2.3.De conclusies van EP 013 luiden in de authentieke Engelse versie als volgt:
1. Process for the preparation of immunoglobulins or fragments thereof directed against determined antigens, comprising the steps of:
- cloning into vectors, especially into phages and more particularly filamentous bacteriophages, DNA or cDNA sequence obtained from lymphocytes of Camelids previously immunized with determined antigens, capable of producing an immunoglobulin, or a fragment thereof, said immunoglobulin comprising two heavy polypeptide chains, each heavy chain being capable of recognizing and binding an antigen, said immunoglobulin containing a variable (VHH) region and a constant region, said constant region being devoid of first constant domain CH1, wherein the immunoglobulin is devoid of polypeptide light chains,
- transforming prokaryotic cells with the above vectors in conditions allowing the production of the immunoglobulins or fragments thereof,
- selecting the appropriate immunoglobulins or fragments thereof by subjecting the transformed cells to antigen affinity selection,
- recovering the immunoglobulins or fragments thereof having the desired specificity.
2. Process according to claim 1, wherein the cloning vector is a plasmid or a eukaryotic virus and the transformed cell is a eukaryotic cell, especially a yeast cell, mammalian cell, plant cell or protozoan cell.
3. Process according to claim 1, wherein the cloning vector is a plasmid capable of expressing the immunoglobulin or fragment in the bacterial membrane.
4. Process according to claim 1, wherein the cloning vector is a plasmid capable of expressing the immunoglobulin or fragment as a secreted protein.
5. Process according to claim 1, with the proviso that the Camelid is not previously immunized with determined antigens.
6. An isolated nucleotide sequence encoding an immunoglobulin or a fragment thereof forming a determined antigen binding site, said immunoglobulin comprising two heavy polypeptide chains capable of recognizing and binding one or several antigens, said immunoglobulin containing a variable (VHH) region and a constant region, said constant region being devoid of a first domain (CH1), said immunoglobulin being devoid of light polypeptide chains, which fragment forms a determined antigen binding site.
7. An isolated nucleotide sequence according to claim 6, wherein the fragment comprises the VHH region of said immunoglobulin.
8. A recombinant vector comprising a nucleotide sequence according to any of claims 6 to 7.
9. Recombinant vector according to claim 8, which is a plasmid, a phage, a virus, a YAC, a cosmid.
10. Recombinant vector according to claim 9, wherein the phage is a bacteriophage.
11. Recombinant cell or organism modified by a vector according to any of claims 8 to 10.
12. Recombinant cell or organism according to claim 11, which is
E. coli, a yeast cell, a mammalian cell, an insect cell, a plant cell or a protozoan cell.
13. A cDNA library comprising nucleotide sequences according to any of claims 6 to 7, wherein said cDNA library is obtained by performing the following steps:
(a) treating a sample containing lymphoid cells from a healthy animal selected among the Camelids in order to separate the lymphoid cells;
(b) separating polyadenylated RNA from other nucleic acids and components of the cells;
(c) reacting the obtained RNA with a reverse transcriptase in order to obtain the corresponding cDNA;
(d) contacting the obtained cDNA with 5’ primers located in the promoter, leader or framework sequences of the VHH sequence of a heavy chain immunoglobulin, which primer contains a determined restriction site, and with 3’ primers located In the hinge, CH2, CH3, 3’ untranslated region or polyA tail;
(e) amplifying the DNA;
(f) cloning the amplified DNA in a vector; and
(g) recovering the obtained clones.
14. A cDNA library comprising nucleotide sequences according to any of claims 6 to 7, wherein said cDNA library is obtained by performing the following steps:
(a) treating a sample containing lymphoid cells from a healthy animal selected among the Camelids in order to separate the lymphoid cells;
(b) separating polyadenylated RNA from other nucleic adds and components of the cells;
(c) reacting the obtained RNA with a reverse transcriptase in order to obtain the corresponding cDNA;
(d) contacting the obtained cDNA with 5’ primers corresponding to mouse VH domain of four-chain immunoglobulins, which primer contains a determined restriction site, and with 3’ primers corresponding to the N-terminal part of a CH2 domain containing a
Kpnl site;
(e) amplifying the DNA;
(f) cloning the amplified DNA in a vector; and
(g) recovering the obtained clones.
15. The cDNA library according to any of claims 13 to 14, wherein the lymphoid cells of step (a) are obtained from an animal previously immunized against a determined antigen and the clones recovered in step (g) encode polypeptide chains having a preselected specificity for the antigen used for immunization.
16. The cDNA library according to any of claims 13 to 15, wherein said lymphoid cells are selected from the group consisting of peripheral lymphocytes, spleen cells, lymph nodes, and other lymphoid tissue.
17. The cDNA library according to any of claims 13 to 16, wherein in step (d) said restriction site is an
Xhol site.
18. The cDNA library according to any of claims 13 to 16, wherein in step (f) said vector is a bluescript™ vector.
19. The cDNA library according to any of claims 13 to 18, wherein the amplification of the cDNA is not performed prior to the cloning of the cDNA.