Patent EA-004329-B1 (EA)
Title: Antibodies to the ed-b domain of fibronectin, their construction and uses
Inventor: NERI DARIO
IPC: A61K38/00
Filed: 1997-05-23
Abstract: 1. A process for the production of a specific binding member, representing an antibody or fragment of antibody, comprising antigen-binding domain of antibody, wherein said antigen-binding domain of antibody is isolated from synthetic molecular kit and specific for and binds directly to the ED-B oncofetal domain of fibronectin (FN), which process comprises: a) screening a peptide or protein library expressed in phage with recombinant antigen derived from the fibronectin protein; b) infecting host bacterial cells with positive clones; c) subjecting positive phage clones to a process of affinity maturation; d) repeating steps a) and b) to select positive phage clones with improved affinity for antigen; e) infecting host cells with positive clones and purifying antibody molecules from said host cells. 2. The process of claim 1, wherein step a) the phage clones are screened with recombinant antigens 7B89 or ED-B. 3. The process of claim 1 or 2, wherein nucleic acid of positive clone is used in producing by expression in host cell as antibody or the fragment of antibody, encoded by nucleic acid, said product in form of antibody or fragment of antibody comprises an antigen-binding domain of antibody, specific for and binds directly to the ED-B oncofetal domain of fibronectin (FN). 4. The process according to claim 3, wherein the product-antibody is in form of integral antibody. 5. The process according to claim 3, wherein the product-antibody is in form of scFv molecule. 6. Isolated specific binding member, representing an antibody or a fragment of antibody, comprising antigen-binding domain of antibody, wherein said antigen-binding domain is isolated from synthetic molecular kit and specific for and binds directly to the ED-B oncofetal domain of fibronectin (FN). 7. A specific binding member according to any claim 6 characterized in that said specific binding member is a variable heavy (VH) chain region of the sequence derived from human germline DP47 (codon 1 Glu codon 98 arg inclusive in Figure 1) and the CDR3 sequence Ser-Leu-Pro-Lys. 8. A specific binding member according to any claim 6 characterized in that in that said specific binding member is a variable heavy (VH) chain region of the sequence derived from human germline DP47 (codon 1 Glu codon 98 arg inclusive in Figure 1) and the CDR3 sequence Gly-Val-Gly-Ala-Phe-Arg-Pro- Tyr-Arg-Lys-His-Glu. 9. A specific binding member according to any claim 6 characterized in that in that said specific binding member is a variable heavy (VL) chain region of the sequence derived from human germline DPL16 (codon 1 Glu codon 90 Ser inclusive in Figure 1) and the remaining part of the CDR3 sequence, in particular, Pro Val Val Leu Asn Gly Val Val. 10. A specific binding member according to any claim 6 characterized in that in that said specific binding member is a variable heavy (VL) chain region of the sequence derived from human germline DPL16 (codon 1 Glu codon 90 Ser inclusive in Figure 1) and the remaining part of CDR3 sequence, in particular, Pro Phe Glu His Asn Leu Val Val. 11. A specific binding member according to any claim 6 characterized in that in that said specific binding member is a variable heavy (VH) chain region of the sequence derived from human germline DP4 (codon 1 Glu codon 98 Ser inclusive in Figure 1) and the CDR3 sequence. 12. A specific binding member according to any one of the claims 6-11 which, when measured as a purified monomer, has a dissociation constant (Kd) of 6 x10<-8>M or less for ED-B FN. 13. A specific binding member according to any one of the claims 6-11, characterized in that said specific binding member is antibody fragment. 14. A specific binding member according to any one of the claims 6-11, characterized in that said specific binding member contains an scFv molecule. 15. A specific binding member according to claim 14, wherein said binding member contains a scFv dimeric molecule. 16. A specific binding member according to claims 6-12, wherein said binding member contains an integral antibody. 17. A nucleic acid, encoding the specific binding member characterized in any of claims 6-16. 18. Phage, containing nucleic acid, characterized in claim 17, and capable the encode the specific binding member, characterized in any of claims 6-15. 19. Host cell, transformed or transfinited nucleic acid, characterized in claim 17.