CN104288765A - New method for applying McAb for neutralizing biological activity of FLT3 to tumor targeting treatment - Google Patents

Abstract

The invention discloses a new method for applying McAb for neutralizing the biological activity of FLT3 to tumor targeting treatment. The method is mainly characterized in that a hybridomas cell strain having the advantages of strong specificity, stable and efficient secretion and anti-FLT3 biological activity is established, and an FLT3 monoclonal antibody secreted by the hybridomas cell strain is applied to suppress the formation of acute lymphoma in order to be used for treating malignant tumors. The method concretely comprises the following steps: immunizing BALB/C mice by the expression protein of gene recombinant FLT3; fusing immune spleen cells with the myeloma cells of the mice by PEG4000; repeatedly carrying out ELISA and WB for screening, and establishing the hybridoma cell strain specifically and stably expressing the FLT3 antibody; carrying out mass amplification, and inoculating the obtained amplified product into the abdominal cavities of mice to prepare ascites; and collecting ascites, carrying out ELISA titer detection, WB detection and biological activity detection for the suppression of leukemia cells from forming tumors.

Description

The new method of the targeted therapy of tumor is applied to the McAb of FLT3 biologic activity during a kind of utilization has

Technical field

This invention belongs to biotechnology and field of biological pharmacy, be specifically related to a kind of utilization have in and the McAb of FLT3 biologic activity be applied to the new method of the targeted therapy of tumor.

Background technology

FLT3 albumen belongs to receptor of the third order TYR kinases (receptor tyrosine kinase, RTK), with platelet derived growth factor (platelet derived growth factor in structure, PDGF), colony stimulating factor (colony stimulating factor 1, CSF1) KIT aglucon is correlated with.RTK albumen generally separates to obtain TYR kinases district in born of the same parents containing the outer similar district of immunoglobulin of 5 born of the same parents and one by the endogenous Insert Fragment of special hydrophilic.The coprecipitated fixed experiment of FLT3 protein immunization of the mankind shows the band that two are positioned at 130KD and 155-160KD place, what wherein the band of high molecular was corresponding is that N holds glycosylated maturation protein type, the low-molecular-weight then corresponding non-adult form albumen containing high-load mannose.

FLT3 full genome comprises 24 exons, altogether 96,982 base pairs.FLT3 albumen comprises 993 aminoacid, and molecular mass is 112.804KD.FLT3 albumen is considered to many and expresses in bone marrow CD34-positive cell (corresponding multipotent bone marrow and B lymphoid progenitor cell) and mononuclear cell, also can express in the fetal liver cell with CD34 high expressed.FLT3 also expresses in the blastocyte in albumen acute nonlymphocytic leukemia and B leukemic lymphoblastoid in addition.

FLT3 is the receptor of FL cytokine, has the function of receptors of TYR protein kinase activity.FL is an early stage function controlling factor, and it guarantees survival, the proliferation and growth of primitive hematopoietic CFU-GM.The combination of FLT3 and part can promote the Dimerized of part and thereafter by the cellular signal transduction of the mediated phosphorylation of multiple cytoplasmic protein, comprise SHC, SHP-2, SHIP, Cbl, Cbl-b, Gab1 and Gab2, can also activate downstream passages such as Ras/Raf/MAPK and the PI3Ras/Raf/MAPK and PI3 kinases etc. of multi-signal conduction.

The sudden change of FLT3 gene is modal genetic distortion, and this variation was found in acute myeloid leukaemia.The length sudden change being positioned at membrane-proximal region is a kind of mutation type the most often occurred, account for 20-25%, next is positioned at the kinase whose enzyme of second TYR to live the sudden change in region, and great majority are that proportion is 7-8% because the codon mutation at this region 835 place and 836 bit codons disappearance cause.Also be once found in the point mutation of membrane-proximal region in addition, and within the scope of full genome, the catastrophe of many newtypes is still increasing.

About have in acute myeloid leukaemia in the FLT3 gene order of 20-25% and occur internal series-connection repetitive sequence, insert and lack the change of (seldom seeing).This situation is also considered to 5-10% genuine myelodysplastic syndrome, refractory anemia with excess of blasts and acute, and a small amount of Lymphocytic leukemia is closely related.In FLT3, repetitive sequence is generally on the 11st exon, but sometimes also appears on 11 introns and 12 exons.It is FLT3-ITD (FLT3 gene internal tandem sequence repeats) that exception in this sequence repeats the most frequently used Terminology.But due to the severely subnormal on molecular structure, FLT3-LM (sudden change of FLT3 length) this description seems more abundant accurate.

Summary of the invention

The present invention adopts and sets up a strain high specificity, and stability and high efficiency secretion has the hybridoma cell strain of anti-FLT3 biologic activity, and the FLT3 monoclonal antibody using it to secrete is applied to the formation thus the treatment that can be used for malignant tumor that suppress acute lymphoma.

Technical scheme of the present invention is: the hybridoma cell strain FLT3E6 filtering out special stably excreting mouse anti human FLT3 antibody.By DNA sequencing and in vitro and in vivo test, confirm that antibody that described hybridoma cell strain is secreted is the monoclonal antibody of a kind of anti-FLT3 completely newly, the formation of acute lymphoma can be suppressed, thus can be used for the treatment of malignant tumor.

Further, concrete steps are: with the FLT3 expressing protein of gene recombinaton, immune balb/c mice; Through PEG 4000, immune spleen cell and murine myeloma cell are merged; Repeatedly carry out ELISA and WB to screen, set up the hybridoma cell strain of the anti-FLT3 of special stably express; A large amount of amplification, Mice Inoculated abdominal cavity, preparation ascites; Collect ascites, carry out the biologic activity detection of ELISA bioactivity, WB detection, Proliferation Ability test.

Monoclonal antibody described in the present invention is produced by the special stably excreting of FLT3E6 monoclonal hybridoma strain, and this cell strain forward China typical culture collection center submits preservation application to.Through genomic DNA order-checking qualification, the antibody gene type sequence of the FLT3 of this monoclonal cell strain coding is unique, and it is encoded to the monoclonal antibody of brand-new anti-FLT3.

Experiment in vivo result shows, with this monoclonal antibody injection tumor-bearing mice, compared with contrast (only injecting normal mouse IgG), obviously extend (>=March, p<0.05) life cycle of injecting the tumor-bearing mice of this monoclonal antibody, tumor body reduce obviously (comparatively matched group on average reduces more than 50%) or disappear.

The experimental result of vivo and vitro all shows, described monoclonal antibody can be used for the formation suppressing acute lymphoma, and the growth of Tumor suppression reduces tumor size, thus reaches the object for the treatment of tumor.

The present invention intends having the hybridoma mRNA of anti-FLT3 biologic activity for template with stably express, mice IgV district degenerate primer amplification VH and VL.Build VH-linker-VL object fragment through overlapping pcr again, be finally built into e. coli bl21/pETa (32+)-VH-linker-VL gene engineering expression bacterium; After conventional fermenting and amplifying, IPTG abduction delivering, fragmentation, purification, carry out medicine In vitro and in vivo activity and pharmacokinetics detect.Carry out pilot scale expression and purification, set up a set of feasible production technology and code; Declare I of medicine, II, III clinical trial phase certification, certificate produced by the national new drug of final acquisition; Carry out products propaganda and introduce to the market, being applied to clinical treatment, for human health makes due contribution.

Use western result in the molecular biology application software DNAMAN6.0.3.99 composition graphs 2 of the Highgrade integration of LynnonBiosoft company of U.S. exploitation, confirm that the former distribution of antibody table of FLT3 albumen is from 871 valines to 993 ends serine (red boxes is indicated), about 122 aminoacid.。

FLT3 variable region of mab nucleotide sequence carries out BLAST in NCBI website, have chosen three kinds of the highest Mus source monoclonal antibody specific 3F9,4C8, KP14 weight chain variabl area sequences of similarity as aligned sequences.Four kinds of sequences analysed and compared at the software vector NTI 8.0 of informaxinc company exploitation, the FLT3 monoclonal antibody in proved invention is the antibody (see Fig. 3) independent of a brand-new sequence of the similar monoclonal antibody of other sequence height.

Detailed description of the invention

The present invention carrys out illustration by following examples, but it should be noted that following examples only for illustration and explanation the present invention, instead of limiting the scope of the invention.

Embodiment 1 prepares the hybridoma cell strain of specificity for FLT3

With the hFL T3 of restructuring for immunogen.According to conventional methods through subcutaneous multiple spot immune balb/c mice, in good time and SP2/0 carries out merging through PEG 4000.Undertaken screening and sub-clone by monoclonal antibody preparation routine, obtaining can the hybridoma cell strain FLT3E6 of efficient, stably excreting specific anti-human FLT3.

This hybridoma is inoculated in after amplification and uses the liquid paraffin sensitization BALB/C mice abdominal cavity of 7 days in advance, within about 10 ~ 14 days, collect mouse ascites and be used for follow-up test research.

Ascites (being later called for short antibody purification) after ammonium sulfate precipitation purification and Protein G-Sepharose 4B affinitive layer purification detects ELISA tires >=and 409600.

With cervical cancer cell (Hela), the cell lysate of Human umbilical vein endothelial cells (HUVEC), leukaemia (HL60 and K562) is that antigen carries out western-blot qualification.Result is except Hela extracellular, and cell lysate is all about 110kdplace's appearance one is band (see Fig. 4) obviously.These results show, and the antibody of described hybridoma secretion has specificity to FLT3.

Embodiment 2 hFL T3 monoclonal antibody is to the therapeutic test of tumor-bearing mice

Routine is in exponential phase HL60 cell (leukaemia) with 0.25% collected by trypsinisation, adjusts cell concentration to be 5 × 107/ml, be inoculated in nude mice oxter, every 0.2ml, totally two ten with normal saline.Inject estrogen (0.15mg/ml) 0.2ml, once a day simultaneously.

After about 20 days, all nude mice oxters all grow the large tubercle body of about Semen phaseoli radiati, are divided into two groups at random, often organize ten.

The anti-FLT3 monoclonal antibody 0.1ml (containing 100 μ g McAb) of tail vein injection normal BALB/C IgG 0.1ml and purification respectively, the estrogen of intramuscular injection simultaneously 0.2ml/ only.

Observed and recorded nude mice survival state and tumorous size.

According to each group of tumorous size and life cycle average make respectively tumorous size bar diagram (see Fig. 5) and existence bar diagram (see Fig. 6).

As shown in Figure 5, the anti-FLT3 mab treatment of the application of the invention, the tumorous size of tumor-bearing mice reduces even gradually until disappear, and the tumorous size of matched group then increases gradually.Fig. 6 then confirms the anti-FLT3 mab treatment of the application of the invention, significantly increases the life cycle of tumor-bearing mice.

Accompanying drawing illustrates:

Fig. 1 is that western identifies FLT3 epitope figure.The gst fusion protein of Zuo Tu: FLT3C1:650-820 amino acids, the gst fusion protein of FLT3C2:650-870 amino acids, the gst fusion protein of FLT3C3:750-998 amino acids.Right figure: anti-FLT3 antibody can only identify the antigen containing FLT3C3.

Fig. 2 is FLT3 epitope sequence analysis chart.

Fig. 3 is three kinds of different antibodies and FLT3 monoclonal antibody heavy variable region nucleic acid sequence alignment figure.

Fig. 4 is that western identifies FLT3 antibody specificity figure.

Fig. 5 is that injection FLT3 antibody mouse and matched group tumor weight contrast block diagram.Wherein 5,14,21 representatives are 0 day with first time injection of antibodies, are separated by thereafter 5,14, within 21 days, take out tumor body afterwards and weigh.The tumor proliferation of FLT3 antibody group is significantly less than matched group.

Fig. 6 is injection FLT3 antibody mouse and matched group comparison diagram life cycle.Wherein 5,14,21 representatives are 0 day with first time injection of antibodies, are separated by thereafter 5,14, within 21 days, calculate the quantity of survival mice.The survival rate of injection FLT3 antibody group is apparently higher than matched group.

Claims (3)

1. during utilization has and the McAb of FLT3 biologic activity be applied to the new method of the targeted therapy of tumor, it is characterized in that: the FLT3 mab treatment tumor secreted by cell strain utilizing new secretion FLT3 monoclonal antibody.

2. during a kind of utilization according to claim 1 has and the McAb of FLT3 biologic activity be applied to the new method of the targeted therapy of tumor, it is characterized in that: set up a strain high specificity, stability and high efficiency secretion has the hybridoma cell strain of anti-FLT3 biologic activity, and the FLT3 monoclonal antibody using it to secrete is applied to the formation thus the treatment that can be used for malignant tumor that suppress acute lymphoma.

3. during a kind of utilization according to claim 2 has and the McAb of FLT3 biologic activity be applied to the new method of the targeted therapy of tumor, it is characterized in that: concrete steps are: with the FLT3 expressing protein of gene recombinaton, immune balb/c mice; Through PEG4000, immune spleen cell and murine myeloma cell are merged; Repeatedly carry out ELISA and WB to screen, set up the hybridoma cell strain of the anti-FLT3 of special stably express; A large amount of amplification, Mice Inoculated abdominal cavity, preparation ascites; Collect ascites, carry out ELISA bioactivity, WB detects and Proliferation Ability test organism Activity determination.