prevention of cancer, and a pharmaceutical composition containing the antibody and other agents for use in the treatment or prevention of cancer.

Further, another object of the present invention is to provide a method of treating or preventing cancer by administering an anti-FGFR2 antibody in combination with other agents or by administering the composition.

The inventors of the present invention have reviewed and found various anticancer agents (for example, chemotherapeutic agents such as doxorubicin, cisplatin, paclitaxel, paclitaxel, fluorouracil, or camptothecin; Or the molecular standard drugs such as crizotinib, dasatinib, lapatinib, etc.; bevacizumab, cetuximab, trastuzumab, ramucirumab, etc. Antibody; anastrozole, exemestane, tamoxifen, etc., and anti-FGFR2 with anticancer activity The antibody is used in combination to obtain an excellent antitumor effect, and the present invention has been completed.

The present invention relates to: (1) a combination or pharmaceutical composition which is "a monoclonal antibody or a functional fragment thereof having the following characteristics (i) to (ix)", and "one or more choices" a combination of a free topoisomerase inhibitor, a microtubule inhibitor, a platinum preparation, an anti-VEGFR2 antibody, and a 5-fluorouracil anticancer agent; the pharmaceutical composition contains the antibody Or a functional fragment thereof, and one or more other pharmaceutical agents selected from the group consisting of topoisomerase inhibitors, microtubule inhibitors, platinum preparations, anti-VEGFR2 antibodies, and 5-fluorouracil anticancer agents Composition: (i) specifically binds to human type 2 fibroblast growth factor receptor (hFGFR2) IIIb and IIIc; (ii) immunoglobulin-like domain 2 or immunoglobulin such as hFGFR2 Protein domain 3 binds; (iii) does not interact with human type 1 fibroblast growth factor receptor (hFGFR1), human type 3 fibroblast growth factor receptor (hFGFR3), and human type 4 fibroblast growth factor receptor ( hFGFR4) specifically binds; (iv) has antibody dependence Cytotoxicity (antibody dependent cellular cytotoxicity); (v) an antibody-dependent cell-mediated phagocytosis (antibody dependent cell phagocytosis) activity; (VI) having hFGFR2 neutralizing activity; (vii) having in vivo antitumor activity; (viii) inhibiting binding of FGF to hFGFR2; (ix) being a chimeric antibody, a humanized antibody or a human antibody; (2) a combination or pharmaceutical composition as described in (1), Wherein the antibody comprises the following light chain and heavy chain: comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 1 (Fig. 1) of the sequence listing, and having the sequence identification number 2 of the sequence listing (Fig. 2 a light chain of CDRL2 consisting of the amino acid sequence shown and an amino acid sequence represented by SEQ ID NO: 3 (Fig. 3) of the sequence listing; and a sequence identifier of the sequence listing CDRH1 consisting of the amino acid sequence shown in Figure 4 (Fig. 4), CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 5 (Fig. 5) of the Sequence Listing, and the sequence identifier of the sequence listing. An amino acid sequence represented by 6 (Fig. 6) or an amino acid in which the first or second amino acid is substituted with another amino acid from the amine terminal of the amino acid sequence a heavy chain of CDRH3 consisting of the sequence; (3) a combination or pharmaceutical composition according to (1) or (2), wherein the anti-system is selected from the following (i) to (vi): (i) comprising the following light And heavy chain humanized antibody (hFR2-14_H19/L1): a light chain comprising amino acid numbers 21 to 235 of the amino acid sequence shown in SEQ ID NO: 8 (Fig. 7), and comprising the sequence identification number 18 (Fig. 12) the heavy chain of amino acid numbers 20 to 467 of the amino acid sequence shown; (ii) the humanized antibody (hFR2-14_H12/L1) comprising the following light and heavy chains: comprising the sequence identification number The light chain of amino acid numbers 21 to 235 of the amino acid sequence shown in Fig. 8 (Fig. 7), and the amino acid number 20 containing the amino acid sequence shown in SEQ ID NO: 16 (Fig. 11) Heavy chain of 467; (i ii) humanized antibody comprising the following light and heavy chains (hFR2-14_H8) /L1): a light chain comprising amino acid numbers 21 to 235 of the amino acid sequence shown in SEQ ID NO: 8 (Fig. 7), and an amino acid represented by SEQ ID NO: 12 (Fig. 9) a heavy chain of amino acid numbers 20 to 467 of the sequence; (iv) a humanized antibody (hFR2-14_H9/L1) comprising the following light and heavy chains: comprising an amino group represented by SEQ ID NO: 8 (Fig. 7) a light chain of amino acid numbers 21 to 235 of an acid sequence, and a heavy chain of amino acid numbers 20 to 467 comprising an amino acid sequence represented by SEQ ID NO: 20 (Fig. 16); (v) comprising the following light Chain and heavy chain humanized antibody (hFR2-14_H11/L1): a light chain comprising amino acid numbers 21 to 235 of the amino acid sequence shown in SEQ ID NO: 8 (Fig. 7), and a sequence identifier The heavy chain of amino acid numbers 20 to 467 of the amino acid sequence shown in Figure 14 (Fig. 10); (vi) the humanized antibody (hFR2-14_H5/L1) comprising the following light and heavy chains: including sequence recognition The light chain of amino acid numbers 21 to 235 of the amino acid sequence shown in No. 8 (Fig. 7), and the amino acid number 20 containing the amino acid sequence shown by SEQ ID NO: 10 (Fig. 8) Heavy chain to 467; (4) combination or medical record as described in (1) a drug composition, wherein the anti-system comprises a heavy chain and a light chain, and binds to human FGFR2: comprises the heavy and light chains of the antibody of any one of (3) to (vi) The amino acid sequence is each a heavy chain and a light chain of 95% or more of the same amino acid sequence; (5) The combination or pharmaceutical composition according to (1), wherein the antibody or a functional fragment thereof is as (2) Or the combination of the antigen on the antigen recognized by the antibody according to any one of (3) to (vi): (6) the combination or pharmaceutical composition according to (1), wherein the antibody or the functional fragment thereof The binding to hFGFR2 competes with the antibody described in any one of (2) to (vi) (2) or (3); (7) A combination or pharmaceutical composition comprising a combination of an antibody or a functional fragment thereof obtained by the method of the following steps (i) and (ii), and a pharmaceutical composition comprising the same An antibody or a functional fragment thereof and other agents: (i) a step of culturing the cells described in (a) or (b) below; (a) introducing a core inserted in any one of the following (a) to (c); A recombinant vector of the nucleotide or a recombinant cell of the nucleotide: (a) an amine comprising one or all of the heavy or light chain of the antibody of any one of (1) to (6) a nucleotide of a base sequence of the acid sequence; (b) an amino acid sequence containing one or all of the heavy or light chain of the antibody of any one of (1) to (6) a nucleotide consisting of a base sequence of the base sequence; (c) an amino acid sequence encoding one or all of the heavy or light chain of the antibody of any one of (1) to (6) (b) a cell which produces the antibody of any one of (1) to (6) or a functional fragment thereof; and (ii) obtained from the aforementioned step (i) Culture recovery as in (1) to (6) The combination of the antibody or the functional fragment thereof according to any one of (1) to (7), wherein the antibody or the pharmaceutical composition is at the amino terminus of the heavy or light chain of the antibody or (1) A combination or a pharmaceutical composition, which is a sugar chain modification of an antibody or a functional fragment thereof according to any one of (1) to (8) Body and " The pharmaceutical composition is a pharmaceutical composition comprising the modified body and another pharmaceutical agent; (10) The combination or pharmaceutical composition according to any one of (1) to (9), which is used in (11) The combination or pharmaceutical composition according to (10), wherein the cancer is hFGFR2 positive; (12) the combination or pharmaceutical composition according to any one of (1) to (11), Wherein the antibody or a functional fragment thereof is further conjugated with a compound; (13) a combination or pharmaceutical composition according to any one of (1) to (12), wherein the other agent is a topoisomerase inhibitor (14) A pharmaceutical composition for use in combination with a topoisomerase inhibitor, and comprising the antibody or the composition contained in the combination or composition according to any one of (1) to (8) or a functional fragment or a modification contained in the combination or composition as described in (9); (15) a pharmaceutical composition comprising a topoisomerase inhibitor, and by as with (1) to (8) The antibody or functional fragment thereof contained in the combination or composition described in any one of the combinations or the composition or composition described in (9) (16) A pharmaceutical composition comprising the composition or composition according to any one of (1) to (8), wherein the antibody, the functional fragment, or the modified body is used in combination; An antibody or a functional fragment thereof, or a modification contained in the combination or composition as described in (9), and which acts in combination with a topoisomerase inhibitor to increase the action of the topoisomerase inhibitor ; (17) A pharmaceutical composition comprising the antibody or the functional fragment thereof contained in the combination or composition according to any one of (1) to (8), or the combination or composition according to (9) (18) The pharmaceutical composition according to any one of (1) to (8), wherein the combination of the above-mentioned (1) to (8) or The antibody or the functional fragment thereof contained in the composition, or the modification contained in the combination or composition described in (9), and the topoisomerase inhibitor are each contained in each preparation as an active ingredient. (19) The pharmaceutical composition according to any one of (1) to (8), wherein the antibody or the functional fragment thereof is contained in the composition or composition according to any one of (1) to (8) Or the modified form and the topoisomerase inhibitor contained in the combination or composition according to (9) are contained in a single preparation as an active ingredient; (20) A method for treating cancer, which is characterized in that The antibody or functional fragment thereof contained in the combination or composition according to any one of (1) to (8), or the combination or composition as described in (9) (21) The pharmaceutical composition according to any one of (13) to (19), wherein the therapeutic composition according to any one of (13) to (19), wherein The topoisomerase inhibitor is selected from one or more of the group consisting of irinotecan, topotecan, and etoposide; (22) as (1) The combination or pharmaceutical composition according to any one of (12), wherein the other agent is a microtubule inhibitor; (23) a pharmaceutical composition for use in combination with a microtubule inhibitor, and containing (1) The combination or composition described in any one of (8) An antibody or a functional fragment thereof, or a modification contained in the combination or composition as described in (9); (24) a pharmaceutical composition comprising a microtubule inhibitor, and by (1) to 8) The antibody or a functional fragment thereof contained in the combination or composition described in any one of the combinations or the modification contained in the combination or composition described in (9), and the antibody, the functional fragment or the antibody (25) A pharmaceutical composition comprising the antibody or the functional fragment thereof contained in the combination or composition according to any one of (1) to (8) or as described in (9) a combination or a modification contained in the composition, and the effect of the microtubule inhibitor is increased by using in combination with a microtubule inhibitor; (26) a pharmaceutical composition characterized by (1) to (1) 8) The antibody or functional fragment thereof contained in the combination or composition described in any one of the above, or the combination or the composition contained in the combination or composition described in (9), and a microtubule inhibitor combination; The pharmaceutical composition according to any one of (1) to (8), wherein the composition or composition according to any one of (1) to (8) The antibody or a functional fragment thereof or the modification or the microtubule inhibitor contained in the combination or composition according to (9) is contained in each preparation as an active ingredient, and is administered at the same time or at different times. The pharmaceutical composition according to any one of (1) to (8), wherein the antibody or functional fragment thereof is contained in the composition or the composition described in (9), or (9) The modified body and the microtubule inhibitor contained in the composition or composition are contained as a active ingredient in a single preparation; (29) A method for treating cancer, which is characterized by being as in (1) to (8) An antibody or a functional fragment thereof contained in the combination or composition described in any one, or a combination or a composition contained in the combination or composition described in (9), and a microtubule inhibitor combination; (30) The pharmaceutical composition according to any one of (22), wherein the microtubule inhibitor is selected from the group consisting of paclitaxel, docetaxel, and vincristine ( One or more of the group consisting of vincristine), vinblastine, eribulin, vindesine, and vinorelbine; (31) as in (1) to (12) A combination or pharmaceutical composition according to any one of (12), wherein the other agent is a platinum preparation; (32) a pharmaceutical composition for use in combination with a platinum preparation, and containing as in (1) to (8) An antibody or a functional fragment thereof contained in the combination or composition described in any one, or a modified composition contained in the combination or composition according to (9); (33) a pharmaceutical composition containing a platinum preparation, and The antibody or the function thereof contained in the combination or composition described in any one of (1) to (8) The fragment or the combination contained in the composition or composition described in (9) is used in combination to increase the action of the antibody, the functional fragment or the modified substance; (34) a pharmaceutical composition containing (1) The antibody or functional fragment thereof contained in the combination or composition described in any one of (8), or the modification contained in the combination or composition according to (9), and used in combination with a platinum preparation Increasing the effect of the platinum preparation; (35) a pharmaceutical composition characterized by being as any of (1) to (8) The antibody or functional fragment thereof contained in the combination or composition described in the composition or the combination or composition contained in the combination or composition described in (9) and the platinum preparation are administered in combination; (36) as described in (35) The pharmaceutical composition, the antibody or the functional fragment thereof contained in the combination or composition according to any one of (1) to (8), or the modification contained in the combination or composition according to (9) And the platinum preparations are each contained in the respective preparations as an active ingredient, and are administered at the same time or at different times; (37) The pharmaceutical composition according to (35), wherein (1) to (8) An antibody or a functional fragment thereof contained in the combination or composition described in the above, or a modification or a platinum preparation contained in the combination or composition according to (9) is contained in a single preparation as an active ingredient; 38. A method for treating cancer, which comprises the antibody or the functional fragment thereof contained in the combination or composition according to any one of (1) to (8), or the combination or composition as described in (9) (39) The pharmaceutical composition described in any one of (31) to (37), wherein the modified product and the platinum preparation are combined and administered; Or the therapeutic method according to (38), wherein the platinum preparation is one or more selected from the group consisting of cisplatin, oxaliplatin, carboplatin and nedaplatin; (40) The combination or pharmaceutical composition according to any one of (1) to (12), wherein the other agent is an anti-VEGFR2 antibody; (41) a pharmaceutical composition for use in combination with an anti-VEGFR2 antibody, and containing The antibody or functional fragment thereof contained in the combination or composition according to any one of (1), or the combination or composition as described in (9) (a) a pharmaceutical composition comprising an anti-VEGFR2 antibody, and the antibody or the composition thereof contained in the combination or composition as described in any one of (1) to (8) or The functional fragment or the combination contained in the combination or composition described in (9) is used in combination to increase the action of the antibody, the functional fragment or the modified substance; (43) a pharmaceutical composition containing, for example, (1) The antibody or the functional fragment thereof contained in the combination or composition described in any one of (8), or the modification contained in the combination or composition according to (9), and used together with the anti-VEGFR2 antibody And (44) a pharmaceutical composition comprising the antibody or the functional fragment thereof contained in the combination or composition according to any one of (1) to (8). Or a combination of the modified form contained in the combination or composition described in (9) and the anti-VEGFR2 antibody; (45) The pharmaceutical composition according to (44), wherein (1) to (8) An antibody or a functional fragment thereof contained in the combination or composition described in any one of the combinations or compositions described in (9) The modified body and the anti-VEGFR2 anti-system are each contained in each preparation as an active ingredient, and administered at the same time or at different times; (46) The pharmaceutical composition as described in (44), wherein (1) to ( 8) The antibody or functional fragment thereof contained in the combination or composition described in any one of the above, or the modification or composition contained in the combination or composition according to (9), and the anti-VEGFR2 anti-system are contained in a single preparation (47) A method for treating cancer, which comprises the antibody or the machine contained in the combination or pharmaceutical composition according to any one of (1) to (8). A pharmaceutical composition according to any one of (40) to (46), or a combination of a modified fragment contained in the combination or composition described in (9) and an anti-VEGFR2 antibody; The therapeutic method according to (47), wherein the anti-VEGFR2 antibody is a remoruzumab; (49) the combination or pharmaceutical composition according to any one of (1) to (12), wherein the other agent is 5-fluorouracil An anti-cancer agent; (50) A pharmaceutical composition for use in combination with a 5-fluorouracil-based anticancer agent, or a composition or composition according to any one of (1) to (8) An antibody or a functional fragment thereof, or a modification contained in the combination or composition as described in (9); (51) a pharmaceutical composition comprising a 5-fluorouracil-based anticancer agent, and by (1) The antibody or the functional fragment thereof contained in the combination or composition described in any one of (8) or the modified composition contained in the combination or composition according to (9), and the antibody and the functional fragment are used in combination. Or the action of the modified body is increased; (52) A pharmaceutical composition comprising the composition or composition as described in any one of (1) to (8) a compound or a functional fragment thereof, or a modified composition contained in the combination or composition according to (9), and used in combination with a 5-fluorouracil-based anticancer agent to increase the action of the 5-fluorouracil-based anticancer agent; (53) A pharmaceutical composition comprising the antibody or the functional fragment thereof contained in the combination or composition according to any one of (1) to (8) or the combination or composition as described in (9) (54) The pharmaceutical composition according to (53), wherein (1) to (8) The antibody or a functional fragment thereof contained in the combination or composition described in the above, or the modified product contained in the combination or composition according to (9), and the 5-fluorouracil-based anticancer agent are each contained in each The pharmaceutical composition according to any one of (1) to (8), which is contained in the composition or composition according to any one of (1) to (8), which is contained in the composition or the composition as described in any one of (1) to (8). The antibody or the functional fragment thereof, or the modification contained in the combination or composition according to (9), and the 5-fluorouracil-based anticancer agent are contained in a single preparation as an active ingredient; (56) Treatment of a cancer The method of the antibody or the functional fragment thereof contained in the combination or composition according to any one of (1) to (8), or the modification or composition contained in the combination or composition according to (9) And a pharmaceutical composition according to any one of (49) to (55), wherein the 5-fluorouracil is a pharmaceutical composition according to any one of (5) to (55) Anticancer agents are fluorouracil, tegafur (tegafur), tegafur. Gimester (gimeracil). Oteracil potassium, tegafur. Uracil, capecitabine, carmofur, doxifluridine, and the like.

The treatment or prevention of various cancers is made possible by using the combination of the antibody and other agents provided by the present invention.

[Fig. 1] Amino acid sequence of CDR1 of humanized FR2-14 antibody light chain (hFR2-14_L1) (SEQ ID NO: 1 in the Sequence Listing)

[Fig. 2] Amino acid sequence of CDR2 of humanized FR2-14 antibody light chain (hFR2-14_L1) (SEQ ID NO: 2 in the Sequence Listing)

[Fig. 3] Amino acid sequence of CDR3 of humanized FR2-14 antibody light chain (hFR2-14_L1) (SEQ ID NO: 3 in the Sequence Listing)

[Fig. 4] Amino acid sequence of CDR1 of humanized FR2-14 antibody heavy chain (hFR2-14_H19) (SEQ ID NO: 4 in the Sequence Listing)

[Fig. 5] Amino acid sequence of CDR2 of humanized FR2-14 antibody heavy chain (hFR2-14_H19) (SEQ ID NO: 5 in the Sequence Listing)

[Fig. 6] Amino acid sequence of CDR3 of humanized FR2-14 antibody heavy chain (hFR2-14_H19) (SEQ ID NO: 6 in the Sequence Listing)

[Fig. 7] Amino acid sequence of hFR2-14_L1 (SEQ ID NO: 8 in the Sequence Listing). Among them, amino acid numbers 1 to 20 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_L1. The variable regions are amino acid numbers 21 to 130 and the constant regions are amino acid numbers 131 to 235.

[Fig. 8] The amino acid sequence of hFR2-14_H5 (SEQ ID NO: 10 in the Sequence Listing). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H5. The variable regions are amino acid numbers 20 to 137 and the constant regions are amino acid numbers 138 to 467.

[Fig. 9] Amino acid sequence of hFR2-14_H8 (SEQ ID NO: 12 in the Sequence Listing). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H8. The variable regions are amino acid numbers 20 to 137 and the constant regions are amino acid numbers 138 to 467.

[Fig. 10] The amino acid sequence of hFR2-14_H11 (SEQ ID NO: 14 in the Sequence Listing). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H11. Variable region The amino acid numbers are 20 to 137, and the constant regions are amino acid numbers 138 to 467.

[Fig. 11] The amino acid sequence of hFR2-14_H12 (SEQ ID NO: 16 in the Sequence Listing). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H12. The variable regions are amino acid numbers 20 to 137 and the constant regions are amino acid numbers 138 to 467.

[Fig. 12] Amino acid sequence of hFR2-14_H19 (SEQ ID NO: 18 in the Sequence Listing). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H19. The variable regions are amino acid numbers 20 to 137 and the constant regions are amino acid numbers 138 to 467.

[Fig. 13] The hFR2-14_H19/L1 alone, the CPT-11 alone, and the hFR2-14_H19/L1 and CPT-11 were administered to human gastric cancer cell line SNU-16 in nude mice. A graph of anti-tumor effects. Unsigned indicates no treatment control; symbol circle indicates hFR2-14_H19/L1 10 mg/kg; symbol triangle indicates CPT-11 40 mg/kg; symbol square indicates hFR2-14_H19/L1 10 mg/kg+CPT-11 40 mg/kg.

[Fig. 14] Presentation of human gastric cancer cell line SNU-16 transplantation in NOD-scid mice, hFR2-14_H19/L1 alone, paclitaxel alone or hFR2-14_H19/L1 combined with paclitaxel A picture of the anti-tumor effect in vivo. Unsigned indicates no treatment control; symbol circle indicates hFR2-14_H19/L1 10 mg/kg; symbol triangle indicates paclitaxel 15 mg/kg; symbol square indicates hFR2-14_H19/L1 10 mg/kg + paclitaxel 15 mg/kg

[Fig. 15] HFR2-14_H19/L1 was administered alone in human lung cancer cell line KNS-62 transplanted in NOD-scid mice, cisplatin alone or A graph of the in vivo antitumor effect of hFR2-14_H19/L1 in combination with cisplatin. Unsigned indicates no treatment control; symbol circle indicates hFR2-14_H19/L1 10 mg/kg; symbol triangle indicates cisplatin 5 mg/kg; symbol square indicates hFR2-14_H19/L1 10 mg/kg + cisplatin 5 mg/kg.

[Fig. 16] Amino acid sequence of humanized FR2-14 heavy chain (hFR2-14_H9) (SEQ ID NO: 20). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H9. The variable regions are amino acid numbers 20 to 137 and the constant regions are amino acid numbers 138 to 467.

[Fig. 17] Presenting in vivo anti-tumor of hFR2-14_H19/L1 administered alone, cisplatin alone or hFR2-14_H19/L1 combined with cisplatin in human gastric cancer cell line SNU-16-transplanted nude mice The map of the effect. Unsigned indicates no treatment control; symbol circle indicates hFR2-14_H19/L1 1 mg/kg; symbol triangle indicates cisplatin 5 mg/kg; symbol square indicates hFR2-14_H19/L1 1 mg/kg + cisplatin 5 mg/kg.

[Fig. 18] In vivo anti-tumor administration of hFR2-14_H19/L1 administered alone, paclitaxel alone or hFR2-14_H19/L1 combined with taxol in vivo in human gastric cancer cell line SNU-16-transplanted nude mice The map of the effect. Unsigned indicates no treatment control; symbol circle indicates hFR2-14_H19/L1 1 mg/kg; symbol triangle indicates European paclitaxel 1 mg/kg; symbol square indicates hFR2-14_H19/L1 1 mg/kg + European paclitaxel 1 mg/kg.

[Fig. 19] Presentation of human gastric cancer cell line SNU-16 transplanted in nude mice hFR2-14_H19/L1 alone, anti-VEGFR2 antibody alone or hFR2-14_H19/L1 and anti-VEGFR2 antibody combined with the in vivo anti-tumor effect. Unsigned indicates no treatment control; symbol circle indicates hFR2-14_H19/L1 1 mg/kg; symbol triangle indicates anti-VEGFR2 antibody 20 mg/kg; symbol square indicates hFR2-14_H19/L1 1 mg/kg + anti-VEGFR2 antibody 20 mg/kg.

[Fig. 20] Presenting hFR2-14_H19/L1 alone, 5-FU alone or hFR2-14_H19/L1 and 5-FU in human gastric cancer cell line SNU-16 transplanted nude mice A graph of anti-tumor effects. Unsigned indicates no treatment control; symbol circle indicates hFR2-14_H19/L1 1 mg/kg; symbol triangle indicates 5-FU 50 mg/kg; symbol square indicates hFR2-14_H19/L1 1 mg/kg+5-FU 50 mg/kg.

[Formation of the Invention]

Definition

In the present invention, "gene" means a nucleotide contained in a nucleotide sequence of an amino acid encoding a protein or a complementary strand thereof, for example, a nucleotide contained in a nucleotide sequence of an amino acid encoding a protein or Polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc., which are complementary to each other, are included in the meaning of "gene". The gene is a single-stranded, double-stranded or triple-stranded nucleotide, an aggregate of a DNA strand and an RNA strand, a ribonucleotide (RNA) and a deoxyriborib nucleus on a single strand of a nucleotide strand. The presence of a glycoside (DNA) mixture and a double-stranded or triple-stranded nucleotide containing such a nucleotide chain are also included in the meaning of "gene". For the "FGFR2" of the present invention The gene includes, for example, DNA, mRNA, cDNA, cRNA, and the like contained in the nucleotide sequence encoding the amino acid sequence of the FGFR2 protein.

In the present invention, "nucleotide", "nucleic acid" and "nucleic acid molecule" are synonymous, for example, DNA, RNA, probe, oligonucleotide, polynucleotide, primer, etc. are also included in "nucleotide" In the meaning. The nucleotide is a nucleotide consisting of a single strand, a double strand or a triple strand, a collection of a DNA strand and an RNA strand, and a ribonucleotide (RNA) on a single strand of the nucleotide strand. The presence of a mixture of deoxyribonucleotides (DNA) and a double or triple chain assembly comprising such a nucleotide chain are also included in the meaning of "nucleotides".

In the present invention, "polypeptide", "peptide" and "protein" are synonymous.

In the present invention, the meaning of "antigen" is sometimes used for "immunogen".

In the present invention, the "cell" also includes various cells derived from an individual animal, subcultured cells, primary cultured cells, cell strains, recombinant cells, and microorganisms.

In the present invention, antibodies such as FGFR2, FGFR2IIIb, FGFR2IIIc, FGFR3, and FGFRs may be labeled as "anti-FGFR2 antibody", "anti-FGFR2IIIb antibody", "anti-FGFR2IIIc antibody", "anti-FGFR3 antibody", and "anti-FGFRs". Antibody, etc. The antibody comprises a chimeric antibody, a humanized antibody, a human antibody, and the like.

The "functional fragment of an antibody" in the present invention means an antibody fragment which exerts at least a part of the function exerted by the original antibody. Examples of the "functional fragment of the antibody" include Fab, F(ab')2, and scFv. , Fab', single-chain immunoglobulin, etc., but are not limited thereto. The functional fragment of the antibody can be obtained by treating the full-length molecule of the antibody protein with an enzyme such as papain or pepsin, or a recombinant protein produced in a suitable host cell using a recombinant gene.

In the present invention, a "site" to which an antibody binds, that is, a "site" recognized by an antibody means a partial peptide or a partial high-order structure on an antigen to which an antibody binds or recognizes. In the present invention, the site is also referred to as an epitope and an antibody binding site. The site on the FGFR2 protein to which the anti-FGFR2 antibody of the present invention binds or recognizes may be a partial peptide or a partial higher order structure on the FGFR2 protein.

It is known that the heavy chain and the light chain of the antibody molecule each have three CDRs (Complemetarity determining regions). The complementarity determining region, also known as the hypervariable domain, is located in the variable region of the heavy and light chains of the antibody, and is a site of extremely high variability in the primary structure, once in the polypeptide chain of the heavy chain and the light chain. Structurally, they are usually separated by three places. In the present invention, regarding the complementarity determining region of the antibody, the complementarity determining region of the heavy chain is labeled from the amino terminal side of the heavy chain amino acid sequence to CDRH1, CDRH2, CDRH3, and the complementarity determining region of the light chain is derived from the light chain amino acid. The amino terminus side of the sequence is labeled as CDRL1, CDRL2, CDRL3. These sites are close to each other in stereostructure and determine the specificity for the bound antigen.

In the present invention, the "antibody variant" means an amine having an amino acid substitution, deletion, addition and/or insertion (hereinafter, collectively referred to as "variation") in the amino acid sequence of the original antibody. A polypeptide having a base acid sequence and binding to the FGFR2 protein of the present invention. Antibody variant The number of variant amino acids in the body is 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40 or 50. This antibody variant is also included in the "antibody" of the present invention.

In the present invention, "a few" in "1 to several" means 3 to 10.

The activity exerted by the antibody of the present invention. The nature is, for example, biological activity, physicochemical property, and the like, and specific examples thereof include various biological activities, binding activity to an antigen or an antigenic epitope, stability at the time of production or storage, thermal stability, and the like. .

In the present invention, "hybridization under stringent conditions" means hybridization under the following conditions or conditions equivalent thereto: hybridization at 65 ° C in a solution containing 5 × SSC, followed by 2 × SSC - 0.1% SDS, respectively. The aqueous solution was washed at 65 ° C for 20 minutes, washed in an aqueous solution containing 0.5 × SSC-0.1% SDS at 65 ° C for 20 minutes, and washed at 65 ° C in an aqueous solution containing 0.2 × SSC-0.1% SDS. minute. SSC is an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and n x SSC means n times the concentration of SSC.

In the present invention, "cell injury" refers to causing a pathological change of a cell in any manner, not only to direct trauma, but also to the cleavage of DNA or the formation of a base dimer, the cleavage of a chromosome, a mitotic device. (mitotic apparatus) damage to all cellular structures or functions such as damage to various enzyme activities. In the present invention, "cytotoxicity" means a person who causes the above-mentioned cell damage.

In the present invention, "antibody-dependent cellular cytotoxicity" means "antibody dependent cellular cytotoxicity (ADCC) activity", which means that NK cells damage the target cells such as tumor cells by antibodies. Activity.

In the present invention, "antibody-dependent cell-mediated phagocytosis activity" means "antibody-dependent cell phagocytosis (ADCP) activity", which means that mononuclear cells or macrophages phagocytose the activity of cells such as tumor cells by antibodies. . Also known as "antibody-dependent phagocytic activity."

In the present invention, "complement-dependent cytotoxic activity" means "complement dependent cytotoxicity (CDC) activity", which means that complement complements the action activity of a target cell such as a tumor cell by an antibody.

In the present invention, "cancer" and "tumor" are used in the same meaning.

In the present invention, the "other active ingredient" or "other agent" means an ingredient having a therapeutic or prophylactic activity of a disease which is used in combination with an anti-FGFR2 antibody or which is contained in the pharmaceutical composition of the present invention together with an anti-FGFR2 antibody or Pharmacy. In a suitable aspect, the combination or pharmaceutical composition is effective for the treatment or prevention of cancer, and in a more suitable aspect, the pharmaceutical composition exhibits a multiplication effect in the treatment or prevention of cancer.

In the present invention, the "multiplication effect" refers to a case where a plurality of active ingredients are combined or a case where a plurality of active ingredients are contained in one pharmaceutical composition, and it is shown that the anticancer activities shown by the respective active ingredients are added. Stronger anti-cancer activity. For example, the anticancer activities of the active ingredients A and B are each a and b, and when A and B are used in combination or A and B are contained in one pharmaceutical composition, the anticancer activity is If the sum of a and b is greater, the combination or pharmaceutical composition can be regarded as a multiplier effect. fruit. In other examples, the anticancer activities of the active ingredients A and B are each a and zero (0: that is, B is a concentration at which anticancer activity is not exerted), and A and B are used in combination or A and B are used. In the case of a pharmaceutical composition contained in one, if the anticancer activity is larger than a, the combination or the pharmaceutical composition can be regarded as exerting a multiplication effect. In another example, any of the anticancer activities exerted by the active ingredients A and B are zero (0: that is, both A and B are concentrations at which anticancer activity is not exerted), and A and B are combined. When the A or B is contained in one of the pharmaceutical compositions, if the anticancer activity is greater than zero (0), the combination or the pharmaceutical composition can be regarded as exerting a multiplication effect.

2. Antigenic protein

(2-1) Characteristics

FGFRs are receptor proteins that bind to fibroblast growth factor (FGF). In the present invention, the FGFRs are derived from a vertebrate, preferably from a mammal, and more preferably from a human. Human FGF and FGFRs are each classified into 22 FGFs (FGF1 to 14, and FGF 16 to 23), and 4 FGFRs (FGFRs 1 to 4) having a tyrosine kinase domain. FGFRs are composed of an extracellular region, a transmembrane region, and an intracellular region containing a ligand binding composed of two or three immunoglobulin domains (IgD1 to 3). The site, the intracellular region contains a tyrosine kinase domain. Among them, two splicing variants called IIIb and IIIc are present in FGFR1, FGFR2 and FGFR3. These isoforms differed in about half of the amino acid sequences in the second half of IgD3, showing different tissue distribution and ligand specificity. FGFRs have the following activities: (1) binding to FGF; (2) dimerization of FGFRs by this binding; (3) by the dimer The specific tyrosine residues of FGFRs are phosphorylated; (4) by this phosphorylation, the recruitment of adapter proteins such as FGFR matrix 2α (FRS2α) is promoted; (5) The cells or tissues of FGFRs convey messages or activate message transmission due to FGF stimulation.

In the present invention, the FGFR2 protein system has the following properties.

(i) combined with FGF.

The FGFR2IIIb protein line usually binds to one or more selected from the group consisting of FGF1, FGF3, FGF7 (KGF), FGF10, FGF22, and FGF23, but can also bind to other FGFs, even in the case of variants, even The FGF contained in the aforementioned group also has an unbound condition.

The FGFR2IIIc protein line is usually bound to one or more selected from the group consisting of FGF1, FGF2, FGF4, FGF6, FGF9, FGF17, FGF18, FGF21, and FGF23. It can also be combined with other FGFs, and in the case of a variant, even if the FGF contained in the aforementioned group is unbound.

(ii) conveying information generated by FGF stimulation in cells or tissues found in FGFR2.

The message transmission by FGF stimulation is not limited, but for example, FGFR2 autophosphorylation, recruitment of FGFR matrix and its promotion, by their MAPK, PI3K, Akt, extracellular Activation of message pathways such as message-controlled kinase (ERK). As the FGFR matrix, FGFR matrix 2α (FRS2α) and the like can be exemplified.

The test method for evaluating the activation of the message and its inhibition is not particularly limited and can be arbitrarily selected by a known method, but for example For the system for evaluating the ERK message transmission, an Elk1 luciferases reporter assay described later can be cited.

(iii) In the present invention, the FGFR2IIIb protein comprises the amino acid sequence described in any one of the following (a) to (d) (hereinafter referred to as "FGFR2IIIb amino acid sequence"), or contains an FGFR2IIIb amine group. The amino acid sequence of the acid sequence consists of or consists of the FGFR2IIIb amino acid sequence: (a) the amino acid sequence of NP_075259; (b) shows that the amino acid sequence with NP_075259 is 80%, 82%, 84% , 86%, 88%, 90%, 92%, 94%, 96%, 98% or 99% or more of the amino acid sequence of the polypeptide having sequence identity and having FGF binding activity; (c) the amino group of NP_075259 Acid sequence, 1 to 50, 40, 35, 30, 25, 20, 15, 12, 10, 8, 6, 5, 4, 3 or 2, or 1 amino acid substituted, deleted, added or An amino acid sequence of a polypeptide inserted and having FGF binding activity; and (d) encoded by a nucleotide sequence possessed by a nucleotide which hybridizes to a nucleotide under stringent conditions, and having FGF binding activity The amino acid sequence of the polypeptide: a nucleotide having a base sequence complementary to the base sequence encoding the amino acid sequence of NP_075259.

The polypeptide described in any one of (b) to (d) may have other activities of FGFR2 in addition to the FGF binding activity.

In the present invention, the FGFR2IIIc protein comprises the amino acid sequence described in any one of the following (a) to (d) (hereinafter referred to as "FGFR2IIIc amino acid sequence"), or comprises an FGFR2IIIc amino acid sequence. The amino acid sequence of the column consists of or consists of the FGFR2IIIc amino acid sequence: (a) the amino acid sequence of NP_000132; (b) shows that the amino acid sequence with NP_000132 is 80% or more, 82% or more, 84% or more 86% or more, 88% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98% or more or 99% or more of the sequence identity, and the amino acid sequence of the polypeptide having FGF binding activity; (c) The amino acid sequence of NP_000132 has 1 to 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 6, 5, 4, 3 or 2, or 1 An amino acid sequence of a polypeptide having a FGF binding activity substituted, deleted, added or inserted; and (d) a base possessed by a nucleotide which hybridizes to the following nucleotide under stringent conditions The amino acid sequence of the polypeptide encoded by the base sequence and having FGF binding activity: a nucleotide having a base sequence complementary to the nucleotide sequence encoding the amino acid sequence of NP_000132.

The polypeptide described in any one of (b) to (d) may have other activities of FGFR2 in addition to the FGF binding activity.

The FGFR2 protein of the present invention may be either a natural type (non-recombinant type) or a recombinant type. Further, fusions with other peptides or proteins such as a carrier or a tag are also included in the meaning of the FGFR2 protein. Further, a modification by performing a chemical modification including a polymer addition of PEG or the like and/or a modification including a sugar chain modification is also included in the meaning of the FGFR2 protein. Further, the FGFR2 protein fragment is also included in the meaning of the FGFR2 protein of the present invention. Among the FGFR2 protein fragments, those having the properties described in (i) and/or (ii) above are referred to as FGFR2 protein functional fragments.

(2-2) antigen gene

In the present invention, the FGFR2IIIb gene system comprises a base sequence encoding the FGFR2IIIb amino acid sequence (hereinafter referred to as "FGFR2 gene sequence"), or a base sequence comprising the FGFR2 gene sequence, or a FGFR2 gene sequence. In the present invention, the FGFR2IIIc gene system comprises a base sequence encoding the FGFR2IIIc amino acid sequence (hereinafter referred to as "FGFR2 gene sequence"), or a base sequence comprising the FGFR2 gene sequence, or a FGFR2 gene sequence.

(2-3) Modulation of antigenic proteins

The FGFR2 protein of the present invention can be prepared by automated tissue (including body fluids), cells derived from the tissue, or purification and isolation of the cell culture, genetic recombination, in vitro translation, chemical synthesis, and the like.

3. Antibody

(3-1) Classification of antibodies

The antibody of the present invention may be any of a single antibody and a plurality of antibodies. Examples of the monoclonal antibodies of the present invention include antibodies derived from non-human animals (non-human animal antibodies), antibodies derived from humans (human antibodies), chimeric antibodies (also referred to as "chimeric antibodies"), and humans. Antibody and the like.

Examples of the non-human animal antibody include antibodies derived from vertebrate animals such as mammals and birds. Examples of the antibody derived from mammals include rodent antibodies such as mouse antibodies and rat antibodies. Examples of the antibody derived from birds include chicken antibodies and the like. Examples of the anti-human FGFR2 rat monoclonal antibody include FR2-10, FR2-13, and FR2-14 (WO2013/154206).

In the case of chimeric antibodies, it can be cited that it will come from non-human movements. The antibody or the like in which the variable region of the antibody binds to the constant region of a human antibody (human immunoglobulin) is not limited thereto. For chimeric antibodies derived from a combination of a variable region of a non-human animal antibody and a human antibody constant region, each of the aforementioned monoclonal antibodies FR2-10, FR2-13 or FR2-14 may be enumerated. The heavy and light chain variable regions, and the human heavy and light chain constant regions are cFR2-10, cFR2-13, cFR2-14 (WO 2013/154206) and the like.

In the case of a humanized antibody, a CDR in a variable region of a non-human animal antibody can be exemplified by a human antibody (a variable region of a human immunoglobulin), and a framework of a non-human animal antibody in addition to the CDR. The sequence of the region is transplanted to a part of human antibodies, one of which is one or more amino acid from non-human animal antibodies, replaced by a human type of amino acid, etc., but is not limited This is the case. For the CDRs in the variable region of the non-human animal antibody, the aforementioned CDRH1 to CDRH3 and the light chain variable region in the heavy chain variable region from FR2-10, FR2-13 or FR2-14 can be exemplified. CDRL1 to CDRL3, one or two amino acids in which the amino acid sequences of the CDRs of the CDRs are substituted with other amino acids, and the like.

The human antibody is not limited as long as it is an antibody which recognizes the antigen of the present invention, but a human antibody which binds to the same site as the antibody having the CDR of the antibody of the present invention, and the aforementioned FR2-10, FR2- are exemplified. 13 or FR2-14 binds to the same site of human antibody or the like on FGFR2.

The antibody of the present invention may have an activity of binding to FGFR2, and may be an antibody composed of a portion derived from a plurality of different antibodies, for example. For example, ones that exchange heavy and/or light chains among a plurality of different antibodies, the entire length of exchange heavy and/or light chains, only exchange variable regions or only exchange constant regions, only exchange all of CDRs. Or part of it. The heavy chain variable region and the light chain variable region of the chimeric antibody may also be derived from a different antibody of the invention. CDRH1 to CDRH3 and CDRL1 to CDRL3 in the variable regions of the heavy and light chains of the humanized antibody may be derived from two or more antibodies of the invention. The CDRH1 to CDRH3 and the CDRL1 to CDRL3 in the variable regions of the heavy and light chains of the human antibody may be a combination of CDRs possessed by the antibodies of the present invention of two or more. Such an antibody composed of a portion derived from a plurality of different antibodies may have one or two or more activities as described in (3-3) to (3-6).

The isotype of the monoclonal antibody of the present invention is not limited, and examples thereof include IgA, IgD, IgE, and the like such as IgG, IgM, IgA1, and IgA2 such as IgG1, IgG2, IgG3, and IgG4. IgG and IgM.

(3-2) Antibody binding specificity

The anti-system of the invention recognizes the FGFR2 protein. In other words, the anti-system of the invention binds to the FGFR2 protein. This antibody was labeled as "anti-FGFR2 antibody". Further, a suitable anti-system of the invention specifically recognizes the FGFR2 protein. In other words, a suitable anti-system of the invention specifically binds to the FGFR2 protein. Furthermore, the more suitable anti-system of the present invention specifically binds to the FGFR2IIIb protein and/or the FGFR2IIIc protein, and further suitable anti-systems and immunoglobulin-like domains possessed by the FGFR2IIIb protein and/or the FGFR2IIIc protein (hereinafter, Specific binding for the "class Ig domain". In the case of this class of Ig domains A class Ig domain 2, a class Ig domain 3, and the like can be exemplified.

In the present invention, "specificity of knowledge", that is, "specific binding" means a combination of adsorption that is not non-specific. The criterion for the determination of the specificity is, for example, a dissociation constant (hereinafter referred to as "KD"). The KD value of the FGFR2 protein of the antibody of the present invention is 1 × 10 ^-5 M or less, 5 × 10 ^-6 M or less, 2 × 10 ^-6 M or less, or 1 × 10 ^-6 M or less, more preferably 5 ×10 ^-7 M or less, 2 × 10 ^-7 M or less, or 1 × 10 ^-7 M or less, further preferably 5 × 10 ^-8 M or less, 2 × 10 ^-8 M or less, or 1 × 10 ^-8 M or less Further preferably, it is 5 × 10 ^-9 M or less, 2 × 10 ^-9 M or less, or 1 × 10 ^-9 M or less, preferably 5 × 10 ^-10 M or less, 2 × 10 ^-10 M or less or 1 ×10 ^-10 M or less.

The binding of the antigen to the antibody in the present invention can be measured or determined by an ELISA method, an RIA method, a surface plasmon resonance (Surface Plasmon Resonance) analysis method, or the like.

(3-3) Antitumor activity of antibodies

The antibody of the present invention has antitumor activity, and preferably has antitumor activity in vivo. In the present invention, antitumor activity is synonymous with anticancer activity.

In the present invention, antitumor activity means inhibiting the activity of proliferation, malignant transformation, infiltration, metastasis, tumor size increase or weight increase of tumor tissues and/or tumor cells.

Antitumor activity can be evaluated according to standard methods. In vivo anti-tumor activity can be evaluated for effects on human tumors by using, for example, a non-human animal model (xenograft) transplanting human cancer tissues or cancer cells. For non-human animals used for xenotransplantation, examples include nude mice and the like. Mouse or rat, etc.

Further, the antitumor activity can also be evaluated as a suppression or inhibitory activity against cell proliferation of cancer cells.

(3-4) cytotoxicity of antibodies

The anti-FGFR2 antibodies of the invention may have antibody-dependent cellular killing (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity and/or antibody-dependent cellular media phagocytosis (ADCP) activity. Suitable anti-systems of the invention have ADCC activity, and preferred anti-systems have ADCC activity on FGFR2 expressing cells. ADCC activity, CDC activity, and ADCP activity can be determined by well-known methods.

(3-5) The role of antibodies in message transmission

The biological activity and properties possessed by the anti-FGFR2 antibodies of the invention can also be assessed by FGF messages via FGFR2. The use of FGF-stimulated signaling via FGFR2 is not limited, but examples thereof include FGFR2 autophosphorylation, recruitment and promotion of FGFR matrix, MAPK, PI3K, Akt, and extracellular messages via them. Activation of a message pathway such as a kinase (ERK).

Further, a suitable anti-system of the present invention has a neutralizing activity against FGFR2, and is more suitably a neutralizing activity against FGFR2IIIb and/or FGFR2IIIc. Neutralizing activity means inhibiting or suppressing the activity of FGFR2 activation due to a ligand of FGFR2. For example, an antibody that inhibits FGF-dependent FGFR2 vector information, message transmission, or the like can be determined to have the neutralizing activity.

(3-6) Inhibition of antibody-receptor-ligand binding activity

Suitable anti-systems of the invention inhibit the binding of FGFR2 to its ligand, more suitably to inhibit the binding of FGFR2IIIb and/or FGFR2IIIc to FGF. The inhibition of binding between the receptor-ligand can be either competitive inhibition or non-competitive inhibition. For the ligands of FGFR2IIIb and FGFR2IIIc, FGF1, FGF3, FGF7, FGF10, FGF22 and FGF23, and FGF1, FGF2, FGF4, FGF6, FGF9, FGF17, FGF18, FGF21 and FGF23 can be exemplified.

(3-7) monoclonal antibody

The present invention provides monoclonal antibodies. The monoclonal antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, and functional fragments thereof derived from a non-human animal such as a rat antibody, a mouse antibody, a rabbit antibody, a chicken antibody, or a fish antibody, and the like. Modifications, etc. Among them, examples of the rat monoclonal antibody include FR2-14 antibody (WO 2013/154206) and the like.

FR2-14 is an anti-human FGFR2 rat monoclonal antibody (WO 2013/154206).

The antibody variant of the present invention is suitable for performing a decrease in sensitivity to decomposition or oxidation of a protein, improvement in biological activity, improvement in antigen binding ability, or imparting physicochemical properties or functional properties. An example of such an antibody variant is an antibody having an amino acid sequence in which an amino acid sequence of an antibody has a substituted amino acid. The preservative amino acid substitution refers to a substitution occurring in a certain amino acid group associated with an amino acid side chain (WO 2013/154206).

The aspartic acid contained in the protein has a small side chain at the end of its C-linked amino acid, and is easily converted to isoaspartic acid by isomerization. On the other hand, aspartame Acidic situation It is easy to change to aspartic acid by deamination, and there is a possibility of further converting to isoaspartic acid by isomerization. When such isomerization or deamination is carried out, the stability of the protein can be affected. Therefore, in order to avoid this isomerization or deamination, it is possible to substitute aspartic acid or aspartic acid in the protein or an amino acid adjacent thereto in the other amino acid. It is preferred that the antibody variant substituted with the amino acid retains the antigen-binding activity possessed by the original antibody.

The amino acid sequence of the antibody of the present invention has an antibody variant having an amino acid sequence substituted with a conservative amino acid, and comprises any one of CDRH1 to CDRH3 and CDRL1 to CDRL3 derived from FR2-14. A mouse antibody, a rat antibody, a chimeric antibody, a humanized antibody, a human antibody or the like which is an amino acid sequence of the amino acid sequence which carries out a conservative amino acid mutation is also included in the present invention.

An antibody variant comprising CDRH1 to CDRH3 and CDRL1 to CDRL3 having the following amino acid sequence, and binding to human FGFR2, is also included in a variant of the antibody of the invention: CDRH1 to CDRH3 from FR2-14 And one or more amino acid sequences of one or more of CDRL1 to CDRL3 having from 1 to several, preferably from 1 to 3, more preferably 1 or 2, most preferably one amino acid An amino acid sequence substituted with another amino acid.

For a suitable variant of FR2-14, for example, a chimeric antibody, a humanized antibody, a human antibody or the like comprising CDRH3 having the following amino acid sequence: 1 or 2 of the amino acid sequence of CDRH3 The amino acid, preferably an amino acid sequence in which the first or second amino acid is substituted with another amino acid from the terminal of the amino acid.

For a suitable variant in which the amino acid of the FR2-14 heavy chain CDRH3 is substituted, for example, (i) a variant of the aspartic acid substituted with glutamic acid of the amino acid of the first amino acid ( SEQ ID NO: 10 and Figure 8: The amino acid of No. 118 is Glu); (ii) Glycine which is the amino acid of No. 2 via tyrosine, alanine, tryptophan, lysine , arginine, aspartic acid, methionine, leucine, lysine, isoleucine, histidine, phenylalanine, glutamic acid or glutamic acid, preferably By phenylalanine, histidine, lysine or leucine (SEQ ID NO: 12, 14, 16, 18, 20 and 9 to 12 and 16: amino acid No. 119 is Phe, Lys , His or Leu), better leucine-substituted variants (SEQ ID NO: 16 or 18 and Figure 11 or 12: amino acid No. 119 is Leu); (iii) amine No. 8 A humanized antibody, a chimeric antibody, a human antibody, a functional fragment thereof, etc., in which a sulphate acid is substituted with alanine. Specifically, CDRH3 consisting of amino acid numbers 118-126 of the amino acid sequence shown in SEQ ID NO: 8, 10, 12, 14, 16, 18 and 20 of the Sequence Listing can be cited.

Further, antibodies having CDRH1 to CDRH3 and CDRL1 to CDRL3 from a plurality of antibodies are also included in the antibody variant. Examples of such variants include antibody variants in which only CDRH3 is derived from an antibody, while CDRH1 and CDRH2 and CDRL1 to CDRL3 are derived from additional antibodies.

In the present invention, antibody variants are also included in "antibodies".

The constant region of the antibody of the present invention is not particularly limited, but a human antibody is preferably used as the antibody of the present invention for treating or preventing a human disease. For the heavy chain constant region of human antibodies, For example, Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, Cε, and the like. Examples of the light chain constant region of the human antibody include, for example, Cκ, Cλ, and the like.

(3-8) Functional fragment of antibody

In one aspect of the invention, a functional fragment of an anti-FGFR2 antibody of the invention is provided. A functional fragment of an antibody means a fragment that retains at least a portion of the function possessed by the antibody. The function of the antibody is generally, for example, an antigen-binding activity, an activity of modulating an activity of an antigen, an antibody-dependent cellular cytotoxicity (ADCC) activity, and an antibody-dependent cell-mediated phagocytosis (ADCP) activity. The function of the anti-FGFR2 antibody of the present invention includes, for example, FGFR2 protein binding activity, ADCC activity, ADCP activity, neutralizing activity against FGFR2, in vivo antitumor activity, and inhibition of binding of FGFR2 to its ligand. Wait.

The functional fragment of the antibody is not particularly limited as long as it is a fragment of the antibody that retains at least a part of the activity of the antibody, and examples thereof include Fab, F(ab') 2, and Fv. Single-chain Fv (scFv), diabodies, linear antibodies, and multispecific antibodies formed by antibody fragments that bind the Fv of the heavy and light chains with an appropriate linker, F(ab')2 is Fab' or the like of one of the variable regions of the variable region of the antibody treated under reducing conditions, but is not limited thereto. For example, a scFv having a linker moiety, a molecule comprising a portion other than the antibody fragment of the present invention is also included in the meaning of the functional fragment of the antibody of the present invention.

The amino acid-terminal and/or carboxy-terminal amino acid of the antibody protein has one to several or more deletions, and the mechanism possessed by the antibody is maintained. At least a portion of the molecules of the molecule are also included in the meaning of the functional fragments of the antibody. For example, it is known that an amino acid residue at the carboxy terminus of a heavy chain of an antibody produced by a mammalian cultured cell is deleted (Journal of Chromatography A, 705: 129-134 (1995)); further, the same heavy chain carboxyl group is known. The terminal glycine acid, the two amino acid residues of the lysine are deleted, and the proline residue at the new carboxy terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, deletions and modifications of such heavy chain sequences have no effect on the antigen binding ability of the antibody and the effector function (activation of complement or antibody-dependent cytotoxicity, etc.). A modified form of the functional fragment of such an antibody is also included in the antibody of the present invention or a functional fragment thereof, or a modified form thereof (described later).

The antibody or functional fragment thereof of the present invention may be a multispecific antibody specific for at least two types of dissimilar antigens. The multispecific antibody is not limited to a bispecific antibody that binds to two kinds of different antigens, and an antibody specific for three or more different antigens is also included in the "multispecific" of the present invention. In the meaning of "antibiotics".

The multispecific antibody of the present invention may be a full length antibody or a functional fragment thereof (e.g., F(ab')2 dual specific antibody). One aspect of the antibody of the present invention is a single-chain antibody (hereinafter referred to as "scFv"). Further, BiscFv or MultiscFv, a single-chain immunoglobulin, a single domain antibody, a nanobody, and the like which are produced by binding two or more scFvs with a polypeptide linker are also functionally contained in the antibody of the present invention. In the meaning of the fragment.

(3-9) Humanized antibodies and human antibodies

The present invention is in one aspect that provides a humanized antibody or a functional fragment thereof.

The anti-FGFR2 humanized antibody of the present invention or a functional fragment thereof has antitumor activity, and preferably has antitumor activity in vivo. Further, the humanized antibody or functional fragment thereof preferably binds specifically to the FGFR2IIIb protein and/or the FGFR2IIIc protein, and more preferably binds to the Ig-like domain possessed by the proteins thereof. Furthermore, the humanized antibody or functional fragment thereof preferably has ADCC activity and/or ADCP activity. Further, the humanized antibody of the present invention or a functional fragment thereof has a neutralizing activity against FGFR2, preferably has a neutralizing activity against FGFR2IIIb and/or FGFR2IIIc, and more preferably has a neutralizing activity against FGFR2IIIb and FGFR2IIIc. Furthermore, the humanized antibody of the present invention or a functional fragment thereof preferably inhibits binding of FGFR2 to its ligand.

Examples of suitable humanized antibodies of the present invention include humanized antibodies consisting of the following light chain and heavy chain, and which recognize the FGFR2 protein of the present invention or a fragment of the antibody which retains the FGFR2 protein binding activity of the antibody. Or a variant thereof or the like: having a CDRL1 comprising an amino acid sequence represented by SEQ ID NO: 1 (Fig. 2) of the sequence listing, represented by sequence identification number 2 (Fig. 2) of the sequence listing The light chain of the variable region of the CDRL3 consisting of the amino acid sequence and the amino acid sequence represented by the sequence identification number 3 (Fig. 3) of the sequence listing, and having the sequence comprising the sequence listing CDRH1 consisting of the amino acid sequence shown in No. 4 (Fig. 4), CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 5 (Fig. 5) of the Sequence Listing, and the sequence of the sequence listing CDRH3 consisting of the amino acid sequence shown in column number 6 (Fig. 6) (except that the first or second amino acid from the end of the amine group may be substituted by other amino acids as described above) The heavy chain of the variable region.

A more suitable humanized antibody of the present invention is a humanized FR2-14 antibody and a variant thereof, and examples thereof include hFR2-14_H1/L1 to hFR2-14_H19/L1 (WO2013/154206), but are not For example, a heavy chain comprising a heavy chain variable region of a humanized antibody comprising any one of hFR2-14_H1/L1 to hFR2-14_H19/L1, and hFR2-14_H1/L1 to hFR2-14_H19/ An antibody to the light chain of the light chain variable region of the humanized antibody of any of L1 is also contained in a more suitable humanized antibody of the present invention.

hFR2-14_H19/L1 is a modified sugar chain modified humanized antibody obtained in Example 9 of WO2013/154206. The base sequence of the light chain of the antibody comprises nucleotide number 61 to 705 of SEQ ID NO: 7, and the amino acid sequence comprises amino acid number 21 to 235 of SEQ ID NO: 8 (Fig. 7), heavy chain The base sequence contains nucleotide numbers 58 to 1401 of SEQ ID NO: 17, and the amino acid sequence contains amino acid numbers 20 to 467 of SEQ ID NO: 18 (Fig. 12).

The base sequence of the light chain of hFR2-14_H12/L1 comprises nucleotide numbers 61 to 705 of SEQ ID NO: 7, and the amino acid sequence contains amino acid numbers 21 to 235 of SEQ ID NO: 8 (Fig. 7). The base sequence of the heavy chain comprises nucleotide number 58 to 1401 of SEQ ID NO: 15, and the amino acid sequence comprises amino acid numbers 20 to 467 of SEQ ID NO: 16 (Fig. 11).

The base sequence of the light chain of hFR2-14_H8/L1 contains the sequence Column number 7 to nucleotide number 61 to 705, amino acid sequence comprising amino acid number 21 to 235 of SEQ ID NO: 8 (Fig. 7), and base sequence of heavy chain comprising SEQ ID NO: 11. Nucleotide numbers 58 to 1401, the amino acid sequence contains amino acid numbers 20 to 467 of SEQ ID NO: 12 (Fig. 9).

The base sequence of the light chain of hFR2-14_H9/L1 comprises nucleotide numbers 61 to 705 of SEQ ID NO: 7, and the amino acid sequence contains amino acid numbers 21 to 235 of SEQ ID NO: 8 (Fig. 7). The base sequence of the heavy chain comprises nucleotide numbers 58 to 1401 of SEQ ID NO: 19, and the amino acid sequence comprises amino acid numbers 20 to 467 of SEQ ID NO: 20 (Fig. 16).

The base sequence of the light chain of the hFR2-14_H11/L1 antibody comprises nucleotide number 61 to 705 of SEQ ID NO: 7, and the amino acid sequence comprises amino acid number 21 of SEQ ID NO: 8 (Fig. 7). 235, the base sequence of the heavy chain comprises nucleotide number 58 to 1401 of SEQ ID NO: 13, and the amino acid sequence comprises amino acid numbers 20 to 467 of SEQ ID NO: 14 (Fig. 10).

The base sequence of the light chain of hFR2-14_H5/L1 comprises nucleotide numbers 61 to 705 of SEQ ID NO: 7, and the amino acid sequence contains amino acid numbers 21 to 235 of SEQ ID NO: 8 (Fig. 7). The base sequence of the heavy chain comprises nucleotide number 58 to 1401 of SEQ ID NO: 9, and the amino acid sequence comprises amino acid numbers 20 to 467 of SEQ ID NO: 10 (Fig. 8).

These humanized anti-systems have high Tm values, excellent stereostructure stability, high binding activity to FGFR2, excellent thermal stability, FGFR2 ligand-dependent FGFR2 message neutralizing activity, ADCC activity, ADCP activity, Inhibition of binding of FGFR2 ligand to FGFR2 Active, in vivo antitumor activity, etc., maintain high antigen binding activity even under severe conditions.

More suitable humanized antibodies of the invention are hFR2-14_H12/L1 and hFR2-14_H19/L1.

The amino acid sequence comprising the heavy or light chain of the antibody described in any one of the humanized hFR2-14_H1/L1 to hFR2-14_H19/L1 antibodies of the present invention is 90% or more, 92% or more, and 94% or more. More than 96%, 98% or more, or more than 99% of the heavy or light chain of the same amino acid sequence, and an antibody that binds to FGFR2 or a functional fragment thereof is also included in the present invention. The sequence identity is preferably 94% or more, more preferably 96% or more, further preferably 98% or more, and most preferably 99% or more. Further, the anti-systems preferably have one or more of the activities described in (3-3) to (3-6).

The identity or identity between the two types of amino acid sequences can be obtained by using Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller). , and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402) system default parameters (default parameter) to determine. The Blast algorithm can also be used by, for example, accessing the Internet http://blast.ncbi.nlm.nih.gov/.

The amino acid sequence containing the heavy or light chain of the antibody described in any one of the humanized hFR2-14_H1/L1 to hFR2-14_H19/L1 antibodies of the present invention has 1 to 10, 8, 6, 5, 4 , 3, or 2, or 1 The heavy or light chain of an amino acid sequence substituted, deleted, added or inserted with an amino acid, and an antibody or a functional fragment thereof which binds to FGFR2 are also included in the present invention. The amino acid variation is preferably a substitution, and the number of the modified amino acids is preferably from 1 to 5, more preferably from 1 to 4, still more preferably from 1 to 3, still more preferably from 1 to 2, the best is 1. Further, the antibodies thereof are preferably ones having one or more of the activities described in (3-3) to (3-6).

An antibody or a functional fragment thereof comprising a heavy chain or a light chain comprising the amino acid sequence described below, and which binds to FGFR2 is also encompassed by the present invention: and has a humanized hFR2-14_H5/L1, hFR2-14_H8/L1 a base complementary to the base sequence of the amino acid sequence of the heavy or light chain of the antibody described in any of the hFR2-14_H9/L1, hFR2-14_H11/L1, hFR2-14_H12/L1, hFR2-14_H12/L1, and hFR2-14_H19/L1 antibodies A nucleotide sequence of a nucleotide sequence encoded by a nucleotide sequence which is hybridized under stringent conditions. Preferably, the anti-systems have one or more of the activities described in (3-3) to (3-6).

In another aspect, the invention provides a human antibody or a functional fragment thereof. The human antibody of the present invention is not limited as long as it is an antibody derived from humans that binds to FGFR2, but preferably has one or more of the activities described in (3-3) to (3-6).

(3-10) Antibodies that bind to epitopes

The "antibody that binds to the same site" to the antibody provided by the present invention is also included in the antibody of the present invention. An antibody that "binds to the same site" as an antibody means another antibody that binds to a site on the antigen molecule recognized by the antibody. If the peptide or part of the antigen molecule bound to the first antibody When the steric structure has a second antibody binding, it can be determined that the first antibody binds to the same site as the second antibody system. Further, the second antibody competes with respect to the binding of the first antibody to the antigen, that is, by confirming that the second antibody interferes with the binding of the first antibody to the antigen, even if the peptide sequence or stereostructure of the specific binding site is not determined. It is also determined that the first antibody binds to the same site as the second antibody. Furthermore, the first antibody binds to the same site as the second antibody, and the first antibody has an effect of an aspect of the antibody of the present invention such as antitumor activity, and the second antibody also has the same activity. The sex system is extremely high. According to this, if the second anti-FGFR2 antibody binds to the site where the first anti-FGFR2 antibody binds, it can be determined that the first antibody and the second antibody bind to the same site on the FGFR2 protein. Further, when it is confirmed that the second anti-FGFR2 antibody competes with the binding of the first anti-FGFR2 antibody to the FGFR2 protein, it can be determined that the first antibody and the second antibody are antibodies that bind to the same site on the FGFR2 protein.

An antibody that binds to a site on the FGFR2 protein recognized by the humanized antibody FR2-10, FR2-13 or FR2-14 of the present invention is also included in the present invention.

(3-11) Modified antibodies

The invention provides modifications of antibodies or functional fragments thereof. The modification of the antibody or functional fragment thereof of the present invention means that the antibody or functional fragment thereof of the present invention is chemically or biologically modified. The chemical modification system comprises a bond to a chemical moiety of an amino acid backbone, a N-bond or a chemical modification of an O-bonded carbohydrate chain, and the like. Biological modifications include post-translational modifications (eg, addition of a sugar chain to an N-bond or O-bond, processing at the N-terminus or C-terminus, deamination, isomerization of aspartic acid, A Oxidation of thiamin, by using a prokaryotic host The cell is made to be expressed, and the methionine residue is added to the N-terminus. Further, in order to detect the isolated antibody or antigen of the present invention, for example, an enzyme marker, a fluorescent marker, and an affinity marker are also included in the meaning of the modifier. Such a modified antibody of the present invention or a functional fragment thereof is useful for improving the stability and blood retention of the antibody or functional fragment thereof of the present invention, the reduction of antigenicity, the detection of the antibody or antigen, or the single Off.

The chemical moiety contained in the chemically modified body may, for example, be a water-soluble polymer such as polyethylene glycol.

Examples of biological modifications include fusions by enzyme treatment or cell treatment, addition of other peptides such as markers by genetic recombination, and expression of intrinsic or extraneous properties. A cell of a sugar chain-modified enzyme is prepared as a host.

Antibody-dependent cytotoxicity can be enhanced by modulating sugar chain modifications (glycosylation, fucose, etc.) that bind to the antibody of the present invention or a functional fragment thereof. For the method of adjusting the sugar chain modification of an antibody, for example, the methods described in WO99/54342, WO00/61739, WO02/31140, etc. are known, but are not limited thereto. The modified antibody of the present invention also includes the glycochain-modified antibody.

The modification can be administered at any position in the antibody or its functional fragment, or at a desired position, and the same or two or more different modifications can be administered at one or two or more positions.

In the present invention, "a modified form of an antibody fragment" also includes "a fragment of a modified form of an antibody".

In the present invention, sometimes a modified antibody, a functional sheet thereof The modified paragraphs are only referred to as "antibody" and "functional fragments of antibodies".

hFR2-14_H19/L1 is a humanized antibody which has been modified by glycosylation, that is, defucosylated (Example 9 of WO2013/154206), and is included in the antibody of the present invention, a modified form of the antibody of the present invention.

4. Method for producing antibody

(4-1) Method of using fusion tumor

As an aspect of the present invention, the anti-FGFR2 antibody can be, for example, according to the method of Kohler and Milstein (Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennet, R. ed., Monoclonal Antibodies, Obtained by p. 365-367, Plenum Press, NY (1980) (WO 2013/154206).

(4-2) Cellular immunoassay

An anti-FGFR2 antibody can be prepared by the fusion method described above by using a cell expressing a natural FGFR2 protein, a cell expressing a recombinant FGFR2 protein or a fragment thereof, or the like as an immunogen (WO 2013/154206).

(4-3) Gene recombination

The antibody of the present invention can be contained in a nucleotide sequence (heavy chain nucleotide) contained in a nucleotide sequence encoding a heavy chain amino acid sequence thereof and a nucleotide sequence encoding the light chain amino acid sequence thereof a nucleotide (light chain nucleotide), or a vector into which the heavy chain nucleotide is inserted, and a vector into which a light chain nucleotide is inserted are introduced into a host cell, and the cell is cultured and recovered from the culture. The antibody is prepared and the antibody thus obtained is also included in the present invention (WO 2013/154206).

(4-4) DNA immunization method

The anti-FGFR2 antibody of the present invention can also be obtained by DNA immunization. The antigen is expressed in an individual, such as a mouse or a rat, by introducing an antigen into a plastid, thereby causing the antigen to be expressed in the individual, thereby inducing immunity against the antigen. In the method of gene introduction, there is a method of directly injecting a plastid into a muscle, a method of introducing a test substance such as a liposome or a polyethyleneimine into an intravenous injection, a method using a viral vector, and attaching a plastid The gold particles are injected by a gene gun, a hydrodynamic method in which a large amount of a plastid solution is rapidly injected intravenously, and the like.

(4-5) Design method and modulation method of humanized antibody

As the humanized antibody, an antibody which binds only the CDR of the non-human animal antibody to the antibody derived from human can be cited (refer to Nature (1986) 321, p. 522-525); the sequence of the CDR is removed by the CDR grafting method. In addition, a part of the framework amino acid residues are also grafted to human antibody (see WO90/07861, US6972323); one or more of the non-human animal antibodies in any of them. An antibody or the like in which an amino acid is substituted with a human type of amino acid, but is not limited thereto.

(4-6) Modulation of human antibodies

Further, as the antibody of the present invention, a human antibody can be cited. An anti-FGFR2 human antibody means an anti-FGFR2 antibody consisting of an amino acid sequence of an antibody derived from human. The anti-FGFR2 human antibody can be obtained by the following well-known methods (WO 2013/154206): a method of producing a mouse using a human antibody, which produces a human gene having a gene containing a heavy chain and a light chain of a human antibody A DNA fragment; a method for obtaining a human antibody derived from phage display, which is selected from a human antibody library, and the like.

(4-7) Modulation of functional fragments of antibodies

Methods for making single-chain antibodies are well known. In the scFv, the heavy chain variable region and the light chain variable region are linked via a linker which is not a conjugate, preferably via a polypeptide linker. The heavy chain variable region and the light chain variable region in the scFv can be from the same antibody or from individual antibodies. The antibody of the present invention can be multiquantified to increase affinity for an antigen. The multi-quantitative antibody may be one type of antibody or a plurality of antibodies that recognize a plurality of epitopes of the same antigen.

The antibody of the present invention may be a mixture of a plurality of anti-FGFR2 antibodies having different amino acid sequences, that is, a plurality of antibodies.

As the modification of the antibody, an antibody that binds to various molecules such as polyethylene glycol (PEG) can also be used.

The antibody of the present invention can also be further conjugated to other agents by such antibodies. Examples of such antibodies include those in which the antibody is bound to a radioactive substance or a pharmacologically active compound (WO 2013/154206).

(4-8) Purification of antibodies

The obtained antibody can be purified to homogeneity. The isolation and purification of the antibody may be carried out by using a separation or purification method used for a usual protein.

For example, if the column is properly selected, the chromatographic column, the filter, the ultrafiltration, the salting out, the dialysis, the preparation of the polypropylene guanamine colloidal electrophoresis, the isoelectric point electrophoresis, etc., the antibody can be isolated and purified, but is not limited thereto. Wait.

(4-9) Recombinant antibody

The antibody or a functional fragment thereof or a modified form thereof obtained by the method for producing an antibody or a functional fragment thereof or a modified form thereof is also included in the present invention, which comprises culturing an antibody or a function thereof encoding the present invention. A step of recovering an antibody or a functional fragment thereof or a modified form thereof from a culture of a nucleotide (antibody gene) or a vector of the vector of the fragment or a modified form thereof.

5. Pharmaceutical composition

The present invention provides a pharmaceutical composition comprising an anti-FGFR2 antibody or a functional fragment thereof or a modified form thereof.

The pharmaceutical composition of the present invention may be used for excessive expression of FGFR2 or a ligand thereof, or abnormality or hyperstimulation of FGFR2 due to mutation or gene amplification of FGFR2, or due to switching of FGFR2 isoforms. The treatment or prevention of diseases (hereinafter, referred to as "FGFR2-related diseases") is particularly useful for the treatment or prevention of various cancers.

For the cause of the onset or deterioration of cancer which is the subject of the treatment or prevention, one base substitution (SNP) in the intron of the FGFR2 gene, high expression of FGFR2, and activation of FGFR2 are exemplified. Missense mutation, increase or overexpression of FGFR2 gene, switching from FGFR2IIIb to FGFR2IIIc.

Examples of the cancer type include lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, and pancreatic cancer such as breast cancer, endometrial cancer, ovarian cancer, and non-small cell lung cancer. Colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, blood cancer, lymphoma, brain tumor, cholangiocarcinoma, malignant melanoma, etc., preferably those which express FGFR2 protein.

In the present invention, the treatment or prevention of the disease comprises the disease, preferably the prevention, the progression or the suppression or inhibition of the onset of the disease in the individual exhibiting the FGFR2 protein, and the presentation of the individual suffering from the disease. The reduction, deterioration, or suppression or remission of one or more symptoms, treatment or prevention of secondary diseases, etc., are not limited thereto.

The pharmaceutical composition of the present invention may contain a therapeutically or prophylactically effective amount of an anti-FGFR2 antibody or a functional fragment thereof, and a pharmaceutically acceptable diluent, carrier, solubilizing agent, emulsifier, preservative and/or Or adjuvant.

The "therapeutically effective amount" means an amount which exerts a therapeutic or prophylactic effect on a specific disease, a form of administration, and a route of administration, and has the same meaning as a "pharmacologically effective amount".

The pharmaceutical composition of the present invention may contain pH, soaking pressure, viscosity, transparency, color, isotonicity, sterility, stability, solubility, and release properties of the composition or the antibody contained therein, A substance that changes, maintains, and maintains absorbency, permeability, dosage form, strength, properties, shape, etc. (hereinafter referred to as "substance for preparation"). The substance to be used for the preparation is not particularly limited as long as it is a pharmacologically acceptable substance. For example, it is a property suitable for a substance for a non-toxic or low-toxicity preparation.

Examples of the substance for preparation include the following, but are not limited thereto: glycine, alanine, glutamic acid, aspartic acid, histidine, arginine or Amino acids such as lysine; antioxidants, ascorbic acid, sodium sulfate or sodium bisulfite; phosphoric acid, citric acid, boric acid buffer, sodium hydrogencarbonate, hydroxymethylamine methane-hydrochloric acid (Tris) -Hcl) a buffer such as a solution; a filler such as mannitol or glycine; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); caffeine, polyvinylpyrrolidine, β-cyclodextrin or hydroxypropyl Base-β-cyclodextrin Mixture; extender such as glucose, mannose or dextrin; other carbohydrates such as monosaccharides, disaccharides or glucose, mannose or dextrin; colorants, fragrances, diluents, emulsifiers or polyvinylpyrrolidine Hydrophilic polymer; low molecular weight polypeptide, salt forming counter ion, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben a preservative such as propyl paraben, chlorhexidine, sorbic acid or hydrogen peroxide; a solvent such as glycerin, propylene glycol or polyethylene glycol; a sugar alcohol such as mannitol or sorbitol a polysorbate of a suspending agent, PEG, sorbitan ester, polysorbate 20 or polysorbate 80; a surfactant such as Triton, tromethamine, lecithin or cholesterol; An anti-reinforcing agent for sucrose or sorbitol; an elastic enhancer such as sodium chloride, potassium chloride, mannitol or sorbitol; a delivery agent, a diluent, an excipient, and/or a pharmaceutically auxiliary Agent.

The amount of the substance to be used in the preparation is 0.001 to 1,000 times, preferably 0.01 to 100 times, more preferably 0.1 to 10 times, based on the weight of the anti-FGFR2 antibody or its functional fragment or a modified form thereof.

Medicine containing an immunoliposome which binds an anti-FGFR2 antibody or a functional fragment thereof or a modified form thereof to a liposome, and an antibody modified body in which an antibody and a liposome are combined (U.S. Patent No. 6214388, etc.) The composition is also included in the pharmaceutical composition of the present invention.

The excipient or carrier is usually a liquid or a solid, and if it is used for injectable water, physiological saline, artificial cerebrospinal fluid, other oral or parenteral administration, the substance is not used. Specially limited. In the case of physiological saline, a neutral person or a serum albumin-containing person can be cited. Wait.

In the case of the buffer, a Tris buffer having a final pH of the pharmaceutical composition of 7.0 to 8.5, an acetate buffer adjusted to a pH of 4.0 to 5.5, and a citric acid buffer adjusted to a pH of 5.0 to 8.0 in the same manner can be exemplified. The liquid was similarly adjusted to a histidine buffer having a pH of 5.0 to 8.0 or the like.

The pharmaceutical composition of the present invention is a solid, a liquid, a suspension or the like. A freeze-dried preparation can be mentioned. In order to mold the freeze-dried preparation, an excipient such as sucrose may be used.

The administration route of the pharmaceutical composition of the present invention may be any of enteral administration, topical administration, and parenteral administration, and examples thereof include intravenous administration, intra-arterial administration, and muscle administration. Internal administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, intra-articular administration, and the like.

The composition of the pharmaceutical composition can be determined depending on the administration method, the FGFR2 protein binding affinity of the antibody, and the like. The higher the affinity of the anti-FGFR2 antibody of the present invention or a functional fragment thereof or a modified form thereof to the FGFR2 protein (the lower the KD value), the less the administration amount can be exerted.

The dose of the anti-FGFR2 antibody of the present invention is not limited as long as it is a pharmacologically effective amount, and may depend on the type of the individual, the type of the disease, the symptom, the sex, the age, the chronic disease, the FGFR2 protein binding affinity of the antibody or The biological activity and other factors are appropriately determined, but usually 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, administered once every 1 to 180 days, or 2 or more times a day. .

In terms of the form of the pharmaceutical composition, an injection can be exemplified ( Including lyophilized preparation, drip agent, suppository, nasal absorption preparation, transdermal absorption preparation, sublingual preparation, capsule, lozenge, ointment, granule, spray, pill, powder, suspension, emulsion, Eye drops, living implantable preparations, and the like.

The anti-FGFR2 antibody or a functional fragment thereof or a modified form thereof (hereinafter referred to as "anti-FGFR2 antibody or the like") can be used in combination with other agents. The pharmaceutical composition which is anti-FGFR2 or the like or contains the active ingredient as an active ingredient can be administered simultaneously or separately with other pharmaceutical agents (that is, a pharmaceutical composition containing an anti-FGFR2 antibody or the like as an active ingredient). For example, a pharmaceutical composition containing an anti-FGFR2 antibody or the like as an active ingredient may be administered after administration of another pharmaceutical agent, or a pharmaceutical composition containing an anti-FGFR2 antibody or the like as an active ingredient may be administered, and another pharmaceutical agent may be administered or administered simultaneously. A pharmaceutical composition containing an anti-FGFR2 antibody or the like as an active ingredient and other pharmaceutical agents. In the case where the single pharmaceutical composition contains an active ingredient such as an anti-FGFR2 antibody or the like, and the case where the two active ingredients are contained in each of the plurality of pharmaceutical compositions, it is referred to as "containing an antibiotic" in the present invention. A pharmaceutical composition such as an FGFR2 antibody and other pharmaceutical agents. In the present invention, the "pharmaceutical composition" is the same as "a pharmaceutical composition which is administered in combination with other agents such as an anti-FGFR2 antibody."

In the present invention, the anti-FGFR2 antibody and the like are administered in combination with other drugs, and it is meant that an anti-FGFR2 antibody or the like is ingested into the body of the subject with another agent for a certain period of time. A preparation in which an anti-FGFR2 antibody or the like and another agent are contained in a single preparation may be administered, and each of them may be separately formulated and administered separately. In the case of separate formulation, the period of administration is not particularly limited, and it may be administered at the same time, or may be different at intervals. Time or difference will be given. When the anti-FGFR2 antibody or the like is administered separately from the other agents at different times or days, the order of administration is not particularly limited. Usually, the respective preparations are administered in accordance with the respective administration methods, and the cases in which they are administered have the same number of times, and the cases are different. Further, in the case of separate formulation, the administration method (administration route) of each preparation can be the same, and it can also be administered by a different administration method (administration route). Further, there is no need for an anti-FGFR2 antibody or the like to be present in the body simultaneously with other agents, and it is necessary to be between a certain period of time (for example, one month, preferably one week, further preferably several days, further preferably one day). It can be ingested into the body, and the active ingredient of the other one can be eliminated from the body when any one of them is administered.

The administration form of "a pharmaceutical composition to be administered by combining an anti-FGFR2 antibody and the like with another agent" includes, for example, 1) administration of a single preparation containing an anti-FGFR2 antibody and the like, and 2) each The two preparations obtained by formulating the anti-FGFR2 antibody and the like in the same administration route, and the preparation of the two kinds of preparations obtained by separately preparing the anti-FGFR2 antibody and the like are prepared. Administration of a time difference of the same administration route, 4) administration of a different administration route of two preparations obtained by separately preparing an anti-FGFR2 antibody and other agents, and 5) administration of an anti-FGFR2 antibody or the like The two preparations obtained by separately formulating the other medicines are administered with different intervals of time difference between the administration routes and the like. The administration amount, the administration interval, the administration form, the preparation, and the like of the pharmaceutical composition containing the anti-FGFR2 antibody and the like, and the administration amount of the pharmaceutical composition containing the anti-FGFR2 antibody are administered. The interval, the dosage form, the preparation, and the like are preferred, but are not limited thereto.

The pharmaceutical composition is a case in which two different preparations are prepared, and may be a kit containing the same.

In the present invention, the "combination" of the anti-FGFR2 antibody and the like and other agents means that the anti-FGFR2 antibody and the like are combined and administered.

In the present invention, the "other agent" is not particularly limited as long as it is an anticancer agent, and is preferably a chemotherapeutic agent, a molecular standard drug, a cancer therapeutic antibody, a hormone agent, more preferably a topoisomerase inhibitor, a microtube. Inhibitor, platinum preparation, anti-VEGFR2 antibody, antimetabolite. Suitable examples of the topoisomerase inhibitor include Elanodic, Topolecan, Etoposide and the like. Suitable examples of the microtubule inhibitor include paclitaxel, paclitaxel, vincristine, vinblastine, vindesine, eribulin, vinorepine, and paclitaxel micelle NK105. Wait. Suitable examples of the platinum preparation include oxaliplatin, carboplatin, cisplatin, nedaplatin, and cisplatin-containing microcapsule NC-6004. Suitable examples of the anti-VEGFR2 antibody include remollozumab and the like. In the case of an antimetabolite, a 5-fluorouracil-based anticancer agent (for example, 5-FU or a compound which is metabolized to 5-FU in vivo to exert an antitumor effect) is preferred, and a 5-fluorouracil-based anticancer agent is preferred. Suitable examples include fluorouracil (5-FU), tegafur, and tegafur. Gimester. Oturacil potassium (S-1 or TS-1), tegafur. Uracil (UFT), capecitabine, carmofur, deoxyfluorouridine, and the like. However, the "other agents" contained in the combination or pharmaceutical composition of the present invention are not limited thereto.

The combination or pharmaceutical composition of the present invention can be further used Other medicines. Further, for other medicines, various treatment methods such as a vaccine and a radiation therapy (radiation therapy agent) can be cited.

The present invention also provides a method for the treatment or prevention of a disease associated with FGFR such as cancer, a use of the antibody of the present invention for modulating a therapeutic or prophylactic pharmaceutical composition of the disease, and an antibody of the present invention for the treatment or prevention of the disease. use. A therapeutic or prophylactic kit comprising an antibody of the invention is also included in the invention.

[Examples]

Hereinafter, the present invention will be described in more detail with reference to examples but the present invention is not limited thereto.

Further, each operation of the following embodiments may be carried out by a method described in an experimental book used by a person having ordinary knowledge in the technical field of the present invention, or a commercially available reagent or kit, unless otherwise specified. In the case, it is carried out in accordance with the instructions of the commercial products.

Example 1. In vivo combination effect of humanized anti-FGFR2 antibody (hFR2-14_H19/L1) and CPT-11 on FGFR2 expression in gastric cancer cell lines

The human gastric cancer cell line SNU-16 (purchased from ATCC) was adjusted to a concentration of 5 × 10 ⁷ cells/mL with a 1:1 mixture of PBS and Matrigel (purchased from BD Japan), and the cell suspension was 0.1. mL was transplanted subcutaneously into the axilla of nude mice (CAnN.Cg-Foxnl ^nu /CrlCrlj, female, 5-6 weeks old, purchased from Charles River, Japan). Grouped using tumor volume values (n=5 for each group), hFR2-14_H19/L1 (WO2013/154206) was administered to the peritoneal cavity at 10 mg/kg, and CPT-11 was administered to the tail vein at 40 mg/kg for mice. Cast. hFR2-14_H19/L1 was administered 10, 17, and 24 days after transplantation, and CPT-11 was administered 10 days after transplantation. The tumor long diameter (mm) and the short diameter (mm) were measured with an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.) over time, and the tumor volume was calculated by the following calculation formula.

Tumor volume ^{(mm 3) = 1/2} × [ tumor long diameter] × [tumor short axis] × [tumor short axis]

The antitumor effect was evaluated by the tumor growth inhibition rate (TGI%) by the calculation formula shown below after 36 days of transplantation.

TGI (%) = (1-A/B) × 100

A: mean tumor volume on the day of judgment of the compound administration group

B: mean tumor volume on the day of judgment of the non-treated control group

The results of administration of hFR2-14_H19/L1 alone, CPT-11 alone, and hFR2-14_H19/L1 in combination with CPT-11 are shown in Fig. 13. Numerical values represent mean ± standard error (SE). TGI after 36 days of transplantation was 24% compared with hFR2-14_H19/L1 alone, 41% for CPT-11 alone, and 80% for hFR2-14_H19/L1 and CPT-11. The anti-tumor effect is enhanced by the combination.

Example 2. In vivo combination effect of humanized anti-FGFR2 antibody (hFR2-14_H19/L1) and paclitaxel on FGFR2 expression in gastric cancer cell lines

The human gastric cancer cell line SNU-16 (purchased from ATCC) was adjusted to a concentration of 5×10 ⁷ cells/mL with a 1:1 mixture of PBS and a basement membrane matrix (purchased from BD Japan), and 0.1 mL of the cell suspension was transplanted to NOD-scid mice (NOD.CB17-Prkdc ^scid / J, females, 5-6 weeks old, purchased from Japan Charles River) of axillary skin. Grouping was performed using tumor volume values (n=4 for paclitaxel alone, n=5 for the other groups), hFR2-14_H19/L1 was administered to the peritoneal cavity at 10 mg/kg, and paclitaxel was administered at 15 mg/kg. The mice were administered as a tail vein. hFR2-14_H19/L1 was administered 10, 17, and 24 days after transplantation, and paclitaxel was administered 10 days after transplantation. The tumor long diameter (mm) and the short diameter (mm) were measured with an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.) over time, and the tumor volume was calculated by the following calculation formula.

Tumor volume ^{(mm 3) = 1/2} × [ tumor long diameter] × [tumor short axis] × [tumor short axis]

The antitumor effect was evaluated by the tumor growth inhibition rate (TGI%) by the calculation formula shown below after 39 days of transplantation.

TGI (%) = (1-A/B) × 100

A: mean tumor volume on the day of judgment of the compound administration group

B: mean tumor volume on the day of judgment of the non-treated control group

The results of administration of hFR2-14_H19/L1 alone, administration of paclitaxel alone, and administration of hFR2-14_H19/L1 in combination with paclitaxel are shown in Fig. 14. Numerical values represent mean ± standard error (SE). The TGI after 39 days of transplantation was 60% compared with hFR2-14_H19/L1 alone, 53% for paclitaxel alone, and 98% for hFR2-14_H19/L1 combined with paclitaxel. The prior art anti-FGFR2 antibody showed enhanced anti-tumor effect by co-administration with paclitaxel, but did not reach tumor shrinkage (US2015/0050273 A1). On the other hand, in the present invention, in combination with CPT-11 or paclitaxel, a large increase in antitumor activity and tumor shrinkage were observed by the combined effect, particularly in combination with paclitaxel, on the 39th, Tumors completely disappeared in 3 of 5 cases.

Example 3. Humanized anti-FGFR2 antibody (hFR2-14_H19) /L1) In vivo combination with cisplatin on FGFR2 expressing lung cancer cell lines

Human lung cancer cell line KNS-62 (purchased from Japan Health Sciences Foundation, Health Science Research Resources Bank) was adjusted to a ratio of PBS to basement membrane matrix (purchased from BD Japan 1:1) to 1.4 × 10 ⁷ cells/mL. , 0.1mL cell suspension was transplanted into NOD-scid mice (NOD.CB17-Prkdc ^scid / J, females, 5-6 weeks old, purchased from Japan Charles River) of a subcutaneous axillary portion. The tumor volume values were used for grouping (n=5 for each group), hFR2-14_H19/L1 was administered to the peritoneal cavity at 10 mg/kg, and cisplatin was administered to the mice at 5 mg/kg for tail vein. hFR2-14_H19/L1 was administered after 7, 14 and 21 days of transplantation, and cisplatin was administered after 7 and 21 days of transplantation. The tumor long diameter (mm) and the short diameter (mm) were measured with an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.) over time, and the tumor volume was calculated by the following calculation formula.

Tumor volume ^{(mm 3) = 1/2} × [ tumor long diameter] × [tumor short axis] × [tumor short axis]

The antitumor effect was evaluated by the tumor growth inhibition rate (TGI%) by the calculation formula shown below after 36 days of transplantation.

TGI (%) = (1-A/B) × 100

A: mean tumor volume on the day of judgment of the compound administration group

B: mean tumor volume on the day of judgment of the non-treated control group

The results of administration of hFR2-14_H19/L1 alone, cisplatin alone, and hFR2-14_H19/L1 in combination with cisplatin are shown in Fig. 15. Numerical values represent mean ± standard error (SE). The TGI after 36 days of transplantation was 16% compared with hFR2-14_H19/L1 alone, 27% for cisplatin alone, and 52% for hFR2-14_H19/L1 combined with cisplatin. . A no significant difference was observed between the untreated control group and the hFR2-14_H19/L1 and the cisplatin-administered group (t-test, p<0.05), showing the combined effect.

Example 4. In vivo combination effect of humanized anti-FGFR2 antibody (hFR2-14_H19/L1) and cisplatin on FGFR2 expression in gastric cancer cell lines

The human gastric cancer cell line SNU-16 (purchased from ATCC) was adjusted to a concentration of 5×10 ⁷ cells/mL with a 1:1 mixture of PBS and a basement membrane matrix (purchased from BD Japan), and 0.1 mL of the cell suspension was transplanted to Nude mice (CAnN.Cg-Foxnl[nu]/CrlCrlj[Foxnlnu/Foxnlnu], female, 5-6 weeks old, purchased from Charles River, Japan) were subcutaneously subcutaneous. The tumor volume values were grouped (n=8 for each group), and hFR2-14_H19/L1 was administered to the mice as a tail vein at 1 mg/kg and cisplatin at 5 mg/kg. hFR2-14_H19/L1 was administered after 6, 13, and 20 days after transplantation, and cisplatin was administered 6 days after transplantation. The tumor long diameter (mm) and the short diameter (mm) were measured with an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.) over time, and the tumor volume was calculated by the following calculation formula.

Tumor volume ^{(mm 3) = 1/2} × [ tumor long diameter] × [tumor short axis] × [tumor short axis]

The antitumor effect was evaluated by the tumor growth inhibition rate (TGI%) by the calculation formula shown below after 27 days of transplantation.

TGI (%) = (1-A/B) × 100

A: mean tumor volume on the day of judgment of the compound administration group

B: mean tumor volume on the day of judgment of the non-treated control group

The administration of hFR2-14_H19/L1 alone, cisplatin alone, and the combination of hFR2-14_H19/L1 and cisplatin are shown in Figure 17. . Numerical values represent mean ± standard error (SE). The TGI after transplantation for 27 days was 68% compared with hFR2-14_H19/L1 alone, 23% for cisplatin alone, and 87% for hFR2-14_H19/L1 combined with cisplatin. A significant difference was observed between the cisplatin-administered group and the hFR2-14_H19/L1 and cisplatin-administered groups (Dunnett-type multiple comparison test, p<0.0001), showing a combined effect.

Example 5. In vivo combination effect of humanized anti-FGFR2 antibody (hFR2-14_H19/L1) and European paclitaxel on FGFR2 expression in gastric cancer cell lines

The human gastric cancer cell line SNU-16 (purchased from ATCC) was adjusted to a concentration of 5×10 ⁷ cells/mL with a 1:1 mixture of PBS and a basement membrane matrix (purchased from BD Japan), and 0.1 mL of the cell suspension was transplanted to Nude mice (CAnN.Cg-Foxnl[nu]/CrlCrlj[Foxnlnu/Foxnlnu], female, 5-6 weeks old, purchased from Charles River, Japan) were subcutaneously subcutaneous. The tumor volume values were grouped (n=8 for each group), hFR2-14_H19/L1 was administered to the mice at 1 mg/kg, and the European paclitaxel was administered at 1 mg/kg each. hFR2-14_H19/L1 was administered after 6, 13, and 20 days after transplantation, and European paclitaxel was administered 6 days after transplantation. The tumor long diameter (mm) and the short diameter (mm) were measured with an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.) over time, and the tumor volume was calculated by the following calculation formula.

Tumor volume ^{(mm 3) = 1/2} × [ tumor long diameter] × [tumor short axis] × [tumor short axis]

The antitumor effect was evaluated by the tumor growth inhibition rate (TGI%) by the calculation formula shown below after 27 days of transplantation.

TGI (%) = (1-A/B) × 100

A: mean tumor volume on the day of judgment of the compound administration group

B: mean tumor volume on the day of judgment of the non-treated control group

The results of administration of hFR2-14_H19/L1 alone, administration of taxol alone, and administration of hFR2-14_H19/L1 in combination with European paclitaxel are shown in Fig. 18. Numerical values represent mean ± standard error (SE). The TGI after transplantation for 27 days was 68% compared with hFR2-14_H19/L1 alone, 16% for European paclitaxel alone, and 73% for hFR2-14_H19/L1 and European paclitaxel. A significant difference was observed between the European paclitaxel alone administration group and hFR2-14_H19/L1 and the European paclitaxel combined administration group (Dunnett type multiple comparison test, p < 0.005), showing a combined effect.

Example 6. In vivo combination effect of humanized anti-FGFR2 antibody (hFR2-14_H19/L1) and anti-VEGFR2 antibody on FGFR2 expression in gastric cancer cell lines

The human gastric cancer cell line SNU-16 (purchased from ATCC) was adjusted to a concentration of 5×10 ⁷ cells/mL with a 1:1 mixture of PBS and a basement membrane matrix (purchased from BD Japan), and 0.1 mL of the cell suspension was transplanted to Nude mice (CAnN.Cg-Foxnl[nu]/CrlCrlj[Foxnlnu/Foxnlnu], female, 5-6 weeks old, purchased from Charles River, Japan) were subcutaneously subcutaneous. Grouped with tumor volume values (n=8 for each group), hFR2-14_H19/L1 was used at 1 mg/kg, anti-mouse VEGFR2 antibody DC101 (purchased from Bio-X-cell) at 20 mg/kg, each for mice It is administered as a tail vein. hFR2-14_H19/L1 was administered after 6, 13, and 20 days after transplantation, and the anti-VEGFR2 anti-system was administered 6, 9, 13, 16, and 20 days after transplantation. The tumor long diameter (mm) and the short diameter (mm) were measured with an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.) over time, and the tumor volume was calculated by the following calculation formula.

Tumor volume ^{(mm 3) = 1/2} × [ tumor long diameter] × [tumor short axis] × [tumor short axis]

The antitumor effect was evaluated by the tumor growth inhibition rate (TGI%) by the calculation formula shown below after 27 days of transplantation.

TGI (%) = (1-A/B) × 100

A: mean tumor volume on the day of judgment of the compound administration group

B: mean tumor volume on the day of judgment of the non-treated control group

The results of administration of hFR2-14_H19/L1 alone, anti-VEGFR2 antibody alone, and hFR2-14_H19/L1 and anti-VEGFR2 antibody were shown in Fig. 19. Numerical values represent mean ± standard error (SE). The TGI after transplantation for 27 days was 68% compared with hFR2-14_H19/L1 alone, 28% for anti-VEGFR2 antibody alone, and 91% for hFR2-14_H19/L1 and anti-VEGFR2 antibody. An anti-VEGFR2 antibody was administered alone, and hFR2-14_H19/L1 and anti-VEGFR2 antibodies were used in combination with the administration group, and a significant difference was observed (Dunnett type multiple comparison test, p < 0.0001), showing a combined effect.

Example 7. In vivo combination effect of humanized anti-FGFR2 antibody (hFR2-14_H19/L1) and 5-FU on FGFR2 expression in gastric cancer cell lines

The human gastric cancer cell line SNU-16 (purchased from ATCC) was adjusted to a concentration of 5×10 ⁷ cells/mL with a 1:1 mixture of PBS and a basement membrane matrix (purchased from BD Japan), and 0.1 mL of the cell suspension was transplanted to Nude mice (CAnN.Cg-Foxnl[nu]/CrlCrlj[Foxnlnu/Foxnlnu], female, 5-6 weeks old, purchased from Charles River, Japan) were subcutaneously subcutaneous. The tumor volume values were grouped (n=8 for each group), and hFR2-14_H19/L1 was administered to the mice as tail veins at 1 mg/kg and 5-FU at 50 mg/kg. hFR2-14_H19/L1 was administered after 7 and 14 days of transplantation, and 5-FU was administered after 7, 10, 14, and 17 days of transplantation. The tumor long diameter (mm) and the short diameter (mm) were measured with an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.) over time, and the tumor volume was calculated by the following calculation formula.

Tumor volume ^{(mm 3) = 1/2} × [ tumor long diameter] × [tumor short axis] × [tumor short axis]

The antitumor effect was evaluated by the tumor growth inhibition rate (TGI%) by the calculation formula shown below after 21 days of transplantation.

TGI (%) = (1-A/B) × 100

A: mean tumor volume on the day of judgment of the compound administration group

B: mean tumor volume on the day of judgment of the non-treated control group

The results of administration of hFR2-14_H19/L1 alone, 5-FU alone, and hFR2-14_H19/L1 and 5-FU were shown in Fig. 20. Numerical values represent mean ± standard error (SE). The TGI after 21 days of transplantation was 49% compared with hFR2-14_H19/L1 alone, 32% for 5-FU alone, and 79% for hFR2-14_H19/L1 and 5-FU. A 5-FU alone administration group, and hFR2-14_H19/L1 and 5-FU were used in combination with the administration group, and a significant difference was observed (Dunnett type multiple comparison test, p<0.0001), showing the combined effect.

[Industrial availability]

The treatment or prevention of various cancers is possible by the combination of the antibody and other agents provided by the present invention.

[sequence table non-keyword text]

SEQ ID NO: 1: Amino acid sequence of CDR1 of humanized FR2-14 light chain (hFR2-14_L1) (Fig. 1)

SEQ ID NO: 2: Amino acid sequence of CDR2 of humanized FR2-14 light chain (hFR2-14_L1) (Fig. 2)

SEQ ID NO: 3: Amino acid sequence of CDR3 of humanized FR2-14 light chain (hFR2-14_L1) (Fig. 3)

SEQ ID NO: 4: Amino acid sequence of CDR1 of humanized FR2-14 heavy chain (hFR2-14_H19) (Fig. 4)

SEQ ID NO: 5: Amino acid sequence of CDR2 of humanized FR2-14 heavy chain (hFR2-14_H19) (Fig. 5)

SEQ ID NO: 6: Amino acid sequence of CDR3 of humanized FR2-14 heavy chain (hFR2-14_H19) (Fig. 6)

SEQ ID NO: 7: The base sequence of the humanized FR2-14 light chain (hFR2-14_L1). Wherein nucleotide numbers 1 to 60 are sequences encoding a message sequence.

SEQ ID NO: 8: Amino acid sequence of humanized FR2-14 light chain (hFR2-14_L1) (Fig. 7). Among them, amino acid numbers 1 to 20 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_L1.

SEQ ID NO: 9: Base sequence of humanized FR2-14 heavy chain (hFR2-14_H5). Wherein nucleotide numbers 1 to 57 are sequences encoding a message sequence.

SEQ ID NO: 10: Amino acid sequence of humanized FR2-14 heavy chain (hFR2-14_H5) (Fig. 8). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H5.

SEQ ID NO: 11: The base sequence of the humanized FR2-14 heavy chain (hFR2-14_H8). Wherein nucleotide numbers 1 to 57 are sequences encoding a message sequence.

SEQ ID NO: 12: Amino acid sequence of humanized FR2-14 heavy chain (hFR2-14_H8) (Fig. 9). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H8.

SEQ ID NO: 13: Base sequence of humanized FR2-14 heavy chain (hFR2-14_H11). Wherein nucleotide numbers 1 to 57 are sequences encoding a message sequence.

SEQ ID NO: 14: Humanized FR2-14 heavy chain (hFR2-14_H11) amino acid sequence (10). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H11.

SEQ ID NO: 15: The base sequence of the humanized FR2-14 heavy chain (hFR2-14_H12). Wherein nucleotide numbers 1 to 57 are sequences encoding a message sequence.

SEQ ID NO: 16: Amino acid sequence of humanized FR2-14 heavy chain (hFR2-14_H12) (Fig. 11). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H12.

SEQ ID NO: 17: The base sequence of the humanized FR2-14 heavy chain (hFR2-14_H19). Wherein nucleotide numbers 1 to 57 are sequences encoding a message sequence.

SEQ ID NO: 18: Amino acid sequence of humanized FR2-14 heavy chain (hFR2-14_H19) (Fig. 12). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H19.

SEQ ID NO: 19: Base sequence of humanized FR2-14 heavy chain (hFR2-14_H9). Wherein nucleotide numbers 1 to 57 are sequences encoding a message sequence.

SEQ ID NO: 20: Amino acid sequence of humanized FR2-14 heavy chain (hFR2-14_H9) (Fig. 16). Among them, amino acid numbers 1 to 19 are message sequences, and are usually not contained in most of the mature amino acid sequence of hFR2-14_H9.

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<120> Compositions containing anti-FGFR2 antibodies and other agents

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Claims (57)

A combination or pharmaceutical composition comprising a monoclonal antibody or a functional fragment thereof characterized by the following (i) to (ix), and one or more selected from the group consisting of topoisomerase inhibitors a combination of a microtubule inhibitor, a platinum preparation, an anti-VEGFR2 antibody, and a 5-fluorouracil anticancer agent; the pharmaceutical composition comprising the antibody or a functional fragment thereof, and one or more A pharmaceutical composition selected from the group consisting of a topoisomerase inhibitor, a microtubule inhibitor, a platinum preparation, an anti-VEGFR2 antibody, and a 5-fluorouracil anticancer agent: (i) a human type 2 fiber matrix Cell proliferation factor receptor (hFGFR2) IIIb and IIIc specifically bind; (ii) bind to immunoglobulin-like domain 2 or immunoglobulin domain 3 such as hFGFR2; (iii) not to human Type 1 fibroblast growth factor receptor (hFGFR1), human type 3 fibroblast growth factor receptor (hFGFR3) and human type 4 fibroblast growth factor receptor (hFGFR4) specifically bind; (iv) antibody-dependent Sex cytotoxicity Ar cytotoxicity); (v) antibody-dependent cell phagocytosis activity; (vi) neutralizing activity against hFGFR2; (vii) antitumor activity in vivo; (viii) inhibition of FGF a combination of hFGFR2; (ix) is a chimeric antibody, a humanized antibody or a human antibody. A combination or pharmaceutical composition according to claim 1, wherein the antibody comprises the following light chain and heavy chain: CDRL1 comprising the amino acid sequence represented by SEQ ID NO: 1 (Fig. 1) of the Sequence Listing The CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 2 (Fig. 2) of the Sequence Listing and the light of the CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 3 (Fig. 3) of the Sequence Listing a CDRH1 comprising an amino acid sequence represented by SEQ ID NO: 4 (Fig. 4) of the Sequence Listing, and an amino acid sequence represented by SEQ ID NO: 5 (Fig. 5) of the Sequence Listing The CDRH2 consisting of and the amino acid sequence shown in SEQ ID NO: 6 (Fig. 6) of the sequence listing or the amino acid of the first or second group is substituted from the amino terminal of the amino acid sequence. A heavy chain of CDRH3 consisting of an amino acid sequence derived from other amino acids. A combination or pharmaceutical composition according to claim 1 or 2, wherein the anti-system is selected from the following (i) to (vi): (i) a humanized antibody comprising the following light and heavy chains (hFR2-14_H19/L1) : a light chain comprising amino acid numbers 21 to 235 of the amino acid sequence shown in SEQ ID NO: 8 (Fig. 7), and an amine comprising the amino acid sequence shown in SEQ ID NO: 18 (Fig. 12) a heavy chain of base acids Nos. 20 to 467; (ii) a humanized antibody (hFR2-14_H12/L1) comprising the following light and heavy chains: comprising the amino acid sequence shown in SEQ ID NO: 8 (Fig. 7) a light chain of amino acid numbers 21 to 235, and a heavy chain comprising amino acid numbers 20 to 467 of the amino acid sequence shown in SEQ ID NO: 16 (Fig. 11); (iii) a humanized antibody (hFR2-14_H8/L1) comprising the following light and heavy chains: a light chain comprising amino acid numbers 21 to 235 of the amino acid sequence shown in SEQ ID NO: 8 (Fig. 7) And a heavy chain comprising amino acid numbers 20 to 467 of the amino acid sequence shown in SEQ ID NO: 12 (Fig. 9); (iv) a humanized antibody comprising the following light and heavy chains (hFR2-14_H9/ L1): a light chain comprising amino acid numbers 21 to 235 of the amino acid sequence shown in SEQ ID NO: 8 (Fig. 7), and an amino acid sequence including SEQ ID NO: 20 (Fig. 16) a heavy chain of amino acid numbers 20 to 467; (v) a humanized antibody (hFR2-14_H11/L1) comprising the following light and heavy chains: amino acid represented by SEQ ID NO: 8 (Fig. 7) a light chain of amino acid numbers 21 to 235 of the sequence, and a heavy chain of amino acid numbers 20 to 467 comprising an amino acid sequence represented by SEQ ID NO: 14 (Fig. 10); (vi) comprising the following light chain And heavy chain humanized antibody (hFR2-14_H5/L1): a light chain comprising amino acid numbers 21 to 235 of the amino acid sequence shown in SEQ ID NO: 8 (Fig. 7), and comprising the sequence identification number 10 (Figure 8) amine of the amino acid sequence shown An acid number from 20 to 467 of the heavy chain. The combination or pharmaceutical composition of claim 1, wherein the anti-system comprises the following heavy and light chains, and binds to human FGFR2: comprising the antibody according to any one of (i) to (vi) of claim 3 The amino acid sequences of the heavy and light chains are each a heavy chain and a light chain of 95% or more of the same amino acid sequence. A combination or pharmaceutical composition according to claim 1, wherein the antibody or a functional fragment thereof binds to a site on an antigen recognized by the antibody according to any one of (i) to (vi) of claim 2 or 3. The combination or pharmaceutical composition of claim 1, wherein the antibody or a functional fragment thereof, in combination with hFGFR2, competes with the antibody of any one of (i) to (vi) of claim 2 or 3. A combination or pharmaceutical composition comprising a antibody or a functional fragment thereof obtained by the method of steps (i) and (ii) below, and a combination thereof, the pharmaceutical composition comprising the antibody or Functional fragment and other agents: (i) a step of culturing the cells described in (a) or (b) below; (a) introducing a nucleotide inserted in any one of the following (a) to (c); Recombinant vector or recombinant cell of the nucleotide: (a) a base comprising an amino acid sequence encoding one or all of the heavy or light chain of the antibody of any one of claims 1 to 6. a sequence of nucleotides; (b) consisting of a base sequence comprising the base sequence of one or all of the amino acid sequences encoding the heavy or light chain of the antibody of any one of claims 1 to 6. (c) a nucleotide consisting of the base sequence of one or all of the amino acid sequences encoding the heavy or light chain of the antibody of any one of claims 1 to 6; B) a cell which produces the antibody of any one of claims 1 to 6 or a functional fragment thereof; and (ii) a culture recovered from the aforementioned step (i) as claimed in claim 1 The step of the antibody or functional fragment thereof according to any one of the six. A combination or pharmaceutical composition according to any one of claims 1 to 7, which has 1 to 5 amino acid deletions at the amino terminus or carboxy terminus of the heavy or light chain of the antibody. A combination or pharmaceutical composition, which is a combination of an antibody of any one of claims 1 to 8 or a functional group thereof, and a combination of other pharmaceutical agents; the pharmaceutical composition comprising the modified body and other pharmaceutical agents Pharmaceutical composition. A combination or pharmaceutical composition according to any one of claims 1 to 9 for use in the treatment or prevention of cancer. A combination or pharmaceutical composition according to claim 10, wherein the cancer is positive for hFGFR2. The combination or pharmaceutical composition of any one of claims 1 to 11, wherein the antibody or a functional fragment thereof is further conjugated to the compound. The combination or pharmaceutical composition of any one of claims 1 to 12, wherein the other agent is a topoisomerase inhibitor. A pharmaceutical composition for use in combination with a topoisomerase inhibitor, and comprising the antibody or functional fragment thereof contained in the combination or composition according to any one of claims 1 to 8 or as claimed A combination of 9 or a modification contained in the composition. A pharmaceutical composition comprising a topoisomerase inhibitor, and the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a combination thereof as claimed in claim 9 Or the modified body contained in the composition is used in combination to make the antibody, the functional fragment or the repair The effect of the trim is rising. A pharmaceutical composition comprising the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification contained in the combination or composition of claim 9 and borrowed The action of the topoisomerase inhibitor is increased by the combination with a topoisomerase inhibitor. A pharmaceutical composition characterized by the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification or a composition contained in the combination or composition of claim 9, And a combination of topoisomerase inhibitors is administered. The pharmaceutical composition of claim 17, wherein the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the modification or composition contained in the combination or composition of claim 9 And the topoisomerase inhibitors are each contained in each preparation as an active ingredient, and administered at the same time or at different times. The pharmaceutical composition of claim 17, wherein the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the modification or composition contained in the combination or composition of claim 9 And topoisomerase inhibitors are contained in a single preparation as an active ingredient. A method of treating cancer, which comprises the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification contained in the combination or composition of claim 9 And a combination of topoisomerase inhibitors are administered. The pharmaceutical composition according to any one of claims 13 to 19, or the therapeutic method of claim 20, wherein the topoisomerase inhibitor is selected from the group consisting of One or more of the group consisting of irinotecan, topotecan, and etoposide. The combination or pharmaceutical composition of any one of claims 1 to 12, wherein the other agent is a microtubule inhibitor. A pharmaceutical composition for use in combination with a microtubule inhibitor, and comprising the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a combination of claim 9 or A modification contained in the composition. A pharmaceutical composition comprising a microtubule inhibitor and comprising or consisting of an antibody or a functional fragment thereof contained in a combination or composition according to any one of claims 1 to 8 or a claim 9 The modified body contained in the substance is used in combination to increase the action of the antibody, the functional fragment or the modified form. A pharmaceutical composition comprising the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification contained in the combination or composition of claim 9 and borrowed The microtubule inhibitor is used in combination with a microtubule inhibitor to increase the action of the microtubule inhibitor. A pharmaceutical composition characterized by the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification or a composition contained in the combination or composition of claim 9, It is administered in combination with a microtubule inhibitor. The pharmaceutical composition of claim 26, wherein the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the modification or composition contained in the combination or composition of claim 9 And microtubule inhibitors are each contained in each preparation as an active ingredient, in the same At the time or at different times. The pharmaceutical composition of claim 26, wherein the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the modification or composition contained in the combination or composition of claim 9 And microtubule inhibitors are contained in a single preparation as an active ingredient. A method of treating cancer, which comprises the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification contained in the combination or composition of claim 9 And administration of microtubule inhibitors in combination. The pharmaceutical composition according to any one of claims 22 to 28, wherein the microtubule inhibitor is selected from the group consisting of paclitaxel, docetaxel, vincristine, One or more of the group consisting of vinblastine, eribulin, vindesine, and vinorelbine. A combination or pharmaceutical composition according to any one of claims 1 to 12, wherein the other agent is a platinum preparation. A pharmaceutical composition for use in combination with a platinum preparation, and comprising the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the combination or composition of claim 9 The modifications contained in the product. A pharmaceutical composition comprising a platinum preparation, and comprising the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a combination or composition as claimed in claim 9 The modified body is used in combination to effect the antibody, the functional fragment or the modified body rise. A pharmaceutical composition comprising the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification contained in the combination or composition of claim 9 and borrowed The effect of the platinum preparation is increased by the use in combination with a platinum preparation. A pharmaceutical composition characterized by the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification or a composition contained in the combination or composition of claim 9, It is administered in combination with a platinum preparation. The pharmaceutical composition of claim 35, wherein the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the modification or composition contained in the combination or composition of claim 9 And the platinum preparations are each contained in each preparation as an active ingredient, and administered at the same time or at different times. The pharmaceutical composition of claim 35, wherein the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the modification or composition contained in the combination or composition of claim 9 And the platinum preparation is contained in a single preparation as an active ingredient. A method of treating cancer, which comprises the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification contained in the combination or composition of claim 9 And a combination of platinum preparations for administration. The pharmaceutical composition according to any one of claims 31 to 37, wherein the platinum preparation is selected from the group consisting of cisplatin, oxaliplatin, carboplatin and nedaplatin. Group of One or two or more. The combination or pharmaceutical composition of any one of claims 1 to 12, wherein the other agent is an anti-VEGFR2 antibody. A pharmaceutical composition for use in combination with an anti-VEGFR2 antibody, and comprising the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a combination or composition of claim 9 A modification contained in the substance. A pharmaceutical composition comprising an anti-VEGFR2 antibody, and the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a combination or composition according to claim 9 The modified body is used in combination to increase the action of the antibody, the functional fragment or the modified body. A pharmaceutical composition comprising the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification contained in the combination or composition of claim 9 and borrowed The effect of the anti-VEGFR2 antibody is increased by the combination with an anti-VEGFR2 antibody. A pharmaceutical composition characterized by the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification or a composition contained in the combination or composition of claim 9, And the anti-VEGFR2 antibody is administered in combination. The pharmaceutical composition of claim 44, wherein the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the modification or composition contained in the combination or composition of claim 9 And the anti-VEGFR2 anti-system are each contained in each preparation as an active ingredient, and administered at the same time or at different times. The pharmaceutical composition of claim 44, wherein the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the modification or composition contained in the combination or composition of claim 9 And the anti-VEGFR2 anti-system is contained as a active ingredient in a single preparation. A method of treating cancer, which comprises the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification contained in the combination or composition of claim 9 And administered in combination with an anti-VEGFR2 antibody. The pharmaceutical composition according to any one of claims 40 to 46, wherein the anti-VEGFR2 antibody is ramucirumab. The combination or pharmaceutical composition according to any one of claims 1 to 12, wherein the other agent is a 5-fluorouracil-based anticancer agent. A pharmaceutical composition for use in combination with a 5-fluorouracil-based anticancer agent, and comprising the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or as claimed in claim 9 A combination or a modification contained in the composition. A pharmaceutical composition comprising a 5-fluorouracil-based anticancer agent, and the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a combination thereof as claimed in claim 9 Or the modified body contained in the composition is used in combination to increase the action of the antibody, the functional fragment or the modified body. A pharmaceutical composition comprising the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification contained in the combination or composition of claim 9 and borrowed By 5-fluorourine The pyrimidine anticancer agent is used in combination to increase the action of the 5-fluorouracil anticancer agent. A pharmaceutical composition characterized by the antibody or a functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification or a composition contained in the combination or composition of claim 9, And a 5-fluorouracil-based anticancer agent is administered in combination. The pharmaceutical composition of claim 53, wherein the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the modification or composition contained in the combination or composition of claim 9 And 5-fluorouracil-based anticancer agents are each contained in each preparation as an active ingredient, and administered at the same time or at different times. The pharmaceutical composition of claim 53, wherein the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or the modification or composition contained in the combination or composition of claim 9 And a 5-fluorouracil-based anticancer agent is contained as a active ingredient in a single preparation. A method of treating cancer, which comprises the antibody or functional fragment thereof contained in the combination or composition of any one of claims 1 to 8 or a modification contained in the combination or composition of claim 9 And a 5-fluorouracil-based anticancer agent is administered in combination. The pharmaceutical composition according to any one of claims 49 to 55, wherein the 5-fluorouracil anticancer agent is fluorouracil, tegafur, tegafur. Gimester (gimeracil). Oteracil potassium, tegafur. Uracil, capecitabine, carmofur, doxifluridine.