CN104804088A - Anti-VEGF monoclonal antibody - Google Patents

Abstract

The invention relates to the field of antibody engineering, discloses a novel anti-human vascular endothelial growth factor (huVEGF) monoclonal antibody, and concretely provides amino acid sequences of a heavy chain variable region and a light chain variable region. The monoclonal antibody can be specifically bound to human VEGF. The invention also provides a DNA molecule for encoding the antibody, and a preparation method of the antibody.

Description

A kind of monoclonal antibody of anti-vegf

Technical field

The invention belongs to antibody engineering field, be specifically related to a kind of mouse monoclonal antibody identifying human vascular endothelial growth factor (huVEGF) epi-position, more specifically, design the heavy chain of this monoclonal antibody and three hypervariable region aminoacid sequences of variable region of light chain.

Background technology

Within 1971, first Folkman observes when gross tumor volume is more than 1-2mm ³, need the formation of neovascularity to maintain growth, thus proposition tumor growth is that blood vessel is dependent the earliest, and points out that angiogenesis inhibitor is the new way controlling tumor growth.There are some researches show, the growth of nearly all solid tumor and transfer all depend on the generation of tumor neovasculature.

Within 1989, Ferrara N has found vascular endothelial growth factor (vascular endothelial growth factor, VEGF), be proved to be one of most important angiogenesis factor found at present afterwards, played an important role in the angiogenic growth of tumour.There is material impact to the invasion and m etastasis of tumour, in the blood vessel generating process of tumour, play extremely important effect.VEGF both may be used for the promising target of oncotherapy, also can as the mark carrying out diagnosing tumor and judging prognosis.

VEGF is the high glycosylation alkalescence homodimer glycoprotein of relative molecular mass 34000-42000, and its encoding gene is positioned at chromosomal 6q21.3, total length 14kb, containing 8 exons and 7 introns, can form 9 kinds of isomer, mainly comprise VEGF ₁₂₁, VEGF ₁₆₅, VEGF ₁₈₉and VEGF ₂₀₆.Wherein most tissue is with VEGF expression ₁₆₅be main.

Genetech company has been proved to be for the Humanized monoclonal antibodies Avastin of VEGF and all has significant inhibition for tumours such as the transitivity rectum cancer, nonsmall-cell lung cancer, mammary cancer since listing in 2004.2006, its optimize after the approval of antibody fragment thunder integument U.S. FDA be used for the treatment of age-related macular distortion, become the drug of first choice of the retinopathy that treatment treating senile maculopathy, diabetes cause.

Due to the diversity of VEGF and acceptor thereof, the VEGF antibody of different epitope or different avidity may have different tumor growth inhibitory effect.Therefore study different VEGF antibody and will contribute to the upgrading of VEGF antibody drug.

Summary of the invention

The object of this invention is to provide the monoclonal antibody of a kind of human vessel endothelium growth factor resisting (huVEGF), can specific identification huVEGF, there is very high avidity to huVEGF.

The invention provides a kind of mouse resource monoclonal antibody for huVEGF.

Present invention also offers preferred VEGF antibody heavy chain variable region, wherein hypervariable region has following amino acid sequences:

CDRH1（SEQ ID NO.1：TYGMS），CDRH2（SEQ ID NO.2：TISNGGSYTFYPDSVKG），CDRH3 （SEQ ID NO.3：HGSVTTRGFDY）。

Present invention also offers preferred VEGF antibody chain variable region, wherein hypervariable region has following amino acid sequences:

CDRL1（SEQ ID NO.4：KASQSVSNDVV），CDRL2（SEQ ID NO.5：YASNRYT），CDRL3（SEQ ID NO.6：LQDYSSPFT）。

Monoclonal antibody of the present invention can be, such as, the derivative of single-chain antibody, double-chain antibody, chimeric antibody, humanized antibody and above-mentioned antibody, function equivalent or homologue, also comprise antibody fragment and any polypeptide containing antigen-binding domains.

Accompanying drawing explanation

Fig. 1 is the collection of illustrative plates that display indirect elisa method measures the relative affinity of monoclonal antibody;

Fig. 2 is that display 16-5 is to the collection of illustrative plates of the hindrance function that VEGF is combined with its acceptor VEGFR1;

Fig. 3 is that display 16-5 is to the collection of illustrative plates of the hindrance function that VEGF is combined with its acceptor VEGFR2.

Embodiment

In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail.It should be noted that embodiment described below is exemplary, only for explaining the present invention, and can not limitation of the present invention be interpreted as.

The preparation of embodiment 1 mouse monoclonal antibody and screening

(1) immunity of mouse

By VEGF ₁₆₅protein dissolution, at l × PBS, gets appropriate protein solution and Freund's complete adjuvant (Sigma) mixes emulsification, gets the female mouse of BALB/c in 8 week age, subcutaneous multi-point injection antigen.After 2 weeks, get appropriate egg and mix emulsification from solution and Freund's incomplete adjuvant (Sigma), subcutaneous mouse, carry out second time immunity.Later every 3 weeks, carry out third and fourth immunity.Latter 3 days of 4th immunity, gets spleen and carries out cytogamy.

(2) cytogamy

Splenocyte is gathered in the crops in immune mouse, greater activity Sp2/0 myeloma cell will be had mix in 1:10-100 ratio with this splenocyte suspension respectively, adding polyoxyethylene glycol makes cell fusion together, in the mixed cell suspension of two kinds of cells, drip nutrient solution, carry out cell cultures with HAT selective medium.

(3) screening of positive colony

When cell cultures to be fused is to 5-10 days, draw in the hole of 96 well culture plates and occur that the culture supernatant enzyme-linked immunosorbent assay method of clone cell bunch detects anti-body contg, three subclone screenings are carried out through limiting dilution, secretion situation according to antibody filters out the antibody-secreting hole of high-titer, cell expansion in hole is cultivated, then carries out antigen specific immune histological chemistry in-site detecting, select high-titer, the cell strain of high specific, called after 16-5.

(4) acquisition of variable region of mab sequence

Utilize Trizol (Invitrogen) method to extract the total serum IgE of hybridoma cell strain 16-5, then use oligo-dT primer to become cDNA with SuperScript II Reverse Transcriptase (lnvitrogen) reverse transcription.Obtain the DNA fragmentation of heavy chain of antibody and light chain through pcr amplification, be connected in cloning vector, picked clones checks order.

(5) purifying of monoclonal antibody

Monoclonal antibody 16-5 adopts Hi-Trap protein A HP(GE Healthcare) affinity column purifying from hybridoma supernatant, SDS-PAGE electrophoresis proves that products therefrom purity is greater than 90%.16-5 after purifying is directly used in following experiment.

Embodiment 2 mouse monoclonal antibody characteristic measurement

(1) qualification of monoclonal antibody immunity immunoglobulin heavy chain and light chain type

Get 16-5 hybridoma cell strain culture supernatant, adopt Rapid ELISA Mouse mAb Isotyping Kit(Pierce company) utilize ELISA method to measure heavy chain of antibody, light chain type.Through qualification, 16-5 hypotype is IgGa2b/kappa.

(2) indirect elisa method measures the relative affinity of monoclonal antibody

With the people VEGF standard substance bag of 0.5 μ g/ml by enzyme mark version, every hole 100 μ l.37 DEG C of incubations 2 hours, with containing after the PBS washing of 1%Tween-20, close 2 hours.Be that 1 μ g/ml does three times of dilutions by monoclonal antibody 16-5 with initial concentration, using commercialization monoclonal antibody avastin as comparison, 37 DEG C of incubations 1 hour.After 5 washings, add that 100 μ l HRP mark respectively corresponding two resist, 37 DEG C of incubations 1 hour.After 5 washings, add 50 μ l nitrite ions, 37 DEG C of incubations 15 minutes, 50 μ l2M sulfuric acid termination reactions, OD ₄₅₀reading.

After four parameter fittings, the EC50 of 16-5 is 40pM.

Table 1: indirect elisa method measures the relative affinity of monoclonal antibody

Figure 1 shows the relative affinity that indirect elisa method measures monoclonal antibody.

(3) specific detection of monoclonal antibody

The each 0.5 μ g/ml wrapper sheet of employment VEGF and mouse VEGF respectively, 4 DEG C are spent the night; Add 16-5 and avastin monoclonal antibody solution 100 μ l/ hole respectively, 37 DEG C of incubations 1 hour.After washing, add that 100 μ l HRP mark respectively corresponding two resist, 37 DEG C of incubations 1 hour.After washing, add 50 μ l nitrite ions, 37 DEG C of incubations 15 minutes, 50 μ l2M sulfuric acid termination reactions, OD ₄₅₀reading.The results are shown in following table 2.

Table 2: the specific detection of monoclonal antibody

16-5	Avastin
		0.195nM	N/A

Visible, 16-5 can simultaneously with people VEGF and mouse VEGF cross reaction, and Avastin only reacts with people VEGF.16-5 can do animal model with mouse.

(4) initial analysis of monoclonal antibody action epi-position.

Whether Inhibition ELISA mensuration 16-5 overlaps with the epitope of Avastine: the enzyme plate of people VEGF antigen is added toward bag the 16-5 that starting point concentration is 10ug/ml, adding simultaneously or do not add concentration is 100ug/ml Avastine.Final according to OD ₄₅₀reading judges that whether 16-5 is identical with the determinant of Avastine.The results are shown in Table 3.

Table 3: Inhibition ELISA measures the epitope of 16-5 with Avastine

In the 5# monoclonal antibody diluent of each concentration corresponding, when adding the Avastine monoclonal antibody of 100ug/ml, as broad as long (the particularly 16-5 monoclonal antibody of lower concentration of colour developing value of the 16-5 under same concentration, its colour developing value is not still subject to the impact adding 100ug/mlAvastine monoclonal antibody), zero lap between the antigen table tail that both explanations identify.

The activity research of embodiment 3 mouse monoclonal antibody

(1) ELISA method measures the hindrance function that 16-5 is combined with its acceptor VEGFR1 VEGF

After VEGFR1 wrapper sheet, add the 16-5 of 40ug/ml VEGF-his and different concns, compare with avastin.OD ₄₅₀after reading, the hindrance function that analysis list clonal antibody is combined with its acceptor VEGFR1 VEGF.

Figure 2 shows the hindrance function that 16-5 is combined with its acceptor VEGFR1 VEGF.

As shown in Figure 2, OD ₄₅₀reading is lower, and the impedance that instruction book clonal antibody combines VEGF and VEGFR1 is more obvious.In 2 concentration selected by us, mouse-anti 16-5 shows the restraining effect stronger than avastin.When not adding monoclonal antibody, OD ₄₅₀be 1.5.

(2) ELISA method measures the hindrance function that 16-5 is combined with its acceptor VEGFR2 VEGF

After VEGFR2 wrapper sheet, add the 16-5 of 40ug/ml VEGF-his and different concns, compare with avastin.OD ₄₅₀after reading, the hindrance function that analysis list clonal antibody is combined with its acceptor VEGFR2 VEGF.

Figure 3 shows the hindrance function that 16-5 is combined with its acceptor VEGFR2 VEGF.

As shown in Figure 3, OD ₄₅₀reading is lower, and the impedance that instruction book clonal antibody combines VEGF and VEGFR2 is more obvious.In 3 concentration selected by us, during high density, mouse-anti 16-5 shows the restraining effect stronger than avastin.During low concentration, mouse-anti 16-5 shows the restraining effect relatively more weak than avastin.When not adding monoclonal antibody, OD ₄₅₀be 1.0.So mouse-anti 16-5 and avastin in higher concentrations, has shown stronger restraining effect.

Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention when not departing from principle of the present invention and aim, revising, replacing and modification.

Claims (6)

1. the monoclonal antibody of an anti-vegf, it is characterized in that, it has the variable region of heavy chain of three hypervariable regions containing, for example lower aminoacid sequence: CDRH1(SEQ ID NO.1:TYGMS), CDRH2(SEQ ID NO.2:TISNGGSYTFYPDSVKG), CDRH3(SEQ ID NO.3:HGSVTTRGFDY).

2. the monoclonal antibody of anti-vegf according to claim 1, it is characterized in that, it is and have the variable region of light chain of three hypervariable regions containing, for example lower aminoacid sequence: CDRL1(SEQ ID NO.4:KASQSVSNDVV), CDRL2(SEQID NO.5:YASNRYT), CDRL3(SEQ ID NO.6:LQDYSSPFT).

3. monoclonal antibody according to claim 1 and 2, it is characterized in that, described monoclonal antibody comprises the derivative of single-chain antibody, double-chain antibody, chimeric antibody, humanized antibody and above-mentioned antibody, function equivalent or homologue, also comprises antibody fragment and any polypeptide containing antigen-binding domains.

4. monoclonal antibody according to claim 1, is characterized in that, its heavy chain amino acid sequence is as shown in SEQ ID NO.7.

5. monoclonal antibody according to claim 2, is characterized in that, its light-chain amino acid sequence is as shown in SEQ ID NO.8.

6. the DNA sequence dna of monoclonal antibody according to claim 1 and 2, is characterized in that, its heavy chain nucleotide sequence is as shown in SEQ ID NO.9; Its light chain nucleotide sequence is as shown in SEQ ID NO.10.