RU2834309C2 - Fgfr2 inhibitors alone or in combination with immunostimulating agents in treating cancer - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are methods of treating stomach cancer in a subject, involving administering an antibody to fibroblast growth factor receptor 2 (FGFR2-IIIb) in a combination with nivolumab or pembrolizumab.

EFFECT: invention can find further application in cancer therapy.

44 cl, 32 dwg, 3 tbl, 8 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to the following three United States Provisional Patent Applications: Ser. No. 62/258,731, filed November 23, 2015, Ser. No. 62/314,174, filed March 28, 2016, and Ser. No. 62/379,094, filed August 24, 2016, each of which is incorporated herein by reference in its entirety.

SEQUENCE LIST

[002] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named "2016-11-17_01134-0046-00PCT_SeqList_ST25.txt" created on November 17, 2016, which has a size of 103,517 bytes. The information in the electronic sequence listing format is incorporated herein by reference in its entirety.

FIELD OF INVENTION

[003] This application relates to the use of fibroblast growth factor receptor 2 (FGFR2) inhibitors in the treatment of cancer, in some cases in combination with immunostimulatory agents such as PD-1 or PD-L1 inhibitors.

LEVEL OF TECHNOLOGY

[004] Members of the fibroblast growth factor (FGF) family bind to four known tyrosine kinase receptors, fibroblast growth factor receptors 1-4 (FGFR1-4), and their isoforms, with different FGFs binding different FGFRs to varying degrees (Zhang et al., J. Biol. Chem. 281:15694, 2006). The human FGFR2 protein sequence is provided, for example, at GenBank Locus AF487553. Each FGFR consists of an extracellular domain (ECD) containing three immunoglobulin (Ig)-like domains (D1, D2, and D3), one transmembrane helix, and an intracellular catalytic kinase domain (Mohammadi et al., Cytokine Growth Factor Revs, 16:107, 2005). FGFs bind to receptors primarily through regions in the D2 and D3 receptors. The link between D1 and D2 contains a continuous stretch of acidic amino acids called the acidic block (AB). The region containing D1 and AB is thought to be involved in autoinhibition of the receptor, which is released by ligand binding.

[005] FGFRs are characterized by multiple alternative splicing of their mRNAs, resulting in multiple isoforms (Ornitz et al., J. Biol. Chem. 271: 15292, 1996; see also Swiss-Prot P21802 and isoforms P21802-1-20 for sequences of FGFR2 and its isoforms). Notably, there are forms containing all three Ig domains (α-isoform) or only the two Ig domains D2 and D3 without D1 (β-isoform). In FGFR1, FGFR2, and FGFR3, all forms contain the first half of D3, designated IIIa, but two alternative exons can be used for the second half of D3, resulting in forms IIIb and IIIc. For FGFR2, they are respectively designated FGFR2-IIIb and FGFR2-IIIc (or just FGFR2b and FGFR2c); the corresponding beta forms are designated FGFR2(beta)IIIb and FGFR2(beta)IIIc. The FGFR2-IIIb form of FGFR2 (also designated K-sam-II) is a high-affinity receptor for both FGF1 and KGF (keratinocyte growth factor) family members (FGF7, FGF10, and FGF22), whereas FGFR2-IIIc (also designated K-sam-I) binds both FGF1 and FGF2 but does not bind KGF family members (Miki et al., Proc. Natl. Acad. Sci. USA 89:246, 1992). Indeed, FGFR2-IIIb is the only receptor for members of the KGF family (Ornitz et al., 1996, op. cit.) and is therefore also designated KGFR.

[006] FGFRs and their isoforms are differentially expressed in different tissues. FGFR2-IIIb (and the IIIb forms of FGFR1 and FGFR3) are expressed in epithelial tissues, whereas FGFR2-IIIc is expressed in mesenchymal tissues (Duan et al., J. Biol. Chem. 267:16076, 1992; Ornitz et al., 1996, op. cit.). Some of the FGF ligands of these receptors have opposite patterns of expression. Thus, members of the KGF subfamily, including FGF7 (KGF), FGF10, and FGF22, bind only to FGFR2-IIIb (Zhang et al., op. cit .) and are expressed in mesenchymal tissues, and so may be paracrine effectors of epithelial cells (Ornitz et al., 1996, op. cit .). In contrast, members of the FGF4 subfamily FGF4-6 bind to FGFR2-IIIc and are expressed in both epithelial and mesenchymal cell lines, and so may have either autocrine or paracrine functions. Based on the expression profiles of FGFR2 isoforms and their ligands, FGFR2 plays a role in epithelial-mesenchymal interactions (Finch et al., Dev. Dyn. 203: 223, 1995), so it is not surprising that knockout of FGFR2-IIIb in mice results in severe embryonic defects and lethality (De Moerlooze et al., Development 127: 483, 2000).

[007] KGF (FGF7) and KGFR (FGFR2-IIIb) are overexpressed in many pancreatic cancers (Ishiwata et al., Am. J. Pathol. 153: 213, 1998), and their coexpression correlates with poor prognosis (Cho et al., Am. J. Pathol. 170:1964, 2007). Somatic mutations in the FGFR2 gene have been found in 12% of a large group of endometrial (uterine) carcinomas and in several cases were required for tumor cell survival (Dutt et al., Proc. Natl. Acad. Sci. USA 105:8713, 2008). In two tumors, the FGFR2 mutation was found to be the same S252W substitution associated with Apert syndrome. Amplification and overexpression of FGFR2 is associated with undifferentiated diffuse type gastric cancer, which has a particularly poor prognosis, and inhibition of FGFR2 activity by small molecules strongly inhibits the proliferation of such cancer cells (Kunii et al., Cancer Res. 68:2340, 2008; Nakamura et al., Gastroenterol. 131:1530, 2006).

[008] FGFR2 inhibitors may include antibodies and FGFR2 ECD domains or FGFR2 ECD fusion molecules. For example, U.S. Patent No. 8,101,723 B2 describes, for example, monoclonal antibodies that bind human FGFR2-IIIb but bind less well or do not bind FGFR2-IIIc, and vice versa. U.S. Patent Publication No. 2015-0050273 A1 describes certain afucosylated antibodies that bind FGFR2-IIIb. U.S. Patent Publication No. US 2013-0324701 A1 describes, for example, specific FGFR2 ECD fusion molecules comprising the extracellular domain of FGFR2-IIIc and a fusion partner. Additional FGFR ECD fusion molecules are described in U.S. Patent No. 8,338,569 B2.

[009] Genetic alterations in cancer provide a diverse repertoire of antigens that can mediate antitumor immunity. Antigen recognition via T cell receptors (TCRs) initiates T cell responses that are regulated by a balance between activating and inhibitory signals. Inhibitory signals, or “immune checkpoints,” play an important role in normal tissues by preventing autoimmunity. Enhanced expression of immune checkpoint proteins may allow cancers to evade antitumor immunity. Two immune checkpoint proteins have been the target of clinical cancer immunotherapies, cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1). An anti-CTLA-4 antibody and an anti-PD-1 antibody have been approved for the treatment of metastatic melanoma and are currently in clinical trials for other cancers. An anti-PD-L1 antibody targeting the ligand for PD-1 is also currently in clinical trials.

[0010] Inhibition of FGFR signaling has been reported to improve antitumor immunity and reduce metastasis in breast cancer. ( See, e.g., T. Ye et al., Breast Cancer Res. Treat. 143: 435-446 (2014)) For example, anti-FGFR2 antibodies have also been studied in gastric cancer models. However, it was unknown whether co-administration of an FGFR2 inhibitor with an immune checkpoint inhibitor, such as a PD-1 or PD-L1 inhibitor, could further improve treatment in a tumor model. The present inventors demonstrated that a combination of an FGFR2 inhibitory antibody and a PD-1 inhibitory antibody exhibited at least additive effects in a mouse breast tumor model. The inventors also demonstrate that treatment with an FGFR2 inhibitory antibody alone results in an increase in PD-L1-expressing cells, NK cells, and CD3+, CD8+, and CD4+ T cells in tumor tissue in a mouse breast tumor model and results in an increase in the ratio of lymphoid to myeloid cells in tumor tissue. Furthermore, a single FGFR2 inhibitor also provides benefit in a human patient with bladder cancer when administered alone. Taken together, the results herein indicate that FGFR2 inhibitors can alter the tumor microenvironment and, therefore, can enhance tumor-killing immune responses, either alone or in combination with a PD-1/PD-L1 inhibitor.

ESSENCE OF THE INVENTION

[0011] In some embodiments, methods are provided for treating a cancer in a subject comprising administering to the subject an FGFR2 inhibitor, such as an anti-FGFR2 antibody or an FGFR2 ECD, or an FGFR2 ECD fusion molecule, in combination with at least one immunostimulatory agent. In some embodiments, the immunostimulatory agent is a PD-1/PD-L1 inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, a PD-1 fusion molecule, or a PD-1 polypeptide. In some embodiments, the immunostimulatory agent includes one or more agents described in the section below entitled "combinations with other immunostimulatory agents." In some embodiments, the FGFR2 inhibitor is an antibody. In some embodiments, the FGFR2 inhibitor is an antibody that recognizes FGFR2-IIIb. In some embodiments, the FGFR2-IIIb antibody binds with lower affinity to FGFR2-IIIc than to -IIIb, or does not detectably bind to FGFR2-IIIc. In some embodiments, the FGFR2 inhibitor is an FGFR2 ECD. In some embodiments, the FGFR2 inhibitor is an FGFR2 ECD fusion molecule comprising an FGFR2 ECD and a fusion partner such as an Fc domain, albumin, or polyethyleneglycol (PEG). In some embodiments, wherein the at least one immunostimulatory agent comprises a PD-1/PD-L1 inhibitor, the PD-1/PD-L1 inhibitor is an antibody. In some embodiments, the PD-1/PD-L1 inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the PD-1/PD-L1 inhibitor is a PD-1 polypeptide, while in some embodiments, the PD-1/PD-L1 inhibitor is a PD-1 fusion molecule.

[0012] In any of the embodiments of the methods and compositions herein, the PD-1/PD-L1 inhibitor may have the following characteristics. In some embodiments, the inhibitor is an anti-PD-1 antibody comprising the CDRs of a heavy chain and a light chain of an antibody selected from nivolumab, pidilizumab, and pembrolizumab. In some embodiments, the anti-PD-1 antibody comprises the variable regions of a heavy chain and a light chain of an antibody selected from nivolumab, pidilizumab, and pembrolizumab. In some embodiments, the anti-PD-1 antibody is selected from nivolumab, pidilizumab, and pembrolizumab. In some embodiments, the PD-1/PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody comprises the heavy chain and light chain CDRs of an antibody selected from BMS-936559, MPDL3280A (atezolizumab), MEDI4736, and MSB0010718C (avelumab). In some embodiments, the anti-PD-L1 antibody comprises the heavy chain and light chain variable regions of an antibody selected from BMS-936559, MPDL3280A, MEDI4736, and MSB0010718C. In some embodiments, the anti-PD-L1 antibody is selected from BMS-936559, MPDL3280A, MEDI4736, and MSB0010718C. In some embodiments, the PD-1/PD-L1 inhibitor is a fusion molecule. In some embodiments, the fusion molecule is AMP-224. In some embodiments, the PD-1/PD-L1 inhibitor is a PD-1 polypeptide, such as AUR-012.

[0013] In any of the compositions or methods described herein that utilize an anti-PD-1 antibody, the anti-PD-1 antibody can be a humanized antibody. In any of the compositions or methods described herein, the anti-PD-1 antibody can be selected from Fab, Fv, scFv, Fab'', and (Fab') ₂ . In any of the compositions or methods described herein, the anti-PD-1 antibody can be a chimeric antibody. In any of the compositions or methods described herein, the anti-PD-1 antibody can be selected from IgA, IgG, and IgD. In any of the compositions or methods described herein, the anti-PD-1 antibody can be IgG. In any of the methods described herein, the antibody can be IgG1 or IgG2.

[0014] In any of the compositions or methods described herein, an FGFR2 inhibitor may have the following characteristics. In some embodiments, the inhibitor is an anti-FGFR2 antibody. In some embodiments, the anti-FGFR2 antibody is an anti-FGFR2-IIIb antibody (also referred to herein as αFGFR2b). In some embodiments, the anti-FGFR2-IIIb antibody binds to FGFR2-IIIb with higher affinity than to FGFR2-IIIc, or alternatively does not bind to FGFR2-IIIc. In some embodiments, the antibody inhibits the binding of FGF2 and/or FGF7 to FGFR2.

[0015] In some embodiments, the FGFR2 antibody has the amino acid sequences of the heavy and light chain hypervariable regions (HVRs) H1, H2, H3, L1, L2, and L3 of the GAL-FR21, GAL-FR22, or GAL-FR23 monoclonal antibodies described in U.S. Patent No. 8,101,723 B2. In some embodiments, the heavy chain variable region of the anti-FGFR2-IIIb antibody comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the light chain variable region comprises: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0016] In some embodiments, an anti-FGFR2 antibody comprises an anti-FGFR2-IIIb antibody wherein the heavy chain variable domain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4, or that comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, an anti-FGFR2 antibody comprises an anti-FGFR2-IIIb antibody wherein the light chain variable domain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 5, or that comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the heavy chain variable domain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4, or that comprises the amino acid sequence of SEQ ID NO: 4, and a light chain variable domain that is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 5, or that comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-FGFR2 antibody comprises an anti-FGFR2-IIIb antibody wherein the heavy chain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2, or that comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-FGFR2 antibody comprises an anti-FGFR2-IIIb antibody wherein the light chain is at least at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 3, or that comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the heavy chain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2, or that comprises the amino acid sequence of SEQ ID NO: 2, and the light chain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 3, or that comprises the amino acid sequence of SEQ ID NO: 3.

[0017] In some embodiments, the heavy chain variable region of the anti-FGFR2-IIIb antibody comprises: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 40; (ii) a CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and (iii) a CDR3 comprising the amino acid sequence of SEQ ID NO: 42; and the light chain variable region comprises: (iv) a CDR1 comprising the amino acid sequence of SEQ ID NO: 44; (v) a CDR2 comprising the amino acid sequence of SEQ ID NO: 45; and (vi) a CDR3 comprising the amino acid sequence of SEQ ID NO: 46.

[0018] In some embodiments, an anti-FGFR2 antibody comprises an anti-FGFR2-IIIb antibody wherein the heavy chain variable domain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 39, or that comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, an anti-FGFR2 antibody comprises an anti-FGFR2-IIIb antibody wherein the light chain variable domain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 43, or that comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the heavy chain variable domain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 39, or which comprises the amino acid sequence of SEQ ID NO: 39, and the light chain variable domain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 43, or which comprises the amino acid sequence of SEQ ID NO: 43.

[0019] In some embodiments, the anti-FGFR2 antibody is afucosylated. In some embodiments, the antibody does not comprise a fucose at Asn297. In some embodiments, the antibody comprises a kappa light chain constant region. In some embodiments, the antibody comprises an IgG1 heavy chain constant region. In some embodiments, the afucosylated antibody has enhanced ADCC (antibody-dependent cellular cytotoxicity) activity in vitro and/or in vivo compared to an antibody having the same amino acid sequence that is fucosylated at Asn297. In some embodiments, the afucosylated antibody has enhanced affinity for Fc gamma RIIIA compared to an antibody having the same amino acid sequence that is fucosylated at Asn297. In some embodiments, the afucosylated antibody is capable of increasing the number of one or more of: PD-L1-positive cells, NK cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and macrophages in tumor tissue in a mouse xenograft tumor model and/or a syngeneic tumor model compared to a control (e.g., compared to a control antibody that does not target FGFR2).

[0020] In some embodiments, the FGFR2 inhibitor is an FGFR2 ECD, such as an FGFR2 ECD fusion molecule. The FGFR2 ECD fusion molecules may comprise fusion partners such as an Fc domain, albumin, or PEG.

[0021] In some embodiments, the FGFR2 inhibitor is capable of binding to FGFR2 as well as to a mutant of FGFR2 with an activating mutation, such as the FGFR2-S252W mutation, which is found in some cancer cells.

[0022] In any of the compositions or methods described herein that utilize an anti-FGFR2 antibody, the anti-FGFR2 antibody can be a humanized antibody. In any of the compositions or methods described herein, the anti-FGFR2 antibody can be selected from Fab, Fv, scFv, Fab'', and (Fab') ₂ . In any of the compositions or methods described herein, the anti-FGFR2 antibody can be a chimeric antibody. In any of the compositions or methods described herein, the anti-FGFR2 antibody can be selected from IgA, IgG, and IgD. In any of the compositions or methods described herein, the anti-FGFR2 antibody can be IgG. In any of the methods described herein, the antibody can be IgG1, IgG2, IgG3, or IgG4.

[0023] In some embodiments, the FGFR2 inhibitor is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 20, or 30 mg/kg, or a range limited by any two of these doses. In some embodiments, the PD-1/PD-L1 inhibitor is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, or 10 mg/kg, or a range limited by any two of these doses, such as a range of 0.5-10 mg/kg. In some embodiments, the FGFR2 inhibitor and at least one immunostimulatory agent, such as a PD-1/PD-L1 inhibitor, are administered at least once every 1, 2, 3, 4, or 5 weeks.

[0024] In some embodiments, the cancer overexpresses FGFR2IIIb, either with or without amplification of the FGFR2 gene. In some embodiments, overexpression of FGFR2IIIb is detected using immunohistochemistry (IHC). For example, overexpression can be detected using an IHC signal of 1+, 2+, or 3+ in at least 10% of the tumor cells, such as at least 20%, 30%, 40%, or 50% of the tumor cells.

[0025] In some embodiments, the cancer is selected from gastric cancer, breast cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, cholangiocarcinoma, esophageal cancer (including gastroesophageal junction adenocarcinoma), and endometrial cancer. In some embodiments, the cancer is recurrent or progressive following therapy selected from surgery, chemotherapy, radiation therapy, or a combination thereof. In some embodiments, the subject is a subject who has insufficiently responded to treatment with a PD-1/PD-L1 inhibitor. In some embodiments, the subject has previously received treatment with a PD-1/PD-L1 inhibitor.

[0026] In some embodiments, the method of treating cancer further comprises administering at least one additional therapeutic agent selected from a platinum-based drug, paclitaxel, ABRAXANE®, docetaxel, gemcitabine, capecitabine, irinotecan, epirubicin, FOLFOX, FOLFIRI, leucovorin, fluorouracil, mitomycin C, and doxorubicin hydrochloride. In some embodiments, the platinum-based drug is selected from cisplatin, oxaliplatin, and carboplatin. In some embodiments, the method of treating cancer further comprises administering paclitaxel. In some embodiments, the method of treating cancer further comprises administering cisplatin and/or 5-FU.

[0027] In some embodiments, the FGFR2 inhibitor and the PD-1/PD-L1 inhibitor are administered simultaneously or sequentially. In some embodiments, the FGFR2 inhibitor and the immunostimulatory agent are administered simultaneously. In some embodiments, one or more doses of the immunostimulatory agent are administered prior to administration of the FGFR2 inhibitor. In some embodiments, the subject has received a full course of therapy with the immunostimulatory agent prior to administration of the FGFR2 inhibitor. In some embodiments, the FGFR2 inhibitor is administered during a second course of therapy with the immunostimulatory agent. In some embodiments, the subject has received at least one, at least two, at least three, or at least four doses of the immunostimulatory agent prior to administration of the FGFR2 inhibitor. In some embodiments, at least one dose of the immunostimulatory agent is administered concomitantly with the FGFR2 inhibitor. In some embodiments, the one or more doses of the FGFR2 inhibitor are administered prior to administration of the immunostimulatory agent. In some embodiments, the subject may receive at least two, at least three, at least three, or at least four doses of the FGFR2 inhibitor prior to administration of the immunostimulatory agent. In some embodiments, at least one dose of the FGFR2 inhibitor is administered concurrently with the immunostimulatory agent.

[0028] In some embodiments, administration of an FGFR2 inhibitor and a PD-1/PD-L1 inhibitor in a mouse xenograft tumor model and/or a syngeneic tumor model results in additive or synergistic inhibition of tumor growth. In some embodiments, the model is a breast cancer model. In some embodiments, the model comprises 4T1 cells.

[0029] In any of the above embodiments, the combination of an FGFR2 inhibitor and a PD-1/PD-L1 inhibitor can inhibit tumor growth in a mouse xenograft tumor model and/or a syngeneic tumor model for at least 1 week, 10 days, or 2 weeks, such as by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In any of the above embodiments of the invention, a combination of an FGFR2 inhibitor and an immunostimulatory agent, such as a PD-1/PD-L1 inhibitor, in a subject can reduce the volume of at least one tumor in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as over at least one month, two months, three months, six months, or one year.

[0030] In any of the above embodiments, administration of an FGFR2 inhibitor to a xenograft tumor model and/or a syngeneic tumor model may demonstrate an increase in NK cells, such as NKp46+ cells, an increase in PD-L1-expressing cells, an increase in macrophages, such as F480+ macrophages, an increase in one or more of CD3+, CD8+, and CD4+ T cells, and/or an increase in the ratio of lymphoid cells to myeloid cells in tumor tissue compared to a control over a period of at least 1 day, at least 4 days, at least 1 week, at least 10 days, or at least 2 weeks, and, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the mouse syngeneic tumor model is a 4T1 mammary tumor model. In some embodiments, the control is a vehicle or is an Ig-Fc molecule or other compound that does not inhibit tumor growth in the model.

[0031] Also provided herein are methods for increasing the number of NK cells, PD-L1-positive cells, and/or CD3+, CD8+, and/or CD8+ T cells, and/or macrophages in tumor tissue of a subject with cancer, and/or methods for increasing the ratio of lymphoid cells to myeloid cells in tumor tissue of a subject with cancer, comprising administering to said subject an effective amount of an inhibitory antibody to FGFR2, such as any of the antibodies to FGFR2 described in the preceding paragraphs. An increase in one or more of: CD3+, CD8+, and CD4+ T cells, and/or an increase in the ratio of lymphoid cells to myeloid cells in tumor tissue compared to a control for at least 1 day, at least 4 days, at least 1 week, at least 10 days, or at least 2 weeks, and such as by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the antibody may have one or more of the following properties: (a) lacks fucose at position Asn297; (b) comprises a κ light chain constant region; (c) comprises an IgG1 heavy chain constant region; (d) has enhanced ADCC activity in vitro compared to an antibody having the same amino acid sequence that is fucosylated at position Asn297; and (e) has enhanced affinity for Fc gamma RIIIA compared to an antibody having the same amino acid sequence that is fucosylated at position Asn297. In some embodiments, the afucosylated antibody is capable of increasing the number of one or more of PD-L1-positive cells, NK cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and macrophages in tumor tissue in a mouse xenograft tumor model and/or a syngeneic tumor model compared to a control (e.g., compared to a control antibody that does not target FGFR2). In some embodiments, the method inhibits tumor growth or reduces the volume of at least one tumor in a subject. In some embodiments, the subject suffers from breast cancer, gastric cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, cholangiocarcinoma, esophageal cancer (including gastroesophageal junction adenocarcinoma), and endometrial cancer. In some embodiments, the method further comprises obtaining at least one tumor sample from the subject, after administration of the anti-FGFR2 antibody, and determining the amount of NK cells, PD-L1-positive cells, and/or CD3+, CD8+, and/or CD4+ T cells in the sample, and if the amount of one or more of these cell types increases relative to the sample before administration of the anti-FGFR2 antibody or relative to a non-tumor sample in the subject, administering a PD-1/PD-L1 inhibitor to the subject. In some embodiments, the method further comprises obtaining at least one tumor sample from the subject, after administration of the anti-FGFR2 antibody, and determining the ratio of lymphoid cells to myeloid cells in the sample and, if the ratio is increased relative to the sample before administration of the anti-FGFR2 antibody or relative to a non-tumor sample in the subject, administering to the at least one subject. The at least one immunostimulatory agent, such as at least one PD-1/PD-L1 inhibitor, of these methods can be any of those described in the preceding paragraphs or described in the section below entitled "combinations with other immunostimulatory agents." In some embodiments, a combination of an FGFR2 inhibitor, a PD-1/PD-L1 inhibitor, and at least one other immunostimulatory agent can be administered to the patient.

[0032] Also provided herein are methods of treating cancer in a subject, comprising administering to the subject an FGFR2 inhibitor and, if it is determined that the subject has an increased number of NK cells, PD-L1-positive cells, macrophages, CD3+ T cells, CD8+ T cells, and/or CD4+ T cells compared to a control, such as a sample before administration of the FGFR2 antibody, or compared to a non-tumor sample in the subject, administering to the subject at least one immunostimulatory agent, such as a PD-1/PD-L1 inhibitor. Also provided herein are methods of treating a cancer in a subject, comprising administering to the subject an FGFR2 inhibitor and, if the subject is determined to have an increased lymphoid to myeloid cell ratio relative to a control, such as a sample prior to administration of the anti-FGFR2 antibody or relative to a non-tumor sample in the subject, administering to the subject at least one immunostimulatory agent, such as a PD-1/PD-L1 inhibitor. In such methods, the FGFR2 inhibitor and the immunostimulatory agent may be any of those described in the preceding paragraphs or in the section below entitled "combinations with other immunostimulatory agents." Additionally, administration of FGFR2 and the immunostimulatory agent may be in accordance with previously described methods, wherein administration of the immunostimulatory agent is not initiated until at least one dose of the FGFR2 inhibitor has been administered. In such cases, a test to determine the number of NK cells, PD-L1-positive cells, macrophages, CD3+, CD8+ and/or CD4+ T cells, lymphoid and/or myeloid cells may, for example, be performed after administration of an FGFR2 inhibitor but before the start of administration of a combination of FGFR2 and an immunostimulatory agent.

[0033] Also provided are compositions comprising any of the FGFR2 inhibitors described herein and any of the immunostimulatory agents described herein. In such compositions, the FGFR2 inhibitor and at least one immunostimulatory agent may be located in separate containers or separate compartments of the same container, or, alternatively, they may be mixed together in the same container or compartment. Such compositions may be used, for example, to treat cancer, such as any of the cancers described above. In some embodiments, instructions for use may also be included, such as instructions for use in the treatment of cancer.

[0034] Also provided herein are methods for increasing the number of one or more of PD-L1 positive cells, NK cells, macrophages, CD3+ T cells, CD4+ T cells, and CD8+ T cells in tumor tissue of a subject suffering from cancer, comprising administering an FGFR2 inhibitor, wherein the inhibitor is an FGFR2 antibody with enhanced ADCC activity. In some such embodiments, the immunostimulatory agent is administered with the FGFR2 antibody. In some such embodiments, administration of an anti-FGFR2 antibody to a mouse xenograft tumor model and/or a syngeneic tumor model increases the number of one or more types of PD-L1-positive cells, NK cells, macrophages, CD3+ T cells, CD8+ T cells, and CD4+ T cells in the tumor tissue compared to a control and/or increases the ratio of lymphoid cells to myeloid cells in the tumor tissue. In some such embodiments, the subject has breast cancer, gastric cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, cholangiocarcinoma, esophageal cancer (including gastroesophageal junction adenocarcinoma), or endometrial cancer such as bladder cancer.

[0035] In the above methods, the anti-FGFR2 antibody may be an anti-FGFR2-IIIb antibody that may have one or more of the following properties: (a) binds to FGFR2-IIIb with higher affinity than to FGFR2-IIIc or does not detectably bind to FGFR2-IIIc; (b) inhibits the binding of FGF2 and/or FGF7 to human FGFR2; (c) inhibits human tumor growth in a mouse xenograft tumor model and/or a syngeneic tumor model; (d) induces ADCC activity; (e) has enhanced ADCC activity; and (f) is afucosylated.

[0036] In some embodiments of the above method, the anti-FGFR2 antibody comprises heavy chain and light chain variable regions, wherein the heavy chain variable region comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8;

and the light chain variable region comprises: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0037] In some cases, the heavy chain variable domain of the anti-FGFR2 antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 and/or the light chain variable domain of the anti-FGFR2 antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5. In some cases, the heavy chain variable domain of the anti-FGFR2 antibody comprises the amino acid sequence of SEQ ID NO: 4 and/or the light chain variable domain of the anti-FGFR2 antibody comprises the amino acid sequence of SEQ ID NO: 5. In some cases, the heavy chain of the anti-FGFR2 antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 and/or the light chain of the anti-FGFR2 antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3. In some cases, the heavy chain of the anti-FGFR2 antibody comprises the amino acid sequence of SEQ ID NO: 2 and/or the light chain of the anti-FGFR2 antibody comprises the amino acid sequence of SEQ ID NO: 3. In some cases, the anti-FGFR2 antibody is chimeric, humanized, or human. In some embodiments, the anti-FGFR2 antibody is selected from Fab, Fv, scFv, Fab'', and (Fab') ₂ . In some embodiments, the anti-FGFR2 antibody has one or more of the following properties: (a) lacks fucose at position Asn297; (b) comprises a κ light chain constant region; (c) comprises an IgG1 heavy chain constant region; (d) has enhanced ADCC activity in vitro compared to an antibody having the same amino acid sequence that is fucosylated at position Asn297; and (e) has an increased affinity for Fc gamma RIIIA compared to an antibody having the same amino acid sequence that is fucosylated at position Asn297. In some embodiments, the afucosylated antibody is capable of increasing the number of one or more of: PD-L1-positive cells, NK cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and macrophages in tumor tissue in a mouse xenograft tumor model and/or a syngeneic tumor model compared to a control (e.g., compared to a control antibody that does not target FGFR2).

[0038] In any of the methods or uses described in the Summary of the Invention, the cancer may be predetermined by overexpression of FGFR2IIIb, either with or without amplification of the FGFR2 gene. Alternatively, in any of the methods or uses described in this section, the method may further comprise examining the cancer in the subject, such as prior to administration of an FGFR2 inhibitor, to determine whether the cancer overexpresses FGFR2IIIb and/or to determine whether the FGFR2 gene is amplified in tumor cells. In either case, FGFR2IIIb may optionally be detected by immunohistochemistry (IHC), and amplification of the FGFR2 gene may optionally be detected by fluorescence in situ hybridization (FISH), such as using probes for the FGFR2 gene locus and the centromere of chromosome 10, where the FGFR2 gene is located. In some embodiments, an IHC signal of 1+, 2+, or 3+ in at least 10% of the tumor cells, such as at least 20%, 30%, 40%, or 50% of the tumor cells, indicates overexpression of FGFR2IIIb. In some embodiments, a ratio of FGFR2 to the centromere of chromosome 10 (CEN10) greater than or equal to 2 indicates amplification of the FGFR2 gene.

[0039] In some embodiments, wherein the patient has gastric or bladder cancer, the subject may be predetermined to have one of the following profiles, or alternatively, the method of treatment comprises determining whether the patient meets one of the following profiles with respect to FGFR2 gene expression/amplification, and which may indicate a level of expected sensitivity to treatment: a) in the case of a subject with gastric cancer, an IHC signal of 3+ in at least 10% of the tumor cells; b) in the case of a subject with gastric cancer, an IHC signal of 3+ in at least 10% of the tumor cells as well as FGFR2 gene amplification; c) in the case of a subject with gastric cancer, an IHC signal of 3+ in at least 10% of the tumor cells without FGFR2 gene amplification; d) in the case of a subject with gastric cancer, an IHC signal of 1+ or 2+ in at least 10% of the tumor cells; e) in the case of a subject with bladder cancer, an IHC signal of 1+ in at least 10% of the tumor cells; f) in the case of a subject with bladder cancer, an IHC signal of 2+ in at least 10% of the tumor cells; g) in the case of a patient with bladder cancer, an H value greater than 20; h) in the case of a subject with bladder cancer, an H value equal to 10-19; i) in the case of a subject with bladder cancer, an H value less than 10.

[0040] This disclosure also provides methods for determining sensitivity to any of the FGFR2 inhibitors, treatments, and uses described above. Such methods may include examining a cancer in a subject to determine whether the cancer overexpresses FGFR2IIIb and/or to determine whether the FGFR2 gene is amplified in tumor cells. Overexpression of FGFR2IIIb may optionally be determined by immunohistochemistry (IHC), and amplification of the FGFR2 gene may optionally be determined by fluorescence in situ hybridization (FISH), such as using probes for the FGFR2 gene locus and the centromere of chromosome 10, where the FGFR2 gene is located. In some embodiments, an IHC signal of 1+, 2+, or 3+ in at least 10% of the tumor cells, such as at least 20%, 30%, 40%, or 50% of the tumor cells, indicates overexpression of FGFR2IIIb. In some embodiments, a ratio of FGFR2 to the centromere of chromosome 10 (CEN10) greater than or equal to 2 indicates amplification of the FGFR2 gene.

[0041] In some embodiments, wherein the patient has gastric or bladder cancer, the method may include determining whether the patient's cancer falls into one of the following categories, which may indicate sensitivity to the treatment or to the FGFR2 inhibitor composition: a) in the case of a subject with gastric cancer, an IHC signal of 3+ in at least 10% of the tumor cells; b) in the case of a subject with gastric cancer, an IHC signal of 3+ in at least 10% of the tumor cells and amplification of the FGFR2 gene; c) in the case of a subject with gastric cancer, an IHC signal of 3+ in at least 10% of the tumor cells without amplification of the FGFR2 gene; d) in the case of a subject with gastric cancer, an IHC signal of 1+ or 2+ in at least 10% of the tumor cells; e) in the case of a patient with bladder cancer, an IHC signal of 1+ in at least 10% of the tumor cells; f) in the case of a subject with bladder cancer, an IHC signal of 2+ in at least 10% of the tumor cells; g) in the case of a patient with bladder cancer, an H value greater than 20; h) in the case of a subject with bladder cancer, an H value equal to 10-19; i) in the case of a subject with bladder cancer, an H value less than 10.

[0042] It is to be understood that both the foregoing general description and the following detailed description of the present invention are illustrative and explanatory only and are not intended to limit the claims. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter of the invention described. All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety for all purposes.

BRIEF DESCRIPTION OF DRAWINGS

[0043] Figures 1a-1b depict changes in the volume of implanted 4T1 mammary tumor cells in BALB/c mice following treatment with an Ig-Fc control, an afucoylated anti-FGFR2b antibody (anti-FGFR2) comprising the heavy and light chain HVRs of SEQ ID NOs: 6-11, or an anti-FGFR2b antibody with the same amino acid sequence except for a Q to N substitution at amino acid position 297 (anti-FGFR2-N297Q) to eliminate effector function. (See SEQ ID NO: 12 in the sequence table below for a description of this mutation.) As shown in Figures 1a and 1b , only the anti-FGFR2 antibody demonstrated inhibition of 4T1 tumor growth. Statistical significance (P < 0.05 = *, P < 0.01 = **, P < 0.001 = ***; P < 0.001 = ****) was determined using 1-way analysis of variance followed by Tukey's multiple comparison test.

[0044] Figures 2a-2d depict the results of staining 4T1 tumor cells for the presence of either NKp46 ( Figures 2a-2b ) or PD-L1 ( Figures 2c-2d ) compared to DAPI (4',6-diamidine-2-phenylindole) staining of cell nuclei either on day 1 or one day after a single dose of vehicle control or anti-FGFR2 ( Figures 2a and 2c ) or on day 4, one day after the second of two therapies administered on day 0 and day 3 of vehicle control or anti-FGFR2 ( Figures 2b and 2d ). Each image was obtained from different tumors, and the images were collected using a 10X objective. Anti-FGFR2 treatment increased the number of NKp46+ cells in 4T1 tumors compared with vehicle at both day 1 and day 4 ( Fig. 2a–2b ) and increased the number of PD-L1+ cells compared with vehicle at both day 1 and day 4 ( Fig. 2c–2d ).

[0045] Figure 3 depicts an analysis of the effect of anti-FGFR2 treatment on the number of NKp46+ cells in 4T1 murine tumors at day 4. Anti-FGFR2 treatment increased the number of NK cells in tumors compared to vehicle control with a P < 0.05 t-test.

[0046] Figures 4a-4b depict the changes in the volume of implanted 4T1 mammary tumors in BALB/c mice after treatment with control Ig-Fc, anti-PD-1 antibody, afucosylated anti-FGFR2b antibody designated anti-FGFR2, and after a combination of treatment with anti-PD-1 antibody and afucosylated anti-FGFR2b antibody. In each graph, tumor volume is depicted as the mean ^mm3 +/- SEM. As shown in Figure 4a , the combination of anti-FGFR2 (10 mg/kg twice weekly) and anti-PD1 antibody (5 mg/kg every other week) resulted in significant inhibition of 4T1 tumor growth compared to control and or antibody alone by day 18. As shown in Figure 4b at day 18 post-tumor implantation, the combination demonstrated statistically significant inhibition of 4T1 tumor growth compared to Ig-Fc or anti-PD1 antibody controls. Statistical significance was determined using 1-way ANOVA followed by Tukey's multiple comparison test.

[0047] Figures 5a-5b depict the results of staining 4T1 tumor cells for the presence of either NKp46 cells or PD-L1+ cells or CD3+ T cells either on day 1 ( Figure 5a ), or one day after a single dose of vehicle control or anti-FGFR2 or anti-FGFR2 N297Q, or on day 4, one day after the second of two therapies administered on day 0 and day 3 with vehicle control or anti-FGFR2 ( Figure 5b ). Each image was obtained from a different tumor and the images were collected using a 10X objective. Anti-FGFR2 treatment increased the number of NKp46+ cells in 4T1 tumors compared with vehicle at both days 1 and day 4 and increased the number of PD-L1+ cells compared with vehicle at both days 1 and day 4, with more cells observed at day 4 than at day 1. CD3+ T cells also infiltrated the tumor at day 4 after anti-FGFR2 treatment.

[0048] Figures 6a-6b depict the results of staining 4T1 tumor cells for the presence of CD3+ and CD8+ T cells ( Figure 6a ) or CD3+ and CD4+ T cells ( Figure 6b ) on day 4 of the treatment protocol, adjacent to cells with DAPI-stained nuclei. The images show that anti-FGFR2 treatment resulted in an increase in the number of all three T cell types in tumor tissue on day 4 compared to control and anti-FGFR2 N297Q treatment.

[0049] Figures 7a-7b depict the results of flow cytometric analysis of tumor cells in the syngeneic 4T1 tumor model at day 1 and day 4, respectively. CD3+ T cells represent a percentage of CD45+ live single cells for each treatment group. As shown in the figures, the anti-FGFR2 group showed an increase in the percentage of CD3+ T cells compared to both the vehicle control and anti-FGFR2 N297Q groups at day 4. This increase was also statistically significant according to Student's t-test, as indicated by ** symbols, denoting P ≤ 0.01.

[0050] Figures 8a-8b depict the results of flow cytometric analysis of tumor cells in the syngeneic 4T1 tumor model at day 1 and day 4, respectively. CD8+ T cells represent for each treatment group a percentage of CD45+ live single cells. As shown in the figures, the anti-FGFR2 group showed an increase in the percentage of CD8+ T cells compared to both the vehicle control and anti-FGFR2 N297Q groups at day 4. This increase was also statistically significant according to Student's t-test, as indicated by ** symbols, denoting P ≤ 0.01.

[0051] Figures 9a-9b depict the results of flow cytometric analysis of tumor cells in the syngeneic 4T1 tumor model at day 1 and day 4, respectively. CD4+ T cells represent for each treatment group a percentage of CD45+ live single cells. As shown in the figures, the anti-FGFR2 group showed an increase in the percentage of CD4+ T cells compared to both the vehicle control and anti-FGFR2 N297Q groups at day 4. This increase was also statistically significant according to Student's t-test, as indicated by * symbols, denoting P ≤ 0.5.

[0052] Figures 10a-10c depict additional flow cytometric analysis results of tumor cells in the syngeneic 4T1 tumor model at day 4. In Figure 10a, NKp46+ cells provide for each treatment group as a percentage of CD45+ live single cells. The figure depicts a statistically significant increase in NKp46+ cells in the anti-FGFR2 group compared to the other groups, as determined by Student's t-test, with * indicating P ≤ 0.5, ** indicating P ≤ 0.01, and *** indicating P ≤ 0.001. In Figure 10b and 10c showed that the number of myeloid cells was significantly decreased in the anti-FGFR2 group, whereas the number of lymphoid cells was significantly increased, indicating that the ratio of lymphoid to myeloid cells increased on day 4 after anti-FGFR2 treatment compared with other groups.

[0053] Figure 11 shows staining of 4T1 tumor tissue in a syngeneic mouse tumor model at days 1 or 4 after treatment with vehicle control (upper panels), anti-FGFR2 antibody (middle panels), or anti-FGFR2-N297Q antibody (lower panels). The figures show staining with anti-F480 antibody to detect infiltration of F480+ macrophages into the tumor tissue and corresponding nuclear staining with DAPI. As can be seen from the figure, after anti-FGFR2 treatment, F480+ macrophages are much more numerous compared to the control (compare upper and middle panels at day 4). No difference was found between the control and anti-FGFR2-N297Q panels (compare upper and lower panels at day 4). Images were collected using a 10X objective.

[0054] Figure 12 shows staining of 4T1 tumor tissue in a syngeneic mouse tumor model 4 days after treatment with different antibodies: control antibody (Fc-G1 antibody) (upper panels), rabbit anti-asialo-GM1 antibody designed to deplete NKp46 cells (second panels), anti-FGFR2 antibody (third panels), and a combination of anti-FGFR2 antibody with anti-asialo-GM1 antibody (lower panels). The tissue was stained with NKp46 reagents or DAPI to stain cell nuclei (left and right panels, respectively). Anti-asialo-GM1 antibody was administered at a dose of 1.25 mg/kg, and anti-FGFR2 antibody was administered at a dose of 10 mg/kg. Images were collected using a 10X objective. As seen in four different NKp46 staining panels, anti-asialo-GM1 antibody depleted NKp46 cells in tumor tissue, whereas anti-FGFR2 antibody increased NKp46 cells. (Compare top and third left panels.) Anti-FGFR2 antibody in combination with anti-asialo-GM1 antibody increased NKp46 cells compared with anti-asialo-GM1 antibody (although not compared with control), indicating that anti-FGFR2 antibody can increase NKp46 in tumor tissue even when these cells are depleted in the presence of a competing antibody (Compare top, second and bottom left panels).

[0055] Fig. 13 shows staining for CD3+ T cells and corresponding DAPI staining of cell nuclei from 4T1 tumor tissue in a syngeneic mouse tumor model for 4 days following treatment with a control antibody (Fc-G1 antibody) (upper panels), rabbit anti-asialo-GM1 antibody (second panels), anti-FGFR2 antibody (third panels), and a combination of anti-FGFR2 antibody with anti-asialo-GM1 antibody (lower panels). Anti-asialo-GM1 antibody was administered at a dose of 1.25 mg/kg, and anti-FGFR2 antibody was administered at a dose of 10 mg/kg. Images were collected using a 10X objective. As can be seen from the comparison of the four left panels of the figure, treatment with anti-FGFR2 antibody increased the number of CD3+ T cells in tumor tissue, but not when administered together with anti-asialo-GM1 antibody.

[0056] Fig. 14 shows staining for PD-L1-positive cells and corresponding DAPI staining of cell nuclei from 4T1 tumor tissue in a syngeneic mouse tumor model for 4 days following treatment with a control antibody (Fc-G1 antibody) (upper panels), rabbit anti-asialo-GM1 antibody (second panels), anti-FGFR2 antibody (third panels), and a combination of anti-FGFR2 antibody with anti-asialo-GM1 antibody (lower panels). Anti-asialo-GM1 antibody was administered at a dose of 1.25 mg/kg, and anti-FGFR2 antibody was administered at a dose of 10 mg/kg. Images were collected using a 10X objective. As can be seen from the comparison of the four left panels of the figure, treatment with anti-FGFR2 antibody increased the number of PD-L1-positive cells in tumor tissue, but not when administered together with anti-asialo-GM1 antibody.

[0057] Figures 15a and 15b depict the growth of orthotopic 4T1 tumors in mice after administration of control phosphate-buffered saline (PBS), anti-FGFR2 antibody, anti-asialo-GM1 antibody, or a combination of anti-FGFR2 and anti-asialo-GM1 antibodies. Figure 15a depicts tumor volume at 12 and 15 days post-administration. Figure 15b depicts a plot of tumor volume in individual mice in each group at 15 days post-administration. The figure shows the statistically significant reduction in tumor volume in the anti-FGFR2 group compared with the control and anti-asialo-GM1 groups according to Student's T-test, with * indicating P ≤ 0.5 and ** indicating P ≤ 0.01, as well as the statistically significant change in tumor volume between the group receiving only anti-FGFR2 antibody and the group receiving a combination of anti-FGFR2 and anti-asialo-GM1 antibodies.

[0058] Figures 16a, b, c, and d depict the growth of orthotopic 4T1 tumors in SCID mice after administration of vehicle control or anti-FGFR2 antibody up to 27 days after tumor cell inoculation. Figure 16a depicts tumor volume as a function of time. Arrows below the graphs indicate the doses of either vehicle or 20 mg/kg anti-FGFR2 antibody on days 12 and 15 after inoculation. An asterisk (*) denotes statistically significant differences between tumor growth in the vehicle- or anti-FGFR2 antibody-treated groups, as determined by Student's t-test at a significance level of P ≤ 0.5. Figure 16b depicts tumor volume in individual mice in each group on day 19 after inoculation. The asterisk (*) indicates statistically significant differences between the tumor growth in the two groups according to the Student's t-test at a significance level of P ≤ 0.5. Fig. 16c shows the tumor volume of individual mice in each group on day 23 after inoculation. The asterisk (*) indicates statistically significant differences between the tumor growth in the two groups according to the Student's t-test at a significance level of P ≤ 0.5. Fig. 16d shows the tumor volume of individual mice in each group on day 27 after inoculation.

Description of specific embodiments of the invention

Definitions

[0059] Unless otherwise defined, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless the context otherwise requires, singular terms include plural forms, and plural terms include singular forms.

[0060] Illustrative techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification methods are known in the art. Many such techniques and procedures are described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), among other sources. In addition, illustrative techniques for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation development, dosage form delivery, and patient treatment are also known in the art.

[0061] In this application, the use of "or" means "and/or" unless otherwise indicated. In the context of a claim dependent on another dependent claim, the use of "or" refers only to several preceding independent or dependent claims in the alternative only. Furthermore, terms such as "element" or "component" cover both elements and components containing a single unit, as well as elements and components that contain more than one subunit, unless otherwise specifically indicated.

[0062] As used in accordance with this disclosure, the following terms, unless otherwise specified, are understood to have the following meanings:

[0063] The terms " nucleic acid molecule " and " polynucleotide " may be used interchangeably and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. " Nucleic acid sequence " refers to a linear sequence of nucleotides that comprise a nucleic acid molecule or polynucleotide.

[0064] The terms " polypeptide " and " protein ," when used interchangeably, refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. The terms also include post-expression modifications of a polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Additionally, for purposes of this invention, a "polypeptide" refers to a protein that contains modifications, such as deletions, additions, and substitutions (usually conservative in nature), relative to the native sequence, so long as the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as through mutations in host proteins or errors associated with PCR amplification.

[0065] " FGFR2 " refers to fibroblast growth factor receptor 2, including any of its alternatively spliced forms, such as splice forms IIIa, IIIb, and IIIc. The term FGFR2 encompasses wild-type FGFR2 and naturally occurring mutant forms, such as FGFR2-activating mutant forms such as FGFR2-S252W, which is found in some cancer cells. " FGFR2-IIIb " or " FGFR2b ," used interchangeably, refers to the splice form IIIb of fibroblast growth factor receptor 2. An exemplary human FGFR2-IIIb is shown in GenBank, accession no. NP_075259.4, accessed July 7, 2013. An exemplary mature amino acid sequence of human FGFR2-IIIb is shown in SEQ ID NO: 1. " FGFR2-IIIc " or " FGFR2c ", used interchangeably, refers to the splice form of fibroblast growth factor receptor 2 IIIc. An exemplary human FGFR2-IIIc is shown in GenBank, accession no. NP_000132.3, July 7, 2013. An exemplary mature amino acid sequence of human FGFR2-IIIc is shown in SEQ ID NO: 12.

[0066] " FGFR2 ECD " refers to the extracellular domain of FGFR2, including naturally occurring and engineered variants thereof. Non-limiting examples of FGFR2 ECD include SEQ ID NOs: 13-23, 29, and 32. " FGFR2 ECD fusion molecule " refers to a molecule comprising an FGFR2 ECD and a fusion partner such as an Fc domain, albumin, or PEG. The fusion partner can be covalently attached, for example, to the N- or C-terminus of the FGFR2 ECD or in an internal region. Non-limiting examples of FGFR2 ECD fusion molecules include SEQ ID NOs: 30, 31, and 33.

[0067] " FGFR2 inhibitor " refers to a molecule, such as an antibody, that binds FGFR2, or such as an FGFR2 ECD or an FGFR2 ECD fusion molecule, that inhibits the binding of FGFR2 to one or more of its ligands, such as FGF1, FGF7, and/or FGF2. In some embodiments, an FGFR2 inhibitor is capable of binding to FGFR2, as well as to a mutant of FGFR2 with an activating mutation, such as the FGFR2-S252W mutation.

[0068] As used herein, the term " immunostimulating agent " refers to a molecule that stimulates the immune system, either by acting as an agonist of an immunostimulatory molecule, including a costimulatory molecule, or by acting as an antagonist of an immunoinhibitory molecule, including a coinhibitory molecule. An immunomodulatory agent may be a biological agent, such as an antibody or antibody fragment, another protein, or a vaccine, or may be a small molecule drug.

[0069] The terms " programmed cell death protein-1 " and " PD-1 " refer to an immune inhibitory receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T cells in vivo and binds to two ligands PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs that share at least one epitope with hPD-1. The complete sequence of hPD-1 can be found in GenBank Accession No. U64863. In some embodiments, PD-1 is human PD-1 having the amino acid sequence of SEQ ID NO: 34 (precursor, with signal sequence) or SEQ ID NO: 35 (mature, without signal sequence).

[0070] The terms " programmed cell death protein-1 ligand 1 " and "PD-L1" refer to one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that regulate T cell activation and cytokine secretion when bound to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs that share at least one epitope with hPD-L1. The complete sequence of hPD-L1 can be found in GenBank Accession No. Q9NZQ7. In some embodiments, the PD-L1 is human PD-L1 having the amino acid sequence of SEQ ID NO: 37 (precursor, with signal sequence) or SEQ ID NO: 38 (mature, without signal sequence).

[0071] The term " PD-1/PD-L1 inhibitor " refers to a moiety that disrupts PD-1/PD-L1 signaling. In some embodiments, the inhibitor inhibits PD-1/PD-L1 signaling by binding to PD-1 and/or PD-L1. In some embodiments, the inhibitor also binds to PD-L2. In some embodiments, the PD-1/PD-L1 inhibitor inhibits PD-1 binding to PD-L1 and/or PD-L2. Non-limiting exemplary PD-1/PD-L1 inhibitors include antibodies that bind PD-1; antibodies that bind PD-L1; PD-1 fusion molecules such as AMP-224; and PD-1 polypeptides such as AUR-012.

[0072] The term " antibody that inhibits PD-1 " refers to an antibody that binds to PD-1 or binds to PD-L1 and thereby inhibits PD-1 and/or PD-L1 signaling. In some embodiments, an antibody that inhibits PD-1 binds to PD-1 and blocks the binding of PD-L1 and/or PD-L2 to PD-1. In some embodiments, an antibody that inhibits PD-1 binds to PD-L1 and blocks the binding of PD-1 to PD-L1. An antibody that inhibits PD-1 and that binds to PD-L1 can be referred to as an anti-PD-L1 antibody. An antibody that inhibits PD-1 and that binds to PD-1 can be referred to as an anti-PD-1 antibody.

[0073] With respect to FGFR2 antibodies, FGFR2 ECDs, and FGFR2 ECD fusion molecules, the terms " blocks binding " or " inhibits binding " of a ligand refer to the ability to inhibit the interaction between FGFR2 and an FGFR2 ligand, such as FGF1 or FGF2. Such inhibition may occur through any mechanism, including direct interference with ligand binding, such as due to overlapping binding sites on FGFR2 and/or antibody-induced conformational changes in FGFR2 that alter ligand affinity, or, such as in the case of FGFR2 ECDs or FGFR2 ECD fusion molecule, by competing for binding to FGFR2 ligands.

[0074] With respect to anti-PD-1 antibodies and PD-1 fusion molecules or polypeptides, the terms " block binding " or " inhibit binding " of a ligand, such as PD-L1, and grammatical variants thereof, refer to the ability to inhibit the interaction between PD-1 and a PD-1 ligand, such as PD-L1. Such inhibition may occur through any mechanism, including direct interference with ligand binding, such as due to overlapping binding sites on PD-1 and/or conformational changes in PD-1 induced by the antibody that alter ligand affinity, etc., or by competing for binding to the PD-1 ligand.

[0075] As used herein, the term "antibody" refers to a molecule comprising at least the hypervariable regions (HVRs) H1, H2, and H3 of the heavy chain and L1, L2, and L3 of the light chain, wherein the molecule is capable of binding an antigen. The term antibody includes, but is not limited to, fragments that are capable of binding an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab'', and (Fab') ₂ . The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies from various species, such as mouse, human, cynomolgus monkey, etc. It also includes antibodies conjugated to other molecules, such as small molecule drugs, bispecific antibodies, and multispecific antibodies.

[0076] The term " heavy chain variable region " refers to a region comprising heavy chain HVR1, framework region (FR) ₂ , HVR2, FR3, and HVR3. In some embodiments, the heavy chain variable region also comprises at least a portion of FR1 and/or at least a portion of FR4.

[0077] The term " heavy chain constant region " refers to a region comprising at least three heavy chain constant domains _CH1 , _CH2 , and _CH3 . Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an α constant region is an IgA antibody. Additionally, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Some isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a γ ₁ constant region), IgG2 (containing a γ ₂ constant region), IgG3 (containing a γ ₃ constant region), and IgG4 (containing a γ ₄ constant region); IgA antibodies include, but are not limited to, IgA1 (containing an α ₁ constant region) and IgA2 (containing an α ₂ constant region); and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0078] The term " heavy chain " refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0079] The term " light chain variable region " refers to a region comprising light chain HVR1, framework region (FR) ₂ , HVR2, FR3, and HVR3. In some embodiments, the light chain variable region also comprises FR1 and/or FR4.

[0080] The term " light chain constant region " refers to a region comprising the light chain constant domain C _L . Non-limiting exemplary light chain constant regions include λ and κ.

[0081] The term " light chain " refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, the light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0082] The term " hypervariable region " or " HVR " refers to each of the regions of the variable domain of an antibody that are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Typically, native four-chain antibodies contain six HVRs; three in the V _H (H1, H2, H3), and three in the V _L (L1, L2, L3). Typically, the HVRs contain amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latter having the highest sequence variability and/or involved in antigen recognition. Illustrative hypervariable loops are located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Illustrative CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 L1, 50-56 L2, 89-97 L3, 31-35B H1, 50-65 H2, and 95-102 H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The terms hypervariable regions (HVRs) and complementarity determining regions (CDRs) refer to the portions of the variable region that form the antigen-binding regions.

[0083] The term "affinity" or " binding affinity " refers to the strength of the total overall non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In some embodiments, "binding affinity" refers to the actual binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant ( _Kd ).

[0084] " Antibody-dependent cell-mediated cytotoxicity " or " ADCC " refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), mediating the specific binding of these effector cytotoxic cells to a target cell that bears the antigen, and the target cell is then destroyed by the cytotoxins. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu . Rev. Immunol 9:457-92 (1991). To assess the activity of To determine the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337, or U.S. Patent No. 6,737,056 (Presta). Suitable effector cells for such assays include PBMCs (peripheral blood mononuclear cells) and NK cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo , such as in an animal model such as that described in Clynes et al . Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Additional antibodies with altered Fc region amino acid sequences and increased or decreased ADCC activity are described, for example, in U.S. Patent No. 7,923,538 and U.S. Patent No. 7,994,290.

[0085] An antibody having " enhanced ADCC activity " refers to an antibody that is more effective at mediating ADCC in vitro or in vivo compared to a parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect and wherein the amounts of such antibody and the parent antibody used in the assay are substantially the same. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is afucosylated while the parent antibody is fucosylated. In some embodiments, ADCC activity will be determined using an in vitro ADCC assay, such as that described in U.S. Publ. No. 2015-0050273-A1, but other assays or methods for determining ADCC activity, such as in an animal model, etc. In some embodiments, an antibody with enhanced ADCC activity also has enhanced affinity for Fc gamma RIIIA. In some embodiments, an antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA (V158). In some embodiments, an antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA (F158).

[0086] " Increased affinity for Fc gamma RIIIA " refers to an antibody that has a greater affinity for Fc gamma RIIIA (also referred to, in some instances, as CD16a) than a parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is afucosylated, while the parent antibody is fucosylated. Any suitable method for determining affinity for Fc gamma RIIIA can be used. In some embodiments, affinity for Fc gamma RIIIA is determined by the method described in U.S. Publ. No. 2015-0050273-A1. In some embodiments, an antibody with increased affinity for Fc gamma RIIIA also has improved ADCC activity. In some embodiments, an antibody with increased affinity for Fc gamma RIIIA has increased affinity for Fc gamma RIIIA (V158). In some embodiments, an antibody with increased affinity for Fc gamma RIIIA has increased affinity for Fc gamma RIIIA (F158).

[0087] As used herein, a " chimeric antibody " refers to an antibody that comprises at least one variable region from a first species (such as a mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as a human, cynomolgus monkey, etc.). In some embodiments, the chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, the chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, the chimeric antibody comprises at least one rat variable region and at least one mouse constant region. In some embodiments, all of the variable regions of the chimeric antibody are from the first species and all of the constant regions of the chimeric antibody are from the second species.

[0088] As used herein, a " humanized antibody " refers to an antibody in which at least one amino acid in a framework region of a non-human variable region has been replaced with a corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody is a Fab, scFv, a (Fab') ₂ , etc.

[0089] As used herein, " human antibody " refers to antibodies produced in humans, antibodies produced in non-human animals that contain human immunoglobulin genes, such as XenoMouse®, and antibodies selected using in vitro methods such as phage display, in which the antibody repertoire is based on human immunoglobulin sequences.

[0090] An " afucosylated " antibody or an " afucose-lacking " antibody refers to an IgG1 or IgG3 isotypic antibody that lacks fucose in its glycosylated constant region. Glycosylation of human IgG1 or IgG3 occurs at Asn297 (N297) as a glycosylation of the oligosaccharide core of the biantennary complex terminating with 2 Gal residues. In some embodiments, the afucosylated antibody lacks fucose at Asn297. These structures are designated G0, G1 (α1,6 or α1,3), or G2 glycan residues, depending on the number of terminal Gal residues. See, e.g., Raju, T. S., BioProcess Int . 1: 44-53 (2003). Glycosylation of the CHO type of Fc antibodies is described, for example, in Routier, FH, Glycoconjugate J. 14: 201-207 (1997). In a population of antibodies, antibodies are considered afucosylated if <5% of the antibodies in the population contain fucose at Asn297.

[0091] " Effector functions " refer to biological activities inherent in the Fc region of an antibody that vary depending on the antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor) and B cell activation.

[0092] " Antibody-dependent cell-mediated cytotoxicity " or " ADCC " refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), mediating specific binding of these effector cytotoxic cells to a target cell that bears the antigen, and the target cell is then destroyed by the cytotoxins. The primary cells to mediate ADCC express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu . Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337, or U.S. Patent No. 6,737,056 (Presta). Suitable effector cells for such assays include PBMCs (peripheral blood mononuclear cells) and NK cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo , such as in an animal model such as that described in Clynes et al . Proc. Natl. Acad. Sci. (USA) 95:652–656 (1998). Additional antibodies with altered Fc region amino acid sequences and increased or decreased ADCC activity are described, for example, in U.S. Patent No. 7,923,538 and U.S. Patent No. 7,994,290.

[0093] An antibody having " enhanced ADCC activity " refers to an antibody that is more effective at mediating ADCC in vitro or in vivo compared to a parent antibody of the same sequence, except for at least one structural change designed to alter ADCC activity, when the amounts of such antibody and the parent antibody used in the assay are substantially the same. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, except for a mutation in the Fc domain, such as an amino acid substitution that results in afucosylation, wherein the parent antibody is fucosylated. In some embodiments, ADCC activity will be determined using an in vitro ADCC assay as described herein, but other assays or methods for determining ADCC activity, such as in an animal model, etc. In some embodiments, an antibody with enhanced ADCC activity has an enhanced affinity for Fc gamma RIIIA. In some embodiments, an antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA (V158). In some embodiments, an antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA (F158).

[0094] " Increased affinity for Fc gamma RIIIA " refers to an antibody that has a greater affinity for Fc gamma RIIIA (also referred to, in some cases, as CD16a) than a parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is afucosylated while the parent antibody is fucosylated. Any suitable method for determining affinity for Fc gamma RIIIA can be used. In some embodiments, affinity for Fc gamma RIIIA is determined by the method described herein. In some embodiments, an antibody with increased affinity for Fc gamma RIIIA has increased ADCC activity. In some embodiments, an antibody with increased affinity for Fc gamma RIIIA has increased affinity for Fc gamma RIIIA (V158). In some embodiments, an antibody with increased affinity for Fc gamma RIIIA has increased affinity for Fc gamma RIIIA (F158).

[0095] The term " leader sequence " refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates secretion of the polypeptide from a mammalian cell. The leader sequence may be cleaved during isolation of the polypeptide from a mammalian cell to form the mature protein. Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached. Non-limiting exemplary leader sequences also include leader sequences from heterologous proteins. In some embodiments, the antibody does not have a leader sequence. In some embodiments, the antibody comprises at least one leader sequence that can be selected from native antibody leader sequences and heterologous leader sequences.

[0096] The term " vector " is used to describe a polynucleotide that can be engineered to contain a cloned polynucleotide or polynucleotides that can be replicated in a host cell. The vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of a polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, such as β-galactosidase). The term " expression vector " refers to a vector that is used to express a polypeptide of interest in a host cell.

[0097] " Host cell " refers to a cell that can be or is a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells and derivatives thereof, such as 293-6E and DG44 cells, respectively.

[0098] As used herein, the term " isolated " refers to a molecule that has been separated from at least some of the components with which it is normally found in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least some of the components of the cell in which it was produced. When a polypeptide is secreted by a cell after expression, the physical separation of the supernatant containing the polypeptide from the cell that produces it is considered to be "isolating" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide (e.g., genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is normally found in nature, or is separated from at least some of the components of the cell in which it was produced, such as in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector within a host cell may be referred to as "isolated" if the polynucleotide is not found in the vector in nature.

[0099] The term " increased level " means a higher level of a protein in a particular tissue of a subject relative to the same tissue in a control, such as an individual or individuals who do not suffer from cancer or other pathological conditions described herein. The increased level may be the result of any mechanism, such as increased expression, increased stability, decreased degradation, increased secretion, decreased clearance, etc. of the protein.

[00100] The terms " decrease " or " decrease " or " increase " or " increase " with respect to a protein or a cell type means a change in the level of that protein or cell type in a particular tissue of a subject, such as a tumor, by at least 10%. In some embodiments, an agent, such as an FGFR2 or PD-1/PD-L1 inhibitor, increases or decreases the level of the protein or cell type in a particular tissue of a subject, such as a tumor, by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. In some embodiments, the level of the protein or cell type is decreased or increased relative to the level of the protein prior to exposure to an agent, such as an FGFR2 or PD-1/PD-L1 inhibitor, or relative to the level of a control therapy.

[00101] The terms " subject " and " patient " are used interchangeably herein to refer to a human. In some embodiments, methods of treating other mammals are also provided, including, but not limited to, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, laboratory mammals, farm mammals, sport mammals, and domestic mammals.

[00102] As used herein, the term " sample " refers to a composition that is obtained or taken from a subject that contains a cellular and/or other molecular compound that is to be characterized, quantified, and/or identified, such as based on physical, biochemical, chemical, and/or physiological characteristics. An exemplary sample is a tissue sample.

[00103] The term " cancer " refers to a malignant proliferative disorder associated with uncontrolled cell proliferation, unlimited cell growth, and decreased cell death through apoptosis. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific, non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer (including gastroesophageal junction adenocarcinoma), astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder carcinoma, stomach cancer, melanoma, and various types of head and neck cancer (including squamous cell carcinoma of the head and neck).

[00104] In some embodiments, the cancer is stomach cancer (which includes gastroesophageal cancer). In some embodiments, the cancer is bladder cancer. Bladder cancer, as defined herein, includes forms of the disease such as urinary bladder cancer (UBC) and transitional cell carcinoma (TCC), which is also known as urothelial carcinoma (UC), as well as non-transitional cell carcinomas that develop in the bladder.

[00105] In some embodiments, the cancer comprises an amplification of the FGFR2 gene, while in some embodiments, the cancer does not comprise an amplification of FGFR2. In some embodiments in which amplification occurs, the FGFR2 amplification comprises an FGFR2:CEN10 (centromere of chromosome 10) ratio> 3. In some embodiments, the FGFR2 amplification comprises an FGFR2:CEN10 ratio ≥ 2. However, in other embodiments, the FGFR2 level comprises an FGFR2:CEN10 ratio between 1 and 2, indicating that FGFR2 is not amplified. In some embodiments, mutations or translocations can cause amplification of the FGFR2 gene. Gene amplification can be detected, for example, by a fluorescence in situ hybridization (FISH) assay.

[00106] In some embodiments, in which the cancer comprises an amplification of the FGFR2 gene, the cancer overexpresses FGFR2-IIIb. In some embodiments, a cancer comprising an amplification of FGFR2 overexpresses FGFR2-IIIb to a greater extent than FGFR2-IIIc. In some embodiments, a cancer comprising an amplification of FGFR2 expresses FGFR2-IIIb at a normalized level that is greater than 2-fold, 3-fold, 5-fold, or 10-fold higher than the normalized expression level of FGFR2-IIIc. In some embodiments, expression levels are normalized to GUSB (beta-glucuronidase). In some embodiments, the cancer overexpresses FGFR2-IIIb but does not comprise an amplification of the FGFR2 gene.

[00107] In some embodiments, the cancer comprises FGFR2, such as overexpression of FGFR2-IIIb protein, while in some other embodiments, the cancer does not include overexpression of FGFR2 or FGFR2-IIIb protein. Overexpression of FGFR2-IIIb protein can be determined by any suitable method in the art, including, but not limited to, antibody-based methods, such as immunohistochemistry (IHC). In some embodiments, IHC staining is assessed according to methods in the art. The terms "overexpression of FGFR2-IIIb protein" and "overexpression of FGFR2IIIb proteins" and the like mean elevated levels of FGFR2-IIIb protein, regardless of the cause of such elevated levels (i.e., whether the elevated levels are the result of increased translation and/or decreased degradation of the protein, another mechanism, or a combination of mechanisms).

[00108] The level of FGFR2 or FGFR2IIIb expression by IHC can be determined by submitting a tumor sample to an IHC score on a scale of 0 to 3, whereby a score of "0" is given if there is no reactivity or there is membrane reactivity only in <10% of the tumor cells; a score of "1+" is given if there is weak or barely noticeable membrane reactivity in at least 10% of the tumor cells or if the cells are reactive in only a portion of their membranes; a score of "2+" is given if there is weak or moderate complete, basolateral, or lateral membrane reactivity in at least 10% of the tumor cells; and a score of "3+" is given if there is strong complete basolateral or lateral membrane reactivity in at least 10% of the tumor cells. In some embodiments, 1+, 2+, or 3+ IHC staining of the tumor cells indicates overexpression of FGFR2IIIb. In some embodiments, 2+ or 3+ IHC staining of the tumor cells indicates overexpression of FGFR2IIIb. In some embodiments, 3+ IHC staining of the tumor cells indicates overexpression of FGFR2IIIb. In some embodiments, the gastric or bladder cancer comprises an amplification of the FGFR2 gene. In some embodiments, the gastric or bladder cancer comprising an amplification of the FGFR2 gene overexpresses FGFR2-IIIb. In some embodiments, the gastric or bladder cancer comprising an amplification of FGFR2 overexpresses FGFR2-IIIb to a greater extent than FGFR2-IIIc. In some embodiments, the gastric or bladder cancer overexpresses FGFR2-IIIb but does not comprise an amplification of the FGFR2 gene. In some embodiments, a gastric or bladder cancer comprising an FGFR2 amplification expresses FGFR2-IIIb at a normalized level that is greater than 2-fold, 3-fold, 5-fold, or 10-fold the normalized expression level of FGFR2-IIIc. In some embodiments, expression levels are normalized to GUSB (beta-glucuronidase). In some embodiments, the overexpression is mRNA overexpression. In some embodiments, the overexpression is protein overexpression.

[00109] " Treatment " as used herein refers to both therapeutic treatment and prophylactic or preventative measures that aim to prevent or slow down (alleviate) a target pathological condition or disorder. In some embodiments, the term " treatment " encompasses any administration or use of a therapeutic agent for a disease in a mammal, including a human, and includes suppressing or slowing down the disease or the progression of the disease; partially or completely alleviating the disease, such as by regressing or restoring or restoring lost, absent or defective function; stimulating an ineffective process; or resulting in a decrease in the severity of the underlying manifestations of the disease. The term " treatment " also includes decreasing the severity of any phenotypic trait and/or reducing the frequency, extent or likelihood of the trait. Those in need of treatment include those who already have the disorder, as well as those who are prone to the disorder or those in whom the disease is to be prevented.

[00110] The term " effective amount " or " therapeutically effective amount " refers to an amount of a drug that is effective in treating a disease or disorder in a subject. In some embodiments, an effective amount refers to an effective amount, at doses and for periods of time necessary to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an FGFR2 inhibitor and/or a PD-1/PD-L1 inhibitor of the invention may vary depending on factors such as the course of the disease, the age, gender, and weight of the individual, and the ability of the antibody or antibodies to elicit a desired response in the individual. A therapeutically effective amount includes an amount in which any toxic or detrimental effects of the antibody or antibodies are outweighed by the therapeutically beneficial effects. In some embodiments, the term "effective amount" refers to an amount of an antibody that is effective in treating cancer.

[00111] Administration " in combination with " one or more additional therapeutic agents includes simultaneous (parallel) and sequential (continuous) administration in any order.

[00112] " Pharmaceutically acceptable carrier " refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, dosage form excipient, or vehicle commonly used in the art for use with a therapeutic agent that together form a " pharmaceutical composition " for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the dosage form employed. For example, if the therapeutic agent is administered orally, the carrier may be a gelatin capsule. If the therapeutic agent is administered subcutaneously, the carrier ideally is non-irritating to the skin and does not cause a reaction at the injection site.

[00113] Additional definitions are provided in the following sections.

Illustrative FGFR2 inhibitors

[00114] FGFR2 inhibitors according to the methods and compositions herein may be FGFR2 antibodies, FGFR2 ECDs, or FGFR2 ECD fusion molecules.

Illustrative antibodies to FGFR2

[00115] In any of the compositions or methods described herein that utilize an anti-FGFR2 antibody, the anti-FGFR2 antibody may be a humanized antibody, a chimeric antibody, or a human antibody. In any of the compositions or methods described herein, the anti-FGFR2 antibody may be selected from Fab, Fv, scFv, Fab'', and (Fab') ₂ . In any of the compositions or methods described herein, the anti-FGFR2 antibody may be selected from IgA, IgG, and IgD. In any of the compositions or methods described herein, the anti-FGFR2 antibody may be IgG. In any of the methods described herein, the antibody may be IgG1 or IgG3.

[00116] Exemplary anti-FGFR2 antibodies include antibodies that bind FGFR2-IIIb. In some embodiments, anti-FGFR2-IIIb antibodies bind FGFR2-IIIc with lower affinity than they bind FGFR2-IIIb. In some embodiments, anti-FGFR2-IIIb antibodies do not detectably bind FGFR2-IIIc.

[00117] An exemplary anti-FGFR2-IIIb antibody for use in embodiments of the present invention is the anti-HuGAL-FR21 antibody described in U.S. Patent No. 8,101,723 B2, issued January 24, 2012, which is specifically incorporated herein by reference. Figures 13 and 14 of U.S. Patent No. 8,101,723 B2 provide the amino acid sequences of the variable regions and chains of full-length mature HuGAL-FR21 antibodies, and are incorporated herein by reference. The sequences of the heavy chain variable region of the HuGAL-FR21 antibody are underlined in Figure 13 of U.S. Patent No. 8,101,723 B2, and are specifically incorporated herein by reference. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not contain fucose at Asn297. Additional antibodies that may be used in embodiments of the present invention include those described in U.S. Patent Publication No. 2015-0050273-A1, which describes certain afucosylated antibodies to FGFR2-IIIb and which are incorporated herein by reference.

[00118] In some embodiments, an anti-FGFR2-IIIb antibody comprises at least one, two, three, four, five, or six hypervariable regions (HVRs, e.g., CDRs) selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise a fucose at Asn297.

[00119] In some embodiments, an anti-FGFR2-IIIb antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, an anti-FGFR2-IIIb antibody comprises at least one heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, an anti-FGFR2-IIIb antibody comprises two heavy chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region. In some embodiments, an anti-FGFR2-IIIb antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, an anti-FGFR2-IIIb antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 and a light chain comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise a fucose at Asn297.

[00120] In some embodiments, an anti-FGFR2-IIIb antibody comprises six HVRs comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-FGFR2-IIIb antibody comprises six HVRs as described above and binds FGFR2-IIIb. In some embodiments, the anti-FGFR-IIIb antibody does not bind FGFR2-IIIc. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not contain fucose at Asn297.

[00121] In one aspect, an anti-FGFR2-IIIb antibody competes with an anti-FGFR2-IIIb antibody that comprises six HVRs comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise a fucose at Asn297.

[00122] In some embodiments, an anti-FGFR2-IIIb antibody comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise a fucose at Asn297.

[00123] In some embodiments, an anti-FGFR2-IIIb antibody comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise a fucose at Asn297.

[00124] In some embodiments, an anti-FGFR2-IIIb antibody comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 8; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not contain fucose at Asn297.

[00125] In some embodiments, an anti-FGFR2-IIIb antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-FGFR2-IIIb antibody comprising the sequence retains the ability to bind to FGFR2-IIIb. In some embodiments, such an anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without detectable binding to FGFR2-IIIc. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 4. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-FGFR2-IIIb antibody comprises the VH sequence of SEQ ID NO: 5, including post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise fucose at Asn297.

[00126] In some embodiments, an anti-FGFR2-IIIb antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-FGFR2-IIIb antibody comprising the sequence retains the ability to bind to FGFR2-IIIb. In some embodiments, the anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without binding to FGFR2-IIIc. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 5. In some embodiments, the substitutions, insertions, or deletions occur outside of the HVR regions (i.e., in the FRs). Optionally, the anti-FGFR2-IIIb antibody comprises the VL sequence of SEQ ID NO: 4, including post-translational modifications of that sequence. In a specific embodiment, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not contain fucose at Asn297.

[00127] In some embodiments, an anti-FGFR2-IIIb antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4 and a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, and a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-FGFR2-IIIb antibody comprising the sequence retains the ability to bind to FGFR2-IIIb. In some embodiments, such an anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without binding to FGFR2-IIIc. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 4. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 5. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-FGFR2-IIIb antibody comprises the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 5, including post-translational modifications of one or both sequences. In a specific embodiment, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise fucose at Asn297.

[00128] In some embodiments, the anti-FGFR2-IIIb antibody is a VH as in any of the embodiments above and a VL as in any of the embodiments above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 4 and SEQ ID NO: 5, respectively, including post-translational modifications of these sequences. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise a fucose at Asn297.

[00129] In some embodiments, an anti-FGFR2-IIIb antibody comprises a heavy chain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, a heavy chain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-FGFR2-IIIb antibody comprising the sequence retains the ability to bind to FGFR2-IIIb. In some embodiments, such an anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without detectable binding to FGFR2-IIIc. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 2. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the heavy chain of the anti-FGFR2-IIIb antibody comprises the VH sequence of SEQ ID NO: 2, including post-translational modifications of that sequence. In a specific embodiment, the heavy chain comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise fucose at Asn297.

[00130] In some embodiments, an anti-FGFR2-IIIb antibody comprises a light chain that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, a light chain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-FGFR2-IIIb antibody comprising the sequence retains the ability to bind to FGFR2-IIIb. In some embodiments, such an anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without detectable binding to FGFR2-IIIc. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 3. In some embodiments, the substitutions, insertions, or deletions occur outside of the HVR regions (i.e., in the FRs). Optionally, the light chain of the anti-FGFR2-IIIb antibody comprises the VL sequence of SEQ ID NO: 3, including post-translational modifications of that sequence. In a specific embodiment, the light chain comprises one, two, or three HVRs selected from (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise fucose at Asn297.

[00131] In some embodiments, an anti-FGFR2-IIIb antibody comprises a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 and a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 99% identity, contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-FGFR2-IIIb antibody comprising the sequence retains the ability to bind to FGFR2-IIIb. In some embodiments, such an anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without detectable binding to FGFR2-IIIc. In some embodiments, a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-FGFR2-IIIb antibody comprising the sequence retains the ability to bind to FGFR2-IIIb. In some embodiments, such an anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without detectable binding to FGFR2-IIIc. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 2. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 3. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the heavy chain of the anti-FGFR2-IIIb antibody comprises the VH sequence of SEQ ID NO: 2, including post-translational modifications of that sequence, and the light chain of the anti-FGFR2-IIIb antibody comprises the VL sequence of SEQ ID NO: 3, including post-translational modifications of that sequence. In a particular embodiment, the heavy chain comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the light chain comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody is afucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise a fucose at Asn297.

[00132] Additional exemplary FGFR2 antibodies are the GAL-FR22 and GAL-FR23 antibodies described in U.S. Patent No. 8,101,723 B2, incorporated herein by reference. For example, the light and heavy chain variable regions of GAL-FR22 are shown as SEQ ID NOs: 7 and 8 in U.S. Patent No. 8,101,723 B2, while the Kabat CDRs and the light and heavy chain variable regions are also shown in Fig. 16 of that patent, which are incorporated herein by reference. GAL-FR21, GAL-FR22, and GAL-FR23 produced by the hybridomas have been deposited with the American Type Culture Collection, PO Box 1549, Manassas VA, USA, 20108, as ATCC Accession Numbers 9586, 9587, and 9408, dated November 6, November 6, and August 12, 2008, respectively. Thus, in some embodiments, an anti-FGFR2 antibody is an antibody comprising an amino acid sequence of an antibody derived from one of these three hybridoma strains.

[00133] The variable regions of the heavy and light chains of GAL-FR22 are also provided herein as SEQ ID NOs: 39 and 43, while the Kabat CDRs are provided herein as SEQ ID NOs: 40-42 and 44-46 herein. Thus, in some embodiments, the heavy chain variable region of an anti-FGFR2-IIIb antibody comprises: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 40; (ii) a CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and (iii) a CDR3 comprising the amino acid sequence of SEQ ID NO: 42; and the light chain variable region comprises: (iv) a CDR1 comprising the amino acid sequence of SEQ ID NO: 44; (v) a CDR2 comprising the amino acid sequence of SEQ ID NO: 45; and (vi) CDR3 comprising the amino acid sequence of SEQ ID NO: 46.

[00134] In some embodiments, an anti-FGFR2 antibody comprises an anti-FGFR2-IIIb antibody wherein the heavy chain variable domain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 39, or that comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, an anti-FGFR2 antibody comprises an anti-FGFR2-IIIb antibody wherein the light chain variable domain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 43, or that comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the heavy chain variable domain is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 39, or which comprises the amino acid sequence of SEQ ID NO: 39, and a light chain variable domain that is at least 95%, such as at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 43, or which comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the antibody is an IgG1 or IgG3 antibody that does not comprise fucose at Asn297.

Afucosylated antibodies to FGFR2

[00135] In some embodiments, anti-FGFR2 antibodies, such as anti-FGFR2-IIIb antibodies as described above, have a carbohydrate structure that does not contain fucose attached (directly or indirectly) to the Fc region (i.e., afucosylated antibodies), i.e., the antibodies are afucosylated. In some embodiments, the afucosylated antibody is an IgG1 or IgG3 antibody that does not contain fucose at Asn297.

[00136] In this regard, antibodies are considered to be afucosylated if a plurality of such antibodies comprises at least 95% afucosylated antibodies. The amount of fucose can be determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all carbohydrate structures attached to Asn 297 (e.g., complex, hybrid, and high-mannose structures). Non-limiting illustrative methods for detecting fucose in an antibody include MALDI-TOF mass spectrometry (see, e.g., WO 2008/077546), HPLC analysis of released fluorescently labeled oligosaccharides (see, e.g., Schneider et al., "N-Glycan analysis of monoclonal antibodies and other glycoproteins using UHPLC with fluorescence detection," Agilent Technologies, Inc. (2012); Lines, J. Pharm. Biomed. Analysis , 14: 601–608 (1996); Takahasi, J. Chrom ., 720: 217–225 (1996)), capillary electrophoresis analysis of released fluorescently labeled oligosaccharides (see, e.g., Ma et al., Anal. Chem., 71: 5185-5192 (1999)) and HPLC with pulsed amperometric detection for the analysis of monosaccharide composition (see, e.g., Hardy, et al., Analytical Biochem., 170: 54-62 (1988)).

[00137] Asn297 refers to the asparagine residue located at position 297 in the Fc region (according to the EU numbering of the Fc region residues of Fc); however, in a given antibody sequence, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. In the anti-FGFR2-IIIb antibody described herein, Asn297 is in the sequence QY N ST and is highlighted in bold and underlined in the sequence table shown below, SEQ ID NO: 2.

[00138] Fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "afucosylated" or "fucose-deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng . 87: 614 (2004). Examples of cell lines that are capable of producing afucosylated antibodies include CHO Lec13 cells, which are defective in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al ., particularly in Example 11), and knockout cell lines, such as cell lines lacking a functional alpha-1,6-fucosyltransferase gene, FUT8, such as knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680–688 (2006); and WO2003/085107).

[00139] Anti-FGFR2 antibodies herein may also comprise divided oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is divided into portions by GlcNAc. Such antibodies may have reduced fucosylation and/or improved ADCC function. Examples of such antibodies are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.,); and U.S. Patent No. 2005/0123546 (Umana et al.). In some embodiments, anti-FGFR2 antibodies have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibodies may have improved CDC function. Such antibodies are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

[00140] In some embodiments, an afucosylated anti-FGFR2 antibody mediates ADCC in the presence of human effector cells more efficiently than an antibody with the same amino acid sequence that contains fucose. Typically, ADCC activity can be determined using the in vitro ADCC assay disclosed in US 2015-0050273 A1, but other assays or methods for determining ADCC activity, e.g., in an animal model, etc.

[00141] In some embodiments, an anti-FGFR2 antibody comprises the heavy and light chain sequences of SEQ ID NOs: 2 and 3. In some embodiments, an antibody comprising the heavy and light chain sequences of SEQ ID NOs: 2 and 3 is afucosylated.

FGFR2 ECD and FGFR2 ECD fusion molecules

[00142] In some embodiments, the FGFR2 inhibitor is an FGFR2 ECD, such as an FGFR2 ECD fusion molecule. FGFR2 ECD fusion molecules can comprise fusion partners such as polymers, polypeptides, lipophilic moieties, and succinyl groups. Exemplary polypeptide fusion partners include serum albumin and an antibody Fc domain. Other exemplary polymeric fusion partners include, but are not limited to, polyethylene glycol, including polyethylene glycols having branched and/or linear chains. Some exemplary fusion partners include, but are not limited to, an immunoglobulin Fc domain, albumin, and polyethylene glycol. The amino acid sequences of some exemplary Fc domains are shown in SEQ ID NOs:24-26.

[00143] Exemplary FGFR2 ECDs and FGFR2 ECD fusion molecules include those described in PCT Publication WO 2007/014123. The FGFR2 ECDs and FGFR2 ECD fusion molecules may comprise a native amino acid sequence of the ECD, including FGFR2-IIIb or FGFR2-IIIc BCD. Alternatively, the FGFR2 ECDs and FGFR2 ECD fusion molecules may comprise an FGFR2 ECD with a C-terminal deletion of one or more and up to 22 amino acid residues measured from the C-terminus, wherein the FGFR2 ECD retains at least one of its FGF ligand binding functions. In some embodiments, the FGFR2 ECD has up to 22 amino acids at the C-terminus deleted. In some embodiments, the deletion does not extend to a valine residue at amino acid residue 357 of native full-length FGFR2-IIIb or amino acid residue 359 of native full-length FGFR2-IIIc.

[00144] For example, in some embodiments, the FGFR2 ECD or FGFR2 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 14, but in which amino acid residues have been removed from the amino terminus and/or the carboxyl terminus, and wherein the resulting molecule is capable of binding to FGF2. In some embodiments, the FGFR2 ECD or FGFR2 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 15, which corresponds to the amino acid sequence of SEQ ID NO: 14, but with the last three carboxyl-terminal amino acid residues, YLE, removed. Other examples of such variants include those in which the 4 C-terminal amino acid residues are deleted (SEQ ID NO: 16), those in which the 5 C-terminal amino acid residues are deleted (SEQ ID NO: 17), those in which the 8 C-terminal amino acid residues are deleted (SEQ ID NO: 18), those in which the 9 C-terminal amino acid residues are deleted (SEQ ID NO: 19), those in which the 10 C-terminal amino acid residues are deleted (SEQ ID NO: 20), those in which the 14 C-terminal amino acid residues are deleted (SEQ ID NO: 21), those in which the 15 C-terminal amino acid residues are deleted (SEQ ID NO: 22), those in which the 16 C-terminal amino acid residues are deleted (SEQ ID NO: 23), those in which the 17 C-terminal amino acid residues are deleted (SEQ ID NO: 24), all when compared to the native sequence FGFR2-IIIb or FGFR2-IIIc. Any of the above FGFR2 ECD fragments can be linked to any of the fusion partners described above to form an FGFR2 ECD fusion molecule.

[00145] In some embodiments, at least one amino acid in the FGFR2 ECD sequence may be mutated to prevent glycosylation at that site in the polypeptide. Non-limiting exemplary amino acids of the FGFR2 ECD that may be glycosylated include N62, N102, N207, N220, N244, N276, N297, and N310 of SEQ ID NO:28.

[00146] Additional exemplary FGFR2 ECDs and FGFR2 ECD fusion molecules include those described in PCT Publication No. WO2010/017198. These include FGFR2 ECDs and FGFR2 ECD fusion molecules with mutations in the "acidic box" region of the FGFR2 ECD. Such an acidic region of the FGFR2 ECD mutein can be used as either an FGFR2 ECD or an FGFR2 ECD fusion molecule. In some embodiments, the FGFR2 ECD or FGFR2 ECD fusion molecule comprises the short acidic box of FGFR1 instead of the short acidic box of FGFR2. For example, FGFR2 ECD residues 111 to 118 (SEQ ID NO: 28) can be replaced with FGFR1 ECD residues 105 to 112 (SEQ ID NO: 29). In some embodiments, the FGFR2 ECD or FGFR2 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the FGFR2 ECD or FGFR2 ECD fusion molecule comprises the amino acid sequence of any of SEQ ID NOs: 31-34. Any of the FGFR2 ECD mutant acidic block sequences, such as SEQ ID NO: 30, may also be fused with any of the FGFR2 ECD C-terminal deletion sequences described above (SEQ ID NOs: 15-24), and optionally with one or more fusion molecules (e.g., SEQ ID NOs: 32-34).

[00147] In some embodiments, the FGFR2 ECD or FGFR2 ECD fusion molecule lacks a signal peptide. In some embodiments, the FGFR2 ECD comprises at least one signal peptide, which can be selected from a native FGFR2 signal peptide and/or a heterologous signal peptide such as FGFR1, FGFR3, or FGFR4.

[00148] In the case of an FGFR2 ECD fusion molecule, the fusion partner may be linked to either the amino terminus or the carboxyl terminus of the polypeptide. In some embodiments, the polypeptide and the fusion partner are covalently linked. If the fusion partner is also a polypeptide (a "polypeptide fusion partner"), the polypeptide and the polypeptide fusion partner may be part of a contiguous amino acid sequence. In such cases, the polypeptide and the polypeptide fusion partner may be translated as a single polypeptide from a coding sequence that encodes both the polypeptide and the polypeptide fusion partner. In some embodiments, the FGFR2 ECD fusion molecule comprises a "GS" (glycine-serine) linker between the FGFR2 ECD or the acidic region of the FGFR2 ECD mutein and the fusion partner. In some embodiments, the polypeptide and the fusion partner are covalently linked by other means, such as, for example, a chemical bond other than a peptide bond. In some embodiments, the polypeptide and the fusion partner are non-covalently linked to each other. In some such embodiments, they can be linked, for example, using binding pairs. Examples of binding pairs include, but are not limited to, biotin and avidin or streptavidin, an antibody and its antigen, etc.

Illustrative PD-1/PD-L1 inhibitors

[00149] Exemplary PD-1/PD-L1 inhibitors include antibodies that inhibit PD-1, such as anti-PD-1 antibodies and anti-PD-L1 antibodies. Such antibodies can be humanized antibodies, chimeric antibodies, murine antibodies, human antibodies, and antibodies comprising the heavy chain and/or light chain CDRs discussed herein. PD-1/PD-L1 inhibitors also include fusion molecules that block the binding of PD-1 to PD-L1, such as AMP-224, and PD-1 inhibitory polypeptides, such as AUR-012, that can compete with PD-1 for binding to PD-L1.

Illustrative antibodies to PD-1/PD-L1

[00150] PD-1 is a key immune checkpoint receptor expressed on activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified, programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2). These ligands are expressed on antigen-presenting cells as well as in many human cancers and have been shown to suppress T cell activation and cytokine secretion upon binding to PD-1. Inhibition of the PD-1/PD-L1 interaction mediates potent antitumor activity in preclinical models.

[00151] Human monoclonal antibodies (HuMAbs) that specifically bind to PD-1 with high affinity are described in U.S. Patent No. 8,008,449. Other anti-PD-1 mAbs are described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509 and in PCT Publication No. WO 2012/145493. Each of the anti-PD-1 HuMAbs described in U.S. Patent No. 8,008,449: (a) binds to human PD-1 with a K _D of 1 x 10 ^-7 M or less, as determined by surface plasmon resonance using a Biacore biosensor system; (b) does not substantially bind to human CD28, CTLA-4, or ICOS; (c) increases T cell proliferation in a mixed lymphocyte culture (MLC) reaction; (d) increases interferon-γ production in a MLC reaction; (e) increases IL-2 secretion in a MLC reaction; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and/or PD-L2 to PD-1; (h) stimulates an antigen-specific memory response; (i) stimulates antibody formation; and/or (j) inhibits tumor cell growth in vivo . Anti-PD-1 antibodies useful in the present invention include antibodies that specifically bind to human PD-1 and exhibit at least one, at least two, at least three, at least four, or at least five of the preceding characteristics (a) through (j).

[00152] In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as "Opdivo ^® ", previously called 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 antibody (S228P), a PD-1 immune checkpoint inhibitor that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of anti-tumor T cell functions (U.S. Patent No. 8,008,449, Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[00153] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as " ^Keytruda® ", lambrolizumab, and MK-3475) is a humanized IgG4 monoclonal antibody directed against the cell surface receptor human PD-1 (programmed death protein-1 or programmed cell death protein-1). Pembrolizumab is described, for example, in U.S. Patent No. 8,900,587; see also "www" dot "cancer" dot "gov" slash "drugdictionary?cdrid=695789" (last accessed: December 14, 2014). Pembrolizumab has been approved by the FDA for the treatment of relapsed or refractory melanoma.

[00154] In other embodiments, the anti-PD-1 antibody is MEDI0608 (formerly AMP-514), which is a monoclonal antibody against the PD-1 receptor. MEDI0608 is described, for example, in U.S. Patent No. 8,609,089 B2 or at the website: "www" dot "cancer" dot "gov" slash "drugdictionary?cdrid=756047" (last accessed: December 14, 2014).

[00155] In some embodiments, the anti-PD-1 antibody is pidilizumab (CT-011), which is a humanized monoclonal antibody. Pidilizumab is described in U.S. Patent No. 8,686,119 B2 or WO 2013/014668 A1.

[00156] Anti-PD-1 antibodies used in the disclosed methods also include isolated antibodies that specifically bind to human PD-1 and cross-compete for binding to human PD-1 with nivolumab (see, e.g., U.S. Patent No. 8,008,449, WO 2013/173223). The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope. These cross-competing antibodies are expected to have functional properties very similar to those of nivolumab by virtue of their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays such as the Biacore assay, ELISA assays or flow cytometry (see, e.g., WO 2013/173223).

[00157] In some embodiments, antibodies that cross-compete for binding to human PD-1 with, or bind to the same epitope region of human PD-1 as nivolumab are monoclonal antibodies. For administration to humans, these cross-competing antibodies may be chimeric antibodies or may be humanized or human antibodies.

[00158] Anti-PD-1 antibodies used in the methods of the disclosed invention also include antigen-binding portions of the above antibodies. Examples include (i) a Fab fragment, a monovalent fragment consisting of the V _L , V _H , _{CL ,} and _CH1 domains; (ii) an F(ab')2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the V _H and _CH1 domains; and (iv) an Fv fragment consisting of the V _L and V _H domains of one arm of an antibody.

[00159] A non-limiting exemplary fusion molecule that is a PD-1/PD-L1 inhibitor is AMP-224 (Amplimmune, GlaxoSmithKline). A non-limiting exemplary polypeptide that is a PD-1/PD-L1 inhibitor is AUR-012.

Illustrative constant regions of an antibody

[00160] In some embodiments, an FGFR2 or anti-PD-1 or anti-PD-L1 antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ.

[00161] In some embodiments, an antibody described herein comprises a human IgG constant region. In some embodiments, if effector function is desired, an antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, an antibody described herein comprises a human IgG1 constant region. In some embodiments, an antibody described herein comprises a human IgG1 constant region, wherein N297 is not fucosylated. In some embodiments, an antibody described herein comprises a human IgG1 constant region and a human κ light chain.

[00162] In this specification and claims, unless explicitly stated or known to one of skill in the art, the numbering of residues in an immunoglobulin heavy chain corresponds to the EU index as described in Kabat et al ., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference. "Kabat EU index" refers to the EU numbering of residues of a human IgG1 antibody.

[00163] In some embodiments, an antibody of the invention comprises a variant Fc region having at least one amino acid substitution compared to an Fc region of a wild-type IgG or a wild-type antibody. In some embodiments, the variant Fc region has two or more amino acid substitutions in the Fc region of a wild-type antibody. In some embodiments, the variant Fc region has three or more amino acid substitutions in the Fc region of a wild-type antibody. In some embodiments, the variant Fc region has at least one, two, or three or more Fc region amino acid substitutions described herein. In some embodiments, the Fc region of a variant herein will have at least about 80% homology to a native sequence Fc region and/or to an Fc region of the parent antibody. In some embodiments, the Fc region of a variant herein will have at least about 90% homology with a native sequence Fc region and/or with an Fc region of a parent antibody. In some embodiments, the Fc region of a variant herein will have at least about 95% homology with a native sequence Fc region and/or with an Fc region of a parent antibody.

[00164] In some embodiments, an antibody provided herein is altered to increase or decrease the degree of glycosylation of the antibody. Addition or removal of glycosylation sites in an antibody can be easily accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[00165] If the antibody comprises an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached via an N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide can comprise a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "core" of the biantennary oligosaccharide structure. In some embodiments, modifications can be made to the oligosaccharide in an antibody of the present invention to create antibodies with certain improved properties.

[00166] Antibodies can also have amino-terminal leader extensions. For example, one or more amino acid residues of an amino-terminal leader sequence are present at the amino terminus of any one or more heavy or light chains of the antibody. An exemplary amino-terminal leader extension comprises or consists of three amino acid residues, VHS, present on one or both light chains of the antibody.

[00167] The in vivo or serum half-life of human FcRn high affinity binding polypeptides can be analyzed, for example, in transgenic mice, in humans, or in non-human primates administered with polypeptides with a variant Fc region. See also, for example, Petkova et al . International Immunology 18(12):1759-1769 (2006).

Illustrative chimeric antibodies

[00168] In some embodiments, an anti-FGFR2 or anti-PD-1 or anti-PD-L1 antibody provided herein is a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al. (1984) Proc Natl. Acad. Sci. USA , 81: 6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate antibody, such as a monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody in which the class or subclass has been altered compared to the parent antibody. The term "chimeric antibodies" includes antigen-binding fragments of such antibodies.

[00169] Non-limiting exemplary chimeric antibodies include anti-FGFR2 or PD-1/PD-L1 chimeric antibodies comprising the heavy chains of HVR1, HVR2, and HVR3 and/or the light chain sequences of HVR1, HVR2, and HVR3 described herein.

[00170] In some embodiments, a chimeric antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ. In some embodiments, a chimeric antibody described herein comprises a human IgG constant region. In some embodiments, a chimeric antibody described herein comprises a human IgG4 heavy chain constant region. In some embodiments, a chimeric antibody described herein comprises a human IgG4 constant region and a human κ light chain.

[00171] As noted above, whether effector function is desirable or undesirable may depend on the particular treatment performed by the antibody. Thus, in some embodiments, if effector function is desirable, a chimeric antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, if effector function is undesirable, a chimeric antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected. In some embodiments, a chimeric antibody described herein comprises a human IgG1 constant region, wherein N297 is not fucosylated. In some embodiments, a chimeric antibody described herein comprises a human IgG1 constant region and a human κ light chain.

Illustrative humanized antibodies

[00172] In some embodiments, humanized antibodies that bind FGFR2 or PD-1/PD-L1 are used. Humanized antibodies are useful as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies (such as the formation of human anti-mouse antibodies (HAMA)), which can lead to an immune response to the therapeutic antibody and a decrease in the effectiveness of the therapy.

[00173] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which the HVRs or CDRs (or fragments thereof) are derived from a non-human antibody and the FRs (or fragments thereof) are derived from human antibody sequences. In some cases, the humanized antibody also comprises at least a fragment of a human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), such as to restore or improve antibody specificity or affinity.

[00174] Humanized antibodies and methods for making them are discussed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and are further described, for example, in Riechmann et al ., (1988) Nature 332:323-329; Queen et al ., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36: 25-34 (describing SDR (a-CDR) grafting); Padlan, (1991) Mol . Immunol. 28:489–498 (describing “surface alteration”); Dall’Acqua et al ., (2005) Methods 36:43–60 (describing “FR shuffling”); and Osbourn et al ., (2005) Methods 36:61–68 and Klimka et al ., (2000) Br. J. Cancer , 83:252–260 (describing a “directed selection” approach to FR shuffling).

[00175] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best match" method (see, e.g., Sims et al . (1993) J. Immunol. 151:2296); framework regions derived from the human antibody consensus sequence of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al . (1992) Proc. Natl. Acad. Sci. USA , 89:4285; and Presta et al . (1993) J. Immunol , 151:2623); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and framework regions obtained from screening of FR libraries (see, e.g., Baca et al ., (1997) J. Biol. Chem . 272: 10678–10684 and Rosok et al ., (1996) J. Biol. Chem. 271: 22611–22618).

[00176] In some embodiments, the humanized antibodies comprise one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ.

[00177] In some embodiments, a humanized antibody described herein comprises a human IgG constant region. In some embodiments, if effector function is desired, the antibody comprises a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region. In some embodiments, a humanized antibody described herein comprises a human IgG1 constant region. In some embodiments, a humanized antibody described herein comprises a human IgG1 constant region, wherein N297 is not fucosylated. In some embodiments, a humanized antibody described herein comprises a human IgG1 constant region and a human κ light chain.

Human antibodies

[00178] Human antibodies to FGFR2 or PD-1/PD-L1 can be produced by any suitable method. Non-limiting illustrative methods include generating human antibodies in transgenic mice that contain human immunoglobulin loci. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993); Jakobovits et al., Nature 362: 255-8 (1993); Lonberg et al., Nature 368: 856-9 (1994); and U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5877397; 5874299; and 5545806.

[00179] Non-limiting illustrative methods also include generating human antibodies using phage display libraries. See, for example, Hoogenboom et al., J. Mol. Biol. 227: 381-8 (1992); Marks et al., J. Mol. Biol. 222: 581-97 (1991); and PCT Publication No. WO 99/10494.

[00180] In some embodiments, a human antibody comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ. In some embodiments, a human antibody described herein comprises a human IgG constant region. In some embodiments, a human antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, a human antibody described herein comprises an S241P mutation in a human IgG4 constant region. In some embodiments, a human antibody described herein comprises a human IgG4 constant region and a human κ light chain.

[00181] In some embodiments, if effector function is desired, a human antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, if effector function is not desired, a human antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected. In some embodiments, the humanized antibody described herein comprises a human IgG1 constant region, wherein N297 is not fucosylated. In some embodiments, the humanized antibody described herein comprises a human IgG1 constant region and a human κ light chain.

Illustrative antibody conjugates

[00182] In some embodiments, an anti-FGFR2 or anti-PD-1/PD-L1 antibody is conjugated to a label and/or a cytotoxic agent. As used herein, a label is a moiety that facilitates detection of the antibody and/or facilitates detection of a molecule to which the antibody binds. Non-limiting exemplary labels include, but are not limited to, radioisotopes, fluorescent groups, enzymatic groups, chemiluminescent groups, biotin, epitope labels, metal binding labels, etc. One of skill in the art can select a suitable label according to the intended use.

[00183] As used herein, a cytotoxic agent is a moiety that reduces the proliferative potential of one or more cells. A cell has reduced proliferative potential if the cell becomes less capable of proliferating, such as because the cell undergoes apoptosis or otherwise dies, the cell fails to go through the cell cycle and/or fails to divide, the cell differentiates, etc. Non-limiting illustrative cytotoxic agents include, but are not limited to, radioisotopes, toxins, and chemotherapeutic agents. One skilled in the art can select an appropriate cytotoxic drug based on the intended use.

[00184] In some embodiments, the label and/or cytotoxic agent is conjugated to the antibody using in vitro chemistry. Non-limiting exemplary conjugation chemistries are known in the art and include equipment, methods, and/or reagents commercially available from, for example, Thermo Scientific Life Science Research Produces (formerly Pierce, Rockford, IL), Prozyme (Hayward, CA), SACRI Antibody Services (Calgary, Canada), AbD Serotec (Raleigh, NC), etc. In some embodiments, if the label and/or cytotoxic agent is a polypeptide, the label and/or cytotoxic agent can be expressed from the same expression vector with at least one antibody chain to produce a polypeptide comprising the label and/or cytotoxic agent fused to an antibody chain. One skilled in the art can select a suitable method for conjugating the label and/or cytotoxic agent to the antibody according to the intended use.

Nucleic acid molecules encoding antibodies

[00185] Nucleic acid molecules comprising polynucleotides that encode one or more chains of an antibody are provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an antibody. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain of an antibody. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.

[00186] In some such embodiments, the heavy chain and the light chain are expressed from a single nucleic acid molecule or from two separate nucleic acid molecules as two separate polypeptides. In some embodiments, for example, if the antibody is an scFv, one polynucleotide encodes one polypeptide comprising both the heavy chain and the light chain linked together.

[00187] In some embodiments, a polynucleotide encoding a heavy chain or a light chain of an antibody comprises a nucleotide sequence that encodes a leader sequence that is located at the N-terminus of the heavy chain or light chain when translated. As discussed above, the leader sequence may be a native leader sequence of the heavy or light chain or may be another heterologous leader sequence.

[00188] Nucleic acid molecules can be constructed using recombinant DNA techniques known in the art. In some embodiments, the nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.

Expression and production of antibodies

Vectors

[00189] Vectors comprising polynucleotides encoding heavy chains and/or light chains of an antibody are provided. Vectors comprising polynucleotides encoding heavy chains and/or light chains of an antibody are also provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and the light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and the light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.

[00190] In some embodiments, the first vector comprises a polynucleotide that encodes a heavy chain and the second vector comprises a polynucleotide that encodes a light chain. In some embodiments, the first vector and the second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a molar or mass ratio of between 5:1 and 1:5 of the first vector and the second vector are transfected into host cells. In some embodiments, a mass ratio of between 1:1 and 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[00191] In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO cells or CHO-derived cells or NSO cells. Illustrative such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[00192] In some embodiments, the vector is selected for in vivo expression of antibody heavy chains and/or antibody light chains in animals, including humans. In some such embodiments, expression of the polypeptide is under the control of a promoter that functions in a tissue-specific manner. For example, liver-specific promoters are described, for example, in PCT Publication No. WO 2006/076288.

Host cells

[00193] In various embodiments, the heavy chains and/or light chains of the antibody can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be accomplished, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express the polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, the heavy chains and/or light chains of the antibody can be expressed in yeast. See, for example, U.S. Publication No. US 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform desired post-translational modifications of the heavy chains and/or light chains of the antibody. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[00194] Introduction of one or more nucleic acids into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting illustrative methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, ^3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into desired host cells according to any suitable method.

[00195] In some embodiments, one or more polypeptides can be produced in vivo in an animal organism that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides according to any suitable method.

Antibody purification

[00196] Antibodies can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include antigen and ligands that bind constant regions of the antibody. For example, protein A, protein G, protein A/G, or an immobilized antibody column can be used to bind the constant region and purify the antibody. Hydrophobic interaction chromatography, such as a butyl or phenyl column, may also be useful for purifying certain polypeptides. Many methods for purifying polypeptides are known in the art.

Cell-free production of antibodies

[00197] In some embodiments, the antibody is produced in a cell-free system. Non-limiting exemplary cell-free systems are described in, for example, Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

Therapeutic compositions and methods

Cancer Treatment Methods

[00198] In some embodiments, methods of treating cancer are provided, comprising administering an effective amount of an FGFR2 inhibitor described herein. Some such embodiments include methods of increasing the number of one or more of: PD-L1-positive cells, NK cells, CD3+ T cells, CD4+ T cells, and CD8+ T cells in tumor tissue of a subject suffering from cancer, comprising administering an FGFR2 inhibitor, wherein the inhibitor is an anti-FGFR2 antibody with enhanced ADCC activity. In some such embodiments, the immunostimulatory agent is administered with the anti-FGFR2 antibody.

[00199] In some other embodiments, methods of treating cancer are provided, comprising administering an effective amount of an FGFR2 inhibitor and an effective amount of at least one immunostimulatory agent. In an exemplary embodiment, the at least one immunostimulatory agent comprises a PD-1/PD-L1 inhibitor. In some embodiments, the FGFR2 inhibitor and at least one immunostimulatory agent, such as a PD-1/PD-L1 inhibitor, are administered simultaneously. In some embodiments, the FGFR2 inhibitor and at least one immunostimulatory agent, such as a PD-1/PD-L1 inhibitor, are administered sequentially. In some embodiments, at least one, at least two, at least three doses, at least five doses, or at least ten doses of the FGFR2 inhibitor are administered prior to administration of the at least one immunostimulatory agent, such as a PD-1/PD-L1 inhibitor. In some embodiments, at least one, at least two, at least three doses, at least five doses, or at least ten doses of at least one immunostimulatory agent, such as a PD-1/PD-L1 inhibitor, are administered prior to administration of the FGFR2 inhibitor. In some embodiments, the last dose of the at least one immunostimulatory agent, such as a PD-1/PD-L1 inhibitor, is administered at least one, two, three, five, or ten days, or one, two, three, five, twelve, or twenty-four weeks prior to the first dose of the FGFR2 inhibitor. In another embodiment, the last dose of the FGFR2 inhibitor is administered at least one, two, three, five, or ten days, or one, two, three, five, twelve, or twenty-four weeks prior to the first dose of the at least one immunostimulatory agent, such as a PD-1/PD-L1 inhibitor. In some embodiments, the subject has received or is receiving a PD-1/PD-L1 inhibitor and an FGFR2 inhibitor is added to the treatment regimen.

[00200] In some embodiments, the cancer is selected from gastric cancer, breast cancer, squamous cell cancer, small cell lung cancer, pituitary cancer, esophageal cancer (including gastroesophageal junction adenocarcinomas), astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder carcinoma, gastric cancer, melanoma, and various head and neck cancers. In some embodiments, the lung cancer is non-small cell lung cancer or squamous cell lung carcinoma. In some embodiments, the leukemia is acute myeloid leukemia or chronic lymphocytic leukemia. In some embodiments, the breast cancer is invasive breast carcinoma. In some embodiments, the ovarian cancer is serous cystadocarcinoma of the ovary. In some embodiments, the kidney cancer is clear cell renal cell carcinoma. In some embodiments, the colon cancer is adenocarcinoma of the colon. In some embodiments, the bladder cancer is urothelial carcinoma of the bladder. In some embodiments, the cancer is selected from bladder cancer, cervical cancer (e.g., squamous cell carcinoma of the cervix), squamous cell carcinoma of the head and neck, rectal adenocarcinoma, non-small cell lung cancer, endometrial cancer, prostate adenocarcinoma, colon cancer, ovarian cancer (such as serous epithelial ovarian cancer), and melanoma. In some embodiments, the cancer is gastric cancer (which includes gastroesophageal cancer) or bladder cancer (such as transitional cell carcinoma, also known as urothelial cancer).

[00201] In some embodiments, the cancer comprises an amplification of the FGFR2 gene, while in some embodiments, the cancer does not comprise an amplification of FGFR2. In some embodiments, a fluorescence in situ hybridization (FISH) assay is used to assess gene amplification, such as with probes for the FGFR2 gene locus and the centromere of chromosome 10. In some embodiments in which amplification occurs, the FGFR2 amplification comprises an FGFR2:CEN10 (centromere of chromosome 10) ratio > 3. In some embodiments, the FGFR2 amplification comprises an FGFR2:CEN10 ratio ≥ 2. However, in other embodiments, the FGFR2 level comprises an FGFR2:CEN10 ratio between 1 and 2, indicating that FGFR2 is not amplified.

[00202] In some embodiments, in which the cancer comprises an FGFR2 gene amplification, the cancer overexpresses FGFR2-IIIb. In some embodiments, a cancer comprising an FGFR2 amplification overexpresses FGFR2-IIIb to a greater extent than FGFR2-IIIc. In some embodiments, the cancer does not comprise a gene amplification, yet FGFR2-IIIb is overexpressed. In some embodiments, a cancer comprising an FGFR2 amplification expresses FGFR2-IIIb at a normalized level that is greater than 2-fold, 3-fold, 5-fold, or 10-fold the normalized expression level of FGFR2-IIIc. In some embodiments, expression levels are normalized to GUSB (beta-glucuronidase). In some embodiments, the cancer overexpresses FGFR2-IIIb but does not comprise an FGFR2 gene amplification. In some embodiments, the gastric or bladder cancer comprises an amplification of the FGFR2 gene. In some embodiments, the gastric or bladder cancer comprises an amplification of the FGFR2 gene that overexpresses FGFR2-IIIb.

[00203] In some embodiments, a gastric or bladder cancer comprising an FGFR2 amplification overexpresses FGFR2-IIIb to a greater extent than FGFR2-IIIc. In some embodiments, the gastric or bladder cancer does not comprise a gene amplification, but FGFR2-IIIb is overexpressed. In some embodiments, a gastric or bladder cancer comprising an FGFR2 amplification expresses FGFR2-IIIb at a normalized level that is greater than 2-fold, 3-fold, 5-fold, or 10-fold the normalized expression level of FGFR2-IIIc. In some embodiments, expression levels are normalized to GUSB (beta-glucuronidase). In some embodiments, the gastric or bladder cancer overexpresses FGFR2-IIIb but does not comprise an FGFR2 gene amplification. In some embodiments, the overexpression is mRNA overexpression. In some embodiments, the overexpression is protein overexpression. In some embodiments, a point mutation or translocation may cause FGFR2 overexpression. Expression levels of FGFR2 types may be determined, for example, using IHC.

[00204] In some embodiments, FGFR2 overexpression is detected using immunohistochemistry (IHC). For example, overexpression can be detected by an IHC signal of 1+, 2+, or 3+ in at least 10% of the tumor cells, such as at least 20%, 30%, 40%, or 50% of the tumor cells. For example, in some such embodiments, the cancer is gastric cancer and patients to be treated may have, for example, an IHC signal for FGFR2b of 3+ in at least 10% of the tumor cells (e.g., in cell membranes). In some embodiments, a patient diagnosed with gastric cancer may have a signal of 2+ or 3+ in at least 10% of the tumor cells. In some embodiments, a patient diagnosed with gastric cancer may have at least a 1+ signal in at least 10% of tumor cells.

[00205] In some embodiments, FGFR2 overexpression may be represented as an "H score." For example, in some such embodiments, the tumor is bladder cancer. To determine the H score, the first membrane staining intensity of cells in a fixed field may be determined, such as by IHC, to obtain scores of 0, 1+, 2+, or 3+, and the H score may be calculated using a formula as follows: 1x (% of cells visualized with IHC intensity of 1+) + 2x (% of cells visualized with IHC intensity of 2+) + 3x (% of cells visualized with IHC intensity of 3+). Theoretically, the H score may range from 0 to 300, and is 300 if all cells in the field have IHC staining of 3+. In some embodiments, the patient to be treated has a baseline H score for an FGFR2, such as FGFR2b (e.g., FGFR2IIIb), of >20, such as >30, >40, >50, or >100, or in the range of 20-300, 20-100, 20-50, 20-40, or 20-30. In some embodiments, the patient has an H score >10, or the H score is in the range of 10-20 or 15-20. In other embodiments, the patient has an H score of 0-10, which may indicate no FGFR2 overexpression. In some such embodiments, the patient is a bladder cancer patient.

[00206] In some embodiments, the cancer has already been determined to overexpress FGFR2IIIb and/or to contain FGFR2 gene amplification. In other embodiments, the methods described herein first assess FGFR2IIIb expression and/or FGFR2 gene amplification before treatment is administered. Additionally, this disclosure provides methods for determining sensitivity to any of the FGFR2 inhibitors, therapies, and uses described above, including assessing FGFR2IIIb expression and/or FGFR2 gene amplification in a cancer patient.

[00207] In some embodiments, wherein the patient has gastric or bladder cancer, the method may include determining whether the patient's cancer falls into one of the following categories, which may indicate sensitivity to the treatment or to the FGFR2 inhibitor composition: a) in the case of a subject with gastric cancer, an IHC signal of 3+ in at least 10% of the tumor cells; b) in the case of a subject with gastric cancer, an IHC signal of 3+ in at least 10% of the tumor cells and amplification of the FGFR2 gene; c) in the case of a subject with gastric cancer, an IHC signal of 3+ in at least 10% of the tumor cells without amplification of the FGFR2 gene; d) in the case of a subject with gastric cancer, an IHC signal of 1+ or 2+ in at least 10% of the tumor cells; e) in the case of a patient with bladder cancer, an IHC signal of 1+ in at least 10% of the tumor cells; f) in the case of a subject with bladder cancer, an IHC signal of 2+ in at least 10% of the tumor cells; g) in the case of a patient with bladder cancer, an H value greater than 20; h) in the case of a subject with bladder cancer, an H value equal to 10-19; i) in the case of a subject with bladder cancer, an H value less than 10.

[00208] In some embodiments of the methods described herein, the subject is a " subject that has insufficiently responded to treatment" with a PD-1/PD-L1 inhibitor. A subject that is a subject that has insufficiently responded to treatment with a PD-1/PD-L1 inhibitor may have previously responded to treatment with a PD-1/PD-L1 inhibitor but may have become less sensitive to the PD-1/PD-L1 inhibitor, or a subject that may have never responded to treatment with a PD-1/PD-L1 inhibitor. An insufficient response to treatment with a PD-1/PD-L1 inhibitor means that aspects of the condition that are expected to improve after a standard dose of the PD-1/PD-L1 inhibitor do not improve, and/or the improvement only occurs when a dose greater than the standard dose is administered. In some embodiments, the subject who has insufficiently responded to treatment with a PD-1/PD-L1 inhibitor has experienced or is experiencing an insufficient response to a PD-1/PD-L1 inhibitor after receiving a standard dose for at least two weeks, at least three weeks, at least four weeks, at least six weeks, or at least twelve weeks. The "standard" dose is determined by a healthcare professional and may depend on age, weight, medical history, severity of disease, frequency of dosing, etc. In some embodiments, the subject who has insufficiently responded to treatment with a PD-1/PD-L1 inhibitor has experienced or is experiencing an insufficient response to an anti-PD-1 antibody and/or an anti-PD-L1 antibody. In some embodiments, the subject who has insufficiently responded to treatment with a PD-1/PD-L1 inhibitor has experienced or is experiencing an insufficient response to AMP-224. In some embodiments, the subject who has insufficiently responded to treatment with a PD-1/PD-L1 inhibitor has experienced or is experiencing an insufficient response to a PD-1/PD-L1 inhibitor selected from nivolumab, pidilizumab, and pembrolizumab.

[00209] In any of the above embodiments, the combination of an FGFR2 inhibitor and at least one immunostimulatory agent, such as a PD-1/PD-L1 inhibitor, can inhibit tumor growth in a mouse tumor model for 1 week, 10 days, or 2 weeks, such as by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In any of the above embodiments of the invention, the combination of an FGFR2 inhibitor and a PD-1/PD-L1 inhibitor in a subject can reduce the volume of at least one tumor in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as for at least one month, two months, three months, six months, or one year.

Anti-FGFR2 antibodies increase the number of NK cells, PD-L1-expressing cells, macrophages, and CD3+, CD8+, and CD4+ T cells, and increase the lymphoid to myeloid cell ratio in tumors

[00210] In any of the above embodiments, administration of an FGFR2 inhibitor can demonstrate an increase in NK cells, such as NKp46+ cells, an increase in PD-L1-expressing cells, an increase in CD3+, CD8+, and/or CD4+ T cells, an increase in macrophages, and/or an increase in the lymphoid to myeloid cell ratio compared to a control in tumors taken from a mouse tumor model, such as a xenograft tumor model or a syngeneic tumor model, for at least 1 day, 4 days, 1 week, 10 days, or 2 weeks, and, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the mouse tumor model is a 4T1 tumor model.

[00211] The data presented in the examples provided herein demonstrate that treatment of syngeneic mouse tumor models with an afucosylated anti-FGFR2b antibody increases the number of NKp46+ cells in mouse tumor tissue and inhibits tumor growth. Similar treatment with an antibody with a mutation at N297 and lacking ADCC activity (anti-FGFR2-N297Q) did not demonstrate an increase in NK cells and did not affect tumor growth. (See Examples 2a-b below.)

[00212] The data presented in the examples herein also demonstrate that treatment of syngeneic mouse models with an afucosylated anti-FGFR2 antibody increases the number of PD-L1-positive cells in tumor tissue. (See Example 2a.) This suggests that an FGFR2 inhibitor may be well suited for combination with a PD-1/PD-L1 inhibitor for the treatment of cancer and that the disclosed combinations may have at least additive and in some cases synergistic effects in combination on tumor volume or tumor growth inhibition. Additional data herein demonstrate that treatment of syngeneic mouse tumor models with an afucosylated anti-FGFR2 antibody also increases the number of CD3+, CD8+, and CD4+ T cells in tumor tissue and increases the ratio of lymphoid to myeloid cells. These results were not observed with an anti-FGFR2 antibody containing the N297Q mutation designed to prevent effector function. (See Example 2b.) Additional data herein demonstrate that treatment of syngeneic mouse tumor models with an afucosylated anti-FGFR2 antibody also increases the number of macrophages in tumor tissue. (See Example 2c.) These data suggest that the tumor growth inhibition observed with the afucosylated anti-FGFR2 antibody is partially mediated by NK cell-mediated ADCC activity. Furthermore, the data suggest that this ADCC activity may increase the number of PD-L1-expressing cells in the tumor, which may lead to T cell infiltration into the tumor. The increased lymphoid to myeloid cell ratio further suggests that afucosylated anti-FGFR2 antibody may have potent antitumor activity as a single agent or when used in combination with a PD-1 inhibitor by altering the tumor microenvironment.

[00213] Accordingly, also included herein is a method for increasing the number of NK cells, PD-L1-positive cells, macrophages, CD3+, CD8+, and/or CD4+ T cells, and/or the ratio of lymphoid cells to myeloid cells in a tumor tissue, comprising administering to said subject an effective amount of an FGFR2 inhibitor, such as an anti-FGFR2 antibody, such as an anti-FGFR2 antibody with enhanced ADCC activity. In some embodiments in which an anti-FGFR2 antibody is administered, the antibody is afucosylated, such as afucosylated at position N297. In some embodiments, the increase may be observed after a period of at least 1 day, 4 days, 1 week, 10 days, or 2 weeks, and may be, for example, an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to a control, such as pre-treatment tumor tissue or non-tumor tissue. The FGFR2 inhibitor may be administered, for example, at the dosing regimens described elsewhere herein.

[00214] Also included herein is a method for increasing the number of NK cells, PD-L1 positive cells, macrophages, CD3+, CD8+, and/or CD4+ T cells, and/or the ratio of lymphoid cells to myeloid cells in tumor tissue of a subject, comprising administering to said subject an effective amount of an antibody with ADCC activity, such as with increased ADCC activity. In some embodiments, the antibody has increased ADCC activity due to afucosylation at position N297. In some embodiments, the increase may be observed after a period of at least 1 day, 4 days, 1 week, 10 days, or 2 weeks, and may be, for example, an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to a control, such as pre-treatment tumor tissue or non-tumor tissue. In general, such an antibody may be administered in an amount of from about 10 μg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, the antibody may be administered in an amount of from about 50 μg/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, the antibody may be administered in an amount of about 100 μg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, the antibody may be administered in an amount of about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the antibody may be administered in an amount of about 0.5 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the antibody may be administered at a dose of 0.1 to 10 mg/kg, such as at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, or 10 mg/kg, or a dose range limited by any two of the preceding numbers.

[00215] This application also includes methods for determining the number of NK cells, PD-L1-positive cells, macrophages, CD3+, CD8+, and/or CD4+ T cells, and/or the ratio of lymphoid cells to myeloid cells in a subject's tumor tissue before and/or after administration of an antibody with ADCC activity or increased ADCC activity, such as to determine whether the antibody has such an effect on at least one tumor of the subject. This application also includes methods for determining the number of NK cells, PD-L1-positive cells, CD3+, CD8+, and/or CD4+ T cells, and/or the ratio of lymphoid cells to myeloid cells in a subject's tumor tissue before and/or after administration of an FGFR2 inhibitor, either alone or as part of a combination with a PD-1/PD-L1 inhibitor. Also included in this document are methods for determining the number of NK cells, PD-L1-positive cells, CD3+, CD8+ and/or CD4+ T cells and/or determining the ratio of lymphoid cells to myeloid cells in tumor tissue of a subject receiving a combination of an FGFR2 inhibitor and a PD-1/PD-L1 inhibitor.

[00216] Determining the number of NK cells, PD-L1-positive cells, macrophages, CD3+, CD8+, and/or CD4+ T cells, and/or determining the ratio of lymphoid cells to myeloid cells can be accomplished, for example, by a tissue biopsy or other method of obtaining a sample from the tumor for such studies. Such a biopsy or other sample can typically be taken, for example, 1, 2, 3, 4, 7, 17, 30, 45, or 90 days after the first administration of an antibody with ADCC activity or an FGFR2 inhibitor. The number of NK cells, PD-L1-positive cells, macrophages, CD3+, CD8+, and/or CD4+ T cells, and/or the ratio of lymphoid cells to myeloid cells can be determined, for example, relative to a control, such as a pre-treatment tumor sample or a non-tumor tissue sample. In some embodiments, the number may be expressed as a percentage of a particular cell type, such as CD45+ individual cells. In some embodiments, the number of particular cell types may be determined using flow cytometry.

[00217] In some embodiments, if an increase in NK cells, PD-L1-positive cells, macrophages, CD3+, CD8+, and/or CD4+ T cells, and/or the ratio of lymphoid cells to myeloid cells is observed in such a test relative to a control, the subject may be further administered a PD-1/PD-L1 inhibitor. In some embodiments, if an increase is not observed or if a significant increase is not observed, the dose of the FGFR2 inhibitor or antibody with ADCC activity may be increased.

[00218] In some embodiments, such an assessment of an increase in NK cells, PD-L1-positive cells, macrophages, CD3+, CD8+, and/or CD4+ T cells, and/or in the ratio of lymphoid cells to myeloid cells can be used to determine whether to administer combination therapy with a PD-1/PD-L1 inhibitor or continue treatment without the addition of a PD-1/PD-L1 inhibitor. For example, following administration of an FGFR2 inhibitor, a tumor sample from a subject may be assessed for the number of NK cells, PD-L1 positive cells, macrophages, CD3+, CD8+ and/or CD4+ T cells, and/or the ratio of lymphoid to myeloid cells compared to a control, and if the sample shows an increase in one or both of these cell types, the PD-1/PD-L1 inhibitor may be administered together with the FGFR2 inhibitor in accordance with any of the embodiments described herein.

Routes of administration, carriers and additional pharmaceutical compositions

[00219] In various embodiments, antibodies can be administered in vivo by various routes, including, but not limited to, oral, intra-arterial, parenteral, intranasal, intravenous, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The disclosed compositions can be formulated as preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. The nucleic acid molecule encoding the antibody can be coated onto gold microparticles and delivered intradermally using a particle bombardment device or "gene gun" as described in the literature (see, e.g., Tang et al., Nature 356: 152-154 (1992)). The appropriate dosage form and route of administration can be selected according to the intended use.

[00220] In various embodiments, compositions comprising antibodies are presented in dosage forms with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus , 20 ^th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 ^th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3 ^rd ed., Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers are available, which include vehicles, adjuvants, and diluents. Additionally, a variety of pharmaceutically acceptable excipients are available, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like. Non-limiting illustrative carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[00221] In various embodiments, compositions containing antibodies can be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other oils, glycerides of synthetic aliphatic acids, esters of higher aliphatic acids, or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In various embodiments, the compositions can be formulated for inhalation, such as using pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. The compositions can also be formulated in various embodiments into sustained-release microcapsules, such as biodegradable or non-biodegradable polymers. A non-limiting exemplary biodegradable formulation includes a copolymer of lactic and glycolic acids. A non-limiting illustrative non-biodegradable composition comprises a polyglycerol fatty acid ester. Some methods for producing such compositions are described, for example, in EP 1 125 584 A1.

[00222] Also provided are pharmaceutical packages and kits comprising one or more containers, each comprising one or more doses of an antibody or antibody combination. In some embodiments, a unit dosage form is provided, wherein the unit dosage form comprises a predetermined amount of a composition comprising an antibody or antibody combination, with or without one or more additional agents. In some embodiments, such a unit dosage form is supplied in a disposable prefilled syringe for injection. In various embodiments, the composition contained in the unit dosage form may comprise a saline solution, sucrose, or the like; a buffer, such as a phosphate, or the like; and/or is formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted upon addition of an appropriate liquid, such as sterile water. In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, the composition of the invention comprises heparin and/or proteoglycan.

[00223] The pharmaceutical compositions are administered in an amount effective to treat or prevent a particular indication. A therapeutically effective amount typically depends on the weight of the patient, the physical or health condition of the patient, the severity of the pathological condition being treated, or the age of the patient being treated. In general, the antibodies can be administered in an amount of from about 10 μg/kg body weight to 100 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 50 μg/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 100 μg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the antibodies may be administered in an amount of from about 0.5 mcg/kg body weight to about 20 mg/kg body weight per dose.

[00224] In some embodiments, a PD-1/PD-L1 inhibitor, such as an antibody or fusion molecule, or a polypeptide, is administered at a dose of 0.1 to 100 mg/kg, such as at a dose of 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 mg/kg, or a dose range limited by any two of the preceding numbers. In some embodiments, an FGFR2 inhibitor, such as an antibody or fusion molecule, or an ECD polypeptide, is administered at a dose of 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, or 10 mg/kg, such as a dose range limited by any two of the preceding numbers.

[00225] Antibody compositions can be administered to subjects as needed. The frequency of administration can be determined by those skilled in the art, such as a physician, based on the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like. In some embodiments, an effective dose of the antibody is administered to the subject one or more times. In various embodiments, an effective dose of the antibody is administered to the subject once a month, less than once a month, such as every two months, or every three months. In other embodiments, an effective dose of the antibody is administered more than once a month, such as every three weeks, every two weeks, or every week. In some embodiments, an effective dose of the antibody is administered once every 1, 2, 3, 4, or 5 weeks. In some embodiments, an effective dose of the antibody is administered two or three times a week. An effective dose of the antibody is administered to the subject at least once. In some embodiments, an effective dose of the antibody may be administered multiple times, including over at least a month, at least six months, or at least one year.

[00226] Also provided are compositions comprising an FGFR2 inhibitor as described herein and a PD-1/PD-L1 inhibitor as described herein. In some embodiments, the FGFR2 inhibitor and the PD-1/PD-L1 inhibitor are contained, for example, in separate containers or in separate compartments of a single container such that they are not mixed. In some embodiments, the FGFR2 inhibitor and the PD-1/PD-L1 inhibitor may be present in the same container or compartment and are thus mixed with each other. In some embodiments, the compositions comprise instructions for use, such as instructions for use in the treatment of cancer.

Combinations with other immunostimulating agents

[00227] In some embodiments, an FGFR2 inhibitor is combined with at least one immunostimulatory agent other than a PD-1/PD-L1 inhibitor. Alternatively, in some embodiments, the combination of an FGFR2 inhibitor and a PD-1/PD-L1 inhibitor may be further combined with an effective amount of at least one additional immunostimulatory agent.

[00228] Immunostimulating agents may include, for example, a small molecule drug or a large molecule drug. Examples of biological immunostimulating agents include, but are not limited to, antibodies, antibody fragments, receptor or ligand polypeptide fragments, for example, that block receptor-ligand binding, vaccines, and cytokines.

[00229] In some embodiments, the at least one immunostimulatory agent comprises an agonist of an immunostimulatory molecule, including a costimulatory molecule, while in some embodiments, the at least one immunostimulatory agent comprises an antagonist of an immunostimulatory molecule, including a co-inhibitory molecule. In some embodiments, the at least one immunostimulatory agent comprises an agonist of an immunostimulatory molecule, including a costimulatory molecule, found on immune cells, such as T cells. In some embodiments, the at least one immunostimulatory agent comprises an antagonist of an immunostimulatory molecule, including a co-inhibitory molecule, found on immune cells, such as T cells. In some embodiments, the at least one immunostimulatory agent comprises an agonist of an immunostimulatory molecule, including a costimulatory molecule, found on cells involved in innate immunity, such as NK cells. In some embodiments, at least one immunostimulatory agent comprises an antagonist of an immunoinhibitory molecule, including a co-inhibitory molecule found on cells involved in innate immunity, such as NK cells. In some embodiments, the combination enhances an antigen-specific T cell response in a subject receiving treatment and/or enhances an innate immune response in a subject.

[00230] In some embodiments, a combination of an FGFR2 inhibitor and at least one immunostimulatory agent results in an improved tumor response in an animal cancer model, such as a mouse xenograft tumor model and/or a syngeneic tumor model, compared to administration of the FGFR2 inhibitor alone. In some embodiments, a combination of an FGFR2 inhibitor with at least one immunostimulatory agent results in an additive or synergistic response in an animal cancer model, such as a mouse xenograft tumor model and/or a syngeneic tumor model, compared to administration of either drug alone.

[00231] In embodiments comprising a combination of an FGFR2 inhibitor, a PD-1/PD-L1 inhibitor, and at least one additional immunostimulatory agent, the combination results in an improved anti-tumor response in an animal cancer model, such as a mouse xenograft tumor model and/or a syngeneic tumor model, compared to administration of the FGFR2 inhibitor alone. In some embodiments, the combination of an FGFR2 inhibitor with additional therapeutic agents results in an additive or synergistic response in an animal cancer model, such as a mouse xenograft tumor model and/or a syngeneic tumor model, compared to the use of the individual therapeutic agents alone.

[00232] In some embodiments, the immunostimulatory agent targets a stimulatory or inhibitory molecule that is a member of the immunoglobulin superfamily (IgSF). For example, the immunostimulatory agent can be an agent that targets (or specifically binds) another member of the B7 polypeptide family. The immunomodulatory agent can be an agent that targets a member of the TNF family of membrane-bound ligands, or a costimulatory or coinhibitory receptor that binds specifically to a member of the TNF family (TNF-tumor necrosis factor). Representative members of the TNF and TNFR (tumor necrosis factor receptor) family that can be targeted by immunostimulatory agents include CD40 and CD40L, OX-40, OX-40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α 1β2, FAS, FASL, RELT, DR6, TROY and NGFR.

[00233] In some embodiments, the immunostimulatory agent may comprise (i) an antagonist of a protein that inhibits T cell activation (e.g., an immune checkpoint inhibitor), such as CTLA4, LAG-3, TIM3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, TIM-4, and ILT4 and/or may comprise (ii) an agonist of a protein that stimulates T cell activation, such as B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, CD40L, DR3 and CD28H.

[00234] In some embodiments, the immunostimulatory agent may include an agent that inhibits or is an antagonist of a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β (transforming growth factor), VEGF (vascular endothelial growth factor), and other immunosuppressive cytokines), and in some embodiments, the immunostimulatory agent may include an agent that is an agonist of a cytokine such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα (e.g., the cytokine itself) that stimulates T cell activation. In some embodiments, the immunostimulatory agents may include an antagonist of a chemokine such as CXCR2 (e.g., MK-7123), CXCR4 (e.g., AMD3100), CCR2, or CCR4 (mogamulizumab).

[00235] In some embodiments, the immunostimulatory agents may include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. In some embodiments, at least one immunostimulatory agent is a KIR antagonist.

[00236] Immunostimulatory agents may also include agents that inhibit TGF-β signaling, agents that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF-secreting cell vaccines, CpG oligonucleotides, and imiquimod, or therapeutic agents that enhance the immunogenicity of tumor cells (e.g., anthracyclines).

[00237] Immunostimulating agents may also include certain vaccines, such as mesothelin-targeted vaccines, or attenuated Listeria-based cancer vaccines, such as CRS-207.

[00238] Immunostimulatory agents may also include agents that deplete or block regulatory T cells, such as agents that specifically bind to CD25.

[00239] Immunostimulating agents may also include agents that inhibit a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase.

[00240] Immunostimulating agents may also include agents that inhibit the formation of adenosine or inhibit the adenosine A2A receptor.

[00241] Immunostimulatory agents may also include agents that alter/prevent T cell anergy or exhaustion that causes innate immune activation and/or inflammation at the tumor site.

[00242] In some embodiments, the immunostimulatory agents may include a CD40 agonist, such as an anti-CD40 agonist antibody. The combination of an FGFR2 inhibitor and a PD-1/PD-L1 inhibitor may also be further combined in a combinatorial approach that targets multiple elements of the immune pathway, such as one or more of the following: at least one agent that enhances tumor antigen presentation (e.g., a dendritic cell vaccine, GM-CSF-secreting cell vaccines, CpG oligonucleotides, imiquimod); at least one agent that inhibits negative immune regulation, such as by inhibiting the CTLA4 pathway and/or depleting or blocking regulatory T cells or other immunosuppressive cells; a therapy that stimulates positive immune regulation, such as with agonists that stimulate the CD-137, OX-40 and/or GITR pathway and/or stimulate T cell effector function; at least one agent that systemically increases the number of anti-tumor T cells; a therapy that depletes or inhibits regulatory T cells, such as regulatory T cells in a tumor, such as using a CD25 antagonist (e.g., daclizumab) or by ex vivo depletion of microbead-bound anti-CD25 antibodies; at least one agent that affects the function of suppressor myeloid cells in a tumor; a therapy that enhances the immunogenicity of tumor cells (e.g., anthracyclines); adoptive transfer of T lymphocytes or NK cells, including genetically modified cells, such as cells modified with chimeric antigen receptors (CAR-T therapy); at least one agent that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase; at least one agent that alters/prevents T cell anergy or exhaustion; a therapy that induces activation of innate immunity and/or inflammation at the tumor site; administration of immunostimulatory cytokines or blockade of immunorepressive cytokines.

[00243] For example, at least one immunostimulatory agent can include one or more agonist agents that ligate positive costimulatory receptors; one or more antagonists (blocking agents) that attenuate signaling through inhibitory receptors, such as antagonists that overcome various immunosuppressive pathways in the tumor microenvironment; one or more agents that systematically increase the frequency of antitumor immune cells, such as T cells, deplete or inhibit regulatory T cells (e.g., by inhibiting CD25); one or more agents that inhibit metabolic enzymes, such as IDO; one or more agents that alter/prevent T cell anergy or exhaustion; and one or more agents that induce innate immune activation and/or inflammation at the tumor site.

[00244] In one embodiment, the at least one immunostimulatory agent comprises a CTLA4 antagonist, such as an antagonist CTLA4 antibody. Suitable CTLA4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab.

[00245] In some embodiments, the at least one immunostimulatory agent comprises a LAG-3 antagonist, such as an anti-LAG-3 antagonist antibody. Suitable anti-LAG-3 antibodies include, for example, BMS-986016 (WO10/19570, WO14/08218) or IMP-731 or IMP-321 (WO08/132601, WO09/44273).

[00246] In some embodiments, the at least one immunostimulatory agent comprises a CD137 (4-1BB) agonist, such as an anti-CD137 agonist antibody. Suitable anti-CD137 antibodies include, for example, urelumab or PF-05082566 (WO12/32433).

[00247] In some embodiments, the at least one immunostimulatory agent comprises a GITR agonist, such as an agonist GITR antibody. Suitable GITR antibodies include, for example, TRX-518 (WO06/105021, WO09/009116), MK-4166 (WO11/028683), or the GITR antibody disclosed in WO 2015/031667.

[00248] In some embodiments, the at least one immunostimulatory agent comprises an OX40 agonist, such as an OX40 agonist antibody. Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469, or MOXR0916 (RG7888, WO06/029879).

[00249] In some embodiments, the at least one immunostimulatory agent comprises a CD27 agonist, such as an anti-CD27 agonist antibody. Suitable CD27 antibodies include, for example, varlilumab (CDX-1127).

[00250] In some embodiments, the at least one immunostimulatory agent comprises MGA271, which targets B7H3 (WO11/109400).

[00251] In some embodiments, the at least one immunostimulatory agent comprises a KIR antagonist, such as lirilumab.

[00252] In some embodiments, the at least one immunostimulatory agent comprises an IDO antagonist. IDO antagonists include, for example, INCB-024360 (WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919 (WO09/73620, WO09/1156652, WO11/56652, WO12/142237), or F001287.

[00253] In some embodiments, the at least one immunostimulatory agent comprises a Toll-like receptor agonist, such as a TLR2/4 agonist (e.g., Bacillus Calmette-Guerin); a TLR7 agonist (e.g., hiltonol or imiquimod); a TLR7/8 agonist (e.g., resiquimod); or a TLR9 agonist (e.g., CpG7909).

[00254] In some embodiments, the at least one immunostimulatory agent comprises a TGF-β inhibitor, such as GC1008, LY2157299, TEW7197, or IMC-TR1.

Adjunctive combination therapy

[00255] Inhibitors can be administered alone or with other treatments. They can be provided before, substantially simultaneously with, or after other treatments, such as surgery, chemotherapy, radiation therapy, or administration of a biological agent such as another therapeutic antibody. In some embodiments, the cancer recurs or progresses after a therapy selected from surgery, chemotherapy, and radiation therapy, or a combination thereof.

[00256] For the treatment of cancer, inhibitors can be administered in combination with one or more additional anticancer agents, such as a chemotherapeutic agent, a growth inhibitor, an antiangiogenesis agent, and/or an antitumor composition. Non-limiting examples of a chemotherapeutic agent, a growth inhibitor, an antiangiogenesis agent, an antitumor agent, and an antitumor composition that can be used in combination with the antibodies of the present invention are provided in the following definitions.

[00257] A " chemotherapeutic agent " is a chemical compound used in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa or cyclophosphamide CITOXAN ^® ; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamin; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183–186 (1994)); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and chenocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carbabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin ^Adriamycin® (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, kelamicin, rodorubicin, streptogreen, streptozocin, tubercidin, ubenimex, cynostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluxuridine; Androgens such as calusterone, dromonostanol propionate, epithiotonol, mepitiostane, testolactone; Antiadrenals such as aminoglutethimide, mitotane, trilostane; Folic acid replacer such as folinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; ^PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine, trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anhydrine), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipropromane, gacytosin, arabinoside ('Ara-C');cyclophosphamide;thiotepa; taxanes such as paclitaxel ^Taxol® (Bristol-Myers Squibb Oncology, Princeton, NJ), ^Abraxane® Cremophor-free, albumin-coated nanoparticle formulations with paclitaxel (American Pharmaceutical Partners, Schaumberg, IL), and doxetaxel ^Taxotere® ( Rorer, Antony, France); chlorambucil; gemcitabine Gemzar ^® ; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinums; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinerelbine Navelbine ^® ; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the regimen of irinotecan, 5-FU, and leucovorin); topoisomerase inhibitor RFS 2000; difluorometallic glycornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (epidermal growth factor receptor) (e.g., erlotinib (Tarceva ^® )) and VEGF-A, which reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[00258] Other non-limiting illustrative chemotherapeutic agents include antihormonal agents that act to regulate or inhibit hormonal action in cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including ^Nolvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene ^Fareston® ; aromatase inhibitors, which inhibit the enzyme aromatase, which regulates the production of estrogens in the adrenal glands, such as, for example, 4 (5)-imidazoles, aminoglutethimide, megestrol acetate Megase ^® , exemestane Aromasin ^® , formestane, fadrozole, vorozole Rivior ^® , letrozole Femara ^® and anastrozole Arimidex ^® ; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as trocacitabine (a 1,3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, especially those that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf, and H-Ras; ribozymes, such as a VEGF expression inhibitor (e.g., ^Angiozyme® ribozyme) and a HER2 expression inhibitor; vaccines, such as a gene therapy vaccine, for example, ^Allovectin® vaccine, ^Leuvectin® vaccine, and ^Vaxid® vaccine; ^Proleukin® rIL-2; the topoisomerase 1 inhibitor ^Lustotecan® ; rmRH ^Abarelix® ; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[00259] " Anti-angiogenic agent " or " angiogenesis inhibitor " refers to a small molecular weight substance, a polynucleotide (including, for example, inhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof that inhibit angiogenesis, vasculogenesis, or undesirable vascular permeability, directly or indirectly. It should be understood that an anti-angiogenic agent includes such agents that bind to and block the angiogenic activity of an angiogenic factor or its receptor. For example, the antiangiogenic agent is an antibody or other antagonist to an angiogenic agent, such as an antibody to VEGF-A (e.g., bevacizumab (Avastin ^® )) or to a VEGF-A receptor (e.g., the KDR receptor or the Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec ^® (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, Sutent ^® /SU11248 (sunitinib malate), AMG706, or those described, for example, in International Patent Application WO 2004/113304). Antiangiogenic agents also include native angiogenesis inhibitors such as angiostatin, endostatin, etc. See, for example, Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217–39; Streit and Detmar (2003) Oncogene 22:3172–3179 (e.g., Table 3 lists antiangiogenic therapies in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12):1359–1364; Tonini et al . (2003) Oncogene 22:6549–6556 (e.g., Table 2 lists known antiangiogenic factors); and Sato (2003) Int. J. Clin. Oncol . 8:200–206 (eg, Table 1 lists antiangiogenic agents used in clinical trials).

[00260] The term " growth inhibitory agent ," as used herein, refers to a compound or composition that inhibits the growth of a cell (such as a cell expressing VEGF) either in vitro or in vivo . Thus, a growth inhibitory agent may be one that significantly reduces the percentage of cells (such as cells expressing VEGF) in S phase. Examples of growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (at a location other than S phase), such as agents that induce G1 arrest and M phase arrest. Classic M phase blockers include the vinca alkaloids (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. G1 blocking agents also cause S-phase arrest, such as DNA alkylating agents such as tamoxifen, prednisolone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. For more information, see Chapter 1, "Cell cycle regulation, oncogenes, and antineoplastic drugs," in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., by Murakami. (WB Saunders, Philadelphia, 1995), for example, p. 13. Taxanes (paclitaxel and docetaxel) are antitumor drugs derived from the yew tree. Docetaxel (TAXOTERE ^® , Rhone-Poulenc Rorer), derived from the European yew tree, is a semisynthetic analogue of paclitaxel (TAXOL ^® , Bristol-Myers Squibb). Paclitaxel and docetaxel stimulate the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in inhibition of mitosis in cells.

[00261] The term " anti-tumor composition " refers to a composition useful for treating cancer, comprising at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, other cancer immunotherapies other than PD-1/PD-L1 inhibitors, apoptotic agents, anti-tubulin agents, and other cancer treatment agents such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonist (e.g., tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (Tarceva ^® ), platelet-derived growth factor inhibitors (e.g., Gleevec ^® (imatinib mesylate)), COX-2 inhibitor (e.g., celecoxib), interferons, CTLA-4 inhibitors (e.g., anti-CTLA antibody ipilimumab (YERVOY®)), PD-L2 inhibitors (e.g., anti-PD-L2 antibodies), TIM3 inhibitors (e.g., anti-TIM3 antibodies), cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, PD-L2, CTLA-4, TIM3 or VEGF receptor(s), TRAIL/Apo2 and other biologically active and organic chemicals, etc. Combinations thereof are also included in the invention.

specific embodiments of the invention

[00262] Some specific embodiments of the present invention include the following:

[00263] 1. A method of treating cancer in a subject, comprising administering to the subject an inhibitor of fibroblast growth factor receptor 2 (FGFR2) and at least one immunostimulatory agent, such as at least one inhibitor of programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1).

[00264] 2. The method of embodiment 1, wherein at least one immunostimulatory agent is a PD-1/PD-L1 inhibitor and wherein the PD-1/PD-L1 inhibitor is an antibody.

[00265] 3. The method of embodiment 2, wherein the PD-1/PD-L1 inhibitor is an anti-PD-1 antibody.

[00266] 4. The method of embodiment 3, wherein the anti-PD-1 antibody comprises the CDRs of the heavy chain and light chain of an antibody selected from nivolumab, pidilizumab, and pembrolizumab.

[00267] 5. The method of embodiment 4, wherein the anti-PD-1 antibody comprises variable regions of a heavy chain and a light chain of an antibody selected from nivolumab, pidilizumab, and pembrolizumab.

[00268] 6. The method of embodiment 5, wherein the anti-PD-1 antibody is selected from nivolumab, pidilizumab, and pembrolizumab.

[00269] 7. The method of embodiment 2, wherein the PD-1/PD-L1 inhibitor is an anti-PD-L1 antibody.

[00270] 8. The method of embodiment 7, wherein the anti-PD-L1 antibody comprises the CDRs of a heavy chain and a light chain of an antibody selected from BMS-936559, MPDL3280A, MEDI4736, and MSB0010718C.

[00271] 9. The method of embodiment 8, wherein the anti-PD-L1 antibody comprises variable regions of a heavy chain and a light chain of an antibody selected from BMS-936559, MPDL3280A, MEDI4736, and MSB0010718C.

[00272] 10. The method of embodiment 9, wherein the anti-PD-L1 antibody is selected from BMS-936559, MPDL3280A, MEDI4736, and MSB0010718C.

[00273] 11. The method of embodiment 1, wherein at least one immunostimulatory agent is a PD-1/PD-L1 inhibitor and wherein the PD-1/PD-L1 inhibitor is a PD-1 fusion molecule.

[00274] 12. The method of embodiment 11, wherein the fusion molecule is AMP-224.

[00275] 13. The method of embodiment 1, wherein the at least one immunostimulatory agent is a PD-1/PD-L1 inhibitor and wherein the PD-1/PD-L1 inhibitor is a PD-1 polypeptide, such as AUR-012.

[00276] 14. The method according to any one of embodiments 1-13, wherein the FGFR2 inhibitor is an antibody to FGFR2.

[00277] 15. The method of embodiment 14, wherein the antibody to FGFR2 is an antibody to FGFR2-IIIb.

[00278] 16. The method of embodiment 15, wherein the antibody to FGFR2-IIIb has one or more of the following properties:

a. binds to FGFR2-IIIb with higher affinity than to FGFR2-IIIc or does not detectably bind to FGFR2-IIIc;

b. inhibits the binding of FGF2 to human FGFR2;

c. inhibits the binding of FGF7 to human FGFR2;

d. inhibits human tumor growth in a mouse tumor model;

e. induces ADCC activity;

f. has increased ADCC activity;

g. is afucosylated; and

h. is able to increase the number of one or more of: PD-L1-positive cells, NK cells, CD3+ T cells, CD4+ T cells, CD8+ T cells and macrophages in tumor tissue in a mouse tumor model compared to controls.

[00279] 17. The method of embodiment 15 or embodiment 16, wherein the anti-FGFR2 antibody comprises heavy chain and light chain variable regions, wherein the heavy chain variable region comprises:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6;

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8;

and the variable region of the light chain contains:

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9;

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[00280] 18. The method of embodiment 17, wherein the heavy chain variable region of the antibody to FGFR2 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4.

[00281] 19. The method of embodiment 17 or 18, wherein the light chain variable region of the FGFR2 antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5.

[00282] 20. The method according to any one of embodiments 17-19, characterized in that the heavy chain variable region of the antibody to FGFR2 comprises the amino acid sequence of SEQ ID NO: 4.

[00283] 21. The method according to any one of embodiments 17-20, characterized in that the light chain variable region of the antibody to FGFR2 comprises the amino acid sequence of SEQ ID NO: 5.

[00284] 22. The method of embodiment 17, wherein the heavy chain of the antibody to FGFR2 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2.

[00285] 23. The method of embodiment 17 or 22, wherein the light chain of the antibody to FGFR2 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3.

[00286] 24. The method according to any one of embodiments 17, 22 or 23, wherein the heavy chain of the antibody to FGFR2 comprises the amino acid sequence of SEQ ID NO: 2.

[00287] 25. The method according to any one of embodiments 17 or 22-24, characterized in that the light chain of the antibody to FGFR2 comprises the amino acid sequence of SEQ ID NO: 3.

[00288] 26. The method according to any one of embodiments 15-25, wherein the antibody to FGFR2 is chimeric, humanized, or human.

[00289] 27. The method according to any one of embodiments 15-26, characterized in that the antibody to FGFR2 is selected from Fab, Fv, scFv, Fab'', and (Fab') ₂ .

[00290] 28. The method according to any one of embodiments 17-27, wherein the FGFR2 antibody has one or more of the following properties:

a. lack of fucose at position Asn297;

b. contains the constant region of the κ light chain;

c. contains the constant region of the IgG1 heavy chain;

d. has increased ADCC activity in vitro compared to an antibody having the same amino acid sequence that is fucosylated at position Asn297;

e. has increased affinity for Fc gamma RIIIA compared to an antibody having the same amino acid sequence that is fucosylated at position Asn297; and

f. is able to increase the number of one or more of: PD-L1-positive cells, NK cells, CD3+ T cells, CD4+ T cells, CD8+ T cells and macrophages in tumor tissue in a mouse tumor model compared to controls.

[00291] 29. The method of any one of embodiments 1-13, wherein the FGFR2 inhibitor is an extracellular domain (ECD) of FGFR2 or an FGFR2 ECD fusion molecule.

[00292] 30. The method of embodiment 29, wherein the FGFR2 inhibitor is a FGFR2 ECD fusion molecule comprising an FGFR2 ECD and at least one fusion partner selected from an Fc domain, albumin, and polyethyleneglycol.

[00293] 31. The method of embodiment 30, wherein the FGFR2 ECD or FGFR2 ECD fusion molecule comprises the amino acid sequence of any one of SEQ ID NOs: 13-33 or 29-33.

[00294] 32. The method according to any one of embodiments 1-31, characterized in that the FGFR2 inhibitor and the immunostimulatory agent are administered simultaneously or sequentially.

[00295] 33. The method of embodiment 32, wherein one or more doses of the immunostimulatory agent are administered prior to administration of the FGFR2 inhibitor.

[00296] 34. The method of embodiment 33, wherein the subject has received a full course of therapy with an immunostimulatory agent prior to administration of the FGFR2 inhibitor.

[00297] 35. The method of embodiment 34, wherein the FGFR2 inhibitor is administered during a second course of therapy with the immunostimulatory agent.

[00298] 36. The method of any one of embodiments 33-35, wherein the subject has received at least one, at least two, at least three, or at least four doses of at least one immunostimulatory agent prior to administration of the FGFR2 inhibitor.

[00299] 37. The method of any one of embodiments 33-36, wherein at least one dose of at least one immunostimulatory agent is administered simultaneously with the FGFR2 inhibitor.

[00300] 38. The method of embodiment 32, wherein one or more doses of the FGFR2 inhibitor are administered prior to administration of the immunostimulatory agent.

[00301] 39. The method of embodiment 38, wherein the subject has received at least two, at least three, at least three, or at least four doses of an FGFR2 inhibitor prior to administration of the at least one immunostimulatory agent.

[00302] 40. The method of embodiment 38 or embodiment 39, wherein at least one dose of the FGFR2 inhibitor is administered simultaneously with the immunostimulatory agent.

[00303] 41. The method of any one of embodiments 1-40, wherein the FGFR2 inhibitor is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, or 30 mg/kg, or a range limited by any two of these mg/kg doses, such as 6-10 mg/kg, 10-15 mg/kg, or 6-15 mg/kg.

[00304] 42. The method according to any one of embodiments 32-41, characterized in that at least one immunostimulatory agent comprises a PD-1/PD-L1 inhibitor.

[00305] 43. The method of embodiment 42, wherein the PD-1/PD-L1 inhibitor is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, or 10 mg/kg.

[00306] 44. The method of any one of embodiments 1-43, wherein the FGFR2 inhibitor and the immunostimulatory agent are administered once every 1, 2, 3, 4, or 5 weeks.

[00307] 45. The method according to any one of embodiments 1-44, characterized in that the cancer is selected from breast cancer, gastric cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, cholangiocarcinoma, esophageal cancer, and endometrial cancer.

[00308] 46. The method of any one of embodiments 1-45, wherein the cancer is recurrent or progressive after therapy selected from surgery, chemotherapy, radiation therapy, or a combination thereof.

[00309] 47. The method of any one of embodiments 1-46, wherein (a) the cancer has previously been determined to overexpress FGFR2IIIb, either with or without amplification of the FGFR2 gene, or (b) the method comprises the additional step of determining whether the cancer overexpresses FGFR2IIIb, and optionally also comprises the additional step of determining whether the FGFR2 gene is amplified in the tumor cells.

[00310] 48. The method of embodiment 47, wherein overexpression of FGFR2IIIb is determined using immunohistochemistry (IHC).

[00311] 49. The method of embodiment 48, wherein overexpression is detected using an IHC signal of 1+, 2+, or 3+ in at least 10% of the tumor cells, such as at least 20%, 30%, 40%, or 50% of the tumor cells.

[00312] 50. The method of any one of embodiments 47-49, wherein the amplification of the FGFR2 gene is determined by obtaining the ratio of FGFR2 to the centromere of chromosome 10 (CEN10) using fluorescence in situ hybridization (FISH method), wherein the FGFR2 gene is considered to be amplified if the ratio of FGFR2/CEN10 determined by the FISH method is greater than or equal to 2.

[00313] 51. The method according to any one of embodiments 47-50, wherein the cancer is gastric cancer or bladder cancer.

[00314] 52. The method according to any one of embodiments 48-50, characterized in that:

a) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of tumor cells;

b) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of the tumor cells, and the FGFR2 gene is amplified;

c) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of the tumor cells, and the FGFR2 gene is not amplified;

d) the cancer is gastric cancer and the cancer has an IHC signal of 1+ or 2+ in at least 10% of the tumor cells;

e) the cancer is bladder cancer and the cancer has an IHC signal of 1+ in at least 10% of the tumor cells;

f) the cancer is bladder cancer and the cancer has an IHC signal of 2+ in at least 10% of the tumor cells;

g) the cancer is bladder cancer and the cancer has an H score of 20 or greater;

h) the cancer is bladder cancer and the cancer has an H value of 10-19; or

i) the cancer is bladder cancer and the cancer has an H score <10.

[00315] 53. The method of any one of embodiments 1-52, wherein the subject is a subject who is insufficiently responsive to treatment with a PD-1/PD-L1 inhibitor.

[00316] 54. The method according to any one of embodiments 1-53, characterized in that administration of an FGFR2 inhibitor and a PD-1/PD-L1 inhibitor in a mouse tumor model results in additive or synergistic inhibition of tumor growth.

[00317] 55. The method of embodiment 54, wherein the cancer is breast cancer and the mouse tumor model comprises 4T1 cells.

[00318] 56. The method of any one of embodiments 1-55, wherein administration of the FGFR2 inhibitor to the mouse tumor model increases the number of NK cells in the tumor tissue compared to a control.

[00319] 57. The method of any one of embodiments 1-56, wherein administration of the FGFR2 inhibitor to the mouse tumor model increases the number of PD-L1-positive cells in the tumor tissue compared to a control.

[00320] 58. The method of any one of embodiments 1-57, wherein administration of an FGFR2 inhibitor to a mouse tumor model increases the number of CD3+, CD8+, and/or CD4+ T cells in the tumor tissue compared to a control.

[00321] 59. The method of any one of embodiments 1-58, wherein administration of an FGFR2 inhibitor to a mouse tumor model increases the ratio of lymphoid cells to myeloid cells in the tumor tissue compared to a control.

[00322] 60. A composition comprising an FGFR2 inhibitor as described in any of embodiments 14-31 and at least one immunostimulatory agent as described in any of embodiments 2-13, such as at least one PD-1/PD-L1 inhibitor.

[00323] 61. The composition of embodiment 60, wherein the FGFR2 inhibitor and at least one immunostimulatory agent are contained in separate containers or compartments.

[00324] 62. The composition of embodiment 60 or 61 further comprising instructions for use in the treatment of cancer.

[00325] 63. The composition of any one of embodiments 60-62 for use in the treatment of cancer.

[00326] 64. The composition of embodiment 63, wherein the cancer is selected from breast cancer, gastric cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, cholangiocarcinoma, esophageal cancer, and endometrial cancer.

[00327] 65. The composition of any one of embodiments 63-64, wherein the cancer overexpresses FGFR2IIIb either with or without amplification of the FGFR2 gene.

[00328] 66. The composition of embodiment 65, wherein the overexpression of FGFR2IIIb is determined using immunohistochemistry (IHC).

[00329] 67. The composition of embodiment 66, wherein overexpression can be detected by an IHC signal of 1+, 2+, or 3+ in at least 10% of the tumor cells, such as at least 20%, 30%, 40%, or 50% of the tumor cells.

[00330] 68. The composition of any one of embodiments 63-67, wherein the cancer has a FGFR2/CEN10 ratio, as determined by FISH, greater than or equal to 2.

[00331] 69. The composition of any one of embodiments 63-68, wherein the cancer is gastric cancer or bladder cancer.

[00332] 70. The composition according to any one of embodiments 63-69, characterized in that:

(a) the cancer is gastric cancer and the cancer has an IHC signal of 3+ in at least 10% of the tumor cells;

b) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of the tumor cells, and the FGFR2 gene is amplified;

c) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of the tumor cells, and the FGFR2 gene is not amplified;

d) the cancer is gastric cancer and the cancer has an IHC signal of 1+ or 2+ in at least 10% of the tumor cells;

e) the cancer is bladder cancer and the cancer has an IHC signal of 1+ in at least 10% of the tumor cells;

f) the cancer is bladder cancer and the cancer has an IHC signal of 2+ in at least 10% of the tumor cells;

g) the cancer is bladder cancer and the cancer has an H score of 20 or greater;

h) the cancer is bladder cancer and the cancer has an H value of 10-19; or

i) the cancer is bladder cancer and the cancer has an H score <10.

[00333] 71. A method for increasing the number of NK cells and/or PD-L1-positive cells in tumor tissue of a subject with cancer, comprising administering to said subject an effective amount of an FGFR2 inhibitor.

[00334] 72. The method of embodiment 71, wherein the FGFR2 inhibitor is an inhibitor of any one of embodiments 14-31.

[00335] 73. The method of embodiment 71 or 72, wherein said method inhibits tumor growth or reduces the volume of at least one tumor in a subject.

[00336] 74. The method of embodiment 73, wherein the cancer is selected from breast cancer, gastric cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, cholangiocarcinoma, esophageal cancer, and endometrial cancer.

[00337] 75. The method of any one of embodiments 71-74, wherein (a) the cancer has previously been determined to overexpress FGFR2IIIb, either with or without amplification of the FGFR2 gene, or (b) the method comprises the additional step of determining whether the cancer overexpresses FGFR2IIIb, and optionally also comprises the additional step of determining whether the FGFR2 gene is amplified in the tumor cells.

[00338] 76. The method of embodiment 75, wherein overexpression of FGFR2IIIb is determined using immunohistochemistry (IHC).

[00339] 77. The method of embodiment 76, wherein overexpression can be detected using an IHC signal of 1+, 2+, or 3+ in at least 10% of the tumor cells, such as at least 20%, 30%, 40%, or 50% of the tumor cells.

[00340] 78. The method of any one of embodiments 75-77, wherein the amplification of the FGFR2 gene is determined by obtaining the ratio of FGFR2 to the centromere of chromosome 10 (CEN10) using fluorescence in situ hybridization (FISH method), wherein the FGFR2 gene is considered to be amplified if the ratio of FGFR2/CEN10 determined by the FISH method is greater than or equal to 2.

[00341] 79. The method according to any one of embodiments 75-78, wherein the cancer is gastric cancer or bladder cancer.

[00342] 80. The method according to any one of embodiments 75-79, characterized in that:

(a) the cancer is gastric cancer and the cancer has an IHC signal of 3+ in at least 10% of the tumor cells;

b) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of the tumor cells, and the FGFR2 gene is amplified;

c) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of the tumor cells, and the FGFR2 gene is not amplified;

d) the cancer is gastric cancer and the cancer has an IHC signal of 1+ or 2+ in at least 10% of the tumor cells;

e) the cancer is bladder cancer and the cancer has an IHC signal of 1+ in at least 10% of the tumor cells;

f) the cancer is bladder cancer and the cancer has an IHC signal of 2+ in at least 10% of the tumor cells;

g) the cancer is bladder cancer and the cancer has an H score of 20 or greater;

h) the cancer is bladder cancer and the cancer has an H value of 10-19; or

i) the cancer is bladder cancer and the cancer has an H score <10.

[00343] 81. The method of any one of embodiments 71-80, wherein the method further comprises obtaining at least one tumor sample from the subject, after administration of the anti-FGFR2 antibody, and determining the number of NK cells and/or PD-L1-positive cells and/or CD8+ T cells in the sample, and if the number of NK cells and/or PD-L1-positive cells and/or CD8+ T cells increases relative to the sample before administration of the anti-FGFR2 antibody, administering to the subject at least one immunostimulatory agent, such as at least one PD-1/PD-L1 inhibitor.

[00344] 82. A method of treating cancer in a subject, comprising administering to the subject an FGFR2 inhibitor and, if it is determined that the subject has an increased number of NK cells and/or PD-L1-positive cells and/or CD8+ T cells compared to a sample before administration of the FGFR2 antibody, administering to the subject at least one immunostimulatory agent, such as at least one PD-1/PD-L1 inhibitor.

[00345] 83. The method of embodiment 82, wherein the FGFR2 inhibitor is an inhibitor of any one of embodiments 14-31.

[00346] 84. The method of embodiment 82 or 83, wherein the at least one immunostimulatory agent comprises at least one PD-1/PD-L1 inhibitor according to any one of embodiments 2-13.

[00347] 85. The method of any one of embodiments 82-84, wherein the FGFR2 inhibitor and at least one immunostimulatory agent are administered according to the method of embodiment 38 or 39.

[00348] 86. A method for increasing the number of one or more of PD-L1-positive cells, NK cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and macrophages in tumor tissue of a subject suffering from cancer, comprising administering an FGFR2 inhibitor, wherein the inhibitor is an FGFR2 antibody with enhanced ADCC activity.

[00349] 87. The method of embodiment 86, wherein the antibody is an antibody according to any one of embodiments 15-28.

[00350] 88. The method of embodiment 86 or 87, wherein administering an antibody to FGFR2 to a mouse tumor model increases the number of one or more of PD-L1-positive cells, NK cells, CD3+ T cells, CD8+ T cells, CD4+ T cells, and macrophages in the tumor tissue compared to a control, and/or increases the ratio of lymphoid cells to myeloid cells in the tumor tissue.

[00351] 89. The method of any one of embodiments 86-88, wherein the subject suffers from breast cancer, gastric cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, cholangiocarcinoma, esophageal cancer, or endometrial cancer.

[00352] 90. The method of any one of embodiments 86-89, wherein (a) the cancer has previously been determined to overexpress FGFR2IIIb, either with or without amplification of the FGFR2 gene, or (b) the method comprises the additional step of determining whether the cancer overexpresses FGFR2IIIb, and optionally also comprises the additional step of determining whether the FGFR2 gene is amplified in the tumor cells.

[00353] 91. The method of embodiment 90, wherein overexpression of FGFR2IIIb is determined using immunohistochemistry (IHC).

[00354] 92. The method of embodiment 91, wherein overexpression can be detected using an IHC signal of 1+, 2+, or 3+ in at least 10% of the tumor cells, such as at least 20%, 30%, 40%, or 50% of the tumor cells.

[00355] 93. The method of any one of embodiments 90-92, wherein the amplification of the FGFR2 gene is determined by obtaining the ratio of FGFR2 to the centromere of chromosome 10 (CEN10) using fluorescence in situ hybridization (FISH method), wherein the FGFR2 gene is considered to be amplified if the ratio of FGFR2/CEN10 determined by the FISH method is greater than or equal to 2.

[00356] 94. The method according to any one of embodiments 89-93, wherein the subject suffers from stomach cancer or bladder cancer.

[00357] 95. The method according to any one of embodiments 90-94, characterized in that:

(a) the cancer is gastric cancer and the cancer has an IHC signal of 3+ in at least 10% of the tumor cells;

b) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of the tumor cells, and the FGFR2 gene is amplified;

c) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of the tumor cells, and the FGFR2 gene is not amplified;

d) the cancer is gastric cancer and the cancer has an IHC signal of 1+ or 2+ in at least 10% of the tumor cells;

e) the cancer is bladder cancer and the cancer has an IHC signal of 1+ in at least 10% of the tumor cells;

f) the cancer is bladder cancer and the cancer has an IHC signal of 2+ in at least 10% of the tumor cells;

g) the cancer is bladder cancer and the cancer has an H score of 20 or greater;

h) the cancer is bladder cancer and the cancer has an H value of 10-19; or

i) the cancer is bladder cancer and the cancer has an H score <10.

[00358] 96. The method of any one of embodiments 86-95, wherein the anti-FGFR2 antibody is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, or 30 mg/kg, or a range limited by any two of these mg/kg doses, such as 6-10 mg/kg, 10-15 mg/kg, or 6-15 mg/kg, every week, every two weeks, every three weeks, or once a month.

[00359] 97. A method for determining whether a patient with gastric cancer or bladder cancer will respond to treatment with an FGFR2 inhibitor, comprising determining whether the gastric or bladder cancer overexpresses FGFR2IIIb using IHC, wherein overexpression is determined by an IHC signal of 1+, 2+, or 3+ in at least 10% of the tumor cells of the cancer, such as at least 20%, 30%, 40%, or 50% of the tumor cells.

[00360] 98. The method of embodiment 97, wherein the method further comprises determining whether the FGFR2 gene is amplified by obtaining a ratio of FGFR2 to the centromere of chromosome 10 (CEN10) using fluorescence in situ hybridization (FISH method), wherein the FGFR2 gene is considered to be amplified if the ratio of FGFR2/CEN10 determined by the FISH method is greater than or equal to 2.

[00361] 99. The method of embodiment 97 or 98, wherein the patient is determined to be responsive to treatment with an antibody to FGFR2IIIb if:

(a) the cancer is gastric cancer and the cancer has an IHC signal of 3+ in at least 10% of the tumor cells;

b) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of the tumor cells, and the FGFR2 gene is amplified;

c) the cancer is gastric cancer, the cancer has an IHC signal of 3+ in at least 10% of the tumor cells, and the FGFR2 gene is not amplified;

d) the cancer is gastric cancer and the cancer has an IHC signal of 1+ or 2+ in at least 10% of the tumor cells;

e) the cancer is bladder cancer and the cancer has an IHC signal of 1+ in at least 10% of the tumor cells;

f) the cancer is bladder cancer and the cancer has an IHC signal of 2+ in at least 10% of the tumor cells;

g) the cancer is bladder cancer and the cancer has an H score of 20 or greater;

h) the cancer is bladder cancer and the cancer has an H value of 10-19; or

i) the cancer is bladder cancer and the cancer has an H score <10.

[00362] 100. The method of any one of embodiments 97-99, wherein the FGFR2 inhibitor is an inhibitor of any one of embodiments 14-31.

[00363] 101. The method of any one of embodiments 97-100, wherein the treating comprises administering an FGFR2 inhibitor at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, or 30 mg/kg, or a range limited by any two of these mg/kg doses, such as 6-10 mg/kg, 10-15 mg/kg, or 6-15 mg/kg.

[00364] 102. The method of any one of embodiments 97-101, wherein the treating comprises performing the method of embodiments 71, 82, or 86.

[00365] 103. The method of any one of embodiments 97-101, wherein the treatment further comprises administering to the subject at least one immunostimulatory agent.

[00366] 104. The method of embodiment 103, wherein the at least one immunostimulatory agent comprises an immunostimulatory agent of any one of embodiments 2-13.

[00367] The method of embodiment 103, wherein the treating comprises administering an FGFR2 inhibitor and at least one immunostimulatory agent according to any one of the methods of embodiments 32-44, or wherein the treating comprises administering a composition according to any one of embodiments 60-62.

EXAMPLES

[00368] The examples provided below are for illustrative purposes only and therefore should not be construed as limiting the invention in any way. The examples are not intended to represent that the experiments provided below are all or the only experiments that were performed. Care has been taken to ensure accuracy with respect to the numbers (e.g., amounts, temperatures, etc.) used, but some experimental errors and variations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1: ADCC activity is required for tumor growth inhibition in a 4T1 mammary tumor mouse model

[00369] Seventy eight-week-old female BALB/c mice (IACUC category: AUP 2011# 01-03) were purchased from Charles River Laboratories (Wilmington, MA, USA). Animals were acclimated for at least 3 days upon arrival and were housed at 5 animals per cage with free access to food and water. After acclimation, they were weighed and shaved for tumor cell implantation.

[00370] The 4T1 mammary tumor line from the BALB/cfC3H mouse strain was used as a tumor model and was obtained from the ATCC (American Type Culture Collection) (Manassas, VA, USA: catalog no. CRL-2539). The cells were cultured at 37°C in RPMI 1640 medium (Mediatech, Inc., Manassas, VA, USA; no. 10-041-CV) with 10% fetal bovine serum, 2 mM L-glutamine, and 1% Penn/Strep (penicillin/streptomycin).

[00371] Each mouse was inoculated with 5 x 10 ⁴ 4T1 cells by orthotopic injection under the 4th mammary nipple (nipple) from the head of the mouse. Tumor volumes and body weights of the mice were then monitored periodically until tumor volumes reached 100 mm ³ +/- 25 mm ³ . When tumors reached 100 mm ³ +/- 25 mm ³ , the mice were sorted by tumor size into four dose groups. The first group was administered 20 mg/kg Fc-G1 antibody (intraperitoneally (i.p.), once every two weeks (BIW)), the second group was administered 20 mg/kg afucosylated antibody to FGFR2 (anti-FGFR2) with HVR of heavy and light chain SEQ ID NO: 6-11 (i.p., BIW) and the third group was administered 20 mg/kg FGFR2 antibody with the N297Q mutation (anti-FGFR2-N297Q), which makes this molecule incapable of stimulating ADCC activity (i.p., BIW).

[00372] Overall, anti-FGFR2 treatment resulted in ~30% inhibition (P < 0.001) of tumor growth compared to control FcG1, whereas anti-FGFR2-N297Q treatment did not inhibit tumor growth compared to control. (See Fig. 1a-b.) These data support a role for ADCC as a mechanism for anti-FGFR2 inhibition of tumor growth.

Example 2a: Anti-FGFR2 antibody exposure results in increased NK cell numbers and increased PD-L1-expressing cells within tumor tissue

[00373] For immunohistochemistry, BALB/cfC3H mice were inoculated with 5 x 10 ⁴ 4T1 cells by orthotopic injection as described above. After tumors reached 100 mm ³ +/- 25 mm 3 (day 0), mice were sorted according to tumor size into two dose groups: vehicle or 10 mg/kg anti-FGFR2 (i.p.). Each group was divided into (a) mice that received one or two doses on day 0 or (b) mice that received one or two doses on day 3, and mice were euthanized 24 hours post-dose on days 1 or 4, respectively, and prepared for histology or flow cytometry.

[00374] For histology, on days 1 and 4, 24 hours after the first and second treatments, respectively, mice were euthanized with CO ₂ and then perfused with phosphate-buffered saline (PBS), pH 7.4. Briefly, the mouse chest was quickly opened and a syringe with a 20-gauge needle was used to inject 40 mL of PBS into the aorta through an incision in the left ventricle. Blood and PBS were drained through a hole in the right atrium. Orthotopic 4T1 tumors were removed and immersed in 10% neutral buffered formalin at 4°C. After 2 hours, tissues were washed 3 times with PBS and then transferred to 30% sucrose in PBS overnight. The next day, tumors were frozen in OCT reagent and stored at -80°C.

[00375] Serial 20-μm-thick sections were prepared from each tumor. Sections were dehydrated on Superfrost Plus slides (VWR) for 1–2 h. Specimens were permeabilized with PBS containing 0.3% Triton X-100 and incubated in 5% normal goat serum in PBS with 0.3% Triton X-100 (blocking solution) for 1 h at room temperature to block nonspecific antibody binding. After 1 h, the blocking solution was removed and sections were incubated with primary antibodies overnight. To detect NK cells, sections were incubated with rat anti-NKp46 (CD335, Biolegend, cat. #137602) diluted 1:500 in blocking solution. For detection of PD-L1, sections were incubated with rat anti-PD-L1 (eBioscience, Cat# 14-5982-82) diluted 1:500 in blocking solution. NK cell and PD-L1 staining were performed in serial sections as both primary antibodies were derived from rats. Secondary antibodies of negative control samples only were incubated in 5% normal serum, unlike primary antibodies.

[00376] The following day, after washing with PBS containing 0.3% Triton X-100, samples were incubated for 4 hours at room temperature with Alexa Fluor 594-labeled goat anti-rat (Jackson Immuno Research, Cat# 112-585-167) and Alexa Fluor 488-labeled goat anti-rabbit (Jackson Immuno Research, Cat# 111-545-144) secondary antibodies diluted 1:400 in PBS. Afterwards, the samples were washed with PBS containing 0.3% Triton X-100, then fixed in 1% paraformaldehyde (PFA), washed again with PBS and fixed in Vectashield with DAPI (4', 6-diamidine-2-phenylindole) (Vector, H-1200). DAPI was used to stain cell nuclei.

[00377] Samples were examined using a Zeiss Axiophot® 2 plus fluorescence microscope equipped with an AxioCam® HRc camera. Representative images for each experimental group showing the number and distribution of NKp46+ and PD-L1+ cells in the tumor were collected and depicted in Figs. 2a-2d .

[00378] In 4T1 tumors from vehicle-treated mice, a small number of scattered NKp46+ NK cells were detected, and most of these cells were located at the tumor periphery ( Fig. 2a ). In comparison, NKp46+ NK cells were more numerous following anti-FGFR2 treatment at 10 mg/kg for 1 day. Although most of these cells were found at the tumor margins, some had infiltrated into the tumor center (See Fig. 2a ). Similar results were observed on day 4 after the second dose of anti-FGFR2 ( Fig. 2b ).

[00379] PD-L1 staining showed that in 4T1 tumors treated with vehicle for 1 day, PD-L1 immunoreactivity was detected in only a few cells in the tumors ( Fig. 2c ). In contrast, 24 hours after 1 dose of anti-FGFR2, PD-L1-positive cells were more numerous in the tumor center ( Fig. 2c ). Similar results were observed 4 days after the start of treatment ( Fig. 2d ).

[00380] As an independent assay for NK cell expansion, we performed flow cytometry on 4T1 tumor-bearing mice that received 2 doses of saline or 10 mg/kg anti-FGFR2 as described above. For flow cytometry, tumors were cut into 1-2 mm pieces and placed in DMEM (Dulbecco's modified Eagle's minimum essential medium) with 10% FBS (fetal bovine serum), 50 U/mL DNase I, and 250 U/mL collagenase I (Worthington Biochemical Corporation, Lakewood, NJ) in a shaking incubator for 30 min at 37°C. Cells were passed through a 70-μm nylon mesh filter, and single-cell suspensions were stained according to standard protocols using antibodies purchased from BD Biosciences (San Jose, CA): CD45 (clone 30-F11) and CD11b (1D3); Affymtrix eBioscience (San Diego, CA): CD16/32 (FC receptor blocking, 93), CD335 (NKp46, 29A1.4), CD8a (53.67), and CD3e (145-2C11); R&D Systems (Minneapolis, MN): EphA2 (233720); or ThermoFisher Scientific (Grand Island, NY): Live/Dead Aqua kit. Cells were fixed and analyzed the next day on a BD LSRII. Results were analyzed using FlowJo (V10, Ashland, OR) with the following gating strategy: CD45+ EphA2-, singlets (FSC-H vs. FSC-A), live cells (Live/Dead negative), and CD11b- to isolate live lymphocytes. NK cells were gated on NKp46+CD3- and expressed as a percentage of CD45+ live singlets.

[00381] As shown in Fig. 3 , tumors treated with anti-FGFR2 demonstrated increased NK cells compared to tumors treated with saline control.

Example 2b: Anti-FGFR2 antibody but not anti-FGFR2 N297Q antibody results in increased NK cells, T cells, and increased PD-L1-expressing cells within tumor tissue

[00382] BALB/cfC3H mice were inoculated with 5 x 10 ⁴ 4T1 cells by orthotopic injection as described in Example 1. After tumors reached 100 mm ³ +/- 25 mm ³ (day 0), mice were sorted by tumor size into three dose groups: vehicle (control group), 10 mg/kg afucosylated anti-FGFR2 antibody (i.p.) (anti-FGFR2 group), and 10 mg/kg anti-FGFR2 N297Q antibody (anti-FGFR2 N297Q group). The N297Q modification is a mutation in the Fc region of the antibody that is designed to abolish the effector function of the antibody.

[00383] Each group was divided into (a) mice that received one or two doses on day 0 or (b) mice that received one or two doses on day 3, and mice were euthanized 24 hours after dosing on days 1 or 4, respectively, and prepared for histology or flow cytometry.

[00384] For histology, on days 1 and 4, 24 hours after the first and second treatments, respectively, mice were euthanized with CO ₂ and then perfused with phosphate-buffered saline (PBS), pH 7.4. Briefly, the mouse chest was quickly opened and a syringe with a 20-gauge needle was used to inject 40 mL of PBS into the aorta through an incision in the left ventricle. Blood and PBS were drained through a hole in the right atrium. Orthotopic 4T1 tumors were removed and immersed in 10% neutral buffered formalin at 4°C. After 2 hours, tissues were washed 3 times with PBS and then transferred to 30% sucrose in PBS overnight. The next day, tumors were frozen in OCT reagent and stored at -80°C.

[00385] Serial 20-μm-thick sections were prepared from each tumor. Sections were dehydrated on Superfrost Plus slides (VWR) for 1–2 h. Specimens were permeabilized with PBS containing 0.3% Triton X-100 and incubated in 5% normal goat serum in PBS with 0.3% Triton X-100 (blocking solution) for 1 h at room temperature to block nonspecific antibody binding. After 1 h, the blocking solution was removed and sections were incubated with primary antibodies overnight. To detect NK cells, sections were incubated with rat anti-NKp46 (CD335, Biolegend, cat. #137602) diluted 1:500 in blocking solution. To detect PD-L1, sections were incubated with rat anti-PD-L1 (eBioscience, Cat# 14-5982-82) diluted 1:500 in blocking solution. To detect CD3+ T cells, sections were incubated with hamster anti-CD3 antibody (BD biosciences, Cat# 553058) at 1:500 in blocking solution. To detect CD4+ T cells, sections were incubated with rat anti-CD4 antibody (AbD Serotec, Cat# MCA4635) at 1:500 in blocking solution. To detect CD8+ T cells, sections were incubated with rat anti-CD8 antibody (Abcam, cat.# ab22378) at 1:500 in blocking solution. NK cell and PD-L1 staining were performed in serial sections since both primary antibodies were derived from rats. CD3 and CD4 positive cells were stained together on the same section. CD3 and CD8 staining were also performed on the same sections. Secondary antibodies of only negative control samples were incubated in 5% normal serum, unlike the primary antibodies.

[00386] The following day, after washing with PBS containing 0.3% Triton X-100, samples were incubated for 4 hours at room temperature with Alexa Fluor 594-labeled goat anti-rat (Jackson Immuno Research, Cat# 112-585-167) and Alexa Fluor 488-labeled goat anti-hamster (Jackson Immuno Research, Cat# 127-545-160) secondary antibodies diluted 1:400 in PBS. Samples were then washed with PBS containing 0.3% Triton X-100, fixed in 1% paraformaldehyde (PFA), washed again with PBS, and fixed in Vectashield with DAPI (Vector, H-1200). DAPI was used to stain cell nuclei.

[00387] Samples were examined using a Zeiss Axiophot® 2 plus fluorescence microscope equipped with an AxioCam® HRc camera. Representative images for each experimental group showing the number and distribution of NKp46+ and PD-L1+ cells in the tumor were collected and depicted in Fig. 5a-5b .

[00388] In 4T1 tumors from mice treated with vehicle or anti-FGFR2 N297Q, a small number of scattered NKp46+ NK cells were detected after 1 day of treatment, and most of these cells were located at the tumor periphery ( Fig. 5a ). In comparison, NKp46+ NK cells were more numerous after treatment with anti-FGFR2 at 10 mg/kg for 1 day. Although most of these cells were found at the tumor margins, some of them infiltrated into the tumor center (See Fig. 5a ). Similar results were observed on day 4 after the second dose of anti-FGFR2 ( Fig. 5b ).

[00389] PD-L1 staining showed that in 4T1 tumors treated with vehicle or anti-FGFR2 N297Q for 1 day, PD-L1 immunoreactivity was detected in only a few cells in the tumors ( Fig. 5a ). In contrast, 24 hours after 1 dose of anti-FGFR2, PD-L1-positive cells were more numerous in the tumor center. Similar results were observed 4 days after treatment ( Fig. 5b ).

[00390] CD3, CD8, and CD4 staining showed that tumors treated with vehicle or anti-FGFR2 N297Q for 1 day or 4 days retained T cell infiltration only at the tumor periphery. In contrast, on day 4, tumors treated with anti-FGFR2 resulted in CD3, CD8, and CD4-positive T cell infiltration into the tumor center ( Fig. 6a-6b ).

[00391] As an independent assay for NK cell expansion, we performed flow cytometry in 4T1 tumor-bearing mice that received 2 doses of saline or 10 mg/kg anti-FGFR2 or anti-FGFR2 N297Q as described above. For flow cytometry, tumors were cut into 1-2 mm pieces and placed in DMEM with 10% FBS, 50 U/mL DNase I, and 250 U/mL collagenase I (Worthington Biochemical Corporation, Lakewood, NJ) in a shaking incubator for 30 min at 37°C. Cells were passed through a 70-μm nylon mesh filter, and single-cell suspensions were stained according to standard protocols using antibodies purchased from BD Biosciences (San Jose, CA): CD45 (clone 30-F11) CD4, (GK1.5), and CD11b (1D3); Affymtrix eBioscience (San Diego, CA): CD16/32 (FC receptor blocking, 93), CD335 (NKp46, 29A1.4), CD8a (53.67), and CD3e (145-2C11); R&D Systems (Minneapolis, MN): EphA2 (233720); or ThermoFisher Scientific (Grand Island, NY): Live/Dead Aqua kit. Cells were fixed and analyzed the next day on a BD LSRII. Results were analyzed using FlowJo (V10, Ashland, OR) with the following gating strategy: CD45+ EphA2-, singlets (FSC-H vs. FSC-A), live cells (Live/Dead negative), and CD11b- to isolate live lymphocytes. NK cells were gated as NKp46+CD3-. CD4 and CD8 cells were gated on CD3+ cells, and each subset was expressed as a percentage of CD45+ live single cells.

[00392] As shown in Fig. 10a , tumors treated with anti-FGFR2 demonstrated an increase in NK cells compared to tumors treated with control saline or anti-FGFR2 N297Q. In addition, CD3, CD8, and CD4 T cells were increased 24 hours after the second dose ( Figs. 7-9 ), and a preferential increase in the lymphoid to myeloid cell ratio was observed with anti-FGFR2 treatment compared to vehicle or anti-FGFR2 N297Q ( Fig. 10b-c ).

Example 2c: Anti-FGFR2 antibody, but not anti-FGFR2 N297Q antibody, increases the number of F480+ macrophages within tumor tissue

[00393] To detect macrophages in 4T1 tumors, sections were incubated with rat anti-F480 antibody (Bio-Rad AbD Serotec Inc, Cat# MCA497R) at 1:500 in blocking solution. NK cell, PD-L1, and F480 staining were performed on serial sections since all of these primary antibodies were derived from rats.

[00394] In 4T1 tumors from control-treated mice, significant numbers of F480+ macrophages were detected in all tumors ( Fig. 11 , upper panels). In comparison, following treatment with anti-FGFR2 at 10 mg/kg for 4 days, the number of F480+ cells detected in tumors was increased ( Fig. 11 , middle panels). This effect was not observed in 4T1 tumor-bearing mice treated with anti-FGFR2 antibody at 10 mg/kg for 1 day or with mutant anti-FGFR2 N297Q antibody for 1 or 4 days ( Fig. 11 , lower panels).

Example 3: Combination of anti-FGFR2 antibody and anti-PD-1 antibody in a syngeneic 4T1 breast tumor model

[00395] In this example, the antitumor effects of a combination of an afucosylated antibody to FGFR2 (anti-FGFR2) and an anti-PD-1 antibody (Bio X Cell, West Lebanon, NH, USA, clone RMP1-14) were assessed in a 4T1 syngeneic mouse model of breast cancer in immunocompetent mice. The 4T1 model exhibits moderate overexpression of FGFR2-IIIb, but not amplified FGFR2.

[00396] Seventy eight-week-old female BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Animals were acclimated for at least 3 days upon arrival and were housed 5 animals per cage with free access to food and water. After acclimation, they were weighed and shaved for tumor cell implantation.

[00397] The 4T1 mammary tumor line from the BALB/cfC3H mouse strain was used as a tumor model and was obtained from the ATCC (American Type Culture Collection) (Manassas, VA, USA: catalog no. CRL-2539). The cells were cultured at 37°C in RPMI 1640 medium (Mediatech, Inc., Manassas, VA, USA; no. 10-041-CV) with 10% fetal bovine serum, 2 mM L-glutamine, and 1% Penn/Strep (penicillin/streptomycin).

[00398] Each mouse was inoculated with 5 x 10 ⁴ 4T1 cells by orthotopic injection under the 4th mammary nipple (nipple) from the head of the mouse. Tumor volumes and body weights of the mice were then monitored periodically until tumor volumes reached 150 mm ³ +/- 25 mm ³ . When tumors reached 150 mm ³ +/- 25 mm ³ , mice were sorted by tumor size into four dose groups. The first group was administered 10 mg/kg control Ig-FC (intraperitoneally (i/p)), once every two weeks (BIW)), the second group - 5 mg/kg anti-PD-1 antibody (i/p, 0, 3 and 7 days), the third group - 10 mg/kg antibody to FGFR2 (i/p, BIW) and the fourth group - a combination of antibodies against PD-1 and FGFR2 at 5 and 10 mg/kg, respectively.

[00399] Tumor volumes were measured at 12 days post-implantation (day 0 of dosing), 15 days post-implantation, and 18 days post-implantation. Figures 4a and b show that on day 18, anti-FGFR2 antibody significantly reduced 4T1 tumor volume in mice (P < 0.001 or P = 0.01, respectively, by t test) compared with the control Ig-FC group. The combination of anti-FGFR2 antibody with anti-PD-1 antibody further reduced tumor volume on day 18 compared with anti-FGFR2 antibody alone (P = 0.08, respectively). (See Figures 4a-b.) And the combination of anti-FGFR2 antibody with anti-PD-1 antibody further reduced tumor volume on day 18 compared with anti-PD-1 antibody alone (P < 0.01).

[00400] Overall, treatment with anti-FGFR2 antibody resulted in ~25% inhibition of tumor growth compared to IgFC, while treatment with anti-PD-1 antibody resulted in 0% inhibition of tumor growth compared to control. Treatment with anti-FGFR2 and anti-PD-1 antibodies inhibited tumor growth by ~40%, suggesting that the combination therapy has at least an additive effect.

[00401] The table below shows the analysis of fractional tumor volumes (FTV) compared to control Fc-G1.

Day ^b FTV anti-FGFR2 FTV anti-PD-1 Expected ^c Observed ^d Expected/ ^e Observed 15 0.80 1.04 0.84 0.69 1.22 18 0.96 0.96 0.73 0.59 1.23

a: FTV=fractional tumor volume=mean TV (tumor volume) in the treatment group/mean TV in the control group

b: Day after tumor cell implantation

c: Expected=(mean FTV for drug 1) x (mean FTV for drug 2)

d: Observed=mean FTV for the combination of drug 1 with drug 2

e: Reported value = expected (c) ÷ observed (d); values > 1 indicate a synergistic response, values = 1 indicate an additive response, and values < 1 indicate an antagonistic response

[00402] In this experiment, the effect of anti-FGFR2 antibodies on NK cell-depleted tumor tissue was studied. The 4T1 mammary tumor line from the BALB/cfC3H mouse strain was used as a tumor model and was obtained from ATCC (Manassas, VA, USA: catalog #CRL-2539). The cells were cultured at 37°C in RPMI 1640 medium (Mediatech, Inc., Manassas, VA, USA; #10-041-CV) with 10% fetal bovine serum, 2 mM L-glutamine, and 1% Penn/Strep (penicillin/streptomycin).

[00403] Each mouse was inoculated with 5 x 10 ⁴ 4T1 cells by orthotopic injection under the 4th mammary nipple (nipple) from the head of the mouse. Tumor volumes and body weights of the mice were then monitored periodically until tumor volumes reached 100 mm ³ +/- 25 mm ³ . When tumors reached 125 mm ³ +/- 25 mm ³ , the mice were sorted by tumor size into four dose groups (day 0). Group 1 was administered 10 mg/kg of human Fc-G1 control antibody (ip) on days 0 and 3). Group 2 was administered 50 mg/kg rabbit anti-asialo-GM1 antibody (Wako Chemicals, Osaka, Japan) intravenously once on day 0, the antibody is designed to deplete NK cells in BalbC mice. Group 3 was administered 10 mg/kg anti-FGFR2 (i.p. on day 0 and day 3). Group 4 was administered 50 mg/kg rabbit anti-asialo-GM1 antibody (day 0) in combination with 10 mg/kg anti-FGFR2 (i.p. on day 0 and day 3).

[00404] For histology on day 4, 24 hours after the second treatment, mice were euthanized with CO ₂ and then perfused with phosphate-buffered saline (PBS), pH 7.4. Briefly, the mouse chest was quickly opened and a syringe with a 20-gauge needle was used to inject 40 ml of PBS into the aorta through an incision in the left ventricle. Blood and PBS were drained through a hole in the right atrium. Orthotopic 4T1 tumors were removed and immersed in 10% neutral buffered formalin at 4°C. After 2 hours, tissues were washed 3 times with PBS and then transferred to 30% sucrose in PBS overnight. The next day, tumors were frozen in OCT reagent and stored at -80°C.

[00405] Serial 20-μm-thick sections were prepared from each tumor. Sections were dehydrated on Superfrost Plus slides (VWR) for 1–2 h. Specimens were permeabilized with PBS containing 0.3% Triton X-100 and incubated in 5% normal goat serum in PBS with 0.3% Triton X-100 (blocking solution) for 1 h at room temperature to block nonspecific antibody binding. After 1 h, the blocking solution was removed and sections were incubated with primary antibodies overnight. To detect NK cells, sections were incubated with rat anti-NKp46 (CD335, Biolegend, cat. #137602) diluted 1:500 in blocking solution. To detect PD-L1, sections were incubated with rat anti-PD-L1 (eBioscience, Cat# 14-5982-82) diluted 1:500 in blocking solution. To detect CD3+ T cells, sections were incubated with hamster anti-CD3 antibody (BD biosciences, Cat# 553058) at 1:500 in blocking solution. NK cell and PD-L1 staining were performed in serial sections since all primary antibodies were derived from rats. Secondary antibodies of negative control samples only were incubated in 5% normal serum, unlike primary antibodies.

[00406] The following day, after washing with PBS containing 0.3% Triton X-100, samples were incubated for 4 hours at room temperature with Alexa Fluor 594-labeled goat anti-rat (Jackson Immuno Research, Cat# 112-585-167) and Alexa Fluor 488-labeled goat anti-hamster (Jackson Immuno Research, Cat# 127-545-160) secondary antibodies diluted 1:400 in PBS. Samples were then washed with PBS containing 0.3% Triton X-100, fixed in 1% paraformaldehyde (PFA), washed again with PBS, and fixed in Vectashield with DAPI (Vector, H-1200). DAPI was used to stain cell nuclei.

[00407] Specimens were examined using a Zeiss Axiophot® 2 plus fluorescence microscope equipped with an AxioCam® HRc camera. Representative images for each experimental group showing the number and distribution of NKp46+, CD3+, and PD-L1+ cells in the tumor were collected and depicted in Figs. 12-14 .

[00408] In 4T1 tumors from mice treated with the control antibody Fc-G1, minimal numbers of scattered NKp46+ NK cells were detected within the tumor ( Fig. 12 , upper panels). Administration of rabbit anti-asialo-GM1 antibody, given at 50 mg/kg, reduced the numbers of NKp46+ NK cells compared to controls ( Fig. 12 , second panels). Following treatment with anti-FGFR2 at 10 mg/kg for 4 days, NKp46+ NK cells were more numerous (see Fig. 12 , third panels). However, this increase was not observed when anti-FGFR2 was combined with rabbit anti-asialo-GM1 antibody and anti-FGFR2. After combination treatment, the number of infiltrating NKp46+NK cells was comparable to the control ( Fig. 12 , fourth panels).

[00409] CD3 staining showed that in 4T1 tumors treated with control for 4 days, few scattered CD3+ T cells were detected in the tumor, and most of them were located at the tumor periphery ( Fig. 13 , upper panels). Treatment with rabbit anti-asialo-GM1 antibody at 50 mg/kg did not affect the number of CD3+ T cells compared with control ( Fig. 13 , second panels). After treatment with anti-FGFR2 at 10 mg/kg for 4 days, the number of infiltrating CD3+ T cells increased ( Fig. 13 , third panels). In comparison, when anti-FGFR2 was combined with rabbit anti-asialo-GM1 antibody, the number of infiltrating CD3+ T cells was comparable to control ( Fig. 13 , fourth panels).

[00410] PD-L1 staining showed that in 4T1 tumors treated with control for 4 days, PD-L1 immunoreactivity was detected only in rare cells within the tumors ( Figure 14 , upper panels). Treatment with rabbit anti-asialo-GM1 antibody did not affect PD-L1 immunoreactivity ( Figure 14 , second panels). Treatment with anti-FGFR2 alone increased the number of PD-L1-positive cells ( Figure 14 , third panels), but when anti-FGFR2 was combined with rabbit anti-asialo-GM1 antibody, PD-L1 staining was similar to control ( Figure 14 , fourth panels).

Example 4b: Inhibition of tumor growth by an anti-FGFR2 antibody is attenuated in the presence of an agent that depletes NK cells

[00411] In this experiment, the effect of NK cell depletion on the efficacy of anti-FGFR2 in a syngeneic 4T1 tumor model was examined. The 4T1 mammary tumor line from the BALB/cfC3H mouse strain was used as a tumor model and was obtained from ATCC (American Type Culture Collection) (Manassas, VA, USA: catalog #CRL-2539). Cells were cultured at 37°C in RPMI 1640 medium (Mediatech, Inc., Manassas, VA, USA; #10-041-CV) with 10% fetal bovine serum, 2 mM L-glutamine, and 1% Penn/Strep (penicillin/streptomycin).

[00412] Each mouse was inoculated with 5 × 10 ⁴ 4T1 cells by orthotopic injection under the 4th mammary nipple (nipple) from the head of the mouse. The tumor volume and body weight of the mice were then monitored periodically until the tumor volumes reached 100 mm ³ +/- 25 mm ³ . When the tumors reached 100 mm ³ +/- 25 mm ³ , the mice were sorted by tumor size into four dose groups (day 0). Group 1 was administered PBS as a control (intraperitoneally (i.p.) on days 0 and 3). Group 2 was administered 50 mg/kg rabbit anti-asialo-GM1 antibody (Wako Chemicals, Osaka, Japan), i.v. once on day 0, the antibody is designed to deplete NK cells in BalbC mice. Group 3 was administered 10 mg/kg anti-FGFR2 (i.p. on day 0 and day 3). Group 4 was administered 50 mg/kg rabbit anti-asialo-GM1 antibody (day 0) in combination with 10 mg/kg anti-FGFR2 (i.p. on day 0 and day 3) and tumor volume was monitored biweekly.

[00413] Overall, anti-FGFR2 treatment resulted in ~35% inhibition (P < 0.01) of tumor growth compared to the PBS control group and the anti-asialo-GM1 antibody group (P < 0.05). Anti-asialo-GM1 antibody treatment did not affect tumor burden compared to the PBS control group. The combination of anti-asialo-GM1 antibody with anti-FGFR2 resulted in attenuated tumor growth inhibition compared to the anti-FGFR2 group (P < 0.05), suggesting that NK cells and ADCC activity are complex in promoting tumor growth inhibition in the syngeneic 4T1 tumor model. When combined with histological data, anti-FGFR2 inhibits 4T1 tumor burden by altering the tumor microenvironment through the innate and adaptive immune systems.

Example 4c: Anti-FGFR2-driven tumor growth inhibition is attenuated in SC17 SCID mice that lack an adaptive immune system

[00414] Eight-week-old female CB17 SCID mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Animals were acclimated for at least 3 days upon arrival and were housed 5 animals per cage with free access to food and water. After acclimation, they were weighed and shaved for tumor cell implantation.

[00415] The syngeneic murine mammary tumor line 4T1 was used as a tumor model and was obtained from the ATCC (American Type Culture Collection) (Manassas, VA, USA: catalog #CRL-2539). The cells were cultured at 37°C in RPMI 1640 medium (Mediatech, Inc., Manassas, VA, USA; #10-041-CV) with 10% fetal bovine serum, 2 mM L-glutamine, and 1% Penn/Strep, which is penicillin and streptomycin.

[00416] Each mouse was inoculated with 5 x 10 ⁴ 4T1 cells by orthotopic injection under the 4th mammary nipple (nipple) from the head of the mouse. Tumor volumes and body weights of the mice were then monitored periodically until tumor volumes reached 80 mm ³ +/- 25 mm ³ . On day 12, if tumors reached 80 mm ³ +/- 25 mm ³ , mice were sorted by tumor size into two dose groups. The first group received vehicle control, the second group received 20 mg/kg afucosylated anti-FGFR2 antibody (intraperitoneally (i.p.), on days 12 and 15).

[00417] In contrast to the ~30%, P<0.001 reduction in tumor volume in BalbC mice (Example 1), treatment with anti-FGFR2 antibody results in ~20% inhibition, P<0.05, of tumor growth compared to vehicle control in CB17 SCID mice, which have an intact innate immune system (NK cells and macrophages) but lack components of the adaptive immune system (T cells and B cells).

[00418] These data suggest that anti-FGFR2 antibody can stimulate innate immune cells to initiate immediate killing of tumor cells, but CB17 SCID mice exhibit a reduced response to anti-FGFR2 antibody treatment, likely because they do not interact with the adaptive immune system. This further demonstrates that anti-FGFR2 antibody works in concert with the innate and adaptive immune systems to alter the tumor microenvironment, resulting in sustained inhibition of tumor growth in immunocompetent mice.

EXAMPLE 5: Open-label, phase I study of patients with advanced solid tumors treated with an anti-FGFR2 antibody

[00419] In a dose escalation study of an afucosylated anti-FGFR2 antibody comprising the heavy and light chain HVRs of SEQ ID NOs: 6-11, approximately 30 patients with any locally advanced or metastatic solid tumor or lymphoma and for whom standard therapies have been ineffective were treated with the antibody every 2 weeks in 28-day cycles. In Part 1A of the study, six cohorts of patients are administered six different dose levels in a dose escalation study: 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, 10 mg/kg, and 15 mg/kg. Patients are evaluated for dose-limiting toxicity on a 28-day cycle. Additional 28-day cycles may be performed if clinically indicated.

[00420] In Part 1B, further evaluation of safety and efficacy is being conducted in up to 30 patients with gastric cancer and/or patients known to have amplified FGFR2 genes or overexpress FGFR2b protein. Subjects in this part will initially receive one dose level below the current maximum dose level of the six cohorts in Part 1A, i.e., 0.3, 1, 3, or 10 mg/kg, escalating to 15 mg/kg in Part 1B if the maximum tolerated dose is not determined in Part 1A.

[00421] Once the reference dose is identified in Parts 1A and 1B of the study, Part 2 is initiated. Part 2 includes patients with histologically confirmed gastric or gastroesophageal cancer, or other histologically or cytologically confirmed solid tumor types with (a) FGFR2b overexpression and FGFR2 amplification; (b) FGFR2b overexpression without FGFR2 amplification; or (c) no FGFR2b overexpression and with locally recurrent or metastatic tumor that has progressed after standard therapy, or is ineligible for standard therapy, and with measurable disease as defined by RECIST version 1.1. Patients are grouped into three cohorts. About 30 patients have gastric cancer with FGFR2b overexpression (defined as 3+ by IHC) and FGFR2 gene amplification (FGFR2 to CEN10 ratio ≥ 2 by FISH). About 30 patients have gastric cancer with FGFR2b overexpression (IHC 3+) but no FGFR2b gene amplification (FISH ratio approximately 1). About 10 patients did not have FGFR2b overexpression (IHC 0 to 2+).

[00422] The detailed objectives, protocol, and inclusion/exclusion criteria for the study include the following:

[00423] The primary objectives are to evaluate the safety profile of escalating doses of the antibody in patients with advanced solid tumors and to determine the maximum tolerated dose (MTD) and recommended dose (RD) ( Part 1A ); and to evaluate the safety profile of escalating doses of the antibody in patients with gastric cancer or advanced gastroesophageal cancer ( Part 1B ), collectively referred to herein as "gastric cancer." The secondary objectives are to (a) characterize the PK profile of single and multiple doses of intravenously administered antibody in patients with gastric cancer and other solid tumor patients; (b) evaluate the safety and tolerability of long-term exposure to the administered antibody; (c) evaluate the objective response rate (ORR) in patients with FGFR2b-established gastric cancer ( Part 2 only ); and (d) evaluate the duration of response in patients with FGFR2b-established gastric cancer who respond to treatment ( Part 2 only ).

[00424] Some of the research objectives are to: (a) evaluate the percentage of patients with stable disease and (b) duration of disease in patients with FGFR2b-overexpressing gastric tumors, or with or without FGFR2 amplification ( Part 2 only ); (c) evaluate progression-free survival (PFS) in patients with FGFR2b-overexpressing gastric tumors, or with or without FGFR2 amplification ( Part 2 only ); and (d) investigate the relationship between the degree of FGFR2b overexpression and FGFR2 amplification in tumor tissue and clinical outcome.

[00425] This is a three-part, open-label, safety, tolerability, and PK study. Patients enroll in either Part 1 (A or B) or Part 2 of the study, but not Parts 1 and 2. Following an initial screening period of up to 28 days (4 weeks), patients are treated with the antibody every 2 weeks in 28-day cycles. In Part 1A, each enrolled patient is observed for 28 days to assess safety and the occurrence of dose-limiting toxicities (EBT observation period). Additional therapies may be administered every 2 weeks in 28-day cycles if clinically indicated (extended treatment period). In Part 1B, patients are treated every 2 weeks in 28-day cycles at the current dose levels calculated from the Part 1A EBT. In Part 2, patients receive anti-FGFR2 antibody treatment every 2 weeks in 28-day cycles at a recommended dose (RD) selected after assessment of the data obtained in Parts 1A and 1B.

[00426] Part 1A is a dose escalation study in patients with any locally advanced or metastatic solid tumor or lymphoma and for whom standard therapies have failed. Approximately 6 dose cohorts are proposed, with each cohort including at least 3 patients. Proposed dose levels are 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, 10 mg/kg, and 15 mg/kg. After assessment of safety and PK parameters, decisions may be made to add cohorts with alternative dose levels or dosing schedules (eg, less frequent dosing) to achieve optimal target exposure. All dose escalation decisions will be based on assessment of EBRT, overall safety, and tolerability and will be made after the last patient enrolled in each cohort completes the first cycle of treatment. Dose escalation decisions will be agreed upon by a cohort review committee (CRC) comprised of the sponsor and the investigators. The maximum tolerated dose (MTD) is defined as the highest dose at which <33% of patients experience EBRT during Cycle 1 (the period during which safety and PK are assessed). If EBRT occurs in 1 of 3 patients, then 3 additional patients will be enrolled at the same dose level. Dose escalation may continue until 2 of 3-6 patients treated at the dose level experience EBRT. The next lower dose is considered the MTD (maximum tolerated dose). Alternatively, an intermediate dose between the last calculated dose level and the dose level resulting in >33% EBRT may be studied before the MTD is determined to have been reached. Once the MTD or RD is reached, 3-10 additional patients with gastric cancer may be added before the start of Part 2 to further study the safety and PK at that dose level.

[00427] The following algorithm is used to make dose escalation decisions in Part 1A:

Number of patients with DLT Action 0/3 Open next cohort 1/3 Include 3 more patients in the same cohort ≥ 2/3 Stop enrollment. Enroll 3 more patients at a lower dose level if only 3 were previously enrolled. 1/6 Open next cohort ≥ 2/6 Stop enrollment. Enroll 3 more patients at a lower dose level if only 3 were previously enrolled.

[00428] If the MTD is not determined, the level of drug exposure exceeds the amount considered necessary based on preclinical pharmacology data or the clinical PK profile, the sponsor and investigators may decide to discontinue dose escalation.

[00429] Upon completion of Cycle 1 (the safety and PK evaluation period) of Part 1A, patients may enter an optional extension treatment period, which begins on the first day of Cycle 2. The anti-FGFR2 antibody is administered every 2 weeks for 4-week cycles until disease progression, unacceptable toxicity, patient or physician request to stop treatment, death, or study termination.

[00430] The purpose of Part 1B is to further evaluate the safety and PK of an anti-FGFR2 antibody in patients with gastric cancer prior to the initiation of Part 2. Some antibodies (eg, bevacizumab and trastuzumab) have been shown to clear more rapidly in patients with gastric cancer than in patients with other solid tumors. Patients included may be gastric cancer patients whose tumors will be studied retrospectively or those known to have an amplified FGFR2 gene or to express the FGFR2b protein. With the step dose escalation in Part 1A, patients in Part 1B are enrolled in a cohort one dose level below the cohort that is at the highest dose being studied in Part 1A. For example, if the current dose level in Part 1A being studied is 3 mg/kg, enrollment of Part 1B patients would be at the 1 mg/kg dose level; If the current dose level being studied in Part 1A is 6 mg/kg, enrollment of Part 1B patients would be at the 3 mg/kg dose level.

[00431] In Part 1B, approximately 3 patients may be enrolled at each dose level, with the sponsor and investigators choosing to enroll up to 6 patients per dose cohort. Dose escalation may continue in Part 1B up to 15 mg/kg if the MTD in Part 1A is not determined.

[00432] Enrollment into Part 2 begins when a recommended dose (RD) has been determined by the CRC based on overall safety, tolerability, PK, and efficacy assessment extrapolated from preclinical data. The RD may or may not be the same as the MTD determined in Part 1A. For example, if the MTD is not reached, or if the exposure at the MTD is much higher than the level believed to be required for efficacy, or if Part 1B patient data or subsequent treatment cycles from both Parts 1 (A and B) provide additional information on the safety profile, then the RD may be different, although not higher than the MTD. Once the RD has been established, gastric cancer patients selected based on FGFR2b expression are enrolled in Part 2 of the study. Part 2 patients are enrolled and treated for the purpose of further evaluating safety and preliminary efficacy in a selected population of cancer patients with the greatest potential for clinical benefit from antibody treatment. Treatment may continue until disease progression, unacceptable toxicity, patient or physician decision to discontinue, death, or termination of the study.

[00433] Patients included in Part 1 (A or B) or Part 2 must meet all of the following inclusion criteria:

1) Understand and sign the approved institutional review board/independent ethics committee informed consent form in accordance with any study-specific assessment;

2) Life expectancy of at least 3 months;

3) General condition score from 0 to 1 according to ECOG criteria;

4) ≥18 years of age at the time of signing the informed consent form, except for patients in Taiwan, where the patient must be ≥20 years of age at the time of signing the informed consent form;

5) In sexually active patients (i.e., women of childbearing potential who are not menopausal as defined by 12 consecutive months of amenorrhea or who have undergone a permanent sterilization procedure and men who have not undergone a permanent sterilization procedure), willingness to use 2 effective methods of contraception, one of which must be a physical barrier method (condom, diaphragm, or cervical cap/vaginal dome cap) for up to 6 months after the last dose of anti-FGFR2 antibody. Other effective forms of contraception include permanent sterilization (hysterectomy and/or bilateral oophorectomy, or bilateral tubal ligation with surgery, or vasectomy) at least 6 months prior to screening. Female patients of childbearing potential must be on continuous oral contraceptive therapy or an intrauterine or implantable contraceptive device for at least 90 days prior to the start of the study or abstain from sexual intercourse as a lifestyle choice.

6) Full hematological and biological function, confirmed by the following laboratory parameters:

a) Bone marrow function

i) ANC (absolute neutrophil count) ≥1.5×10 ⁹ /l

ii) Platelets >100×10 ⁹ /l

iii) Hemoglobin ≥9 g/dl

b) Liver function

i) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤3 x upper limit of normal (ULN); if liver metastases, then ≤5 x ULN

ii) Bilirubin ≤1.5 x ULN

c) Kidney function

i) Serum creatinine ≤1.5 x ULN

7) Tumor tissue available for determination of FGFR2b expression and FGFR2 amplification ( optional for Part 1A patients ).

[00434] Patients included in Part 1A (dose escalation) of the study must also meet the following inclusion criteria:

8) Histologically or cytologically confirmed solid tumor or lymphoma that is locally recurrent or metastatic and progresses after standard treatment or is refractory to standard treatment;

9) Measurable or unmeasurable manifestation of the disease.

[00435] Patients included in Part 1B of the study must also meet the following inclusion criteria:

10) Histologically confirmed gastric cancer or gastroesophageal cancer;

11) Tumor tissue for presumptive or retrospective determination of FGFR2b expression and FGFR2 amplification;

12) Locally recurrent or metastatic tumor that has progressed after standard treatment or is not suitable for standard treatment;

13) Measurable manifestation of disease as defined in RECIST version 1.1.

[00436] Patients included in Part 2 (dose escalation) of the study must also meet the following inclusion criteria:

14) Histologically confirmed gastric cancer or gastroesophageal cancer with

a) overexpression of FGFR2b and amplification of FGFR2, or

b) overexpression of FGFR2b without amplification of FGFR2, or

c) without FGFR2b overexpression;

15) Locally recurrent or metastatic tumor that has progressed after standard treatment or is not suitable for standard treatment;

16) Measurable manifestation of disease as defined in RECIST version 1.1.

[00437] Patients included in Part 1 (A or B) or Part 2 will be excluded if any of the following criteria apply:

1) Uncontrolled or symptomatic central nervous system (CNS) metastases: Patients with asymptomatic CNS metastases are eligible if they are clinically stable for at least 4 weeks and do not require intervention such as surgery, radiation, or any corticosteroid therapy for the treatment of symptoms associated with the CNS disease.

2) Impaired cardiac function or clinically significant cardiac disease, including any of the following:

a) Unstable angina ≤6 months prior to first scheduled dose of antibody

b) Acute myocardial infarction ≤6 months before the first scheduled dose of antibody

3) QTc interval > 470 msec

4) Known human immunodeficiency virus (HIV) infection or acquired immunodeficiency syndrome (AIDS)-related diseases, or history of chronic hepatitis B or C.

5) Treatment with any anticancer therapy or participation in another therapeutic clinical trial with experimental drugs ≤14 days (≤28 days for patients in Korea) before the first dose of the antibody.

6) Current adverse events from previous treatment >1 grade according to the CTCAE NCI (National Cancer Institute of the USA) classification.

a) Retinal disease or history of retinal disease or detachment, or, in the opinion of an ophthalmologist, an increased risk of retinal detachment

b) Current evidence or history of retinal vein occlusion (RVO) or central serous retinopathy.

c) Glaucoma was diagnosed within 1 month before the first day of the study.

d) Continuing drug therapy for glaucoma.

e) Previous intraocular injection or laser treatment for macular degeneration.

f) Corneal deficiency, corneal ulceration, keratitis, keratoconus, history of corneal transplantation, or other known corneal abnormalities that may, in the opinion of an ophthalmologist, pose a risk to treatment with an anti-FGFR2 antibody.

g) Patients with NSCLC (non-small cell lung cancer) with EGFR exon 19 or 21 mutation or ALK (anaplastic lymphoma kinase) amplification who have not received an EGFR or ALK TKI (tyrosine kinase inhibitor), respectively ( part 1A only ).

h) Patients with HER2-overexpressing gastric cancer and breast cancer who have not received anti-HER2-targeted therapy.

i) Major surgery is not allowed ≤28 days before administration of anti-FGFR2 antibody. In all cases, the patient must be sufficiently healthy and stable before treatment.

j) Women who are pregnant or breastfeeding; women of childbearing potential should not consider becoming pregnant during the study.

k) The presence of any serious or unstable concomitant systemic disorder incompatible with the clinical trial (e.g. substance abuse, psychiatric disorder or uncontrolled intercurrent illness including active infection, arterial thrombosis and symptomatic pulmonary embolism).

l) The presence of any other pathological condition that may increase the risk associated with participation in the study or may interfere with the interpretation of the study results and, in the opinion of the investigator, makes the patient ineligible to participate in the study.

7) Known allergy or hypersensitivity to any component of the medicinal product containing anti-FGFR2 antibody, including polysorbate.

8) History of previous malignancy, except:

a) Skin cancer other than melanoma that has been treated with radical methods or

b) The solid tumor was treated with radical methods more than 5 years ago without signs of recurrence or

c) History of other malignancy that, in the opinion of the investigator, will not affect the determination of the effect of the treatment.

9) In parts 1B and 2 (dose escalation), prior treatment with any selective inhibitor (eg, AZD4547, BGJ398, JNJ-42756493, BAY1179470) of the FGF-FGFR pathway.

[00438] No deviations from these inclusion or exclusion criteria will be granted.

[00439] Part 2 will include the following study cohorts A, C, D, E, and F. Cohort A will include approximately 30 patients with gastric cancer with strong FGFR2b overexpression, defined as IHC 3+ ≥ 10% tumor membrane staining. Cohort C will include approximately 10-30 patients with gastric cancer without FGFR2b overexpression, as defined by IHC=0. Cohorts D-F may also be included. Cohort D will include approximately 30 patients with gastric cancer with moderate FGFR2b overexpression, defined as IHC 2+ ≥ 10% and/or IHC 3+ < 10% tumor membrane staining. Cohort E will include approximately 30 patients with gastric cancer with low FGFR2b overexpression, defined as IHC 1+ and/or IHC 2+ <10% tumor membrane staining. Cohort F will include approximately 30 patients with non-gastric solid tumors for each tumor type tested. For bladder cancer patients, there will be two subgroups. Subgroup 1 will have FGFR2b H values of 10-19 and subgroup 2 will have FGFR2b H values of 20 or more.

[00440] Study Drug: Anti-FGFR2 antibody is supplied in a sterile vial for reconstitution into an infusion bag for administration to the study site. In Part 1A, patients will receive 2 doses of anti-FGFR2 antibody separated by 2 weeks. If tolerated without disease progression by the end of the first cycle, patients may continue the study in an extended treatment period and receive anti-FGFR2 antibody every 2 weeks until disease progression or other reasons for discontinuing the study.

[00441] Dose Adjustments: Dose reductions may be permitted for patients treated outside the EBRT period in Part 1A or for any patient in Parts 1B or 2 upon discussion and approval by the sponsor. Patients may miss up to 2 consecutive doses (up to 6 weeks between doses) for adverse or other events; failure to take additional doses for more than 6 weeks for adverse or other events will require withdrawal of the patient from the study, if approved by the study sponsor. Individual dose escalations above the starting dose for each patient in Part 1 (A and B) and Part 2 are not permitted. If a patient's dose is reduced for a reason that is no longer relevant, dose escalation to the originally prescribed dose may occur upon discussion and approval by the sponsor.

[00442] Concomitant Drug Therapy: Maintenance therapy (e.g., antiemetics, analgesics for pain relief) may be used at the discretion of the investigator and in accordance with institutional procedures. Hematopoietic immunostimulatory agents may be used when indicated. Concomitant anticancer therapy of any kind is not allowed, except for long-term maintenance therapies such as luteinizing hormone-releasing hormone (LHRH) modulating agents for breast or prostate cancer, which may be continued if the patient has been taking these agents and 1) continued use is unlikely to result in additional tumor shrinkage and 2) is considered standard of care for the patient.

[00443] Exclusion Criteria: The patient must be withdrawn from protocol-prescribed therapy if any of the following occurs:

Revocation of informed consent at the request of the patient or his legally authorized representative;

Patient disease progression. Patients who experience clinical benefit despite disease progression may continue the study after discussion with their medical monitor;

Any event that poses an unacceptable risk to patient safety;

Concomitant illness that will significantly affect the assessment of the clinical condition;

A positive pregnancy test at any time during the study;

At the specific request of the sponsor or its authorized representative (e.g. if the study is terminated for patient safety reasons.

[00444] Pharmacokinetic Parameter Assessment: Patients enrolled in Parts 1A and 1B will have blood samples collected to measure serum anti-FGFR2 antibody concentrations throughout Cycle 1 on Days 1, 2, 4, and 8. In addition, blood samples will be collected both before and at the end of infusion on Day 15 of Cycle 1 and Day 1 of Cycles 2-5, and Day 1 of every other cycle beginning with Cycle 5, as well as at the end of treatment. For patients in Part 2, blood samples from Cycle 1 were collected both before and at the end of Day 1 of infusion and on Day 8. Blood samples are collected on day 15 of cycle 1 and day 1 of cycles 2–5 and day 1 of every other cycle beginning with cycle 5 both before and at the end of each infusion and at the end of treatment for PK studies in selected gastric cancer patients with FGFR2b-overexpressing tumors with or without FGFR2 amplification. Standard PK parameters are determined based on serum anti-FGFR2 antibody concentrations over time.

[00445] Immunogenicity: All patients in the study had pre-dose samples collected on day 1 of cycles 1-5 and throughout each cycle from cycle 5 onwards to measure anti-FGFR2 antibodies.

[00446] Efficacy assessments: Efficacy measures include tumor assessments consisting of clinical examination and appropriate imaging, preferably computed tomography (CT) scans of the chest, abdomen, and pelvis with appropriate slice thicknesses per RECIST; other assessments (magnetic resonance imaging [MRI], x-ray, positron emission tomography (PET), and ultrasound) as needed. Tumor assessments are performed at screening, then every 6 weeks after the first dose for 24 weeks, and then approximately every 12 weeks thereafter. After an initial complete response (CR) or partial response (PR) is noted, confirmatory scans should be performed at 4 to 6 weeks.

[00447] Safety assessments: Safety measures include AEs (adverse events), complete blood count, clinical chemistry, urinalysis, vital signs, body weight, concomitant medications/procedures, ECOG performance status, targeted physical examination, ECG, ophthalmologic/retinal examination, and anti-FGFR2 antibody dose modifications.

[00448] The total enrollment planned for this study is approximately 100-130 patients: approximately 20-30 patients are enrolled in Part 1A. Up to 30 patients with gastric cancer are enrolled in Part 1B. For Part 2, study activity is assessed by enrolling one or more of:

Cohorts A: Approximately 30 patients with gastric cancer with FGFR2b overexpression (IHC 3+) and FGFR2 amplification (FISH ratio ≥ 2);

Cohort B: Approximately 30 patients with gastric cancer with FGFR2b overexpression (IHC 3+) and no FGFR2 amplification (FISH ratio=1) to characterize the prognostic value of FGFR2 selection.

Cohorts C: Approximately 10-30 patients with gastric cancer without FGFR2b overexpression as determined by IHC=0 to 2+.

Cohort D: Approximately 30 patients with gastric cancer with moderate FGFR2b overexpression, defined as IHC 2+ ≥ 10% and/or IHC 3+ <10% tumor membrane staining.

Cohort E: Approximately 30 patients with gastric cancer with low FGFR2b overexpression, defined as IHC 1+ and/or IHC 2+ <10% tumor membrane staining.

Cohorts F: About 30 patients with non-gastric solid tumors for each tumor type tested. For bladder cancer patients, there will be two subgroups. Subgroup 1 will have FGFR2b H values of 10-19, and subgroup 2 will have FGFR2b H values of 20 or more.

Example 6: Treatment of a patient with bladder cancer using an anti-FGFR2 antibody

[00449] This example describes the treatment of a 76-year-old male enrolled in the above study (see Example 5) who was diagnosed with bladder cancer in July 2014 after presenting to his primary care physician with hematuria using an afucosylated FGFR2 antibody containing the heavy and light chain HVRs of SEQ ID NOs: 6-11 of the study described in Example 5. The patient underwent diagnostic cystoscopy and biopsy, and then underwent resection of the primary tumor. He was staged as a T2, N2, M0 muscle-invading tumor, and 3 of 6 lymph nodes sampled were positive, making him a stage 4 subject. He received 4 cycles of gemcitabine and cisplatin (SOC) in the adjuvant setting. In March 2015, approximately 6 months after completion of adjuvant chemotherapy, the patient was followed up regularly with PET and CT. CT demonstrated multiple enlarged lymph nodes in the pelvis and retroperitoneum, while PET confirmed that they were metabolically active and consistent with recurrent metastatic bladder cancer (UBC). The patient was started on anti-FGFR2 antibodies at a dose of 3 mg/kg approximately every two weeks. The largest lymph node was then monitored for assessment and overall adenopathy. At initial screening and first dose of FGFR2 in April 2015, the largest lymph node measured 18 × 12 mm. Six weeks later, the largest node measured was 15 × 11 mm, and 12 weeks later it measured 9 × 7 mm, thus decreasing by approximately half. As of August 2014, no significant adenopathy was observed. Subsequent follow-up scans confirmed the absence of detectable lymphadenopathy, and a PET scan performed in November 2015 showed no abnormal metabolic activity. The patient is currently continuing anti-FGFR2 antibody therapy.

Example 7: Immunohistochemical analysis of bladder cancer samples for FGFR2b overexpression

[00450] Immunohistochemistry (IHC) was used to assess the degree of FGFR2b overexpression in the bladder cancer population. Immunohistochemistry (IHC) was performed on normal bladder specimens and archival urothelial cancer (UC) specimens using a mouse anti-αFGFR2b antibody containing the mouse variable regions of GAL-FR21 (see U.S. Patent No. 8,101,723 B2). 422 formalin-fixed, paraffin-embedded UC sections, both primary and metastatic, in whole section or tissue array format, were stained and detected using a chromogenic substrate. Tumor cell staining intensity was scored on a 0-3 scale, with "0" being given if no reactivity was observed or there was membrane reactivity in only <10% of tumor cells; A score of "1+" is given if there is weak or barely noticeable membrane reactivity in at least 10% of the tumor cells or if the cells are reactive in only a portion of their membranes; a score of "2+" is given if there is weak or moderate complete, basolateral, or lateral membrane reactivity in at least 10% of the tumor cells; and a score of "3+" is given if there is strong complete basolateral or lateral membrane reactivity in at least 10% of the tumor cells. Tumors with 1+ membrane reactivity in ≥ 10% of the tumor cells were considered positive in this experiment.

[00451] Normal bladder has weak transitional epithelial staining (<1+). However, IHC analysis of 422 archival primary UC samples showed that FGFR2b is overexpressed in >10% of samples with an expression intensity of at least 1+.

[00452] Additionally, in a primary tumor specimen from a patient with anti-FGFR2-sensitive bladder cancer (see Example 6), there was 15% 2+ IHC staining and 35% 1+ staining with the FPR2-D antibody from surgical resection. This is in contrast to previous data from patients with gastric cancer, in which tumor specimens were collected from patients with 3+ staining and objective responses were observed. The response of this patient to the anti-FGFR2 antibody (see Example 6) and the positive IHC staining in the UC specimens together suggest that this bladder cancer is an additional indicator that it may be sensitive to anti-FGFR2 antibody treatment.

Example 8: Open-label phase I study of anti-FGFR2 antibody in combination with nivolumab in patients with advanced solid tumors

[00453] A two-part, open-label, multicenter, dose-escalation, enrollment-expanding study will be conducted to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of an afucosylated FGFR2 antibody containing the heavy and light chain HVRs of SEQ ID NOs: 6-11 in combination with nivolumab in patients with advanced solid tumors. Each part of the study will consist of 3 periods: screening (up to 28 days), treatment, and follow-up (up to 100 days). The anti-FGFR2 antibody and nivolumab will be given on day 1 of each 14-day treatment cycle. The anti-FGFR2 antibody will be administered as an IV infusion over 30 minutes followed by a 30-minute rest period, followed by nivolumab as an IV infusion over 30 minutes. If any grade 3 or higher infusion reaction is observed during the infusion of either drug at the proposed infusion rate, the infusion rate will be increased to 60 minutes for all current and subsequent patients throughout this study. Survival follow-up will be conducted for all patients after completion of treatment and subsequent study periods.

[00454] The study will include a dose-escalation Part 1 and an expansion Part 2. Part 1 will consist of two planned dose cohorts of anti-FGFR2 antibodies in combination with nivolumab in patients with gastric cancer or gastroesophageal junction cancer (collectively referred to as gastric cancer). Phenotypic characterization will not be required for entry into the dose-escalation portion of the study, but will be performed retrospectively. This phenotypic characterization will include, but is not limited to, analysis of FGFR2b and PD-L1 expression by immunohistochemistry (IHC). Each patient enrolled in Part 1 will be followed for 28 days (or completion of two 14-day cycles) to assess safety and the occurrence of dose-limiting toxicities (EBRT observation period). At the first occurrence of a delayed DLT in any patient enrolled in Part 1 (defined as any AE that occurs between 4-6 weeks after study drug administration), all current and subsequent DLT periods will be extended to 42 days (or after completion of three 14-day cycles) for all remaining and subsequent patients enrolled in Part 1. Additional therapies may be administered every 2 weeks in 14-day cycles if clinically indicated (extended treatment period).

[00455] Part 2 consists of two expansion cohorts of patients with advanced gastric cancer. Enrollment in Part 2 of the study will require prospective IHC analysis of FGFR2b expression using a validated assay. Patients whose tumors are positive for FGFR2b by IHC will be eligible for enrollment if other inclusion criteria are met. The two cohorts in Part 2 of the study will be defined by the level of FGFR2b IHC positivity in the patient's tumor. Cohort 2a will include patients whose tumors stain with an intensity of 1+ or 2+ in at least 10% of tumor cells. Cohort 2a will include patients whose tumors stain with an intensity of 3+ in at least 10% of tumor cells. Opening of each of these cohorts will be at the discretion of the sponsor. The expansion portion of the Part 2 study in patients receiving MTD is open-label. Patients who meet all eligibility criteria will be treated every 2 weeks in 14-day cycles with the anti-FGFR2 antibody in combination with 3 mg/kg nivolumab at the recommended dose (RD) selected after assessment of data obtained in Part 1. Patients will be enrolled in Part 1 or Part 2 of the study, but not both.

[00456] Part 1 entails two dose cohorts, with at least 6 patients with gastric cancer included in each cohort. The planned dose levels are as follows: the first patient will be enrolled in dose level 1:

Dose level -1: 6 mg/kg anti-FGFR2 antibody + 3 mg/kg nivolumab (once every 2 weeks)

Dose level 1: 10 mg/kg anti-FGFR2 antibody + 3 mg/kg nivolumab (once every 2 weeks)

Dose level 2: 15 mg/kg anti-FGFR2 antibody + 3 mg/kg nivolumab (once every 2 weeks)

[00457] All dose escalation decisions will be based on assessment of EBRT, overall safety and tolerability, and will be made after the last patient enrolled in each cohort has completed the prescribed EBRT observation period. Dose escalation decisions will be agreed upon by a cohort review committee (CRC) consisting of the sponsor and the investigators. Following assessment of safety and PK parameters, decisions can be made to add cohorts with alternative dose levels to achieve optimal target exposure. Dose level -1 will only be explored in detail if EBRT is observed at dose level 1, necessitating exploration of a lower dose of anti-FGFR2 antibody. The highest dose level with ≤1 observed EBRT will be considered the recommended phase 2 dose (RPD2). An additional 8 patients will be enrolled in Part 1 of the study once RPD2 is determined, bringing the total number of patients enrolled in Part 1 to approximately 20.

[00458] Following completion of the DLT observation period, Part 1 patients may enter an optional extension treatment period beginning on Day 1 of Cycle 2. The anti-FGFR2 antibody will continue to be administered in combination with nivolumab at the same dose every 2 weeks for 4-week cycles until disease progression, unacceptable toxicity, patient or physician request to stop treatment, death, or study termination.

[00459] To further characterize the safety and efficacy of the anti-FGFR2 antibody in combination with nivolumab, Part 2 will enroll approximately 40 patients with gastric cancer in two cohorts of 20 patients per cohort. The two cohorts will differ in the degree of tumor IHC positivity for FGFR2b. Patients whose tumors score 1+ or 2+ in ≥ 10% of tumor cells by central validation (low or moderate overexpression) will be included in Cohort 2A; patients whose tumors score 3+ in ≥ 10% of tumor cells will be included in Cohort 2B. Enrollment into Part 2 begins when the recommended dose (RD) has been determined by the CRC based on overall safety and tolerability. The RD may or may not coincide with the MTD defined in Part 1. The anti-FGFR2 antibody will be administered in combination with nivolumab on an RD of 2 weeks in 4-week cycles until disease progression, unacceptable toxicity, patient or physician request to stop treatment, death, or study termination.

[00460] If, after inclusion and evaluation of the first 20 patients, sufficient activity is deemed to merit further investigation, then inclusion will be opened for an additional 20 patients per cohort. Opening of each cohort and addition of 20 patients to any cohort will be at the discretion of the sponsor.

[00461] Patients enrolled in Part 2 will have their tumor tissue (archival and/or recent) retrospectively tested for PD-L1 expression using a validated immunohistochemistry (IHC) assay. Biopsies of the primary tumor lesion or metastatic lesion will be obtained (to the extent possible) before and during treatment to examine immune infiltrates and expression of selected tumor markers. Optional biopsies may be obtained from tumors that responded and/or progressed during or after treatment to understand mechanisms of resistance.

[00462] Dose reductions for anti-FGFR2 antibodies may be permitted for patients during treatment outside the DLT period in Part 1 or for any patient in Part 2 in accordance with recommendations set out in the protocol.

[00463] This study is planned to enroll up to 60 subjects in North America and Europe. It includes 20 patients in Part 1, a dose escalation study, and approximately 40 patients in Part 2, an expansion study, to receive MTD treatment.

Inclusion Criteria

[00464] Patients included in Part 1 or Part 2 must meet all of the following inclusion criteria:

1. Understand and sign the approved institutional review board/independent ethics committee informed consent form in accordance with any study-specific assessment

2. Life expectancy of at least 3 months

3. General condition score from 0 to 1 according to ECOG criteria

4. ≥18 years of age at the time of signing the informed consent form

5. In sexually active patients (i.e., women of childbearing potential who are not menopausal as defined by 12 consecutive months of amenorrhea or who have undergone a permanent sterilization procedure and men who have not undergone a permanent sterilization procedure), willingness to use 2 effective methods of contraception, one of which must be a physical barrier method (condom, diaphragm, or cervical cap/vaginal dome cap) for up to 6 months after the last dose of anti-FGFR2 antibody. Other effective forms of contraception include permanent sterilization (hysterectomy and/or bilateral oophorectomy, or bilateral tubal ligation with surgery, or vasectomy) at least 6 months prior to screening. Female patients of childbearing potential must be on stable oral contraceptive therapy or an intrauterine or implantable device for at least 90 days prior to study entry or abstain from sexual intercourse as a lifestyle choice.

6. Full hematological and biological function, confirmed by the following laboratory parameters:

a) Bone marrow function

ACN ≥ 1.5×10 ⁹ /l

Platelets >100×10 ⁹ /l

Hemoglobin ≥ 9 g/dL

b) Liver function

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 3 x upper limit of normal (ULN); if liver metastases, then ≤ 5 x ULN

Bilirubin ≤ 1.5 x ULN

c) Kidney function

Serum creatinine ≤ 1.5 x ULN

7. Histologically or cytologically confirmed gastric or gastroesophageal cancer that is locally recurrent or metastatic and progresses after standard treatment or is refractory to standard treatment

Patients included in Part 1 (dose escalation) of the study must also meet the following inclusion criteria:

7. Measurable or assessable disease

Patients included in Part 2 (dose escalation) of the study must also meet the following inclusion criteria:

8. Measurable manifestation of disease as defined in RECIST version 1.1.

9. The tumor is positive for FGFR2b expression as determined by a validated IHC assay

10. Tumor tissue (archival or recent) for retrospective determination of PD-L1 expression

Exclusion criteria

[00465] Patients included in Part 1 or Part 2 will be excluded if any of the following criteria apply:

1. Uncontrolled or symptomatic central nervous system (CNS) metastases. Patients with asymptomatic CNS metastases are eligible if they are clinically stable for at least 4 weeks and do not require intervention such as surgery, radiation, or any corticosteroid therapy for the treatment of symptoms associated with CNS disease.

2. Impaired cardiac function or clinically significant cardiac disease, including any of the following:

o Unstable angina ≤ 6 months prior to first scheduled dose of anti-FGFR2 antibody

o Acute myocardial infarction ≤ 6 months prior to first scheduled dose of anti-FGFR2 antibody

3. QTc interval > 470 msec

4. Known history of testing positive for human immunodeficiency virus (HIV) 1 or 2 or known acquired immunodeficiency syndrome (AIDS)

5. A positive test for hepatitis B surface antigen (HBsAg) or detectable hepatitis C virus ribonucleic acid (HCV RNA), indicating acute or chronic infection

6. Positive test for latent tuberculosis (TB) on screening (QuantiFERON test) or symptoms of active TB

7. Symptomatic interstitial lung disease or inflammatory pneumonitis

8. Active, known or suspected autoimmune disease. Patients with type 1 diabetes mellitus, hypothyroidism requiring only hormone replacement therapy, skin diseases (such as vitiligo, psoriasis or alopecia) not requiring systemic treatment, or pathological conditions that are not expected to recur without an external trigger are eligible to participate in the study.

9. Any uncontrolled inflammatory gastrointestinal disease, including Crohn's disease and ulcerative colitis

10. History of antibodies to the drug, severe allergic, anaphylactic or other infusion-related reaction to a previously received biological agent

11. The use of immunosuppressive doses of systemic agents for treatment, such as steroids or locally applied absorbable steroids (doses> 10 mg/day of prednisone or equivalent daily) should be discontinued at least 2 weeks before administration of the drug, except in cases of treatment of tumor-related AEs. Patients with a pathological condition requiring chronic systemic treatment or corticosteroids (inhaled or locally applied steroids and replacement therapy with adrenal steroid doses> equivalent to prednisone at a dose of 10 mg/day) or other immunosuppressants for 2 weeks of treatment are allowed in the absence of active autoimmune disease (except patients with glioma).

12. Non-oncologic vaccine therapies for the prevention of infectious diseases (e.g., HPV vaccines) within 4 weeks after drug administration. Inactivated seasonal influenza vaccine may be administered to patients before and during therapy without restrictions. Live virus influenza vaccines or other clinically indicated vaccinations for infectious diseases (e.g., pneumovax, varicella, etc.) may be allowed but should be discussed with the sponsor's medical monitor and a washout period for the investigational drug before and after vaccine administration may be required.

13. Treatment with any anticancer therapy or participation in another therapeutic clinical trial with investigational drugs ≤ 14 days before the first dose of the drug.

14. Current severe adverse events from previous treatment > grade 1 according to the NCI CTCAE classification.

15. Patients with HER2-overexpressing gastric cancer who did not receive anti-HER2-targeted therapy.

16. Major surgery is not allowed ≤28 days before FPA144 administration. In all cases, the patient must be sufficiently healthy and stable before starting treatment.

17. Women who are pregnant or breastfeeding; women of childbearing potential should not consider becoming pregnant during the study.

18. Presence of any serious or unstable concomitant systemic disorder incompatible with the clinical trial (e.g. substance abuse, psychiatric disorder, or uncontrolled intercurrent illness including active infection, arterial thrombosis, and symptomatic pulmonary embolism).

19. The presence of any other pathological condition that may increase the risk associated with participation in the study or may interfere with the interpretation of the study results and, in the opinion of the investigator, makes the patient ineligible to begin participation in the study.

20. Known allergy or hypersensitivity to any component of the anti-FGFR2 antibody or nivolumab formulations, including polysorbate.

21. Prior treatment with any selective inhibitor (eg, AZD4547, BGJ398, JNJ-42756493, BAY1179470) of the FGF-FGFR pathway.

22. History of previous malignancy, except:

a) Skin cancer other than melanoma that has been treated with radical methods or

b) The solid tumor was treated with radical methods more than 5 years ago without signs of recurrence or

c) History of other malignancy that, in the opinion of the investigator, will not affect the determination of the effect of the treatment.

[00466] Blood samples will be collected from all patients for pharmacokinetic analysis. Standard PK parameters will be determined based on anti-FGFR2 antibody concentration-time data. Blood samples will also be collected for anti-drug antibody (ADA) analysis against the anti-FGFR2 antibody and nivolumab.

[00467] Efficacy endpoints include tumor assessments that include clinical examination and appropriate imaging, preferably computed tomography (CT) scans of the chest, abdomen, and pelvis with appropriate slice thickness per RECIST 1.1; other assessments (magnetic resonance imaging (MRI), x-ray, positron emission tomography (PET), and ultrasound) as needed. Tumor imaging will be performed at screening, then every 6 weeks after the first dose for 24 weeks, and then approximately every 12 weeks thereafter. After an initial complete response (CR) or partial response (PR) is noted, confirmatory scans should be performed 4 to 6 weeks later.

[00468] Tumor biopsies, mandatory if possible, will be performed pre-treatment and either 15 days or 29 days post-treatment for all patients in Part 2. Implementation at each time point will be assessed by the investigator and should include an assessment of patient safety. Patients may also have an optional on-treatment biopsy following confirmed tumor response and/or an optional post-treatment biopsy following confirmed tumor progression following discussion with the sponsor.

[00469] The sample size of up to approximately 60 subjects is based on the dose escalation study design in Part 1 and 20 patients per arm per treatment group in Part 2. Each individual arm in Part 2 of this study will continue with a two-stage Simons design. The estimated observed response rate of nivolumab monotherapy in PD-L1 unselected advanced gastric cancer is 12%; the ORR (objective response rate) for the anti-FGFR2 antibody may vary depending on the level of FGFR2b expression observed in the tumor. For cohort 2A, the estimated ORR for the anti-FGFR2 antibody monotherapy is 0%. For cohort 2B, the ORR for the anti-FGFR2 antibody monotherapy is 30%. Thus, cohort 2A will not be expanded by 20 patients if <2 responses are observed in the first 20 patients. For cohort 2B, the 20-patient expansion will not be opened if <6 patients achieve an objective response among the first 20 enrolled patients.

sequence table

[00470] The table below provides a list of specific sequences provided in this document.

SEQ. ID. NO. DESCRIPTION Subsequence 1 Mature human FGFR2-IIIb RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKHSGINSS NAEVLALFNV TEADAGEYIC KVSNYIGQAN QSAWLTVLPK QQAPGREKEI TASPDYLEIA IYCIGVFLIA CMVVTVILCR MKNTTKKPDF SSQPAVHKLT KRIPLRRQVT VSAESSSSMN SNTPLVRITT RLSSTADTPM LAGVSEYELP EDPKWEFPRD KLTLGKPLGE GCFGQVVMAE AVGIDKDKPK EAVTVAVKML KDDATEKDLS DLVSEMEMMK MIGKHKNIIN LLGACTQDGP LYVIVEYASK GNLREYLRAR RPPGMEYSYD INRVPEEQMT FKDLVSCTYQ LARGMEYLAS QKCIHRDLAA RNVLVTENV MKIADFGLAR DINNIDYYKK TTNGRLPVKW MAPEALFDRV YTHQSDVWSF GVLMWEIFTL GGSPYPGIPV EELFKLLKEG HRMDKPANCT NELYMMMRDC WHAVPSQRPT FKQLVEDLDR ILTLTTNEEY LDLSQPLEQY SPSYPDTRSS CSSGDDSVFS PDPMPYEPCL PQYPHINGSV KT 2 αFGFR2b heavy chain; Asn297 is bold and underlined QVQLVQSGAE VKKPGSSVKV SCKASGYIFT TYNVHWVRQA PGQGLEWIGS IYPDNGDTSY NQNFKGRATI TADKSTSTAY MELSSLRSED TAVYYCARGD FAYWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK 3 αFGFR2b light chain DIQMTQSPSS LSASVGDRVT ITCKASQGVS NDVAWYQQKP GKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ HSTTPYTFGQ GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 4 αFGFR2b heavy chain variable region QVQLVQSGAE VKKPGSSVKV SCKASGYIFT TYNVHWVRQA PGQGLEWIGS IYPDNGDTSY NQNFKGRATI TADKSTSTAY MELSSLRSED TAVYYCARGD FAYWGQGTLV TVSS 5 αFGFR2b light chain variable region DIQMTQSPSS LSASVGDRVT ITCKASQGVS NDVAWYQQKP GKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ HSTTPYTFGQ GTKLEIK 6 HVR1 heavy chain (HC) αFGFR2b TYNVH 7 HVR2HCαFGFR2b SIYPDNGDTS YNQNFKG 8 HVR3HCαFGFR2b GDFAY 9 HVR1 light chain (LC) αFGFR2b KASQGVSNDV A 10 HVR2 LC αFGFR2b SASYRYT 11 HVR3 LC αFGFR2b QQHSTTPYT 12 αFGFR2b heavy chain N297Q; N297Q point mutation is shown in bold and underlined QVQLVQSGAE VKKPGSSVKV SCKASGYIFT TYNVHWVRQA PGQGLEWIGS IYPDNGDTSY NQNFKGRATI TADKSTSTAY MELSSLRSED TAVYYCARGD FAYWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYQSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK 13 Mature human FGFR2-IIIc RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEIT ASPDYLEIAI YCIGVFLIAC MVVTVILCRM KNTTKKPDFS SQPAVHKLTK RIPLRRQVTV SAESSSSMNS NTPLVRITTR LSSTADTPML AGVSEYELPE DPKWEFPRDK LTLGKPLGEG CFGQVVMAEA VGIDKDKPKE AVTVAVKMLK DDATEKDLSD LVSEMEMMKM IGKHKNIINL LGACTQDGPL YVIVEYASKG NLREYLRARR PPGMEYSYDI NRVPEEQMTF KDLVSCTYQL ARGMEYLASQ KCIHRDLAAR NVLVTENNVM KIADFGLARD INNIDYYKKT
TNGRLPVKWM APEALFDRVY THQSDVWSFG VLMWEIFTLG GSPYPGIPVE ELFKLLKEGH RMDKPANCTN ELYMMMRDCW HAVPSQRPTF KQLVEDLDRI LTLTTNEEYL DLSQPLEQYS PSYPDTRSSC SSGDDSVFSP DPMPYEPCLP QYPHINGSVK T 14 FGFR2 VKD RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEIT ASPDYLE 15 FGFR2 Δ3 VCD RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEIT ASPD 16 FGFR2 Δ4 VCD RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEIT A.S.P. 17 FGFR2 VCD Δ5 RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEIT AS 18 FGFR2 Δ8 VCD RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEI 19 FGFR2 Δ9 VCD RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKE 20 FGFR2 VCD Δ10 RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREK 21 FGFR2 VCD Δ14 RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAP 22 FGFR2 VCD Δ15 RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PA 23 FGFR2 Δ16 VCD RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL P 24 FGFR2 VCD Δ17 RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL 25 Fc C237S EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 26 Fc ERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 27 Fc ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 28 FGFR2(111-118) DDEDDTDG 29 FGFR1(105-112) EDDDDDDD 30 VKD FGFR2 with R2(111-118) replaced by R1(105-112) RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS G EDDDDDDD A EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEIT ASPDYLE 31 ECD FGFR2 with R2(111-118) replaced by R1(105-112)+Fc RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS G EDDDDDDD A EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEIT ASPDYLE EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 32 ECD FGFR2 Δ3+GS linker+ Fc
(also called FGFR2ECD(delta3)-GS linker-Fc and FGFR2-Fc) RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGA EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEIT ASPD GS EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 33 ECD FGFR2 Δ3 with R2(111-118) replaced by R1(105-112) RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS G EDDDDDDD A EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEIT ASPD 34 ECD of FGFR2 Δ3 with R2(111-118) replaced by R1(105-112)+GS linker+Fc
(also called FGFR2ECD(FGFR2(111-118):FGFR1(105-112):delta3)-GS linker-Fc and R2(111-118):R1(105-112)) RPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV RCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPR DSGLYACTAS RTVDSETWYF MVNVTDAISS G EDDDDDDD A EDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQA GLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSK YGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEY TCLAGNSIGI SFHSAWLTVL PAPGREKEIT ASPD GS EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 35 human PD-1 precursor (with signal sequence) UniProtKB/Swiss-Prot: Q15116.3, 01-OCT-2014 MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL 36 human PD-1 (mature, no signal sequence) PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL 37 human PD-L1 precursor (with signal sequence) UniProtKB/Swiss-Prot: Q9NZQ7.1, 01-OCT-2014 MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH LVILGILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET 38 human PD-L1 (mature, no signal sequence) FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH LVILGILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET 39 Anti-FGFR2 heavy chain variable region Gal-FR22 QVQLKQSGPG LVQPSQSLSI TCTVSGFSLT SFGVHWVRQS PGKGLEWLGV IWSGGSTDYN ADFRSRLSIS KDNSKSQIFF KMNSLQPDDT IAYCANFYYG YDDYVMDYWG QGTSVTVSS 40 Anti-FGFR2 Gal-FR22 Heavy Chain CDR1 SFGVH 41 Anti-FGFR2 Gal-FR22 Heavy Chain CDR2 VIWSGGSTDYNADFRS 42 Anti-FGFR2 Gal-FR22 Heavy Chain CDR3 FYYGYDDYVMDY 43 Anti-FGFR2 Gal-FR22 light chain variable region DIQMTQSPSS LSASLGGRVT ITCKASQDIK NYIAWYQHKP GKSPRLLIHY TSTLQPGVPS RFSGSGSGRD YSFSISNLEP EDIATYYCLQ YDDDLYMFGG GTKLDIK 44 Anti-FGFR2 Gal-FR22 Light Chain CDR1 KASQDIKNYIA 45 Anti-FGFR2 Gal-FR22 Light Chain CDR2 YTSTLQP 46 Anti-FGFR2 Gal-FR22 Light Chain CDR3 LQYDDLYM

--->

SEQUENCE LIST

<110> FIVE PRIME THERAPEUTICS, INC.

<120> FGFR2 INHIBITORS ALONE OR IN COMBINATION WITH

IMMUNOSTIMULATING AGENTS IN CANCER TREATMENT

<130> 01134-0046-00PCT

<150> US 62/258731

<151> 2015-11-23

<150> US 62/314174

<151> 2016-03-28

<150> US 62/379094

<151> 2016-08-24

<160> 46

<170> PatentIn version 3.5

<210> 1

<211> 801

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> Mature human FGFR2-IIIb

<400> 1

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys His Ser Gly Ile Asn Ser Ser Asn Ala Glu Val Leu

290 295 300

Ala Leu Phe Asn Val Thr Glu Ala Asp Ala Gly Glu Tyr Ile Cys Lys

305 310 315 320

Val Ser Asn Tyr Ile Gly Gln Ala Asn Gln Ser Ala Trp Leu Thr Val

325 330 335

Leu Pro Lys Gln Gln Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser

340 345 350

Pro Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile Gly Val Phe Leu Ile

355 360 365

Ala Cys Met Val Val Thr Val Ile Leu Cys Arg Met Lys Asn Thr Thr

370 375 380

Lys Lys Pro Asp Phe Ser Ser Gln Pro Ala Val His Lys Leu Thr Lys

385 390 395 400

Arg Ile Pro Leu Arg Arg Gln Val Thr Val Ser Ala Glu Ser Ser Ser

405 410 415

Ser Met Asn Ser Asn Thr Pro Leu Val Arg Ile Thr Thr Arg Leu Ser

420 425 430

Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser Glu Tyr Glu Leu

435 440 445

Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys Leu Thr Leu Gly

450 455 460

Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala

465 470 475 480

Val Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val Thr Val Ala Val

485 490 495

Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu Ser Asp Leu Val

500 505 510

Ser Glu Met Glu Met Met Lys Met Ile Gly Lys His Lys Asn Ile Ile

515 520 525

Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro Leu Tyr Val Ile Val

530 535 540

Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg

545 550 555 560

Pro Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu Glu

565 570 575

Gln Met Thr Phe Lys Asp Leu Val Ser Cys Thr Tyr Gln Leu Ala Arg

580 585 590

Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala

595 600 605

Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val Met Lys Ile Ala Asp

610 615 620

Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys Thr

625 630 635 640

Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe

645 650 655

Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser Phe Gly Val Leu

660 665 670

Met Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro

675 680 685

Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys

690 695 700

Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met Met Arg Asp Cys Trp

705 710 715 720

His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp

725 730 735

Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu Tyr Leu Asp Leu

740 745 750

Ser Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr Pro Asp Thr Arg Ser

755 760 765

Ser Cys Ser Ser Gly Asp Asp Ser Val Phe Ser Pro Asp Pro Met Pro

770 775 780

Tyr Glu Pro Cys Leu Pro Gln Tyr Pro His Ile Asn Gly Ser Val Lys

785 790 795 800

Th

<210> 2

<211> 444

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2b heavy chain

<220>

<221>MISC_FEATURE

<222> (294)..(294)

<223> Asn297

<400> 2

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Thr Tyr

20 25 30

Asn Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Asp Asn Gly Asp Thr Ser Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Arg Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

165 170 175

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

180 185 190

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

195 200 205

Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 3

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> anti-FGFR2b light chain

<400> 3

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Gly Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Ser Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 4

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2b heavy chain variable region

<400> 4

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Thr Tyr

20 25 30

Asn Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Asp Asn Gly Asp Thr Ser Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Arg Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 5

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2b light chain variable region

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Gly Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Ser Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 6

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> HVR1 heavy chain (HC) anti-FGFR2b

<400> 6

Thr Tyr Asn Val His

1 5

<210> 7

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> HVR2 HC anti-FGFR2b

<400> 7

Ser Ile Tyr Pro Asp Asn Gly Asp Thr Ser Tyr Asn Gln Asn Phe Lys

1 5 10 15

Gly

<210> 8

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> HVR3 HC anti-FGFR2b

<400> 8

Gly Asp Phe Ala Tyr

1 5

<210> 9

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> HVR1 light chain (LC) anti-FGFR2b

<400> 9

Lys Ala Ser Gln Gly Val Ser Asn Asp Val Ala

1 5 10

<210> 10

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> HVR2 LC anti-FGFR2b

<400> 10

Ser Ala Ser Tyr Arg Tyr Thr

1 5

<210> 11

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> HVR3 LC anti-FGFR2b

<400> 11

Gln Gln His Ser Thr Thr Pro Tyr Thr

1 5

<210> 12

<211> 444

<212> Protein

<213> Artificial sequence

<220>

<223> anti-FGFR2b heavy chain N297Q

<220>

<221>MISC_FEATURE

<222> (294)..(294)

<223> N297Q

<400> 12

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Thr Tyr

20 25 30

Asn Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Asp Asn Gly Asp Thr Ser Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Arg Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

165 170 175

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

180 185 190

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

195 200 205

Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 13

<211> 800

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> Mature human FGFR2-IIIc

<400> 13

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro

340 345 350

Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile Gly Val Phe Leu Ile Ala

355 360 365

Cys Met Val Val Thr Val Ile Leu Cys Arg Met Lys Asn Thr Thr Lys

370 375 380

Lys Pro Asp Phe Ser Ser Gln Pro Ala Val His Lys Leu Thr Lys Arg

385 390 395 400

Ile Pro Leu Arg Arg Gln Val Thr Val Ser Ala Glu Ser Ser Ser Ser

405 410 415

Met Asn Ser Asn Thr Pro Leu Val Arg Ile Thr Thr Arg Leu Ser Ser

420 425 430

Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser Glu Tyr Glu Leu Pro

435 440 445

Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys Leu Thr Leu Gly Lys

450 455 460

Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Val

465 470 475 480

Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val Thr Val Ala Val Lys

485 490 495

Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu Ser Asp Leu Val Ser

500 505 510

Glu Met Glu Met Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn

515 520 525

Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro Leu Tyr Val Ile Val Glu

530 535 540

Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg Pro

545 550 555 560

Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu Glu Gln

565 570 575

Met Thr Phe Lys Asp Leu Val Ser Cys Thr Tyr Gln Leu Ala Arg Gly

580 585 590

Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala

595 600 605

Arg Asn Val Leu Val Thr Glu Asn Asn Val Met Lys Ile Ala Asp Phe

610 615 620

Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys Thr Thr

625 630 635 640

Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp

645 650 655

Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Met

660 665 670

Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val

675 680 685

Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro

690 695 700

Ala Asn Cys Thr Asn Glu Leu Tyr Met Met Met Arg Asp Cys Trp His

705 710 715 720

Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu

725 730 735

Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu Tyr Leu Asp Leu Ser

740 745 750

Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr Pro Asp Thr Arg Ser Ser

755 760 765

Cys Ser Ser Gly Asp Asp Ser Val Phe Ser Pro Asp Pro Met Pro Tyr

770 775 780

Glu Pro Cys Leu Pro Gln Tyr Pro His Ile Asn Gly Ser Val Lys Thr

785 790 795 800

<210> 14

<211> 356

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> FGFR2 VKD

<400> 14

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro

340 345 350

Asp Tyr Leu Glu

355

<210> 15

<211> 353

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> EVA FGFR2 delta-3

<400> 15

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro

340 345 350

Asp

<210> 16

<211> 352

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> EVA FGFR2 delta-4

<400> 16

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro

340 345 350

<210> 17

<211> 351

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> EVA FGFR2 delta-5

<400> 17

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser

340 345 350

<210> 18

<211> 348

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> VKD FGFR2 delta-8

<400> 18

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile

340 345

<210> 19

<211> 347

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> VKD FGFR2 delta-9

<400> 19

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu

340 345

<210> 20

<211> 346

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> EVA FGFR2 delta-10

<400> 20

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys

340 345

<210> 21

<211> 342

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> VKD FGFR2 delta-14

<400> 21

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro

340

<210> 22

<211> 341

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> VKD FGFR2 delta-15

<400> 22

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala

340

<210> 23

<211> 340

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> VKD FGFR2 delta-16

<400> 23

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro

340

<210> 24

<211> 339

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> VKD FGFR2 delta-17

<400> 24

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu

<210> 25

<211> 232

<212> Protein

<213> Artificial sequence

<220>

<223> Fc C237S

<400> 25

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 26

<211> 228

<212> Protein

<213> Artificial sequence

<220>

<223> Fc

<400> 26

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

65 70 75 80

Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro

100 105 110

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Pro Gly Lys

225

<210> 27

<211> 229

<212> Protein

<213> Artificial sequence

<220>

<223> Fc

<400> 27

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 28

<211> 8

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> FGFR2(111-118)

<400> 28

Asp Asp Glu Asp Asp Thr Asp Gly

1 5

<210> 29

<211> 8

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> FGFR1(105-112)

<400> 29

Glu Asp Asp Asp Asp Asp Asp Asp

1 5

<210> 30

<211> 356

<212> Protein

<213> Artificial sequence

<220>

<223> ECD FGFR2 with R2(111-118) replaced by R1(105-112)

<400> 30

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Glu Asp

100 105 110

Asp Asp Asp Asp Asp Asp Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro

340 345 350

Asp Tyr Leu Glu

355

<210> 31

<211> 588

<212> Protein

<213> Artificial sequence

<220>

<223> ECD FGFR2 with R2(111-118) replaced by R1(105-112) + Fc

<400> 31

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Glu Asp

100 105 110

Asp Asp Asp Asp Asp Asp Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro

340 345 350

Asp Tyr Leu Glu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro

355 360 365

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

370 375 380

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

385 390 395 400

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

405 410 415

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

420 425 430

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

435 440 445

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

450 455 460

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

465 470 475 480

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

485 490 495

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

500 505 510

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

515 520 525

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

530 535 540

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

545 550 555 560

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

565 570 575

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

580 585

<210> 32

<211> 587

<212> Protein

<213> Artificial sequence

<220>

<223> FGFR2 delta-3 VCD + GS linker + Fc

<400> 32

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Asp Asp

100 105 110

Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro

340 345 350

Asp Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

355 360 365

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

370 375 380

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

385 390 395 400

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

405 410 415

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

420 425 430

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

435 440 445

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

450 455 460

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

465 470 475 480

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

485 490 495

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

500 505 510

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

515 520 525

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

530 535 540

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

545 550 555 560

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

565 570 575

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

580 585

<210> 33

<211> 353

<212> Protein

<213> Artificial sequence

<220>

<223> FGFR2 delta-3 VKD with R2(111-118) replaced by R1(105-112)

<400> 33

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Glu Asp

100 105 110

Asp Asp Asp Asp Asp Asp Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro

340 345 350

Asp

<210> 34

<211> 587

<212> Protein

<213> Artificial sequence

<220>

<223> FGFR2 delta-3 VKD with R2(111-118) replaced by R1(105-112) + GS

linker + Fc (also called FGFR2ECD(FGFR2(111-118):FGFR1(105-112):

delta3)-GS linker-Fc and R2(111-118):R1(105-112))

<400> 34

Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr Leu Glu Pro Glu Glu

1 5 10 15

Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu Val Tyr Val Ala Ala

20 25 30

Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu Lys Asp Ala Ala Val

35 40 45

Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly Pro Asn Asn Arg Thr

50 55 60

Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp

65 70 75 80

Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr Val Asp Ser Glu Thr

85 90 95

Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile Ser Ser Gly Glu Asp

100 105 110

Asp Asp Asp Asp Asp Asp Ala Glu Asp Phe Val Ser Glu Asn Ser Asn

115 120 125

Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg

130 135 140

Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala

145 150 155 160

Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu

165 170 175

Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His

180 185 190

Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr

195 200 205

Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His

210 215 220

Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly

225 230 235 240

Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val

245 250 255

Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His

260 265 270

Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu

275 280 285

Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu

290 295 300

Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr

305 310 315 320

Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu

325 330 335

Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro

340 345 350

Asp Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

355 360 365

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

370 375 380

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

385 390 395 400

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

405 410 415

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

420 425 430

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

435 440 445

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

450 455 460

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

465 470 475 480

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

485 490 495

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

500 505 510

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

515 520 525

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

530 535 540

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

545 550 555 560

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

565 570 575

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

580 585

<210> 35

<211> 288

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> human PD-1 precursor (with signal sequence)

<400> 35

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys

180 185 190

Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro

195 200 205

Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly

210 215 220

Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro

225 230 235 240

Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly

245 250 255

Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg

260 265 270

Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

<210> 36

<211> 268

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> human PD-1 (mature, no signal sequence)

<400> 36

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly Leu Leu Gly Ser

145 150 155 160

Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys Ser Arg Ala Ala

165 170 175

Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro Leu Lys Glu Asp

180 185 190

Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly Glu Leu Asp Phe

195 200 205

Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro Cys Val Pro Glu

210 215 220

Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly Met Gly Thr Ser

225 230 235 240

Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg Ser Ala Gln Pro

245 250 255

Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

260 265

<210> 37

<211> 290

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> human PD-L1 precursor (with signal sequence)

<400> 37

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr

245 250 255

Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys

260 265 270

Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu

275 280 285

GluThr

290

<210> 38

<211> 272

<212> Protein

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> Human PD-L1 (mature, no signal sequence)

<400> 38

Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser

1 5 10 15

Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu

20 25 30

Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln

35 40 45

Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg

50 55 60

Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala

65 70 75 80

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys

85 90 95

Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val

100 105 110

Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro

115 120 125

Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys

130 135 140

Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys

145 150 155 160

Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr

165 170 175

Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr

180 185 190

Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile

195 200 205

Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His Leu Val

210 215 220

Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr Phe Ile

225 230 235 240

Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys Gly Ile

245 250 255

Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu Glu Thr

260 265 270

<210> 39

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2 heavy chain variable region Gal-FR22

<400> 39

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Asp Phe Arg

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Ile Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Pro Asp Asp Thr Ile Ala Tyr Cys Ala Asn

85 90 95

Phe Tyr Tyr Gly Tyr Asp Asp Tyr Val Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 40

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2 Gal-FR22 heavy chain CDR1

<400> 40

Ser Phe Gly Val His

1 5

<210> 41

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2 Gal-FR22 heavy chain CDR2

<400> 41

Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Asp Phe Arg Ser

1 5 10 15

<210> 42

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2 Gal-FR22 heavy chain CDR3

<400> 42

Phe Tyr Tyr Gly Tyr Asp Asp Tyr Val Met Asp Tyr

1 5 10

<210> 43

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2 Gal-FR22 light chain variable region

<400> 43

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Asn Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Ser Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Asp Leu Tyr

85 90 95

Met Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys

100 105

<210> 44

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2 Gal-FR22 light chain CDR1

<400> 44

Lys Ala Ser Gln Asp Ile Lys Asn Tyr Ile Ala

1 5 10

<210> 45

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2 Gal-FR22 light chain CDR2

<400> 45

Tyr Thr Ser Thr Leu Gln Pro

1 5

<210> 46

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Anti-FGFR2 Gal-FR22 light chain CDR3

<400> 46

Leu Gln Tyr Asp Asp Leu Tyr Met

1 5

<---

Claims (60)

1. A method of treating gastric cancer in a subject, comprising administering to the subject an afucosylated antibody to fibroblast growth factor receptor 2 IIIb (FGFR2-IIIb) and nivolumab or pembrolizumab, wherein the antibody to FGFR2-IIIb comprises heavy chain and light chain variable regions, wherein the heavy chain variable region comprises: (i) hypervariable region 1 (HVR-H1) comprising the amino acid sequence of SEQ ID NO: 6; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the variable region of the light chain contains: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. 2. The method of claim 1, comprising administering nivolumab to the subject. 3. The method of claim 1, comprising administering pembrolizumab to the subject. 4. The method according to claim 1, wherein the antibody to FGFR2-IIIb lacks fucose at position Asn297. 5. The method according to claim 1, wherein the variable domain of the heavy chain of the antibody to FGFR2-IIIb comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4. 6. The method according to claim 5, wherein the variable domain of the light chain of the antibody to FGFR2-IIIb comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5. 7. The method according to claim 1, wherein the variable domain of the heavy chain of the antibody to FGFR2-IIIb comprises the amino acid sequence of SEQ ID NO: 4. 8. The method according to claim 7, wherein the variable domain of the light chain of the antibody to FGFR2-IIIb comprises the amino acid sequence of SEQ ID NO: 5. 9. The method according to claim 1, wherein the heavy chain of the antibody to FGFR2-IIIb comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2. 10. The method according to claim 9, wherein the light chain of the antibody to FGFR2-IIIb comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3. 11. The method according to claim 1, wherein the heavy chain of the antibody to FGFR2-IIIb comprises the amino acid sequence of SEQ ID NO: 2. 12. The method according to claim 11, wherein the light chain of the antibody to FGFR2-IIIb comprises the amino acid sequence of SEQ ID NO: 3. 13. The method according to any one of claims 1-12, wherein the antibody to FGFR2-IIIb and nivolumab or pembrolizumab are administered simultaneously or sequentially. 14. The method of claim 13, wherein the subject has received a full course of nivolumab or pembrolizumab therapy prior to administration of the FGFR2-IIIb antibody. 15. The method according to any one of claims 1-12, wherein the antibody to FGFR2-IIIb is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 mg/kg or in a range limited by any two of these mg/kg doses, such as 6-10 mg/kg, 10-15 mg/kg or 6-15 mg/kg. 16. The method of claim 15, wherein nivolumab or pembrolizumab is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5 or 10 mg/kg. 17. The method according to any one of claims 1-12, wherein the antibody to FGFR2-IIIb and nivolumab or pembrolizumab are administered once every 1, 2, 3, 4 or 5 weeks. 18. The method according to any one of claims 1-12, wherein the gastric cancer is recurrent or progressive after surgery, chemotherapy, radiation therapy, or a combination thereof. 19. The method of any one of claims 1-12, wherein (a) the gastric cancer has previously been determined to overexpress FGFR2IIIb either with or without amplification of the FGFR2 gene, or (b) the method comprises the additional step of determining whether the cancer overexpresses FGFR2IIIb, and optionally also comprises the additional step of determining whether the FGFR2 gene is amplified in the tumor cells. 20. The method according to claim 19, wherein the overexpression of FGFR2IIIb is determined using immunohistochemistry (IHC). 21. The method according to claim 19, wherein the amplification of the FGFR2 gene is determined by obtaining the ratio of FGFR2 to the centromere of chromosome 10 (CEN10) using fluorescent in situ hybridization (FISH method), wherein the FGFR2 gene is considered to be amplified if the ratio of FGFR2/CEN10 determined by the FISH method is greater than or equal to 2. 22. The method according to item 20, where: a) gastric cancer has an IHC signal of 3+ in at least 10% of tumor cells; b) gastric cancer has an IHC signal of 3+ in at least 10% of tumor cells and the FGFR2 gene is amplified; or c) gastric cancer has an IHC signal of 3+ in at least 10% of tumor cells and the FGFR2 gene is not amplified. 23. A method of treating gastric cancer in a subject, comprising administering to the subject an antibody to FGFR2-IIIb comprising the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 3, and nivolumab. 24. The method according to claim 23, wherein the antibody to FGFR2-IIIb and nivolumab are administered simultaneously or sequentially. 25. The method of claim 24, wherein the subject has received a full course of nivolumab therapy prior to administration of the FGFR2-IIIb antibody. 26. The method of claim 23, wherein the antibody to FGFR2-IIIb is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 mg/kg or in a range limited by any two of these mg/kg doses, such as 6-10 mg/kg, 10-15 mg/kg or 6-15 mg/kg. 27. The method of claim 26, wherein nivolumab is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, or 10 mg/kg. 28. The method according to claim 23, wherein the antibody to FGFR2-IIIb and nivolumab are administered once every 1, 2, 3, 4, or 5 weeks. 29. The method according to claim 23, wherein the gastric cancer is recurrent or progressive after surgery, chemotherapy, radiation therapy, or a combination thereof. 30. The method of claim 23, wherein (a) the gastric cancer has previously been determined to overexpress FGFR2IIIb either with or without amplification of the FGFR2 gene, or (b) the method includes the additional step of determining whether the cancer overexpresses FGFR2IIIb, and optionally also includes the additional step of determining whether the FGFR2 gene is amplified in the tumor cells. 31. The method according to claim 30, wherein the overexpression of FGFR2IIIb is determined using immunohistochemistry (IHC). 32. The method according to claim 30, wherein the amplification of the FGFR2 gene is determined by obtaining the ratio of FGFR2 to the centromere of chromosome 10 (CEN10) using fluorescent in situ hybridization (FISH method), wherein the FGFR2 gene is considered to be amplified if the ratio of FGFR2/CEN10 determined by the FISH method is greater than or equal to 2. 33. The method according to item 31, where: a) gastric cancer has an IHC signal of 3+ in at least 10% of tumor cells; b) gastric cancer has an IHC signal of 3+ in at least 10% of tumor cells and the FGFR2 gene is amplified; or c) gastric cancer has an IHC signal of 3+ in at least 10% of tumor cells and the FGFR2 gene is not amplified. 34. A method of treating gastric cancer in a subject, comprising administering to the subject an antibody to FGFR2-IIIb comprising the heavy chain amino acid sequence of SEQ ID NO: 2 and the light chain amino acid sequence of SEQ ID NO: 3, and pembrolizumab. 35. The method according to claim 34, wherein the antibody to FGFR2-IIIb and pembrolizumab are administered simultaneously or sequentially. 36. The method of claim 35, wherein the subject has received a full course of pembrolizumab therapy prior to administration of the FGFR2-IIIb antibody. 37. The method of claim 34, wherein the antibody to FGFR2-IIIb is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 mg/kg or in a range limited by any two of these mg/kg doses, such as 6-10 mg/kg, 10-15 mg/kg or 6-15 mg/kg. 38. The method of claim 37, wherein pembrolizumab is administered at a dose of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, or 10 mg/kg. 39. The method according to claim 34, wherein the antibody to FGFR2-IIIb and pembrolizumab are administered once every 1, 2, 3, 4 or 5 weeks. 40. The method according to claim 34, wherein the gastric cancer is recurrent or progressive after surgery, chemotherapy, radiation therapy, or a combination thereof. 41. The method of claim 34, wherein (a) the gastric cancer has previously been determined to overexpress FGFR2IIIb, either with or without amplification of the FGFR2 gene, or (b) the method comprises the additional step of determining whether the cancer overexpresses FGFR2IIIb, and optionally also comprises the additional step of determining whether the FGFR2 gene is amplified in the tumor cells. 42. The method according to claim 41, wherein the overexpression of FGFR2IIIb is determined using immunohistochemistry (IHC). 43. The method according to claim 41, wherein the amplification of the FGFR2 gene is determined by obtaining the ratio of FGFR2 to the centromere of chromosome 10 (CEN10) using fluorescent in situ hybridization (FISH method), wherein the FGFR2 gene is considered to be amplified if the ratio of FGFR2/CEN10 determined by the FISH method is greater than or equal to 2. 44. The method according to item 43, where: a) gastric cancer has an IHC signal of 3+ in at least 10% of tumor cells; b) gastric cancer has an IHC signal of 3+ in at least 10% of tumor cells and the FGFR2 gene is amplified; or c) gastric cancer has an IHC signal of 3+ in at least 10% of tumor cells and the FGFR2 gene is not amplified.

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