CN100430418C - Method of preparing monoclonal antibody, monoclonal antibody, pharmaceutical composition and diagnostic reagent - Google Patents

Abstract

The present invention relates to an efficient method of producing monoclonal antibodies against surface antigens of cells and viruses. The method accommodates for antigens which are present in only relatively small amounts, or antigens of which only very small amounts are available or antigens which easily lose their in vivo conformation. Thus the method according to the invention comprises a series of steps, comprising a step in which B-cells from a mammal injected with surface antigen-comprising material are enriched with respect to the relative number of specific B-cells and a step which comprises a small-scale fusion technique.

Description

Preparation monoclonal antibody method, monoclonal antibody, pharmaceutical composition and diagnostic reagent

The present invention relates to prepare the method for anti-surface antigen monoclonal antibodies, especially only constitute a spot of surface antigen that is injected in the total antigenic surface antigen in the Mammals and loses conformation in its body easily.A small amount of antigen, takes place when mammiferous antigenic substance is derived from other mammal species when being used to inject for example with respect to the antigenic problem of total amount.

For example, when needs prepared the monoclonal antibody of anti-T-cell receptors (TCR) specificity deciding section, the problem relevant with antigen described above just became clear.This (mono-clonal) antibody is called anti-clonotype (anti-clonotype) antibody because they discern the antigen-specific part (or clonotype structure) of a kind of specific T-cell clone T-cell receptors.Although (mouse) monoclonal anti-clonotype antibody of anti-mouse T-cell receptors (TCR) only has odd report, known anti-human T-cell receptors (mouse) monoclonal anti-clonotype antibody still less.The T-cell is difficult to cultivate, and causes the antigen that is difficult to obtain with the purifying q.s, that is, and and T-cell receptors albumen.This causes conversely and is difficult to obtain immunne response and also makes the screening difficulty.Among wherein a kind of in anti-human TCR monoclonal anti-available seldom several examples of clonotype antibody, these problems can not occur because this antigen is present in (reference 1) among the Jurkat leukemia cell.Because the leukemia cell can cultivate endlessly, the antigen shortage problem can not occur.Therefore, also do not obtain preparing anti-monoclonal antibody rare and/or very labile antigen up to now and need not to screen unfriendly the method that a large amount of hybridomas are cloned.

The monoclonal antibody method that the purpose of this invention is to provide preparation anti-cell surface antigen under above-mentioned rough sledding, described method reduces hybridoma to be screened clone's number significantly, makes the method according to this invention more effective or when failing according to the method for the state of the art even success.

For this purpose, the method according to this invention may further comprise the steps

1) to comprise the material injection Mammals of cell-surface antigens, described material is selected from i) whole cell and ii) by handling the membrane portions that whole cell obtains;

2) from described Mammals spleen, separate the cell part that contains the B-cell;

3) by following steps enriching step 2 in the B-cell special to described cell-surface antigens) the middle described cell part that obtains, with contacting of cell part and the cell relevant with carrier-bound material with described whole cell, described cell with the described cell-surface antigens of carrier-bound material want, and the enrichment from be ready to use in next step unconjugated contain separation and combination in the cell part of B-cell to relevant cell with carrier bound substances on the B-cell;

The cell that contains the B-cell of the enrichment that 4) will be in front obtains in the step partly carries out limited dilution and carries out clonal expansion then;

5) screening B-cell clone also infinitely breeds the described B-cell clone that filters out with the small-scale integration technology; With

6) screening and clone can produce the hybridoma of specificity in conjunction with described cell-surface antigens antibody, separate the part that comprises monoclonal antibody then from the supernatant liquor of this hybridoma.

In order to keep specification sheets and claims readability and understandable purpose, the term cell here is used to not only represent that mammalian cell also represents virus, and especially comprises the virus of film.

The term cell comprise the membrane portions of complete or whole cell (and virus), described complete whole cell (and virus) in conjunction with carrier substance, or surface antigen or its mixture of purifying basically.

If not special in addition explanation, the whole cell of term refers to have the cell of surface antigen interested.The term relevant cell refers to such cell: promptly they are preferably only lacking difference aspect the interested surface antigen, or more specifically they lack immunologic determinants at least, need to obtain monoclonal antibody just because of this anti-this determinant.

Surprisingly contact with the material of cell by cell part with the isolating B-of containing cell, described cell is from the species identical with the material that therefrom obtains to comprise cell-surface antigens but lack cell-surface antigens, proof is under the situation of not using cell-surface antigens, and enrichment of cell partly is possible in the special B-cell of pair cell surface antigen.In other words, by with the relevant cell of B-cell nothing to do with or cellular material contact and remove bonded B-cell, enrichment is accomplished.

Therefore, the monoclonal antibody of the anti-less important cell-surface antigens of preparation is possible, even described cell-surface antigens configuration instability.

European patent EP 0488470 and described a kind of method based on the reference 3 of this European patent, I wherein) Mammals is injected with antigen, II) separate the part that contains the B-cell, III) screening antigen-specific b-cell, IV) part of enrichment is carried out clonal expansion and at V) screening B-cell clone and make VI after its unlimited breeding with integration technology on a small scale) screen and then separate the part that comprises monoclonal antibody with the clone hybridization knurl.In Step II I, screen the B-cell by it being incorporated into the antigenic frosting of coating or forming rosettes (rosetting) by paramagnetic (paramagnetic) globule with the antigen coating.Non-specific B-cell is removed by washing.Therefore, except other difference, this method depends on sufficient operability and antigenic use (during the screening with 2 μ g/ml antigens with the antigen spread plate), the invention solves the problem of this situation on the contrary, can obtain very limited amount antigen and even highly impure.

Embodiment preferred is characterised in that the Mammals with the surface antigen injection is and the surface antigen source different species of mammalian species wherein.

The method according to this invention is suitable for preparing monoclonal antibody very much when having the antigen of many kinds energy induce immune responses.

Especially have constant region and variable region when surface antigen, wherein when the described variable region of small part defined the specificity deciding section of described surface antigen, this situation existed.

The material that comprises acceptor molecule according to embodiment preferred is as the material that comprises cell-surface antigens.

The method according to this invention is particularly useful for preparing the monoclonal antibody of anti-acceptor molecule specificity deciding section.The specificity deciding section of acceptor molecule only is the less important part of acceptor molecule, and acceptor itself is all molecules and so antigenic less important integral part on the cell surface.

Preferred target is the T-cell clone according to the present invention, and the T-cell clone is used to prepare the material that comprises acceptor molecule.

The T-cell can be used as whole cell, or is used to prepare its membrane portions.Therefore, in the previous case, the term preparation here, may relate to and only separate the T-cell or be resuspended in different substratum.

Membrane portions according to preferred embodiment step 1 obtains by the whole cell of mechanical treatment, and comprises that the material of cell-surface antigens is injected in Mammals when lacking adjuvant.

Two kinds of measures help to prevent the forfeiture of conformation in the surface antigen body.

In addition, the cell part that contains the B-cell of enrichment is preferably by with this cell part further enrichment with being selected from the containing that material in conjunction with the carrier cell surface antigen contact of following material (step 3): j) whole cell jj) available from the membrane portions and the jjj of described whole cell) cell-surface antigens of purifying basically, and separate not to be attached to describedly combining B-cell on the carrier substance with being attached to this subsequently, be attached to this comprises further enrichment in conjunction with the B-cell on the carrier substance cell part in conjunction with the B-cell on the carrier substance.

But this further enrichment called after positive-selecting technology, the B-cell that specificity screening is special.Therefore, though almost can not get any antigen, further enrichment can be done.

Preferentially be used as carrier according to preferred embodiment paramagnetic globule.

The use of paramagnetic globule helps separating that want and undesired B-cell in enrichment process.

The antigen of inessential amount also goes wrong in screening process.According to embodiment preferred, screening in step 5 and 6 at least one step is carried out with the aggegation analysis, wherein the supernatant liquor of B-cell clone contacts with the carrier that is coated with antibody, this antibody can and have the whole cell of cell-surface antigens in conjunction with the antibody of the injection mammalian species that is used for step 1, and detects aggegation.

Mix B-cell culture supernatant, whole cell simply and with allowing very sensitive in conjunction with the carrier of the antibody coating of the mammalian species antibody that is used for step 1 and apace suitable clone's detection being need not to wash.

Relevant with whole cell but whole cell that lack surface antigen can be preferably used as contrast.

This allows to abandon the false positive clone, and saves time by avoiding unnecessary small-scale to merge.

According to the preferred embodiment of the inventive method, the B-cell clone that filters out in step 5 mixes with the myeloma cell and carries out trace-electricity and merges (mini-electrofusion).

Trace-electricity merge and to allow seldom to measure (as, hundreds of) effective fusion of cell.

The present invention also relates to pharmaceutical composition, comprising prepared in accordance with the present invention and with appropriate excipients blended monoclonal antibody.

The present invention relates to the monoclonal antibody that reacts with T-cell receptors clonotype structure in addition.

This monoclonal antibody can be used for diagnostic purpose and is used for preparation of drug combination.

More particularly T-cell receptors be with self-Immunological diseases, and the relevant T-cell receptors of rheumatoid arthritis especially.

The T-cell receptors of the reactive T-cell clone of monoclonal antibody and HCgp-39 preferably, and especially with the T-cell clone H.243 the T-cell receptors of (ECACC preserving number 96103122) react.

The specific examples of suitable monoclonal antibody for those by being selected from the monoclonal antibody that following hybridoma produces, comprise TCR69 (ECACC preserving number, 96103118), TCR70 (ECACC preserving number 96103119), TCR72 (ECACC preserving number 96103120) and TCR83 (ECACC preserving number 96103121).

As the above, the present invention also relates to be applicable to the pharmaceutical composition of rheumatoid arthritis treatment, comprise according to monoclonal antibody of the present invention and with appropriate excipients and mixing.

The present invention relates at last to be selected from monoclonal antibody prepared according to the methods of the invention with according to the diagnostic uses of the monoclonal antibody of monoclonal antibody of the present invention.The diagnostic reagent that comprises antibody also is embodiment of the present invention.

Now the present invention is further explained in detail with reference to the following examples, described embodiment demonstrates and finishes the optimal mode of the present invention that is used to prepare to the special mouse monoclonal antibody of human T-cell receptors clonotype.

Description of drawings

Fig. 1: by the evaluation of the H.243T-cell surface molecule that resists MAb immunoprecipitation H.243.SDS/PAGE finishes in 10% gel under non-reduced condition (2,3 and 4 road) or reductive condition (5,6 and 7 road).

1 road: 10KD ladder mark; 2 roads and 5 roads: contrast MAb; 3 roads and 6 roads: TCR83; 4 roads and 7 roads: anti--CD3, OKT3.

Fig. 2: anti-clonotype MAb discerns TCR α β in the Western trace.H.243 immunoprecipitation TCR/CD3 mixture is separating in the 10%SDS-PAGE gel under the non-reduced condition and is being transferred on the pvdf membrane subsequently.With this film and MAb incubation and last by sheep anti mouse Ig second antibody detection bonded MAb with the combined alkali acid phosphatase.

1 road: 10KD ladder mark, 2 roads: TCR64,3 roads: TCR66,4 roads: TCR69,5 roads: TCR70,6 roads: TCR72,7 roads: TCR73,8 roads: TCR76,9 roads: TCR78,10 roads: TCR79,11 roads: TCR83,12 roads: contrast MAb, 13 roads: substratum contrast.

Fig. 3: fixed resists-TCR MAb stimulating human T-cell clone propagation H.243.By anti-MAb inductive propagation (TCR64 is to TCR83) H.243 with shown in contrast MAb and make comparisons with TCR44 for the anti-clonotype MAb that resists other TCR.The average counter of per 5 minutes quadruplicate cultures of each value representative+/-standard deviation.

Fig. 4: suppress or stop the propagation of human T-cell clone antigen-driving H.243 with the preincubation of anti-TCR MAb.A) anti-inhibition (TCR64 is to TCR83) H.243MAb with shown in contrast MAb and make comparisons with TCR44 for the anti-clonotype MAb that resists other TCR.The average counter of per 5 minutes quadruplicate cultures of each value representative+/-standard deviation.B) suppress the dose response curve of MAb by force; TCR64 (open circle), TCR70 (sealing trilateral), TCR78 (open square), TCR83 (sealing square) and contrast MAb1 (sealing circle).The average counter of per 5 minutes triplicate cultures of each value representative.

Fig. 5: anti-clonotype MAb induces anergy in H.243 human Th1-clones.Anti-clonotype MAb of fixed or contrast MAb1 cross liquid with cell incubation H.243T.Remove cell from the anergy stimulator after, giving cell increases concentration peptide and DRB gradually ^*The stimulation fully of the APC of 0401 coupling.Propagation is passed through ³The H-thymus pyrimidine mixes to be calculated.The average counter of per 5 minutes triplicate cultures of each value representative+/-standard deviation.

Fig. 6

Anergy is cell inhibited reaction (non-anergic) replying of cell H.243T H.243T.

Fixed TCR76 or contrast MAb1 are incubated overnight with cell H.243T.Then, two kinds of T-cell colonys with the peptide (or PHA) that increases concentration gradually and APC with every hole 2 * 10 ⁴Individual T cell is used for proliferation assay.In addition, the anergy cell of the various amounts that obtain with the TCR76 incubation with and the MAb1 incubation and obtain 2 * 10 ⁴Individual reactive cytomixis is also carried out proliferation assay.A/N: the anergy cell is to the ratio of reactive cell; 1=2 * 10 ⁴Individual cell.Propagation is passed through ³Mixing of H-thymus pyrimidine and calculating.The per 5 minutes average counter+standard deviation of each value triplicate culture of representative.

Embodiment

Material and method

Reagent

Substratum: DMEM/HAMShi F12 (Gibco catalog number (Cat.No.) 32500) replenishes with the 2500mg/l sodium bicarbonate, 2.3mg/l 2 mercapto ethanol, 55mg/l Sodium.alpha.-ketopropionate, 1.22mg/l thanomin, 360mg/l L-glutaminate, 4.5 * 10 ^-4The mg/l Sodium Selenite, 62.5mg/l benzylpenicillin sodium and 62.5mg/l Vetstrep (streptomycin sulphate).In fusion experiment, substratum further replenishes with 13.61mg/l xanthoglobulin and 3.83mg/l thymus pyrimidine.This substratum is called DMEM/HAMShi F12/HT.The screening of hybridoma contains among the DMEM/HAMShi F12/HT that IL-6 goes up cleer and peaceful 0.4 μ M aminopterin and finishes replenishing human bladder cancer cell lines T24 (T24CM) with 1% (V/V).

Merge substratum: 280mM inositol, 0.1mM calcium acetate, 0.5mM magnesium acetate and 1mM Histidine; Resistivity: 1.1110 ⁴Ω .cm.Composition be dissolved in the Milli-Q water and specific conductivity subsequently with Milli-Q water or contain the 1mM calcium acetate and the solution of 5mM magnesium acetate transfers to 90 μ S/cm.

Cell cultures

H.243, the T-cell clone is derived from the patient of rheumatoid arthritis.The T-cell clone identification DRB1 of this Th1 sample ^*The epi-position RSFTLASSETGVG (SEQ ID NO:1) of human cartilage gp-39 in 0401 (belonging to MHC II class) (HC is-39).This clone's TCR is characterized as the V α 8 and V β 9 positives.Cell routine is incubated at and replenishes with 10%FCS, 20U/mlIL-2, is delivery cell (APC) or with phytohemagglutinin (PHA) and periodically stimulation again of feeder cell among the DMEM/HAMShi F12 of 5U/ml IL-4 and with antigen and histocompatibility antigen.For proliferation assay foetal calf serum (FCS) replaces with normal human serum (NHS).

The preparation of T-cellular lysate and with TCR/CD3 mixture load globule

Again stimulated the back 10 to 14 days, the T-cell is washed with PBS and is passed through them with 10 ⁸The concentration of cell/ml is in extraction damping fluid (10mM trolamine, 150mM NaCl, 1mM Na according to (reference 2) such as Oettgen ₂EDTA, 1% digitonin, 100 μ g/ml leupeptins, 10 μ g/ml, aprotinin, 1 μ g/ml pepstatin, 1mM

AEBSF and 1,8mg/ml iodine ethamine pH7.8) incubation (30 minutes, 0 ℃) in and dissolving.Gentle sanitising agent digitonin allows to extract complete TCR/CR3 mixture.Nuclear and cell debris be by 16,000g4 ℃ centrifugal 15 minutes and removal and supernatant packing are stored in-20 ℃ up to screening that is used for the B-cell or immunoprecipitation.

In order to screen specific b-cell, the paramagnetic globule is with the load of TCR/CD3 mixture.

In brief, 100 μ l sheep anti mouse Ig link coupled paramagnetic globule (SAM-globules;

110.02) with have 22 μ g among the PBS of 0.1%BSA anti--CD3, OKT3 incubation (spending the night 4 ℃).After washing globule with PBS/BSA, add and be equivalent to 1.4 * 10 ⁸The T-cellular lysate of individual cell and equal volume (1.4ml) have the PBS-BSA of 1% normal mouse serum.Add the latter and be and prevent that the B-cell from combining with free SAM binding site on the globule.The globule suspension was also washed with PBS/BSA in 4 ℃ of incubations in 2 to 4 hours before use.

Immunity

The female BALB/C mice in 6 ages in week with the interval in 3-7 week with 5 * 10 ⁶Individual whole T-cell (invention) or to be equivalent to 5 * 10 ⁷Paramagnetic globule (control experiment) immunity of the TCR/CD3 mixture load of individual cell.Use different route of administration as shown in Table I.In the example (control experiment) that uses adjuvant, injection is for the first time finished with Freund's complete adjuvant, and injection is subsequently finished with Freund's incomplete adjuvant and last reinforcement adjuvant useless is finished.

The generation of anti--clonotype MAb

The female BALB/C mice in 6 ages in week uses 5 * 10 with the interval peritoneal injection in 3 to 7 weeks 4 times at every turn ⁶Tranquillization T-cell among the individual PBS.Last injection is after 5 days, kill mouse and as described above (reference 3,4) preparation remove red corpuscle and monocytic splenocyte colony.This obtains being rich in the cell part of B-cell, but does not obtain the enrichment to the special B-cell of described cell-surface antigens with respect to other non-specific B-cell of the step 2 according to the present invention.

For the B-cell of preclearing and human CD3 from these splenocyte colonies or the reaction of TCR α β constant region, about 3 * 10 ⁷Individual cell and 20 μ l are with the SAM globule incubation of irrelevant T-cell clone (V α 3V β 14 positive T-cell clones, SCRO.08A, but any other irrelevant T-cell is with V α 3V β 14 feminine genders (will do)) TCR/CD3 mixture load 2 times.Then, the special B-cell in TCR variable region screens by the cell suspension that will obtain and SAM globule incubation with the TCR/CD3 mixture load of T-cell clone interested (H.243).Each incubation in replenish with 10% calf serum (

) and the DMEM/HAMShi F12 of 0.2% normal mouse serum in room temperature carried out 90 minutes.Between these incubation period, suspension did even in per 5 minutes carefully, and after the last incubation, globule is with DMEM/HAMShi F12, and identical substratum is washed 5 times carefully and be resuspended in to 10% calf serum.

Merge (3) by clonal expansion and aforesaid trace-electricity, the hybridoma that produces monoclonal antibody produces from the B-cell of these screenings.In brief, the EL-4B5-cell of the B-cell of screening and T-cell conditioned medium (TSN) and 50,000 irradiations (2500 rad) mixes in the flat tissue culturing plate in 96 holes with the DMEM/HAMShi F12 that final volume 200 μ l replenish with 10%FCS.At the 8th day, detect supernatant by a following step T-cell agglutination analytical procedure with interested T-cell clone or irrelevant T-cell clone.Generation has the B-cell culture of recognition capability MAb to be used for trace-electric fusion steps.Content and 10 with these B-cell cultures ⁶NS-1 myeloma cell (5) mixes and removes serum by washing with DMEM/HAMShi F12/HT.Then, cell is with PRONASE A solution-treated 3 minutes and wash with the fusion substratum subsequently.Electricity merges to merge in the cell in 50 μ l by the following method to be finished, and comprises that the alternating-electric field of 30S, 2MHz, 400V/cm is then carried out 10 μ s, high field pulse of 3KV/cm rectangle and the alternating-electric field of 30S, 2MHz, 400V/cm once more.At last, the content in the fusion cell is transferred in the 20ml selection substratum and coats on the 96 hole microtitre flat boards.Merge after 8 days, check the growth of hybridoma in the culture and go on foot the screening of T-cell agglutination analytical method with one once more.

The T-cell agglutination is analyzed

Anti--the screening of T-cell antibody hybridoma culture and the mensuration of MAb and other T-cell cross reactivity are finished with a step agglutination assay.Mix with 20 μ l globule/cell suspensions with the hybridoma supernatant of the long-pending microtitre flat board of demifacet, contain 2 * 10 50 μ l ⁵Individual have the paramagnetic globule of covalent attachment sheep-anti--mouse Ig and in DMEM/HAMShi F12 5 * 10 ⁴Individual interested T-cell.After 2 to 3 hours, check aggegation by the gathering of seeking cell and globule in 37 ℃ of incubations at microscopically.

Immunoprecipitation and Western trace

Again after stimulating 10 to 14 days, wash the T-cell and with the biotin labeling cell surface protein.In brief, the T-cell is with 5 * 10 ⁶The concentration of individual cells/ml and 0.5mg/ml

Sulfo--NHS-vitamin H incubation 30 minutes under the room temperature in PBS.Subsequently, wash cell and once more by above-mentioned dissolving.Be used for before the immunoprecipitation experiment, by with SAM-paramagnetic globule incubation preclearing cellular lysate once.Immunoprecipitation will be by being equivalent to 10 ⁷The preclearing lysate of individual cell and 15 μ l finish with the SAM-paramagnetic globule incubation of MAb (1ml hybridoma supernatant) preload.Then, immunoprecipitate is given a baby a bath on the third day after its birth time with PBS, boils in sample-loading buffer and under reductive condition or non-reduced condition, carries out SDS-PAGE with Laemmli (reference 5) and Mini-protean II system (Bole) in 10% gel.Immunoblotting is finished according to (reference 6) such as Towbin with pvdf membrane.Nonspecific binding site is by being sealed with additional film with 5% skimmed milk incubation among the PBS of 0.5% polysorbas20.After washing, trace is with PBS, 0.5% polysorbas20, and 1%BSA, the streptavidin in 1% normal sheep serum-alkaline phosphatase room temperature was detected in 1 hour.At last, trace develops the color as chromogenic substrate with BCIP and NBT.For Western trace research, paramagnetic SAM-globule by above-mentioned with OKT3 as immunoprecipitating antibody and normal T-cellular lysate with the load of TCR/CD3 mixture.The reduction or non-reduced condition under behind the SDS-PAGE on the 10%PAA-gel, trace and 2.5ml hybridoma supernatant incubation.Bonded antibody is detected by above-mentioned with alkaline phosphatase bonded goat-anti-mouse Ig and BCIP/NBT.

Antibody-mediated T-cell proliferation

The coating of flat microtitre hole is with the 100 μ l goat-anti-mouse Ig (crossing liquid, room temperature) of 40 μ g/ml among the PBS.Wash hole and then with PBS in the 100 μ l MAb incubations 2 hours of 2 μ g/ml concentration.Excessive free MAb replenishes with 2.5 * 10 among the 200 μ l DMEM/HAMShi F12 of 10%NHS by washing to remove and add ⁴Individual tranquillization T-cell.37 ℃ of incubations are after 2 days, and cell is with 0.5 μ Ci[ ³H]-the thymus pyrimidine pulse labelling and again incubation 16 by 18 hours.At last, cell harvesting to glass fiber filter (glass fibre filters) and [ ³H]-thymus pyrimidine be incorporated in Matrix 96 ^TM(Packard) upward detected by the β counting.Each variable is to be detected in quadruplicate.

Antibody-mediated antigen induction inhibition of proliferation

Flat microtitre hole inoculation is with 20 μ l hybridoma supernatants, in replenishing with the 150 μ lDMEM/HAMShi F12 of 10%NHS 2 * 10 ⁴Individual tranquillization T-cell and 1 * 10 ⁵Tissue compatible MNC.37 ℃ of incubations added 50 μ l peptide RSFTLASSETGVG after 3 hours in culture.Culture once more in 37 ℃ of incubations 2 days and [ ³H]-mixing by above-mentioned of thymus pyrimidine detected.Each variable is to be detected in quadruplicate.

With special Mab the inducing of clonotype to anergy

The coating of 24 well culture plates is with the 1ml goat-anti-mouse Ig (spending the night room temperature) of 40 μ g/ml among the PBS.Wash hole and then with PBS in the 1ml MAb incubation 2 hours of 2 μ g/ml concentration, excessive free MAb removes and is incorporated in 2ml DMEM/HAMShi F12 by washing, among the 10%NHS washed 2 * 10 ⁶Individual tranquillization T-cell.The cyclosporin A that wherein needs (Sandoz, Bazel Switzerland), rIL-2, cycloheximide (Sigma, St. Louis, MO, the U.S.) or HLA-DRB1 ^*The APC (irradiation of 3000 rads) of 0401 coupling adds when cultivation is initial.After being incubated overnight, dull and stereotyped resuspended by suction pipe (pipetting) in cooled on ice and T cell.Cell is washed 1 time with perfect medium and by the following T-of being used for analysis of cell proliferation, cytokine analysis and facs analysis.

The specific proliferative response of T cell antigen detects in flat micropore culture, contains 2 * 10 in the culture ⁴Individual T cell, 1 * 10 ⁵HLA-DRB1 ^*0401 coupling, the PBMC of (3000 rad) of irradiation and in 200 μ lDMEM/HAMShi F12, the various antigen concentrations among the 10%NHS.37 ℃ of incubations are after 2 days, and cell is with 0.5 μ Ci[ ³H]-thymus pyrimidine pulse labelling and incubation 16-18 hour once more.At last, cell harvesting to glass fiber filter and [ ³H]-mix on Matrix 96 (Packard, Meriden, CT U.S.) of thymus pyrimidine detected by gas scintillation.Each variable is to be detected in triplicate.

The result

Immunity

Do not produce any serum that to distinguish H.243 with the antibody of irrelevant T-cell (table 1: immune group II, III and IV) that contains with the immunity (control experiment) of adjuvant.The serum titer of group V low (in aggegation is analyzed is 1: 100), the serum titer much higher (1: 1200) of those group I and II on the contrary.

The generation of anti-clonotype Mab

With the splenocyte nothing to do with T-cell clone of group I, be that the paramagnetic globule incubation of TCR/CD3 mixture load H.258 obtains many Roses annular cells here, show and successfully removed many CD3-and TCR α β-reactive B-cell.When the TCR/CD3-mixture that the cell that forms when nothing-rosettes has nothing to do with identical T-cell clone carries out once more preclearing, almost fail to observe any Rose annular cell.

After the paramagnetic globule incubation of T-cell clone interested, microscopy is failed to see rosettes and is formed.If the expected frequence of specific b-cell, this is not at all surprising.After limiting dilution and clonal expansion 36B-cell, find H.243 and the not irrelevant T-cell clone of aggegation H.258 (Table II) of supernatant aggegation.Some B-cell conditioned mediums can two kinds of clones of aggegation, show that they have escaped the preclearing program.

In addition, the major portion of from Table II, inferring specific b-cell culture be non-clonotype because the B-cell that reacts with any T-cell does not grow out yet.Yet this is not that a problem is because after trace-electricity merged, the hybridoma of these non-specific B-cells can screen.Carrying out trace-electricity with 18 in specific b-cell culture merges.Some fusion can not get the hybridoma and the other of T-cell-specific can not be cloned into stable clone.Finally obtained stable hybridoma among 11 from 18 trace-electricity merges.These 11 MAb are further characterized.The isotypes of these MAb are listed in Table III.

The specificity of MAb

In order further to study the specificity of MAb, carry out immunoprecipitation and T-cell agglutination and detect and the Western trace.

SDS/PAGE under non-reduced and sex change condition shows that all MAb can be settled out the main band of about 85KD and the less important band of 21KD in T-cell clone digitonin lysate H.243.This is consistent with the molecular weight of TCR α β-mixture and CD3 chain ε/δ respectively.The band of 85KD is dissociated into 40 and two bands of 50KD under reductive condition, and it is respectively with TCR α-and the molecular weight of beta chain is consistent.The immunoblotting of one of them anti-H.243 specificity MAb is with being shown in Fig. 1 as the OKT3 of positive control with as the irrelevant MAb of negative control.

Table III shows MAb and 6 V β 9 positive T-cell clones, and 2 V α 8 positive T-cell clones have other the cross reactivity of T-cell clone of different V α and V β with 8.Antibody TCR74 should be counted as specific because all the V β 9 positive T-cells of its aggegation of V β 9 and other cell of not aggegation.Other TCR antibody only with reaction H.243 therefore probably these antibody anti-H.243 antigen-binding portion thereof because with V α 8, the reaction of V β 9, TCR α β constant region, CD3 and other common T-cell surface molecule is excluded.Fig. 2 shows can dye TCR (85KD band) on the non-reducing Western trace of the antibody from hybridoma clone TCR64, TCR66, TCR69, TCR70, TCR73 and TCR76MAb.The non-specific dark band at figure top is derived from the antibody SAM and the OKT3 of immunoprecipitation.Having obtained in the Western trace under reductive condition does not have painted situation (result does not show) with anti-clonotype MAb, shows the conformational epitope that MAb identification is formed by complete TCR α β complex body.

MAb inductive T-cell proliferation

Whether can induce the H.243T-propagation of cell in order to study MAb, MAb is fixed on the flat microtitre flat board and with T-cell incubation.Though have very big difference between the MAb, Fig. 2 shows that all can induce the H.243 propagation of cell in them.The propagation that is similar to anti--CD3 (OKT3) obtains with TCR64, TCR66, TCR70, TCR76 and TCR79.Think other TCR anti--TCR44 of clonotype MAb do not obtain propagation.

The antigen of MAb mediation-the induce inhibition of T-cell proliferation

Studied the propagation whether the MAb group can suppress the cell H.243T-of antigen-driving in addition.In this experiment, MAb and T-cell and APC preincubation and after 3 hours, with the peptide burst process (pulsed) of different concns.Fig. 4 has shown that TCR64, TCR70, TCR76, TCR78 and TCR83 suppress the propagation of antigen-driving consumingly.Observe nearly 98% inhibition with TCR83.Dose response curve shows available few TCR64, TCR70 and the TCR83 and obtaining to 100ng/ml of maximum inhibition.TCR78 validity reduces about 10 times.Suppression mode does not rely on the peptide (result does not show) that whether adds the following or saturation concentration of best appropriateness.The anti-clonotype MAb of anti-other TCR (TCR44) does not demonstrate any inhibition.

Anti--clonotype MAb induces the H.243 unresponsiveness to stimulating with antigen and APC subsequently of human T-cell clone

Taking of known T-cell receptors induced anergy when lacking collaborative the stimulation.We found that the anti-clonotype MAb that is fixed in H.243 can have the TCR that excites this clone functionally in the past.Therefore, whether identical antibody can be induced anergy is interesting in research.For this reason, 10 different anti--clonotype MAb is fixed on the 24 hole flat boards and is incubated overnight with cell H.243 to be stimulated to produce anergy.Then, from flat board remove the T cell and whether detect they can to increase gradually concentration by irradiation, DRB1 ^*The HCgp-39 that the APC of 0401-coupling presents ^262-277(SEQ ID NO:3) makes and replying.Fig. 5 shows that this replys by 8 among 10 MAb and offsets fully and still the peptide that APC presents is replied well with the H.243 cell of contrast MAb1 incubation.When higher peptide concentration and the t cell response of TCR69 and TCR83 incubation significantly reduce but do not offset fully.

Replying of anergy cell inhibited reaction sexual cell

Whether anergy H.243 cell can inhibited reaction H.243 replying also of cell be studied.Anergy induce by TCR76 and reactive cell available from the contrast MAb1 incubation.Subsequently, proliferation assay is with peptide HCgp-39 ^261-275(SEQ ID NO:2) finishes, and having used different T-cell colonys is every hole 2 * 10 ⁴Individual anergy T cell, every hole 2 * 10 ⁴The anergy T cell (4 * 10 of concentration successively decreases for individual reaction-ive T cell or every hole ⁴, 2 * 10 ⁴, 1 * 10 ⁴With 0.5 * 10 ⁴) and every hole 2 * 10 ⁴The mixture of individual reaction-ive T cell.By adding the anergy cell, replying in the relevant mode of dosage of reactive cell significantly reduces (Fig. 6).Having obtained the minimizing of about 90% propagation and anergy with the antigen concentration of 1 μ g/ml is 2: 1 to the ratio of reactive cell.Observe less propagation with higher antigen concentration and reduce when 72% and 25 μ g/ml (during 5 μ g/ml 39%).Therefore this embodiment show produce anti-human T-cell receptors anti--the clonotype monoclonal antibody is possible.This experiment obtains 10 hybridomas, wherein 4 be preserved in ECACC (Salisbury, UK), as shown in Table III.This strategy does not need a large amount of T-cells or the purifying TCR of capacity.The method according to this invention is that tediously long recombination method is attractive alternative, and the soluble T CR that wherein expresses in the homology mouse cell is used for immune step by generation.Therefore, relevant with conformational stability possible problem is also avoided.Because the T-of antibody induction cell proliferation detect and antibody-mediated to the detection of antigen-drivings T-cell inhibitory effect (inhibition of observation arrival 98%) in monoclonal antibody demonstrate different replying, this shows on identical clonotype structure and recognizes different epi-positions.

If be crosslinked, all anti--clonotype monoclonal antibodies can be induced the H.243T-propagation of cell.

Proved in a small amount fixed anti--clonotype MAb can induce anergy in autonomic response (autoreactive) clone.After anergy stimulated, owing to lack the IL-2 gene transcription, the T cell failed to breed when stimulating again.In addition, found the IFNg output that descends.

The facs analysis of anergy cell shows that anergy is not descending adjusting of TCR or the result who lacks free TCR.In co-culture experiments, find anergy T cell inhibitory phase replying with the cloning reaction sexual cell.This onlooker (bystander) suppresses to cause 90% amplification inhibition.This digital proof the antibody of clonotype-special in external reactionless ability.This MAb can be used for inducing and keeping the tolerance of antigen-specificity T in the rheumatoid arthritis-cell.

Mice immunized (the Table I be coupled to TCR/CD3 immunocomplex quite pure on the paramagnetic globule by injection; Immune group IV) in the serum, mice serum records the specificity T-cell that is used for immunity more positive than irrelevant T-cell.Yet the hybridoma that acquisition produces anti--clonotype monoclonal antibody is impossible.Although do not having to prove the specificity of T-cell interested to irrelevant T-cell from the serum of the whole T-cellular immunization mouse of a small amount of, the method according to this invention has produced the hybridoma that can produce anti--clonotype monoclonal antibody.

Much less Fab can for example be made with papoid by monoclonal antibody according to the present invention.Monoclonal antibody or its Fab also can be labeled the detection and the scope of the present invention that are beneficial to them and comprise all these forms.

The irrelevant cell that also much less is used to remove non--specific b-cell is preferably very alike with the cell that has cell-surface antigens.

Table I: the feature description of immunization schedule and serum

2 BALB/C mice groups are with whole T cell or be incorporated into TCR/CD3 mixture immunity on the paramagnetic globule.Immunity is by finishing under existence of being shown in adjuvant or the non-existent situation.Used different immunization routes, that is: the ip=intraperitoneal; The im=intramuscular; Sc=is subcutaneous; In the is=spleen (intrasplenic).

The combination of feature description mice serum in the analysis of T-cell agglutination, FACS-dyeing and Western trace.Functional character is described with antibody-mediated T-analysis of cell proliferation and the antibody-mediated mensuration to antigen driving T-cell inhibitory effect and is finished.

^* T cell 1=is used for the T-cell clone of immunity; The T-cell clone that T cell 2=is irrelevant.

#+ means the inhibition of T-cell proliferation.

Table II: the outgrowth of specific b cells in EL-4 B-cell culture of anti--T cell

The hole count of inoculation d	Coating density	Hole count with B-cell outgrowth	With aggegation H.243	With aggegation H.258
					384	x	384	68	40
192	1/6x	171	13	7
					192	1/36x	81	3	1

In order to screen the special B cell of anti-clonotype, to 28 * 10 ⁶Individual lymphocyte carries out Immunobead (immunobead) screening procedure.The B cell that filters out increases in the EL-4B cell culture system with different coating concentrations and in the 8th day, measures in the supernatant liquor agglutinating antibody with T cell interested (H.243) and irrelevant T cell (H.258).X: the undeterminate cell count that filters out

Table III: the aggegation of different sorts T cell and anti--TCRmAb

¹⁾H.243:V α 8 (V α 8 en V β 9positive) in V β 9 positives

Reference

1.Moretta A. waits (1985) anti-screening and feature description that produces human leukemia T-clone idiotype (idiotype) the spline structure monoclonal antibody of interleukin II.International journal of cancer 36:253-259 page or leaf.

2.Oetgen, the same albumen composition of T3 that H.C etc. (1986) are relevant with mouse T cell antigen receptor.Nature 320:272-275 page or leaf.

3.Steenbakkers P.G.A. waits (1994) to produce monoclonal antibody effectively from the antigen-specific b cells of prescreen.Molecular biology report 19, the 125-134 page or leaf.

4.Steenbakkers, a kind of novel method that produces human and murine antibody hybridoma of P.G.A. etc. (1992).Immunological method magazine 152, the 69-77 page or leaf.

5.Laemmli, the cutting of U.K. (1970) structural protein in the assembling process of phage T4 head.Nature 277:680-685 page or leaf.

6.Towbin H. etc. (1979) albumen is from the electrophoretic transfer of polyacrylamide gel to nitrocellulose filter: method and some application.Institute of American Academy of Sciences reports the 76:4350-4354 page or leaf.

Shortenings

The PBS phosphate buffered saline (PBS)

BCIP 5-bromo-4-chloro-3-indyl phosphoric acid ester (indoylphospate) is right-the toluene amine salt

NBT is right-nitroblue tetrazolium muriate (p-nitro blue tetrazoliumChloride)

The BSA bovine serum albumin

The FCS foetal calf serum

The SAM sheep anti mouse

TSN T-cell conditioned medium liquid

MAb monoclonal antibody (antibody)

Manufacturers

The Bole, Richmond, CA, the U.S.

Gibco, Paisley, Scotland

Hyclone, Logan, the U.S.

Packard, Meriden, CT, the U.S.

Sequence table

(1) general information:

(I) applicant

(A) title: Akzo Nobel N.V.

(B) street: Velperweg 76

(C) city: Arnhem

(E) country: Holland

(F) postcode (ZIP): 6824BM

(G) phone: 0,412 666379

(H) fax: 0,412 650592

(ii) denomination of invention: preparation monoclonal antibody method, monoclonal antibody, pharmaceutical composition and diagnostic reagent

(iii) sequence number: 3

(iv) computer-reader form:

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, Version#1.30 (EPO)

(2) information of SEQ ID NO:1:

(i) sequence signature:

(A) length: 13 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: protein

(v) clip types: inner

(xi) sequence description: SEQ ID NO:1

Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly

1 5 10

(2) information of SEQ ID NO:2:

(i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: protein

(v) clip types: inner

(xi) sequence description: SEQ ID NO:2

Phe Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly

1 5 10 15

(2) information of SEQ ID NO:3:

(i) sequence signature:

(A) length: 16 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: protein

(v) clip types: inner

(xi) sequence description: SEQ ID NO:3

Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly Ala Pro

1 5 10 15

Claims (5)

1. with the monoclonal antibody of T-cell receptors clonotype structural response, it is characterized in that T-cell receptors is the T-cell receptors relevant with autoimmune disease, this autoimmune disease is a rheumatoid arthritis, and the T-cell receptors reaction of described monoclonal antibody and the reactive T-cell clone of HC gp-39.

2. according to the monoclonal antibody of claim 1, it is characterized in that the T-cell clone is an ECACC preserving number 96103122.

3. according to the monoclonal antibody of claim 2, it is characterized in that it is by being selected from following hybridoma preparation: ECACC preserving number 96103118, ECACC preserving number 96103119, ECACC preserving number 96103120 and ECACC preserving number 96103121.

4. pharmaceutical composition, it comprise according in the claim 1 to 3 any one with appropriate excipients blended monoclonal antibody, be suitable for treating rheumatoid arthritis.

5. the diagnostic reagent that comprises monoclonal antibody, this monoclonal antibody are selected from according to any one monoclonal antibody in the claim 1 to 3.

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