TWI267517B - Method for producing human antibodies with properties of agonist, antagonist, or inverse agonist - Google Patents

Abstract

A method for obtaining agonist, antagonist and inverse agonist, to a given physiological receptor is disclosed. For the method, use is made of in silico design synthetic immunogens, which are caused to act in vitro on human lymphocyte-containing cell populations. A preferred receptor is human CD152, particularly the regions of CDR1, CDR2 and CDR3 that elicit antibodies serving as antagonist, inverse agonist and agonist, respectively. Also provided is a method for use in the screening of CD152 ligands that yield pharmacological effects.

Description

</ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; 5 [Prior Art] In general, drugs must reach the cell surface receptor site in the body to interact with drug molecules. The interaction of the drug with the receptor or the result of the binding y can activate the receptor or block the receptor, so in pharmacokinetics, a group of 1 or 2 ligands or ligands having the same or similar physiological effects are "synergy agents". (ag=st)". Similarly, when the efficacy of a drug is opposite to other drugs or ligands, it is called an "antagonist." When a ligand acts against a companion opposite to a synergist, the ligand is called an "inverse agonist". 15 Human antibodies have been successfully used as therapeutic drugs for various diseases, especially as Traditional infectious diseases such as respirat〇ry syncytial virus (rsv). Although not fully used clinically, there have been recent reports revealing a small number of antibodies with pharmacokinetic activity. Anti-mouse CD152 (killer lymphocyte antigen-4, CTLA-4) is disclosed in US Patent No. 5, 811, No. 97, U.S. Patent No. 5,855,887, and U.S. Patent No. MR227. The murine monoclonal antibody is a mouse hamster which is a fusion protein of mouse cm52 舁 human IgG1. Since cd152 belongs to the immunoregulatory receptor called CD28 co-exciting receptor superfamily, it has the function of negatively regulating antibody and cellular immune response, so it will inject this antibody with 1267517 antibody. After tumor mice, the experimental mice will develop an anti-tumor immune response' to inhibit tumor growth (Leach Zheng, Science 271:1734, 1996). The concept of "ctla_4 (antibody) blockade" comes from the fact that the negative function of cDl52 can be blocked by antibodies, so antibodies can act as antagonists to activate an existing but weak anti-tumor immune response. Human monoclonal antibodies against human CD152 have only been used as antagonists, and have limitations in clinical applications. The invention discloses a method for synthesizing human antibody molecules having different binding and signal transmissibility to the cD152 receptor, and discloses a method for screening such 10 molecules for the tau cell reaction in an in vitro test manner, so as to facilitate treatment, prevention and vaccination. application. SUMMARY OF THE INVENTION The present invention can be applied to a specific receptor or a specific part of a receptor, and can be used in humans in the future without causing a side effect such as a human anti-small allergic reaction. The present invention also provides a simple and effective method. a method for producing a synergistic agent, an antagonist, and a reverse synergist for a specific human CD152 receptor, and having fewer allergic reactions than other methods, and identifying at least two different antigenic regions of human CD152; The invention also provides a method for confirming the pharmacological efficacy of an antibody which can act as an in vitro synergist, an antagonist 盥/or a reverse 20 synergist of a receptor; the invention further provides a computer information for constructing and predicting an epitope amino acid A method of sequencing to induce an antibody response. . Several inventions include a method of identifying the pharmacological efficacy of an in vitro synergist, an antagonist, and/or a reverse synergist antibody as a receptor, comprising the steps of: 25(4) providing a human lymphocyte, a cleavage promoter, a ligand, or a plurality of strains Activator; 1267517 (曲b) Adding a splitting promoter or multiple activators to individual containers and increasing the concentration of the ligand's formation-mixture; (4) providing - the first control group containing the splitting promoter, with the natural receptor-containing a second control group culture of the synergist; (d) cultivating human lymphocytes in the first control group medium, the control group 5 medium, and the plurality of ligands and the split promoter mixture; (e) The extent of apoptosis and/or differentiation of human lymphocytes in each medium and mixture; and (f) programmed cell death (apoptosis) and/or degree of differentiation using the concentration of the ligand in the mixture determines the ligand pair The utility of the body. Furthermore, the invention further provides a method of producing a human antibody, 10 which recognizes at least three different human CD152 antigen positions. Therefore, a fully human antibody recognizing a physiologically intact receptor can be obtained by immunizing lymphocytes with an antigen and screening the immunized lymphocytes with the recombinant CD 152 antigen. Anyone can use the common techniques of prolonging the production capacity of antibodies, such as transfection of Epstein-Barr virus or fusion with heteromyeloma cells to form a 15-fold binary tumor (tri〇ma) cell, which can be used for a long time. Survival and stable production of antibodies. Alternatively, various genetic recombination methods can be applied to produce such antibodies in bacteria, yeast or mammalian cells. Accordingly, the present invention further provides a method of preparing a fully human antibody that recognizes a physiological receptor, the steps comprising: (using a lymphocyte derived from a human donor that is resting (not stimulated by the antigen); (b) Computer-assisted life

The information is designed to immunize the lymphocyte in vitro; (c) to add the eB virus to the immune lymphocyte; (d) to confirm that the eb virus-infected cell produces an antibody that recognizes the receptor; and (1) a screening step ((1) The pharmacological function of the produced antibody. 1267517 The method can further add a step (al) before the step (b) to remove CD8 + and CD56+ cells from the lymphocytes. The removal of CD8 + and CD56+ cells can be based on any Conventional techniques have been established, such as the use of specific CD8 &amp; cd56 antibodies to remove such cells. In an embodiment, these antibodies can be ligated to 5 magnetic beads. Additionally, the method can additionally be followed by step (d) In one step (e), one or two 7G fusion tumor cells or a recombinant immunoglobulin are formed to obtain a complete human monoclonal antibody, wherein a plurality of antibodies can be prepared by the method of the present invention. In the present invention, the antibody recognition antigen is preferably An antigen having a Kd value of about ι〇〇nM or less, about 30 nM or less, about 1 〇 or less, about 3 vesicles or less, or 10 about luM or less; the antibody is an IgG antibody, Jia Wei (10)i and Gong Yang. Confirmation Screening methods can also use other cleavage promoters or multiple strains of activation? d such as concanvalin A (ConA), Pokeweed mit〇gen (pWM), 14 酉夂佛波月曰-13_ acetate (ph〇rb〇1仏零匕 her (3) café's just (4) 15 and superantigens such as staphylococcal enterotoxin A (SEA) to replace or be used for bloody sputum Phyc〇hemagglutinin (pHA) conjugate. The present invention provides a pharmaceutical composition comprising an antibody, which may comprise a pharmaceutically acceptable carrier or adjuvant. 20 [Embodiment] "Unless otherwise specified" used in the present invention The term is defined as follows: "heart" is a compound that binds to another molecule, such as a receptor protein; "fork body" refers to a protein that interacts with extracellular physiological signals and converts it into an intracellular effect; "--, person, "70 king human antibody" means that an antibody contains only human sequences; 25 "nai've human donor" means a person who is not exposed to the desired antigen, 1267517 whose blood can be used as a source of immune cells or factors. Resting donor blood test Circulating antibodies to antigens, typical resting human donors are healthy and normal donors, whose HIV antibody detection is persistently negative; "immunization" of cells with antigens refers to the action of exposing cells or animals to antigens, cells戋5 animals can be immunized in any way, as long as the cells or animals are exposed to =. "In vitro localized immunity" refers to the in vitro lymphocyte stimulation process to achieve antibody response to the desired protein fragment, based on synthetic heterogeneous coupling Determines a 10-site immunogen that contains a peptide of a sputum cell and a 6-cell epitope that triggers a humoral immune response against the entire protein; "treating" or "improving" a disease or condition refers to reducing or eliminating disease or medicine. Symptoms γ or the progression of the disease/medical condition; "prevention" of a disease or medical condition refers to the detection of a subject who does not have a disease or medical condition, wherein the last subject does not develop the disease/medical condition , or the disease/medical condition has only evolved slightly. An effective amount means an amount of a medicament sufficient to achieve the desired effect. In the case of treatment or improvement of an antibody, an effective amount means an amount of an antibody sufficient to reduce or eliminate the disease or to alleviate the progression of the disease; A ligand has the same or similar effect as another naturally occurring endogenous ligand or ligand group; the "antagonist" is a ligand or a drug that blocks a synergistic J The term "reverse synergist" refers to a ligand which produces a useful phase = the same receptor can be occupied by the synergist; "receptor blockade" refers to the use of a straw to resist binding to the receptor. Blocks the effects of natural endogenous ligands (such as hormones or divine, two-conducting factors) binding to the receptor. 1267517 "sample" means a representative part of a substance or substance, material or group, which may be a sample of water, dirt, oil, sand, blood, biological tissue, urine or flank, and "biological sample" means A sample collected by a biological subject such as an animal, plant, or microorganism. 5 The present invention also encompasses a pharmaceutical composition comprising one or more antibodies which may be used as an active ingredient in association with a pharmaceutically acceptable carrier or adjuvant. In the case of the composition of the invention, the active ingredient/antibody is usually mixed with an adjuvant, diluted with an adjuvant or encapsulated with a carrier, and the form can be made into a capsule, a pouch, a paper or a container. 1 § When a pharmaceutically acceptable adjuvant is a diluent, it may be a solid, semi-solid or liquid material and act as a vehicle, carrier or vehicle for the active ingredient. Thus, the composition can be a solution (especially a sterile injectable solution), a tablet, a pill, a powder, a key, a sachet, a capsule, an ugly agent, a suspension, an emulsion, a syrup, an aerosol (such as a solid or liquid medium). ), containing, for example, 1% by weight of anti-15 body ointment, soft or hard gel sac, suppository, and aseptic packaging powder. Some examples of suitable adjuvants include lactose, glucose, sucrose, sorbitol, mannitol, house powder, gum arabic, squamous acid, algae, jaundice powder, gel, shixi _, microcrystalline cellulose , polyethylene, scent, sun, cellulose, sterile water, syrup, methyl cellulose. The composition may further comprise: lubricating 20 agents such as talc, magnesium stearate and mineral oil; humectant; emulsifier and suspending agent; preservatives such as methyl- and propyl-benzoate; sweetener; and flavoring agent . The compositions of the present invention may be configured to provide rapid, slow or delayed release of the active ingredient after administration by a patient, as shown in the prior art. The liquid form of the novel composition of the present invention may be formulated to be administered orally or by injection to 1267517, including an aqueous solution (e.g., PBS), a suitable flavored syrup, an aqueous or oily suspension, and a flavored emulsion containing an edible oil such as corn oil, Cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as tinctures and similar pharmaceutically acceptable solvents. Other suitable formulations of the present invention can be found in Remington&apos;s Pharmaceutical Science. [The following examples are provided to illustrate the invention and are not intended to limit the scope of the invention. For example, similar studies have also targeted other members of the CD28 receptor superfamily and such as CD28 isoform (CD28i), inducible eukaryotic protein (ICOS), B and T lymphocyte attenuating protein (BTLA), and programmed cell death protein-l (PD-l). 10 Similarly, it can be applied to other receptor families, including ion channels, G-protein coupled receptors, tyrosine kinase-linked receptors, and transcription factors. Table 1 Amino acid sequence of the peptide of the present invention Amino acid sequence of the peptide SEQ ID NO TT N5—QYIKANSKFIGITEL—C5 1 CDR1 (ext) N'-EYASPGKATEVRVTV—C' 2 CDR2(ext) Ν'AATYMMGNELTFLDD-C' 3 CDR3 (ext N'-KVELMYPPPYYLGIG-C' 4 TT-CDRl(ext) Ν'-QYIKANSKFIGITELEYASPGK ATEVRVTV-C' 5 TT-CDR2(ext) Ν'-QYIKANSKFIGITELAATYMM GNELTFLDD-C' 6 TT-CDR3(ext) Ν'-QYIKANSKFIGITELKVELMY PPPYYLGIG - C' 7 Example 1. Preparation of peptide antigen The overall structure of the human CD152 receptor is depicted in Figure 1A. The amino acid sequence conforming to human 15 CD152 is shown in Figure 1B. The important correlation between the CDR3 region and the native CD80 and CD86 ligands was established by integrating information from the scientific literature. Additional evidence suggests that amino acids located in the CDR1 region play a role in CD80/CD86 interaction. However, the synergistic binding of CDR2 to the 1267517 mechanism has not been fully explored to date. In order to elicit an antibody that binds to a bulk protein, the peptide of the protein used must have properties similar to its structure. Thus, the complete sequence of the CDR1, CDR2 and CDR3 regions is retained to design a synthetic immunogen. In addition, each CDR peptide portion is extended to conform to the aforementioned epitope pattern. As an effective immunogen, if the antibody produced is to be actually used, the peptide must be selected from the accessible region of the protein. The protein-prone region is exposed to the outside of the structure. Since these areas are in contact with the aqueous environment, they are often hydrophilic. Then I used GeneWorks8 Software 10 (IntelliGenetics) to create a hydrophobic map to determine the direction of extension. Chain variability or surface likelihood is also calculated using Gene Works®, and the area near the CDR is also considered a secondary parameter for peptide design (Figure 2). The high score CDR peptide obtained after the calculation was used to link the tetanus toxin "helper" sequence with an amino acid position of 830-844 (SEQ ID NO: 1). 15 Example 2: Production of anti-CD152 human antibodies Healthy blood donors were screened negative for HIV-1/2, HTLV-I/II, HCV, HbsAg and contained normal amounts of alanine transferase (ALT) blood. From the China Blood Foundation Tainan Blood Donation Center (Tainan, Taiwan). Peripheral blood mononuclear cells (PBMC) were separated by density centrifugation (400 x g) onto 20 Ficoll-Paque (Amersham Biosciences); the cells were then washed twice with PBS and collected by centrifugation at 100 x g. The resulting PBMCs were first magnetically calibrated with CD45RO MACS microbeads (Miltenyi Biotec) and separated by VarioMACS (Miltenyi Biotec). After magnetic calibration, the cells were passed through a separation column placed on a strong 1267517 permanent magnet. The matrix of the magnetic column produces a high order magnetic field. Magnetically labeled cells will stay in the column and filter out uncalibrated cells. After the magnetic field of the column is released, the cells are flushed. The proposed CD45RO+ cells were obtained by centrifugation at 1 〇〇 xg and used immediately. CD45RO+T-cells were cultured in tissue culture flasks, 5 RPMI_1640 (HyQTM, HyClone) with a density of 2×10 6 cells/ml, and supplemented with lx non-essential amino acids (Life Technologies), 10% human serum, 50 pg/ml Qingda Pharmaceutics/Kanamycin (China Chemical &amp; Pharmaceutical), 50 μΜ 2-ethylthiol and 10 pg/ml Pokeweed Splitting Promoter (PWM, Sigma). After 24 hours of culture, the cells were removed from the heart at 400 xg and removed. Finally, the supernatant was collected to prepare a CD45RO+ T-cell replacement factor, filtered through a 0.45 mm filter and stored frozen at -20 °C. The PBMC was removed by magnetic cell removal to remove the cytotoxic group to suppress the immune response in vitro. The use of colloidal supermagnetic microbeads to bind to monoclonal anti-mouse CD8 and anti-CD5 6 antibodies (Miltenyi Biotech) is as described above. Cytotoxicity 15 Cell-removed PBMC was immunized in vitro by a two-step immunoassay: primary immunization, which was cultured in medium containing 1 OnM peptide antigen for 6 days, ie TT_CDRl(ext), TT_CDR2(ext) and TT(iv) CDR3(ext) in 50 μΜ 2-ethylthiol, 10% heat deactivated human serum, 〇.〇5 ng/ml rIL2 (Calbiochem) and 25% (v/v) CD45RO+ T cell replacement factor, in 7 Primary immune cells were harvested for 20 days and centrifuged at 40% Ficoll-Paque; for secondary immune responses, 3xl07 cells were mixed with peptide antigens and fixed overnight at 5/ig/ml CD40L (CD154, Vinci-Biochem). The flask was cultured; the cells were cultured for 3-5 days in a medium containing 5% human serum, 50 μM 2-ethylthiol and 1 〇nM peptide antigen. 1267517 In vitro immunized cells were subsequently infected with Epstein-Barr virus: 107 lymphocytes were cultured for 2 hours at 37 ° C, and resuspended in 1 ml of prion-containing supernatant, which was derived from the EB virus-producing apes Cell line B95-8 (ATCC CRL 1612). The infected cells were seeded in a 96-well plate at a number of 105/well and 5 PBMCs treated with mitomycin (Kyowa Hakko Kogyo) were added as the feeder cells (104/well). The antibody-specific ELISA was performed by first expressing recombinant human CD152 (CTLA-4)-muIg fusion protein (Ancell Corporation) in 1 Mg/ml BHK cells at room temperature, 1 /xg/ml of single mouse IgG2a ( Ancell), 10 10 gg/well of bovine serum albumin (BSA, Sigma) or tetanus toxoid (ADImmune Corporation) was applied overnight to a microtiter plate. The culture supernatant was diluted to a desired concentration with a buffer of 0.5 mM sodium and 0.1% Tween-20 in 10 mM sodium ore buffer, pH 8.0. The coated titration plate was cultured with the diluted culture supernatant. After washing, the human IgG (Zymed Laboratories) was identified as a emulsified age standard, and 100 μ of the chromogenic original substrate o-phenylenediamine was added. OPD, Sigma) developed for 15 min; 30 min, 1 Μ sulfuric acid was added to stop the reaction, and the results were obtained at 490 nm absorbance. The anti-CD 152 antibody released by EB virus-infected lymphocytes was confirmed by the above solid ELIS A. The integration of the amount of specific antibody 20 produced by lymphocytes per well was performed according to the following criteria: (a) the recombinant human CD152-mulg fusion protein had an OD value at least five times the OD value of the negative control group; and (b) The reactivity index (RI) is >2, where RI = [ODcm52-muIg - OD medium control group vs. CD152-muIg] / f〇D murine igG2a_ 〇 D medium control group vs. mouse igG2a]. If the lymphocyte-containing wells were positive in the above analysis, the supernatant of 1267517 was collected and quantified and standardized by ELISA for further study. Confirmation of CDR region reactivity is the use of individual peptides for ELIS A competition. These wells were transferred by limiting dilution and cryopreserved. Example 3. The ability to alter anti-CD 152 antibodies to induce apoptosis and fine cell differentiation and compared with natural synergists. The identification of different anti-CD 152 human antibody binding sites is based on the synthesis of the peptide corresponding to the primary alkyl group. Amine-derived cellulose film (Rapp Polymere GmbH). To further evaluate the pharmacological effects of different anti-CD 152 antibodies and the preferred natural synergist CD80, cell growth and PBMC culture of human peripheral lymphocytes in vitro after plant 10 hemagglutinin stimulation were performed. Briefly, prepare a 96-well microtiter plate with a flat bottom and add 50 μM cell suspension (105 cells), 60 μM barium-containing medium (final concentration of 1.25 pg/ml, Amersham Biosciences AB), 40 μΐ autologous Plasma and 50 μΐ RPMI-1640 medium containing human anti-CD152 antibody or monomer 15 CD80-muIg fusion protein (Ancell) at a concentration ranging from 0.2 to 5 pg/ml. In the €〇80 stimulation response, 5 48/1111 goat anti-mouse 18〇2 &amp; (8〇111:1161*11 Biotechnology Associates) was added to provide a signal for cross-linking. The total volume of the culture is 200 μΐ. Incubate at 5% of 37CC (: 〇2 in a humid environment for 96 h. Add 50 μl of a gasified thymus 20 ϋ ϋ ϋ ( ( ( 306 306 (Amersham, specific activity 2) per well 20 hours before collection Ci/mol). The medium was collected with a glass fiber filter in combination with a semi-automatic multiple collector (PHD, Cambridge Technology Inc.). The cell binding [3h]-thymidine was determined by LKB liquid flash counter. In the final stage, the cell viability was measured by Trypan blue dye. The same three replicate cultures were carried out in 1267517, and the calculation was performed at the median of the three. The percentage of apoptosis was evaluated, and the cells were treated at 200xg. Centrifuge, resuspend in ice-cold 80% alcohol and mix vigorously until the final density is 1 X 106/ml. The cells are cultured at 4 ° C for at least 30 min. The alcohol-fixed cells are centrifuged with 5 and 1 ml of propidium iodide. Resuspension in (PI, Sigma) stain (PBS 0.15 M pH 7.4, 0.1% Triton X-100, 0.1 m M EDTA disodium salt, 50 /xg/ml RNase A and 50 /xg/ml PI). Store at room temperature in the dark until analysis and at 24 hours The amount of DNA in the nucleus was determined by carbonized bite staining and FACScan cytometer with Lysis II software 10 (Becton Dickinson). The decision of the dead cell was to measure the double set of DNA after propidium iodide staining by flow cytometry. Percentage. A panel of anti-CD152 human antibodies was provided to determine the function of these ligands on human T cells under PHA activation, resulting in moderate cell differentiation and apoptosis (Fig. 3A). Cross-linked CD80 induced Apoptosis was not observed to have significant cell differentiation (Fig. 3B). Similarly, antibodies induced by the CDR3-containing peptide immunogen also elicited rapid cell death rather than differentiation, thus confirming synergistic activity (Fig. 3E). The stimulatory effect of CDR1-induced human antibodies was significantly reduced, but PHA-driven cell death was not completely removed (Fig. 3C). When PHA-activated PBMC were cultured alone with CDR2-induced human antibodies, 20 to height and reproducible Sexual cell differentiation, and cell death caused by PHA can be completely removed (Fig. 3D). The driving of CD152 caused by anti-CDR2-induced cell differentiation is similar to that of 5 ng/ml IL-2 treatment. Form and cause the disappearance of typical apoptotic cell type changes (eg, cell membrane foaming and cell incompleteness and nucleation of vesicles). 1267517 The above examples are merely examples for convenience of explanation, and the claimed rights of the present invention The scope is subject to the scope of the patent application, and is not limited to the above embodiments. 5 [Simplified Schematic] Fig. 1A shows the relative positions of the homodimeric protein structure of the CD 152 receptor on the surface of T cells and three immunoglobulin CDR regions. Figure 1B is a human CD152 amino acid sequence translated into cDNA (GenBank No. L15006, NCBI Protein No. P16410). The asterisk shows the beginning of the mature 10 peptide, the intermembrane region and the intracellular region. Similar regions of CDR1, CDR2 and CDR3 are indicated by squares. Figures 2A, 2B and 2C depict humans, respectively.疏水 152 〇 〇 111 and the hydrophobicity, chain variability and surface possibility of the connected zone. Figures 2D, 2E and 2F depict the hydrophobicity, chain variability and surface likelihood of CDR2 and associated regions, respectively. 15 Figures 21, 2J and 2K depict the hydrophobicity, chain variability and surface likelihood of the CDR3 and associated regions, respectively. Box type refers to a CDR-like region whose amino acid sequence is shown below. The arrows indicate the chain structure of the predicted amino acid composition. Figure 3 shows the reaction of PHA-stimulated human PBMC cell differentiation (□) with apoptosis (). Figures 3A, 3B, 3C, 3D and 3E are PHA alone 20 stimuli (1.25, 5 and 20 gg/ml), 1.25 jitg/ml PHA with cross-linking CD80 (0.2, 1 and 5 / xg/ml), Results obtained for anti-CDR1, anti-CDR2 and anti-CDR3 antibodies (0.1, 1 and 10 gg/ml). [Explanation of main component symbols] 25 No 17 1267517 Sequence Listing &lt;110&gt; Chin, Li-Te Hsu, Shu-Ching &lt;120&gt; Human antibody production method with synergistic agent, antagonist or reverse synergist property &lt;130 〉 P7676/0600 &lt;160> &lt;170&gt;&lt;210&gt; 8 Patentln version 3.3 1 10 &lt;211&gt; 15 &lt;212> PRT &lt;213&gt;&lt;220> tetanus toxin &lt;221&gt; peptide 15 &lt;;222&gt;&lt;300&gt; (1)..(15) &lt;301&gt; Stephane Demotz, Antonio Lanzavecchia, Ulrich

Eisel, Heiner Niemann, Christian Widmann, and

Giampietro Corradin 20 &lt;302&gt; describes various DR-restricted T cell epitopes &lt;306> 394-402 &lt;307&gt; 1989-01-15 &lt;313&gt;&lt;400&gt;(1)&quot;(15) 1 25 Gin Tyr lie Lys Ala Asn Ser Lys Phe lie Gly lie Thr Glu Leu 15 10 15 &lt;210&gt; 2 18 1267517 &lt;211> 15 &lt;212&gt; PRT &lt;213> Modern Man &lt;220&gt; 5 &lt;221> Win Peptide &lt;222&gt;(1)&quot;(15)&lt;400&gt; 2 Glu Tyr Ala Ser Pro 1 5 10 &lt;210> 3 &lt;211> 15 &lt;212> PRT &lt;213> Modern Man &lt;220> 15 &lt ;221> Peptide &lt;222&gt;(1)&quot;(15)&lt;400&gt; 3 Ala Ala Thr Tyr Met 1 5 20 &lt;210&gt; 4 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Modern Man&lt;220&gt; 25 &lt;221> peptide &lt;222&gt; (1)..(15) &lt;400> 4 10 15 ❿ 10 15 19 1267517

Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly lie Gly 1 5 10 15 &lt;210&gt; 5 &lt;211&gt; 30

5 &lt;212&gt; PRT &lt;213&gt; Synthetic &lt;220&gt;&lt;223&gt; The CDR peptide was synthesized and used to prepare a compatible epitope to link the sequence derived from tetanus virus.

10 &lt;220〉 &lt;221&gt; peptide &lt;222> (1)..(30) &lt;400&gt; 5

Gin Tyr lie Lys Ala Asn Ser Lys Phe lie Gly lie Thr Glu Leu Glu 15 U 5 10 15

Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr Val 20 25 30

&lt;210〉 6 &lt;211&gt; 30

20 &lt;212&gt; PRT &lt;213&gt; Synthetic &lt;220&gt;&lt;223&gt; The CDR peptide was synthesized and used to prepare a compatible epitope to link the sequence derived from tetanus virus. 25 &lt;220&gt;&lt;221&gt; peptide &lt;222&gt; (1)..(30) 20 1267517 &lt;400&gt; 6

Gin Tyr lie Lys Ala Asn Ser Lys Phe lie Gly lie Thr Glu Leu 1 5 10 15

Ala Ala Thr Tyr Met Met Gly Asn Glu Leu 5 Thr Phe Leu Asp Asp 20 25 30 &lt;210&gt; 7 &lt;211&gt; 30 &lt;212&gt; PRT 10 &lt;213&gt;&lt;220&gt; Synthetic &lt;223&gt; Synthesis The CDR peptide is used to prepare a compatible epitope to link the sequence derived from the tetanus virus 'fTTff. &lt;220&gt; 15 &lt;221> peptide &lt;222&gt;&lt;400&gt;(1)&quot;(30) 7

Gin Tyr lie Lys Ala Asn Ser Lys Phe lie Gly lie Thr Glu Leu 1 5 10 15 20 Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly lie Gly 20 25 30 &lt;210&gt;&lt;211&gt; 8 223 &lt;212&gt; PRT 25 &lt;213&gt;&lt;220> Modern Man &lt;221&gt; Peptide &lt;222&gt;&lt;400&gt;(1)&quot;(223) 8 21 1267517

Met Ala Cys Leu Gly Phe Gin Arg His Lys Ala Gin Leu Asn Leu 1 5 10 15

Ala Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe 20 25 30 5 lie Pro Val Phe Cys Lys Ala Met His Val Ala Gin Pro Ala Val Val 35 40 45

Leu Ala Ser Ser Arg Gly lie Ala Ser Phe Val Cys Glu Tyr Ala 50 55 60

Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gin 10 65 70 75

Ala Asp Ser Gin Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met 80 85 90

Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser He Cys Thr Gly Thr 95 100 105 15 Ser Ser Gly Asn Gin Val Asn Leu Thr lie Gin Gly Leu Arg Ala 110 115 120

Met Asp Thr Gly Leu Tyr lie Cys Lys Val Glu Leu Met Tyr Pro 125 130 135

Pro Pro Tyr Tyr Leu Gly lie Gly Asn Gly Thr Gin lie Tyr Val 20 140 145 150

Lie Asp Pro Glu Pro Cys Pro Asp Ser Asp Phe Leu Leu Trp lie 155 160 165

Leu Ala Ala Val Ser Ser Gly Leu Phe Phe Tyr Ser Phe Leu Leu 170 175 180 25 Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys Arg Ser Pro Leu 185 190 195

Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu Pro Glu Cys 200 205 210

Glu Lys Gin Phe Gin Pro Tyr Phe lie Pro lie Asn 30 215 220 22

Claims (1)

1267517 public Patent application Van VA 1 A method for producing a human antibody, wherein the antibody has the characteristics of a synergist, an antagonist and/or a reverse synergist to the receptor, and comprises the following steps: 5 (a) using the receptor The bioinformatics characteristic of the external position of each cell defines a peptide fragment external to the individual cell of the receptor; (b) coupling the peptide fragment to a helper cell epitope, the resulting peptide contains at least a T helper cell epitope; (c) preparing an immunogen having the amino acid sequence of the coupled fragment; 10 (d) stimulating the human lymphocyte in vitro with the immunogen; (e) identifying and screening to produce the recognizable receptor The human lymphocyte of the antibody; and (0) the method of the invention, wherein the amino acid sequence of the peptide fragment having the same as the extracellular 15 ° 卩 has the same SEq ID N 〇s: 2 7 at least 75% of the amino acid sequence identity. 3. The method of claim 3, wherein the subject human CD152. 20. The method of claim 1, wherein the method of the present invention comprises the method of the amino acid sequence shown in SEQ ID NO: 1 The bioinformatics characteristics of the site are hydrophobic, variability or surface. An antibody that binds to a coupled fragment, wherein the coupled fragment has a 23 1267517-t helper epitope and a peptide fragment that corresponds to the outer position of each cell of the receptor. 7. A method for identifying the pharmacological efficacy of an in vitro synergist, antagonist and/or reverse synergist antibody as a receptor, comprising the steps of: 5 (4) providing a human lymphocyte, a cleavage, and a ligand And optionally a plurality of activators; (8) adding the splitting promoter or the plurality of activators to the individual containers and increasing the ligand concentration to form a mixture; (4) providing the first-control group medium containing the - splitting promoter, and a second control group medium for a natural synergist of the receptor; (4) cultivating human lymphocytes in the _ control group medium, the second control group medium, and the plurality of ligands and a cleavage promoter; 15 20 (4) determining the degree of apoptosis and/or replication activation of human lymphocyte programmers in each medium and each mixture; and (f) using the mixture of the ligands, the programmed apoptosis and/or replication of increased mobility The degree of activation determines the effect of the ligand on the receptor. 8. As described in item 7 of the patent application, ^ ^ m method, wherein the human lymphoblast, peripheral blood mononuclear cells. 9. The method of claim 5, wherein the splitting promotes d or the buddha activator is hemagglutination, ^^v „ 刀 丑 血 blood cell agglutination A, American Pokeweed as a crack promoter, 12-decanoic acid phorbol-13-acetate and superantigen are used as early or mixed. 10· A method for preparing human antibodies, 兮&gt; Γ 'The antibody recognizes the physiological receptor of 24 1267517 and includes the following steps: (a) providing a lymphocyte derived from a resting human donor; (b) synthesizing a synthetic antigen based on bioinformatics characteristics Inoculation of the lymphocytes in vitro; 5 (0 addition of Epstein-Barr virus to the immune lymphocytes; (d) identification of cells infected with Epstein-Barr virus to produce the recognizable antibody; and (e) screening step (d) Pharmacological functions of the antibodies produced. 25