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US-12624100-B2 - Humanized CLDN18.2 antibodies

US12624100B2US 12624100 B2US12624100 B2US 12624100B2US-12624100-B2

Abstract

The invention provides humanized antibodies binding to CLDN18.2 with a high affinity. Further, the antibodies do not exhibit cross-reactivity to CLDN18.1. The invention also provides nucleic acids, vectors, host cells and medical uses.

Inventors

  • Lukas Bammert
  • Lenka Kyrych Sadilkova
  • Lorenz Waldmeier
  • Roger Beerli
  • Ulrich Moebius

Assignees

  • Sotio Biotech A.S.

Dates

Publication Date
20260512
Application Date
20201207
Priority Date
20191206

Claims (17)

  1. 1 . An antibody or fragment thereof binding to claudin 18.2 (CLDN18.2), which comprises: a. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 18, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; b. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 19, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; c. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 20, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 30, respectively; d. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 12 and SEQ ID NO: 21, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 26, SEQ ID NO: 5 and SEQ ID NO: 30, respectively; e. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 13 and SEQ ID NO: 18, respectively and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 31, respectively; f. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 8, SEQ ID NO: 14 and SEQ ID NO: 22, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; g. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 15 and SEQ ID NO: 23, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 27, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; h. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 16 and SEQ ID NO: 23, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; or i. HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 8, SEQ ID NO: 17 and SEQ ID NO: 24, respectively and LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 28, SEQ ID NO: 5 and SEQ ID NO: 31, respectively.
  2. 2 . The antibody or fragment thereof of claim 1 , comprising: a. a VH sequence of SEQ ID NO: 32; b. a VH sequence of SEQ ID NO: 34; c. a VH sequence of SEQ ID NO: 35; d. a VH sequence of SEQ ID NO: 37; e. a VH sequence of SEQ ID NO: 39; f. a VH sequence of SEQ ID NO: 41; g. a VH sequence of SEQ ID NO: 42; h. a VH sequence of SEQ ID NO: 44 or i. a VH sequence of SEQ ID NO: 45; and j. a VL sequence of SEQ ID NO: 33; k. a VL sequence of SEQ ID NO: 36; l. A VL sequence of SEQ ID NO: 38; m. a VL sequence of SEQ ID NO: 40; n. a VL sequence of SEQ ID NO: 43; or o. a VL sequence of SEQ ID NO: 46.
  3. 3 . The antibody or fragment thereof of claim 1 , comprising: a. a VH sequence of SEQ ID NO: 32 and a VL sequence of SEQ ID NO: 33; b. a VH sequence of SEQ ID NO: 34 and a VL sequence of SEQ ID NO: 33; c. a VH sequence of SEQ ID NO: 35 and a VL sequence of SEQ ID NO: 36; d. a VH sequence of SEQ ID NO: 37 and a VL sequence of SEQ ID NO: 38; e. a VH sequence of SEQ ID NO: 39 and a VL sequence of SEQ ID NO: 40; f. a VH sequence of SEQ ID NO: 41 and a VL sequence of SEQ ID NO: 33; g. a VH sequence of SEQ ID NO: 42 and a VL sequence of SEQ ID NO: 43; h. a VH sequence of SEQ ID NO: 44 and a VL sequence of SEQ ID NO: 33; or i. a VH sequence of SEQ ID NO: 45 and a VL sequence of SEQ ID NO: 46.
  4. 4 . The antibody or fragment thereof of claim 1 , consisting of: a. the heavy chain sequence of SEQ ID NO: 49 and light chain sequence of SEQ ID NO: 50; b. the heavy chain sequence of SEQ ID NO: 51 and light chain sequence of SEQ ID NO: 50; c. the heavy chain sequence of SEQ ID NO: 52 and light chain sequence of SEQ ID NO: 53; d. the heavy chain sequence of SEQ ID NO: 54 and light chain sequence of SEQ ID NO: 55; e. the heavy chain sequence of SEQ ID NO: 56 and light chain sequence of SEQ ID NO: 57; f. the heavy chain sequence of SEQ ID NO: 58 and light chain sequence of SEQ ID NO: 50; g. the heavy chain sequence of SEQ ID NO: 59 and light chain sequence of SEQ ID NO: 60; h. the heavy chain sequence of SEQ ID NO: 61 and light chain sequence of SEQ ID NO: 50; or i. the heavy chain sequence of SEQ ID NO: 62 and light chain sequence of SEQ ID NO: 63.
  5. 5 . The antibody or fragment thereof of claim 1 , wherein the format of the antibody or fragment thereof is selected from the group consisting of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, synthetic IgG, IgM, F(ab) 2 , scFv, IgGΔCH2, F(ab′) 2 , scFvCH3, Fab, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv) 2 , a non-depleting IgG, a diabody, and a bivalent antibody, or Fc-engineered versions thereof.
  6. 6 . The antibody or fragment thereof of claim 1 , wherein the antibody or fragment thereof (i) is humanized; (ii) is isolated; and/or (iii) does not bind to claudin 18.1 (CLDN18.1).
  7. 7 . The antibody or fragment thereof of claim 1 , wherein the antibody or fragment thereof exhibits increased binding to claudin 18.2 (CLDN18.2) as compared to a reference antibody, wherein increased binding is measured as EC50 value and/or maxMFI value by flow cytometry titration on cells expressing claudin 18.2 (CLDN18.2), wherein the cells are HEK293T cells or PA-TU-8988S cells, wherein the reference antibody comprises a heavy chain sequence of SEQ ID NO: 47 and a light chain sequence of SEQ ID NO: 48.
  8. 8 . The antibody or fragment of claim 7 , wherein (i) the measured EC50 value of the antibody is at least 10% lower than the EC50 value of the reference antibody; and/or (ii) the measured maxMFI value of the antibody is at least 10% higher than the maxMFI value of the reference antibody.
  9. 9 . A nucleic acid encoding the antibody or fragment thereof of claim 1 .
  10. 10 . A vector comprising the nucleic acid of claim 9 .
  11. 11 . A host cell comprising the nucleic acid of claim 9 .
  12. 12 . A method of treating a subject suffering from a neoplastic disease overexpressing claudin 18.2 (CLDN18.2), comprising administering the antibody or fragment thereof of claim 1 , to the subject.
  13. 13 . The method of claim 12 , wherein the neoplastic disease overexpressing claudin 18.2 (CLDN18.2) is selected from the group consisting of pancreatic, gastric, esophageal, ovarian and lung cancer.
  14. 14 . A host cell comprising the vector of claim 10 .
  15. 15 . A method of treating a subject suffering from a neoplastic disease overexpressing claudin 18.2 (CLDN18.2), comprising administering the nucleic acid of claim 9 to the subject.
  16. 16 . A method of treating a subject suffering from a neoplastic disease overexpressing claudin 18.2 (CLDN18.2), comprising administering the vector of claim 10 to the subject.
  17. 17 . A method of treating a subject suffering from a neoplastic disease overexpressing claudin 18.2 (CLDN18.2), comprising administering the host cell of claim 11 to the subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Application No. PCT/EP2020/084831, filed Dec. 7, 2020 and titled “HUMANIZED CLDN18.2 ANTIBODIES,” which in turn claims priority from a European Patent Application having Ser. No. 19/214,104.2, filed Dec. 6, 2019, both applications of which are incorporated herein by reference in their entireties. REFERENCE TO SEQUENCE LISTING This application contains a Sequence Listing submitted via EFS-Web. The entire contents of the sequence listing in ASCII text file entitled “S12415_PCT_Sequence_Listing.txt” created on Dec. 3, 2020, and having a size of 110 kilobytes, is incorporated herein by reference. BACKGROUND Tight junctions are multiprotein complexes connecting adjacent epithelial or endothelial cells to form a barrier, preventing molecules from passing in between the cells, and helping to maintain the cell and tissue polarity. Tight junctions consist of three main groups of transmembrane proteins: claudins and occludin, cytoplasmic plaque proteins, and cingulin. They also contain cytoskeletal and signaling proteins, e.g. actin, myosin II, and PKCζ. These proteins interact to maintain tight junction structure (Yu and Turner 2008). Claudins form a family of 23 proteins (Hewitt, Agarwal, and Morin 2006). Claudin 18 is human protein encoded by the CLDN18 gene, which forms tight junction strands in epithelial cells. The human CLDN18 can be alternatively spliced with two alternative first exons, resulting in two protein isoforms, CLDN18.1 (or Claudin 18.1) and CLDN18.2 (or Claudin 18.2). CLDN18.2 was first disclosed as Zsig28 protein in WO2000/015659. The two isoforms differ in the N-terminal 69 amino acids, encompassing the first extracellular loop. The first extracellular domain spans from amino acid 28 to amino acid 80. Within this stretch there are 8 amino acid differences between CLDN18.1 and CLDN18.2. The two different isoforms are expressed in different tissues, with CLDN18.1 being predominantly expressed in lung tissue whereas CLDN18.2 displays stomach specificity (Niimi et al. 2001). CLDN18.2 expression in normal stomach is restricted to the differentiated short-lived cells of stomach epithelium. CLDN18.2 expression has further been identified in various tumor tissues. For example, CLDN18.2 has been found to be expressed in pancreatic, esophageal, ovarian, and lung tumors, correlating with distinct histologic subtypes (Sahin et al. 2008). In view of its restricted expression pattern in normal tissues, and its ectopic expression in human cancers, CLDN18.2 is an attractive pan-cancer target for antibody therapy of epithelial tumors. A number of studies have been made towards such an antibody therapy. WO2004/047863 identified the splice variants of CLDN18 and screened antibodies against different peptides derived from CLDN18.2: peptide DQWSTQDLYN (SEQ ID NO: 68), N-terminal extracellular of CLDN18.2, independent of glycosylation; peptide NNPVTAVFNYQ (SEQ ID NO: 69), N-terminal extracellular of CLDN18.2, mainly unglycosylated; and peptide STQDLYNNPVTAVF (SEQ ID NO: 70), N-terminal extracellular domain of CLDN18.2, unglycosylated. It also disclosed polyclonal rabbit antibodies screened with a pan-CLDN18 peptide TNFWMSTANMYTG (SEQ ID NO: 71) in the C-terminal extracellular domain common to both CLDN18.1 and CLDN18.2 isoforms. WO2005/113587 discloses antibodies against specific epitopes on CLDN18.2 defined by the following peptide sequences: ALMIVGIVLGAIGLLV (SEQ ID NO: 72) and RIGSMEDSAKANMTLTSGIMFIVS (SEQ ID NO: 73). WO200/7059997 discloses CLDN18.2 specific monoclonal antibodies obtained by immunization with the peptide METDTLLLWVLLLWVPGSTGDAAQPARRARRTKLGTELGSTPVWWNSADGRMDQ WSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRAAIQH SGGRSRRARTKTHLRRGSE (SEQ ID NO: 74), including the first extracellular domain of CLDN18.2 with N- and C-terminal extensions. Antibodies obtained by this immunization mediate cell killing by complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Antibody IMAB362, also known as Claudiximab or Zolbetuximab, is disclosed in WO2007/059997 and WO2016/165762. IMAB362 is an IgG1 antibody derived from a murine monoclonal antibody and has been chimerized to display the human IgG1 constant region for clinical use. WO2008/145338 also discloses antibodies binding to overlapping peptides within the first extracellular domain (MDQWSTQDLYNNPVT (SEQ ID NO: 75), LYNNPVTAVFNYQGL (SEQ ID NO: 76), VFNYQGLWRSCVRES (SEQ ID NO: 77), QGLWRSCVRESSGFT (SEQ ID NO: 78), and RSCVRESSGFTECRG (SEQ ID NO: 79)). In an effort to produce antibodies targeting the C-terminal portion of CLDN18.2 for diagnostic purposes to detect CLDN18.2 expression in cells of cancer tissue sections, WO2013/167259 discloses antibodies binding to C-terminal epitopes of CLDN18.2. The sequences of the two epitopes are TEDEVQSYPSKHDYV (SEQ ID NO: 80) and EVQSYPSKHDYV (SEQ ID NO: 81). WO2013/174509 presents combinat