CN101856497A - Conjugate of hapten and amino-containing carrier and preparation method and usage thereof - Google Patents
Conjugate of hapten and amino-containing carrier and preparation method and usage thereof Download PDFInfo
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- CN101856497A CN101856497A CN200910049174A CN200910049174A CN101856497A CN 101856497 A CN101856497 A CN 101856497A CN 200910049174 A CN200910049174 A CN 200910049174A CN 200910049174 A CN200910049174 A CN 200910049174A CN 101856497 A CN101856497 A CN 101856497A
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Abstract
The invention belongs to the field of drug synthesis, and relates to a combined conjugate of a hapten and an amino-containing carrier, and a preparation method as well as the usage thereof. In the invention, the combined conjugate of the hapton and the amino-containing carrier is obtained via Sanger's reaction. A signal can be amplified during micro determination by the immunity of the hapten and the function of the carrier which are combined with the monoclonal antibody for the hapten, and the sensitivity of determination is obviously improved. Amino-containing dinitrobenzol structure is taken as a war head with a killing function when inducing cellular immunity in vivo, is combined with the carrier with a targeting function, has peculiar discrimination on cancer and chronic infection, locally induces multiple immunity activating factors at targeting lesion parts, improves local immunity micro environment, and treats cancer or infection by strengthening immunization. The conjugate can be used for micro determination of immunization and the preparation of drug for preventing and curing tumor.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, relate to a kind of hapten and contain conjugate of amino carrier and its production and use.
Background technology
Known hapten is an imperfection unicity antigen, body skin is put on the skin outward, or animal can be conjugated protein after oral, after 7-10 days, induce intensive cell immune response in vivo, hapten such as dinitrofluorobenzene and dinitrofluorobenzene all can not generate in vivo naturally, in all biologies of nature, do not contain the dinitro benzene structure in the proteinic amino acid residue, unless artificial labelling is arranged yet.There is research to disclose and passes through Sanger ' s reaction, i.e. hydrogen on the dinitro benzene of DNFB or the DNCB displacement amino, under the alkalescence condition, protein reaches with the DNFB mixing and combines.This method is a large amount of covalent bond labellings to the antibody that detects usefulness or detects on the antigen of usefulness, utilizes at the dinitro benzene monoclonal antibody, produces the signal amplification of immunoassay, and with after targeting vector combines, induce in body, drive, reinforced immunological provides chemical fundamentals.At present, extensively use the mensuration of micro-antigenicity substance at the monoclonal antibody of single antigenic determinat, though use the method for immunity of monoclonal antibody or antibody can reach the nanogram level at present, but still can not satisfy the needs of clinical and scientific research, seek more responsive and special method is the tireless pursuit of research worker always.Over past ten years, have research with the targeting of monoclonal antibody to tumor as auxiliary treatment, as anti-HER-2 monoclonal antibody He Saiding (Herceptin) treatment mastocarcinoma, women's head-ornaments portion cancer, anti-CD22 monoclonal antibody Mabthera (Rituximab) treatment B cell lymphoma, and confirmed to have certain curative effect.But practice shows, uses the inductive targeting immunization of monoclonal antibody institute limited generally merely, far can not reach the control chronic infection, especially prevents and treats malignant tumor.Seek a kind ofly can bring into play bigger immunization at lesions position, prevention effect is good, and the medicine that can satisfy prevention and treatment needs is the focus of attention of this area research worker always.
Prior art related to the present invention has following list of references:
List of references:
[1]. Yang Guoliang Wang Xiasheng edits the 1st edition the 1st printing May in 1996 of modern dermatological
[2].Maja?Aleksic，Camilla?K，David?A?Basketter?et?al.Investigating?protein?haptenation?mechanisms?of?skin?sensitisers?using?human?serum?albumin?as?a?modal?protein.Toxicology?in?vitro.2007，21，723-73
[3].Bernard?Metz，Gideon?F.A.Kerstent，Peter?Hoogerhout?et?al.Identification?of?formaldehyde-induced?modifications?of?protein.The?journal?of?biological?chemistry.2003，279(8)：6235-6243
[4]. the reorganization nuclear antigen indirect ELISA method of detection avian influenza virus antibodies such as the luxuriant Xiao's Hu Sishun transport power of Wu Ren set up journal of animal science and veterinary medicine 2006,37 (10): 1067-72
[5]. Wang Xian thanks to the progress biology magazine 2008,25 (1) of HER_2_neu gene in breast carcinoma: 5-8 such as little strong Cao Liang
[6]. the flat Liu Hui bevacizumab of Sun Tingting Xu Jiang influences PLA's medical journal 2008,33 (12): 1438-1440 to drug resistance nasopharyngeal carcinoma cell apoptosis
[7]. the pretty monoclonal antibody related application of Wang Lei warrior Hua Wangsong is analyzed Chinese preventive medicine magazine 2006,7 (6): 569-570
[8]. fight progress China tumor 2008,17 (11): 942 of the molecular targeted medicine of refined monarch of Liu Duanqi
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of hapten and the conjugate that contains amino carrier are provided.
Hapten of the present invention has the structure of formula (I) with the conjugate that contains amino carrier.
Described structure comprises its isomers;
Described hapten is dinitrofluorobenzene (DNFB) or dinitrochlorobenzene (DNCB);
Describedly contain amino carrier, it can be the material of monoclonal antibody or antibody or antigen or immune epitope or specific bond immunoglobulin, as SP (being SPA), and other have the bonded material of targeting, as hepatocarcinoma is had higher affinity, manually combine the iodate grease etc. of ethylaminoethanol (as cross structure).
Described hapten is attached to hapten dinitro benzene structure on the immunizing antigen of chronic infection pathogen with the conjugate that contains amino carrier, induces thereby reach, and reinforced immunological is prevented curing oncoma, the chronic infection purpose.
Among the present invention, described hapten DNFB or DNCB combine by the amino on the carrier with described carrier, and DNFB is under the alkalescence condition, and dinitro benzene is partly replaced the hydrogen on the amino, combines with amino covalence.
Further purpose of the present invention provides described hapten and the preparation method that contains the conjugate of amino carrier.
The present invention our experiments show that within the specific limits, the combination of DNFB is to the not influence of antibody titer of HBIG, and immunoglobulin (antibody) antibody can be used as the effective carrier of hapten dinitrofluorobenzene immunity targeted therapy.The present invention adopts Sanger ' s reaction, and hapten DNFB wherein or DNCB are by the amino on the carrier, and DNFB is under the alkalescence condition, and dinitro benzene is partly replaced the hydrogen on the amino, combines the conjugate that makes hapten and contain amino carrier with amino covalence.
Carrier of the present invention can be antibody or the antigen that is used for immunoassay, in conjunction with the monoclonal antibody that utilizes at single antigen (as DNFB), measured signal is amplified; The recurrence interval recycles, and can make the measured signal amplification; Be connected relevant quantification display packing, the sensitivity of the targeting thing (as antigenicity, the antibody material) that detects micro-targeting vector is improved.
Among the present invention, each carrier is determined by the free ammonia radix that carrier contained in conjunction with the hapten molecule number, carrier must not influence the targeting combined function of carrier when maximum is in conjunction with hapten molecule, this maximum effectively is the biggest factor of the immune amplification of signal of decision in conjunction with number.
Further purpose of the present invention provides described above material in trace detection and anti-curing oncoma, the purposes of chronic infection aspect.
The present invention utilizes hapten within the specific limits, in albumen, antigen or an antibody in conjunction with the constant characteristic of its immunity behind the 2-200 hapten DNFB molecule, and measure the method that can improve measured signal with the monoclonal antibody of anti-DNFB, provide a kind of immunoassay (as ELISA) are obviously improved sensitivity 1-200 method and diagnosis reagent doubly.
Among the present invention, hapten dinitro benzene structure is covalently bound to targeting antibodies, antigen or monoclonal antibody, or tumor antigen, or the tumor antigen epi-position, or the antigen of pathogen infection, or the pathogen antigen epi-position, or the material of specific bond immunoglobulin, as SP (SPA), tumor high-affinity albumen or small peptide reach in the material that contains amino in other structures with guiding or targeting and produce couplet, can bring into play the targeted therapy effect of hapten immunity, can be used as therapeutic agent, or prevent and treat and use vaccine, can induce, drive, strengthen specific immune, to anti-curing oncoma, chronic infectious disease is implemented immunoprophylaxis or treatment.
Described chronic infection disease can be a chronic hepatitis B, chronic hepatitis C etc.
Conjugate of the present invention also is used for micro-qualitative and quantitative analysis and detects organizational groups dyeing.
Among the present invention, contain hapten dinitro benzene structure in vivo the inducing cell immunity as the lethal effect bullet, in conjunction with carrier with targeting, to carcinoma, chronic infection has specific recognition power, at the targeting lesions position, utilize its strong hapten cellular immunization, induce the panimmunity activation factor in the part, improve the local immunity microenvironment, realize executing reinforced immunological treatment carcinoma or infection.
The present invention shows through experiment, described hapten dinitrofluorobenzene does not have under the situation that immune system participates in external, can directly suppress hepatoma carcinoma cell, measuring its IC50 with mtt assay is 32.22 μ M, IC90 is 49.53 μ M, though simultaneously 5 fluorouracil (5-FU) of Ce Dinging though IC50 be evident as little than DNFB, be 3.62 μ M, but IC90＞200 μ M, prompting hapten dinitro benzene structure can be used as prodrug (prodrug) structural design medicine, shows that the chemical compound after the dinitro benzene structure covalent bond has direct antitumaous effect.Hapten DNFB of the present invention or DNCB and the conjugate that contains amino carrier can further prepare the medicine of anti-curing oncoma.
Compare with existing anticarcinogen, the present invention can obviously have activation, induce, strengthen specific immune and play a role,, be applicable to hepatocarcinoma, rectal cancer, esophageal carcinoma, gastric cancer, cancer of pancreas, breast carcinoma and pulmonary carcinoma and lymphoma by injection or vascular interventional treatment, the treatment of local infiltration sexual type leukemia etc., also can directly or with other drug be mixed for topical therapeutic, abdominal cavity, lacuna treatments such as thoracic cavity etc.
Description of drawings
Fig. 1 is a rat liver cancer intratumor injection tumor killing effect,
Wherein show, DNFB intratumor injection mouse subcutaneous transplanting tumor result, D solution is the molten 0.1ml phosphate buffer of DNFB 1mg (PBS), and dehydrated alcohol (ETH) is 0.1ml, and matched group is the PBS solution of 0.1ml.D solution and ETH group tumour inhibiting rate are respectively 74.43%, 59.2%, and both compare with matched group, and all there were significant differences (p＜0.01 reaches＜0.05 respectively).
Fig. 2 is that absorbance changes,
Wherein shown, in 500ul dibastic sodium phosphate (PBS) buffer solution system, 2mg, 4mg, 0mg (being matched group) DNFB and the 13mg height anti-hepatis B immunoglobulin (HBIG) of tiring combines potency ratio that double serial dilution in back measure, through two-way analysis of variance zero difference (p＜0.05), with determined by ultraviolet spectrophotometry, in conjunction with interior scalar quantity, 2mg, in the 4mg articulated system, there is 6.12%, 7.86% amino acid residue to realize and the combining of HBIG respectively.
The specific embodiment
Followingly further specify the present invention, but can not limit content of the present invention by embodiment.
Monoclonal antibody with hapten dinitrofluorobenzene and resistance of hepatitis B surface antigen is a reaction raw materials, under weak basic condition, pass through Sanger ' s reaction, fluorine comes off in conjunction with the hydrogen on the amino in the monoclonal antibody, dinitro benzene partly replaced be covalently bound on the amino, obtain the covalently bound hepatitis B surface antigen monoclonal antibody of target product dinitro benzene, in one of them target product, combination rate is: dinitro benzene has 1-200, and this binding capacity does not influence the special identification function of monoclonal antibody.
Above-mentioned hapten also can adopt dinitrochlorobenzene, binitro bromobenzene, or dinitro iodobenzene.
Through immunoassay (as ELISA), the result shows that the immunoreaction measurement signal amplifies up to 200 times, obviously improves the sensitivity of immunoassay.
Following of room temperature in conjunction with anti-hepatis B immunoglobulin (HBIG (13mg/500ul PBS) aqueous solution respectively with various dose (2,4, hapten dinitrofluorobenzene (DNFB) 8mg) mixes, form two milligrams of pipes respectively, four milligrams of pipes, eight milligrams of pipes, centrifugal after mixing spends the night, precipitation, two milligrams of pipes appear in the HBIG aqueous solution of rarely seen eight milligrams of pipes, four milligrams of pipes reach the HBIG aqueous solution control tube that does not add DNFB, half amount of respectively getting is done after the half-and-half dilution respectively with the desalination of HiTrap molecular sieve column, and three eluents are made double serial dilution respectively 11 times again, make two milligrams of pipes, four milligrams of pipes and matched group are respectively formed 12 specimen respectively, totally 36 specimen.Detect tiring of hepatitis B surface antibody respectively with the ELISA method.Get two milligrams of pipes, second half of four milligrams of pipes got supernatant after giving saturated ammonium sulphate respectively, measures A with the ultraviolet light photometric analyzer respectively
340, make interior target control tube supernatant with five series concentration that add various dose DNFB and compare, calculate two milligrams of pipes, four milligrams of residue content of managing DNFB in the supernatant, thus HBIG is in conjunction with the DNFB amount of falling in the calculating respective tube.
The result shows: two milligrams of pipes, four milligrams of pipes and matched group, through two-way analysis of variance and q check, between three groups with extinction difference (OD between dilution factor
450-OD
630) zero difference (p＞0.05), highly significant difference (p＜0.01) is then arranged between each dilution factor.Saturated ammonium sulphate confirms two milligrams of pipes in conjunction with spectrophotometry, in four milligrams of pipes 6.5mg HBIG respectively combine the DNFB of 0.67mg and 0.86mg, the HBIG that is equivalent to every mg combines the DNFB of 0.104mg and 0.132mg respectively, be that each HBIG molecule combines 88.72 and 113.48 DNFB hapten molecules respectively, account for 6.12% and 7.86% of HBIG amino acid residue.Experiment confirm: within the specific limits, the combination of DNFB is to the not influence of antibody titer of HBIG, and immunoglobulin (antibody) can be used as a kind of effective carrier of hapten DNFB immunity targeted therapy chronic viral infection or tumor.
Press the preparation method of embodiment 1, adopt the covalently bound tumor of dinitro benzene or infect targeting preparation, preparation is by the inductive immune targeted therapy tumour medicine of hapten, or/and the antitumaous effect that is directly had as former medicine by dinitrofluorobenzene.The result shows that combining ratio is with embodiment 1; DNFB intratumor injection mouse subcutaneous transplanting tumor tumour inhibiting rate is good.
Described tumor or infection targeting preparation can be selected from anti-HER-2 monoclonal antibody He Saiding or hepatitis B monoclonal antibody.
Press the preparation method of embodiment 1, adopt the immunizing antigen of covalently bound tumor of dinitro benzene or chronic infection pathogen or epitope preparation to induce foundation, or strengthen the control medicine of specific immune by hapten.In conjunction with hepatitis B surface antigen, DNFB is in conjunction with melanoma tumor regression epitope etc. as DNFB, can be used for the specificity prevention or as tumor therapeutic vaccine etc., the result shows that its combining ratio is with embodiment 1.Described targeted therapy tumor or chronic infection medicine is characterized in that with chemical compound with targeting as the targeting part in conjunction with tumor, with the haptenic group part with covalent bond, implement tumor or chronically infected targeted therapy.
Table 1: external to SMMC-7721 hepatoma cell strain 48 hour cell survival rates (mtt assay, the mean of six holes value).
Test final concentration dinitrofluorobenzene (DNFB) five fluorouracil (5-FU)
0uM 100 100
2uM 102.99 53.03
10uM 101.32 38.08
50uM 8.92 17.82
100uM 1.54 16.55
200uM 1.34 13.37
Estimate IC50 32.22uM 3.62uM
Estimate IC90 49.53uM＞200uM
Table 2:DNFB and 5-FU are respectively to SMMC-7721 hepatoma cell strain 48 hour cell survival rates: (0,2,10,50,100, during 200uM, the concrete numerical value in six holes that mtt assay is measured)
The DNFB group
1.062?1.072?1.066?1.124?1.213?1.231
1.107?1.114?1.176?1.145?1.121?1.297
1.126?1.074?1.210?1.144?1.135?1.164
0.192?0.148?0.147?0.145?0.142?0.140
0.065?0.079?0.085?0.067?0.074?0.070
0.063?0.067?0.076?0.071?0.076?0.074
The 5-FU group
1.204?1.190?1.156?1.222?1.282?1.391
0.662?0.675?0.666?0.677?0.726?0.702
0.484?0.507?0.476?0.480?0.520?0.579
0.235?0.250?0.279?0.258?0.279?0.306
0.223?0.244?0.244?0.241?0.270?0.295
0.200?0.214?0.207?0.228?0.222?0.220
Acellular matched group
0.054?0.057?0.060?0.051?0.054?0.065。
Claims (9)
1. hapten and the conjugate that contains amino carrier is characterized in that described hapten and the described structure of conjugate that contains amino carrier comprise its isomers, have the structure of formula (I):
2. by described hapten of claim 1 and the conjugate that contains amino carrier, it is characterized in that described hapten is dinitrofluorobenzene or dinitrochlorobenzene.
3. by described hapten of claim 1 and the conjugate that contains amino carrier, it is characterized in that the described amino carrier that contains, be selected from the material of monoclonal antibody or antibody or antigen or immune epitope or specific bond immunoglobulin, and other have the bonded material of targeting.
4. by described hapten of claim 3 and the conjugate that contains amino carrier, the material that it is characterized in that described specific bond immunoglobulin is a protein staphylococcus.
By the described hapten of claim 3 with contain the conjugate of amino carrier, it is characterized in that described other have the bonded material of targeting be selected from hepatocarcinoma is had a higher affinity or manually combine ethylaminoethanol the iodate grease.
6. claim 1 or 2 hapten and the preparation method that contains the conjugate of amino carrier, it is characterized in that described hapten and combining of carrier are by the amino on the carrier, under the alkalescence condition, dinitro benzene is partly replaced the hydrogen on the amino, obtains with the amino covalence association reaction.
The hapten of claim 1 with contain the application of conjugate in micro determination of immunization of amino carrier.
8. the hapten of claim 1 is preparing the purposes of preventing and treating in the tumour medicine with the conjugate that contains amino carrier.
9. the hapten of claim 1 is preparing the purposes of preventing and treating in the chronic infection disease medicament with the conjugate that contains amino carrier.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019127346A1 (en) * | 2017-12-29 | 2019-07-04 | 江南大学 | Bifunctional molecule that recruits antibodies and targets tumor cells |
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Non-Patent Citations (1)
| Title |
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| 戴兆云等: "二硝基氯苯的结合不影响乙肝免疫球蛋白的抗体功能", 《中华医学会全国第九次感染病学学术会议论文汇编》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019127346A1 (en) * | 2017-12-29 | 2019-07-04 | 江南大学 | Bifunctional molecule that recruits antibodies and targets tumor cells |
| CN111971060A (en) * | 2017-12-29 | 2020-11-20 | 江南大学 | Bifunctional molecules for recruiting antibodies and targeting tumor cells |
| CN111971060B (en) * | 2017-12-29 | 2023-06-13 | 江南大学 | Bifunctional molecules that recruit antibodies and target tumor cells |
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Application publication date: 20101013 |