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WO-2026092717-A1 - HUMANIZED ANTI-MSLN ANTIBODY AND USE THEREOF

WO2026092717A1WO 2026092717 A1WO2026092717 A1WO 2026092717A1WO-2026092717-A1

Abstract

The present invention provides a humanized anti-MSLN antibody and a use thereof. The humanized MSLN antibody of the present invention is modified to improve affinity, and the affinity thereof for binding to a human MSLN antigen is equal to or better than that of a commercially available humanized anti-MSLN antibody.

Inventors

  • HUANG, KE
  • LI, YUHANG

Assignees

  • 深圳市济因生物科技有限公司

Dates

Publication Date
20260507
Application Date
20251031
Priority Date
20241031

Claims (20)

  1. A humanized antibody or antigen-binding fragment, characterized in that it comprises VH and VL, wherein; VH includes one or more of the following: The amino acid sequence of CDR-H1 is shown in SEQ ID NO:39: X 1 YTMN, where X 1 is Y, G or N; The amino acid sequence of CDR-H2 is shown in SEQ ID NO:4: LITPYNGASSYNQKFRG; and/or, The amino acid sequence of CDR-H3 is shown in SEQ ID NO:6: GGYDGRGFDY; And/or, VL includes one or more of the following: The amino acid sequence of CDR-L1 is shown in SEQ ID NO:40: SASSX 2 VSYMH, where X2 is S or Y; The amino acid sequence of CDR-L2 is shown in SEQ ID NO:11: DTSKLAS; and/or, The amino acid sequence of CDR-L3 is shown in SEQ ID NO:13: QQWSKHPLT.
  2. The antibody or antigen-binding fragment as described in claim 1, wherein X1 is G.
  3. The antibody or antigen-binding fragment as described in claim 1, wherein X2 is S.
  4. The antibody or antigen-binding fragment as described in any one of claims 1 to 3 is characterized in that, The antibody or antigen-binding fragment targets MSLN, is humanized, and has more than 80% sequence homology with any one of the following amino acid sequences: The VH region is selected from one of the following: SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; and/or, The VL region is selected from one of the following: SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26.
  5. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that the VH comprises one or more of the following: The amino acid sequence of FR-H1 is shown in SEQ ID NO:1; the amino acid sequence of FR-H2 is shown in SEQ ID NO:3; the amino acid sequence of FR-H3 is shown in SEQ ID NO:5; and/or, the amino acid sequence of FR-H4 is shown in SEQ ID NO:7.
  6. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that the VL comprises one or more of the following: The amino acid sequence of FR-L1 is shown in SEQ ID NO:41: DIX 3 LTQSPSX 4 X 5 SASX 6 GDRVTITC, where X 3 is E or Q, X 4 is A or S, X 5 is M or L, and X 6 is V or P; The amino acid sequence of FR-L2 is shown in SEQ ID NO:42: WYQQKPGX 7 X 8 PKRWIY, where X 7 is T or K and X 8 is S or A; The amino acid sequence of FR-L3 is shown in SEQ ID NO:43: GVPSRFSGSGSGNX 9 YTLTISSX 10 QPEDFATYY, where X 9 is S or D, X 10 is L or V; and/or, The amino acid sequence of FR-L4 is shown in SEQ ID NO:44: FGX 11 GTKVEIK, where X 11 is Q or G.
  7. A scFv targeting MSLN, characterized in that it comprises an antibody or antigen-binding fragment as claimed in any one of claims 1 to 6; The VH and VL are connected by a flexible linker peptide.
  8. An isolated polynucleotide, characterized in that the polynucleotide encodes an antibody or antigen-binding fragment of any one of claims 1 to 6 or the scFv of claim 7.
  9. The use of an antibody or antigen-binding fragment of any one of claims 1 to 6, the scFv of claim 7, or the polynucleotide of claim 8 in the preparation of a targeting vector.
  10. A targeting vector, characterized in that the surface of the targeting vector contains a recombinant targeting molecule, the recombinant targeting molecule containing a targeting binding region, the targeting binding region containing an antibody or antigen binding fragment of any one of claims 1 to 6 or the scFv of claim 7.
  11. The targeting vector as described in claim 10 is characterized in that the vector is selected from one or more of lipid nanoparticles, virus-like particles, extracellular vesicles, adenovirus, adeno-associated virus, pseudolentiviral vectors, and retroviral vectors.
  12. The targeting vector according to claim 11 is characterized in that the vector is a pseudolentiviral vector and a retroviral vector.
  13. According to any one of claims 10 to 12, the recombinant targeting molecule further comprises a transmembrane region, and the targeting binding region is directly or indirectly connected to the transmembrane region and is displayed on the surface of the targeting carrier.
  14. The targeting vector as described in claim 13, wherein the transmembrane region is selected from the transmembrane regions of the following proteins: CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1 BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B 4. BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D and CS1.
  15. According to claim 13, the targeting vector is characterized in that the recombinant targeting molecule further comprises a linker domain, and the targeting binding region is indirectly connected to the transmembrane region through the linker domain; the linker domain is selected from: (a) Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions; (b) Hinge region, wherein the hinge region is selected from the wild-type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154; (c) All or part of the Fc domain, wherein the Fc domain is selected from one or more of the CH1, CH2, and CH3 domains; and (d) Stem regions of type II C-lectins, wherein the type II C-lectins are selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A and NKG2D.
  16. The use of an antibody or antigen-binding fragment of any one of claims 1 to 6, the scFv of claim 7, or the polynucleotide of claim 8 in the preparation of a chimeric antigen receptor.
  17. A chimeric antigen receptor, characterized in that it comprises: (a) Extracellular antigen-binding region; (b) Transmembrane region; and (c) Intracellular signal transduction domains; The extracellular antigen-binding region comprises an antibody or antigen-binding fragment of any one of claims 1 to 6 or the scFv of claim 7.
  18. The chimeric antigen receptor as claimed in claim 17, characterized in that the transmembrane region is selected from the transmembrane regions of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244. 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcεRIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.
  19. The chimeric antigen receptor as claimed in claim 17, wherein the intracellular signal transduction domain is selected from the intracellular signal transduction domains of the following proteins: CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79b, FcεRIγ, FcεRβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12, and other intracellular signal transduction domains of proteins whose intracellular signal transduction domains include at least one ITAM.
  20. The chimeric antigen receptor as described in any one of claims 17 to 19, characterized in that the chimeric antigen receptor further comprises a hinge region connecting the extracellular antigen-binding region and the transmembrane region; The hinge region is selected from the hinge regions of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

Description

A humanized anti-MSLN antibody and its application Technical Field This invention relates to the field of biomedicine, specifically to a humanized MSLN antibody and its applications. Background Technology Mesothelin (MSLN) is a glycoprotein found on cell surfaces and in serum. Its gene encodes a 69 kDa precursor protein, which is enzymatically hydrolyzed during maturation into a membrane-bound protein of approximately 40 kDa, retaining its C-terminus. This mature mesothelin is the precursor. The approximately 30 kDa N-terminal fragment of the hydrolyzed protein is a fragment of megakaryocyte-promoting factor (MPF) that detaches and is released extracellularly into blood and urine. Under normal circumstances, MSLN expression is limited to mesothelial cells (peritoneum, pericardium, and pleural cavity) at low levels, and is essentially absent in other tissues and cells. However, MSLN has been found to be significantly overexpressed in various tumor types, including mesothelioma, pancreatic cancer, non-small cell lung cancer, lung adenocarcinoma, fallopian tube cancer, head and neck cancer, cervical cancer, and ovarian cancer. Studies have shown that abnormal MSLN expression promotes tumor cell proliferation, invasion, and metastasis. The fact that MSLNs are not expressed or are expressed at low levels in normal cells gives them a potential advantage as a specific target for targeted cancer therapy. Clinical studies have shown that MSLN expression is correlated with tumor severity and survival. Summary of the Invention Beneficial effects In view of this, the present invention provides a humanized antibody or antigen-binding fragment comprising VH and VL, wherein; VH includes one or more of the following: The amino acid sequence of CDR-H1 is shown in SEQ ID NO:39: X 1 YTMN, where X 1 is Y, G or N; The amino acid sequence of CDR-H2 is shown in SEQ ID NO:4: LITPYNGASSYNQKFRG; and/or, The amino acid sequence of CDR-H3 is shown in SEQ ID NO:6: GGYDGRGFDY; And/or, VL includes one or more of the following: The amino acid sequence of CDR-L1 is shown in SEQ ID NO:40: SASSX 2 VSYMH, where X2 is S or Y; The amino acid sequence of CDR-L2 is shown in SEQ ID NO:11: DTSKLAS; and/or, The amino acid sequence of CDR-L3 is shown in SEQ ID NO:13: QQWSKHPLT. Preferably, X1 is G. Preferably, X2 is S. The aforementioned antibody or antigen-binding fragment, which targets MSLN, is humanized and has more than 80% sequence homology with any one of the following amino acid sequences: a. The VH region is selected from one of the following: SEQ ID NO:21: EVQLVQSGAEVKKPGASVKVSCKASGYSFTYYTMNWVRQAPGQSLEWIGLITPYNGASSYNQKFRGRATLTVDKSASTAYMELSSLRSEDMAVYFCARGGYDGRGFDYWGQGTTVTVSS; SEQ ID NO:23: EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQSLEWIGLITPYNGASSYNQKFRGRATLTVDKSASTAYMELSSLRSEDMAVYFCARGGYDGRGFDYWGQGTTVTVSS; SEQ ID NO:25: EVQLVQSGAEVKKPGASVKVSCKASGYSFTNYTMNWVRQAPGQSLEWIGLITPYNGASSYNQKFRGRATLTVDKSASTAYMELSSLRSEDMAVYFCARGGYDGRGFDYWGQGTTVTVSS; b. The VL zone is selected from one of the following: SEQ ID NO:22: DIELTQSPSAMSSVGDRVTITCSASSSVSYMHWYQQKPGTSPKRWIYDTSKLASGVPSRFSGSGSGNSYTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKVEIK; SEQ ID NO:24: DIELTQSPSAMSSASVGDRVTITCSASSYVSYMHWYQQKPGTSPKRWIYDTSKLASGVPSRFSGSGSGNSYTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKVEIK; SEQ ID NO:26: DIQLTQSPSSLSASPGDRVTITCSASSYVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGNDYTLTISSVQPEDFATYYCQQWSKHPLTFGGGTKVEIK. The antibody or antigen-binding fragment of any of the above, wherein the VH includes one or more of the following: The amino acid sequence of FR-H1 is shown in SEQ ID NO:1: EVQLVQSGAEVKKPGASVKVSCKASGYSFT; The amino acid sequence of FR-H2 is shown in SEQ ID NO:3: WVRQAPGQSLEWIG; The amino acid sequence of FR-H3 is shown in SEQ ID NO:5: RATLTVDKSASTAYMELSSLRSEDMAVYFCAR; and/or, The amino acid sequence of FR-H4 is shown in SEQ ID NO:7: WGQGTTVTVSS. The antibody or antigen-binding fragment described in any of the above-mentioned embodiments, wherein the VL comprises one or more of the following: The amino acid sequence of FR-L1 is shown in SEQ ID NO:41: DIX 3 LTQSPSX 4 X 5 SASX 6 GDRVTITC, where X 3 is E or Q, X 4 is A or S, X 5 is M or L, and X 6 is V or P; The amino acid sequence of FR-L2 is shown in SEQ ID NO:42: WYQQKPGX 7 X 8 PKRWIY, where X 7 is T or K and X 8 is S or A; The amino acid sequence of FR-L3 is shown in SEQ ID NO:43: GVPSRFSGSGSGNX 9 YTLTISSX 10 QPEDFATYY, where X 9 is S or D, X 10 is L or V; and/or, The amino acid sequence of FR-L4 is shown in SEQ ID NO:44: FGX 11 GTKVEIK, where X 11 is Q or G. The present invention also provides an scFv targeting MSLN, comprising any of the above-mentioned antibody or antigen-binding fragments; The VH and VL are connected by a flexible linker peptide. More preferably, the flexible linker peptide is selected from (G4S)n linker peptide and linker peptide 1 with an amino acid sequence as shown in SEQ ID NO:33; wherein n=1 to 4. Also provided is an isolated polynucleotide encoding an antibody or antigen-binding fragment of any one of the above opt