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WO-2026092428-A1 - NON-FUCOSYLATED ANTI-FGFR2B MONOCLONAL ANTIBODY

WO2026092428A1WO 2026092428 A1WO2026092428 A1WO 2026092428A1WO-2026092428-A1

Abstract

The present invention provides a non-fucosylated anti-FGFR2-IIIb antibody. Specifically, the present invention provides a non-fucosylated anti-FGFR2-IIIb antibody, a method for expressing the non-fucosylated FGFR2-IIIb antibody by using FUT8 knockout cell lines, and an application of the antibody in the treatment of diseases. Compared with conventionally expressed antibodies, the non-fucosylated antibody of the present invention exhibits enhanced FcyRllla binding affinity. The antibody of the present invention can enhance ADCC effect, and has potential for applications in the field of oncotherapy.

Inventors

  • ZHANG, XUEJUN
  • LIU, LIFEI
  • CHENG, Zhikui
  • YANG, JUN
  • LI, Lie
  • HE, Kun
  • HU, Lingli

Assignees

  • 湖北生物医药产业技术研究院有限公司

Dates

Publication Date
20260507
Application Date
20251028
Priority Date
20241028

Claims (14)

  1. An antibody against FGFR2Ⅲb, said antibody comprising a heavy chain and a light chain, wherein the variable region of said heavy chain has a complementarity-determining region (CDR) selected from the group consisting of: (1) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:7, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rules; (2) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:16, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rules; (3) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:19, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rules; (4) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:19, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rules; (5) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:21, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rules; and (6) VH-CDR1 shown in SEQ ID NO:9, VH-CDR2 shown in SEQ ID NO:10, and VH-CDR3 shown in SEQ ID NO:11, wherein the CDRs are defined according to the IMGT rules; Furthermore, the variable region of the light chain has a complementary determinant region (CDR) selected from the following group: (1) VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:13, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDR is defined according to the Kabat rule; (2) VL-CDR1 shown in SEQ ID NO:17, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDRs are defined according to the Kabat rules; (3) VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, wherein the CDRs are defined according to the Kabat rules; and (4) VL-CDR1 shown in SEQ ID NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDRs are defined according to the IMGT rules; Furthermore, any amino acid sequence in the above-mentioned CDR sequence also includes a derivative sequence which optionally involves the addition, deletion, modification and/or substitution of 1-2 amino acids, such that the derivative antibody composed of the heavy chain and light chain containing the derived CDR sequence can retain the binding affinity of FGFR2Ⅲb or its derivative protein. Wherein, the fucosylation ratio of the antibody is ≤10%, preferably, the fucosylation ratio of the antibody is ≤5%, ≤3%, ≤2%, or ≤1%, and more preferably, the antibody is not fucosylated.
  2. The antibody as claimed in claim 1, characterized in that the antibody has a heavy chain variable region CDR (VH-CDR) and a light chain variable region CDR (VL-CDR) selected from the group consisting of: (1) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:7, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:13, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDRs are defined according to the Kabat rules; (2) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:16, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:17, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDRs are defined according to the Kabat rules; (3) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:19, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, wherein the CDRs are defined according to the Kabat rules; (4) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:19, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, wherein the CDRs are defined according to the Kabat rules; (5) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:21, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, wherein the CDRs are defined according to the Kabat rules; (6) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:16, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, wherein the CDRs are defined according to the Kabat rules; and (7) VH-CDR1 shown in SEQ ID NO:9, VH-CDR2 shown in SEQ ID NO:10, VH-CDR3 shown in SEQ ID NO:11, VL-CDR1 shown in SEQ ID NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDRs are defined according to the IMGT rules.
  3. The antibody according to claim 1, characterized in that the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 31 or 4 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with it; and/or the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 25, 27, 29 or 5 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with it.
  4. The antibody according to claim 1, characterized in that the antibody has: (1) The heavy chain variable region as shown in SEQ ID NO:24 and the light chain variable region as shown in SEQ ID NO:25; (2) The heavy chain variable region as shown in SEQ ID NO:26 and the light chain variable region as shown in SEQ ID NO:27; (3) The heavy chain variable region as shown in SEQ ID NO:28 and the light chain variable region as shown in SEQ ID NO:29; (4) The heavy chain variable region as shown in SEQ ID NO:30 and the light chain variable region as shown in SEQ ID NO:29; (5) The heavy chain variable region as shown in SEQ ID NO:31 and the light chain variable region as shown in SEQ ID NO:29; (6) The heavy chain variable region as shown in SEQ ID NO:26 and the light chain variable region as shown in SEQ ID NO:29; or (7) The heavy chain variable region as shown in SEQ ID NO:4 and the light chain variable region as shown in SEQ ID NO:5.
  5. The antibody according to claim 1, characterized in that the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 32, 34, 36, 38, 39, or 22, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with it; and/or the amino acid sequence of the light chain is as shown in SEQ ID NO: 33, 35, 37, or 23, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with it.
  6. The antibody according to claim 1, characterized in that the antibody has: (1) The heavy chain as shown in SEQ ID NO:32 and the light chain as shown in SEQ ID NO:33; (2) The heavy chain as shown in SEQ ID NO:34 and the light chain as shown in SEQ ID NO:35; (3) The heavy chain as shown in SEQ ID NO:36 and the light chain as shown in SEQ ID NO:37; (4) The heavy chain as shown in SEQ ID NO:38 and the light chain as shown in SEQ ID NO:37; (5) The heavy chain as shown in SEQ ID NO:39 and the light chain as shown in SEQ ID NO:37; (6) The heavy chain as shown in SEQ ID NO:34 and the light chain as shown in SEQ ID NO:37; or (7) The heavy chain as shown in SEQ ID NO:22 and the light chain as shown in SEQ ID NO:23.
  7. A composition comprising the antibody as described in any one of claims 1-6, characterized in that at least 95% of the anti-FGFR2Ⅲb antibody in the composition is unfucosylated.
  8. A recombinant protein, said recombinant protein comprising: (i) the antibody as claimed in claim 1; and (ii) Optional tag sequences to assist in expression and/or purification.
  9. A host cell, characterized in that its genome integrates a polynucleotide encoding an antibody as described in claim 1 or a recombinant protein as described in claim 8, or the host cell contains a vector comprising the polynucleotide. The host cells are engineered to lose their protein fucosylation modification function.
  10. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: (i) an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 8, the host cell of claim 9, or a combination thereof; and (ii) Pharmaceutically acceptable carriers.
  11. Use of the antibody of claim 1, the recombinant protein of claim 8, the host cell of claim 9, or the pharmaceutical composition of claim 10 in the preparation of a medicament for the prevention and/or treatment of diseases associated with abnormal expression or function of FGFR2Ⅲb.
  12. The use as described in claim 12, wherein the disease is cancer.
  13. The use as described in claim 12 is characterized in that the cancer is selected from: breast cancer, gastric cancer, esophageal cancer, colorectal cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, cholangiocarcinoma, lung cancer, and non-small cell lung cancer.
  14. A detection plate, characterized in that the detection plate comprises: a substrate and a test strip, wherein the test strip contains an antibody as described in claim 1, or a recombinant protein as described in claim 8, or a combination thereof.

Description

Fucosylated anti-FGFR2b monoclonal antibody This application claims priority to Chinese Patent Application No. 202411516256.8, filed on October 28, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field This invention relates to the field of biomedicine. Specifically, this invention relates to a fucosylated anti-FGFR2IIIb monoclonal antibody. Background Technology Fibroblast growth factor receptor (FGFR) belongs to a subfamily of the tyrosine kinase receptor superfamily. There are four FGFR genes (FGFR1-4) in the human genome. FGFR-mediated signaling pathways are essential for normal cell growth and differentiation, participating in physiological processes such as angiogenesis, cell proliferation and migration, regulation of organ development, and wound healing. However, mutations or overexpression of FGFR can lead to excessive activation of the FGFR signaling pathway, further inducing the transformation of normal cells into cancerous cells. Specifically, excessive activation of RAS-RAF-MAPK can stimulate cell proliferation and differentiation; excessive activation of PI3K-AKT inhibits apoptosis; SATA is closely related to promoting tumor invasion and metastasis and enhancing tumor immune escape capabilities; and the PLCγ signaling pathway is an important pathway for regulating tumor cell metastasis. FGFRs contain three extracellular immunoglobulin-like domains (D1-D3), a hydrophobic single-transmembrane helical structure, and an intracellular tyrosine kinase domain, with an 8-residue acid cassette between D1 and D2. In FGFRs 1-3, ligand binding specificity largely depends on alternative splicing at the C-terminus of the D3 domain. There are two alternative splices at the C-terminus of the D3 domain, encoded by exon 8 or 9 to generate FGFRb or FGFRc isoforms. These b and c isoforms are typically confined to epithelial and mesenchymal tissues, respectively. In this way, selective splicing of the receptor allows the ligand to activate the receptor in adjacent mesenchymal or epithelial tissues without activating autocrine signals. The primary ligands for the FGFR2IIIb (or FGFR2b) form of FGFR2 are FGF1, FGF7, FGF10, and FGF22. FGFR2b is highly expressed in tumors through FGFR2 gene amplification or transcriptional upregulation of FGFR2b subtypes, with FGFR2 amplification being the most common FGFR2 gene aberration. In immunohistochemical section analysis of gastric cancer patients, FGFR2b expression was detected in approximately 60% of tumor samples, with 2%–9% of these samples showing FGFR2b overexpression at levels ranging from 31% to 61%. These gastric cancer samples often exhibit diffuse overexpression and are associated with aggressive characteristics of gastric cancer, including higher-grade T stages, more frequent lymph node metastasis, and lower overall survival. Further research has established that overexpression of the FGFR2b receptor or FGFR2 gene amplification is directly related to poor prognosis in gastric cancer patients. Besides gastric cancer, aberrant activation of the FGF/FGFR2 signaling pathway has also been observed in other cancers, including but not limited to esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, lung cancer (e.g., non-small cell lung cancer), and cholangiocarcinoma. Therefore, inhibiting FGFR2 signaling may be an effective mechanism for treating various cancers. Modifying the Fc domain of antibodies can enhance antibody-dependent cell-mediated cytotoxicity (ADCC), thereby increasing antitumor activity. Previous studies have found that mutations in the Fc domain can enhance the binding affinity of antibodies to Fc receptors, thus strengthening ADCC activity; for example, the S239D/A330L/I332E mutant can enhance cytotoxicity. Furthermore, alterations in the glycosylation of the Fc domain can also affect its effector function. It has been reported that a reduction in core fucosylation in antibodies alters the function of Fc effectors, particularly Fcγ receptor binding and ADCC activity. Therefore, there is a need in this field to develop an anti-FGFR2IIIb monoclonal antibody with a stronger ADCC effect. Summary of the Invention The purpose of this invention is to provide an anti-FGFR2IIIb monoclonal antibody with a strong ADCC effect. In a first aspect of the invention, an antibody against FGFR2Ⅲb is provided, the antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementarity-determining region (CDR) selected from the group consisting of: (1) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:7, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rules; (2) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:16, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rules; (3) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID N