CN101407545A - 131I labeled anti-tumor humanized monoclonal antibody 1E2 and use thereof - Google Patents

Abstract

The invention provides an anti-lung cancer human resource monoclonal antibody 1E2 marked by <131>I. The human resource 1E2 antibody, Na <131>I and chloramine T are used as raw materials, and purified 1E2 antibody marked by the <131>I can be prepared by using the chloramine T method, wherein, the human source 1E2 antibody is obtained by taking paracanser lymph nodes of a non-small cell lung cancer patient, extracting total RNA in lymph node tissues, and then a phage antibody library containing the 1E2 antibody is obtained by using the constructing technology of the phage antibody library, and the phage antibody 1E2 is adsorbed by using a carbamyl phosphate synthetase antigen, and a human source 1E2 antibody which is specific to the carbamyl phosphate synthetase is finally obtained by washing, screening and amplifying. The invention applies radiation immunity treatment technology, combines advantages of radionuclide and anti-tumor cell human source monoclonal antibody to one, improves the targeting ability and curative effect of medicines to tumor cells, reduces side effects and poor immune reactions, and is wider in application range and suitable for treatments of the non-small cell lung cancer of each stage.

Description

131The anti-lung cancer Humanized monoclonal antibodies 1E2 and the application thereof of I mark

Technical field

The present invention relates to a kind of radioactivity anti-tumour antibody, particularly a kind of usefulness ¹³¹The anti-lung cancer Humanized monoclonal antibodies 1E2 and the application thereof of I mark.

Background technology

Along with the continuous development of medical technology, radioimmunotherapy (RIT) is the another oncotherapy means except that operation, chemicotherapy.RIT is injected into the radioisotope labeling anti-tumour antibody in the body, monoclonal antibody and labelled nuclide combine (constant-bearing guidance) specifically with tumour cell, radionuclide is carried to tumor locus, the ray that sends in the radionuclide decay process causes damage to tumour cell DNA, finally causes growth of tumour cell to suppress and necrosis.Radioimmunotherapy is an another brand-new methods of treatment behind chemotherapy of tumors and radiotherapy, and it combines the targeting of monoclonal antibody and the strong lethal effect of internal irradiation of radioactive nuclide, has good effect, the low characteristics of side effect.Existing both at home and abroad scholar utilizes nucleic glioma, lymphoma, mammary cancer, liver cancer, peritoneal cancer, lung cancer, bone and soft tissue neoplasm and prostate cancer bone transfer etc. diagnosed and to be treated.Used in the market monoclonal antibody mostly is the antibody of mouse source property, and these antibody are not suitable for clinical application because of the anti-mouse immune response of people.Radioimmunotherapy (RIT) at lung cancer has the anti-lung cancer monoclonal antibody of human Yttrium-90 mark LC-1 to adopt the administering mode of topical therapeutic, and tumour inhibiting rate is lower; The new drug of unique listing ¹³¹Its mechanism of action of I-chTNT is: late period, the characteristic feature of noumenal tumour was to have a large amount of degeneration necrosis cells, occur in the process of degeneration necrosis at cell, many cracks can appear in cytolemma, a large amount of ingredients from lossing are outside born of the same parents in the endochylema, intracellular chromosomal three-dimensional structure is destroyed, produce a large amount of denatured DNA strand complex bodys, this strand complex body is exactly ¹³¹I-chTNT acts on the target spot of noumenal tumour, so this medicine is only applicable to put, the advanced lung cancer patient of chemotherapy failure.

Summary of the invention

The invention provides a kind of tumor-targeting good, that tumour inhibiting rate is higher, be applicable to each phase nonsmall-cell lung cancer ¹³¹The anti-lung cancer Humanized monoclonal antibodies 1E2 of I mark.

The technical scheme that adopts is as follows: with concentration is the humanization 1E2 antibody, specific radioactivity of the 200 μ g/ml Na greater than 2.5GBq/ml ¹³¹I, concentration are that the chlorine ammonia T of 1 μ g/ μ l is a raw material, and three's volume ratio is to take a sample at 1: 2: 1, uses conventional chloramine-t method to make; Humanization 1E2 antibody prepares preservation for the inventor, and being diluted to concentration with pH7.5, concentration as the phosphate buffered saline buffer of 0.5mol/L before using is 200 μ g/ml; All the other all starting material preparations all can be bought and obtain.

The preparation method of humanization 1E2 antibody is as follows: cut the other lymphoglandula of cancer that pathology turns out to be the nonsmall-cell lung cancer patient in the operation, total RNA in the extracting lymph node tissue, utilize the phage antibody library constructing technology to obtain containing the phage antibody library of 1E2 antibody, utilize carbamyl phosphate synthetase antigen absorption phage antibody 1E2, obtain the humanized antibody 1E2 special through washing, screening, amplification, identify at last and the analysis of antibody vigor CPS1.

Being chosen in the radioimmunotherapy of monoclonal antibody is very important one side.The present invention through a large amount of screening with the humanization 1E2 antibody that serves as a mark.1E2 is the monoclonal antibody that suppresses the lung carcinoma cell growth, and it combines with tumor cell membrane, and anticancer is bred, and unites with monoclonal antibody and radionuclide and carries out targeted therapy, uses conventional chloramine-t method mark on the preparation method ¹³¹I-1E2 is simply effective, and mark rate is 77.73%, and radiochemicsl purity is 95.63%, and specific radioactivity is 432MBq/ μ g.Its advantage is that advantage and Humanized monoclonal antibodies with radionuclide is integrated in one on result of treatment, on the one hand, the radioactive breakdown sphere of action of nucleic can reach the dozens of cell dia, overcome the blind area that the expression heterogeneity of tumour antigen is caused to a certain extent, and nucleic does not need the internalization process of immunotoxin or immune drug to oncocyte energy direct killing.The lethal effect of nucleic does not rely on synthetic (the silent tumor cell is also had lethal effect) of DNA.On the other hand, antibody targeted treatment advantage is arranged, improved the target of medicine, thereby improve curative effect,, be applicable to the treatment of each phase nonsmall-cell lung cancer from its mechanism of action to tumour cell.Owing to used Humanized monoclonal antibodies, have advantages such as efficient, low toxicity, patient are difficult for developing immunity to drugs, significantly reduce the anti-mouse immune response of people, thereby reduced side effect, be more suitable for clinical application.While has overcome mouse monoclonal antibody transformation period weak point again, application can be introduced patient's shortcomings such as HAMA repeatedly, and humanized's monoclonal antibody has become the medicine of another treatment tumour after excision, radiotherapy and chemotherapy.

In order to prove ¹³¹The anti-lung cancer Humanized monoclonal antibodies 1E2 of I mark has done following test to the result of treatment of nonsmall-cell lung cancer:

Set up Lewis lung cancer mice with tumor model earlier, 40 tumor-bearing mices are divided into four groups after becoming knurl at random: physiological saline control group (NS 0.1ml); Simple 1E2 antibody group (injection 1E2 3 μ g); ¹³¹(the injection of I mark IgG group ¹³¹I-IgG 18.5MBq); ¹³¹(the injection of I mark 1E2 antibody group ¹³¹I-1E218.5MBq), 1 time/2 days, totally 10 times, preceding 2 days of injection of labelled antibody, mouse is drunk 0.1%KI solution with the sealing Tiroidina.Adopt vernier callipers to measure a tumour maximum diameter (a) and maximum perpendicular diameter (b) in per 3 days.Put to death mouse after 21 days, peel off the knurl body, measure tumor tissues weight.Gross tumor volume size and weight are carried out one-way analysis of variance.The gross tumor volume calculation formula is: V (mm ³)=1/2 * ab ²Calculate unit of length mm; Gross tumor volume tumour inhibiting rate: tumour inhibiting rate=(V ₀-V _n)/V ₀* 100% (V ₀Be control group volume, V _nBe treatment group volume).

Observe pathological change, put to death mouse after 21 days, get treatment group and control group tumor tissues 10% formalin fixed, the HE dyeing of paraffin embedding section back, mirror is observed pathological change down.The result shows:

1, ¹³¹I-1E2 is in the intravital distribution of mouse

Every gram of 36h tumour organizes percentage injection dose rate (%ID/g) to be up to 11.61 behind the tumor-bearing mice injection of labelled antibody, this moment knurl/blood ratio=4.73, knurl/liver ratio=7.44 is behind knurl/spleen ratio=7.07,24 hour ¹³¹I-1E2 concentration in tumor tissues proves than other each position height (P＜0.05) ¹³¹I-1E2 antibody is dense poly-(table 1) in tumor tissues.

Table 1. ¹³¹The intravital distribution situation of different time mouse behind the I-1E2 intratumor injection (x ± S)

Unit: %ID/g

2, respectively organize mouse tumor weight, volume, tumour inhibiting rate relatively

Measure gross tumor volume and weight (table 2) after 21 days, the SNK-q check shows ¹³¹I mark 1E2 antibody group and other three groups are variant, and no significant difference relatively between other three groups, explanation ¹³¹I-1E2 obviously suppresses tumor growth, and tumour inhibiting rate is up to 78.3%.

Table 2 is respectively organized tumor weight and volume ratio and tumour inhibiting rate (x ± s)

* compare P＜0.05 with control group

3, mirror down as seen ¹³¹The large stretch of tumour cell necrosis of I mark 1E2 antibody group, nuclear concentrates, nuclear fragmentation, is vitreous degeneration.And other three groups of pathological section nucleus divisions are seen mutually, and obviously atypia is not seen large stretch of tissue necrosis.

Hence one can see that: injection ¹³¹Tumor growth rate is starkly lower than control group behind the I-1E2, ¹³¹Though I-IgG group also local injection high dosage ¹³¹I, tumor growth rate obviously faster than ¹³¹The I-1E2 group, though single have certain restraining effect with the monoclonal antibody group to tumor growth, DeGrain this shows to have only monoclonal antibody is combined with radionuclide, just can play the effect of good killing tumor cell, and tumour inhibiting rate is greatly enhanced.

The present invention does not have tangible clinical toxic side effect, can carry out the local injection treatment to tumour and can intravenous injection treat yet, but the local injection effect is better than intravenous injection, is applicable to the treatment of each phase nonsmall-cell lung cancer.

The invention has the beneficial effects as follows: the present invention uses ¹³¹The anti-lung cancer Humanized monoclonal antibodies of I mark 1E2 is integrated in one the advantage and the Humanized monoclonal antibodies of radionuclide, and both synergies improve the target to tumour, and tumor growth is had the obvious suppression effect, have improved tumour inhibiting rate greatly.What the present invention simultaneously adopted is Humanized monoclonal antibodies, has significantly reduced immune response, and the scope of application is more extensive, and is all effective to each phase nonsmall-cell lung cancer.

Specific embodiment (totally 5 examples)

The anti-lung cancer monoclonal antibody of embodiment 1 preparation humanization 1E2

(1) structure of phage antibody library:

1. the extraction of total RNA: operation cuts other lymphoglandula (pathology the turns out to be nonsmall-cell lung cancer) 10g of patients with lung cancer cancer, it is frozen to put into liquid nitrogen after sterile gauze is wrapped rapidly, in filling liquid nitrogen stone roller alms bowl, rapidly Lymphoid tissue is crushed into powder, and then utilize RNA extraction agent box to extract, obtain total RNA.

2. the amplification of VH and VL gene segment: as the synthetic cDNA of primer, the PCR condition is: 50 ℃ of 30min, 99 ℃ of 5min, 5 ℃ of 5min, a circulation with Oligo dT-Adaptor primer.Be template then with cDNA, the pairing of above downstream primer increase respectively light chain gene segment and heavy chain gene segment, the PCR condition is: 94 ℃ of 2min, a circulation, 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 90sec, 30 circulations.Utilize Linker primer increase respectively VH-Linker gene segment and VL-Linker gene segment at last, the PCR condition is: 94 ℃ of 2min, a circulation, 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 90sec, 35 circulations.Obtain VH-Linker gene segment and VL-Linker gene segment.

3. the connection of Scfv gene fragment and pcr amplification: the VH-Linker gene segment that glue is reclaimed purifying is connected with the VL-Linker gene segment, and reaction conditions is: 94 ℃ of 5min, a circulation, 94 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 90sec, five circulations.Thereby VH gene segment and VL gene segment are connected to form the scfv gene segment.And then in reaction system, adding the primer (having restriction enzyme site) that scfv increases, reaction conditions is: 94 ℃ of 30sec, 60 ℃ of 60sec, 72 ℃ of 90sec, five circulations.Form the scfv gene segment that two ends have restriction enzyme site respectively.

4. construction of recombinant plasmid: connect through sfi I restriction enzyme and Not I restriction enzymes double zyme cutting ScFv gene segment and PCANTAB-5E carrier with the T4 dna ligase, transform TG with electroporation ₁Competent cell is laid on penbritin and selects on the substratum, and overnight incubation is calculated clone's number.TG1 bacterium colony after 24 conversions of picking at random joins in the 2-YT nutrient solution shaking culture respectively and spends the night, and extracts plasmid, checks with PCR to have or not the ScFv gene to insert among the DNA.

5. the structure of antibody library: the positive bacteria clone is laid on the dull and stereotyped last 37 ℃ of overnight incubation of SOBAG, adds 10ml 2-YT nutrient solution pinching bacterium liquid again.Be diluted to A600=0.3 with the 2-YT nutrient solution.Add penbritin, final concentration is 100ug/ml, and the final concentration of glucose is 2%.37 ℃ of 150rpm shaking culture 1h add 5.1 * 10 again ¹⁰The M13K07 of pfu to final concentration be 4 * 10 ⁹, 37 ℃ of 250rpm shaking culture 1h.The centrifugal 20min of 4000g abandons supernatant.Precipitation is resuspended in the 100ml-YTAK nutrient solution, and 30 ℃ of 250rpm shaking culture are spent the night.The centrifugal 10min precipitum of 10000g is got the PEG/NaCl of supernatant adding 1/5, ice bath 60min.4 ℃ of centrifugal 20min of 10000g abandon supernatant.Precipitation is resuspended in the 1ml2-YT nutrient solution, 10000g recentrifuge 10min, and the adding final concentration is 0.02% sodium azide in the supernatant.4 ℃ of preservations.

(2) the affine screening of phage antibody library: with 1ml scFv phage antibody library solution, adding concentration is 300 μ g/ml carbamyl phosphate synthetase (CPS1) antigen 1 ml, static 30 minutes.2. the non-specific binding thing is removed in the PBS washing, collects and antigens c PS1 bonded phage antibody 1E2; 3. ehec infection makes special phage antibody 1E2 amplification enrichment, just can obtain the humanized antibody 1E2 special to CPS1.

(3) positive bacteriophage mono-clonal PCR identifies the mono-clonal that the picking third round filters out, extract the mono-clonal plasmid and carry out antibody cloning. 20 mono-clonals of picking at random, adding 3mL contains the 2-YT nutrient solution incubated overnight of 100mg/L penbritin, the conventional plasmid that extracts. with the plasmid is that template is carried out the PCR reaction, identifies whether amplify goal gene with the 10g/L agarose gel electrophoresis then.

(4) positive bacteriophage mono-clonal enzyme is cut and is identified 10 mono-clonal plasmids of picking identify whether contain goal gene with Nhe I and BssH II double digestion with the 10g/L agarose gel electrophoresis at random.

(5) positive monoclonal antibody activation analysis picking positive monoclonal, with the carrier that does not contain goal gene is contrast. carry out phage surface by preceding method and present, collect supernatant and be phage antibody. there are 96 orifice plates of human fibroblasts and lung carcinoma cell to spend the night monoclonal antibody and contrast supernatant adding inoculation, add M1337 ℃ of incubation 1h of HRP-goat-anti again, add TMB colour developing 10min, adding 2mol/L sulfuric acid termination reaction. microplate reader is read plate, and is positive with P/N＞2.1.

The preparation of embodiment 2 usefulness chlorine ammonia T methods ¹³¹I mark 1E2 antibody

Method is as follows:

1, iodination reaction: get the humanization 1E2 antibody 10 μ g of embodiment 1 preparation, with pH7.5, concentration be the phosphate buffered saline buffer of 0.5mol/L to be diluted to concentration be 200 μ g/ml, it is standby accurately to measure 50 μ l; Get the Na of specific radioactivity 5.5GBq/ml ¹³¹I 100 μ l; Getting concentration again is the chlorine ammonia T 50 μ l of 1 μ g/ μ l, and with three's mixing, adding is placed with in the glass test tube of magnetic stir bar, places on the magnetic stirring apparatus fully to shake up, and at room temperature reacts 60s;

2, stop iodination reaction: add Sodium Metabisulfite (Na behind the 60s immediately ₂S ₂O ₅)) 100 μ g, volume 100 μ l stop iodination reaction;

3, separation and purification:

(1), labeled reactant liquid is injected the Sephadex G50 chromatography column of 1cm * 15cm specification, PBS solution (pH=7.4) wash-out, flow velocity is 10/min, use the test tube Fractional Collections, 10/pipe, collects 100 and manages;

(2), (counts per minute cpm), draws the purifying curve, and twice purifying curve compared, and determines Na to measure the per minute radiocounting of every pipe on Γ-calculating instrument ¹³¹The position at I radiation peak.Collect the reaction solution at first radiation peak of purifying for the first time, obtain purifying ¹³¹The 1E2 antibody of I mark.

The purifying that 3 couples of embodiment of embodiment 2 are prepared ¹³¹The 1E2 antibody of I mark carries out the evaluation of mark rate and radiochemicsl purity

Trichoroacetic acid(TCA) is a developping agent, is upholder with No. 1 filter paper of Xinhua, and the reaction solution before and after the separation and purification is analysed certification mark rate and radiochemicsl purity as ply of paper, formula:

$=77.73\%$

$=432\mathrm{MBq}/\mathrm{\&mu;g}$

Embodiment 4 ¹³¹I mark 1E2 antibody concentration is measured:

Become 20 μ g/100 μ l, 10 μ g/100 μ l, 5 μ g/100 μ l, 2.5 μ g/100 μ l, 1.25 μ g/100 μ l, 0.625 μ g/100 μ l, 0.3125 μ g/100 μ l as normal concentration and to be measured 1E2 antibody doubling dilution ¹³¹Each 100 μ l of I mark 1E2 antibody are respectively charged in the 5ml test tube, add Xylene Brilliant Cyanine G 100 μ l mixings;

Measure the absorbancy (OD of each test tube after 30 minutes at wavelength 595 with spectrophotometer ₅₉₅); Measure twice with above same procedure;

Set up regression equation according to absorbancy:

$\hat{y}=a+\mathrm{bx}$

$b=\frac{\mathrm{\&Sigma;xy}-\left(\mathrm{\&Sigma;x}\right)\left(\mathrm{\&Sigma;y}\right)/n}{{\mathrm{\&Sigma;x}}^2-{\left(\mathrm{\&Sigma;x}\right)}^2/n}=\frac{\mathrm{\&Sigma;}(x-\stackrel{\&OverBar;}{x})(y-\stackrel{\&OverBar;}{y})}{\mathrm{\&Sigma;}{(x-\stackrel{\&OverBar;}{x})}^2}=\frac{{\mathrm{SP}}_{\mathrm{xy}}}{{\mathrm{SS}}_x}$ a＝y-bx

Calculating the concentration that is labeled thing is 18.65 μ g/100 μ l.

Embodiment 5 ¹³¹The calculating of the specific activity of I mark 1E2 antibody

The radioactivity of measuring mark thing, obtain specific radioactivity then, formula:

Claims (2)

1, a kind of ¹³¹The anti-lung cancer Humanized monoclonal antibodies 1E2 of I mark is characterized in that with concentration be the humanization 1E2 antibody, specific radioactivity of the 200 μ g/ml Na greater than 2.5GBq/ml ¹³¹I, concentration are that the chlorine ammonia T of 1 μ g/ μ l is a raw material, and three's volume ratio is to take a sample at 1: 2: 1, uses conventional chloramine-t method to make; The preparation method of described humanization 1E2 antibody is as follows: cut the other lymphoglandula of cancer that pathology turns out to be the nonsmall-cell lung cancer patient in the operation, total RNA in the extracting lymph node tissue, utilize the phage antibody library constructing technology to obtain containing the phage antibody library of 1E2 antibody, utilize carbamyl phosphate synthetase antigen absorption phage antibody 1E2, obtain the humanized antibody 1E2 special through washing, screening, amplification, identify at last and the analysis of antibody vigor CPS1.

2, as claimed in claim 1 ¹³¹The application of the anti-lung cancer Humanized monoclonal antibodies 1E2 of I mark in the medicine of preparation treatment nonsmall-cell lung cancer.