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.
2.11.
In en omstreeks 2014 hebben Ablynx en [gedaagde] gesprekken gevoerd over een deelneming van Ablynx in (de aandelen van) [gedaagde] . In dat kader heeft [C] op 12 mei 2014 namens [gedaagde] aan de heer [G] van Ablynx (hierna: [G] ) - onder meer - het volgende gemaild2:
Allereerst mijn dank voor het goede gesprek van jl vrijdag. I[k] denk dat vrijwel alle aspecten aan de orde gekomen zijn, maar voor de goed[e] orde som ik ze hieron[d]er nog even op:
a. Business Plan [gedaagde] doorgenomen met nadruk op relatie met BAC, de fase nul trials en het unieke van [gedaagde] tags;
b. Terugverdien model van GMP geproduceerde VHH/nanobodies besproken. Gezien de kennis van gisten en ferme[nt]atieprocessen met deze gisten zal [gedaagde] per 20 liter run voldoende functionaliseerde VHH maken voor 4-5 tphase 0 trials. De productiekosten [worden] (behalve de labeling) geheel betaald door de oorspronkelijke opdrachtgever. Dat betekent dat [gedaagde] 3-4 keer voor K€ 60-100 / phase 0 trial aan andere UMC kan verkopen, terwijl de kosten van functionalisatie onder GMP omstandigheiden en de zuivering daarvan niet meer dan K€ 10-15 zullen bedragen, derhalve per GMP geproduceerd VHH ongeveer een winst van 3-4 * {K€ 40-K€60}. Indien alle 9 VHH gemaakt zouden worden moet dit bedrag dus vanaf 2018 met 9 vermenigvuldigd worden;
c. Er is twijfel aan de Research markt voor VHH. Inderdaad zijn de cijfers tot nu toe teleurstellend, maar er wordt hard gewerkt aan VHH in combinatie met microfluidics (zie bijgevoegde publicatie), VHH voor digital pathology (die info kan nog niet gegeven worden ivm met Philips en VHH voor MRI en spierziekten en MRI geleide biopsies[)]. Daar en boven komt dus de ontwikkeling van FACS analysen mbv. VHH samen met de BAC;
d. Ablynx heeft interesse in nanobodies geselecteerd door [gedaagde] die aanvullend zijn op haar eigen verzameling nanobodies;
e. Ablynx heeft interesse wat [gedaagde] met chimeren gedaan heeft, in het bijzonder het opruimen van virusdeeltjes mbv. anti EGRF gekoppeld aan HIV en/of HBV (maar bruikbaar ook bruikbaar voor andere zaken op te ruimen);
f. [gedaagde] geeft aan dat ook als Ablynx [gedaagde] niet wil overnemen, er meerdere mogelijkheden tot samenwerking kunnen zijn, te weten contract research als Ablynx nieuwe nanobodies wil isoleren en gebruik van voor therapie niet geschikte nanobodies door [gedaagde] als Research en lmaging moleculen (zie bijgevoegde lijst wat ooit met [D] en [I] is doorgepraat). Ook zou samengewerkt kunnen worden om de efficacy van nanobodies mbv imaging technieken te bepalen.