RU99101121A

RU99101121A - POLYEPEPTIDS ABLE TO FORM ANTIGEN-BINDING STRUCTURES SPECIFIC TO RES-D ANTIGENS, DNA ENCODING THEM, METHODS FOR PRODUCING AND APPLICATION

Info

Publication number: RU99101121A
Application number: RU99101121/13A
Authority: RU
Inventors: Сильвия Мишер; Моник Вожель; Беда Штадлер; Андреас Морелль; Мартин Имбоден; Ханспетер Амштутц
Original assignee: Роткройцштифтунг Центральлабораториум Блутшпендединст СРК
Priority date: 1996-06-24
Filing date: 1997-06-20
Publication date: 2000-12-27

Claims

1. Polypeptides capable of forming antigen-binding structures with specificity for rhesus D antigens, containing rhesus D specific regions of CDR 1, CDR 2 and CDR 3 pairs of amino acid sequences V _H and V _L , with the same or different identification numbers corresponding to certain figures, as shown in table 1, shown in the graphic part.

2. The polypeptides according to claim 1, including the rhesus-D-specific region of CDR 1, CDR 2 and CDR 3 pairs of amino acid sequences of V _H and V _L , with the same identification numbers corresponding to certain figures, as shown in table 1 in paragraph 1 .

3. The polypeptides according to claim 1, including regions with amino acid sequences of V _H and V _L and having identification numbers corresponding to certain figures, as shown in table 1 in claim 1.

4. The polypeptides according to claims 1, 2 or 3, characterized in that they represent antigen-binding Fab fragments.

5. The polypeptides according to claims 1, 2 or 3, containing the heavy and light chains of immunoglobulin, capable of forming complete anti-Rhesus D antibodies.

6. DNA sequences encoding polypeptides capable of forming antigen-binding structures with specificity for rhesus D antigens, containing regions with rhesus D specific segments of CDR 1, CDR 2 and CDR 3 pairs of DNA sequences V _H and V _L , with the same or different identification numbers corresponding to certain figures, as shown in table 2, shown in the graphic part and their functional equivalents.

7. DNA sequences according to claim 6, encoding polypeptides capable of forming antigen-binding structures with specificity for rhesus D antigens, including regions with rhesus D specific segments of CDR 1, CDR 2 and CDR 3 pairs of V _H DNA sequences and V _L , with the same identification numbers corresponding to certain figures, as shown in table 2 in paragraph 6.

8. DNA sequences according to claim 6 or 7, including regions with DNA sequences V _H and V _L , with identification numbers corresponding to certain figures, as shown in table 2 in paragraph 6.

9. DNA sequences according to claim 6 or 7 or 8, encoding polypeptides capable of forming antigen-binding Fab fragments.

10. DNA sequences according to claim 6 or 7 or 8, encoding polypeptides capable of forming complete anti-Rhesus D antibodies.

11. A method for producing recombinant polypeptides capable of forming antigen-binding structures, for example, Fab fragments, with specificity for Rh-D antigens, comprising, in this order, stages
a) re-immunizing an individual capable of generating anti-Rhesus D antibodies with Rhesus D positive red blood cells,
b) isolating mononuclear cells from an individual,
c) isolation of complete RNA from mononuclear cells,
d) obtaining cDNA through the use of oligo (dT) seed and reverse transcription of mRNA with reverse transcriptase M-MuLV and amplification of the cDNA kit by polymerase chain reaction using a family of specific seeds of immunoglobulin genes,
f) creating a phage representative library by inserting DNA encoding the heavy and light chain of the Fab polypeptide into the phage vector; DNA for the heavy chain is inserted into the frame of the gene encoding phage protein pIII, which allows the expression of the fused protein Fab-pIII on the surface of the phage,
f) transforming the bacterial cells with the obtained recombinant plasmids, culturing the transformed bacterial cells and coexpressing the heavy and light chain Fab on the filamentous phage particles,
g) amplification of the Fab-bearing phage in bacteria,
h) selection of individual phage clones through several cycles of panning on Rh-D-positive red blood cells,
i) isolation of plasmid DNA from selected clones and excision of the cpIII gene,
j) transforming the bacterial cells with the obtained plasmid, culturing the transformed bacterial cells expressing Fab, and isolating the Fab fragments.

12. A method for the selection of recombinant polypeptides capable of forming antigen-binding structures with specificity for Rh-D antigens, and, in particular, showing reactivity with respect to the incomplete Rh-DVI variant, and without any signs of reactivity with Rh-negative red blood cells phenotypes, in particular, without any signs of reactivity against the rhesus alleles C, c, E and e, including, in that order, stages
a) the implementation of several negative removals on the following erythrocytes: phenotype 1 (rr ', Ccddee), treated with bromelase, phenotype 1, untreated bromelase, phenotype 2 (ryry, CCddEE) treated with bromelase, and phenotype 2, untreated bromelase,
b) the implementation of positive absorption on DVI + red blood cells with or without treatment with bromelase,
c) determining the titer of phage binding to DVI + red blood cells,
d) repeating steps a), b) and c) until the titer of the phage binding to DVI + red blood cells reaches a satisfactory level.

13. The method according to p. 12, characterized in that the recombinant polypeptides capable of forming antigen-binding structures, represent Fab fragments.

14. Anti-Rhesus D antibodies with variable regions of the heavy and light chains containing Rhesus D-specific sequences of CDR 1, CDR 2 and CDR 3 pairs of amino acid sequences V _H and V _L , with the same or different identification numbers in accordance with table 3 given in the graphic part.

15. Anti-Rhesus D antibodies with variable regions of the heavy and light chains containing Rhesus D-specific sequences of CDR 1, CDR 2 and CDR 3 pairs of amino acid sequences V _H and V _L , with the same identification numbers, as shown in the table in p .fourteen.

16. The anti-Rhesus D antibody of claim 14 or 15, comprising pairs of amino acid sequences of V _H and V _L , with identification numbers corresponding to certain figures, as shown in the table in paragraph 14.

17. Antirhesus-D antibodies according to claims. 14, 15 or 16, characterized in that the immunoglobulin constant regions represent at least one of the specific phenotypes IgG1, IgG2, IgG3 or IgG4.

18. The method of obtaining complete anti-Rhesus D antibodies according to any one of claims 14-17, comprising, in the indicated order, stages
a) amplification, individually, of a pair of members from the heavy chain V-gene segment and the light chain V-gene segment containing the Rhesus D-specific regions of CDR 1, CDR 2 and CDR 3, as shown in figures 1a-16b and 1b- 16b, respectively, from an anti-Rhesus-D-Fab encoding plasmid by means of a specific primed polymerase chain reaction,
b) obtaining, separately, the genes of the heavy chain of the full anti-Rhesus D-immunoglobulin and the light chain of the full anti-Rhesus-D-immunoglobulin in suitable plasmids containing segments of the immunoglobulin constant region gene encoding any of the heavy chains of the human immunoglobulin γ1, γ2, γ3 and γ4 and \ the light chain κ or λ of a human immunoglobulin, and the transformation, individually, of the obtained plasmids into suitable E. coli bacteria, and
c) cotransfection of the obtained plasmids into suitable eukaryotic host cells, culturing the cells, separating the untransformed cells, cloning the cultures, selecting the best producing clone, using it as a production culture, and isolating complete antibodies from the cell culture supernatant.

19. A pharmaceutical composition comprising at least one polypeptide according to claims. 1, 2 or 3, or at least one anti-Rhesus D antibody according to any one of claims 14-17, for the prophylaxis of hemolytic disease of newborns, for the treatment of idiopathic thrombocytopenic purpura and for errors in transfusion of Rh incompatible blood.

20. A diagnostic composition for Rh-D typing containing the Fab fragment according to claim 4 or an anti-Rhesus-D antibody according to any one of claims 14-17.