CN101168067A - Method for decreasing immune cell surface antigenic sites immunogenicity and use - Google Patents

Abstract

The invention provides a method of reducing immunogenicity of antigen site of the surface of immune cells. 188Re (Rhenium) is marked on monoclonal antibody of 2E9/13, and is dialyzed, purified, filtered and finally prepared to 188Re-2E9/13F(ab)2 which is realized by being utilized on reaction model of external unidirectional mixed lymphocyte. The invention combines biology and nuclear medicine, utilizes the technology of introducing 2E9/13F (ab)2 fragments of monoclonal antibody resisting MHC-II marked with 188Re to externally provide liver to regionally infusing, thereby solving the problem of permanently reducing immunogenicity of homologous allogenous MHCII-type antigen. The method has the advantages of safety, small influence on recipients, full functions and strong practicability and validity.

Description

A kind of method and application that reduces immune cell surface antigenic sites immunogenicity

Technical field

The present invention relates to biotechnology, relate generally to and reduce the immunogenic method of immunocyte surface MHC-II antigen site, application that can be in rejection.

Background technology

By tissue, the transplanting of cell or organ, replacing the tissue of loss of function, cell and organ is one of important means of modern medicine treatment.Provide the individuality of graft to be called donor, and the individuality of accepting graft is called the receptor.According to donor and receptor's combination, can have following three kinds to transplant type: autotransplantation is meant giving the receptor from the tissue of transplanting receptor itself or organ transplantation.Syngraft is meant the transplanting between the identical or closely similar individuality of hereditary constitution.Allotransplantation is meant the transplanting between the same animal inbred genetic structure Different Individual.Xenotransplantation is meant the transplanting between the individuality of different animals kind, as the transplanting between pig and the people.Rejection can not take place in preceding two kinds transplanting.Current, no matter be or domestic concrete truth from the world, allotransplantation (allogenic transplanting) be main clinically also be feasible implantation method.Studies show that, in allotransplantation, the receptor is to transplant successful major obstacle to the repulsion of graft, and the dominant mechanism of generation rejection has also obtained the common recognition of educational circles, promptly cell-mediated by receptor T, at the immunne response of allogenic antigen.

At present, the patient who accepts organ transplantation increases year by year, but the incidence rate of the acute rejection of organ is very high after transplanting, and has influenced the success rate of transplanting and patient's survival rate.The main method of existing anti-immunological rejection still depends on various immunosuppressant, not only cost an arm and a leg, and because immunosuppressive action infects, the chance of occurrence of tumor also obviously increases.In addition, by relying on In vitro culture, freezing, outside X-ray therapy, means such as transplantings reduce the method that the graft immunogenicity prolongs the transplant organ life span and have obtained certain effect thereby animal is adopted, but there is no the using value of reality in organ transplantation, for example, outer X-ray therapy makes for the inside and outside line of departure of accepting of organ often inhomogeneous, measure too small falling flat, and measures excessively then may cause extensive infringement to the confession organ.Therefore, up to now, do not find the strong and definite effective means of clinical usability as yet.

Find that when carrying out tissue transplantation between different genera or other individuality of not homology of the same race, rejection can occur, its essence is the inductive a kind of immunne response of the allogenic antigen of cell surface 20 century.This individual specificity's of representative allogenic antigen claims transplantation antigen or histocompatibility antigen, can cause that wherein the antigen of rejection claims major histocompatibility antigen by force and rapidly, causes that the antigen of more weak rejection claims minor histocompatibility antigen.Major histocompatibility antigen is the antigen systems of a complexity, the gene of this system of encoding is positioned on the same chromosome segment, be one group of closely linked gene group, be called major histocompatibility complex (major histocompatibility complex, MHC).Its MHC of the animal of different genera has different names, claims H-2 complex as the MHC of mice, and the MHC of pig claims SLA complex, and people's MHC claims HLA complex etc.Mhc gene comprises classical MHC-I and MHC-II gene, synthetic respectively MHC-I and MHC-II quasi-molecule antigen, and it has characteristics separately at molecular structure on combination distribution and the function.The I quasi-molecule is made up of heavy chain and β m chain, is distributed in all nucleated cell surfaces, mainly discerns and offer the endogenous antigen peptide, with accessory receptor CD8 ⁺The cell combination provides constraints to the identification of CTL cell.The II quasi-molecule is made up of a chain and β chain, only be expressed in the various cell surfaces in the lymphoid tissue, (comprise the B cell as full-time antigen presenting cell, dendritic cell, macrophage etc.), the exogenous antigen peptide is mainly discerned and offered to thymic epithelial cell and activated T cell etc., with accessory receptor CD4 ⁺In conjunction with, the identification of Th is provided constraints.

The MHC molecular antigen participates in the cell-mediated immunologic rejection response pathway of T two: a direct identification approach, the complete allotype MHC molecule of being offered by donor APC cell on the receptor T cell Direct Recognition graft and the complex of peptide are the main causes of acute grafing rejection.Two indirect identification approach, receptor T cell recognition by receptor APC offer with processed after the allotype MHC molecular peptide of the donor that comes off from graft and the complex of receptor MHC molecule, relevant with chronic rejection.

Zoopery confirms: the antigen-MHC-II quasi-molecule that causes the acute grafing immunological rejection to take place mainly is from the passenger leukocyte in the graft, it wherein mainly is sophisticated dendritic cell, sophisticated lymphocyte and mononuclear phagocyte, T lymphocytic cell surface in vascular endothelial cell surface and part also has expression simultaneously, before transplanting, the cell that carries MHC-II is removed in the experiment, the transplant rejection of MHC-I mispairing is slowed down to be alleviated, even do not take place, can rebuild rejection and re-enter the donor's cells who carries the MHC-II quasi-molecule, its mechanism is that the passenger leukocyte that carries allotype MHC-II molecule can activate receptor CD4 ⁺The Th cell, activated T h emiocytosis cytokine, the CD8 that auxiliary allotype is replied ⁺The CTL cell is fully grown and is broken up, thereby excites rejection.As before transplanting, removing passenger leukocyte, the CD8 that replys of allotype then ⁺CTL is difficult to amplification and differentiation, and rejection just weakens, and therefore, using anti-MHC-II monoclonal antibody can combine by the directed MHC-II antigen with the target cell surface, reduces the immunogenicity of donor, effectively reduces the chance of post-transplantation generation rejection.

¹⁸⁸Re is ⁹⁹The congeners of Tc, ⁹⁹The present application of Tc is comparatively general, and ¹⁸⁸Re is as a kind of emerging nucleic, with ⁹⁹Tc compares has better nuclear physics character, the β of emission 2.12MeV ^-Ray, the 155MeV gamma-rays, 16.9 hours half-life, can in body, get rid of rapidly, can not accumulate in vivo.Maximum range in the tissue is 0.97cm, can effectively kill the passenger leukocyte that combines with it, can not cause large-scale damage to other histiocyte again, has good effect and safety.

Summary of the invention

The purpose of this invention is to provide a kind of immunocyte surface MHC-II antigen site immunogenicity that promptly reduces, reduce its method again the effect of stimulating proliferation of the T cell in the reactive cell.The inventive method is passed through will ¹⁸⁸Re is tagged on the 2E9/13 monoclonal antibody through dialysis, and purification concentrates, and is prepared into after the filtration ¹⁸⁸Re-2E9/13F (ab) ₂, be applied to realize that concrete steps are in the external unidirectional mixed lymphocyte reaction model:

(1) by extracting F (ab) wherein behind the papain hydrolysis 2E9/13 monoclonal antibody ₂Fragment, and through filtering, purification concentrates, and places-20 ℃ of refrigerators to preserve;

(2) from ¹⁸⁸W- ¹⁸⁸Obtain in the Re generator ¹⁸⁸ReO ₄ ^-Eluent will by direct preparation method ¹⁸⁸Re is marked on F (ab) ₂On the fragment, be prepared into ¹⁸⁸Re-2E9/13F (ab) ₂Fragment.

(3) establish the normal saline matched group respectively, monoclonal antibody group and isotopic labeling antibody group, every group of laboratory animal be some carries out the liver transplantation experiment, taking off the external normal saline that pours into respectively in back for liver, monoclonal antibody and isotopic labeling monoclonal antibody be a large amount of normal saline flushings after 30 minutes, wash quiet potting compound, implant then in the laboratory animal recipient's body;

(4) observe each hepatic and renal function index of organizing of different time respectively, the liver organization pathological section is observed in the variation of serum cytokines level at last;

(5) result shows: warp ¹⁸⁸Re-2E9/13F (ab) ₂The probability that early stage acute grafing rejection takes place for liver in recipient's body behind the fragment ex vivo perfusion and degree and simple antibody with normal saline perfusion compared more significantly reduction.

Another object of the present invention is that this method is used in external unidirectional mixed lymphocyte reaction model.The inventive method combines biology and two kinds of methods of nuclear medicine, takes the lead in having introduced ¹⁸⁸The monoclonal antibody 2E9/13F (ab) of the anti-MHC-II molecule of Re labelling ₂Fragment is successfully applied in the zoopery first.

Mechanism is by 2E9/13F (ab) ₂Fragment sealing immunocyte surface MHC-II antigen site reduces its immunogenic while, utilizes ¹⁸⁸This antigenic immunocyte is expressed in the ray deactivation of Re, contains the secretion again of its MHC-II antigen and collaborative stimulating factor, and the immunogenicity of nonvolatil downward modulation passenger leukocyte further reduces its effect of stimulating proliferation to the T cell in the reactive cell.

The present invention has following characteristics: the present invention collects biology and two kinds of methods of nuclear medicine are one, has introduced in zoopery liver transplantation of the same race ¹⁸⁸The anti-MHC-II monoclonal antibody 2E9/13F (ab) of Re labelling ₂Fragment is in the external regional perfusion technique of liver that supplies, solved permanent downward modulation allogeneic mhc class ii immunogenicity of antigens problem, this perfusion technique method feasibility is strong, with direct perfusion in vivo or after transplanting again method such as perfusion compare, have saferly, the receptor is influenced less, act on advantages such as more abundant, the use of this nucleic antibody and method for filling has stronger practicality and effectiveness than other pure biological means that prolongs the life span of graft.The effect of verified this method of zoopery in liver transplantation also will have important value for clinical application and vast potential for future development aspect other filed of organ transplantation and the mankind's the clinical practice.

Description of drawings

Fig. 1 is before matched group and the experimental group art and the postoperative total serum bilirubin changes broken line graph.

Fig. 2 is before matched group and the experimental group art and postoperative serum glutamic oxalacetic transaminase level changes broken line graph.

The specific embodiment

The present invention is further described by embodiment.

Embodiment 1

1. by extracting F (ab) wherein behind the papain hydrolysis 2E9/13 monoclonal antibody ₂Fragment, and through filtering, purification concentrates, and places-20 ℃ of refrigerators to preserve;

From ¹⁸⁸W- ¹⁸⁸Obtain in the Re generator ¹⁸⁸ReO ₄ ^-Eluent will by direct preparation method ¹⁸⁸Re is marked on F (ab) ₂On the fragment, be prepared into ¹⁸⁸Re-2E9/13F (ab) ₂Fragment.

3. carry out the allograft experiment: establish the normal saline matched group respectively, monoclonal antibody group and isotopic labeling antibody group, every group of laboratory animal be some carries out the liver transplantation experiment, taking off the external normal saline that pours into respectively in back for liver, monoclonal antibody and isotopic labeling monoclonal antibody be a large amount of normal saline flushings after 30 minutes, wash quiet potting compound, implant then in the laboratory animal recipient's body;

4. observe the hepatic and renal function index of each group of different time respectively, the liver organization pathological section is observed in the variation of serum cytokines level at last;

5. sum up above each result of experiment, drawn to draw a conclusion: warp ¹⁸⁸Re-2E9/13F (ab) ₂The probability that early stage acute grafing rejection takes place for liver in recipient's body behind the fragment ex vivo perfusion and degree and simple antibody with normal saline perfusion compared more significantly reduction.

Embodiment 2

1. Antibody Preparation:

(1) by extracting F (ab) wherein behind the papain hydrolysis 2E9/13 monoclonal antibody ₂Fragment, and through filtering, purification concentrates, and places-20 ℃ of refrigerators to preserve.

(2) Na ¹⁸⁸ReO ₄Reduction:

Get citric acid-tartaric acid mixed solution (pH4.4, citric acid 0.015M, tartaric acid 0.15M) 0.5mL, add the SnCl that newly joins ₂Solution (SnCl ₂2H ₂O20g/L), add newly from ¹⁸⁸W- ¹⁸⁸The Na that drip washing is got off on the Re generator ¹⁸⁸ReO ₄Normal saline solution 0.3mL (～1.3mCi, 50,000kBq), under room temperature the reaction 40min promptly ¹⁸⁸The Re reducing solution.

(3) reduction of 2E9/13 antibody:

Get 2E9/13F (ab) 2 solution (1mg/mL) 0.3mL, (pH5.2,40mgVc/mL) 0.2mL react 30min to the PBS solution of adding ascorbic acid under room temperature, promptly get 2E9/13F (ab) 2 antibody also

Stock solution.

2.2E9/13F (ab) 2 antibody ¹⁸⁸The Re labelling:

Merge in the above-mentioned steps 1,2 ¹⁸⁸Re reducing solution and 2E9/13F (ab) 2 antibody reducing solutions react 2h, promptly under room temperature ¹⁸⁸Re labelling 2E9/13F (ab) 2 monoclonal antibody solution, the radioactivity of activity meter (FJ-391-A2 type) measurement markers solution.

3. the mensuration of radiochemical purity:

Adopt paper chromatography, get ¹⁸⁸Re labelling 2E9/13F (ab) 2 monoclonal antibody solution are an amount of, drop on No. 1 filter paper of Xinhua, and be developing solvent with the normal saline, launch 10cm according to ascending method.Taking-up is dried, in the increased radioactivity that detects under the thin layer chromatogram scanner (BioscanAR-2000) on the chromatographic paper. ¹⁸⁸The Rf=0.0 of Re labelling 2E9/13F (ab) 2 monoclonal antibodies, datum point place radioactivity accounts for the percentage ratio of radioactivity total amount, is ¹⁸⁸The radiochemical purity of Re labelling 2E9/13F (ab) 2 monoclonal antibodies.

4. labelling result:

Three batches have been prepared altogether ¹⁸⁸Re labelling 2E9/13F (ab) 2 monoclonal antibodies, its radioactivity and radiochemical purity (wherein radioactivity all is calculated to back 17 hours of preparation) as shown in the table:

Preparation time	Radioactivity	Radiochemical purity
			06.6.12	13,200kBq	97.4％
06.6.19	15,400kBq	96.9％
			06.6.26	15,700kBq	98.3％

Embodiment 3 young Hepar Sus domestica transplantation experiments:

Cut and repair for liver: the big otch of abdominal part stringer advances abdomen, and all ligaments of free liver are separated and cut common bile duct, Hepatic artery and portal vein, insert intrusion pipe to main portal vein through the splenic vein, the ventral aorta intrusion pipe.The whole body heparinization is through portal vein and ventral aorta 4 ℃ of Ru Suanlingeshi of perfusion and 4 ℃ of UW liquid, through common bile duct broken ends of fractured bone intubate flushing biliary tract.The continuous icing bits of liver surface are to help the liver cooling in the filling process, and to the limpid depletion of blood of effluent, the liver cooling after the surface is even yellow, from all blood vessels of disconnected liver, cuts for liver, puts in 4 ℃ of UW liquid and repairs.Prune on the liver and infrahepatic vena cava, the excision gallbladder, remove hepatic portal portion, portal vein and Hepatic artery connective tissue and lymph node on every side, after each pipeline has been repaired, water filling is checked, after ligation or suture repair are wanted in leakage place, will be placed 4 ℃ of UW liquid to preserve for liver, this group does not have hot ischemia for liver, and the cold holding time was controlled in 3 hours.

Matched group for liver external through portal vein and hepatic arterial infusion Ru Suanlingeshi 500ml; Simple antibody group: 500mI Ru Suanlingeshi adds the anti-MHC-II monoclonal antibody of 300ug (2E9/13 monoclonal antibody F (ab) ₂Fragment); Isotopic labeling antibody group: 500ml Ru Suanlingeshi add the anti-MHC-II monoclonal antibody of isotope-labeled 300ug ( ¹⁸⁸Re-2E9/13 monoclonal antibody F (ab) ₂Fragment), and folder closes for liver all efferent tracts, keeps perfusion half an hour separately.Three groups of operating times, no liver phase, cold ischemia time etc. all do not have significant difference.

Receptor operation abdominal part center longitudinal incision, bladder stoma in advance, all ligaments of free then liver and blood vessel, from disconnected Hepatic artery, with vascular occlusion clamp respectively on blocking door vein, the liver and the liver postcava be close to liver and on disconnected portal vein, liver, reach the liver postcava and shift out liver, repair each ends of vessels, to insert the receptor abdominal cavity for the liver original position, sew up continuously with the 5-0Plolene needlework, the identical continuously portal vein of end to end anastomosis suprahepatic vena cava, 6-0Plolene needlework recovers liver blood stream, with the identical continuously liver postcava of 5-0Plolene needlework, open liver postcava blood flow.When coincideing suprahepatic vena cava from 4 ℃ of Ru Suanlingeshi of portal vein perfusion in case liver heat up.With the identical Hepatic artery of 7-0Plolene needlework, common bile duct is inserted thin tube for transfusion and is fixed in stomach wall, drains external.Flushing is closed abdomen behind the abdominal cavity.

The operation beginning, the no liver phase, new liver period and art finish, postoperative venous blood samples 5ml 1-9 days every days, survey glutamic oxaloacetic transaminase, GOT (AST), glutamate transaminase (ALT), total bilirubin (total bilirubin), electrolyte, inosine, blood urea nitrogen.

Experimental result is referring to Fig. 1 and Fig. 2.Among the figure: included 4 refer to the operation beginning respectively before the art, and the no liver phase, new liver period and art finish the fourth phase, and postoperative is represented postoperative 1-9 days, matched group: represent the normal saline matched group; Experimental group 1: represent simple antibody group; Experimental group 2: represent isotopic labeling monoclonal antibody group.

Sum up above each result of experiment, drawn to draw a conclusion: in zoopery, warp ¹⁸⁸Re-2E9/13F (ab) ₂The probability that early stage acute grafing rejection takes place for liver in receptor's animal body behind the fragment ex vivo perfusion and degree are poured into normal saline with simple antibody and have been compared tangible reduction, this just provides important evidence for further clinical research, lays a solid foundation for adopt this novel technical method in human body.

Embodiment 4 cultivates in (MLR) model at the external unidirectional mixed lymphocytes of construction and uses

Get embodiment 1 preparation ¹⁸⁸Re labelling 2E9/13F (ab) 2 monoclonal antibodies: 1. establish positive controls A (irritation cell+reacting cells after normal saline is handled) respectively, monoclonal antibody group B (irritation cell+reacting cells after monoclonal antibody is handled), isotopic labeling monoclonal antibody group C (irritation cell+reacting cells after the isotopic labeling monoclonal antibody is handled) and negative control group D (reacting cells), each group is made as 48 again, 72,96,120 hours (H) differential responses time, do the MTT test behind the harvesting; 2. grouping is as above established 72 and 120 hours differential responses time, is RT-PCR behind the harvesting, and is analyzed for every group.

The result:

1.MTT result

Group	24H		48H		72H		96H
										A490	Suppression ratio (%)	A490	Suppression ratio (%)	A490	Suppression ratio (%)	A490	Suppression ratio (%)
A B C D	0.49±0.02 0.508±0.1 0.427±0.06 0.486±0.08		0.512±0.03 0.52±0.05 0.46±0.06* 0.48±0.03	10.2	0.83±0.18 0.64±0.09* 0.482±0.11** 0.477±0.05	22.9 41.9	1.358±0.46 0.829±0.12** 0.441±0.21** 0.457±0.21	39 67.5

Annotate: positive controls A, monoclonal antibody group B, isotopic labeling monoclonal antibody group C, negative control group D, each group is compared with positive controls, ^*P＜0.05, ^*P＜0.01

2.RT-PCR the result is referring to Fig. 3, wherein A is IL-2, band length 337; B is IL-10, band length 526; C is TNF-α, band length 385; D is IFN-γ, band length 410; Band 1 negative matched group (reacting cells); Band 2 is antibody group (irritation cell+reacting cells after the antibody treatment); Band 3 is isotopic labeling antibody group (irritation cell+reacting cells after the isotopic labeling antibody treatment); Band 4 positive matched groups (normal saline is handled back irritation cell+reacting cells); Band M is maker; The upper strata light belt is a genes of interest; Lower floor's light belt is β-actin gene.

Claims (2)

1. method that reduces immune cell surface antigenic sites immunogenicity will be by will ¹⁸⁸Re is tagged on the 2E9/13 monoclonal antibody through dialysis, and purification concentrates, and is prepared into after the filtration ¹⁸⁸Re-2E9/13F (ab) ₂, be applied to realize that concrete steps are in the external unidirectional mixed lymphocyte reaction model:

(1) by extracting F (ab) wherein behind the papain hydrolysis 2E9/13 monoclonal antibody ₂Fragment, and through filtering, purification concentrates, and places-20 ℃ of refrigerators to preserve;

(2) from ¹⁸⁸W- ¹⁸⁸Obtain in the Re generator ¹⁸⁸ReO ₄ ^-Eluent will by direct preparation method ¹⁸⁸Re is marked on F (ab) ₂On the fragment, be prepared into ¹⁸⁸Re-2E9/13F (ab) ₂Fragment.

(3) establish the normal saline matched group respectively, monoclonal antibody group and isotopic labeling antibody group, every group of laboratory animal be some carries out the liver transplantation experiment, taking off the external normal saline that pours into respectively in back for liver, monoclonal antibody and isotopic labeling monoclonal antibody be a large amount of normal saline flushings after 30 minutes, wash quiet potting compound, implant then in the laboratory animal recipient's body;

(4) observe each hepatic and renal function index of organizing of different time respectively, the liver organization pathological section is observed in the variation of serum cytokines level at last;

(5) result shows: warp ¹⁸⁸Re-2E9/13F (ab) ₂The probability that early stage acute grafing rejection takes place for liver in recipient's body behind the fragment ex vivo perfusion and degree and simple antibody with normal saline perfusion compared more significantly reduction.

2. a kind of application of method in external unidirectional mixed lymphocyte reaction model that reduces immune cell surface antigenic sites immunogenicity according to claim 1.

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