New monoclonal anti-idiotypic antibodies, useful for treating hemophilia A, are directed against human antibodies that inhibit factor VIII

摘要

#CMT# #/CMT# Monoclonal anti-idiotypic antibodies (Ab) directed against a human antibody (Ab2) that inhibits factor VIII (F8), where Ab2 is directed against the C1 domain of F8 is new. In Ab (i) at least one complementarity-determining region (CDR) of each light chain includes a peptide sequence at least 70% identical with SEQ ID NOs: 12, 13 or 14 and (ii) at least one CDR of each heavy chain includes a peptide sequence at least 70% identical with SEQ ID NOs: 9, 10 or 11. #CMT# : #/CMT# Arg-Ala-(Ser) 3-Val-Ser-Tyr-Met-Asn (12) Ala-Thr-Ser-Asn-Leu-Ala-Ser (13) Gln-Gln-Trp-Ser-Ser-Asn-Pro-Pro-Met-Leu-Thr (14) Gly-Tyr-Thr-Phe-Thr-Thr-Tyr-Trp-Met-His (9) Tyr-Ile-Asn-Pro-Thr-Ser-Gly-Tyr-Thr-Glu-Tyr-Asn-Gln-Asn-Phe-Lys (10) Ser-Gly-Ala-Tyr-Tyr-Arg-Tyr-Asp-Asp-Ala-Met-Asp-Ser (11). Independent claims are included for the following: (1) stable cell lines that produce Ab; (2) hybridoma 18D6 (CNCM I-3559); and (3) DNA fragments Seq. (15) and (16) (of 325 and 367 bp; reproduced) that encode the variable regions of, respectively, the heavy and light chains of Ab. #CMT#ACTIVITY : #/CMT# Hemostatic. No details of tests for hemostatic activity are given. #CMT#MECHANISM OF ACTION : #/CMT# Inhibition of autoantibodies that inactivate factor VIII. #CMT#USE : #/CMT# Ab are used (i) for treatment of hemophilia A, particularly with inhibitors; (ii) for in vitro neutralization of Ab2 and (iii) for in vitro detection and/or purification of Ab2 (all claimed). #CMT#BIOTECHNOLOGY : #/CMT# Preferred Antibodies: In Ab, each CDR in each heavy/light chain sequence has at least 70% identity with Seq. (9)-(14), and these chains are encoded by sequences at least 70, best 100, % identical with Seq. (16) and (15). Sequences encoded by Seq. (15) and (16) are reproduced (122 and 108 amino acids, respectively). Particularly Ab is a murine antibody of class IgG1kappa or it is a chimera (particularly a human hybrid) or humanized antibody. Ab may be used as a fragment (F(ab') 2, Fab', Fab, CDR or their modified forms) and most especially is the product of hybridoma 18B6. Preparation: Ab are made by essentially standard methods for preparing chimeric antibodies, e.g. recombinant DNA techniques are used to replace the sequences encoding the constant region of a non-human antibody chain with the corresponding human sequence. #CMT#EXAMPLE : #/CMT# The antibody RHD5, directed against the C1 domain of factor VIII (F8) was used to immunize mice, then spleen cells from the immunized animals that produced anti-RHD5 antibodies were fused with SP2/0 myeloma cells. The resulting hybridomas were screened by enzyme-linked immunosorbent assay to identify antibodies able to inhibit binding of RHD5 to insolubilized recombinant F8. One antibody, 18B6, at concentration 4 mu g/ml gave 89.5% neutralization of 0.4 mu g/ml RHD5.