CN100491398C - Method of manufacturing human antibody possessing bioreceptor synergist, antagonist and/or inverse synergist - Google Patents
Method of manufacturing human antibody possessing bioreceptor synergist, antagonist and/or inverse synergist Download PDFInfo
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- CN100491398C CN100491398C CNB2004100798893A CN200410079889A CN100491398C CN 100491398 C CN100491398 C CN 100491398C CN B2004100798893 A CNB2004100798893 A CN B2004100798893A CN 200410079889 A CN200410079889 A CN 200410079889A CN 100491398 C CN100491398 C CN 100491398C
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Abstract
A method for developing the synergist, antagon and reverse synergist of a particular bioreceptor includes using computer to design and synthesize the immunogen and externally acting on human lymphocyte cluster. Said receptor is human CD 152, especially the CDR1, CDR2 and CDR3 regions able to respectively trigger the antibody to play the role of synergist, antagon and reverse synergist. A method for determining their pharmacologic efficacy is also disclosed.
Description
Technical field
The invention relates to a kind of method of making complete human antibodies, its antibody has synergist, antagonist and/or the reverse character such as synergist of this receptor.
Background technology
Generally speaking, medicine must arrive cells in vivo surface receptor position, just can with the drug molecule effect.The interaction of medicine and acceptor or bonded the possibility of result can activated receptor or blocking-up acceptors, and therefore in pharmacokinetics, what one group of medicine or part had same or similar physiology effectiveness is called synergist (agonist).In like manner, when the effect of a medicine during, then be called as antagonist (antagonist in contrast to other medicines or part.And when a part for the effect of same acceptor during in contrast to synergist, this part is called as reverse synergist (inverse agonist).
Human antibodies successfully as the medicine of various diseases, particularly merges virus (respiratory syncytial virus, RSV) the heritage infectious diseases of Denging as breathing.Though use fully as yet clinically, still some report has disclosed minority and has had the active antibody of pharmacokinetics recently.As WO00/32231, United States Patent (USP) the 5th, 811, No. 097, United States Patent (USP) the 5th, 855, No. 887 with United States Patent (USP) disclosed anti-mouse CD152 (killer T lymphocyte antigen-4 for the 6th, 051, No. 227, CTLA-4) mouse source monoclonal antibody is the fusion protein immunization hamster with mouse CD152 and IgG 1.Because being under the jurisdiction of, CD152 is called the immunoregulation acceptor that CD28 swashs (costimulation) receptor superfamily altogether, effect with negative regulation antibody and cell immune response, so after will having the mouse of tumour to the single-minded antibody injection of this acceptor, experimental mouse can produce anti tumor immune response, and then suppress tumor growth (people such as Leach, Science 271:1734,1996).The notion of this kind " CTLA-4 (antibody) blocking-up " comes from the negative sense function of CD152 can be by antibody blocking, so antibody can be used as antagonist, has activated existing but faint anti tumor immune response.
The human monoclonal antibody of existing now anti-human CD152 only can be used as the antagonism blocker, and its restriction is arranged in clinical application.The open human antibody molecule's synthetic method that the CD152 acceptor is had different combinations and message transmission capacity of the present invention, disclose simultaneously in the in vitro tests mode at the method that t cell responses screens this quasi-molecule, be beneficial to various application such as treatment, prevention and vaccine inoculation.
Summary of the invention
The present invention can be at the privileged site manufacturer antibody-like of special receptor or acceptor, and can not produce as side effects such as the anti-mouse anaphylaxis of people in be used in the mankind in the future; The present invention provides a kind of simple and easy, effective means simultaneously, can make synergist, antagonist and reverse synergist at specific human CD152 acceptor, and anaphylaxis than antibody that alternate manner produced for few, but and at least three kinds of different antigenic regions of the human CD152 of identification; In addition, the present invention also provides a kind of method, with the pharmacology effect of the antibody of the external synergist of confirming can be used as acceptor, antagonist and/or reverse synergist; The present invention also provides a kind of method with computing information construction and prediction epitope aminoacid sequence, to induce antibody response.
The present invention includes a kind of discriminating as the external synergist of acceptor, antagonist and/or the reverse method of synergist antibody pharmacological effect, include the following step:
(a) provide people's quasi-lymphocyte, division promotor, part and many strains activator;
(b) will divide promotor or many strains activator and add individual containers and increase ligand concentration, form a mixture;
(c) provide one to contain the first control group substratum that divides promotor, with the second control group substratum that contains the natural synergist of an acceptor;
(d) people's quasi-lymphocyte is incubated at this first control group substratum, this second control group substratum and this several parts and divides in the accelerator mixture;
(e) determine the degree of human Lymphocyte Apoptosis in each substratum and each mixture and differentiation: and
(f) apoptosis (apoptosis) and the differentiation degree that utilizes this mixture ligand concentration to increase determines the effectiveness of part to acceptor.
In addition, the present invention further provides a kind of method that produces human antibodies, but at least three kinds of different human CD152 antigen positions of this antibody identification.Therefore, screen the lymphocyte that is subjected to immunity, can obtain the complete human antibodies of complete acceptor on the identification physiology with an antigen immune lymphocyte and with the CD152 antigen of recombinating.Anyone can utilize the known technology of common prolongation antibody producing ability, as: merge to form ternary via the commentaries on classics shape of Epstein-Barr virus or with different myeloma cell and to merge knurl (trioma) cell, it can be survived for a long time and stably produce antibody.Perhaps can use the range gene recombination method, this antibody-like is produced in bacterium, yeast or cells of mamma animals.
Therefore, but the method that a kind of complete human antibodies of preparation identification physiology acceptor is provided again of the present invention, and its step includes:
(a) use one is derived from the human contributor's of tranquillization (not being subjected to antigenic stimulation) lymphocyte;
(b) design antigen with the auxiliary biological information of computer and carry out this lymphocyte of external immunity;
(c) add Epstein-Barr virus in this immunity lymphocyte;
(d) but confirm that the cell of ebv infection produces the antibody of identification this receptor; And
(f) the pharmacology function of screening step (d) antibody that produces.
Present method also can add a step (a1) before in step (b), by removing CD8 in the lymphocyte
+And CD56
+Cell.CD8
+And CD56
+Removing of cell can remove this class cell as utilizing specific CD8 and CD56 antibody according to any known technology of having set up.In one embodiment, these antibody can be connected in magnetic bead.In addition, present method can comprise a step (e) in addition after step (d), forms a ternary hybridoma cell or gene recombination immunoglobulin (Ig) to obtain human completely monoclonal antibody.Wherein polyclonal antibody can utilize the inventive method to be prepared.
Among the present invention, antibody identification antigen is preferably has the about 100nM of Kd value or littler, about 30nM or littler, about 10nM or littler, about 3nM or littler, or about 1nM or littler antigen; Antibody is IgG antibody, is preferably IgG1 and IgG4.
Affirmation or screening method also can use other division promotor or many strains activator, Canavalia ensiformis homo agglutinin A (concanvalin A for example, ConA), dyers' grapes division promotor (Pokeweedmitogen, PWM), 12-TETRADECONIC ACID phorbol ester-13-acetate (phorbol 12-myriState13-acetate, PMA) and superantigen such as staphylococcal enterotoxin A (SEA), to replace or to be used for and wherein phytohaemagglutinin (phycohemagglutinin, PHA) joint.The present invention provides a pharmaceutical compositions that comprises antibody simultaneously, and it can comprise pharmaceutically acceptable carrier or adjuvant.
Description of drawings
Figure 1A represents the heterodimeric protein matter structure of CD152 acceptor on the T cell surface and the relative position in three similar immunoglobulin (Ig) CDR districts.
Figure 1B is the human CD152 aminoacid sequence of translating in cDNA (gene pool numbering L15006, NCBI protein numbering P16410).Asterisk shows the starting point of mature polypeptide, intermembranous district and intracellular region.The similar district of CDR1, CDR2 and CDR3 is with box indicating.
Fig. 2 A, 2B and 2C describe hydrophobicity, chain mobility and the surperficial possibility of CDR1 and the contiguous area of human CD152 respectively.Fig. 2 D, 2E and 2F describe hydrophobicity, chain mobility and the surperficial possibility of CDR2 and contiguous area respectively.
Fig. 2 G, 2H and 2I describe hydrophobicity, chain mobility and the surperficial possibility of CDR3 and contiguous area respectively.The box district refers to the similar district of CDR, and its aminoacid sequence is shown in the below.Arrow indicates the chain structure that institute's predicted amino acid is formed.
Fig. 3 shows the human PBMC cytodifferentiation () of PHA stimulation and the reaction situation of apoptosis (■).Fig. 3 A, 3B, 3C, 3D and 3E are respectively PHA stimulates separately that (1.25,5 and 20 μ/ml), 1.25 μ g/ml PHA follow the result of bridging property CD80 (0.2,1 and 5 μ g/ml), anti--CDR1, anti--CDR2 and anti--CDR3 antibody (0.1,1 and 10 μ g/ml) gained.
Embodiment
Unless otherwise, nominal definition used in the present invention is as follows:
Part is meant the compound that is bonded to another molecule, for example receptor protein;
Acceptor is meant with extracellular physiological signal effect and is converted to the albumen of cell interior effect;
Human antibodies is meant that an antibody only contains the human sequence fully;
Tranquillization (
) human contributor is meant and is not exposed to the antigenic people of institute's desire, its blood can be used as the source of the immunocyte or the factor, tranquillization contributor blood does not detect antigenic circulating antibody, and the human contributor of typical tranquillization is healthy normal blood donating person, and its HIY antibody detecting is negative for continuing; Be meant with antigen immune cell or animal cell or animal are exposed to antigenic action, cell or animal can carry out immunity by any way, as long as cell or animal touch antigen.
The immunity of external location is meant external LS process, to reach antibody response to desired protein fragments, according to the heterogeneous coupling decision of synthetic position immunogen, the polypeptide that it contains T cell and B cell antigen decision position can cause humoral immune reaction to resist whole albumen;
Treatment or improve disease or medical conditions and be meant and reduce or eliminate disease or symptom medically, or relax the process of disease/medical conditions;
Preventing disease or medical symptom are meant and detect a testee of not having disease or a medical symptom that wherein, last testee does not develop into and has this disease/medical condition, the perhaps only a little development of disease/medical conditions.
Significant quantity be meant the amount of a medicament be enough to reach desire effectiveness, with the treatment of antibody or improve a disease, a significant quantity means to be enough to reduce or the symptom or relax the antibody amount of disease process of eliminating a disease;
Synergist is that a part has endogenous part or the same or similar effectiveness person of part group with another natural generation;
Antagonist is that a synergist person is blocked in the effect of a part or medicine;
Oppositely synergist is meant a part, and the effectiveness of its generation but can take identical acceptor in contrast to synergist;
Receptor blocking is meant that utilizing the pharmacology antagonist to be bonded to acceptor blocks natural endogenous part (as the hormone or the nerve conduction factor) and be bonded to the effectiveness that is taken place behind the acceptor.
Sample is meant the representative part of aliquot or material, material or group, and a sample can be the sample of water, dirt, oil, sand, blood, biological tissue, urine or ight soil;
Biological specimen is meant the sample of being collected by biological testee such as animal, plant or Institute of Micro-biology.
The present invention also comprises pharmaceutical compositions, comprises one or more antibody that can be used as active ingredient, and cooperates pharmaceutically acceptable carrier or adjuvant.Aspect preparation constituent of the present invention, activeconstituents/antibody mixes with adjuvant usually, seals with the adjuvant dilution or with carrier, and its pattern can be made into capsule, pouch, pattern or other container.
When pharmaceutically acceptable adjuvant is a thinner, can be solid, semisolid or fluent material, and as solvent, carrier or the media of activeconstituents.Therefore, this constituent can be solution (referring to aseptic Injectable solution especially), tablet, pill, powder, lozenge, pouch, capsule, elixir, suspension, emulsion, syrup, aerosol (as solid or liquid media), contains the ointment just like 10% weight percent antibody, soft or forms such as hard gel capsule, suppository and aseptic packaging powder.
Some example that is suitable for adjuvant comprises lactose, glucose, sucrose, sorbyl alcohol, N.F,USP MANNITOL, starch, gum arabic, calcium phosphate, algae polysaccharide, yellow rubber powder, gel, Calucium Silicate powder, Microcrystalline Cellulose, polyvinylpyrrolidone, Mierocrystalline cellulose, sterilized water, syrup, methylcellulose gum.This composition can also comprise: lubricant is talcum powder, Magnesium Stearate and mineral oil for example; Wetting Agent for Printing Inks; Emulsifying agent and suspension agent; Sanitas such as methyl-with the propyl group hydroxybenzoate; Sweetener; And seasonings.Constituent of the present invention can be configured to quick, slow or its activeconstituents of delay release of supplying after sufferer is offerd medicine, and its mode is as shown in known technology.
The liquid form of the new constituent of the present invention can be configured to oral or drug administration by injection, comprise the aqueous solution (for example PBS), suitably flavored syrup, water-based or oiliness suspension, reach emulsion through seasoning, it contains edible oil such as Semen Maydis oil, Oleum Gossypii semen, sesame oil, Oleum Cocois or peanut oil, and elixir and similar solvent pharmaceutically.Of the present invention other is suitable for joins, and can see Lei Shi pharmacy complete works (Remington ' sPharmaceutical Science).
Following example just is used for the present invention is explained, and is non-as limitation category of the present invention.For example, similarly study also at other CD28 receptor superfamily member and as CD28 abnormal shape (CD28i), the common shock protein (ICOS) that can bring out, B and T lymphocyte decay albumen (BTLA) and program necrocytosis albumen-1 (PD-1).Similarly, also can be applicable to other receptor family, comprise that ionic channel, G-protein-coupled receptor, Tyrosylprotein kinase connect acceptor and transcription factor etc.
Table one:
The preparation of embodiment one, polypeptide antigen
The one-piece construction of human CD152 acceptor is plotted in Figure 1A.The aminoacid sequence that accords with human CD152 is then listed in Figure 1B.By the information of integrating the scientific literature gained, important cognation thereby foundation between CDR3 district and natural CD80 and the CD86 part.Other evidence shows, the amino acid that is positioned at the CDR1 district is played the part of the role of certain and CD80/CD86 effect.Yet the synergist binding mechanism of CDR2 is not so far but as yet by complete discussion.
Want to cause to be bonded to the proteic antibody of body, the proteic polypeptide that uses must have the characteristic of similar its structure.Therefore, the complete sequence in CDR1, CDR2 and CDR3 district is retained with design synthetic immunogen.In addition, prolong each CDR polypeptide portion to meet aforementioned epitope pattern.As an effective immunogen, if desire the actual antibody that is produced that uses, then this polypeptide must be selected from proteic easy structure district (accessible region).Proteic easy structure district is the position that is exposed to the structure outside.Because these zones contact with aqueous environment, so be wetting ability often.Then people use
Software (IntelliGenetics) is set up hydrophobicity and is drawn with the decision extending direction.Chain mobility or surperficial possibility also with
Calculate, and the CDR near zone also is considered as the secondary parameters (Fig. 2) of polypeptide design.
The high score CDR polypeptide of gained is used to connect tetanus toxin and " assists " sequence after computing, and its amino acid position is 830-844 (SEQ ID N0:1).
The generation of embodiment two, anti-CD152 human antibodies
Healthy blood contributor is negative through HIV-1/2, HTLV-I/II, HCV, HbsAg screening, and contains the blood of the Beta Alanine transferring enzyme (ALT) of normal amount, contributes blood center (platform south, Taiwan) from Chinese blood foundation platform south.Peripheral blood monocyte (PBMC) is located away from Ficoll-Paque (Amersham Biosiences) in density centrifugation (400xg) mode; Cell is subsequently with the PBS washed twice and with the centrifugal collection of 100xg.
Gained PBMC at first carries out magnetic with CD45RO MACS microballon (Miltenyi Biotec) and demarcates, and separates with VarioMACS (Miltenyi Biotec) again.After magnetic was demarcated, by separating tubing string, it placed on the powerful permanent magnet with cell.The matrix of logical magnetic tube post can produce high-order magnetic field.Magnetic demarcation cell can stay in the tubing string and leach does not demarcate cell.After remove in tubing string magnetic field, dash and propose cell.Dash the CD45RO that proposes
+Cell must be existed side by side with the 100xg centrifuging and taking and promptly be used.With CD45RO
+The T-cell cultures is in tissue culture flasks, and its density is 2 x 10
6Cell/mL RPMI-1640 (HyQ
TM, HyClone), and additional 1x non-essential amino acid (Life Technologies), 10% human serum, 50 μ m/ml gentamicin/kantlex (China Chemical ﹠amp; Pharmaceutical), 50 μ M 2-ethanethios and 10 μ g/ml dyers' grapes division promotor (PWM, Sigma).Cultivate after 24 hours, cell is centrifugal and shift out with 400xg.At last, collect supernatant liquor with preparation CD45RO
+The T-cell replacement factor is with the 0.45mm membrane filtration and in-20 ℃ of refrigerated storage.
PBMC is removed cytotoxicity group with magnetic cell removal method, with the vitro inhibition immune response.The usage that the super magnetic micro-beads of colloid is bonded to anti-mouse CD8 of individual plant and anti-CD56 antibody (MiltenyiBiotech) as described above.The PBMC that cytotoxic cell is removed carries out immunization in external with two step immunizations: elementary immunization, be with cell cultures in the substratum that contains the 10nM polypeptide antigen 6 days, that is TT-CDR1
(ext), TT-CDR2
(ext)With TT-CDR3
(ext)In containing 50 μ M2-ethanethios, 10% heat deactivate human serum, 0.05ng/mlrIL2 (Calbiochem) and 25% (v/v) CD45RO
+T cell replacing factor was collected elementary immunocyte and centrifugal with 40%Ficoll-Paque at the 7th day; At secondary immune response, with 3 * 10
7Individual cell and polypeptide antigen are mixed in prior CD40L with 5 μ g/ml (CD154, Vinci-Biochem) fixing culturing bottle overnight; Again with the culture medium culturing cell that contains 5% human serum, 50 μ M 2-ethanethios and 10nM polypeptide antigen 3-5 days.
The cell of external immunity is subsequently with ebv infection: with 10
7Individual lymphocyte was cultivated 2 hours down in 37 ℃, and the supernatant liquor that contains Epstein-Barr virus with 1ml carries out resuspending, and this supernatant liquor is derived from the marmoset MC strain B95-8 (ATCC CRL 1612) that produces Epstein-Barr virus.With the cell that infects with 10
5The quantity kind in/hole is in 96 porose discs and add PBMC that mitomycin (Kyowa Hakko Kogyo) handles with as feeder cell (10
4/ hole).
The carrying out of antibody specificity ELISA, at first under room temperature with the bovine serum albumin in recombinant human CD152 (the CTLA-4)-muIg fusion rotein (Ancell Corporation) of 1 μ g/ml bhk cell performance, 1 μ g/ml individual plant muroid IgG2a (Ancell), 10 μ g/ holes (BSA, Sigma) or Toxoid,tetanus (ADImmune Corporation) microtiter plates that is coated on overnight.With the supernatant liquor cultivated containing the 10mM sodium phosphate buffer of 0.5M sodium vapor and 0.1%Tween-20, pH8.0 be diluted to desire concentration.The titration dish that applies is cultivated with the medium supernatant of dilution, the goat antibody identification IgG (Zymed Laboratories) that the washing back is demarcated with peroxidase, and add 100 μ l chromogen matrix ortho-phenylene diamine (OPD, Sigma) development 15min; Add 1M sulfuric acid stopped reaction behind the 30in, sentence read result under the 490nm light absorption value.
Confirm that with above-mentioned solid-state ELISA the lymphocyte of ebv infection is disengaged possible anti--CD152 antibody.The integration of the contained lymphocytic specificity antibody growing amount in every hole is to carry out with following standard: (a) the OD value of recombinant human CD152-muIg fusion rotein is at least five times of negative control group OD value; And (b) reactivity indexes (R1)〉2, RI=[OD wherein
CD152-muIg-OD
Substratum The control group is to CD152-muIg]/[OD
Muroid IgG2a-OD
Substratum control group is to muroid IgG2a].
Contain lymphocytic Kong Ruo and in above-mentioned analysis, be positive, then collect its supernatant liquor, and quantitatively reach stdn with further research with ELISA.Reactive being confirmed to be in CDR district utilizes indivedual polypeptide to carry out the ELISA competing reaction.These culture hole are grown and freezing preservation with the commentaries on classics of limit number dilution method.
The ability of embodiment three, the anti-CD152 antibody of change is to bring out apoptosis and cytodifferentiation and to compare with natural synergist
Being confirmed to be the synthetic polypeptide of different resisting-CD152 human antibodies binding site corresponding to one-level alkylamine deutero-cellulose membrane (Rapp Polymere GmbH).For further assessment different anti--the pharmacology effectiveness of CD152 antibody and preferable natural synergist CD80, carry out post-stimulatory cell growth of the external phytohaemagglutinin of human peripheral lymphocyte and PBMC cultivation.Briefly, prepare 96 flat hole microtiter plates and add 50 μ l mountain cell suspending liquids (10
5Individual cell), 60 μ l contain the PHA substratum (ultimate density of cultivation are 1.25 μ g/ml, Amersham Biosciences AB), 40 μ l autologous plasmas and 50 μ l RPMI-1640 substratum, it contains anti--CD152 antibody or monomeric human CD80-muIg fusion rotein (Ancell) concentration range by 0.2 to 5 μ g/ml.In the CD80 irritant reaction, add 5 μ g/ml goat anti-mouse IgG 2a (Southern BiotechnologyAssocetes) so that the signal of cross-linked form to be provided.The cumulative volume of cultivating is 200 μ l.Be incubated at 37 ℃ 5%CO
2Reach 96h in the wet environment.In collection preceding 204 hours, every hole added the substratum (TRA 306, Amersham, given activity 2Ci/mol) that 50 μ l contain the tritiated thymidine of 0.5 μ Ci.Cooperate semi-automatic multiple collector (PHD, Cambridge Technology Inc.) to collect substratum with glass fiber filter.The cell combination [
3H]-decision of thymus pyrimidine is to count with the LKB liquid scintillation counter.In some was cultivated, carried out the measurement of cell survival rate with Trypan Blue (Trypan blue) stain latter stage.Carry out identical three and repeat to cultivate, and carry out computing with three's intermediate value.
Assessment natural death of cerebral cells per-cent is that cell is centrifugal with 200xg, and resuspending mixes up to final densities 1 x 10 in 80% ice-cold alcohol and concuss
6/ ml.With cell cultures in 4 ℃ of 30min at least.The cell of alcohol fixation centrifugal subsequently and in the 1ml propidium iodide (PI Sigma) carries out resuspending in the stain (PBS0.15M pH7.4,0.1%Triton X-100,0.1mM EDTA disodium salt, 50 μ g/ml RNase A and 50 μ g/ml PI).Sample is dark the preservation up to analysis under room temperature, and carries out in 24 hours.Endonuclear DNA amount cooperates Lysis H software (Becton Dicknson) decision with propidium iodide dyeing and FACScan cell instrument.The decision of apoptotic cell is time two cover DNA per-cents that utilize after flow cytometer is measured propidium iodide dyeing.
Provide a dish anti--human antibodies of CD152, to determine these parts function to human T cell under the PHA activation, can produce moderate cytodifferentiation and apoptosis (Fig. 3 A).The crosslinked apoptosis that CD80 brought out has not been observed obvious cytodifferentiation (Fig. 3 B).Similarly, the antibody that polypeptide immunogen brought out that contains CDR3 also causes necrocytosis fast and undifferentiated, thereby has confirmed synergist activity (Fig. 3 E).The hormesis of the human antibodies that CDR1 brings out obviously reduces, but does not remove the necrocytosis (Fig. 3 C) that PHA drives fully.When PHA activatory PBMC separately brings out human antibodies and cultivates with CDR2, can be observed highly and the cytodifferentiation situation of tool reproducibility, and the necrocytosis that PHA causes can be removed (Fig. 3 D) fully.Driving situation by anti--CD152 that cytodifferentiation causes that CDR2 brings out is similar to 5ng/ml IL-2 processing situation, and causes the disappearance (as, cytolemma foamed imperfect with cell and karyomorphism become vesicle) of typical apoptotic cell kenel variation phenomenon.
The foregoing description only is to give an example for convenience of description, and the interest field that the present invention advocated should be as the criterion so that claim is described certainly, but not only limits to the foregoing description.
Sequence table
<110>Chin，Li-Te
Hsu，Shu-Ching
＜120〉have synergist, antagonist or the reverse human antibodies manufacture method of synergist characteristic
<130>P7226/0600
<160>8
<170>PatentIn?version?3.3
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<211>15
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＜213〉tetanus toxin
<220>
＜221〉polypeptide
<222>(1)..(15)
<300>
<301>Stephane?Demotz，Antonio?Lanzavecchia，Ulrich?Eisel，Heiner
Niemann，Christian?Widmann，and?Giampietro?Corradin
＜302〉the T cell antigen decision position that multiple DR-limits is described
<306>394-402
<307>1989-01-15
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<400>1
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<211>15
<212>PRT
＜213〉modern
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＜213〉modern
<220>
＜221〉polypeptide
<222>(1)..(15)
<400>4
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<211>30
<212>PRT
＜213〉synthetic
<220>
＜223〉synthetic CDR polypeptide and be used to prepare the epitope that is harmonious and be derived from tetanus virus " TT " sequence with connection.
<220>
＜221〉polypeptide
<222>(1)..(30)
<400>5
<210>6
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<220>
＜223〉synthetic CDR polypeptide and be used to prepare the epitope that is harmonious and be derived from tetanus virus " TT " sequence with connection.
<220>
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<222>(1)..(30)
<400>6
<210>7
<211>30
<212>PRT
＜213〉synthetic
<220>
＜223〉synthetic CDR polypeptide and be used to prepare the epitope that is harmonious and be derived from tetanus virus " TT " sequence with connection.
<220>
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<222>(1)..(30)
<400>7
<210>8
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Claims (9)
1. method of making human CD152 receptor antibody, wherein this antibody has synergist, antagonist or the reverse characteristic of synergist to acceptor, includes the following step:
(a) utilize the biological information characterizing definition at each position, extracellular of this receptor to go out the polypeptide fragment at position, this receptor extracellular out of the ordinary;
(b) with this polypeptide fragment and a t helper cell antigen decision bit coupling, the complex polypeptide that is produced contains a t helper cell antigen decision bit at least;
(c) preparation one has the immunogen of this coupling fragment aminoacid sequence;
(d) carry out stimulated in vitro people quasi-lymphocyte with this immunogen;
(e) but discriminated union screening produces people's quasi-lymphocyte of the antibody of identification this receptor; And
(f) collect this antibody.
2. the method for claim 1 is characterized in that, wherein this polypeptide fragment aminoacid sequence that is consistent with this position, extracellular is made up of arbitrary described aminoacid sequence among the SEQ ID Nos:2 to 7.
3. the method for claim 1 is characterized in that, wherein the aminoacid sequence of this t helper cell antigen decision bit is shown in SEQ ID NO:1.
4. the method for claim 1 is characterized in that, wherein the biological information at each position, extracellular of this receptor is characterized as hydrophobicity, mobility or surperficial possibility.
5. one kind is incorporated into the segmental antibody of coupling, and wherein this coupling fragment is made up of a t helper cell epitope and the polypeptide fragment that is coincident with each position, extracellular of CD152 acceptor; Wherein:
The aminoacid sequence of this t helper cell antigen decision bit is shown in SEQ ID NO:1; This polypeptide fragment aminoacid sequence is made up of arbitrary described aminoacid sequence among the SEQ ID Nos:2 to 7.
6. a discriminating includes the following step as the external synergist of acceptor, antagonist or the reverse method of the human CD152 receptor antibody pharmacological effect of synergist:
(a) provide people's quasi-lymphocyte, a division promotor, a part to reach optionally many strains activator;
(b) should divide promotor or many strains activator and add individual containers and increase ligand concentration, form a mixture;
(c) provide the first control group substratum that contains a division promotor, with the second control group substratum that contains the natural synergist of an acceptor;
(d) people's quasi-lymphocyte is incubated at this first control group substratum, this second control group substratum and these a plurality of parts and divides in the accelerator mixture;
(e) determine in each substratum and each mixture procedural apoptosis of people's quasi-lymphocyte and duplicate the activatory degree; And
(f) utilize procedural apoptosis that this mixture ligand concentration increases and duplicate activation degree and determine the effectiveness of part acceptor.
7. method as claimed in claim 6 is characterized in that, wherein this people's quasi-lymphocyte is peripheral blood mononuclear cell.
8. method as claimed in claim 6, it is characterized in that, wherein this division promotor or many strains activator are phytohaemagglutinin, Canavalia ensiformis homo agglutinin A, dyers' grapes division promotor, 12-TETRADECONIC ACID phorbol ester-13-acetate or superantigen, use with independent or hybrid mode.
9. method for preparing human CD152 receptor antibody, but the physiological acceptor of this antibody identification includes the following step:
(a) provide the people quasi-lymphocyte;
(b) get the synthetic antigen of complying with biological information characteristic Design, this lymphocyte is carried out immune response external;
(c) add Epstein-Barr virus in this immunity lymphocyte;
(d) but differentiate that the weary cell of Epstein-Barr virus infection produces the antibody of identification this receptor; And
(e) the pharmacology function of screening step (d) antibody that produces.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100798893A CN100491398C (en) | 2004-09-24 | 2004-09-24 | Method of manufacturing human antibody possessing bioreceptor synergist, antagonist and/or inverse synergist |
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| CNB2004100798893A CN100491398C (en) | 2004-09-24 | 2004-09-24 | Method of manufacturing human antibody possessing bioreceptor synergist, antagonist and/or inverse synergist |
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| CN1752106A CN1752106A (en) | 2006-03-29 |
| CN100491398C true CN100491398C (en) | 2009-05-27 |
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