Humanized anti clever-1 antibodies and their use
This invention relates to an agent and a humanized antibody or single chain Fv or Fab fragment capable of binding to human CLEVER-1 recognizing an epitope of CLEVER-1, wherein the epitope is discontinuous and comprises the sequences: PFTVLVPSVSSFSSR and QEITVTFNQFTK. This invention relates also an agent capable of binding to an epitope of human CLEVER-1 for use in removing tumour or antigen induced immunosuppression. Further, the invention relates to a pharmaceutical composition comprising the agent capable of binding to human CLEVER-1 and an appropriate excipient.
1. A humanized antibody or single chain Fv or Fab fragment capable of binding to an epitope of human CLEVER-1, wherein
said antibody or single chain Fv or Fab fragment comprises
a human IgG heavy chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO 18 and SEQ ID NO: 20, and a human IgG light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 30 with the following sequences of complementarity determining regions (CDRs)
i) of the heavy chain
CDR 1:
(SEQ ID NO: 7)
TSGMGIG,
CDR 2:
(SEQ ID NO: 8)
HIWWDDDKRYNPALKS,
and
CDR 3:
(SEQ ID NO: 9)
HYGYDPYYAMDY;
and
ii) of the light chain
CDR 1:
(SEQ ID NO: 10)
TASSSVSSSYLH,
CDR 2:
(SEQ ID NO: 11)
RTSNLAS,
and
CDR 3:
(SEQ ID NO: 12)
HQYHRSPPT.
2. The humanized antibody or single chain Fv or Fab fragment according to claim 1 capable of binding to human CLEVER-1, wherein the antibody or single chain Fv or Fab fragment binds to one or more sequences selected from the group consisting of
(SEQ ID NO: 3)
ATQTGRVFLQ,
(SEQ ID NO: 4)
DSLRDGRLIYLF,
(SEQ ID NO: 5)
SKGRILTMANQVL,
and
(SEQ ID NO: 6)
LCVYQKPGQAFCTCR.
3. The humanized antibody or single chain Fv or Fab fragment according to claim 1 capable of binding to human CLEVER-1, wherein the human IgG heavy chain variable region sequence comprises SEQ ID NO: 16, SEQ ID NO 18 or SEQ ID NO: 20.
4. The humanized antibody or single chain Fv or Fab fragment according to claim 1 capable of binding to human CLEVER-1, wherein the human IgG light chain variable region sequence comprises SEQ ID NO: 28 or SEQ ID NO: 30.
5. The humanized antibody or single chain Fv or Fab fragment capable of binding to human CLEVER-1 according to claim 1 , wherein the combination of the human IgG heavy and light chain variable regions is selected from the group consisting of the following combinations:
SEQ ID NO: 14 and SEQ ID NO: 22,
SEQ ID NO: 16 and SEQ ID NO: 22,
SEQ ID NO: 16 and SEQ ID NO: 24,
SEQ ID NO: 16 and SEQ ID NO: 26,
SEQ ID NO: 16 and SEQ ID NO: 28,
SEQ ID NO: 16 and SEQ ID NO: 30,
SEQ ID NO: 18 and SEQ ID NO: 22,
SEQ ID NO: 18 and SEQ ID NO: 24,
SEQ ID NO: 18 and SEQ ID NO: 28,
SEQ ID NO: 18 and SEQ ID NO: 30,
SEQ ID NO: 20 and SEQ ID NO: 24, and
SEQ ID NO: 20 and SEQ ID NO: 30
wherein said antibody, single chain Fv or Fab fragment is capable of binding to human CLEVER-1 with a relative IC50<1.0 in comparison to the IC50 of monoclonal antibody 3-372 (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH on Aug. 21, 2001).
6. An adjuvant for a vaccine comprising an agent capable of binding to human CLEVER-1 according to claim 1 , wherein said adjuvant is present in an amount sufficient to remove immune suppression against the vaccine antigens by modulating M2 macrophages into M1 macrophages.
7. A pharmaceutical composition comprising an agent capable of binding to human CLEVER-1 according to claim 1 in combination with an appropriate excipient.
8. The humanized antibody or single chain Fv or Fab fragment according to claim 1 , wherein the antibody or single chain Fv or Fab fragment binds to the epitope sequences:
PFTVLVPSVSSFSSR (SEQ ID NO: 1), and
QEITVTFNQFTK (SEQ ID NO: 2) on human CLEVER-1.
9. The humanized antibody or single chain Fv or Fab fragment according to claim 1 , wherein the human IgG heavy chain variable region sequence comprises SEQ ID NO: 18 and the human IgG light chain variable region sequence comprises SEQ ID NO: 30.
10. The humanized antibody or the single chain FV or Fab fragment according to claim 1 , wherein said antibody or single chain FV or Fab fragment comprises constant regions of human IgG4 heavy chain and kappa light chain.
11. The humanized antibody or the single chain FV or Fab fragment according to claim 1 , wherein said antibody or single chain FV or Fab fragment comprises constant regions of human IgG4 heavy chain and kappa light chain with the mutations L248E and/or S241P.
12. The humanized antibody or the single chain Fv or Fab fragment according to claim 1 capable of binding to human CLEVER-1 with a relative IC50<1.0 in comparison to the IC50 of monoclonal antibody 3-372 (DSM ACC2520 deposited at DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH on Aug. 21, 2001).
13. The humanized antibody or the single chain Fv or Fab fragment according to claim 12 capable of binding to human CLEVER-1 with a relative IC50<0.8, <0.6 or <0.5 in comparison to the IC50 of monoclonal antibody 3-372.
14. The humanized antibody or the single chain Fv or Fab fragment according to claim 13 capable of binding to human CLEVER-1 with a relative IC50<0.5 in comparison to the IC50 of monoclonal antibody 3-372.