CN1265840C - Immunity coupler of containing monoclonal antibody Fab' segment of anti TV type collagenase poly glutamic acid-blemycin A5 - Google Patents
Immunity coupler of containing monoclonal antibody Fab' segment of anti TV type collagenase poly glutamic acid-blemycin A5 Download PDFInfo
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Abstract
本发明涉及一种免疫偶联物Fab’-PLG-PYM，该偶联物是由抗IV型胶原酶单克隆抗体3G11的Fab’片段、多聚谷氨酸(PLG)分子以及抗肿瘤抗生素平阳霉素(PYM)构成的，该偶联物具有靶向杀伤肿瘤细胞和显著抑制肿瘤的效果，可作为新型、高效抗肿瘤导向药物应用于肿瘤治疗。
The invention relates to an immunoconjugate Fab'-PLG-PYM, which is composed of the Fab' fragment of the anti-IV type collagenase monoclonal antibody 3G11, polyglutamic acid (PLG) molecules and antitumor antibiotic Pingyang Composed of PYM, the conjugate has the effect of targeting and killing tumor cells and significantly inhibiting tumors, and can be used as a new type of high-efficiency anti-tumor-oriented drug for tumor treatment.
Description
Technical field:
The present invention relates to a kind of immune conjugate Fab '-PLG-PYM, this conjugate is to be made of the Fab ' fragment of type-IV collagenase-resisting monoclonal antibody 3G11, polyglutamic acid (PLG) molecule and antitumor antibiotics Bleomycin A5 (PYM).Said conjugate has target killing tumor cell and significantly suppresses the effect of tumor, can be used as novel, high efficiency anti-tumor targeted drug and is applied to oncotherapy.
Background technology:
Bleomycin A5 (PYM) is a kind of glycopeptide class antitumor antibiotics of China's independent research, belongs to the bleomycin A5 component, promptly is used for the clinical treatment of tumors such as incidence scale cancer and malignant lymphoma the beginning of the eighties in last century in China.The domestic existing many reports of immune conjugate that prepare itself and monoclonal antibody at Bleomycin A5, Jiang Min etc. with PYM and rat monoclonal antibody 3A5 with glucosan T-40 (DT-40) coupling after, conjugate has strong killing hepatoma cell activity (Chinese microbiology and Journal of Immunology, 1991,11:230); With PYM with DT-40 and anti-nasopharyngeal carcinoma monoclonal antibody BAC5 coupling, the external activity of conjugate be 6.8 times of free PYM (cancer, 2003,22:831).This research department had before also prepared type-IV collagenase-resisting monoclonal antibody 3G11 (culture presevation number: active Fab ' fragment CGMCCNo.0831), its molecular weight is 1/3 of a complete monoclonal antibody, and be the conjugate that cross-linking agent has prepared monoclonal antibody Fab ' fragment and PYM equally with DT-40, the conjugate molecular weight is 170kDa, also embodied the antitumor action (Chinese Medical Journal stronger than free drug, 2001,81:201).Although it is little many that the conjugate that the more complete anti-molecule of Fab ' fragment itself and PYM form has been cut out,, the further miniaturization of whole conjugate molecule is very limited because the molecule of coupling agent DT-40 is huge.Therefore be necessary to seek of the coupling of littler molecular application,, monoclonal antibody targeted drug efficiently more small-sized to obtain in monoclonal antibody Fab ' fragment and PYM.
Polyglutamic acid is the single amino acid straight chain polymerizable molecular that is formed by the glutamic acid molecule aggregation, and (poly-α-L-glutamic acid PLG) can be connected to form polymer with small-molecule drug to poly-α wherein-L-glutamic acid.External existing report is with PLG and different antitumor drug such as daunorubicin (Journal of National Cancer Institute, 1984,73:721), paclitaxel (Cancer Research, 1998,58,2404), camptothecine (International Journal of Oncology, 2001,18:331) wait medicine to carry out coupling, the conjugate inside and outside all presents the good antitumor effect, and some of them have entered the clinical research of different phase.Many evidences show, PLG because of its avirulence, have excellent biological compatibility and biological degradability to be widely used in the coupling of antitumor drug.But up to now, there is no relevant polyglutamic acid both at home and abroad and be used for Bleomycin A5 and the link coupled report of homologue bleomycin thereof.Size that it should be noted that PLG in these conjugates of having reported is between 21～46kDa, and the difference with DT-40 with regard to molecular weight is not remarkable, and its mean molecule quantity of PLG of the present invention only is 7, and 500Da is significantly smaller than the PLG of present report.Aspect the PYM coupling, to compare with used in the past cross-linking agent DT-40, the very little and structure of the PLG molecule that the present invention uses is the straight chain shape, sterically hindered little, react single-minded complete, simple controllable, the single easy purification of product may be the suitable PYM target medicine carrier of a class.Of the present invention studies show that, select for use PLG to add the type-IV collagenase-resisting active fragment of monoclonal antibody Fab ' of SPDP structure and conjugate Fab '-PLG-PYM of PYM, its preparation route is clear and definite, reacts completely and easy to control, and coupled product is single, by-product is few, be convenient to downstream purification, conjugate not only reduced more greatly, and molecular weight only is 66kDa, also have obvious antineoplastic, be expected to become novel antitumor monoclonal antibody targeted drug.
Summary of the invention:
The said Fab ' of the present invention-Bleomycin A5 conjugate is as the medicine intermediate carrier with polyglutamic acid (PLG), together with cross-linking agent N-butanimide-3-(2-pyridine dimercapto) propionic ester (SPDP) molecule the Fab ' active fragment of monoclonal antibody 3G11 and antitumor antibiotics Bleomycin A5 (PYM) are carried out that coupling constitutes, the molecular weight of conjugate is about 66kDa, and wherein the molecular proportion of Fab ', PLG and PYM is about 1: 1: 4.2.
Particular content comprises:
Fab ' produced in fragments and the purification of monoclonal antibody 3G11;
The preparation of Fab ' fragment and PYM conjugate and purification;
The evaluation of conjugate Fab '-PLG-PYM and the molecular proportion of each component are calculated;
The immunocompetence of conjugate Fab '-PLG-PYM is measured;
Conjugate Fab '-PLG-PYM detects the growth inhibited effect of the tumor cell of In vitro culture;
Conjugate Fab '-PLG-PYM is to the therapeutical effect evaluation of mouse subcutaneous transplanting hepatocarcinoma 22.
The invention effect:
The new targeting immune conjugate that advantage of the present invention and good effect have been to provide a kind of Bleomycin A5 and monoclonal antibody to make up.Bleomycin A5 is clinical antitumor drug commonly used, the Bleomycin A5 that makes up as pharmaceutical carrier with the polyglutamic acid molecule has with monoclonal antibody activity Fab ' fragment conjugate that molecular weight is little, the clear and definite controlled advantage of technology path, it is external to have targeting killing effect to tumor cell, the anti-tumor in vivo effect obviously is better than free Bleomycin A5, life cycle that can also the significant prolongation animal, being expected to exploitation becomes the new antitumoral targeted drug, has a good application prospect.
Description of drawings:
The SDS-PAGE of Fig. 1: Fab '-PLG-PYM conjugate after gel-purified detects
Wherein: 1 is the molecular weight of albumen standard
2 is F (ab ')
2
3 is Fab '
4 is Fab '-PLG-PYM
Fig. 2: Fab '-PLG-PYM conjugate is to the immunoreactivity of antigen IV Collagen Type VI enzyme
Wherein: 1 is Fab '
2 is Fab '-PLG-PYM
Fig. 3: PYM and Fab '-PLG-PYM conjugate is to the cytotoxicity of KB cell
Wherein: 1 is PYM
2 is Fab '-PLG-PYM
Fig. 4: PYM and Fab '-PLG-PYM conjugate is to the cytotoxicity of PG cell
Wherein: 1 is PYM
2 is Fab '-PLG-PYM
Fig. 5: PYM and Fab '-PLG-PYM conjugate is transplanted the inhibitory action of hepatocarcinoma 22 to mice
Wherein: 1 is contrast
2 is the PYM of 10mg/kg dosage
3 is the PLG-PYM of 10mg/kg dosage
4 is the Fab '-PLG-PYM of 5mg/kg dosage
5 is the Fab '-PLG-PYM of 10mg/kg dosage
Fig. 6: PYM and Fab '-PLG-PYM conjugate is to transplanting the prolongation effect of hepatocarcinoma 22 mice life spans
Wherein: 1 is contrast
2 is the PYM of 10mg/kg dosage
3 is the PLG-PYM of 10mg/kg dosage
4 is the Fab '-PLG-PYM of 5mg/kg dosage
5 is the Fab '-PLG-PYM of 10mg/kg dosage
The specific embodiment:
Below listed embodiment be for those skilled in the art understand the present invention better, but do not limit the present invention.
Embodiment 1: Fab ' produced in fragments and the purification of monoclonal antibody 3G11
The monoclonal antibody 3G11 of affinity purification (contains 2mM EDTA with 0.05M Tris-HCl, pH7.5) fully dialysis back concentration is adjusted into 4～6mg/ml, behind 37 ℃ of pre-temperature 30min, press enzyme/substrate (E/S, w/w)=3: 110 consumption adds the ficin Ficin (available from Sigma company) of same pre-temperature, and to add final concentration be the cysteine activating reaction of 1mM, puts 37 ℃ of slow stirring reaction 4h.Product obtains F (ab ') behind Sephadex G-150 gel chromatography
2Fragment.F (ab ')
2Fragment (contains 10mM EDTA with 0.1M Tris-HCl, pH7.5) dialysis back ultrafiltration and concentration becomes 2～5mg/ml, β-MeSH room temperature reduction 1h with 10mM, reactant liquor is immediately through the desalination of PD-10 post, (culture presevation number: CGMCC No.0831), molecular weight is about 53kDa (Fig. 1) to obtain Fab ' fragment with the PBS-EDTA buffer solution elution of blowing over nitrogen in advance.
Preparation and the purification of embodiment 2:Fab ' fragment and Bleomycin A5 conjugate Fab '-PLG-PYM
Choosing mean molecule quantity is 7,500Da, and the degree of polymerization is that 50 PLG (Sigma company product) is used for the coupling experiment.With PLG and 5～8 times of excessive SPDP (Sigma company product) reaction, PD-10 post desalting and purifying behind the stirring at room 1h, the PLG of the SPDP derivatization that generates carries out condensation by dehydrant EDC and an amount of PYM (Tianjin pharmaceutical factory product), PYM to PLG about 5 times excessive, room temperature reaction 2h, (contain 150mM NaCl, 2mM EDTA, pH7.4) buffer is fully dialysed, and obtains conjugate PLG-PYM with 20mM PBS-EDTA.This conjugate is mixed with the Fab ' fragment of prepared fresh, the former is excessive to 3 times of Fab ' approximately, room temperature inflated with nitrogen reaction 1h, reactant liquor concentrates through centrifugal, carry out Sephacyl S-200 gel chromatography 0.45 μ m filter membrane is removed post precipitation, the end-product that obtains is Fab '-PLG-PYM immune conjugate.
Embodiment the 3:Fab '-evaluation of PLG-PYM conjugate and the molecular proportion of each component are calculated
Fab '-PLG-PYM is carried out non-reduced SDS-PAGE electrophoresis detection (Fig. 1), adopt 12% separation gel, the result shows that conjugate reaches more than 80% through purity after the gel-purified, and its mean molecule quantity is about 66kDa.With each components contents in the spectrophotometry conjugate and calculate molecular proportion.The result shows that the molecular proportion of Fab ', PLG and PYM is about 1: 1: 4.2 in the conjugate.Because Fab ' molecular weight is about 53kDa, the PLG mean molecule quantity of selecting for use is 7,500Da, so the conjugate molecular weight of trying to achieve by above-mentioned molecular proportion also is 66kDa, the result is consistent with the electrophoresis gained.
The immunocompetence of embodiment 4:Fab '-PLG-PYM conjugate is measured
Fab ' and Fab '-PLG-PYM are adjusted to protein concentration measure immunocompetence (Fig. 2) with conventional indirect elisa method after consistent.The result shows that conjugate has kept the immunoreactivity to antigen IV Collagen Type VI enzyme, but with Fab ' mutually specific activity decrease.
Embodiment 5:Fab '-PLG-PYM conjugate detects the growth inhibited effect of the tumor cell of In vitro culture
Detect human mouth scale cancer KB cell and the people high cytotoxicity that shift giant cell carcinoma of lung PG cell of conjugate Fab '-PLG-PYM with clone forming method and tetrazole indigo plant (MTT) method respectively to In vitro culture.The result shows that Fab '-PLG-PYM is to the half clone inhibition concentration IC of KB and PG cell
50Be respectively 1.91 μ M and 0.27 μ M, and the IC of free PYM
50Be respectively 0.27 μ M and 0.18 μ M (table 1), the inhibitory action of tumor cell be weaker than free PYM, the cytotoxicity of target cell PG obviously is better than non-target cell KB, embodied selective killing effect target cell although show Fab '-PLG-PYM.Mtt assay has proved the difference (Fig. 3, Fig. 4, table 1) of conjugate to two kinds of cytosiies equally.
The external inhibited proliferation of table 1 Fab '-PLG-PYM conjugate to different cells
| Group | IC 50(μM) | |||
| KB # | PG # | KB ## | PG ## | |
| PYM Fab’-PYM | 1.98 7.47 | 2.47 4.94 | 0.27 1.91 | 0.18 0.27 |
# is that mtt assay: ## is a clone forming method
Embodiment 6:Fab '-PLG-PYM conjugate is to the therapeutical effect evaluation of mouse subcutaneous transplanting hepatocarcinoma 22
With the female kunming mice random packet in 6～8 ages in week, test and got rat liver cancer H22 ascites on the 0th day, being diluted to cell number with normal saline is 5 * 10
6/ ml, it is subcutaneous only to be inoculated in the kunming mice axillary fossa by 0.2ml/.Test the 3rd day begin treatment, administration every other day totally 6 times only is tail vein injection 0.2ml/.Matched group gives normal saline, PYM (10mg/kg), PLG-PYM (10mg/kg) and Fab '-PLG-PYM (5mg/kg and 10mg/kg) that all the other each groups give respectively.Experimental session was measured the major diameter a and the minor axis b of a tumor in per 3～4 days, with formula V=ab
2/ 2 calculate the tumor volume, draw tumor growth curve, calculate tumour inhibiting rate.Observe animal to 100 day, draw the animal survival curve.The result shows that the gross tumor volume of administration treated animal is significantly less than matched group, and Fab ' in the administration group-PLG-PYM conjugate obviously is better than PYM and PLG-PYM (Fig. 5) to the inhibitory action of tumor.Tested the 22nd day, the matched group tumor is 6.59 ± 3.43cm
3, the PYM group is 2.80 ± 2.52cm
3, suppression ratio is 60.6%, Fab '-PLG-PYM 5mg/kg and 10mg/kg dosage group tumor are respectively 1.15 ± 0.90cm
3With 0.65 ± 0.65cm
3, suppression ratio is respectively 82.5% and 90.1%, has shown the antitumor action (table 2) of the efficient targeting of conjugate.
Table 2 Fab '-PLG-PYM conjugate is to the tumor inhibition effect of rat liver cancer 22
#
| Group | Dosage (mg/kg) | Average weight changes (g) | Gross tumor volume (cm 3)(X±SD) | Suppression ratio (%) |
| Contrast PYM PLG-PYM Fab '-PLG-PYM | - 10 10 5 | +17.6 +11.5 +10.7 +9.4 | 6.59±3.43 2.80±2.52 1.23±1.01 1.15±0.90 | - 60.6 * 81.3 ** 82.5 ** |
| Fab’-PLG-PYM | 10 | +8.0 | 0.65±0.65 | 90.1 ** |
The 22nd day record result behind the # tumor inoculation, animal does not have death.
*Compare with matched group p＜0.01
*Compare with the PYM group p＜0.01
Observe animal to inoculating back 100 days, the result shows, but the life span (Fig. 6) of conjugate significant prolongation animal, the Fab ' of 10mg/kg dosage in the time of 100 days-PLG-PYM group still has 2 animals survived, median survival time reaches 80 days, prolonged 73.9% than matched group (median survival time is 46 days) life, and the median survival time of PYM group is 51 days, has only prolonged 10.9%.The body weight normal growth of animal is in good condition during the whole test, shows that animal can tolerate all dosage.
Claims (6)
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