Anti-FGFR2 antibodies and methods of use thereof

Abstract

Provided herein are monoclonal antibodies and antigen-binding fragments thereof that bind to fibroblast growth factor receptor 2 (FGFR2), and methods of using same. Also included are antibody-drug conjugates (ADCs) comprising anti-FGFR2 antibodies or antigen-binding fragments thereof linked to cytotoxic agents, radionuclides, or other moieties, and methods of treatment using same.

Description

[Technical Field]

[0001] The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to fibroblast growth factor receptor 2 (FGFR2), as well as antibody-drug conjugates of such antibodies and methods of use thereof.

[0002] Sequence Listing A public copy of the Sequence Listing has been submitted electronically via EFS-Web contemporaneously herewith as an ASCII-formatted Sequence Listing with the filename "10680WO01_Sequence_Listing_ST25.TXT," created on October 21, 2021, and approximately 72 kilobytes in size. The Sequence Listing contained in this ASCII-formatted document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] The fibroblast growth factor (FGF) receptor tyrosine kinase (RTK) family, consisting of fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3, and FGFR4, encompasses high-affinity receptors for up to 18 different FGF ligands. The receptors are transmembrane tyrosine kinases, and FGFR signaling drives downstream pathways, including the mitogen-activated protein kinase (MAPK) and AKT pathways, which are important for cell proliferation, differentiation, survival, and migration. Binding of FGF ligands to the receptor induces dimerization of the FGF:FGFR complex. Dimerization leads to kinase activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor, and activation of downstream signaling pathways including the phosphoinositide 3-kinase (PI3K)-AKT and MAPK-extracellular signal-regulated kinase (ERK) pathways. Alternative splicing of the IgIII loop in FGFR1-3 generates the FGFRIIIb or FGFRIIIc isoforms.

[0004] FGFR2 amplification has been reported in various cancers. It affects signal transduction without altering the receptor's intrinsic kinase activity. Evidence suggests that signaling through overexpressed FGFR2 is ligand-independent and sensitive to FGFR inhibitors (Lorenzi, 1997, "Ligand-independent activation of fibroblast growth factor receptor-2 by carboxyl terminal alterations," Oncogene; Takeda, 1999, "AZD2171 shows potent antitumor activity against gastric cancer over-expressing fibroblast growth factor receptor 2 / keratinocyte growth factor receptor," Clin Cancer Research; Cha, 2009, "Aberrant receptor internalization and enhanced FGFR2-dependent signaling contribute to the transforming activity of the fibroblast growth factor receptor 2 IIIb C3 isoform," J. Biol. Chem.).

[0005] Furthermore, FGFR2 protein is overexpressed in approximately 3% of breast cancers, including triple-negative breast cancer, and approximately 10% of gastric / esophageal cancers. FGFR2 overexpression is also seen in other cancers, including colon cancer, hepatocellular carcinoma, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, glioma, and head and neck cancer. Furthermore, mutations in the FGFR2 gene have been reported in approximately 12% of endometrial cancers. FGFR2 overexpression is associated with poor survival in gastric cancer patients.

[0006] There is considerable interest in FGFR2 as a therapeutic target across tumor types. In gastric cancer patients with FGFR2 amplification, therapeutic intervention using small molecule FGFR inhibitors such as TAS-120 and AZD4547 has been suggested.

[0007] Both preclinical and clinical studies have demonstrated that FGFR-amplified tumors are sensitive to FGFR inhibition and therefore amenable to therapeutic targeting. Treatment of FGFR2-positive tumors with anti-FGFR2 therapies, such as bemarituzumab (also known as HGS1036, HGS1036, FP-1039, FPA144, GSK3052230), has been reported to result in improved early survival and advanced disease. (Gemo et al. (2014) “FPA144:A Therapeutic Antibody for Treating Patients with Gastric Cancers Bearing FGFR2 Gene Amplification”AACR Abstract ID 5446, Powers et al. (2016) “FPA144,A Therapeutic Monoclonal Antibody Targeting the FGFR2b Receptor,Promotes Antibody Dependent Cell-Mediated Cytotoxicity and Stimulates Sensitivity to PD-1 in the 4T1 Syngeneic Tumor Model”AACR Abstract ID 1407, and Lee et al. (2016) “Antitumor Activity and Safety of FPA144, an ADCC-Enhanced, FGFR2b Isoform-Selective Monoclonal Antibody, in Patients with FGFR2b+Gastric Cancer and Advanced Solid Tumors” ASCO Abstract ID 2502).

[0008] Some FGFR2 antibodies have been conjugated to cytotoxic payloads, including thorium-227 (apurtumab, also known as BAY1179470, BAY2304058) and auristatins (apurtumab ixadotin, also known as BAY1187982, BAY1179470 ADC). Aprutumab-TTC (an anti-FGFR2 antibody, a chelator moiety covalently attached to the antibody, and the alpha-particle-emitting radionuclide thorium-227) has reportedly inhibited tumor growth in several xenograft models. (Wickstroem et al. (2019) “Preclinical Combination Studies of an FGFR2 Targeted Thorium Conjugate and the ATR Inhibitor Bay 1895344” Int J Radiat Oncol Biol Phys 105(2):410-422; Wittemer-Rump et al. (2014) “Pharmacokinetic and Pharmacodynamic (PK / PD) Modeling of Preclinical Data of a Novel Anti-Fibroblast Growth Factor Receptor 2 (FGFR2) Antibody (BAY 1179470) to Guide Dosing in Phase 1” AACR Abstract ID 672.) In clinical trials, aprutumab ixodotin was found to be poorly tolerated, with the minimum tolerated dose below the preclinical estimated therapeutic threshold, leading to early termination of the study.(Sommer et al. (2016) “Preclinical Efficacy of the Auristatin-Based Antibody-Drug Conjugate BAY 1187982 for the Treatment of FGFR2-Positive Solid Tumors” Cancer Res 76(21):6331-6339, Sommer et al. (2014) “FGFR2-ADC Potently and Selectively Inhibits Growth of Gastric and Breast Cancer Xenograft Models” AACR Abstract ID 4491).

[0009] There remains a significant unmet medical need for improved anti-cancer drugs that are effective against FGFR2-expressing cancers. Summary of the Invention [Means for solving the problem]

[0010] Provided herein are antibodies and antigen-binding fragments thereof that bind to the human FGF receptor 2 protein (FGFR2). The antibodies are particularly useful for targeting tumor cells that express FGFR2. The anti-FGFR2 antibodies and antigen-binding portions thereof may be used alone in unmodified form or may be included as part of an antibody-drug conjugate.

[0011] Other embodiments will become apparent from consideration of the detailed description that follows. [Brief explanation of the drawings]

[0012] [Figure 1A] Figure 1A illustrates a monoclonal antibody with a click moiety spacer, a cathepsin B cleavable linker, and a tubulysin payload. Figure 1B illustrates the process for site-specific conjugation on an antibody. [Figure 1B] Same as above. [Figure 2]1 provides SEC-HPLC analysis of anti-FGFR2 antibody-Tubulysin conjugates. [Figure 3] 1 shows the changes in tumor volume and body weight over time in human gastric cancer xenografts upon treatment with different doses of an anti-FGFR2 antibody-Tubulysin conjugate. [Figure 4] 1 shows the changes in tumor volume and body weight over time in mice bearing human gastric cancer xenografts upon treatment with different doses of an anti-FGFR2 antibody-maytansinoid conjugate. [Figure 5] 1 shows the changes in tumor volume and body weight over time in mice bearing human gastric cancer PDX GA0033 tumors following treatment with different doses of an anti-FGFR2 antibody-tubulysin conjugate and an anti-FGFR2 antibody-maytansinoid conjugate. On days 0 and 7, the mice were administered the respective ADCs or a control antibody. [Figure 6] 1 shows the changes in tumor volume and body weight over time in mice bearing SNU-16 human gastric cancer xenografts upon treatment with different doses of an anti-FGFR2 antibody-camptothecin analog conjugate (DAR8). On day 0, mice were administered each conjugate and dose. [Figure 7] 1 shows the changes in tumor volume and body weight over time in mice bearing SNU-16 human gastric cancer xenografts following treatment with different doses of an anti-FGFR2 antibody-camptothecin analog conjugate (DAR4). Mice were administered each dose on days 0 and 7. [Figure 8] 1 illustrates the basic component structures of non-limiting exemplary azidoamine linkers. [Figure 9] 1 illustrates the basic component structures of exemplary, non-limiting branched alkyl azidoamine linkers. [Figure 10] Figure 1 shows the changes in tumor volume and body weight over time in mice bearing GA1224 human PDX tumors treated with different doses of the anti-FGFR2 antibody camptothecin ADC (DAR4). Mice were administered each dose on days 0 and 7. [Figure 11] 1 shows the changes in tumor volume and body weight over time in mice bearing SNU-16 human gastric cancer xenografts (FGFR2b positive) following treatment with an anti-FGFR2 antibody camptothecin ADC (DAR4) and an anti-FGFR2 antibody maytansinoid ADC. [Figure 12] 1 shows the changes in tumor volume and body weight over time in mice bearing SNU-5 human gastric cancer xenografts (FGFR2b negative) following treatment with an anti-FGFR2 antibody camptothecin ADC (DAR4) and an anti-FGFR2 antibody maytansinoid ADC. [Figure 13] Figure 1 shows changes in tumor volume and body weight in mice co-implanted with SNU-16 (FGFR2b-positive) and SNU-5 (FGFR2b-negative) human gastric cancer xenografts at a 2:1 ratio following treatment with an anti-FGFR2 antibody camptothecin ADC (DAR4) and an anti-FGFR2 antibody maytansinoid ADC. DETAILED DESCRIPTION OF THE INVENTION

[0013] Before describing the present invention, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. As used herein, the term "about," when used in connection with a specific recited numerical value, means that the value may vary by 1% or less from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0015] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are now described. All patents, patent applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0016] FGFR2 protein As used herein, terms such as "FGFR2" refer to a tyrosine protein kinase that functions as a cell surface receptor for fibroblast growth factors. This protein is also known as keratinocyte growth factor receptor and CD332. FGFR2b may refer to the amino acid sequence set forth in SEQ ID NO: 55 and / or the amino acid sequence set forth in NCBI accession number NP_075259.4, and is encoded by FGFR2 located on chromosome 10.

[0017] The epithelial splicing regulatory protein 1 (ESRP1) isoform is involved in FGFR2 splicing and the resulting expression of the epithelial FGFR2b isoform.

[0018] FGFR2 is a tyrosine protein kinase that acts as a cell surface receptor for fibroblast growth factors and plays an essential role in regulating cell proliferation, differentiation, migration, and apoptosis, as well as embryonic development. Activation of FGFR2 leads to the phosphorylation of PLCG1, FRS2, and PAK4, and the activation of several signaling cascades. PLCG1 activation leads to the production of the cell signaling molecules diacylglycerol and inositol 1,4,5-triphosphate. Phosphorylation of FRS2 triggers the recruitment of GRB2, GAB1, PIK3R1, and SOS1, mediating the activation of the RAS, MAPK1 / ERK2, MAPK3 / ERK1, and MAP kinase signaling pathways, as well as the AKT1 signaling pathway. FGFR2 signaling is downregulated by ubiquitination, internalization, and degradation. Mutations that lead to constitutive kinase activation or impair normal FGFR2 maturation, internalization, and degradation lead to aberrant signaling. Similarly, overexpressed FGFR2 promotes the activation of STAT1.

[0019] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless expressly specified as being from a non-human species. Thus, the term "FGFR2" refers to human FGFR2, unless specified as being from a non-human species, e.g., "mouse FGFR2," "monkey FGFR2," etc.

[0020] As used herein, the phrase "cell surface-expressed FGFR2" refers to one or more FGFR2 proteins or extracellular domains thereof that are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the FGFR2 protein is exposed to the extracellular side of the cell membrane and accessible to the antigen-binding portion of an antibody. "Cell surface-expressed FGFR2" may include or consist of an FGFR2 protein expressed on the surface of a cell that normally expresses FGFR2 protein. Alternatively, "cell surface-expressed FGFR2" may include or consist of an FGFR2 protein expressed on the surface of a cell that does not normally express human FGFR2 on its surface but has been artificially engineered to express FGFR2 on its surface.

[0021] Anti-FGFR2 antibodies and antigen-binding fragments thereof As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex that comprises at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., FGFR2). The term "antibody" includes immunoglobulin molecules that comprise four polypeptide chains, two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules consisting of two HCs and two LCs, four polypeptide chains interconnected by disulfide bonds. Each HC comprises a heavy chain variable region (HCVR or VCR) and a heavy chain variable region (VCR) that are connected to each other by disulfide bonds. H The heavy chain constant region is made up of three domains, C H1 , C H2 , and C H3 Each LC comprises a light chain variable region (herein referred to as LCVR or V L The light chain constant region comprises one domain (C L1 ) included. V H Area and V LThe regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with regions that are relatively conserved called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of an anti-FGFR2 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0022] Similarly, the term "antibody" includes immunoglobulin molecules comprising eight polypeptide chains, four HC and four LC, interconnected by disulfide bonds, i.e., bispecific antibodies. The term "antibody" also includes immunoglobulin molecules consisting of eight polypeptide chains, four HC and four LC, interconnected by disulfide bonds.

[0023] The term "antibody" further includes functionalized immunoglobulin molecules comprising at least one HC, wherein the HC comprises an azido-PEG3-amine. In some embodiments, the azido-PEG3-amine is located at the Q295 position on the antibody HC. In some embodiments, the azido-PEG3-amine is located at the Q297 position on the antibody. In some embodiments, an antibody has two HCs functionalized with azido-PEG3-amines located at both Q295 positions of the HC. In some embodiments, an antibody has two HCs functionalized with azido-PEG3-amines located at both Q297 positions of the HC. In some embodiments, an antibody has two HCs functionalized with azido-PEG3-amines located at both Q295 positions and both Q297 positions of the HC. The Q297 position in the HC is obtained by modifying N297 to Q297, also referred to herein as an N297Q modification.

[0024] According to one embodiment, an anti-FGFR2 antibody is provided. Exemplary anti-FGFR2 antibodies according to this embodiment are listed in Tables 3 and 4 herein. Table 3 lists the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), heavy chain (HC), and light chain (LC) of the anti-FGFR2 antibody or antigen-binding fragment thereof disclosed herein. Table 4 lists the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HC, and LC of exemplary antibodies.

[0025] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a CDR within the HCVR that comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 3, or a substantially similar sequence thereof that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0026] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0027] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a CDR within the LCVR that comprises an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 3, or a substantially similar sequence thereof that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0028] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0029] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a CDR within an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 3 paired with any of the LCVR amino acid sequences listed in Table 3. Also provided herein is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising an HCVR / LCVR amino acid sequence pair comprising any of the HCVR amino acid sequences listed in Table 3 paired with any of the LCVR amino acid sequences listed in Table 3. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 28, and 40 / 44.

[0030] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0031] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0032] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0033] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0034] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0035] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0036] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising an HCDR1 and LCDR1 amino acid sequence pair (HCDR1 / LCDR1) comprising any of the HCDR1 amino acid sequences listed in Table 3 paired with any of the LCDR1 amino acid sequences listed in Table 3. According to certain embodiments, the antibody or antigen-binding fragment thereof comprises at least the HCDR1 / LCDR1 amino acid sequence pair contained in any of the exemplary anti-FGFR2 antibodies listed in Table 3. In certain embodiments, the HCDR1 / LCDR1 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 4 / 12, 24 / 30, and 24 / 46.

[0037] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising an HCDR2 and LCDR2 amino acid sequence pair (HCDR2 / LCDR2) comprising any of the HCDR2 amino acid sequences listed in Table 3 paired with any of the LCDR2 amino acid sequences listed in Table 3. According to certain embodiments, the antibody or antigen-binding fragment thereof comprises at least the HCDR2 / LCDR2 amino acid sequence pair contained in any of the exemplary anti-FGFR2 antibodies listed in Table 3. In certain embodiments, the HCDR2 / LCDR2 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 6 / 14 and 6 / 32.

[0038] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 3 paired with any of the LCDR3 amino acid sequences listed in Table 3. According to certain embodiments, the antibody or antigen-binding fragment thereof comprises at least an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-FGFR2 antibodies listed in Table 3. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 8 / 16, 26 / 34, and 42 / 34.

[0039] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to FGFR2, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-FGFR2 antibodies listed in Table 3. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 24-6-26-30-32-34, and 24-6-42-46-32-34.

[0040] In related embodiments, an antibody or antigen-binding fragment thereof that specifically binds to FGFR2 comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-FGFR2 antibodies listed in Table 3. For example, an anti-FGFR2 antibody or antigen-binding fragment thereof can comprise a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 28, and 40 / 44.

[0041] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the particular HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0042] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a heavy chain (HC) comprising an amino acid sequence selected from any of the HC amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. For example, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising an amino acid sequence selected from any of the HC amino acid sequences listed in Table 3, but comprising an HC that comprises an N297Q modification or an equivalent modification. Exemplary, provided herein is an antibody or antigen-binding fragment thereof having a heavy chain that comprises an N297Q modification within an HC amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 36, and 49. In some aspects, the antibody or antigen-binding fragment thereof comprises an HC that comprises an N297Q modification within the HC amino acid sequence of SEQ ID NO: 18. In some aspects, the antibody or antigen-binding fragment thereof comprises an HC that comprises an N297Q modification within the HC amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody or antigen-binding fragment thereof comprises a HC that comprises a N297Q modification within the HC amino acid sequence of SEQ ID NO:49.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HC amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 53, and 54. Such HC sequences include those equivalent to the N297Q modification.

[0044] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising a light chain (LC) comprising an amino acid sequence selected from any of the LC amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0045] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2, comprising an HC and LC amino acid sequence pair (HC / LC) comprising any of the HC amino acid sequences listed in Table 3 paired with any of the LC amino acid sequences listed in Table 3. According to certain embodiments, the antibody comprises two HC / LC amino acid sequence pairs contained within any of the exemplary anti-FGFR2 antibodies listed in Table 3. In certain embodiments, the HC / LC amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 18 / 20, 36 / 38, and 49 / 51. In some aspects, the HC comprises the N297Q modification provided above. Thus, the HC / LC amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 52 / 20, 53 / 38, and 54 / 51.

[0046] Also provided herein are nucleic acid molecules encoding anti-FGFR2 antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 3, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0047] Also provided are nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 3, which in certain embodiments comprise a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0048] Also provided is a nucleic acid molecule encoding any of the HCDR1 amino acid sequences listed in Table 3, which in certain embodiments comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0049] Also provided are nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 3, which in certain embodiments comprise a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0050] Also provided are nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 3, which in certain embodiments comprise a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0051] Also provided is a nucleic acid molecule encoding any of the LCDR1 amino acid sequences listed in Table 3, which in certain embodiments comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0052] Also provided are nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 3, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0053] Also provided are nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 3, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0054] Also provided is a nucleic acid molecule encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti-FGFR2 antibodies listed in Table 3.

[0055] Also provided is a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary anti-FGFR2 antibodies listed in Table 3.

[0056] Also provided are nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Table 3, and the LCVR comprises the amino acid sequence of any of the LCVR amino acid sequences listed in Table 3. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same anti-FGFR2 antibody listed in Table 3.

[0057] Also provided are nucleic acid molecules that encode any of the HC amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. For example, the nucleic acid molecule can encode any of the HC amino acid sequences listed in Table 3, wherein the HC has an N297Q modification.

[0058] Also provided are nucleic acid molecules that encode any of the LC amino acid sequences listed in Table 3, or that encode a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0059] Also provided herein are recombinant expression vectors capable of expressing polypeptides comprising the heavy or light chain variable regions of anti-FGFR2 antibodies. For example, the recombinant vector comprises any of the nucleic acid molecules referred to herein, i.e., a nucleic acid molecule encoding any of the HCVR, LCVR, and / or CDR sequences as described in Table 3. Host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions that allow the production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced, are also within the scope of the present disclosure.

[0060] Provided herein are anti-FGFR2 antibodies or antigen-binding fragments thereof with modified glycosylation patterns. In some embodiments, modifications to remove undesired glycosylation sites or antibodies lacking fucose moieties present on the oligosaccharide chains may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be performed to alter complement-dependent cytotoxicity (CDC).

[0061] antigen-binding domain As used herein, the phrase "antigen-binding domain" refers to any peptide, polypeptide, nucleic acid molecule, scaffold molecule, peptide display molecule, or polypeptide-containing construct that can specifically bind to a particular antigen of interest (e.g., human FGFR2). Terms such as "specifically bind," as used herein, refer to an antigen-binding domain that binds specifically to a target antigen with a dissociation constant (K) of 500 pM or less. D ) and does not bind other unrelated antigens under typical test conditions. "Unrelated antigens" are proteins, peptides, or polypeptides that share less than 95% amino acid identity with each other.

[0062] Exemplary classes of antigen-binding domains that may be used in the context of the present disclosure include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising the ligand-binding portion of a receptor that specifically binds to a particular antigen, antigen-binding scaffolds (such as, for example, DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins [see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein]), and aptamers or portions thereof.

[0063] Methods for determining whether two molecules specifically bind to one another are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and similar methods. For example, as used in the context of the present specification, an antigen-binding domain has a K of less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM, as measured by a surface plasmon resonance assay. D The present invention also includes polypeptides that bind to specific antigens (e.g., target molecules [T] or internalization effector proteins [E]) or portions thereof, having the following structure:

[0064] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, in a BIAcore® system (GE Healthcare's Biacore Life Sciences division, Piscataway, NJ).

[0065] "K D The term "" as used herein refers to the equilibrium dissociation constant of a particular protein-protein interaction (e.g., an antibody-antigen interaction). D Values ​​are K values ​​measured at 25 °C by surface plasmon resonance assay. D Points to a value.

[0066] As indicated above, an "antigen-binding domain" may comprise or consist of an antibody or an antigen-binding fragment of an antibody. The term "antibody," as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a specific antigen (e.g., human FGFR2). The term "antibody" includes immunoglobulin molecules comprising two polypeptide chains, one heavy (H) chain and one light (L) chain, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). The heavy chain comprises a heavy chain variable region (referred to herein as HCVR or VL). H The heavy chain constant region is made up of three domains, C H1 , C H2 , and C H3 The light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L 1) V H Area and V L The regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with regions that are relatively conserved called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments, the FRs (or antigen-binding portions thereof) of the antibodies provided herein may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0067] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any natural, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from intact antibody molecules using any suitable standard method, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding antibody variable regions and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0068] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0069] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V associated with domain H In an antigen-binding fragment having a domain, V H Domain and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region may be a dimer, with V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0070] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a VH -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L、 (viii)V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments may be homodimers or heterodimers (or other multimers) of any of the above-listed variable and constant domain configurations, non-covalently linked to each other and / or to one or more monomeric V H Domain or V LIt may be included in non-covalent association with the domain (eg, by a disulfide bond).

[0071] The antibodies provided herein may comprise or consist of human antibodies and / or recombinant human antibodies, or fragments thereof. The term "human antibody," as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, and particularly in the CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0072] The antibodies or antigen-binding fragments thereof of the present disclosure may comprise or consist of recombinant human antibodies or antigen-binding fragments thereof. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more detail below), antibodies isolated from a recombinant combinatorial human antibody library (described in more detail below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), thereby increasing the V of the recombinant antibody. H Area and V L The amino acid sequence of the region is human germline V H Sequence and V L While the sequences are derived from and related to the sequences, they may not naturally occur in the human antibody germline repertoire in vivo.

[0073] The anti-FGFR2 antibodies or antigen-binding fragments thereof provided herein may be "isolated." As used herein, an "isolated anti-FGFR2 antibody" refers to an antibody or antigen-binding fragment thereof that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody or antigen-binding fragment thereof that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody was produced, is an "isolated anti-FGFR2 antibody" for purposes of this disclosure. An isolated anti-FGFR2 antibody or antigen-binding fragment thereof also includes molecules in situ within recombinant cells. An isolated antibody or antigen-binding fragment thereof is a molecule that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated anti-FGFR2 antibody may be substantially free of other cellular material and / or chemicals.

[0074] variant The anti-FGFR2 antibodies and antigen-binding fragments thereof disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibody or antigen-binding fragment thereof is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies or antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations").

[0075] Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate many antibodies or antigen-binding fragments thereof that contain one or more individual germline mutations or combinations thereof. H and / or VL All of the framework and / or CDR residues within a domain are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a different germline sequence from the germline sequence from which the antibody was originally derived).

[0076] Furthermore, antibodies or antigen-binding fragments thereof of the present disclosure may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies or antigen-binding fragments thereof containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies or antigen-binding fragments thereof obtained in this general manner are encompassed by the present disclosure.

[0077] Typically, antibodies or antigen-binding fragments provided herein that have been modified in some way retain the ability to specifically bind to FGFR2, e.g., retain at least 10% of their FGFR2 binding activity (compared to the parent antibody), when activity is expressed on a molar basis. In some embodiments, antibodies or antigen-binding fragments of the present disclosure retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the FGFR2 binding affinity of the parent antibody. It is also contemplated that antibodies or antigen-binding fragments of the present disclosure may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of an antibody) that do not substantially alter their biological activity.

[0078] A polynucleotide "variant" is a polynucleotide that is a matched variant of a reference nucleotide sequence described herein (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13) when the comparison is performed using the BLAST algorithm and the algorithm parameters are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expectation threshold: 10, word size: 28, maximum match in query range: 0, match / mismatch score: 1, -2, gap cost: linear). , 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 48, and 50).

[0079] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or GAP, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0080] "Variants" of polypeptides such as immunoglobulin chains (e.g., mAb1 V H , V L , HC, or LC, mAb2 V H , V L , HC, or LC, or mAb3 V H , VL , HC, or LC) when the comparison is performed using the BLAST algorithm and the algorithm parameters are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expectation threshold: 10, word size: 3, maximum match in query range: 0, BLOSUM62 matrix, gap cost: extent 11, extension 1, conditional composition score matrix adjustment), as described herein. It refers to a polypeptide comprising an amino acid sequence that is at least about 70 to 99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical to or similar to the amino acid sequence (e.g., SEQ ID NO: 2, 10, 18, 20, 22, 28, 36, 38, 40, 44, 49, or 51).

[0081] As applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned using predetermined gap weights, such as by the programs GAP or BESTFIT, share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity. Preferably, residue positions that are not identical differ only by conservative amino acid substitutions. "Conservatively modified variants" or "conservative substitutions" refer to variants in which there is a substitution of one or more amino acids in a polypeptide with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure and rigidity, etc.). Such changes can often be made without significantly impairing the biological activity of the antibody or fragment. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). Moreover, substitutions of structurally or functionally similar amino acids are unlikely to significantly destroy biological activity.

[0082] When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of homology may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. (See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331.) Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45. A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0083] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences provided herein to databases containing multiple sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0084] The present invention also includes anti-FGFR2 antibodies and antigen-binding fragments thereof comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. Exemplary variants included in this embodiment include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more substitutions, e.g., conservative substitutions. In some embodiments, the present disclosure includes anti-FGFR2 antibodies and antigen-binding fragments thereof having HCVR, LCVR, and / or CDR amino acid sequences that include, for example, 10 or less, 8 or less, 6 or less, 4 or less, 3 or less, 2, or 1 amino acid substitution relative to any of the HCVR, LCVR, and / or CDR amino acid sequences listed in Table 3 herein, wherein the modified antibodies and antigen-binding fragments thereof retain their binding activity to FGFR2.

[0085] Anti-FGFR2 antigen binding proteins, e.g., antibodies and antigen-binding fragments thereof, of the present disclosure, in one embodiment, comprise a heavy chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 95%, 99%) amino acid sequence identity to the amino acids set forth in SEQ ID NO: 2, 22, or 40, and / or a light chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 95%, 99%) amino acid sequence identity to the amino acids set forth in SEQ ID NO: 10, 28, or 44.

[0086] Furthermore, variant anti-FGFR2 antigen binding proteins may include polypeptides comprising an amino acid sequence described herein, except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations, such as, for example, missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. For example, the present disclosure includes antigen binding proteins comprising an immunoglobulin heavy chain variant comprising the amino acid sequence set forth in SEQ ID NO: 2, 22, or 40, but with one or more such mutations, and / or an immunoglobulin light chain variant comprising the amino acid sequence set forth in SEQ ID NO: 10, 28, or 44, but with one or more such mutations. In one embodiment of the present disclosure, the variant anti-FGFR2 antigen binding protein comprises an immunoglobulin heavy chain variant comprising HCDR1, HCDR2, and HCDR3, in which one or more (e.g., one, or two, or three) of such CDRs have one or more of such mutations (e.g., conservative substitutions), and / or an immunoglobulin light chain variant comprising LCDR1, LCDR2, and LCDR3, in which one or more (e.g., one, or two, or three) of such CDRs have one or more of such mutations (e.g., conservative substitutions).

[0087] The disclosure further provides variant anti-FGFR2 antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof, comprising one or more variant CDRs (e.g., any one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3) described herein that have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% sequence identity or sequence similarity to, e.g., SEQ ID NOs: 4, 6, 8, 12, 14, and / or 16, or 24, 6, 26, 30, 32, and / or 34, or 24, 6, 42, 46, 32, and / or 34.

[0088] Embodiments of the present disclosure also include the corresponding V specifically described herein. H , V Limmunoglobulin V, comprising an amino acid sequence having 70% or more (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) overall amino acid sequence identity or similarity to the amino acid sequence of immunoglobulin V, HC, or LC. H and V L or variant antigen binding proteins, such as anti-FGFR2 antibodies and antigen-binding fragments thereof, comprising the HC and LC, but wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 of such immunoglobulins are not variant and comprise the amino acid sequence set forth in SEQ ID NOs: 4, 6, 8, 12, 14, and / or 16, or 24, 6, 26, 30, 32, and / or 34, or 24, 6, 42, 46, 32, and / or 34, respectively. Thus, in such embodiments, the CDRs within the variant antigen binding protein are not themselves variant.

[0089] In some aspects, provided herein is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 90% identical thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 90% identical thereto.

[0090] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 with no more than two amino acid substitutions, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 with no more than two amino acid substitutions, HCDR3 comprises the amino acid sequence of SEQ ID NO: 8 or the amino acid sequence of SEQ ID NO: 8 with no more than two amino acid substitutions, LCDR1 comprises the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence of SEQ ID NO: 12 with no more than two amino acid substitutions, LCDR2 comprises the amino acid sequence of SEQ ID NO: 14 or the amino acid sequence of SEQ ID NO: 14 with no more than two amino acid substitutions, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence of SEQ ID NO: 16 with no more than two amino acid substitutions.

[0091] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, LCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0092] In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence at least 95% identical thereto, and an LCVR comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 95% identical thereto.

[0093] In some embodiments, the anti-FGFR2 antibody, or antigen-binding fragment thereof, comprises an HCVR comprising the amino acid sequence of SEQ ID NO:2 and an LCVR comprising the amino acid sequence of SEQ ID NO:10.

[0094] In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is at least 90% identical thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 90% identical thereto.

[0095] In some aspects, HCDR1 comprises the amino acid sequence of SEQ ID NO:24 or the amino acid sequence of SEQ ID NO:24 with no more than two amino acid substitutions, HCDR2 comprises the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:6 with no more than two amino acid substitutions, HCDR3 comprises the amino acid sequence of SEQ ID NO:26 or the amino acid sequence of SEQ ID NO:26 with no more than two amino acid substitutions, LCDR1 comprises the amino acid sequence of SEQ ID NO:30 or the amino acid sequence of SEQ ID NO:30 with no more than two amino acid substitutions, LCDR2 comprises the amino acid sequence of SEQ ID NO:32 or the amino acid sequence of SEQ ID NO:32 with no more than two amino acid substitutions, and LCDR3 comprises the amino acid sequence of SEQ ID NO:34 or the amino acid sequence of SEQ ID NO:34 with no more than two amino acid substitutions.

[0096] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, HCDR3 comprises the amino acid sequence of SEQ ID NO: 26, LCDR1 comprises the amino acid sequence of SEQ ID NO: 30, LCDR2 comprises the amino acid sequence of SEQ ID NO: 32, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 34.

[0097] In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 95% identical thereto, and an LCVR comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence at least 95% identical thereto.

[0098] In some embodiments, the anti-FGFR2 antibody, or antigen-binding fragment thereof, comprises an HCVR comprising the amino acid sequence of SEQ ID NO:22 and an LCVR comprising the amino acid sequence of SEQ ID NO:28.

[0099] Function-conservative variants of anti-FGFR2 antibodies and antigen-binding fragments thereof are also part of the present invention. Any variant of an anti-FGFR2 antibody and antigen-binding fragment thereof (discussed herein) may be a "function-conservative variant." Such function-conservative variants may also be characterized as conservatively modified variants in some instances. As used herein, "function-conservative variant" refers to a variant of an anti-FGFR2 antibody or antigen-binding fragment thereof in which one or more amino acid residues have been altered without significantly altering one or more functional properties of the antibody or fragment. In one embodiment of the present invention, a function-conservative variant of an anti-FGFR2 antibody or antigen-binding fragment thereof of the present disclosure comprises a variant amino acid sequence and exhibits one or more of the following functional properties: 2.5 x 10 as measured by surface plasmon resonance -8 K below M D Binding to FGFR2b at selectively binds to hFGFR2b over hFGFR2c, and / or · Have a half-life (t1 / 2) of greater than about 10 minutes as measured by surface plasmon resonance.

[0100] Anti-FGFR2 antibodies or antigen-binding fragments thereof containing Fc variants According to certain embodiments provided herein, there are provided anti-FGFR2 antibodies comprising an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides an Fc domain comprising a C H Area 2 or C HThe present invention includes anti-FGFR2 antibodies containing mutations in the three regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes, where the pH ranges from about 5.5 to about 6.0). Such mutations can extend the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D, or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F and / or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0101] For example, provided herein are anti-FGFR2 antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T and 256E (e.g., M252Y, S254T and T256E), 428L and 434S (e.g., M428L and N434S), and 434S (e.g., M428L and N434S). S), 257I and 311I (e.g., P257I and Q311I), 257I and 434H (e.g., P257I and N434H), 376V and 434H (e.g., D376V and N434H), 307A, 380A and 434A (e.g., T307A, E380A and N434A), and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations in antibody variable domains disclosed herein, are contemplated as being within the scope of the present disclosure.

[0102] The present disclosure provides chimeric heavy chain constant (C H ) region, and chimeric C H The region is composed of C H For example, the antibodies of the present disclosure may comprise a segment derived from a C region derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule. H C derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule in combination with part or all of the 3 domains H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibodies provided herein may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216-227 according to EU numbering) derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues at positions 228-236 according to EU numbering) derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric C described herein H Antibodies comprising the region, in certain embodiments, exhibit altered Fc effector functions without negatively impacting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Provisional Patent Application No. 61 / 759,578, filed February 1, 2013).

[0103] multispecific antibodies The antibodies provided herein may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide, or may contain antigen-binding domains specific for multiple target polypeptides. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.

[0104] Any of the multispecific antigen-binding molecules or variants thereof provided herein can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) that will be known to those skilled in the art.

[0105] In some embodiments, FGFR2-specific antibodies are produced in a bispecific format (bispecific), in which variable regions that bind to distinct domains of FGFR2 are linked to confer dual antigen specificity within a single binding molecule. Properly designed bispecifics can enhance overall FGFR2 inhibitory efficacy by increasing both specificity and binding affinity. Variable regions with specificity for individual domains, or variable regions that can bind to different regions within a single domain, are paired on a structural scaffold, allowing each region to simultaneously bind to distinct epitopes or different regions within a single domain. In one example, for bispecificity, a heavy chain variable region (V) from a binding molecule with specificity for one domain is combined with a heavy chain variable region (V) from a binding molecule with specificity for another domain. H ) with a light chain variable region (V) derived from a series of binding domains with specificity for the second domain. L ) and recombined it to the original V H and its V H Non-cognate V that can be paired without disturbing the original specificity of L Identify your partner. In this way, a single V L Segments (e.g., V L 1) Two different V H Domain (e.g., V H 1 and V H 2) to form two binding "arms" (V H 1-V L 1 and V H 2-V L 1) can be generated. L The use of segments reduces the complexity of the system, thereby simplifying and improving the efficiency of the cloning, expression, and purification processes used to generate bispecifics (see, e.g., USSN 13 / 022759 and US2010 / 0331527).

[0106] Alternatively, antibodies that bind to two or more domains and a second target, such as, but not limited to, a second, different anti-FGFR antibody, may be prepared in a bispecific format using the methods described herein or other techniques known to those skilled in the art. An antibody variable region that binds to a separate domain can be linked to a variable region that binds to a related site on FGFR2, for example, to confer dual antigen specificity within a single binding molecule. This property of properly designed bispecific antibodies serves dual functions: a variable region with specificity for one antigen is combined with a variable region with specificity for FGFR2 and paired on a structural scaffold, allowing each variable region to bind to a different antigen.

[0107] An exemplary bispecific antibody format that can be used in the context of the present disclosure is a first immunoglobulin (Ig) C H 3 domain and second Ig C H 3 domains, in which the first and second Ig C H The three domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. H The 3 domain binds to protein A and the second Ig C H The 3 domain contains a mutation that reduces or eliminates Protein A binding, for example, the H95R (according to IMGT exon numbering; H435R in EU numbering) modification. H 3 may further contain a Y96F modification (according to IMGT, Y436F according to EU). HFurther modifications that may be found within 3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I in IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT). Variations on the bispecific antibody formats described above are contemplated within the scope of this disclosure.

[0108] Other exemplary bispecific formats that may be used in the context of the present disclosure include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, duobodies, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 Bispecific formats include those described in Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of such formats. Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012].)

[0109] Therefore, the present invention contemplates a bispecific antibody comprising at least one antigen-binding domain that binds to FGFR2.Methods for producing bispecific antibodies are known in the art and can be used to construct the bispecific antigen-binding molecules disclosed herein.

[0110] Exemplary antigen-binding domains that can be included in anti-FGFR2 bispecific antigen-binding molecules include one or more antigen-binding domains derived from any of the anti-FGFR2 sequences disclosed herein. For example, the present disclosure includes FGFR2xFGFR2 bispecific antigen-binding molecules comprising a D1 and / or D2 antigen-binding domain comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. Also provided herein are FGFR2xFGFR2 bispecific antigen-binding molecules comprising a D1 and / or D2 antigen-binding domain comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0111] Biological characteristics of the antigen-binding molecules provided herein Provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that bind to human FGFR2 (e.g., hFGFR2b.mmh) with high affinity. For example, the present disclosure provides antibodies and antigen-binding fragments thereof that have a K of less than about 25 nM as measured by surface plasmon resonance at 25° C., e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. Dand bind to human FGFR2 at a K of less than about 25 nM, less than about 20 nM, less than about 18 nM, less than about 15 nM, less than about 12 nM, less than about 10 nM, less than about 9 nM, or less than about 8 nM, as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. D The present invention provides an anti-FGFR2 antibody and an antigen-binding fragment thereof that bind to human FGFR2 at 25°C.

[0112] Also provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that bind to human FGFR2 (e.g., hFGFR2b.mmh) with a dissociation half-life (t) of greater than about 10 minutes, as measured by surface plasmon resonance at 25° C., e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. According to certain embodiments, provided are anti-FGFR2 antibodies and antigen-binding fragments thereof that bind to human FGFR2 at 25° C. with a t of greater than about 12 minutes, greater than about 14 minutes, greater than about 16 minutes, greater than about 18 minutes, greater than about 20 minutes, greater than about 22 minutes, greater than about 24 minutes, or greater than about 25 minutes, or longer, as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay.

[0113] Thus, provided herein is an isolated monoclonal antibody or antigen-binding fragment thereof that binds to FGFR2, wherein the antibody or antigen-binding fragment thereof exhibits one or more of the following characteristics: (a) It is a fully human monoclonal antibody; (b) 2.5 × 10 as measured by surface plasmon resonance -8 K below M D Binding to FGFR2b at (c) selectively binds to hFGFR2b over hFGFR2c; and (d) comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 22, or an amino acid sequence that is at least 90% identical thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 28, or an amino acid sequence that is at least 90% identical thereto.

[0114] Also provided herein is an anti-FGFR2 antibody or antigen-binding fragment thereof having one or more of the following characteristics: (a) It is a fully human monoclonal antibody; (b) 2.5 × 10 as measured by surface plasmon resonance -8 K below M D Binding to FGFR2b at (c) having a half-life (t) of greater than about 10 minutes as measured by surface plasmon resonance; and (d) selectively binds to hFGFR2b over hFGFR2c;

[0115] Also provided herein is an anti-FGFR2 antibody or antigen-binding fragment thereof having one or more of the following characteristics: (a) It is a fully human monoclonal antibody; (b) 8.9 × 10 as measured by surface plasmon resonance -9 K below M D Binding to FGFR2b at (c) having a half-life (t) of greater than about 25 minutes as measured by surface plasmon resonance; and (d) selectively binds to hFGFR2b over hFGFR2c;

[0116] Also provided herein is a cytotoxin-conjugated anti-FGFR2 antibody or antigen-binding fragment thereof, having one or more of the following characteristics: (a) being selectively cytotoxic to hFGFR2b-expressing cells over hFGFR2c-expressing cells; (b) 4.8 × 10 as measured by surface plasmon resonance -8 K below M D Binding to FGFR2b at (c) having a half-life (t) of greater than about 7 minutes as measured by surface plasmon resonance; and (d) Inducing regression of FGFR2b-positive tumors in a dose-dependent manner.

[0117] Also provided herein are anti-FGFR2 antibodies or antigen-binding fragments thereof that, when conjugated to a cytotoxin, are less cytotoxic to cells with low FGFR2b expression but high FGFR2c expression; are less cytotoxic to non-FGFR2-expressing cells; and / or are cytotoxic to FGFR2b-positive cells. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin, are weakly cytotoxic against low FGFR2b but high FGFR2c expressing NCI-H716 cells, i.e., have an IC50 of greater than about 20 nM; are weakly cytotoxic against non-FGFR2-expressing IM-9 cells, i.e., have an IC50 of greater than about 20 nM; and / or are cytotoxic against FGFR2b-positive MFM-223 or SNU-16 cells, i.e., have an IC50 of less than about 270 pM, e.g., less than about 268 pM, less than about 214 pM, less than about 206 pM, less than about 172 pM, less than about 125 pM, less than about 63 pM, less than about 43.4 pM, or less than about 37 pM, using the in vitro cytotoxicity assay format defined in Example 6 herein, or a substantially similar assay.

[0118] Also provided herein is an anti-FGFR2 antibody or antigen-binding fragment thereof that, when conjugated to a cytotoxin, reduces tumor growth in FGFR2b-positive tumors. Also provided herein is an anti-FGFR2 antibody or antigen-binding fragment thereof that, when conjugated to a cytotoxin, causes tumor regression in FGFR2b-positive tumors. Reduction in tumor growth and tumor regression can be confirmed using tumor xenografts implanted in SCID mice, as described in Example 7 herein, or a substantially similar assay.

[0119] According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as Tubulysin 1 ALP, reduce SNU-16 xenograft tumor growth by at least 16.2% in mice administered a single subcutaneous injection of 0.1 mg / kg of the anti-FGFR2 antibody. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as Tubulysin 1 ALP, reduce SNU-16 xenograft tumor growth by at least 72.7% in mice administered a single subcutaneous injection of 0.3 mg / kg of the anti-FGFR2 antibody. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as Tubulysin 1 ALP, reduce SNU-16 xenograft tumor growth by at least 132.5% in mice administered a single subcutaneous injection of 1.0 mg / kg of the anti-FGFR2 antibody. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as Tubulysin 1 ALP, reduce SNU-16 xenograft tumor growth by at least 139% in mice administered a single subcutaneous injection of 3.0 mg / kg of the anti-FGFR2 antibody.

[0120] According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as maytansinoid 1ALP, reduce SNU-16 xenograft tumor growth by at least 63.9% in mice administered a single subcutaneous injection of 1.0 mg / kg of the anti-FGFR2 antibody. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as maytansinoid 1ALP, reduce SNU-16 xenograft tumor growth by at least 89.2% in mice administered a single subcutaneous injection of 3 mg / kg of the anti-FGFR2 antibody. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as maytansinoid 1ALP, reduce SNU-16 xenograft tumor growth by at least 102.3% in mice administered a single subcutaneous injection of 10 mg / kg of the anti-FGFR2 antibody. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as maytansinoid 1ALP, reduce SNU-16 xenograft tumor growth by at least 100.4% in mice administered a single subcutaneous injection of 15 mg / kg of the anti-FGFR2 antibody.

[0121] According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as camptothecin LP1 (8DAR), reduce SNU-16 xenograft tumor growth by at least 99% in mice administered a single subcutaneous injection of 1.0 mg / kg of the anti-FGFR2 antibody. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as camptothecin LP1 (8DAR), reduce SNU xenograft tumor growth by at least 131% in mice administered a single subcutaneous injection of 3 mg / kg of the anti-FGFR2 antibody. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as camptothecin LP1 (8DAR), reduce SNU xenograft tumor growth by at least 131% in mice administered 10 mg / kg of the anti-FGFR2 antibody as a single subcutaneous injection.

[0122] According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as camptothecin LP2 (4DAR), reduce SNU-16 xenograft tumor growth by at least 72% in mice administered 0.3 mg / kg of the anti-FGFR2 antibody by subcutaneous injection on days 0 and 7. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as camptothecin LP2 (4DAR), reduce SNU-16 xenograft tumor growth by at least 106% in mice administered 1 mg / kg of the anti-FGFR2 antibody by subcutaneous injection on days 0 and 7. According to certain embodiments, provided herein are anti-FGFR2 antibodies and antigen-binding fragments thereof that, when conjugated to a cytotoxin such as camptothecin LP2 (4DAR), reduce SNU xenograft tumor growth by at least 137% in mice administered 3 mg / kg of the anti-FGFR2 antibody by subcutaneous injection on days 0 and 7.

[0123] The antigen-binding proteins of the present disclosure may possess one or more of the foregoing biological characteristics, or any combination thereof. The foregoing list of antibody biological characteristics is not intended to be comprehensive. Other biological characteristics of the antibodies provided herein will become apparent to those of skill in the art from a review of this disclosure, including the Examples herein.

[0124] Antibody-drug conjugates (ADCs) Provided herein are antibody-drug conjugates (ADCs) comprising an anti-FGFR2 antibody or antigen-binding fragment thereof conjugated to a therapeutic moiety, such as a cytotoxic agent (i.e., a cytotoxin), a chemotherapeutic agent, or a radioisotope.

[0125] Cytotoxic agents include any agent that is detrimental to cell growth, viability, or proliferation, including, but not limited to, tubulin-interacting agents and DNA-damaging agents. In some embodiments, the cytotoxic agent is a tubulin inhibitor. In certain embodiments, the tubulin inhibitor inhibits tubulin polymerization. In some embodiments, the cytotoxic payload is a topoisomerase I inhibitor. In some embodiments, the cytotoxic agent is a maytansinoid, auristatin, hemiasterin, vinblastine, vincristine, pyrrolobenzodiazepine, paclitaxel, docetaxel, cryptophycin, tubulysin, or a camptothecin analog.Additionally, examples of suitable cytotoxic and chemotherapeutic agents that can be conjugated to anti-FGFR2 antibodies according to this aspect of the disclosure include, for example, 1-(2chloroethyl)-1,2-dimethanesulfonylhydrazide, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-one, 1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, actinomycin D, amanitin, aminopterin, anguidine, and arginine. anthracycline, anthramycin (AMC), bleomycin, busulfan, butyric acid, calicheamicin (e.g., calicheamicin gamma 1), camptothecin, carminomycin, carmustine, cemadotin, cisplatin, colchicine, combretastatin, cyclophosphamide, cytarabine, cytochalasin B, Dxd or its derivatives, dactinomycin, daunorubicin, decarbazine, diacetoxypentyldoxorubicin, dibromomannitol, dihydroxyanthracin dione, disorazole, dras statins (e.g., dolastatin 10), doxorubicin, duocarmycin, echinomycin, eleutherobin, emetine, epothilones, esperamicin, estramustine, ethidium bromide, etoposide, fluorouracil, geldanamycin, gramicidin D, glucocorticoids, irinotecan, kinesin spindle protein (KSP) inhibitors, leptomycin, leurosine, lidocaine, lomustine (CCNU), maytansinoids, mechlorethamine, melphalan, mercaptopurine, methopterone phosphate, methotrexate, mithramycin, mitomycin, mitoxantrone, N8-acetylspermidine, podophyllotoxin, procaine, propranolol, pteridine, puromycin, pyrrolobenzodiazepines (PBDs), rhizoxin, streptozotocin, tallysomycin, taxol, tenoposide, tetracaine, thioepachlorambucil, tomaymycin, topotecan, tubulysin, vinblastine, vincristine, vindesine, vinorelbine, and derivatives of any of the above.According to certain embodiments, the cytotoxic agent conjugated to the anti-FGFR2 antibody is a maytansinoid such as DM1 or DM4, a tomaymycin derivative, or a dolastatin derivative. According to certain embodiments, the cytotoxic agent conjugated to the anti-FGFR2 antibody is an auristatin such as MMAE, MMAF, or a derivative thereof. In some embodiments, the cytotoxic agent is Dxd or a derivative thereof. In some embodiments, the cytotoxic agent is AZ13599185 (see, e.g., Li et al., 2016 Cancer Cell 29, 117-129). Other cytotoxic agents known in the art are also contemplated as being within the scope of the present disclosure, including protein toxins such as ricin, C. difficile toxin, Pseudomonas exotoxin, ricin, diphtheria toxin, botulinum toxin, bryodin, saponin, pokeweed toxin (i.e., phytolaccatoxin and phytolacchigenin), and other toxins such as those described in Sapra et al., Pharmacol. & Therapeutics, 2013, 138:452-469. In some embodiments, the cytotoxic agent is tubulysin, maytansinoid, or camptothecin or an analog thereof.

[0126] In certain embodiments, the cytotoxic agent is a tubulysin. Suitable tubulysins include those described in U.S. Patent Application No. 16 / 724,164, filed December 20, 2019. In some embodiments, the tubulysin is compound IVa, IVa', IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVj, IVk, IV-l, IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt, IVu, IVvA, IVvB, IVw, IVx, IVy, Va, Va', Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, Via, VIb, VIc, VId, VIe, VIf, VIg, VIh, VI, Vii, VII, VIII, IX, X, D-5a, or D-5c cited in U.S. patent application Ser. No. 16 / 724,164, filed December 20, 2019. In certain embodiments, the tubulysin is compound Ve of U.S. Patent Application No. 16 / 724,164, filed December 20, 2019. In some embodiments, the tubulysin has the following structure: [ka]

[0127] Tubulysin 1A can be prepared using the methods disclosed in U.S. Patent Application No. 16 / 724,164, filed December 20, 2019.

[0128] In some embodiments, the payload of the present disclosure is camptothecin. In certain embodiments, the payload of the present disclosure is a camptothecin analog and / or derivative. [ka]

[0129] In some embodiments, a suitable camptothecin analog is topotecan, irinotecan, belotecan, or deruxtecan (Dxd).

[0130] In one embodiment, the payload of the present disclosure is deruxtecan (Dxd): [ka]

[0131] In another embodiment, the payload is exatecan: [ka]

[0132] In certain embodiments, the payload of the present disclosure is a camptothecin analog having the structure PI, [ka] wherein R1, R2, and R3, and R4 are independently hydrogen or alkyl, e.g., C1-C 12 alkyl, or C1-C8 alkyl, or C1-C6 alkyl, or C1-C4 alkyl, or R2 and R3 together form a 5- or 6-membered ring; or a pharmaceutically acceptable salt thereof.

[0133] In one embodiment, R1 is hydrogen. In one embodiment, R2 is hydrogen. In one embodiment, R2 is C1-C4 alkyl. In one embodiment, R3 is hydrogen. In one embodiment, R3 is C1-C4 alkyl. In one embodiment, R4 is hydrogen. In one embodiment, R4 is C1-C4 alkyl. In one embodiment, R1, R2, and R3, and R4 are hydrogen at each occurrence. In one embodiment, the compound of the present disclosure is P-IA, [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, P-IA is converted to dxd. In some aspects, P-IA is converted to dxd in vivo. In some aspects, the ADC delivers a payload to an FGFR2b-expressing tissue, such as an FGFR2b-expressing tumor, and in some aspects, P-IA is converted to dxd in the local environment. In some aspects, the ADC comprises P-1A, and the ADC is converted to dxd in the local environment.

[0134] In one embodiment, R2 and R3 together form a 5-membered ring. In one embodiment, R2 and R3 together are -(CH2)3-.

[0135] In one embodiment, R2 and R3 together form a 6-membered ring. In one embodiment, R2 and R3 together are -(CH2)4-.

[0136] In one embodiment, R1 is hydrogen and R2 and R3 together form a five-membered ring.

[0137] In one embodiment, the compound of the present disclosure has a structure according to formula (P-II): [ka] (P-II) wherein R is hydrogen or alkyl, e.g., C-C 12 alkyl, or C1-C8 alkyl, or C1-C6 alkyl, or C1-C4 alkyl, or a pharmaceutically acceptable salt thereof.

[0138] It should be understood by those skilled in the art that the above-described compound P-II is also intended to include all isomers (e.g., enantiomers, diastereomers, and geometric (or conformational) forms) of the structure. For example, R and S configurations for each asymmetric center are within the scope of the present disclosure. As an example, the two isomers depicted below are within the scope of the present disclosure. [ka]

[0139] In one embodiment, the compound of the present disclosure is [ka] or a pharmaceutically acceptable salt thereof.

[0140] In one embodiment, a payload according to the present disclosure is conjugated to form a protein-drug conjugate (e.g., an anti-FGFR2 antibody-drug conjugate). In one embodiment, the payload is covalently attached to a moiety M. In one embodiment, the payload is M-Dxd. In one embodiment, M-Dxd is [ka] wherein R is hydrogen or C1-C4 alkyl; [ka] represents the attachment point to L2.

[0141] In one embodiment, the payload according to the present disclosure comprises: [ka] and During the ceremony, [ka] represents the point of attachment to the linker.

[0142] Provided herein is an anti-FGFR2 antibody, or antigen-binding fragment thereof, conjugated via a linker to a payload having the following structure: [ka] The anti-FGFR2 antibody or antigen-binding fragment thereof comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 2 / 10, 22 / 28, and 40 / 44.

[0143] Provided herein is an anti-FGFR2 antibody, or antigen-binding fragment thereof, conjugated via a linker to a payload having the following structure: [ka] The anti-FGFR2 antibody or antigen-binding fragment thereof comprises a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set selected from the group consisting of: SEQ ID NOs: 4-6-8-12-14-16, 24-6-26-30-32-34, and 24-6-42-46-32-34.

[0144] Provided herein is an anti-FGFR2 antibody, or antigen-binding fragment thereof, conjugated via a linker to a payload having the following structure: [ka] The anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 2 / 10, 22 / 28, and 40 / 44.

[0145] Provided herein is an anti-FGFR2 antibody, or antigen-binding fragment thereof, conjugated via a linker to a payload having the following structure: [ka] The anti-FGFR2 antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence selected from the group consisting of: SEQ ID NO: 18, 36, 49, 52, SEQ ID NO: 53, and SEQ ID NO: 54. In some embodiments, the anti-EGFR2 antibody or antigen-binding fragment thereof comprises a heavy chain / light chain amino acid sequence pair selected from the group consisting of: SEQ ID NO: 18 / 20, 36 / 38, 49 / 51, 52 / 20, 53 / 38, and 54 / 51.

[0146] In certain embodiments, the cytotoxic agent is a maytansinoid, e.g., a derivative of maytansine. Suitable maytansinoids include DM1, DM4, or derivatives, stereoisomers, or isotopologues thereof. Suitable maytansinoids also include, but are not limited to, those disclosed in WO2014 / 145090A1, WO2015 / 031396A1, US2016 / 0375147A1, US10,570,151 (e.g., compound 6 therein), and US2017 / 0209591A1 (incorporated herein by reference in their entireties). In some embodiments, the maytansinoid is DM1.

[0147] In some embodiments, the maytansinoid has the structure: [ka] wherein A is an optionally substituted arylene or heteroarylene.

[0148] In some embodiments, the maytansinoid has the structure: [ka] wherein A is an optionally substituted arylene or heteroarylene.

[0149] In some embodiments, the maytansinoid has the structure: [ka] In the formula, n is an integer from 1 to 12, and R 1 is alkyl.

[0150] In some embodiments, the maytansinoid is: [ka] [ka] [ka] [ka]

[0151] In some embodiments, the maytansinoid is: [ka]

[0152] In some embodiments, the maytansinoid is a compound having the formula maytansinoid 1A. [ka]

[0153] Provided herein is an antibody-drug conjugate comprising an anti-FGFR2 antibody or antigen-binding fragment thereof conjugated to maytansinoid 1A, wherein the antibody-drug conjugate has the structure: [ka] wherein Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof, and L is a linker. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34.

[0154] Provided herein is an antibody-drug conjugate comprising an anti-FGFR2 antibody or antigen-binding fragment thereof conjugated to Tubulysin 1A, wherein the antibody-drug conjugate has the structure: [ka] wherein Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof, and L is a linker. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34.

[0155] Provided herein is an antibody-drug conjugate comprising an anti-FGFR2 antibody or antigen-binding fragment thereof conjugated to a camptothecin analog, wherein the antibody-drug conjugate has the structure: [ka] wherein Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof, and n is 2 or 4. In a specific embodiment, n is 2. In a specific embodiment, n is 4. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the Ab is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the Ab is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the Ab is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. n is a value from 2 to 12, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0156] Also provided herein are antibody-radionuclide conjugates (ARCs) comprising anti-FGFR2 antibodies conjugated to one or more radionuclides. Examples of radionuclides that may be used in the context of embodiments of the present disclosure include, but are not limited to, the following: 225 Ac, 212 Bi, 213 Bi, 131 I, 186 Re, 227 Th, 222 Rn, 223 Ra, 224 Ra, and 90 Examples include Y.

[0157] In certain embodiments, provided herein are ADCs comprising an anti-FGFR2 antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent (e.g., any of the cytotoxic agents disclosed above) via a linker. A linker is any group or moiety that links, connects, or bonds an antibody or antigen-binding protein described herein to a therapeutic moiety, such as a cytotoxic agent. Suitable linkers can be found, for example, in Antibody-Drug Conjugates and Immunotoxins; Phillips, G.L., Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates; Ducry, L., Ed.; Humana Press, 2013; and Antibody-Drug Conjugates; Wang, J., Shen, W.-C., and Zaro, J.L., Eds.; Springer International Publishing, 2015, the contents of which are incorporated herein by reference in their entireties. Generally, binder linkers suitable for the antibody conjugates described herein are linkers that are stable enough to take advantage of the circulating half-life of the antibody while simultaneously being able to release their payload after antigen binding and / or antigen-mediated internalization of the conjugate. The linker may be cleavable or non-cleavable. Cleavable linkers include linkers that are cleaved by intracellular metabolism after internalization, such as cleavage via hydrolysis, reduction, or enzymatic reaction. Non-cleavable linkers include linkers that release the attached payload via lysosomal degradation of the antibody after internalization. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolytically labile linkers, enzyme-cleavable linkers, reduction-labile linkers, self-immolative linkers, and non-cleavable linkers.Suitable linkers also include, but are not limited to, linkers that are or include peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units. In some embodiments, the linker includes more than one of the PEG groups.

[0158] Any linker molecule or linker technology known in the art can be used to generate or construct the ADCs of the present disclosure. In certain embodiments, the linker is a cleavable linker, e.g., a cathepsin B-cleavable linker. According to other embodiments, the linker is a non-cleavable linker. Exemplary linkers that may be used in the context of the present disclosure include, for example, linkers comprising or consisting of MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), a dipeptide moiety in a protease-cleavable linker, ala-phe (alanine-phenylalanine), a dipeptide moiety in a protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio)pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SIAB (N-succinimidyl(4-iodo-acetyl)aminobenzoate), and variants and combinations thereof. Additional exemplary linkers that may be used in the context of the present disclosure are provided, for example, in U.S. Pat. No. 7,754,681, and Ducry, Bioconjugate Chem., 2010, 21:5-13, the contents of which are incorporated herein by reference in their entireties, and references cited therein.

[0159] In certain embodiments, the linker is stable under physiological conditions. In certain embodiments, the linker is cleavable, e.g., capable of releasing at least the payload portion in the presence of an enzyme or at a particular pH range or value. In some embodiments, the linker comprises an enzyme-cleavable moiety. Exemplary enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester bonds, hydrazones, and disulfide bonds. In some embodiments, the linker comprises a cathepsin-cleavable linker.

[0160] In some embodiments, the linker comprises a non-cleavable moiety.

[0161] Suitable linkers also include, but are not limited to, linkers that are chemically bonded to two cysteine ​​residues of a single binding agent, such as an antibody, and can serve to mimic the disulfide bonds of antibodies that are disrupted as a result of the conjugation process.

[0162] In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker comprises two amino acids. In some embodiments, the linker comprises three amino acids. In some embodiments, the linker comprises four amino acids. Suitable amino acids include natural, unnatural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D-alpha amino acids. In some embodiments, the linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, derivatives thereof, or combinations thereof. In certain embodiments, one or more amino acid side chains are linked to the side chains described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the linker comprises lysine, valine, and citrulline. In some embodiments, the linker comprises lysine, valine, and alanine. In some embodiments, the linker comprises valine and alanine. In some embodiments, the linker comprises a dipeptide, tripeptide, or tetrapeptide. In some embodiments, the linker comprises a peptide, wherein the peptide is valine-citrulline (val-cit or VC), glutamic acid-valine-citrulline (EVC), glycine-glycine-phenylalanine-(GGF), or glycine-glycine-phenylalanine-glycine (GGFG).

[0163] In some embodiments, the linker comprises a self-immolative group. The self-immolative group can be any such group known to one of skill in the art. In certain embodiments, the self-immolative group is p-aminobenzyl (PAB), or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). In some embodiments, the linker comprises a moiety having the following structure: [ka]

[0164] In some embodiments, the linker is: [ka] During the ceremony, [ka] is the bond to an antibody or antigen-binding protein (e.g., via a lysine residue), [ka] is a linkage to a cytotoxic agent (e.g., DM1). In some embodiments, the linker is: [ka] During the ceremony, [ka] is the bond to an antibody or antigen-binding protein (e.g., via a lysine residue), [ka] is a linkage to a cytotoxic agent (e.g., DM1). In certain embodiments, the linker is: [ka]

[0165] In certain embodiments, the linker is: [ka]

[0166] In some embodiments, the linker is derived from maleimidylmethyl-4-trans-cyclohexanecarboxysuccinate: [ka]

[0167] In some embodiments, the linker is: [ka] During the ceremony, [ka] is the bond to an antibody or antigen-binding protein (e.g., via a lysine residue), [ka] is a conjugate with a cytotoxic agent (e.g., a compound having the formula: [ka]

[0168] Suitable linkers include, but are not limited to, linkers comprising one or more cyclic moieties. In some embodiments, the cyclic moiety is derived from a cycloaddition reaction. In certain embodiments, the cyclic moiety is derived from a 1-3-cycloaddition reaction between an azide and an alkyne, such as a cycloalkyne. In some embodiments, the cyclic moiety is [ka] or a cycloaddition positional isomer thereof. [ka]

[0169] As used herein, "positional isomer," "positional isomers," or "mixture of positional isomers" refers to the product of a 1,3-cycloaddition or strain-promoted alkyne-azide cycloaddition (SPAAC) (also known as a click reaction) derived from a suitable azide (e.g., an -N3, or -PEG-N3 derivatized antibody) treated with a suitable alkyne. In certain embodiments, for example, positional isomers and mixtures of positional isomers are characterized by the click reaction products shown below. [ka]

[0170] In certain embodiments, two or more suitable azides and two or more suitable alkynes may be utilized within the synthetic scheme en route to the product, and each azide-alkyne pair may participate in one or more independent click reactions to generate a mixture of regioisomeric click reaction products. For example, one skilled in the art will recognize that a first suitable azide may independently react with a first suitable alkyne, and a second suitable azide may independently react with a second suitable alkyne en route to the product, resulting in the generation of four possible click reaction regioisomers or a mixture of four possible click reaction regioisomers.

[0171] In certain embodiments, such cycloaddition reactions facilitate the conjugation of a payload comprising a linker moiety containing an alkyne to an antibody functionalized with one or more azide groups. In some embodiments, the anti-FGFR2 antibodies described herein are functionalized with one or more azide groups. When an antibody is specifically functionalized with an azide group at a particular amino acid residue, e.g., Q295, such an antibody can be site-specifically conjugated to a payload having a linker moiety containing an alkyne capable of undergoing a cycloaddition reaction with the azide group. In some embodiments, an anti-FGFR2 antibody is functionalized at Q295 by reacting the antibody with a primary amine compound containing an azide group and transglutaminase. An antibody conjugated via a glutamine residue is bound, for example, via a -CONH- moiety resulting from the reaction of the glutamine residue with a primary amine compound. In some embodiments, the bond connecting the linker-payload to such a conjugated glutamine residue is [ka] and both represent the resulting -C(O)NH- moiety that links the antibody to the linker payload described herein. In some embodiments, the primary amine compound comprising an azide group comprises a PEG group. In certain embodiments, the primary amine compound is: [ka] wherein n is 1 to 12. In certain embodiments, the primary amine compound is: [ka]

[0172] In some embodiments, the linker comprises one or more spacers. Suitable spacers include, for example, a moiety that connects two linker moieties, a linker moiety with a payload, or a linker moiety with an antibody, covalently or via ionic interactions. In certain embodiments, the spacer is a PEG group.

[0173] The present disclosure includes ADCs in which a linker connects an anti-FGFR2 antibody or antigen-binding fragment thereof to a drug or cytotoxin via attachment at a specific amino acid within the antibody or antigen-binding molecule. Exemplary amino acid additions that can be used in the context of this embodiment include, for example, lysine (see, e.g., U.S. Pat. No. 5,208,020; U.S. Patent Application Publication No. 2010 / 0129314; Hollander et al., Bioconjugates). Chem., 2008, 19:358-361, WO2005 / 089808, U.S. Patent No. 5,714,586, U.S. Patent Application Publication No. 2013 / 0101546, and U.S. Patent Application Publication No. 2012 / 0585592), cysteine ​​(see, e.g., U.S. Patent Application Publication No. 2007 / 0258987, WO2013 / 055993, WO2013 / 055990, WO2013 / 053873, WO2013 / 053872, WO2011 / 130598, U.S. Patent Application Publication No. 2013 / 0101546, and U.S. Patent No. 7,750,116), selenocysteine ​​(see, e.g., WO2008 / 122039; and Hofer et al. al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formylglycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51, and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), unnatural amino acids (see, e.g., WO2013 / 068874 and WO2012 / 166559), and acidic amino acids (see, e.g., WO2012 / 05982).Linkers may also be conjugated to antigen-binding proteins via attachment to carbohydrate (see, e.g., U.S. Patent Application Publication No. 2008 / 0305497, WO2014 / 065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide linkers (see, e.g., WO2013 / 085925, WO2010 / 010324, WO2011 / 018611, and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313). Site-specific conjugation techniques may also be employed to directly conjugate to specific residues of antibodies or antigen-binding proteins (see, e.g., Schumacher et al., J Clin Immunol (2016) 36(Suppl 1):100). Site-specific conjugation techniques include, but are not limited to, transglutaminase-mediated glutamine conjugation (see, for example, Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0174] According to certain embodiments, the present disclosure provides an ADC in which an anti-FGFR2 antibody or antigen-binding fragment thereof described herein is conjugated to a linker-drug composition described in International Patent Application Publication No. WO2014 / 145090 (the disclosure of which is incorporated herein by reference in its entirety) (e.g., compound "7" disclosed therein). [ka]

[0175] Also provided herein is an antibody-drug conjugate comprising an anti-FGFR2 antibody disclosed herein, wherein the antibody is conjugated to a cytotoxic agent. In certain embodiments, the cytotoxic agent is a maytansinoid. In certain embodiments, the maytansinoid is a compound having the following formula: [ka] In the formula, n is an integer from 1 to 12, and R 1 is alkyl. In certain embodiments, the maytansinoid is: [ka] In some embodiments, the maytansinoid is 1A. [ka] In certain embodiments, the cytotoxic agent is a maytansinoid, and the maytansinoid is covalently attached to the antibody via a non-cleavable linker. In certain embodiments, the cytotoxic agent is a maytansinoid, and the maytansinoid is covalently attached to the anti-FGFR2 antibody or antigen-binding fragment thereof via a cleavable linker.

[0176] An exemplary cleavable linker is a cathepsin B cleavable linker. In certain embodiments, the cytotoxic agent is a maytansinoid, and the maytansinoid is covalently attached to the anti-FGFR2 antibody or antigen-binding fragment thereof via a cathepsin B cleavable linker. In one embodiment, the anti-FGFR2 antibody or antigen-binding fragment thereof is conjugated to the maytansinoid via a cleavable linker, wherein the linker comprises a dipeptide. In another embodiment, the anti-FGFR2 antibody or antigen-binding fragment thereof is conjugated to the maytansinoid via a cleavable linker, wherein the linker comprises a valine-citrulline dipeptide. In another embodiment, the anti-FGFR2 antibody or antigen-binding fragment thereof is conjugated to the maytansinoid via a cleavable linker, wherein the linker comprises a valine-citrulline dipeptide and a PAB group. In one embodiment, the anti-FGFR2 antibody or antigen-binding fragment thereof is [ka] is conjugated to [ka] is binding to the antibody. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34.

[0177] In one embodiment, the anti-FGFR2 antibody or antigen-binding fragment thereof [ka] is conjugated to [ka] is binding to the antibody. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34.

[0178] In one embodiment, the anti-FGFR2 antibody or antigen-binding fragment thereof [ka] is conjugated to [ka] is binding to the antibody. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34.

[0179] In one embodiment, the anti-FGFR2 antibody or antigen-binding fragment thereof [ka] is conjugated to [ka] is binding to the antibody. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34.

[0180] In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof is conjugated to BAY1187982, see Sommer et al., 2016, Cancer Res, 76(21), 6631-6639, doi:10.1158 / 0008-5472.CAN-16-0180. In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof is conjugated to: [ka]

[0181] In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof [ka] wherein L is a linker; [ka] is binding to the antibody. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34.

[0182] In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof [ka] or a cycloaddition regioisomer thereof, wherein SP is a spacer; [ka] is the binding to the antibody. In some embodiments, [ka] is a bond to a glutamine residue of the antibody. In certain embodiments, the glutamine residue is Q295. In some embodiments, the antibody is conjugated at Q295 and Q297. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34.

[0183] In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof [ka] or conjugated to its cycloaddition regioisomer, [ka] During the ceremony, [ka] is a bond with a glutamine residue of the antibody. In certain embodiments, the glutamine is heavy chain Q295 glutamine. In certain embodiments, the anti-FGFR2 antibody comprises a heavy chain having Q295 and Q297, wherein Q297 is derived from an N297Q mutation. In some aspects, the anti-FGFR2 antibody comprises a heavy chain (HC) selected from the group consisting of SEQ ID NOs: 52, 53, and 54. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising an HCDR1 amino acid sequence of SEQ ID NO: 24, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 26, an LCDR1 amino acid sequence of SEQ ID NO: 30, an LCDR2 amino acid sequence of SEQ ID NO: 32, and an LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody is an anti-FGFR2 antibody or an antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 24, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 42, an LCDR1 amino acid sequence of SEQ ID NO: 46, an LCDR2 amino acid sequence of SEQ ID NO: 32, and an LCDR3 amino acid sequence of SEQ ID NO: 34.

[0184] In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof is linked via a linker to: [ka] is conjugated to [ka] represents a point of attachment to the linker. In some embodiments, the linker is attached to one or more glutamine residues of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34.

[0185] In some embodiments, the conjugate has the structure: Ab-[L-Pay] n During the ceremony: Ab is an anti-FGFR2 antibody or antigen-binding fragment thereof described herein; L is a linker, Pay is a cytotoxic agent, n is an integer from 1 to 12.

[0186] In some embodiments, n is 2. In some embodiments, n is 4. In some embodiments, Pay is: [ka]

[0187] In some embodiments, L is a cleavable linker. In some embodiments, L comprises a peptide. In some embodiments, L comprises val-cit. In some embodiments, L comprises: [ka] In some embodiments, L comprises a PEG group.

[0188] In some embodiments, L comprises a cyclic moiety. In certain embodiments, the cyclic moiety is the product of a 1,3-cycloaddition of an azide with a cycloalkyne. In some embodiments, -L-Pay is [ka] and SP 1 and SP 2 are each independently a spacer, [ka] is a cyclic moiety, [ka] is a glutamine residue of the antibody. In some embodiments, the glutamine is Q295 glutamine. In some embodiments, n is 2, and two L-Pays are conjugated to Q295. In some embodiments, n is 4, and two L-Pays are conjugated to Q295 and two L-Pays are conjugated to Q297. In some embodiments, the cyclic moiety is the product of a cycloaddition reaction. In certain embodiments, the cyclic moiety is the product of a cycloaddition reaction between an azide and a cycloalkyne. In certain embodiments, the cyclic moiety is: [ka] In certain embodiments, SP 1 comprises a PEG moiety. In some embodiments, the PEG comprises 1 to 12 ethylene glycol units. In certain embodiments, SP 2 In certain embodiments, SP2 comprises a dipeptide. In certain embodiments, SP2 comprises val-cit. 2 teeth, [ka] In certain embodiments, SP 2 comprises a PEG moiety. In some embodiments, SP2-Pay is: [ka] In certain embodiments, [ka] teeth, [ka] or a cycloaddition positional isomer thereof.

[0189] In certain embodiments, [ka] teeth, [ka] or a positional isomer thereof, [ka] During the ceremony, [ka] is a bond with a glutamine residue of the antibody. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 26, the LCDR1 amino acid sequence of SEQ ID NO: 30, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody is an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 24, the HCDR2 amino acid sequence of SEQ ID NO: 6, the HCDR3 amino acid sequence of SEQ ID NO: 42, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 32, and the LCDR3 amino acid sequence of SEQ ID NO: 34.

[0190] In one aspect, the disclosure provides a compound having a structure according to formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A), in the formula, BA is an anti-FGFR2 antibody or an antigen-binding fragment thereof; Gln is a glutamine residue, L1 is absent or is a first linker; B is a branching unit comprising at least one adduct of a group B' and a group B" wherein one of the groups B' and B" is -N3 and [ka] and the other of groups B' and B" is selected from [ka] wherein Q is C or N; L2 is a second linker covalently attached to branching unit B via at least one group B″; M is absent or has the structure [ka] wherein R', R', and R" are independently, at each occurrence, hydrogen or C1-C4 alkyl, or R' and R" together form a 5- or 6-membered ring; Dxd is an antitumor agent having a structure according to formula (P): [ka] k is an integer from 1 to 12, m is an integer from 1 to 30; n is an integer of 1 to 30.

[0191] In one embodiment, an anti-FGFR2 antibody-drug conjugate according to the present disclosure comprises an anti-FGFR2 antibody or antigen-binding fragment thereof and a linker-payload, wherein the linker-payload has the following structure: [ka] or a pharmaceutically acceptable salt thereof, wherein: [ka] Represents the point of attachment to the antibody, either directly or via a second linker.

[0192] In one embodiment, a compound according to the present disclosure has the structure: [ka] wherein BA is an anti-FGFR2 antibody or an antigen-binding fragment thereof.

[0193] In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof, i.e., a BA, comprises a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof comprises a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCDR1 amino acid sequence of SEQ ID NO: 24, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 26, an LCDR1 amino acid sequence of SEQ ID NO: 30, an LCDR2 amino acid sequence of SEQ ID NO: 32, and an LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCDR1 amino acid sequence of SEQ ID NO: 24, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 42, an LCDR1 amino acid sequence of SEQ ID NO: 46, an LCDR2 amino acid sequence of SEQ ID NO: 32, and an LCDR3 amino acid sequence of SEQ ID NO: 34.

[0194] In one embodiment, a compound according to the present disclosure has the structure: [ka] wherein BA is an anti-FGFR2 antibody or an antigen-binding fragment thereof.

[0195] In one embodiment, a compound according to the present disclosure has the structure: [ka] wherein BA is an anti-FGFR2 antibody or an antigen-binding fragment thereof.

[0196] In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof, i.e., a BA, comprises a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCDR1 amino acid sequence of SEQ ID NO: 4, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 8, an LCDR1 amino acid sequence of SEQ ID NO: 12, an LCDR2 amino acid sequence of SEQ ID NO: 14, and an LCDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof comprises a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCDR1 amino acid sequence of SEQ ID NO: 24, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 26, an LCDR1 amino acid sequence of SEQ ID NO: 30, an LCDR2 amino acid sequence of SEQ ID NO: 32, and an LCDR3 amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCDR1 amino acid sequence of SEQ ID NO: 24, an HCDR2 amino acid sequence of SEQ ID NO: 6, an HCDR3 amino acid sequence of SEQ ID NO: 42, an LCDR1 amino acid sequence of SEQ ID NO: 46, an LCDR2 amino acid sequence of SEQ ID NO: 32, and an LCDR3 amino acid sequence of SEQ ID NO: 34.

[0197] In one embodiment, a compound according to the present disclosure has the structure: [ka] where BA is an anti-FGFR2 antibody or an antigen-binding fragment thereof).

[0198] Linker L1 In certain embodiments, the linker L1 is absent.

[0199] In certain embodiments, the linker L1 is present and is covalently attached to the amine of a glutamine residue of the anti-FGFR2 antibody.

[0200] In certain embodiments, the linker L is alkyl (e.g., C 1-20 Alkyl, or C 1-12 Alkyl or C 1-6 alkyl), -NH-, -C(O)-, -(CH2) u -NH-C(O)-, -(CH2) u -C(O)-NH-, -(CH2-CH2-O) v -, -(CH2) u -(O-CH2-CH2) v -C(O)-NH-, a peptide unit containing 2 to 4 amino acids, or a combination thereof, each of which may be optionally substituted with one or more of -S-, -S(O2)-, -C(O)-, -C(O2)-, or -CO2H, where the subscripts u and v are independently integers from 1 to 8.

[0201] In certain embodiments, the free (unconjugated) linker L1 comprises a primary amine for attachment to a glutamine residue via a transglutamation reaction.

[0202] In one embodiment, the linker L1 comprises one or more polyethylene glycol (PEG) units, hi one embodiment, L1 comprises 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 PEG units.

[0203] In one embodiment, the linker L1 comprises a disulfide (-SS-) bond.

[0204] In one embodiment, the linker L1 comprises a -S(O2)- moiety.

[0205] In one embodiment, one or more carbons on the linker L1 are substituted with -CO2H.

[0206] In one embodiment, the linker L1 comprises a peptide unit comprising 2 to 4 amino acids, or a peptide unit comprising 2 amino acids, a peptide unit comprising 3 amino acids, or a peptide unit comprising 4 amino acids.

[0207] In one embodiment, the linker L1 comprises a peptide unit comprising two amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, and citrulline, and combinations thereof. In one particular embodiment, the linker L1 comprises a valine-citrulline unit.

[0208] In one embodiment, the linker L1 is [ka] wherein R A is a group including alkyne, azide, tetrazine, trans-cyclooctene, maleimide, amine, ketone, aldehyde, carboxylic acid, ester, thiol, sulfonic acid, tosylate, halide, silane, cyano group, carbohydrate group, biotin group, lipid residue (wherein the subscripts x, n, p, and q are independently integers from 0 to 12), and combinations thereof.

[0209] Branch unit B In one aspect, branching unit B comprises at least one adduct of group B'. In certain embodiments, B comprises one adduct of group B'. In certain embodiments, B comprises two adducts of group B'. In certain embodiments, B comprises three adducts of group B'.

[0210] In certain embodiments, B comprises at least four adducts of group B'. In certain embodiments, B comprises four adducts of group B'. In certain embodiments, B comprises five adducts of group B'. In certain embodiments, B comprises six adducts of group B'.

[0211] In general, adducts of group B' according to the present disclosure encompass any moiety that comprises the product of the addition reaction of group B', regardless of the synthetic steps taken to produce the moiety.

[0212] In some embodiments, the adduct of group B' is a substituted maleimide and, for example, a thiol, or a substituted trans-cyclooctene (e.g., [ka] wherein n is an integer from 0 to 12), and may be, for example, a product of tetrazine.

[0213] In some embodiments, the adduct of group B' may be the product of a 1,3-cycloaddition reaction between an azide and an alkyne moiety. Without being bound by theory, the azide-alkyne cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3-triazole.

[0214] More specifically, -N3, [ka] (wherein Q is C or N), the adducts of group B' selected from 1,3-cycloaddition adducts of group B', as well as -N3, [ka] wherein Q is C or N, and the group B" is complementary to the group B' to form a 1,3-cycloaddition adduct.

[0215] As a non-limiting example, group B' can be an azide (-N3) and group B" can be an alkyne-containing group, such as [ka] may be.

[0216] As another non-limiting example, group B' can be an alkyne-containing group, such as [ka] and the group B″ may be an azide.

[0217] In one embodiment, the adduct of group B′ and group B″ comprises a triazole moiety. In one particular embodiment, the adduct of group B′ and group B″ comprises [ka] wherein Q is C or N.

[0218] As noted above, in one embodiment, B includes one appendage of group B'.

[0219] In certain embodiments, L1-B is [Table A] wherein: [ka] is the amino attachment point to the glutamine residue of the anti-FGFR2 antibody, and (B') is the attachment of group B'.

[0220] In one embodiment, group B' is an azide (-N3) and the adduct of group B' comprises a triazole.

[0221] According to one embodiment of the present disclosure, the linker L1-B may be an azidoamine linker (AL) comprising an amine group directly attached to the antibody, a PEG-containing base structure, and an azide functional group B' (n=1).

[0222] The basic component structures of non-limiting exemplary azidoamine linkers are listed and shown in Figure 8. Specific structures that have been synthesized as examples are provided in Table 1.

[0223] In one embodiment, B comprises at least two adducts of group B'. In certain embodiments, B is [ka] where (B') comprises the point of attachment of the addition of group B'.

[0224] In certain embodiments, B is selected from the group consisting of: [Table B]

[0225] In certain embodiments, L1-B is [Table C-1] [Table C-2] wherein: [ka] is the amino attachment point to the glutamine residue of the anti-FGFR2 antibody.

[0226] According to another embodiment of the present disclosure, the linker L1-B may be a branched alkyl azidoamine linker (BL) comprising an amine group that directly binds to the anti-FGFR2 antibody, a branched alkyl PEG-containing base structure, and 2 to 6 azide functional groups B' (n=2 to 6).

[0227] The basic component structures of exemplary, non-limiting branched alkyl azidoamine linkers are listed in Figure 9. Specific structures that have been synthesized as examples are provided in Table 2.

[0228] Linker L2 In certain embodiments of the present disclosure, L2 has a structure according to formula (L2): B”-SP1-B2-(-SP2-AA-SP3) p (L2) During the ceremony, B" is a group capable of covalently bonding to group B'; SP1 is absent or is the first spacer unit, B2 is absent or is a branching unit; SP2 is absent or a second spacer unit, AA is a peptide unit containing 2 to 4 amino acids or none at all, SP3 is absent or a third spacer unit; p is an integer from 1 to 12.

[0229] In certain embodiments of the present disclosure, L2 has a structure according to formula (L2'): H2N-SP1-B2-(-SP2-AA-SP3) p (L2') During the ceremony, SP1 is absent or is the first spacer unit, B2 is absent or is a branching unit; SP2 is absent or a second spacer unit, AA is a peptide unit containing 2 to 4 amino acids or none at all, SP3 is absent or a third spacer unit; p is an integer from 1 to 12.

[0230] In certain embodiments of the present disclosure, L2 has a structure according to formula (L2"): Maleimide-N-SP1-B2-(-SP2-AA-SP3) p (L2”) During the ceremony, SP1 is absent or is the first spacer unit, B2 is absent or is a branching unit; SP2 is absent or a second spacer unit, AA is a peptide unit containing 2 to 4 amino acids or none at all, SP3 is absent or a third spacer unit; p is an integer from 1 to 12.

[0231] In certain embodiments where linker L2 has the structure L2' or L2", linker L2 may be directly covalently attached to the anti-FGFR2 antibody, for example, via TGase-mediated glutamine ligation (linker L2') or cysteine ​​ligation (linker L2").

[0232] In certain embodiments, the linker L2 comprises a B" group that can be covalently bonded to a B' group, as described above.

[0233] In certain embodiments, the group B″ is —N 3、 , [ka] wherein Q is C or N.

[0234] As a non-limiting example, the group B″ can be an alkyne-containing group, such as [ka] may be.

[0235] As another non-limiting example, the group B″ can be an azide.

[0236] In one embodiment, the adduct of group B′ and group B″ comprises a triazole moiety. In one particular embodiment, the adduct of group B′ and group B″ comprises [ka] wherein Q is C or N.

[0237] In one embodiment, the first spacer SP1 is absent.

[0238] In another embodiment, SP1 is [ka] is selected from the group consisting of:

[0239] In one embodiment, the second spacer SP2 is absent.

[0240] In another embodiment, SP2 is alkyl (e.g., C 1-20 Alkyl or C 1-12 Alkyl or C 1-10 Alkyl or C 1-8 Alkyl or C 1-6 alkyl), -(CH2-CH2-O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -, -NH-(CH2) u -C(O)-, -NH-(CH2-CH2-O) v -, -NH-(CH2-CH2-O) v -C(O)--, -NH-(CH2-CH2-O) v -(CH2) u -, -NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u -C(O)-NH-, or combinations thereof, and the subscripts u and v are independently integers from 1 to 8.

[0241] In certain embodiments, AA is a peptide unit comprising two to four amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, phenylalanine, and citrulline, and combinations thereof.

[0242] In one embodiment, AA is a peptide unit comprising 2 amino acids. In one embodiment, AA is a peptide unit comprising 3 amino acids. In one embodiment, AA is a peptide unit comprising 4 amino acids.

[0243] In one particular embodiment, AA is valine-citrulline, valine-alanine, or phenylalanine-lysine.

[0244] In another specific embodiment, AA is selected from the group consisting of glycine-glycine-glycine (GGG), glycine-glycine-glycine-glycine-glycine (GGGG), glycine-glycine-phenylalanine (GGF), and glycine-glycine-phenylalanine-glycine (GGFG), and glutamic acid-valine-citrulline (EVC).

[0245] In one embodiment, the third spacer SP3 is absent.

[0246] In another embodiment, SP3 is [ka] and combinations thereof, wherein R c is independently, in each occurrence, absent, or [ka] is a group selected from

[0247] In one embodiment, the spacer SP3 is covalently attached to the camptothecin analog, eg, Dxd or M-Dxd.

[0248] In one embodiment, the linker L2 comprises from about 1 to about 12, or from about 1 to about 10, or from about 1 to about 8, or from about 1 to about 6, or from about 1 to about 4, or from about 1 to about 2 (SP2-AA-SP3) moieties, and the linker-payload L2-Dxd comprises from about 1 to about 12, or from about 1 to about 10, or from about 1 to about 8, or from about 1 to about 6, or from about 1 to about 4, or from about 1 to about 2 Dxd payload molecules. Part M

[0249] In certain embodiments, the moiety M is absent.

[0250] In certain embodiments, M is present and has the structure [ka] wherein R, R', and R" are independently, at each occurrence, hydrogen or alkyl, or R' and R" together form a ring, e.g., a 3- to 8-membered ring.

[0251] In certain embodiments, M is present and has the structure [ka] wherein R, R', and R" are independently, at each occurrence, hydrogen or C1-C4 alkyl, or R' and R" together form a 5- or 6-membered ring.

[0252] In one embodiment, R is hydrogen.

[0253] In one embodiment, R' is hydrogen. In one embodiment, R' is C1-C4 alkyl.

[0254] In one embodiment, R" is hydrogen. In one embodiment, R" is C1-C4 alkyl.

[0255] In one embodiment, R' and R" together form a 5-membered ring. In one embodiment, R' and R" together are -(CH2)3-.

[0256] In one embodiment, R' and R" together form a 6-membered ring. In one embodiment, R' and R" together are -(CH2)4-.

[0257] In one embodiment, R, R', and R" are hydrogen in each occurrence, i.e., M is [ka] is.

[0258] In another embodiment, R is hydrogen, and R′ and R″ together form a 5-membered ring, e.g., R′ and R″ together are —(CH 2 ) 3 —, and M is [ka] is.

[0259] Linker-Payload (L2-P) In another aspect, the disclosure provides a compound according to formula (L2-P), (L2'-P), or (L2"-P), B”-SP1-(-SP2-AA-SP3-M-Dxd)p(L2-P), H2N-SP1-(-SP2-AA-SP3-M-Dxd)p(L2'-P), or Maleimide-N-SP1-(-SP2-AA-SP3-M-Dxd)p(L2'-P) During the ceremony, B” is -N3, [ka] is selected from the group consisting of SP1 is not present or [ka] is a first spacer unit selected from the group consisting of: SP2 is not present or C 1-6 Alkyl, -(CH2-CH2-O) v-, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -, -NH-(CH2) u -C(O)-, -NH-(CH2-CH2-O) v -, -NH-(CH2-CH2-O) v -C(O)-, -NH-(CH2-CH2-O) v -(CH2) u -, -NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u a second spacer selected from the group consisting of: —C(O)—NH—, —C(O)—NH—, or combinations thereof; and the subscripts u and v are independently integers from 1 to 8; AA is a peptide unit containing 2 to 4 amino acids or none at all, SP3 does not exist or [ka] and a third spacer unit selected from the group consisting of: c is independently, in each occurrence, absent, or [ka] is a group selected from M is absent, or [ka] wherein R', R', and R" are independently, at each occurrence, hydrogen or C1-C4 alkyl, or R' and R" together form a 5- or 6-membered ring; Dxd is an antitumor agent having a structure according to formula (P): [ka] p is an integer from 1 to 12.

[0260] In certain embodiments where L2-P has the structure L2'-P or L2"-P, the linker-payload may be directly covalently attached to the anti-FGFR2 antibody, for example, via TGase-mediated glutamine ligation (L2'-P) or cysteine ​​ligation (L2"-P).

[0261] In one embodiment, the product of direct attachment of L2'-P to an anti-FGFR2 antibody (BA) may have the following structure: BA-(Gln-NH-SP1-B2-(-SP2-AA-SP3-M-Dxd) p ) n

[0262] In certain embodiments, the linker-payload L2-P, L2′-P, or L2″-P according to the present disclosure is [Table D-1] [Table D-2] [Table D-3] or a pharmaceutically acceptable salt thereof.

[0263] Branched Linker 2-Payload (BL2P) In another aspect, the present disclosure provides L2-P comprising one or more branching units. Exemplary branching units B1-B5 according to the present disclosure are illustrated in Table 1 below. [Table 1]

[0264] Table 2 below provides exemplary branched linker 2-payload (BL2P) structures according to the present disclosure. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0265] In one embodiment, a compound (i.e., linker-payload) according to the present disclosure has the following structure: [ka] or a pharmaceutically acceptable salt thereof.

[0266] In some embodiments, the Ab is conjugated to a linker-payload or payload disclosed in WO2015 / 157592, such as compound T32 disclosed therein. In some embodiments, the Ab is conjugated to deruxtecan (DXd), optionally via a linker comprising GGFG.

[0267] In some embodiments, the Ab is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 10. In some aspects, the Ab is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising the HCVR amino acid sequence of SEQ ID NO: 2 and the LCVR amino acid sequence of SEQ ID NO: 10.

[0268] In some embodiments, the Ab is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 22 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 28. In some aspects, the Ab is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising the HCVR amino acid sequence of SEQ ID NO: 22 and the LCVR amino acid sequence of SEQ ID NO: 28.

[0269] In some embodiments, the Ab is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising a CDR within the HCVR amino acid sequence of SEQ ID NO: 40 and a CDR within the LCVR amino acid sequence of SEQ ID NO: 44. In some aspects, the Ab is an anti-FGFR2 antibody, or antigen-binding fragment thereof, comprising the HCVR amino acid sequence of SEQ ID NO: 40 and the LCVR amino acid sequence of SEQ ID NO: 44.

[0270] In some embodiments, L is a cleavable linker. In some embodiments, L is a non-cleavable linker. In some embodiments, L comprises a dipeptide. In some embodiments, L comprises a PAB moiety.

[0271] In some embodiments, L comprises a moiety having the structure: [ka]

[0272] In some embodiments, L comprises a moiety having the structure: [ka]

[0273] In some embodiments, L comprises a moiety having the structure: [ka]

[0274] In some embodiments, L comprises a moiety having the structure: [ka]

[0275] In some embodiments, Pay is tubulysin.

[0276] In some embodiments, Pay is a camptothecin analog.

[0277] In some embodiments, Pay is a maytansinoid.

[0278] In some embodiments, Pay is: [ka] In the formula, R 1 is alkyl.

[0279] In some embodiments, Pay is: [ka]

[0280] In some embodiments, Pay is: [ka]

[0281] In some embodiments, n is an integer from 2 to 5.

[0282] In some embodiments, -L-Pay is: [ka] During the ceremony, [ka] is the binding to the antibody.

[0283] In some embodiments, -L-Pay is: [ka] During the ceremony, [ka] is the binding to the antibody.

[0284] In some embodiments, -L-Pay is: [ka] During the ceremony, [ka] is the binding to the antibody.

[0285] In some embodiments, -L-Pay is: [ka] During the ceremony, [ka] is the binding to the antibody.

[0286] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein: Ab is (i) an anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 4, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 8, an LCDR1 having the amino acid sequence of SEQ ID NO: 12, an LCDR2 having the amino acid sequence of SEQ ID NO: 14, and an LCDR3 having the amino acid sequence of SEQ ID NO: 16; L-Pay is: [ka] (Maytansinoid 1ALP) wherein: [ka] is the bond to the antigen-binding protein, and n is an integer of 2 to 5.

[0287] In some embodiments, the conjugate has the structure: Ab-[L-Pay] n wherein: Ab is (ii) an anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 24, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 26, an LCDR1 having the amino acid sequence of SEQ ID NO: 30, an LCDR2 having the amino acid sequence of SEQ ID NO: 32, and an LCDR3 having the amino acid sequence of SEQ ID NO: 34; L-Pay is: [ka] (Maytansinoid 1ALP) wherein: [ka] is the bond to the antigen-binding protein, and n is an integer of 2 to 5.

[0288] In some embodiments, the conjugate has the structure: Ab-[L-Pay] n wherein: Ab is (i) An anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 4, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 8, an LCDR1 having the amino acid sequence of SEQ ID NO: 12, an LCDR2 having the amino acid sequence of SEQ ID NO: 14, and an LCDR3 having the amino acid sequence of SEQ ID NO: 16, wherein Pay is: [ka] During the ceremony, [ka] is the bond to the antigen-binding protein, and n is an integer of 2 to 5.

[0289] In some embodiments, the conjugate has the structure: Ab-[L-Pay] n wherein: Ab is (ii) an anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 24, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 26, an LCDR1 having the amino acid sequence of SEQ ID NO: 30, an LCDR2 having the amino acid sequence of SEQ ID NO: 32, and an LCDR3 having the amino acid sequence of SEQ ID NO: 34; Pay is below, [ka] During the ceremony, [ka] is the bond to the antigen-binding protein, and n is an integer of 2 to 5.

[0290] In some embodiments, the conjugate has the structure: Ab-[L-Pay] n wherein: Ab is (i) an anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 4, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 8, an LCDR1 having the amino acid sequence of SEQ ID NO: 12, an LCDR2 having the amino acid sequence of SEQ ID NO: 14, and an LCDR3 having the amino acid sequence of SEQ ID NO: 16; L-Pay is [ka] or a positional isomer thereof, wherein: [ka] is a bond to the heavy chain glutamine. In some embodiments, n is 2. In certain embodiments, n is 2 and L-Pay is bonded to Q295 glutamine. In some embodiments, n is 4. In certain embodiments, n is 4 and L-Pay is bonded to Q295 glutamine and Q297 glutamine.

[0291] In some embodiments, the conjugate has the structure: Ab-[L-Pay] n wherein: Ab is (ii) an anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 24, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 26, an LCDR1 having the amino acid sequence of SEQ ID NO: 30, an LCDR2 having the amino acid sequence of SEQ ID NO: 32, and an LCDR3 having the amino acid sequence of SEQ ID NO: 34; L-Pay is [ka] or a positional isomer thereof, wherein: [ka] is a bond to the heavy chain glutamine. In some embodiments, n is 2. In certain embodiments, n is 2 and L-Pay is bonded to Q295 glutamine. In some embodiments, n is 4. In certain embodiments, n is 4 and L-Pay is bonded to Q295 glutamine and Q297 glutamine.

[0292] In some embodiments, the conjugate has the structure: Ab-[L-Pay] n wherein: Ab is (i) An anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 4, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 8, an LCDR1 having the amino acid sequence of SEQ ID NO: 12, an LCDR2 having the amino acid sequence of SEQ ID NO: 14, and an LCDR3 having the amino acid sequence of SEQ ID NO: 16, wherein Pay is: [ka] [ka] During the ceremony, [ka] represents the point of attachment to the antibody via the linker, and n is an integer from 2 to 8.

[0293] In some embodiments, the conjugate has the structure: Ab-[L-Pay] n wherein: Ab is (ii) an anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 24, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 26, an LCDR1 having the amino acid sequence of SEQ ID NO: 30, an LCDR2 having the amino acid sequence of SEQ ID NO: 32, and an LCDR3 having the amino acid sequence of SEQ ID NO: 34; Pay is below, [ka] [ka] During the ceremony, [ka] represents the point of attachment to the antibody via the linker, and n is an integer from 2 to 8.

[0294] In some embodiments, the conjugate has the structure: Ab-[L-Pay] n wherein: Ab is (i) an anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 4, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 8, an LCDR1 having the amino acid sequence of SEQ ID NO: 12, an LCDR2 having the amino acid sequence of SEQ ID NO: 14, and an LCDR3 having the amino acid sequence of SEQ ID NO: 16; L-Pay is: [ka] wherein L-Pay is attached to Ab via the heavy chain glutamine. In some embodiments, n is 2. In certain embodiments, n is 2 and L-Pay is attached to Q295 glutamine. In some embodiments, n is 4. In certain embodiments, n is 4 and L-Pay is attached to Q295 glutamine and Q297 glutamine. In some embodiments, n is 6. In some embodiments, n is 8. In some embodiments, n is 8, L is branched, and L-Pay is attached to Q295 and Q297.

[0295] In some embodiments, the conjugate has the structure: Ab-[L-Pay] n wherein: Ab is (ii) an anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 24, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 26, an LCDR1 having the amino acid sequence of SEQ ID NO: 30, an LCDR2 having the amino acid sequence of SEQ ID NO: 32, and an LCDR3 having the amino acid sequence of SEQ ID NO: 34; L-Pay is: [ka] wherein L-Pay is attached to Ab via the heavy chain glutamine. In some embodiments, n is 2. In certain embodiments, n is 2 and L-Pay is attached to Q295 glutamine. In some embodiments, n is 4. In certain embodiments, n is 4 and L-Pay is attached to Q295 glutamine and Q297 glutamine. In some embodiments, n is 6. In some embodiments, n is 8. In some embodiments, n is 8, L is branched, and L-Pay is attached to Q295 and Q297.

[0296] The antibody-drug conjugates described herein can be prepared using conjugation conditions known to those skilled in the art (see, e.g., Doronina et al. Nature Biotechnology 2003, 21, 7, 778, which is incorporated herein by reference in its entirety). In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof drug conjugate is prepared by contacting an anti-FGFR2 antibody or antigen-binding fragment thereof described herein with a compound comprising a desired linker and a cytotoxic agent, wherein the linker bears a moiety reactive with the antibody or antigen-binding protein, e.g., at a desired residue of the antibody or antigen-binding protein.

[0297] In some embodiments, the antibody-drug conjugates provided herein are conjugated at Q295 glutamine and / or Q297 glutamine in the heavy chain. Antibodies containing Q297 glutamine in the heavy chain can be prepared using techniques known in the art, for example, via an N297Q mutation. See, for example, Bioconjugate Chem. 2014, 25, 3, 569 (2014). Such conjugation methods can provide antibody-drug conjugates with a DAR of 2 or 4. In such embodiments in which the linker is branched, the conjugation methods can provide antibody-drug conjugates with a DAR of 6 or 8.

[0298] In some embodiments, such methods include introducing a first reactive moiety at a heavy chain glutamine by reacting the antibody with a primary amine compound containing the first reactive moiety in the presence of transglutaminase to produce an antibody containing a first reactive moiety at Q295 and optionally Q297. This product can then be reacted with a linker and a payload having a complementary second reactive moiety to provide an antibody-drug conjugate provided herein. In some embodiments, the first reactive moiety is an azide. In some embodiments, the second reactive moiety is a cycloalkyne. In some embodiments, the payload having a linker and a complementary second reactive moiety is: [ka] It can be prepared using the methods described in U.S. Patent Application No. 16 / 724,164, filed December 20, 2019 (e.g., compound LP4 described therein). In certain embodiments, the primary amine compound is: [ka]

[0299] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof described herein can be administered in combination with the compound [ka] wherein the anti-FGFR2 is functionalized with an azide.

[0300] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof described herein are provided herein as compounds having the following formula A: 1 A compound having [ka] and an aqueous diluent.

[0301] In some embodiments, Formula A 1 In some embodiments, the compound of formula A is present in stoichiometric excess. 1 The compound is present in a 5-6 fold stoichiometric excess. In some embodiments, the aqueous diluent comprises HEPES. In some embodiments, the aqueous diluent comprises DMA.

[0302] In some embodiments, Formula A 1 The compound of formula A 2 or A 3 is a compound of: [ka]

[0303] In some embodiments, Formula A 2 The compound of formula A is stereoisomerically pure. 3 In some embodiments, the compound of formula A 1 The compound of formula A 1 or A 2 A 1 or A 2 The compound is present in a diastereomeric excess of greater than 50%. In certain embodiments, the diastereomeric excess is greater than 70%. In certain embodiments, the diastereomeric excess is greater than 90%. In certain embodiments, the diastereomeric excess is greater than 95%.

[0304] The term "diastereomeric excess" refers to the difference in the molar fraction of a desired single diastereomer compared to the remaining diastereomers in a composition. Diastereomeric excess is calculated as follows: (amount of single diastereomer) - (amount of other diastereomer) / 1. For example, a composition containing 90% 1 and 10% 2, 3, 4, or a mixture thereof has an 80% diastereomeric excess [(90 - 10) / 1]. A composition containing 95% 1 and 5% 2, 3, 4, or a mixture thereof has a 90% diastereomeric excess [(95 - 5) / 1]. A composition containing 99% 1 and 1% 2, 3, 4, or a mixture thereof has a 98% diastereomeric excess [(99 - 1) / 1]. Diastereomeric excess can be calculated similarly for any one of 1, 2, 3, or 4.

[0305] In some embodiments, Formula A 1 The compound of formula (a) is: [ka] with a compound of formula (b): [ka] in the presence of silica gel and a diluent. In some embodiments, the diluent comprises an organic solvent and water.

[0306] Also provided herein is a product prepared by the following process: (i) A compound of formula (a): [ka] with a compound of formula (b): [ka] in the presence of silica gel and a diluent to synthesize an intermediate; and (ii) contacting an anti-FGFR2 antibody or antigen-binding fragment thereof described herein with the intermediate and an aqueous diluent;

[0307] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof described herein are administered with a compound having the following formula B: [ka] wherein LG is a leaving group, and an aqueous diluent.

[0308] In some embodiments, the compound of Formula B is present in stoichiometric excess. In some embodiments, the compound of Formula B is present in a 5-6 fold stoichiometric excess. In some embodiments, the aqueous diluent comprises HEPES. In some embodiments, the aqueous diluent comprises DMA. In some embodiments, -C(O)-LG is an ester, e.g., NHS or a trifluorophenyl ester.

[0309] In some embodiments, the compound of formula B has the following formula B 1 is a compound of: [ka]

[0310] In some embodiments, Formula B 1 The compound of formula C is: [ka] with N-hydroxysuccinimide (NHS), a peptide coupling reagent, and an organic diluent. Suitable peptide coupling reagents include those that activate or render reactive carboxylic acid moieties for reaction with nucleophiles. In certain embodiments, the peptide coupling reagent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). In some embodiments, the organic solvent is dichloromethane.

[0311] In some embodiments, the compound of formula C is a compound of formula D: [ka] Compound D can be prepared by contacting adipic acid, a peptide coupling agent, and an organic solvent. In certain embodiments, the peptide coupling agent is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ). In certain embodiments, the organic solvent comprises dichloromethane. Compound D can be prepared as described in WO2014 / 145090.

[0312] Epitope mapping and related technologies The term "epitope" refers to a site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed from contiguous amino acids and from non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Typically, an epitope contains at least 3, more usually at least 5, or 8-10 amino acids in a unique spatial conformation.

[0313] Provided herein are anti-FGFR2 antibodies that interact with one or more amino acids present in the FGFR2 protein.

[0314] The epitope to which the antibody or antigen-binding fragment thereof binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the FGFR2 protein. Alternatively, the relevant epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of FGFR2 (e.g., a conformational epitope).

[0315] Various techniques known to those skilled in the art can be used to determine the epitope on FGFR2 with which the antibodies and antigen-binding domains of the present disclosure interact. Exemplary techniques that can be used to determine the epitope or binding domain of a particular antibody or antigen-binding domain include, for example, point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection, and peptide cleavage analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids in a polypeptide that interact with an antibody is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves labeling a protein of interest with deuterium, followed by binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids not part of the interface. As a result, amino acids that form part of the protein / antibody interface may retain deuterium and therefore exhibit a relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein undergoes protease cleavage and mass spectrometry analysis to reveal deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography can also be used to identify amino acids within a polypeptide with which an antibody interacts.

[0316] Further provided herein are anti-FGFR2 antibodies or antigen-binding fragments thereof that bind to the same epitope as any of the specific exemplary antibodies or antigen-binding fragments thereof described herein (e.g., antibodies comprising any of the amino acid sequences described in Table 3 herein). Also provided herein are anti-FGFR2 antibodies or antigen-binding fragments thereof that compete for binding to FGFR2 with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences described in Table 3 herein).

[0317] By using routine methods known in the art and exemplified herein, it is easy to determine whether an antibody binds to the same epitope as a reference anti-FGFR2 antibody or competes for binding with the reference anti-FGFR2 antibody. For example, to determine whether a test antibody binds to the same epitope as a reference anti-FGFR2 antibody provided by the present invention, the reference antibody is bound to an FGFR2 protein. The ability of the test antibody to bind to an FGFR2 molecule is then evaluated. If the test antibody can bind to FGFR2 after saturation binding with the reference anti-FGFR2 antibody, it can be concluded that the test antibody binds to a different epitope from the reference anti-FGFR2 antibody. On the other hand, if the test antibody cannot bind to an FGFR2 molecule after saturation binding with the reference anti-FGFR2 antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-FGFR2 antibody. Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits binding of the other antibody by at least 50%, but preferably by 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody.

[0318] To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-FGFR2 antibody, the above-described binding method is carried out in two ways: in the first way, the reference antibody is bound to FGFR2 molecules under saturating conditions, and then the binding of the test antibody to the FGFR2 molecule is evaluated. In the second way, the test antibody is bound to FGFR2 molecules under saturating conditions, and then the binding of the reference antibody to the FGFR2 molecule is evaluated. In both ways, if only the first (saturating) antibody can bind to the FGFR2 molecule, it is concluded that the test antibody and the reference antibody compete for binding to FGFR2. As will be understood by those skilled in the art, an antibody that competes for binding to a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0319] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present disclosure to generate human antibodies that specifically bind to FGFR2. Antibodies against FGFR2 can be generated using an immunogen comprising any one of the following: In certain embodiments, the antibodies of the present disclosure are obtained from mice immunized with FGFR2b, e.g., GenBank Accession No. NP_075259.4 (SEQ ID NO: 55), or the extracellular domain of a recombinant human FGFR2b isoform (Accession No. NP_075259.4) fused to a mouse Fc domain (Accession No. P01863) (SEQ ID NO: 56). Alternatively, FGFR2 protein or a fragment thereof can be produced, modified, and used as an immunogen using standard biochemical techniques. In one embodiment, the immunogen is a recombinant FGFR2 protein or a fragment thereof. In certain embodiments, the immunogen may be a commercially available FGFR2 protein. In certain embodiments, one or more booster injections may be administered. In certain embodiments, booster injections may include one or more commercially available FGFR2 proteins. In certain embodiments, the immunogen may be recombinant FGFR2 protein expressed in E. coli or any other eukaryotic or mammalian cells, such as Chinese hamster ovary (CHO) cells.

[0320] Using VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, a high-affinity chimeric antibody against FGFR2 having a human variable region and a mouse constant region is first isolated. VELOCIMMUNE® technology involves generating transgenic mice whose genomes contain human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antibodies containing the human variable region and the mouse constant region in response to antigenic challenge. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0321] Generally, VELOCIMMUNE® mice are challenged with an antigen of interest, and lymphocytes (such as B cells) are collected from the mice that express antibodies. Lymphocytes may be fused with myeloma cell lines to prepare immortalized hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions may be isolated and linked to constant regions of the desired heavy and light chain isotype. Such antibody proteins may be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the light and heavy chain variable domains may be isolated directly from antigen-specific lymphocytes.

[0322] First, a high-affinity chimeric antibody having a human variable region and a mouse constant region is isolated. As in the experimental section below, the antibody is characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant region is replaced with a desired human constant region to generate a fully human antibody, such as a wild-type or modified IgG1 or IgG4. While the constant region selected can vary depending on the specific application, the characteristics of high-affinity antigen binding and target specificity reside in the variable region. In some instances, fully human anti-FGFR2 antibodies are isolated directly from antigen-positive B cells.

[0323] biological equivalent The anti-FGFR2 antibodies or antigen-binding fragments thereof provided herein include proteins having amino acid sequences that differ from the amino acid sequences of the described antibodies but retain the ability to bind to human FGFR2. Such variant antibodies and antibody fragments contain one or more amino acid additions, deletions, or substitutions compared to the parent sequence, but exhibit essentially the same biological activity as the described antibody. Similarly, DNA sequences encoding the anti-FGFR2 antibodies of the present disclosure include sequences that contain one or more nucleotide additions, deletions, or substitutions compared to the disclosed sequences, but encode anti-FGFR2 antibodies or antibody fragments that are essentially biologically equivalent to the anti-FGFR2 antibodies or antibody fragments of the present disclosure. Examples of such variant amino acid sequences and DNA sequences are discussed above.

[0324] Two antigen-binding proteins or antibodies are considered to be bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in the rate and extent of absorption when administered in the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies will be considered equivalents or pharmaceutical substitutes if their extent of absorption is equivalent but their rate is not, and they may still be considered bioequivalent because the difference in absorption rate is intentional and reflected in the labeling, is not essential to achieving effective body drug levels, e.g., with chronic use, and is not considered medically significant for the particular pharmaceutical product being tested.

[0325] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0326] In one embodiment, two antigen binding proteins are bioequivalent if a patient can make one or more switches between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or a decrease in efficacy, compared to continuous therapy without such switches.

[0327] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms and to the known extent of such mechanisms for one or more conditions of use.

[0328] Bioequivalence may be demonstrated by in vivo and in vitro methods, including, for example, (a) in vivo tests in humans or other mammals that measure the concentration of the antibody or its metabolites in blood, plasma, serum, or other biological fluid as a function of time, (b) in vitro tests that correlate with and reasonably predict in vivo bioavailability data in humans, (c) in vivo tests in humans or other mammals that measure the relevant acute pharmacological effects of the antibody (or its target) as a function of time, and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0329] Biologically equivalent variants of the anti-FGFR2 antibodies or antigen-binding fragments thereof provided herein may be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent unnecessary or incorrect intramolecular disulfide bridge formation during renaturation. In other contexts, biologically equivalent antibodies may include anti-FGFR2 antibody variants containing amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that abolish or eliminate glycosylation.

[0330] Species selectivity and species cross-reactivity According to certain embodiments, the present disclosure provides anti-FGFR2 antibodies (and antigen-binding molecules comprising anti-FGFR2 antigen-binding domains) that bind to human FGFR2 but not to FGFR2 from other species. The present disclosure also includes anti-FGFR2 antibodies (and antigen-binding molecules comprising anti-FGFR2 antigen-binding domains) that bind to human FGFR2 and FGFR2 from one or more non-human species. For example, anti-FGFR2 antibodies and antigen-binding molecules may bind to human FGFR2, but may or may not, in some cases, bind to one or more FGFR2 from mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee.

[0331] Therapeutic Formulations and Administration Provided herein are pharmaceutical compositions comprising anti-FGFR2 antibodies, including anti-FGFR2 ADCs of the present invention. The pharmaceutical compositions may be formulated with suitable carriers, excipients, and other agents that improve transportation, delivery, tolerability, etc.

[0332] In some aspects, a pharmaceutical composition comprising a therapeutically effective amount of one or more isolated human monoclonal antibodies, or antigen-binding fragments thereof, described herein, together with one or more pharmaceutically acceptable excipients.

[0333] Therapeutic uses of antibodies Provided herein are methods comprising administering to a subject in need thereof a therapeutic composition comprising an anti-FGFR2 antibody or antigen-binding fragment thereof, including an anti-FGFR2 antibody ADC, comprising any of the sequences described herein. The therapeutic composition can comprise an anti-FGFR2 antibody or antigen-binding fragment thereof, including an anti-FGFR2 antibody ADC, disclosed herein, and a pharmaceutically acceptable carrier or diluent. In some embodiments, the therapeutic composition is used in the manufacture of a medicament for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by FGFR2 expression, signaling, or activity, or treatable by blocking FGFR2 signaling or otherwise inhibiting FGFR2 activity and / or signaling. In some embodiments, the therapeutic composition is used in the manufacture of a medicament for treating cancer.

[0334] Anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, are useful, inter alia, for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by FGFR2 expression, signaling, or activity, or treatable by blocking FGFR2 signaling or otherwise inhibiting FGFR2 activity and / or signaling.

[0335] For example, anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs of the present disclosure, are useful for treating tumors that express (or overexpress) FGFR2, particularly anti-FGFR2b. In some embodiments, anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, bind to FGFR2b-positive cells, e.g., SNU-16 and MFM-223 cells. In some embodiments, anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, minimally bind to FGFR2c-positive cells, e.g., NCI-H716 cells. In some embodiments, anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, poorly bind to cells lacking FGFR2 expression, e.g., IM-9 cells.

[0336] In some embodiments, anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, exhibit cell killing of FGFR2b-positive cells but not low FGFR2 expressers. In some aspects, cell killing is less than about 15%, less than about 10%, less than about 5%, or less than about 2% of cells expressing low levels of FGFR2.

[0337] In some embodiments, anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, exhibit cell killing of FGFR2b-positive cells but not FGFR2c-positive cells. In some aspects, cell killing is less than about 35%, less than about 30%, less than about 20%, less than about 10%, or less than about 1% of cells expressing FGFR2c.

[0338] In certain embodiments, anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, are used to treat one or more of the following cancers: astrocytoma, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cervical cancer, renal clear cell carcinoma, colorectal cancer, microsatellite-intermediate colorectal cancer, cutaneous squamous cell carcinoma, diffuse large B-cell lymphoma, endometrial cancer, esophageal cancer, fibroid cancer, and ovarian cancer. Sarcoma, gastric cancer, glioblastoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, leukemia, liver cancer, leiomyosarcoma, lung cancer, lymphoma, melanoma, mesothelioma, myeloma, nasopharyngeal carcinoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, primary and / or recurrent carcinoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, squamous cell carcinoma, synovial sarcoma, thyroid cancer, triple-negative breast cancer, uterine cancer, and Wilms' tumor. In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic and / or recurrent cancer.

[0339] In some embodiments, anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, may be used to treat primary and / or metastatic tumors positive for FGFR2b, i.e., tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood-forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye.

[0340] In the context of the therapeutic methods described herein, the anti-FGFR2 antibody or antigen-binding fragment thereof may be administered as monotherapy (i.e., as the only therapeutic agent), in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein), or as an ADC (examples of which are described elsewhere herein).

[0341] Accordingly, provided herein are methods of treating cancer in a subject suffering from an FGFR2-overexpressing tumor. The methods comprise administering to the subject an anti-FGFR2 antibody or antigen-binding fragment thereof provided herein. In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises (i) an HCDR1 having the amino acid sequence of SEQ ID NO: 4, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 8, an LCDR1 having the amino acid sequence of SEQ ID NO: 12, an LCDR2 having the amino acid sequence of SEQ ID NO: 14, and an LCDR3 having the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises (ii) an HCDR1 having the amino acid sequence of SEQ ID NO: 24, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 26, an LCDR1 having the amino acid sequence of SEQ ID NO: 30, an LCDR2 having the amino acid sequence of SEQ ID NO: 32, and an LCDR3 having the amino acid sequence of SEQ ID NO: 34.

[0342] In certain aspects, the cancer is selected from the group consisting of breast invasive ductal carcinoma, gastric fundic adenocarcinoma, esophageal adenocarcinoma, colon adenocarcinoma, and gastroesophageal junction adenocarcinoma.

[0343] Also provided herein are methods for treating cancer, reducing tumor growth, and / or causing tumor regression in a subject. The methods include administering to a subject in need thereof an antibody-drug conjugate (ADC) comprising an anti-FGFR2 antibody or antigen-binding fragment thereof and a cytotoxin, wherein the anti-FGFR2 antibody or antigen-binding fragment thereof comprises: (i) an HCDR1 having the amino acid sequence of SEQ ID NO: 4, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 8, an LCDR1 having the amino acid sequence of SEQ ID NO: 12, an LCDR2 having the amino acid sequence of SEQ ID NO: 14, and an LCDR3 having the amino acid sequence of SEQ ID NO: 16; or (ii) an HCDR1 having the amino acid sequence of SEQ ID NO: 24, an HCDR2 having the amino acid sequence of SEQ ID NO: 6, an HCDR3 having the amino acid sequence of SEQ ID NO: 26, an LCDR1 having the amino acid sequence of SEQ ID NO: 30, an LCDR2 having the amino acid sequence of SEQ ID NO: 32, and an LCDR3 having the amino acid sequence of SEQ ID NO: 34. In some embodiments, the cytotoxin is tubulysin. In some embodiments, the cytotoxin is maytansinoid. In some embodiments, the cytotoxin is a camptothecin analog, hi some embodiments, the cytotoxin is an auristatin.

[0344] Combination Therapies and Formulations Provided herein are compositions and therapeutic formulations comprising any of the anti-FGFR2 antibodies, including anti-FGFR2 antibody ADCs described herein, or antigen-binding fragments thereof, in combination with one or more additional therapeutically active ingredients, as well as methods of treatment comprising administering such combinations to a subject in need thereof.

[0345] Anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, may be co-formulated with and / or administered in combination with one or more additional therapeutically active ingredients selected from the group consisting of: another antagonist of FGFR2, e.g., an antagonist of FGFR2b (e.g., bemarituzumab, or aprtuzumab, or aprtuzumab ixadotin), an antagonist of HER2 / ErbB2 (e.g., anti-ErbB2 [e.g., trastuzumab]), or T-DM1 {KADCYLA®}, or trastuzumab deruxtecan {T-SXD; a DNA topoisomerase 1 inhibitor}], or a small molecule inhibitor of ErbB2 activity), an antagonist of another EGFR family member such as ErbB3 or ErbB4 (e.g., an anti-ErbB3 or anti-ErbB4 antibody, or a small molecule inhibitor of ErbB3 or ErbB4 activity), a MET antagonist (e.g., an anti-MET antibody [e.g., onartuzumab, emtuzumab, betuzumab, and H4H14639D], or small molecule inhibitors of MET), EGFR antagonists (e.g., anti-EGFR antibodies [e.g., cetuximab or panitumumab] or small molecule inhibitors of EGFR [e.g., gefitinib or erlotinib]), EGFRvIII antagonists (e.g., anti-EGFRvIII antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-08 79, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies, or small molecule kinase inhibitors such as imatinib mesylate or sunitinib malate), PDGF ligand inhibitors (e.g., anti-PDGF-A, -B, -C, or -D antibodies, aptamers, siRNA, etc.), VEGF antagonists (e.g., VEGF-Trap such as aflibercept, e.g., US 7,087,See, e.g., 411 (also referred to herein as "VEGF-inhibitory fusion proteins"), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US 2009 / 0142354, such as REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US 2011 / 0027286, such as H1H685P), FOLH1 antagonists (e.g., anti-FOLH1 antibodies), antagonists of STEAP1 or STEAP2 (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies). , MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA9 antibodies), uroplakin antagonists (e.g., anti-uroplakin [e.g., anti-UPK3A] antibodies), MUC16 antagonists (e.g., anti-MUC16 antibodies), Tn antigen antagonists (e.g., anti-Tn antibodies), CLEC12A antagonists (e.g., anti-CLEC12A antibodies), TNFRSF17 antagonists (e.g., anti-TNFRSF17 antibodies), LGR5 antagonists (e.g., anti-LGR5 antibodies), monovalent CD20 antagonists (e.g., monovalent anti-CD20 antibodies such as rituximab), CD20×CD3 bispecific antibodies, PD-1 blockers (e.g., anti-PD-1 antibodies such as pembrolizumab or nivolumab), and the like. Other agents that may be beneficially administered in combination with the antibodies provided herein include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, and IL-18, or their respective receptors.

[0346] For example, a PD-1 inhibitor, such as an anti-PD-1 antibody, may be combined with an anti-FGFR2 antibody or antigen-binding fragment thereof, or antibody-drug conjugate described herein. Target patient populations include, in particular, patients with tumors that overexpress FGFR2 mutations or express mutant FGFR2, such as patients with breast cancer that expresses FGFR2.

[0347] Provided herein are compositions and therapeutic formulations comprising an anti-FGFR2 antibody or antigen-binding fragment thereof, including the anti-FGFR2 antibody ADCs described herein, in combination with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine (trimethylol melamine). ethylenimines and methylamelamines, including methylolomelamine; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, and the like; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, etc.; antibiotics, such as aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esophagus, rubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, and the like; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU), and the like;Folic acid analogues, such as denopterin, methotrexate, pteropterin, trimetrexate, etc.; purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.; androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane, etc.; folic acid replenishers, such as folinic acid, etc.; aceglatone; aldophosphamide glycosides glycoside); aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarubicin; podo phyllic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g., paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere™, Aventis, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum;Examples include etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included within this definition are antihormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens, including tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0348] Anti-FGFR2 antibodies or antigen-binding fragments thereof, including anti-FGFR2 antibody ADCs, may also be administered in combination with and / or co-formulated with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-gamma, and / or NSAIDs.

[0349] The additional therapeutically active ingredient, for example, any of the agents listed above or their derivatives, may be administered immediately prior to, simultaneously with, or immediately following administration of an anti-FGFR2 antibody or antigen-binding fragment thereof, including an anti-FGFR2 antibody ADC (for purposes of this disclosure, such administration regimens are considered administration of the antibody "in combination with" the additional therapeutically active ingredient). The present disclosure includes pharmaceutical compositions in which an anti-FGFR2 antibody or antigen-binding fragment thereof, including an anti-FGFR2 antibody ADC, is co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein.

[0350] Dosing regimen According to certain embodiments, multiple doses of an anti-FGFR2 antibody or antigen-binding fragment thereof, including an anti-FGFR2 antibody ADC, or a pharmaceutical composition comprising a combination of an anti-FGFR2 antibody or anti-FGFR2 antibody ADC and any of the additional therapeutically active agents mentioned herein, may be administered to a subject over a defined time course. The method according to this aspect comprises sequentially administering to a subject multiple doses of an anti-FGFR2 antibody or antigen-binding fragment thereof, including an anti-FGFR2 antibody ADC provided herein. As used herein, "sequentially administering" means that each dose of the antibody or ADC is administered to the subject at different time points, e.g., on different days, separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering to a patient a single initial dose of an anti-FGFR2 antibody or antigen-binding fragment thereof, including an anti-FGFR2 antibody ADC, followed by one or more secondary doses of the anti-FGFR2 antibody or antigen-binding fragment thereof or anti-FGFR2 antibody ADC, and optionally followed by one or more tertiary doses of the anti-FGFR2 antibody or antigen-binding fragment thereof or ADC.

[0351] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration of an anti-FGFR2 antibody or antigen-binding fragment thereof, or an anti-FGFR2 antibody ADC. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-FGFR2 antibody or antigen-binding fragment thereof, or anti-FGFR2 antibody ADC, but may generally differ from one another in terms of administration frequency. However, in certain embodiments, the amount of antibody contained in the initial, secondary, and / or tertiary doses differs from one another (e.g., adjusted accordingly) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of a treatment regimen, with subsequent doses administered less frequently (e.g., "maintenance doses"). Diagnostic uses of antibodies

[0352] The anti-FGFR2 antibodies or antigen-binding fragments thereof of the present disclosure can also be used, for example, for diagnostic purposes, to detect and / or measure FGFR2, e.g., FGFR2b, or FGFR2b-expressing cells in a sample. For example, an anti-FGFR2 antibody or a fragment thereof can be used to diagnose a condition or disease characterized by abnormal expression (e.g., overexpression, underexpression, lack of expression, etc.) of FGFR2. An exemplary diagnostic assay for FGFR2 includes, for example, contacting a sample obtained from a patient with an anti-FGFR2 antibody or antigen-binding fragment thereof, where the antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-FGFR2 antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125 The FGFR2 probe can be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Specific examples of assays that can be used to detect or measure FGFR2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immuno-PET (e.g., 89 Zr, 64 Cu), and fluorescence-activated cell sorting (FACS). In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof is labeled as described in WO2018 / 044540, the entire contents of which are incorporated herein by reference in their entirety. In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof is labeled as follows: [ka]

[0353] In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCVR amino acid sequence of SEQ ID NO: 2 and the LCVR amino acid sequence of SEQ ID NO: 10 is labeled to biosensor 1.

[0354] In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCVR amino acid sequence of SEQ ID NO: 22 and the LCVR amino acid sequence of SEQ ID NO: 28 is labeled to biosensor 1.

[0355] In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof comprising the HCVR amino acid sequence of SEQ ID NO: 40 and the LCVR amino acid sequence of SEQ ID NO: 44 is labeled to biosensor 1.

[0356] Samples that can be used in the FGFR2 diagnostic assay according to the present disclosure include any tissue or body fluid sample that can be obtained from a subject. Generally, FGFR2 levels are measured in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal FGFR2 levels or activity) to first establish a baseline or standard FGFR2 level. The baseline FGFR2 level can then be compared with the FGFR2 level measured in a sample obtained from an individual suspected of having an FGFR2-related disease or condition. [Example]

[0357] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions provided herein, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. 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.

[0358] The two comparative antibodies used in the examples are referred to as Comp1 and Comp2. Comp1 (Five Prime Therapeutics) is a humanized anti-FGFR2b isoform bispecific antibody identified by Catenacci et al. (2020, Phase I Escalation and Expansion Study of Bemarituzumab (FPA144) in Patients With Advanced Solid Tumors and FGFR2b-Selected Gastroesophageal Adenocarcinoma, J Clin Onc, DOI https: / / doi.org / 10.1200 / JCO.19.01834). This same bispecific antibody is referred to as HuGAL-FR21 in U.S. Patent No. 8,603,987 (Galaxy Biotech). Comp2 (Bayer) is a humanized IgG1 antibody that binds to both FGFR2b and FGFR2c at the N-terminus. The Comp2 antibody is mentioned in Sommer et al. (2016, Preclinical Efficacy of Auristatin-based antibody-drug conjugate BAY 1187982 for the treatment of FGFR2-positive solid tumors, Cancer Res, 76(21), 6631-6639, doi:10.1158 / 0008-5472.CAN-16-0180) and in U.S. Patent Publication No. 2014 / 0322220. In that patent publication, the Comp2 antibody is referred to as TPP-1402, a variant of MO48-D01, which, according to U.S. Publication No. 2014 / 0322220, refers to heavy chain SEQ ID NO: 133 and light chain SEQ ID NO: 124.

[0359] The isotype control antibodies used herein are referred to as IC1 and IC2. Such antibodies may be conjugated to any of the linker payloads, including maytansinoid 1LP, which delivers a maytansinoid 1A payload; tubulysin 1LP, which delivers a tubulysin 1A payload; DXd1 LP (branched) and DXd2 LP (unbranched), which deliver DXd-G or DXd; and BAY-LP (BAY1187982; see Sommer et al., 2016, Preclinical Efficacy of Auristatin-based Antibody-Drug Conjugate BAY 1187982 for the Treatment of FGFR2-Positive Solid Tumors, Cancer Res, 76(21), 6631-6639, doi:10.1158 / 0008-5472.CAN-16-0180).

[0360] Example 1. Generation of human antibodies against FGFR2 Human antibodies against FGFR2 were obtained by immunizing genetically engineered mice containing DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions with an immunogen containing the extracellular domain of recombinant human FGFR2b isoform (accession number NP_075259.4) fused to a mouse Fc domain according to SEQ ID NO: 56 (accession number P01863).

[0361] The antibody immune response was monitored by an FGFR2-specific immunoassay. When the desired immune response was obtained, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines producing FGFR2-specific antibodies. Using this technique, several anti-FGFR2 chimeric antibodies (i.e., antibodies having a human variable domain and a mouse constant domain) were obtained. Furthermore, several fully human anti-FGFR2 antibodies were isolated directly from antigen-positive B cells without fusion with myeloma cells, as described in U.S. Patent No. 7,582,298, which is expressly incorporated herein by reference in its entirety. Using this method, fully human anti-FGFR2 antibodies (i.e., antibodies having a human variable domain and a human constant domain) were obtained.

[0362] Certain biological properties of exemplary anti-FGFR2 antibodies generated according to the methods of this example are detailed in the Examples set forth below.

[0363] Example 2. Heavy and Light Chain Variable Region Amino Acid Sequences, Heavy and Light Chain Amino Acid Sequences, and Nucleotide Sequences Table 3 lists the heavy and light chain variable regions and CDRs of exemplary anti-FGFR2 antibodies, as well as the amino acid sequence identifiers for the heavy and light chain sequences. The corresponding nucleic acid sequence identifiers are listed in Table 4. [Table 3] [Table 4]

[0364] The antibodies disclosed herein have fully human variable regions but can have mouse constant regions (e.g., mouse IgG1 Fc or mouse IgG2 Fc (a or b isotype)) or human constant regions (e.g., human IgG1 Fc or human IgG4 Fc). As will be understood by one of skill in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a mouse IgG1 Fc can be converted to an antibody having human IgG4, etc.), but in either case, the variable domains (including CDRs) indicated by the numerical identifiers shown in Tables 3 and 4 remain the same, and the binding characteristics to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain. In some examples, the antibodies disclosed herein have identical heavy chain variable region sequences but may or may not have the N297Q modification within the Fc domain of the complete heavy chain amino acid sequence.

[0365] Example 3. Biacore binding affinity and rate constants of human monoclonal anti-FGFR2 antibodies Equilibrium dissociation constants (K) for hFGFR2b.mmh binding to anti-FGFR2 antibodies conjugated with tubulysin ("tubulysin 1ALP"), auristatin ("auristatin LP1"), or mc-VC-PAB-NMe-C3-May ("maytansinoid 1ALP") were calculated. DThe binding activity (p < 0.05) was determined using a real-time surface plasmon resonance biosensor assay on a Biacore 2000 or Biacore 4000 instrument. The Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567 or Jackson catalog number 109-005-098) to capture anti-FGFR2 ADCs expressed with human constant regions and unmodified parent antibodies. Biacore binding studies were performed in HEPES-EP running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20). Human FGFR2b and human FGFR2c expressing C-terminal myc-myc-hexahistidine tags (hFGFR2b-MMH-SEQ ID NO: 57, hFGFR2c-MMH-SEQ ID NO: 58) were prepared in-house. Different concentrations (3-fold dilutions) of hFGFR2b-MMH or hFGFR2c-MMH (ranging from 30 nM to 1.1 nM) prepared in HBS-EP running buffer were injected over the anti-FGFR2 ADC or antibody capture surface at a flow rate of 50 μL / min. Association of hFGFR2b-MMH or hFGFR2c-MMH to the capture ADC and monoclonal antibody, respectively, was monitored for 4 min. Dissociation was then monitored for 10 min in HBS-EP running buffer. The anti-human Fc surface was regenerated with a short injection of 20 mM H3PO4. All binding kinetic experiments were performed at 25 °C. Kinetic association (k) was calculated by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve-fitting software. a ) and dissociation (k d ) rate constant was determined. All sensorgrams were double-referenced by subtracting the signal of the buffer injection sensorgram from the corresponding analyte sensorgram, which eliminated artifacts caused by dissociation of the antibody from the capture surface. The binding-dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the reaction rate constants as follows:

number

[0366] The binding kinetic parameters for the binding of hFGFR2b.mmh to anti-FGFR2 monoclonal antibodies at 25° C. are shown in Tables 5a and 5b. [Table 5a] [Table 5b]

[0367] At 25°C, the anti-FGFR2 monoclonal antibodies had K values ​​of 8.92 nM and 25.0 nM, respectively. D The anti-FGFR2 monoclonal antibody conjugates bound to hFGFR2b.mmh with K values ​​ranging from 12.8 nM to 48.1 nM. D Comp2 bound to hFGFR2b.mmh at 100 ng / mL. The isotype control antibody IC1 showed no binding. See Table 5a. In contrast, the anti-FGFR2 monoclonal antibody did not bind to hFGFR2c.mmh, demonstrating specificity for the FGFR2b protein. As expected, Comp2 bound to both hFGFR2b.mmh and hFGFR2c.mmh.

[0368] Example 4: Site-specific conjugation of anti-FGFR2 antibodies with Tubulysin 1A In this example, an anti-FGFR2 antibody was conjugated to the cytotoxin Tubulysin 1A, which has the following structure: [ka] The antibodies were conjugated via the linker moieties described in the procedures below to give ADCs and / or cyclization regioisomers thereof having the following structures: [ka] wherein Ab is an anti-FGFR2 antibody.

[0369] Site-specific conjugates of cyclooctyne-spacer-payload to wild-type antibodies or their antigen-binding fragments were produced in three steps. The first step involved deglycosylation of the wild-type antibody. The second step involved enzymatic attachment of a spacer moiety suitable for subsequent purification to the Q295 site of the deglycosylated antibody via microbial transglutaminase (MTG)-based cycloaddition, e.g., an azide-PEG3-amine spacer (hereafter referred to as "MTG-based" conjugation). The third step involved attachment of the cyclooctyne-spacer-payload to the azide-functionalized antibody via a [2+3] cycloaddition, e.g., a 1,3-dipolar cycloaddition between azide and cyclooctyne (also known as copper-free click chemistry). See Baskin, JM; Prescher, JA; Laughlin, ST; Agard, NJ; Chang, PV; Miller, IA; Lo, A.; Codelli, JA; Bertozzi, CRPNAS 2007, 104(43), 16793-7. Figure 1A shows an example of a linker-spacer-payload complex bearing a DIBAC moiety conjugated to an azide-functionalized antibody via a [2+3] cycloaddition. This process provides site-specific and stoichiometric conjugates in approximately 50-80% isolated yield. Figure 1B shows an example of a three-step site-specific conjugation procedure performed as follows:

[0370] Step 1: Preparation of deglycosylated antibody. Deglycosylation was performed to expose the conjugation sites. Anti-FGFR2 human IgG4 antibody (30 mg, 40 mg / mL in PBS, pH 5.5-8.0) was mixed with PNGase F enzyme (New England BioLabs, 500,000 U / mL, 2 μL of enzyme per mg of antibody, for a total of 60 μL). The reaction mixture was incubated overnight at 37°C with gentle stirring. Deglycosylation was monitored by ESI-MS. Upon completion of the reaction, the reaction mixture was used directly in the next step.

[0371] Step 2: Preparation of azide-functionalized antibody. Deglycosylated FGFR2 antibody (30 mg) in 1.5 mL of PBS (pH 7.2) was incubated with 200 molar equivalents of azido-PEG3-amine (MW = 218.26 g / mol) in the presence of MTG (ACTIVA TI, Ajinomoto, Japan) (0.06 mg MTG / mg antibody). The reaction was incubated at 37°C for 4 hours with gentle mixing, then at 25°C overnight. The reaction was monitored by ESI-MS. Upon completion of the reaction, excess azido-PEG3-amine and MTG were removed by SEC (Superdex 200 PG, GE Healthcare) to generate the azide-functionalized antibody. Azido-PEG3-amine was added to two Q295 sites on the antibody, resulting in a 404 Da increase in the 2DAR of the antibody-PEG3-azide conjugate.

[0372] This process can also be performed on antibodies with N297Q modifications in one or both heavy chains. In this example, azido-PEG3-amine is added to two Q295 sites and at least one 297Q site on the antibody, resulting in a 3DAR or 4DAR antibody-PEG3-azide conjugate.

[0373] Step 3: Preparation of site-specific conjugates by [2+3] click reaction between azide-functionalized glutaminyl-modified antibodies and cyclooctynes ​​containing linker-payloads (LPs). Generally, the azide-functionalized glutamyl-modified antibody is reacted with ≥6 molar equivalents of LP, such as a compound of the following structure: [ka] Azide-functionalized antibody-LP conjugates were prepared by incubating with mAb-PEG3-N3 at 25°C to 37°C for 3 to 24 hours. The progress of the reaction was monitored by ESI-MS. The absence of azide-functionalized antibody (mAb-PEG3-N3) indicated completion of the conjugation. Excess linker-payload (LP) and organic solvent were removed by desalting column or size-exclusion chromatography (SEC). The purified conjugates were analyzed by SEC-HPLC and ESI-MS. The monomer purity of the conjugates was >99% by SEC-HPLC analysis.

[0374] As a specific example, azide-functionalized anti-FGFR2 antibodies, such as mAb1 and mAb2 (23 mg) in 3.2 mL of PBS, were treated with 6 equivalents of Tubulysin 1A-LP (in DMA at a concentration of 10 mg / mL) overnight at 30 °C. Excess linker payload (LP) was removed by SEC (Superdex 200 PG, GE Healthcare). The final product was characterized by UV, SEC-HPLC (see Figure 2), and ESI-MS.

[0375] A list of DAR (ESI-MS) values ​​for antibody-tubulysin conjugates (ADCs) is shown in Table 6. Characterization of antibodies and ADCs by SEC-HPLC and LC-ESI-MS

[0376] The purified conjugates were analyzed by SEC-HPLC and ESI-MS using representative SEC and ESI-MS techniques.

[0377] Analytical SEC experiments were performed using a Waters 1515 instrument on a Superdex™ 200 Increase (1.0 x 30 cm) column at a flow rate of 0.80 mL / min using PBS pH 7.2 and monitored at λ = 280 nm using a Waters 2998 PDA. Analytical samples consisted of 30-80 μL of test sample. SEC results in Figure 2 show typical retention times for the monomeric mAb and its conjugates with minimal aggregation or degradation.

[0378] Intact mass measurements of ADC samples were performed by LC-ESI-MS to determine the drug-payload distribution profile and calculate the average DAR. Each test sample (20-50 ng, 5 μL) was reduced with DTT and then loaded onto an Acquity UPLC Protein BEH C4 column (10 Kpsi, 300 Å, 1.7 μm, 75 μm x 100 mm, catalog number 186003810). After desalting for 3 minutes, the protein was eluted and mass spectra were acquired using a Waters Synapt G2-Si mass spectrometer. [Table 6]

[0379] The ADCs generated in this experiment were used in the following examples.

[0380] Example 5: Conjugation of anti-FGFR2 antibodies with maytansinoid linker payloads In this example, an anti-FGFR2 antibody was conjugated to the cytotoxin maytansinoid 1A, which had the following structure: [ka] The antibodies were conjugated via the linker moieties described in the procedure below to yield ADCs with the following structures: [ka] wherein Ab is an anti-FGFR2 antibody.

[0381] Antibodies (10–20 mg / ml) in 50 mM HEPES, 150 mM NaCl, pH 8.0, and 10–15% (v / v) DMA were conjugated with a 5–6-fold excess of the following compounds for 2 h at ambient temperature: [ka] Conjugates were purified by size-exclusion chromatography or extensive ultrafiltration and sterile filtered. Protein concentrations were determined by UV spectroscopy. All conjugates used were confirmed to be >90% monomeric by size-exclusion HPLC and to have less than 1% unconjugated linker payload by RP-HPLC. All conjugated antibodies were analyzed for linker-payload loading by UV and / or by mass difference between native and conjugated forms according to Hamblett et al. (American Association for Cancer Research. 2004 Oct 15;10(20):7063-70). Payload-to-antibody ratios (DAR) are shown in Table 7. [Table 7]

[0382] Characterization of the conjugate by liquid chromatography-mass spectrometry To determine the loading of linker-payload on the antibody, the conjugate was deglycosylated and analyzed by LC-MS.

[0383] For this assay, 50 μg of conjugate was diluted with Milli-Q water to a final concentration of 1 mg / mL. 10 μL of PNGase F solution (PNGase F solution was prepared by adding 150 μL of PNGase F stock (New England Biolabs, catalog no. P0704L) and 850 μL of Milli-Q water and mixing thoroughly) was added to the diluted conjugate solution, which was then incubated overnight at 37°C. 5 μL of each sample was injected onto an LC-MS (Waters Synat G2-Si) and eluted with a 20-40% mobile phase gradient at 0.1 mL / min over 25 min (Mobile Phase A: 0.1% v / v in HO, Mobile Phase B: 0.1% v / v FA in acetonitrile). LC separation was performed on a Waters Acquity BEH C4 column (1.0 × 50 mm, 1.7 μM) at 80°C.

[0384] Mass spectrometry spectra were deconvoluted using Masslynx software and the drug-to-antibody ratio (DAR) was calculated using the following equation: 1. Relative percentage (%) of drug (Dn) by distribution peak intensity (PI): Dn%=PIn / Σ(PI0+PI1+PI2…….+PIi)×100,(n=0,1,2,3,…,i) 2. Calculating the average DAR: DAR=Σ(1×D1%+2×D2%+3×D3%+……+i×Di%)

[0385] Example 6: In vitro cytotoxicity assay of anti-FGFR2 antibody drug conjugates in tumor cell lines To test the ability of the anti-FGFR2 antibody-drug conjugates (ADCs) disclosed herein to kill human cell lines, in vitro cytotoxicity assays were performed. The in vitro cytotoxicity of the anti-FGFR2 ADCs, isotype control ADCs, and reference free payloads was assessed using the CellTiter-Glo 2.0 Assay Kit (Promega, Catalog No. G9243), which uses the amount of ATP present to determine the number of viable cells in culture. The payloads maytansinoid 1A, maytansinoid 1A* (a cell-permeable equivalent of maytansinoid 1A), and tubulysin 1A were tested as free payloads, and unconjugated antibodies, including comparison antibodies, were used as controls.

[0386] For the assay, SNU-16, MFM-223, NCI-H716, or IM-9 cells were seeded at 2000 cells / well in poly-D-lysine-coated white 96-well BioCoat plates (Corning #356693) in complete growth medium and grown overnight at 37°C with 5% CO2. Five-fold serial dilutions of anti-FGFR2 ADCs or isotype control ADCs were prepared in dilution medium (Optimem + 0.1% BSA) and added to cells at final concentrations ranging from 20 nM to 0.051 pM (concentrations were corrected for DAR and dosed based on effective payload concentration). Five-fold serial dilutions of free payload were prepared in 100% DMSO, transferred to fresh dilution medium, and then added to cells at a final constant DMSO concentration of 0.2% and final payload concentrations ranging from 20 nM to 0.051 pM. The last well in each dilution series served as an untreated control and contained medium alone or medium + 0.2% DMSO, plotted as a series of 5-fold serial dilutions. After 6 days, 100 μL of CellTiter Glo 2.0 was added to each well, the plate was mixed on an orbital shaker for 2 minutes, and the plate was incubated at room temperature for 10 minutes. Relative light units (RLU) were measured on an Envision luminometer (PerkinElmer), and cell viability was expressed as a percentage of untreated (100% viable) cells. IC 50Values ​​were determined using a four-parameter logistic equation over a 10-point dose-response curve (GraphPad Prism). Maximum % killing was determined for each test article as follows: 100 - minimum survival. IC of each test article 50 The values ​​and maximum % mortality are shown in Table 8.

[0387] As shown in Table 8, mAb2-Tubulysin1A-LP and mAb1-Tubulysin1A-LP (anti-FGFR2 ADCs conjugated to a tubulysin payload) had IC values ​​ranging from 37.0 pM to 125 pM. 50 These same anti-FGFR2 ADCs killed FGFR2b-positive SNU-16 and MFM-223 cells with IC values ​​of 1.0 and 9.0%, and maximum killing percentages ranging from 90.0% to 95.4%. These same anti-FGFR2 ADCs had IC values ​​of >20 nM in NCI-H716 cells (a low FGFR2b but high FGFR2c expressing cell line) and non-FGFR2-expressing IM-9 cells. 50 The free payload Tubulysin 1A (the payload of Tubulysin 1A-LP) showed weak cytotoxicity with IC values ​​ranging from 3.69 pM to 74.3 pM. 50 A non-binding control antibody conjugated to the Tubulysin payload (IC1-Tubulysin1A-LP) killed all cells tested with IC values ​​ranging from 1.98 nM to over 20 nM. 50 In addition, the mAb2-maytansinoid 1A-LP and mAb1-maytansinoid 1A-LP (anti-FGFR2 ADC conjugated to a maytansinoid payload) antibody conjugates showed IC values ​​ranging from 172 pM to 268 pM. 50 values ​​and maximum % killing ranging from 85.5% to 95.0%. Free payload maytansinoid 1A (payload in maytansinoid 1A-LP) and a non-binding control antibody conjugated to a maytansinoid payload (IC1-maytansinoid 1A-LP) killed FGFR2b-positive SNU-16 and MFM-223 cells with IC values ​​ranging from 4.63 nM to over 20 nM. 50values, which had little or no killing activity at the concentrations tested. Cell-permeable equivalents of maytansinoid 1A (information about bystander effects) ranged from 113 pM to 5.45 nM. 50 values ​​killed all lines tested, suggesting that none of the cell lines tested were generally resistant to the class of maytansinoid payloads.

[0388] A comparative anti-FGFR2 ADC that recognizes both FGFR2b and FGFR2c and is conjugated to an auristatin payload (Comp2-BAY-LP) was also tested for cytotoxicity. Similar to the other tested anti-FGFR2 ADCs, Comp2-BAY-LP exhibited IC values ​​of 87.1 pM and 63.8 pM, respectively. 50 In contrast to the other tested anti-FGFR2 ADCs that bind only to FGFR2b, the dual FGFR2b+FGFR2c binder, Comp2-BAY-LP, killed SNU-16 and MFM-223 cells with an IC value of 349 pM. 50 Comp2-BAY-LP also killed NCI-H716 cells (which express high FGFR2c levels but low FGFR2b expression) with IC values ​​of 6.06 nM to >20 nM. Comp2-BAY-LP was weakly cytotoxic in FGFR2-negative IM-9 cells, whereas the non-binding control conjugated to an auristatin payload (IC2-BAY-LP) exhibited IC values ​​ranging from 6.06 nM to >20 nM. 50 All unconjugated antibodies, including the FGFR2b-selective comparison antibody, Comp1, were weakly cytotoxic in all tested strains, with EC50s above 20 nM. [Table 8]

[0389] Co-cultures and bystander effects We also evaluated the ability of anti-FGFR2 ADCs to induce cytotoxicity in FGFR2-negative cells via payload release in anti-FGFR2-positive cells, i.e., bystander activity. In this assay, 10,000 FGFR2-positive SNU-16 cells, 10,000 FGFR2-negative NALM-6 cells, or a 1:1 mixture of 5,000 SNU-16 and NALM-6 cells were seeded into a 96-well assay plate (Corning #355691) and incubated overnight at 37°C and 5% CO2. Three-fold serial dilutions were performed for all test articles at concentrations ranging from 100 nM to 15.2 pM, and cytotoxicity was assessed as previously described for tumor cell lines, except that cells were incubated with the test articles for 5 days.

[0390] As shown in Table 9, mAb2-Tubulysin 1 ALP and mAb1-Tubulysin 1 ALP antibody conjugates killed FGFR2-positive SNU-16 monocultures with 97.2 and 97.1% maximum killing, respectively, and were not cytotoxic in FGFR2-negative NALM-6 monocultures. mAb2-Tubulysin 1 ALP and mAb1-Tubulysin 1 ALP antibody conjugates also killed SNU-16 + NALM-6 cocultures with 97.7 and 87.8% maximum killing, suggesting bystander killing activity. The non-binding ADC control (IC1-Tubulysin 1 ALP) was less cytotoxic in SNU-16 + NALM-6 cocultures with 17.0% maximum killing. mAb2-Maytansinoid 1 ALP was cytotoxic in FGFR2-positive SNU-16 cell monocultures (EC of 64.5 pM). 50mAb2-maytansinoid 1ALP exhibited approximately 18.3% maximal cytotoxicity in cocultures of SNU-16 and NLAM-6 cells (94.8% maximal killing), but had no cytotoxic activity in FGFR2-negative NALM-6 cell monocultures (5.0% maximal killing). In cocultures of SNU-16 and NLAM-6 cells prepared at a 1:1 ratio, mAb2-maytansinoid 1ALP exhibited approximately 18.3% maximal cytotoxicity, suggesting little or no bystander killing of the FGFR2-negative NALM-6 cell population. The nonbinding control IC1-maytansinoid 1ALP was weakly cytotoxic (less than 10% maximal killing) under all test conditions. The comparative ADC, Comp2-BAY-LP, also exhibited weak bystander activity, as evidenced by its robust killing activity in SNU-16 cells (95.6% maximal killing%) and weak killing activity in SNU-16 + NALM-6 cocultures (21.2% maximal killing%). [Table 9]

[0391] Example 7: In vivo efficacy of anti-FGFR2 antibody drug conjugates (ADCs) on the growth of SNU-16 gastric cancer xenografts The FGFR2 tubulysin ADC mAb1-tubulysin1ALP was tested in experiment A (Figure 3), and the FGFR2 maytansinoid ADC mAb2-maytansinoid1ALP was tested in experiment B (Figure 4) for their ability to inhibit the growth of SNU-16 human gastric cancer xenografts at different doses. Briefly, 5 × 10 FGFR2 tubulysin ADC mAb1-tubulysin1ALP mixed with Matrigel (BD Biosciences) was used. 6 SNU-16 cells (ATCC) were subcutaneously implanted into the flanks of male BALB / c SCID mice (6-8 weeks old, Jackson Laboratory). Tumors grew to 200-250 mm. 3After reaching an average tumor volume of 1000 mg / kg, mice were randomized into treatment groups (n = 5-6 mice per group in Experiment A, n = 6-7 mice per group in Experiment B). In Experiment A, mice were administered the human Fc isotype control tubulysin ADC IC1-tubulysin 1ALP (3.0 mg / kg) or FGFR2 tubulysin ADC mAb1-tubulysin 1ALP (0.1, 0.3, 1.0, or 3.0 mg / kg), and in Experiment B, mice were administered the human Fc isotype control maytansinoid ADC IC1-maytansinoid 1ALP or FGFR2 maytansinoid ADC mAb2-maytansinoid 1ALP (1.0, 3.0, 10, or 15 mg / kg). All ADCs were administered once via subcutaneous injection. Tumor volumes were measured twice weekly throughout the course of the experiment. The mean tumor growth (mean ± standard deviation) (change in tumor volume from the start of treatment to the end of the experiment) was calculated for each treatment group. The percent reduction in tumor growth was calculated compared to the isotype control group, and the percent tumor regression at the end of the experiment was calculated compared to the tumor volume at the start of treatment. The results are shown in Table 10. [Table 10]

[0392] These results demonstrate that ADCs targeting the FGFR2b isoform induced complete regression of SNU-16 tumor xenografts in a dose-dependent manner, with maximal efficacy achieved by the 1.0 mg / kg dose of the tubulysin FGFR2 ADC and the 10 mg / kg dose of the maytansinoid FGFR2 ADC.

[0393] Example 8: In vivo efficacy of anti-FGFR2b antibody drug conjugates (ADCs) on the growth of human gastric cancer PDX GA0033 tumors In this experiment, the FGFR2 maytansinoid ADC mAb1-maytansinoid 1ALP and the FGFR2 tubulysin ADC mAb1-tubulysin 1ALP were tested for their ability to inhibit the growth of human gastric cancer PDX GA0033 tumors at different doses (Figure 5). Briefly, HuPrime® primary human gastric cancer PDX GA0033 tumor fragments (Crown Bioscience) measuring 2-3 mm in diameter were subcutaneously implanted into the right flank of female BALB / c nude mice (5-9 weeks old, GemPharmatech). Tumors were grown to 100-200 mm. 3 After reaching an average tumor volume of 1000 mg / kg, mice were randomized into treatment groups (n = 6 mice per group). Mice were administered human Fc isotype control maytansinoid ADC IC-maytansinoid 1ALP (10 mg / kg), FGFR2 maytansinoid ADC mAb1-maytansinoid 1ALP (3 or 10 mg / kg), human Fc isotype control tubulysin ADC IC-tubulysin 1ALP (3 mg / kg), FGFR2 tubulysin ADC mAb1-tubulysin 1ALP (1 or 3 mg / kg), human Fc isotype control naked antibody IC (15 mg / kg), or FGFR2 naked antibody mAb1 (15 mg / kg). All ADCs were administered once per week via subcutaneous injection for two treatments, and all naked antibodies were administered twice per week via subcutaneous injection throughout the entire experiment. Tumor volumes were measured twice per week throughout the course of the experiment. The mean tumor growth (mean ± standard deviation) (change in tumor volume from the start of treatment to the end of the experiment) was calculated for each treatment group. The percent reduction in tumor growth was calculated compared to the corresponding isotype control group, and the percent tumor regression at the end of the experiment was calculated compared to the tumor volume at the start of treatment. The results are shown in Table 11. [Table 11]

[0394] These results demonstrate that ADCs targeting the FGFR2b isoform induced complete regression of human gastric cancer PDX GA0033 tumors in a dose-dependent manner, with maximal efficacy achieved by the 3.0 mg / kg dose of the tubulysin FGFR2 ADC and the 10 mg / kg dose of the maytansinoid FGFR2 ADC.

[0395] Example 9: Synthesis of camptothecin derivatives (payload) [Table 12]

[0396] Payload P1, exatecan mesylate, was commercially obtained from MCE. Payloads P2 and P3 were synthesized as described in WO2015 / 155998, which is incorporated herein by reference, and camptothecin derivative payload P4 was synthesized as described in Scheme 1A and following the synthetic steps outlined in Examples 9A-1E.

[0397] Scheme 1: Synthesis of camptothecin derivatives (payloads) P3 and P4 [ka] Example 9A: Synthesis of 9H-fluoren-9-ylmethyl N-[2-(2-hydroxypyrrolidin-1-yl)-2-oxoethyl]carbamate (P4-2) [ka] To a mixture of Fmoc-Gly-Pro-OH P4-1 (0.10 g, 0.26 mmol) in dry DMF (1 mL) was added lead tetraacetate (0.14 g, 0.31 mmol). The resulting mixture was stirred at room temperature for 30 minutes, and the reaction progress was monitored by LCMS. The resulting mixture was filtered through Celite, and the filtrate was diluted with ethyl acetate, washed with water and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (0-10% ethyl acetate in petroleum ether) to give compound P4-2 (50 mg, 53% yield) as a white solid, with no acetic acid intermediate. ESI m / z: 389 (M+23). + . 1 H NMR(400MHz,DMSO)δ 7.90(d,J=7.4Hz,2H),7.73(d,J=7.5Hz,2H),7.47-7.37(m,3H),7.33(t,J=7.3Hz,2H),5.86(br s,1H),5.48(d,J=4.0Hz,0.25H),5.39(d,J=4.0Hz,0.75H),4.33-4.18(m,3H),3.96(d,J=6.0H z,1.5H),3.75(d,J=6.0Hz,0.5H),3.59-3.33(m,1H),3.22-3.11(m,1H),2.00-1.59(m,4H)ppm.

[0398] Example 9B: Synthesis of 2-{[1-(2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}acetyl)pyrrolidin-2-yl]oxy}benzyl acetate (P4-3) [ka] To a solution of compound P4-2 (0.30 g, 0.82 mmol) in DCM (25 mL) was added chlorotrimethylsilane (TMSCl) (0.27 g, 2.5 mmol). The reaction mixture was stirred at room temperature for 3 h, and the reaction progress was monitored by LCMS. The resulting mixture was concentrated in vacuo, and the residue was diluted with DCM (25 mL). Benzyl glycolate (0.27 g, 1.6 mmol) and DIPEA (0.21 g, 1.6 mmol) were added to the solution, and the reaction mixture was stirred at room temperature for 1 h. The completion of the reaction was monitored by LCMS. The resulting mixture was concentrated in vacuo, and the residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous ammonium bicarbonate (0.05%)) to give compound P4-3 (0.11 g with a purity of >99%, 25% yield, and 50 mg with a purity of 75%) as a white solid. ESI m / z: 537.3 (M+Na). + . 1 H NMR (400 MHz, DMSO d6 )δ 7.91-7.89(m,2H),7.74-7.69(m,2H),7.63-7.48(m,1H),7.42-7.25(m,9H),5.51-5.09(m,2H),4 .35-4.21(m,5H),4.00-3.77(m,2H),3.52-3.38(m,2H),3.30-3.18(m,1H),2.19-1.64(m,4H)ppm.

[0399] Example 9C: Synthesis of 2-{[1-(2-{[(9H-fluoren-9-ylmethyl)carbonyl]amino}acetyl)pyrrolidin-2-yl]oxy}acetic acid (P4-4) [ka] To a solution of compound P4-3 (89 mg, 0.17 mmol) in methanol (3 mL) and THF (7 mL) was added wet palladium on carbon (10% Pd, 20 mg) under nitrogen protection. The mixture was degassed and stirred under hydrogen balloon pressure at room temperature for 2 hours, and the completion of the reaction was monitored by LCMS. The reaction mixture was filtered through Celite, and the filtrate was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous ammonium bicarbonate (0.05%)) to give compound P4-4 (36 mg, 49% yield) as a white solid. ESI m / z: 447.1 (M+Na). + .

[0400] Example 9D: 9H-Fluoren-9-ylmethyl N-{2-[2-({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)pyrrolidin-1-yl]-2-oxoethyl}carbamate (P4-5) [ka] To a mixture of compound P4-4 (63 mg, 0.15 mmol) and exatecan mesylate (66 mg, 0.12 mmol) in DMF (2 mL), HATU (61 mg, 0.16 mmol) and DIPEA (46 mg, 0.36 mmol) were added, and the mixture was stirred at room temperature for 2 h. The reaction completion was monitored by LCMS. The reaction mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give compound P4-5 (45 mg, 44% yield) as a yellow solid. ESI m / z: 842.3 (M+H). + .

[0401] Example 9E: 2-{[1-(2-aminoacetyl)pyrrolidin-2-yl]oxy}-N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]acetamide (P4) [ka] To a solution of compound P4-5 (45 mg, 54 mol) in DCM (4 mL) was added diethylamine (20 mg, 0.27 mmol), and the mixture was stirred at room temperature overnight. Reaction completion was monitored by LCMS. The reaction mixture was concentrated in vacuo, and the residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to give compound P4 (9.5 mg, 28% yield) as a colorless oil. ESI m / z: 620.3 (M+H). + .

[0402] Example 10: Synthesis of vcPAB-carbamate linker-payload Linker-payloads LP1 and LP2 were synthesized as described below in Scheme 2 and Examples 10A-10C (for LP1) and 10D (for LP2). Starting materials L1-1 (CAS 2226472-26-8) and L2-3 (CAS 2226472-28-0) were synthesized according to WO2018 / 089373A2, the entire contents of which are incorporated herein by reference.

[0403] Scheme 2. Synthesis of vcPAB-carbamate linker-payload [ka] Example 10A: N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]-1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amide (L1-2) [ka] To a solution of compound L1-1 (0.17 g, 0.33 mmol) in DMF (10 mL), DIPEA (0.13 g, 1.0 mmol) and vcPAB (0.13 g, 0.34 mmol) were added successively, and the reaction mixture was stirred at room temperature for 1 h. The reaction completion was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-80% acetonitrile in water) to give compound L1-2 (0.18 g, 70% yield) as a colorless oil. ESI m / z: 791.3 (M+H). + . 1 H NMR (400 MHz, DMSO d6 )δ 9.91(s,1H),8.11(d,J=8.4Hz,1H),7.89(d,J=8.8Hz,1H),7.61(t,J=5.6Hz,1H),7.5 5(d,J=8.4Hz,2H),7.23(d,J=8.4Hz,2H),5.98(t,J=5.6Hz,1H),5.42(s,2H),5.10(br s,1H),),4.43(s,2H),4.39-4.37(m,1H),4.30-4.21(m,2H),3.87(d,J=14.8Hz,1H),3.7 5(d,J=14.8Hz,1H),3.62-3.58(m,2H),3.50-3.46(m,12H),3.43(t,J=6.0Hz,2H),3.27- 3.22(m,2H),3.06-2.92(m,2H),2.41-2.32(m,2H),2.26-2.05(m,3H),1.99-1.66(m,6H) ,1.62-1.55(m,3H),1.44-1.35(m,3H),0.89(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H)ppm.

[0404] Example 10B: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl 4-nitrophenyl carbonate (L1-3) [ka] A suspension of compound L1-2 (80 mg, 0.10 mmol), DMAP (12 mg, 0.10 mmol), and DIPEA (26 mg, 0.20 mmol) in dry DMF (5 mL) was stirred at room temperature for 10 minutes, after which bis(4-nitrophenyl)carbonate (61 mg, 0.20 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. Reaction completion was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-80% acetonitrile in water) to give compound L1-3 (53 mg, 55% yield) as a white solid. ESI m / z: 956.3 (M+H). + .

[0405] Example 10C: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1) [ka] To a yellow solution of compound L1-3 (16 mg, 17 μmol) and exatecan mesylate (12 mg, 17 μmol) in dry DMF (2 mL), DIPEA (6.5 mg, 51 μmol) was added, and the clear reaction solution was stirred at room temperature for 2 h. Reaction completion was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0 to 60% acetonitrile in aqueous TFA (0.01%)) to afford linker-payload LP1 (15 mg as the TFA salt, 63% yield) as a white solid. ESI m / z: 698.8 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ 9.99(s,1H),8.80(t,J=6.8Hz,1H),8.50(d,J=9.2Hz,1H),8.12(d,J=7.2Hz,1H),7.87(d,J=8.8Hz,1H),7. 79(d,J=10.8Hz,1H),7.62-7.58(m,3H),7.42(t,J=6.0Hz,1H),7.31(s,1H),7.28(d,J=8.4Hz,2H),6.53(br s,1H),5.98(t,J=5.2Hz,1H),5.63-5.57(m,1H),5.46-5.37(m,3H),5.21(s,2H),4.93(s,2H),4.63(d,J=6.4Hz,2H),4 .41-4.35(m,1H),4.29-4.21(m,2H),4.02(s,2H),3.87(d,J=14.4Hz,1H),3.75(d,J=14.8Hz,1H),3.63-3.58(m,4H),3 0.50-3.48 (m, 12H), 3.46-3.41 (m, 2H), 3.27-3.24 (m, 2H), 3.23-3.12 (m, 2H), 3.07-2.91 (m, 2H), 2.47-2.45 (m, 0.5H), 2.41-2.33 (m, 4.5H), 2.25-2.04 (m, 5H), 1.99-1.69 (m, 9H), 1.63-1.54 (m, 3H), 1.44-1.33 (m, 3H), 0.88-0.82 (m, 9H) ppm. (TFA protons were not observed.) 19 F NMR (376 MHz, DMSOd6 ) δ-74(TFA),-111(Ar-F)ppm.

[0406] Example 10D: {4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecan-15-amido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP2) [ka] Following the procedure for making LP1, except replacing L1-3 with L2-3, the linker-payload LP2 (12 mg, 46% yield) was obtained as a mixture of the lactone product (LP2 in the upper diagram) and the ring-opened product (LP2-RO in the lower diagram) as a white solid after purification by reverse-phase flash chromatography (0–100% methanol in 10 mM aqueous ammonium bicarbonate). [ka]

[0407] Lactone LP2: HPLC purity: 67%, retention time: 7.41 min, ESI m / z: 507.3 (M / 3+H) + ,760.5(M / 2+H) +Ring-opening product LP2-RO: HPLC purity: 33%, holding time: 6.61 min, ESI m / z: 513.3 (M / 3+H) + 769.5 (M / 2+H) + .

[0408] Mixture of lacton products and open-ring products 1 H NMR (400MHz, DMSO) d6 )δ 9.99(s,1H),8.80(t,J=6.4Hz,1H),8.50(d,J=8.8Hz,1H),8.12(d,J=7.2Hz,1H),7.87(d,J =8.4Hz,1H),7.80-7.75(m.2H),7.69-7.67(m,1H),7.63-7.58(m,3H),7.51-7.46(m,3H),7. 45-7.33(m,3H),7.32-7.26(m,4H),6.53(s,1H),5.98(t,J=6.0Hz,1H),5.63-5.57(m,1H),5 .42(s,4H),5.21(s,2H),5.03(d,J=14.0Hz,1H),4.93(s,2H),4.63(d,J=6.8Hz,2H),4.41-4 .35(m,1H),4.25-4.21(m,1H),4.02(s,2H),3.62-3.57(m,5H),3.48-3.45(m,12H),3.31-3. 28(m,2H),3.23-3.14(m,2H),3.11-3.07(m,2H),3.05-2.98(m,1H),2.96-2.91(m,1H),2.60 -2.55(m,1H),2.46-2.44(m,1H),2.39(s,3H),2.35-2.33(m,1H),2.26-2.15(m,3H),2.03-1 .94(m,2H),1.88-1.67(m,4H),1.63-1.57(m,1H),1.46-1.33(m,2H),0.88-0.81(m,9H)ppm. 19 F NMR (376MHz, DMSO) d6 )δ-111ppm.

[0409] Example 11: FGFR2b-カンプトテシンコンジュゲーション This example generally illustrates a method for site-specific conjugation of a payload to an anti-FGFR2b antibody or antigen-binding fragment thereof according to embodiments of the present disclosure. The first step was the attachment of a linker 1 (L1-B'), such as bis-azido-alkyl-substituted amine (BL7) or azido-PEG3-amine (AL1), to the FGFR2b antibody via microbial transglutaminase (MTG). Excess amine reagent was used to avoid potential cross-linking of antibody chains. The second step was the attachment of an alkyne-linked payload linker payload (L2P) to the N3-tagged conjugate via strain-promoted azide-alkyne cycloaddition (SPAAC). The number of L2P molecules added to the antibody depends on the number of conjugation sites and the number of azide functional groups (n) in L1 (AL, n = 1; BL, n ≥ 2). For an antibody with a WT Fc domain that has been enzymatically deglycosylated or has the N297D Fc mutation and then azide-functionalized with an AL or BL linker, the expected DAR is 2×n×m, where n is the number of azide functional groups B′ on each L1 linker and m is the number of respective L2P payloads. For an antibody with the N297Q Fc mutation and then azide-functionalized with an AL or BL linker, the expected DAR is 4×(n)×(m).

[0410] Example 11A: Step 1: Preparing site-specific azide-functionalized antibody drug conjugates containing 2, 4, or 8 azide groups. Anti-FGFR2b human IgG antibody containing the N297Q mutation or an isotype control antibody was mixed with 150 molar equivalents of azido-PEG3-amine (AL1, MW 218.26 g / mol) or bisazido-alkyl-substituted amine (BL7, MW 325.38 g / mL). The resulting solution was mixed with transglutaminase (25 U / mL, 1 U mTG / mg antibody, Zedira, Darmstadt, Germany) to obtain a final antibody concentration of 1–20 mg / mL. The reaction mixture was incubated at 25–37°C for 4–24 hours with gentle shaking while monitoring by ESI-MS. Upon completion, excess amine and mTG were removed by size-exclusion chromatography (SEC) or protein A column chromatography. The conjugates were characterized by UV-Vis, SEC, and ESI-MS. An azide linker was attached to the antibody, resulting in a mass increase of 804 Da or 1232 Da for the DAR=4 conjugates with AL1 and BL7, respectively. The monomer purity of the conjugates was greater than 99% by SEC.

[0411] Example 11B: Step 2: Creating site-specific conjugates via a 1,3-cycloaddition ("click") reaction between an azide-functionalized antibody and an alkyne containing linker-payload. Site-specific antibody-drug conjugates were prepared by incubating azide-functionalized antibody (1–20 mg / mL) in PBS (pH 7.4) with 6 or more molar equivalents of linker-payload dissolved in an organic solvent, such as DMSO or DMA (10 mg / mL), with a reaction mixture containing 5–15% organic solvent (v / v) for 1–48 h at 25–37 °C with gentle shaking. The reaction was monitored by ESI-MS. Upon completion, excess linker-payload and protein aggregates were removed by size-exclusion chromatography (SEC). The purified conjugate was concentrated, sterile-filtered, and characterized by UV-Vis, SEC, and ESI-MS. The monomer purity of the conjugate exceeded 99% by SEC.

[0412] Example 12: ADC Conjugation: Three Approaches The present disclosure illustrates three exemplary approaches for attaching a branched linker-payload to the anti-FGFR2b antibody Q295 / 297 site.

[0413] Approaches I and II involve a two-step process for antibody-drug conjugation. The first step is the attachment of a small amine, such as AL1 or BL2, to the mAb-Q site mediated by microbial transglutaminase (MTG), using an excess of amine reagent to avoid potential crosslinking of antibody chains (WO 2017 / 147542, incorporated herein by reference in its entirety). The second step is the attachment of an alkyne-linked linker payload (L2P) to the N3-tagged conjugate, for example, via strain-promoted azide-alkyne cycloaddition (SPAAC, also known as copper-free click chemistry). When the reactive group (RG) is a DIBAC or COT moiety, conjugation is carried out with an azide-functionalized antibody via [2+3] cycloaddition. This process results in site-specific and stoichiometric conjugates. The number of L2P molecules added to an antibody depends on the number of conjugation sites and the number of azide functional groups (n) in L1 (e.g., for AL, n=1; for BL, n≧2).

[0414] Approach I is to conjugate a small molecule amine linker L1 (e.g., AL1) to the antibody Q295 / 297 moiety to generate an antibody-azide tag (Ab-N3), which is then covalently reacted (e.g., via "click" cycloaddition) with an alkyne tether linear linker-payload (LL2P) to generate a 4DAR ADC, and with an alkyne tether branched linker-payload (BL2P) to generate an 8DAR ADC.

[0415] Approach II is to conjugate a small molecule branched azide-amine (e.g., BL2) to the antibody Q295 / 297 site to generate an antibody-branched azide tag (Ab-branched-2N3), which can then be covalently reacted (e.g., via "click" cycloaddition) with a linear linker-payload to generate an 8DAR ADC, with an alkyne tether branched linker-2 payload to generate a 16DAR ADC, or with a branched tether 3 payload to generate a 24DAR ADC. Similarly, site-specific ADC conjugation on the antibody Q295 site with a linear or branched linker-payload can generate DAR2-DAR12 ADCs.

[0416] In Approach III conjugation, MTG-mediated attachment of an amine-branched linker-payload to the antibody Q295 / 297 site was achieved using 20 or more molar equivalents of the amine reagent in a single-step MTG-mediated reaction.

[0417] For an antibody with a WT Fc domain that has been enzymatically deglycosylated or has the N297D Fc mutation and then azide-functionalized with an AL or BL linker, the expected DAR per azide-tag on 2Fc is 2n. For an antibody with the N297Q Fc mutation that has been azide-functionalized with an AL or BL linker, the expected DAR per azide-tag on 2Fc is 4n. m × payload (P m For antibodies conjugated with each linker-payload with m, the expected ADC-DAR for the N297D mutant antibody is (2n×m) and for the N297Q mutant antibody is (4n×m).

[0418] General procedure for making site-specific conjugates in two steps:

[0419] Example 12A: Step 1: Making site-specific azide-functionalized antibody drug conjugates containing 2, 4, or 8 azide groups. Aglycosylated human anti-FGFR2b antibody IgG (e.g., IgG1, IgG4) containing the N297Q or N297D mutation in 800kJ / pH 7.4 buffer was mixed with up to 150 molar equivalents of azido-PEG3-amine (AL1) or bisazido-alkyl-substituted amine (BL2). The resulting solution was mixed with transglutaminase (25 U / mL; 1 U mTG / mg antibody, Zedira, Darmstadt, Germany, or 10 U / mL; 5.5 U MTG / mg antibody, Modernist Pantry-ACTIVA TI containing maltodextrin, Ajinomoto, Japan) to obtain a final antibody concentration of 0.5–20 mg / mL. The reaction mixture was incubated at 25–37°C for 24 hours with gentle shaking while monitored by ESI-MS. Upon completion, excess amine and mTG were removed by size-exclusion chromatography (SEC) or protein A column chromatography. The conjugates were characterized by UV-Vis, SEC, and ESI-MS. An azide linker was attached to the antibody, resulting in a mass increase of 804 Da or 1232 Da for the 4DAR conjugates with AL1 and BL2, respectively, and an increase of 2768 Da for the 8DAR antibody-BL2-(azide)8 conjugate. The monomer purity of the conjugates was greater than 99% by SEC.

[0420] Example 12B: Step 2: Creating site-specific conjugates via a [2+3] click reaction between an azide-functionalized antibody and an alkyne containing linker-payload. Site-specific antibody-drug conjugates with human IgG (e.g., IgG1, IgG4) were prepared via a [2+3] azide-alkyne "click" reaction between an azide-functionalized antibody and an alkyne-functionalized linker-payload. Azide-functionalized antibody (1–20 mg / mL) in PBS (pH 7.4) was incubated with 6 or more molar equivalents of linker-payload (LP) dissolved in an organic solvent, such as DMSO or DMA (10 mg / mL), for 1–48 h at 25–37 °C with gentle shaking, resulting in a reaction mixture containing 5–15% organic solvent (v / v). The reaction was monitored by ESI-MS. Upon completion, excess LP and organic solvent were removed by desalting the column with PBS (pH 7.4), and protein aggregates (if any) were removed by size-exclusion chromatography (SEC). The purified conjugate was concentrated, sterile-filtered, and characterized by UV-Vis, SEC, and ESI-MS. The monomer purity of the conjugate was greater than 99% by SEC.

[0421] Example 13: Effect of FGFR2b antibody drug conjugates (ADC) on the growth of SNU-16 gastric cancer xenografts To evaluate the antitumor activity of the FGFR2b camptothecin ADC against SNU16 xenografts (human gastric cancer xenografts), 5 × 10 6 SNU-16 cells (ATCC) were subcutaneously implanted into the flanks of male BALB / c SCID mice (6-8 weeks old, Jackson Laboratory). Tumors grew to 200-250 mm. 3 After reaching an average volume of 100 mg / kg, mice were randomized into groups for treatment (n=6 mice per group). Camptothecin-LP1 and camptothecin-LP2 are shown in Table 13. All ADCs were administered via subcutaneous injection at the doses shown in Table 14. Mice treated with the anti-FGFR2 camptothecin-LP1 ADC (DAR8) were dosed on day 0, and mice treated with the anti-FGFR2 camptothecin-LP2 ADC (DAR4) were dosed twice, first on day 0 and then again on day 7. [Table 13]

[0422] The construct comprises a camptothecin analog. [ka] [Table 14]

[0423] Tumor volumes were measured twice per week over the course of the experiment. The mean tumor growth (mean ± standard deviation) (change in tumor volume from the start of treatment to the end of the experiment) was calculated for each treatment group. The percent reduction in tumor growth was calculated compared to the isotype control group, and the percent tumor regression at the end of the experiment was calculated compared to the tumor volume at the start of treatment. The results of two separate experiments utilizing ADCs with different DARs are shown in Table 15 and Figures 6 and 7. The results demonstrate that FGFR2b camptothecin derivative ADCs induce complete regression of SNU-16 tumor xenografts in a dose-dependent manner. [Table 15]

[0424] Example 14: Effect of FGFR2b antibody drug conjugate (ADC) on the growth of gastric cancer patient-derived xenografts (GA1224) To evaluate the antitumor activity of the FGFR2b camptothecin-LP2 ADC (Table 13) against patient-derived gastric cancer xenografts (GA1224), tumor fragments (2-3 mm in diameter) were subcutaneously implanted into the flanks of female BALB / c nude mice (6-9 weeks old, GemPharmatech Co., Ltd.). Tumors were grown to 100-150 mm. 3 After reaching an average tumor volume of 1000 mg / kg, the mice were randomized into groups for treatment (n=6 mice per group). Tumor-bearing mice were administered once per week for 2 weeks via intravenous injection at the doses shown in Table 16. [Table 16]

[0425] Tumor volumes were measured twice per week over the course of the experiment. The mean tumor growth (mean ± standard deviation) (change in tumor volume from the start of treatment to the end of the experiment) was calculated for each treatment group. The percent reduction in tumor growth was calculated compared to the isotype control antibody group, and the percent tumor regression at the end of the experiment was calculated compared to the tumor volume at the start of treatment. The results are shown in Table 17 and Figure 10. These results demonstrate that DXd ADCs targeting the FGFR2b isoform induced complete regression of GA1224 PDX tumors at all doses tested. [Table 17]

[0426] Example 15: In vivo bystander killing effect of anti-FGFR2b antibody drug conjugates (ADCs) on the growth of coinoculated SNU-16 / SNU-5 gastric cancer xenografts The ability of the anti-FGFR2 ADCs to induce tumor regression in FGFR2-negative cells via payload release in anti-FGFR2-positive cells, i.e., bystander activity, was also evaluated. The ability of the FGFR2b camptothecin ADC and FGFR2b maytansinoid ADC to inhibit tumor growth was tested against FGFR2-amplified SNU-16 human gastric cancer xenografts (FIG. 11), FGFR2-negative SNU-5 human gastric cancer xenografts (FIG. 12), or coinoculated SNU-16 and SNU-5 xenografts implanted at a 2:1 ratio (FIG. 13).

[0427] Briefly, 5 × 10 cells were mixed with Matrigel (BD Biosciences). 6 SNU-16 and / or SNU-5 cancer cells (ATCC) were subcutaneously implanted into the flanks of male BALB / c SCID mice (6-8 weeks old, Jackson Laboratory). Tumors were grown to 200-250 mm. 3After reaching an average volume of 1000 mg / kg, the mice were randomized into groups for treatment (n=6 mice per group). Tumor-bearing mice were administered a single dose via subcutaneous injection at the doses shown in Table 18. [Table 18]

[0428] Tumor volumes were measured twice per week over the course of the experiment. The mean (mean ± standard deviation) tumor growth (change in tumor volume from the start of treatment to the end of the experiment) was calculated for each treatment group. The percent reduction in tumor growth was calculated compared to the isotype control group, and the percent tumor regression at the end of the experiment was calculated compared to the tumor volume at the start of treatment. The results are shown in Table 19.

[0429] These results suggest that camptothecin ADCs targeting the FGFR2b isoform induced complete regression of SNU-16 xenografts (FGFR2b-positive) but did not inhibit the growth of SNU-5 xenografts (FGFR2b-negative), but induced complete regression of SNU-16 and SNU-5 co-inoculated xenografts, with significant bystander killing of the FGFR2b-negative SNU-5 cell population. Maytansinoid ADCs targeting the FGFR2b isoform induced complete regression of SNU-16 xenografts, did not inhibit the growth of SNU-5 xenografts, and only partially inhibited the growth of SNU-16 and SNU-5 co-inoculated xenografts, with little or no bystander killing of the FGFR2b-negative SNU-5 cell population. [Table 19] [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27]

[0430] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The present invention provides, for example, the following items. (Item 1) An isolated monoclonal antibody or antigen-binding fragment thereof that binds to FGFR2, wherein the antibody or antigen-binding fragment thereof has the following characteristics: (a) It is a fully human monoclonal antibody; (b) 2.5 × 10 as measured by surface plasmon resonance -8 K below M D Binding to FGFR2b at (c) selectively binds to hFGFR2b over hFGFR2c; and (d) An isolated monoclonal antibody or antigen-binding fragment thereof, which exhibits one or more of the following characteristics: it comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 22, or an amino acid sequence that is at least 90% identical thereto; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 28, or an amino acid sequence that is at least 90% identical thereto. (Item 2) 2. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 1, comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 90% identical thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 90% identical thereto. (Item 3) The anti-FGFR2 antibody or antigen-binding fragment thereof has the following characteristics: (a) It is a fully human monoclonal antibody; (b) 2.5 × 10 as measured by surface plasmon resonance -8 K below M D Binding to FGFR2b at (c) having a half-life (t) of greater than about 10 minutes as measured by surface plasmon resonance; and (d) selectively binds to hFGFR2b over hFGFR2c. (Item 4) 3. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 2, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 with not more than two amino acid substitutions; HCDR2 comprises the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 with not more than two amino acid substitutions; HCDR3 comprises the amino acid sequence of SEQ ID NO: 8 or the amino acid sequence of SEQ ID NO: 8 with not more than two amino acid substitutions; LCDR1 comprises the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence of SEQ ID NO: 12 with not more than two amino acid substitutions; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14 or the amino acid sequence of SEQ ID NO: 14 with not more than two amino acid substitutions; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence of SEQ ID NO: 16 with not more than two amino acid substitutions. (Item 5) 3. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 2, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, LCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16. (Item 6) 3. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 2, wherein the anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 95% identical thereto, and an LCVR comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical thereto. (Item 7) 3. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 2, wherein the anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 2 and an LCVR comprising the amino acid sequence of SEQ ID NO: 10. (Item 8) 2. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 1, comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is at least 90% identical thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 90% identical thereto. (Item 9) The anti-FGFR2 antibody or antigen-binding fragment thereof has the following characteristics: (a) It is a fully human monoclonal antibody; (b) 8.9 × 10 as measured by surface plasmon resonance -9 K below M D Binding to FGFR2b at (c) having a half-life (t) of greater than about 25 minutes as measured by surface plasmon resonance; and (d) selectively binds to hFGFR2b over hFGFR2c. (Item 10) 9. The anti-FGFR2 antibody or antigen-binding fragment thereof according to Aspect 8, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 24 or the amino acid sequence of SEQ ID NO: 24 with not more than two amino acid substitutions, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 with not more than two amino acid substitutions, HCDR3 comprises the amino acid sequence of SEQ ID NO: 26 or the amino acid sequence of SEQ ID NO: 26 with not more than two amino acid substitutions, LCDR1 comprises the amino acid sequence of SEQ ID NO: 30 or the amino acid sequence of SEQ ID NO: 30 with not more than two amino acid substitutions, LCDR2 comprises the amino acid sequence of SEQ ID NO: 32 or the amino acid sequence of SEQ ID NO: 32 with not more than two amino acid substitutions, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 34 or the amino acid sequence of SEQ ID NO: 34 with not more than two amino acid substitutions. (Item 11) 9. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 8, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, HCDR3 comprises the amino acid sequence of SEQ ID NO: 26, LCDR1 comprises the amino acid sequence of SEQ ID NO: 30, LCDR2 comprises the amino acid sequence of SEQ ID NO: 32, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 34. (Item 12) 9. The anti-FGFR2 antibody or antigen-binding fragment thereof according to Item 8, wherein the anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is at least 95% identical thereto, and an LCVR comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 95% identical thereto. (Item 13) 9. The anti-FGFR2 antibody or antigen-binding fragment thereof according to Item 8, wherein the anti-FGFR2 antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 22 and an LCVR comprising the amino acid sequence of SEQ ID NO: 28. (Item 14) 14. A pharmaceutical composition comprising a therapeutically effective amount of one or more isolated human monoclonal antibodies or antigen-binding fragments thereof according to any one of items 1 to 13, together with one or more pharmaceutically acceptable excipients. (Item 15) 14. A nucleic acid molecule encoding a human monoclonal antibody or a fragment thereof that binds to FGFR2 according to any one of items 1 to 13. (Item 16) Item 16. An expression vector comprising a nucleic acid molecule encoding a human monoclonal antibody or a fragment thereof that binds to FGFR2 according to Item 15. (Item 17) A host cell containing the expression vector according to item 11. (Item 18) 14. The anti-FGFR2 antibody or antigen-binding fragment thereof according to any one of items 1 to 13, wherein the anti-FGFR2 antibody or antigen-binding fragment thereof is conjugated to a cytotoxin. (Item 19) The anti-FGFR2 antibody or antigen-binding fragment thereof has the following characteristics: (a) being selectively cytotoxic to hFGFR2b-expressing cells over hFGFR2c-expressing cells; (b) 4.8 × 10 as measured by surface plasmon resonance -8 K below M D Binding to FGFR2b at (c) having a half-life (t) of greater than about 7 minutes as measured by surface plasmon resonance; and (d) inducing regression of FGFR2b-positive tumors in a dose-dependent manner. (Item 20) 19. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 18, wherein the cytotoxin is selected from the group consisting of a biotoxin, a chemotherapeutic agent, and a radioisotope. (Item 21) 21. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 20, wherein the cytotoxin is tubulysin, maytansinoid, or camptothecin. (Item 22) 14. The anti-FGFR2 antibody or antigen-binding fragment thereof according to any one of items 1 to 13, wherein the anti-FGFR2 antibody or antigen-binding fragment thereof is conjugated to a cytotoxin via a linker. (Item 23) 23. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 22, wherein the cytotoxin is tubulysin. (Item 24) The anti-FGFR2 antibody or antigen-binding fragment thereof is azido-PEG 3 24. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 23, conjugated to tubulysin via an amine linker. (Item 25) 24. The anti-FGFR2 antibody or antigen-binding fragment thereof according to item 23, wherein the tubulysin is:

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Claims

1. 1. An isolated monoclonal antibody or antigen-binding fragment thereof that selectively binds to the FGFR2b isoform, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 16; or (b) An isolated monoclonal antibody or antigen-binding fragment thereof, comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 26, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 32, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

34.

2. 2. The anti-FGFR2b antibody or antigen-binding fragment thereof of claim 1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, LCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

16. (a) the anti-FGFR2b antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence at least 95% identical thereto, and an LCVR comprising the amino acid sequence of SEQ ID NO:10, or an amino acid sequence at least 95% identical thereto; or (b) The anti-FGFR2b antibody or antigen-binding fragment thereof according to claim 2, wherein the anti-FGFR2b antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 2 and an LCVR comprising the amino acid sequence of SEQ ID NO:

10.

4. 2. The anti-FGFR2b antibody or antigen-binding fragment thereof of claim 1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, HCDR3 comprises the amino acid sequence of SEQ ID NO: 26, LCDR1 comprises the amino acid sequence of SEQ ID NO: 30, LCDR2 comprises the amino acid sequence of SEQ ID NO: 32, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

34. (a) the anti-FGFR2b antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 95% identical thereto, and an LCVR comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence at least 95% identical thereto; or (b) The anti-FGFR2b antibody or antigen-binding fragment thereof according to claim 4, wherein the anti-FGFR2b antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 22 and an LCVR comprising the amino acid sequence of SEQ ID NO:

28.

6. The anti-FGFR2b antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment is fully human.

7. An antibody-drug conjugate (ADC) comprising the anti-FGFR2b antibody or antigen-binding fragment thereof described in any one of claims 1 to 6, wherein the anti-FGFR2b antibody or antigen-binding fragment thereof is conjugated to a cytotoxin via a linker. (a) the cytotoxin is selected from the group consisting of a biotoxin, a chemotherapeutic agent, and a radioisotope; or (b) the cytotoxin is tubulysin, maytansinoid, or camptothecin, or an analog thereof. (a) the anti-FGFR2b antibody or antigen-binding fragment thereof is azide-PEG- ３ - conjugated to said cytotoxin via an amine linker, and / or (b) the anti-FGFR2b antibody or antigen-binding fragment thereof is conjugated to the cytotoxin at residues Q295 and / or Q297 of the heavy chain.

10. The ADC described in claim 7, wherein the linker is a cleavable linker.

11. The ADC of claim 10, wherein the linker comprises a valine-citrulline moiety and a PAB moiety.

12. the anti-FGFR2b antibody or antigen-binding fragment thereof, 【Chemical Engineering 201】 The ADC of any one of claims 7 to 11, which is conjugated to

13. the anti-FGFR2b antibody or antigen-binding fragment thereof, 【Chemical Engineering 202】 or a positional isomer thereof, wherein: 【Chemical 203】 is a bond to heavy chain glutamine.

14. the anti-FGFR2b antibody or antigen-binding fragment thereof, 【Chemical 204】 is conjugated to 【Chemical 205】 is the bond to the linker.

15. the anti-FGFR2b antibody or antigen-binding fragment thereof, 【Chemical 206】 is conjugated to 【Chemical 207】 is the bond to the linker.

16. The linker is 【Chemical 208】 wherein: 【Chemical Engineering 209】 refers to binding to the antibody or antigen-binding fragment thereof, 【Chemical 210】 16. The ADC of claim 15, wherein the bond marked represents a bond to the maytansinoid.

17. The cytotoxin is 【Chemistry 211】 wherein: 【Chemical Engineering 212】 represents the point of attachment to the linker.

18. the antibody or antigen-binding fragment thereof 【Chemistry 213】 is conjugated to 【Chemical 214】 is a bond to a glutamine residue of the antibody or antigen-binding fragment thereof, and n is a value between 2 and 12.

19. the antibody or antigen-binding fragment thereof 【Chemical 215】 is conjugated to 【Chemical 216】 is a bond to a glutamine residue of the antibody or antigen-binding fragment thereof, and n is a value between 2 and 12.

20. An ADC described in claim 18 or 19, wherein n = 4.

21. A nucleic acid molecule encoding a human monoclonal antibody or antigen-binding fragment thereof that binds to FGFR2b according to any one of claims 1 to 6.

22. An expression vector comprising the nucleic acid molecule described in claim 21.

23. A host cell containing the expression vector described in claim 22.

24. A pharmaceutical composition for use in treating cancer, reducing tumor growth, and / or causing tumor regression in a subject, comprising an ADC according to any one of claims 7 to 20.

25. 25. The pharmaceutical composition of claim 24, wherein the cancer is selected from the group consisting of breast invasive ductal carcinoma, gastric fundic gland adenocarcinoma, esophageal adenocarcinoma, colon adenocarcinoma, and gastroesophageal junction adenocarcinoma.

26. The pharmaceutical composition described in claim 24, wherein the cancer is gastric cancer.

27. The pharmaceutical composition of any one of claims 24 to 26, wherein the cancer or tumor overexpresses FGFR2b or expresses mutant FGFR2b.

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