LU86752A1 - PROCESS FOR PRODUCING HUMAN MONOCLONAL ANTIBODIES (IGG) ANTICYTOMEGALOVIRUS AND MONOCLONAL ANTIBODIES THUS OBTAINED - Google Patents

Description

ΊΟΓ & "7 K 9 GRAND-DUCHY OF LUXEMBOURG BL-3991 v Patent N ° W 0 .......... / p iCy ^ Mister the Minister of January 30, 1987 jfaaL of Economy and Average Qasses

Title issued Intellectual Property Service

î re e vre ..........— pgS LUXEMBOURG

? , 'V Invention Patent Application -________________, _____________ di I. Request υΗΧ1ΖΕΒΕΧΤΕ .._ ΕΙΒΕΕ ... DF, E ~ RTTKF.TTEfi} ._ 5Q -, _ AurP-tme-IR .-' D., Ppnaeyel tB = 1-05Q ......... (2)

BruxellesT Belgique__________________________ * represented-by · —E ^ T ^ ÆceyliBger. AE »lfeyegSy - î-ag-r - ooas ^ —en-propr-v — i-ad-â-s- (3) .46, t ._. Rne_dn. · Cemetery, Luxombow-g, -ma - shareholders ________; _ deposit (s) this —thirty ..... January - mid-nine — one hundred and eighty-seven --- (4) at ... 15.0.0.— hours, at the Ministry of 'Economy and the Middle Classes, in Luxembourg: 1. this request for obtaining a patent for invention concerning:

Erocédé-de prodnction - dlantiGorps-rDonoG4enanx ~ brmains - ^ IgG) - anti- (5) cytomegalovirus ...... and ..... antibodiesmrmQeltmany »tust nTifpmig ..____ 2. description in language ________________ French -_______ of the invention in triplicate; 3. ................— / .............................. ... drawing boards, in triplicate; 4. the receipt of the taxes paid to the Luxembourg Registration Office, 3Q .._ .j.anvier _._ L9.B7_; 5. the delegation of power, dated ------. Brussels --------- on 20-j-anvier — 490? -; 6. the successor document (authorization); declare (s) assuming responsibility for this declaration, that the inventor (s) is (are): {6)

Dr BB.0N Dominique, 28a, ...... avenue — de --- la ~ Sapisiêres ·· —B ^ lL-SD -'- Bruxel-les-- M ”.... STRIJÇK ^ _Meise_____ M, ..... DELFORGE -Alain "...... 64ï '" avenue - des - Grands - Prixr-B-ll-50-Brt3xell-es- claims (nt) for the above patent application priority of one (s) request (s) from (7) _______..... .....___________. / _________ filed (s) in. (8) _JL- le (9) ------ ----------------- / -: --- under N ° (10) ____________________________________ / __________ on behalf of (11) ----------- ------------------------------------------------ J- ____________ elect (elect) domicile for him / her and, if designated, for his / her representative, in Luxembourg-- 46, ...... rue du ..... Cimetière, ..... Luxembourg______ (12) request (s) the grant of a patent for the invention for the subject described and represented in the abovementioned appendices, with postponement of this grant to _______ dix-irurt ___________ months. “(13)

The depositor / agent: -------____ X_____________________________ (14) s. \ Π. Deposit Minutes

The above application for a patent for an invention has been filed with the Ministry of the Economy and Middle Classes, Service de la Propriété Intelleefqdfe âdfcîjxïhqbourg, dated: January 30, 1987 [* / \ ^ * \ Pr. The Minister of Economy and Average Qasses, at ..... 15.00 - ·· hours _ I ÿ 'w. * § & \ Ί 1 p. d γ ° ν \ Lj ^ 'The head of the intellectual property service, y' / A 68007 __ ^ [___ ^ _ / / _ EXPLANATIONS RELATING TO THE DEPOSIT FORM. J ^ \ / (Ijs'iiy takes place "Request for certificate of addition to the main patent, at the patent application prindpaJNa .......... tnàu. Y / ........" - (2) enter the surname, first name, prciessioii, address of the applicant, when the latter is an individual opts corporate name, legal form, address of the registered office, if the applicant is a legal person - (5; register *

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Process for the production of anti-cytomegalovirus human monoclonal antibodies (IgG) and monoclonal antibodies thus obtained.

Technological background 5 1: Cytomegalovirus (CMV) is responsible for infections especially affecting newborns (most often transmitted from mother to child), and 'les. immunocompromised adult subjects.

10 Cytomegalovirus is responsible in humans for: 1 ° mononucleotic syndromes, which occur readily in subjects who have received massive transfusions; 2 ° of hepatic damage, with a clinical picture superimposable on that of the usual-15 viral hepatitis; 3 ° generalized infections, occurring in subjects presenting with a malignant tumor or subjected to immunosuppressive therapies; 4 ° newborn disease, classic disease of cytomegalic inclusions, due to infection of the fetus in utero. Evolution is, as a rule, deadly.

All these pathologies are brought together by the same elements of diagnosis: sometimes by the isolation of the virus in; urine, saliva or any tissue removed by biopsy or postmortem; above all "2 by serodiagnosis, which shows an increase in the level of antibodies between two samples taken 10 or 15 days apart" The use of human anti-cytomegalovirus monoclonal antibodies 5 is therefore of great interest for such serodiagnostics " EMANUEL D. et al. Human monoclonal antibody to cytomegalovirus (CMV) - J. Immunol. 4: 2202-2206, 1984 have already described a human monoclonal antibody.

10 This antibody could not find clinical and pharmaceutical use, mainly due to the fact that it conserves the genome of the Epstein-Barr virus. (EBV) and that it is of a low title, which makes it diff icilemënt marketable.

15 Object of the present invention

The present invention aims to provide an improved production process which in particular makes it possible to get rid of the EBV genome and which provides · therefore a monoclonal antibody useful not only in seromegagnos-20 tic tests of cytomegalovirus (CMV) but also potentially useful in vivo in the prophylaxis and / or treatment of CMV infections. The invention also extends to the detection of the virus in various biopsy tissues or bronchoalveolar lavage by this monoclonal antibody. 25 Characteristic elements of the invention

The technique used according to the present invention essentially comprises the following operational steps: a) enrichment in B lymphocytes of a mononuclear cell population taken from the peripheral blood of a donor having a recent CMV infection; b) transformation of the enriched cell population by the Epstein-Barr virus; C) establishment of lymphoblastoid lines ♦ 3 and cloning of those producing a strong positive reaction to the anti-CMV specificity test; d) culturing a chosen line and fusion with a heteromyeloma, preferably the SHM-D33 cell line and selection of the hybrids producing IgG (class 2), Xanti-CMV and cloning to ensure monoclonality.

The monoclonal antibodies by this method recognize all of the CMV strains in the possession of the Applicant and in particular the three reference strains: TOWN; DAVIS and AD 169.

The various operating steps used are known per se, various operating details can be modified by the practitioner implementing these techniques, depending on the circumstances.

From a practical point of view, the Applicant 20 however recommends the execution of the different operational stages of production under the following conditions: The separation of stage a) preferably takes place in two stages, namely first separation of the mononuclear cells from the blood of the red blood cells on a Ficoll-Hypaque gradient and then separation in the collected lymphocytes, of the B lymphocytes and of the T lymphocytes by rosetting with red blood cells of sheep treated with AET (2-amino-ethylisothio-monium hydrobromide bromide).

Step b) of transformation of the cell population enriched in B lymphocytes by EBV is advantageously carried out using the culture supernatant of the Marmoset line (B95-8) containing the transforming and noninfecting EBV virus.

The anti-CMV specificity test of step 4 c) is preferably an ELISA test.

The selection of the hybrids of step d) is carried out, preferably, with a medium containing hypoxantine-aminopterin-thymidine 5 (HAT) and * ouabain.

Example of an embodiment of the method of the invention.

Donor: patient, age: 25 years old, having undergone a recent CMV infection.

10 1st part: production technique of an anti-CMV lymphoblastoid line 30 cc of peripheral blood are taken during a routine blood test and collected on calparinized tubes in August 1985. The 15-nucleated cells of the blood are separated red blood cells * 7 on a Ficoll-Hypaque gradient and 4 x 10 lymphocytes are collected. The B lymphocytes were then separated from the T lymphocytes by rosetting with red blood cells from sheep treated with AET (2-amino-20 ethylisothio-monium bromide hydrobromide). The cell population enriched in B lymphocytes (5 × 10) is then transfected with EBV from the culture supernatant of the Marmoset line (B95-8) containing the transforming and non-infecting EBV virus, this surna-25 giant being added at a concentration of 20% in the culture medium (Iscove's + 20% FCS).

After 2 weeks in culture, several lymphoblastoid lines are established and tested for anti CMV specificity. Four of these lines are positive for the production of anti-CMV antibodies and are cloned by successive dilutions up to 1 cell per well in a 96-well dish. More than 50 anti-CMV lymphoblastoid lines are thus obtained (September 1985), 35> 5

2nd part: production of anti-CMV human hybridomas

One of these lines producing a strong positive reaction to the ELISA against CMV. is cultured and 4 x 10 of these cells are fused 7 5 with 2 x 10 heteromyeloma cells (SHM-D33) in the presence of polyethylene glycol. After fusion, the cells are placed in a 5 to 96-well culture dish at a rate of 2 × 10 cells per well.

The hybrids are selected by a medium containing 10 of the hypoxantine-aminopterin-thymidine (HAT) and -7 × 10 5 of ouabain. In October 1985, the hybrids appeared in 37% of the wells (141/384) of which 66% produced a specific anti-CMV monoclonal antibody (ELISA-BEHRING). All the hybrids produce 15 IgG (class 2), anti-CMV λ whose rate of * 6 production varies from 0.1 to 20 (median 2). ug / ml / 10 cell / day. Two hybridomas are cloned to ensure monoclonality and 1 clone (8E9H5) is selected for more specific tests. Multiple clones 20 are frozen and will be tested in a second step.

The monoclonal antibody produced is an IgG2, λ which recognizes all the strains of CMV (including the 3 reference strains: TOWN - DAVIS - AD169) whose production has been stable for more than 12 months in continuous culture, including Cytotoxic activity for fibroblasts infected with CMV has been demonstrated 51 by a Cr test in the presence of complement and whose EBV genome sought by probe after hybridization of DNA has disappeared after fusion.

This latter characteristic is particularly important since it is the cause of the specificity of the antibody and of the high anti-CMV titer.

These antibodies can be used according to all known immunological diagnostic techniques and can in particular be presented under '' *

V

6 form of detection kit »

Claims (8)

1. Method for producing human monoclonal antibodies (IgG) anti-cytomegalovirus characterized by the operating steps; A) enrichment in B lymphocytes of a mononuclear cell population taken from the peripheral blood of a donor having undergone a recent CMV infection; b) transformation of the enriched cell population with Epstein-Barr virus; c) establishment of lymphoblastoid lines. and cloning of those producing a strong positive reaction to the anti-CMV specificity test; d) culturing a chosen line and fusion with a heteromyeloma, preferably the cell line SHM-D33 and selection of the hybrids producing IgG (class 2), anti-CMV and cloning to ensure monoclonality. 2. Method according to claim 1, charac-20 terized in that the separation of step a) takes place in two stages, namely first separation of the mononuclear cells of the blood from the red cells on a Ficoll-Hypaque gradient and then separation in the collected lymphocytes, of B lymphocytes and T lymphocytes by rosetting with red blood cells of sheep treated with AET (2-amino-ethyliso-thio-monium bromide hydrobromide). 3. Method according to claim 1, characterized in that the transformation of step b) of the cell population enriched in B lymphocytes by EBV is carried out from the culture supernatant of the Marmoset line (B95- 8) containing transforming and non-infecting EBV virus. 4. Method according to claim 1, charac-35 terized in that the anti-CMV specificity test of t • * · 8 step c) is an ELISA test. 5. Method according to claim 1, characterized in that the selection of the hybrids of step d) is carried out by a medium containing 05 11hypoxantine-aminoptërine-thymidine (HAT) and ouabain. 6. Monoclonal antibody obtained by the method of any one of the preceding claims, characterized by the human nature of immunoglobulins (gamma globulin type Ig G) on the one hand and on the other hand by its capacity to recognize the reference reference strain CMV ·. TOWN, DAVIS and AD 169 and the absence of the EBV genome. 7. Cytomegalovirus detection kit 15 characterized in that it contains at least one monoclonal antibody according to claim 6, 7 fr 8. Pharmaceutical product applicable in vivo for the prophylaxis and / or treatment of cytomegalovirus infections.