CN103965355A - Fully human-derived antibody heavy chain and light chain variable regions aiming at VEGFR2, corresponding Fab and full-length antibody - Google Patents

Abstract

The present invention relates to the heavy chain and light chain variable regions of fully human antibodies against VEGFR2 and the corresponding Fab and full-length antibodies. More specifically, the present invention uses a human monoclonal antibody phage display library to screen the heavy chain and light chain variable regions of a monoclonal antibody against human VEGFR2, and uses the variable regions to prepare the corresponding Fab fragments and complete long antibody. The antibodies of the present invention can effectively affect the VEGF/VEGFR2 interaction.

Description

Fully human antibody heavy and light chain variable regions and corresponding Fab and full-length antibody against VEGFR2

technical field

The present invention relates to the heavy chain and light chain variable regions of fully human antibodies against VEGFR2 and the corresponding Fab and full-length antibodies. More specifically, the present invention relates to the heavy chain and light chain variable regions of fully human antibodies against VEGFR2 screened by phage display technology, as well as antibody Fab fragments and full-length antibodies containing the variable regions.

Background technique

Angiogenesis not only plays a key role in normal development, but also plays an important role in some diseases, such as tumors. Tumor cell proliferation and tumor volume enlargement must be accompanied by angiogenesis to provide nutrients and oxygen for tumor cells while removing metabolic waste.

Angiogenesis is a complex physiological process, and existing evidence shows that uncontrolled angiogenesis is the main reason for tumor proliferation and metastasis. Angiogenesis is also closely related to other diseases, including diabetic retinopathy, rheumatoid arthritis and psoriasis. Therefore, inhibition of angiogenesis has become a feasible method for the treatment of these diseases.

In the process of angiogenesis, vascular endothelial cell growth factor VEGF (vascular endothelial growth factor) plays an extremely important role, and VEGF can strongly stimulate the proliferation of vascular endothelial cells. VEGF is also called VEGF-A, and the VEGF family also includes members such as PIGF, VEGF-B, VEGF-C, and VEGF-D. VEGF regulates the growth of normal and pathological blood vessels, while VEGF-C and VEGF-D mainly regulate lymphatic angiogenesis. Among them, VEGF gene expression produces four isoforms through selective splicing of its exons, namely VEGF ₁₂₁ (containing 121 amino acids), VEGF ₁₆₅ (containing 165 amino acids), VEGF ₁₈₉ (containing 189 amino acids) and VEGF ₂₀₆ (contains 206 amino acids). Among them, VEGF ₁₆₅ is the main expression isoform of VEGF, which is closely related to various physiology and pathology. It is a secreted protein, but it can also bind to the cell surface and extracellular matrix through heparin. Secreted VEGF exists as a dimer in which two monomers are linked together by two disulfide bonds. VEGF is a multifunctional cytokine that is induced during hypoxia and oncogenic mutations. VEGF is a strong inducer of vascular penetration, endothelial cell chemoattractant, endothelial cell survival and proliferation growth factor.

The receptors to which VEGF binds include VEGFR1 (also known as Flt-1 ) and VEGFR2 (also known as Flk-1 ). Existing evidence suggests that VEGFR2 mediates multiple effects of VEGF, including endothelial cell proliferation, angiogenesis, and infiltration, although VEGF binds to VEGFR1 with greater affinity. While VEGFR1 does not appear to be directly involved in endothelial cell proliferation and angiogenesis, VEGFR1 is involved in VEGF-induced migration of monocytes and macrophages.

VEGFR1 and VEGFR2 belong to the type III receptor tyrosine kinase family, which is characterized by seven IgG-like domains in the extracellular region, a cell transmembrane region in the middle, and a tyrosine kinase domain in the cell. VEGFR3 binds to VEGF-C and VEGF-D and plays a role in the development of lymphatic vessels. VEGF dimer binding to VEGFR2 induces receptor dimerization, tyrosine phosphorylation of the intracellular tyrosine kinase domain, thereby activating downstream signaling pathways, including activating phospholipase C, increasing intracellular calcium ion concentration, etc. .

Therefore, VEGF and VEGFR2 are good therapeutic targets. Inhibiting this signaling pathway can theoretically inhibit angiogenesis, thereby achieving the treatment of angiogenesis-related diseases. At present, there are many methods to inhibit VEGF signaling pathway, including VEGF humanized monoclonal antibody, VEGFR small molecule inhibitor and so on. Given the important role of VEGFR2 in the VEGF signaling pathway, inhibition of VEGFR2 should be a strategy to inhibit angiogenesis.

Inhibitory antibodies against VEGFR2 can be used to treat angiogenesis-related diseases, including tumors such as solid and non-solid tumors, neovascular glaucoma, coronary artery disease, inflammatory diseases such as rheumatoid arthritis, ophthalmic diseases such as diabetic retinopathy and Age-related macular degeneration and other diseases.

Antibodies are an important class of protein molecules used to treat human diseases. Monoclonal antibodies have been widely used clinically as drugs due to their high specificity for targets. Antibodies are a class of glycoproteins that share the same structural features. Natural antibodies are usually a class of tetrameric glycoproteins with a molecular weight of 150kD, consisting of two identical light chains and two identical heavy chains. Each light chain is connected to a heavy chain by a disulfide bond, and the two heavy chains are also connected by a disulfide bond. Each heavy and light chain is composed of variable and constant regions, respectively. The heavy chain constant partitions are α, δ, ε, γ, and μ, and the light chain constant partitions are kappa (κ) and lambda (λ). The variable region determines the specificity of the antibody and its affinity for the antigen. The variable region includes 3 hypervariable regions (CDR regions) and 4 framework regions (FR regions), and a hypervariable region connects the two framework regions. The constant region of the heavy chain contains three domains (CH1, CH2 and CH3), and the constant region of the light chain contains one domain, CL.

Since antibodies derived from animals will produce an immune response when used in humans, antibodies derived from animals must be humanized. In addition, with the development of technology, fully human monoclonal antibodies can be obtained through phage display technology and other technologies, such as ribosome display technology and antibody humanized mouse technology. Therapeutic monoclonal antibodies currently used clinically include chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. Among them, the fully human monoclonal antibody is the most popular because of its high affinity and low immunity.

The phage display technology of antibodies is to display antibody V region fragments fused to phage surface protein (g3) on the surface of phages, and then screen the phage library against different antigens to finally obtain specific antibodies. The display and screening of antibody fragments on the surface of phages is actually a simulation of immune screening of the body, and antibodies can be screened from artificially established phage libraries. The same library can screen multiple different monoclonal antibodies against different antigens. The diversity of antibody molecules included in the phage antibody library and the size of the library capacity determine the affinity of the screened antibody. At present, there are many methods to construct antibody libraries, including directly amplifying the V region fragments of heavy chain and light chain from B cell cDNA by PCR, and cloning them into different vectors after random combination in vitro to obtain scFv or Fab libraries. These B cells can be derived from individuals immunized with antigens or from non-immunized individuals. This antibody library directly derived from individuals can screen high-affinity antibodies against different antigens, including self-antigens and toxic antigens. At the same time, different regions of antibody V region fragments can also be amplified or artificially synthesized in vitro to obtain antibody scFv or Fab libraries.

The function of antibody phage display can not only screen specific antibodies against different antigens, but also further mutate the hypervariable regions of the obtained antibodies to improve their affinity and enhance their therapeutic efficacy.

Therefore, the present invention attempts to use phage display technology to screen the heavy chain and light chain sequences of fully human monoclonal antibodies against VEGFR2, and then use genetic engineering methods to obtain complete antibodies, thereby providing corresponding anti-angiogenesis disease treatment. Drug.

Contents of the invention

Therefore, the technical purpose of the present invention is to use phage display technology to obtain the heavy chain and light chain sequences of a fully human monoclonal antibody against VEGFR2, and then use genetic engineering methods to obtain the complete antibody or its functional fragments.

Therefore, the first aspect of the present invention relates to a fully human monoclonal antibody for mammals including human VEGFR2, its functional fragments or derivatives thereof, the sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain are respectively the same as SEQ The sequences shown in ID NO.31, SEQ ID NO.32 and SEQ ID NO.33 have at least 80% identity, and the sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the light chain are respectively selected from the following groups The sequences share at least 80% identity:

Group 1: SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36;

Group 2: SEQ ID NO.37, SEQ ID NO.38 and SEQ ID NO.39;

Group 3: SEQ ID NO.40, SEQ ID NO.41 and SEQ ID NO.42;

Group 4: SEQ ID NO.43, SEQ ID NO.44 and SEQ ID NO.45;

Group 5: SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48;

Group 6: SEQ ID NO.49, SEQ ID NO.50 and SEQ ID NO.51;

Group 7: SEQ ID NO.52, SEQ ID NO.53 and SEQ ID NO.54;

Group 8: SEQ ID NO.55, SEQ ID NO.56 and SEQ ID NO.57;

Group 9: SEQ ID NO.58, SEQ ID NO.59 and SEQ ID NO.60;

Group 10: SEQ ID NO.61, SEQ ID NO.62 and SEQ ID NO.63;

Group 11: SEQ ID NO.64, SEQ ID NO.65 and SEQ ID NO.66;

Group 12: SEQ ID NO.67, SEQ ID NO.68 and SEQ ID NO.69;

Group 13: SEQ ID NO.70, SEQ ID NO.71 and SEQ ID NO.72; or

Group 14: SEQ ID NO.73, SEQ ID NO.74 and SEQ ID NO.75.

Preferably, said identity is at least 85%, preferably at least 90%, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, Most preferably, 100%.

Preferably, the heavy chain constant region of the fully human monoclonal antibody is selected from α, δ, ε, γ or μ or a sequence having at least 80% identity with α, δ, ε, γ or μ, and the light chain is constant The region is selected from kappa or lambda or a sequence having at least 80% identity to kappa or lambda, preferably said identity is at least 85%, preferably at least 90%, more preferably at least 91%, 92%, 93% %, 94%, 95%, 96%, 97%, 98% or 99%, most preferably, 100%.

Preferably, the identity of its heavy chain sequence to the sequence shown in SEQ ID NO.2 is at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably, 100%.

Preferably, its light chain sequence is at least 80% identical to a sequence such as one of the following sequences: SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28 or SEQ ID NO.30, preferably at least 85%, preferably at least 90%, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 %, most preferably, 100%.

Preferably, the type of the antibody is IgA, IgD, IgE, IgG or IgM.

Preferably, the type of the antibody is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

Preferably, the functional fragment or derivative is (Fab')2, Fab, single-chain antibody, single-chain sv, multivalent single-chain antibody, bispecific antibody or trispecific antibody.

The second aspect of the present invention relates to a nucleotide sequence encoding a fully human monoclonal antibody against mammals including human VEGFR2 as described in the first aspect above, a functional fragment thereof or a derivative thereof.

Preferably, the coding sequence of its heavy chain is at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 91%, 92% identical to the sequence shown in SEQ ID NO.1 %, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably, 100%; the sequence of the light chain is at least 80% identical to a sequence selected from the group consisting of: SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27 or SEQ ID NO.29, preferably at least 85%, preferably at least 90%, more preferably , at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably, 100%.

The third aspect of the present invention relates to a fully human monoclonal antibody against mammals including human VEGFR2 according to the above first aspect, its functional fragments or derivatives thereof or the nucleotides according to the above second aspect Use of the sequence in the preparation of a medicament for blocking VEGF/VEGFR2 interaction.

The fourth invention of the present invention relates to a fully human monoclonal antibody against mammals including human VEGFR2 according to the above first aspect, its functional fragment or its derivative or the nucleoside according to the above second aspect Use of the acid sequence in the preparation of medicines for treating tumor and non-tumor diseases.

Preferably, the tumor is primary, metastatic and refractory various solid tumors and non-solid tumors, preferably, the solid tumor is selected from breast cancer, lung cancer, colon cancer, pancreatic cancer, glioma , head and neck cancer, prostate cancer, ovarian cancer, liver cancer, melanoma, stomach cancer, kidney cancer or skin cancer, non-solid tumors are selected from various leukemias, lymphomas or myeloma; preferably, the non-tumor diseases are selected from neovascularization glaucoma, coronary artery disease, inflammatory disease preferably rheumatoid arthritis, ophthalmic disease preferably diabetic retinopathy or age-related macular degeneration.

The fifth aspect of the present invention relates to a kind of main active ingredient is the fully human monoclonal antibody against mammals including human VEGFR2 according to the above first aspect, its functional fragment or its derivative or according to the above second aspect composition of nucleotide sequences.

Preferably, the composition further contains chemotherapeutic drugs, tumor suppressors, preferably small molecule compound inhibitors, or monoclonal antibodies, preferably anti-EGFR, IGF1R or PDGFR monoclonal antibodies.

Preferably, the fully human monoclonal antibody against mammals including human VEGFR2, its functional fragment or its derivative according to the first aspect above is coupled with cytotoxin.

In other words, the heavy chain and light chain (including framework region and CDR region) of the VEGFR2 monoclonal antibody of the present invention are fully human sequences obtained by phage display technology (by screening phage libraries containing fully human sequences). The antibody can bind to the extracellular region of VEGFR2, block the binding of VEGF to VEGFR2, inhibit the activity of VEGF in promoting the proliferation of endothelial cells, and thus inhibit the growth of tumors.

The heavy chain and light chain sequences screened and identified in the present invention can be used to obtain complete antibody IgA, IgD, IgE, IgG or IgM by means of genetic engineering. The heavy chain constant regions of these antibodies correspond to α, δ, ε, γ, and μ, respectively, and the light chain constant regions of these antibodies may be kappa (κ) and lambda (λ). These antibodies may include subtypes thereof, such as IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, and the like. Other forms of monoclonal antibodies can also be obtained by genetic engineering, including (Fab')2, Fab, single-chain antibody, single-chain sv (SCFV), multivalent single-chain antibody, bispecific antibody, trispecific antibody and any other form of antibody.

Those skilled in the art know that the key factors that determine the specificity and other properties of antibodies are the variable region sequences of their heavy and light chains (especially the sequences of CDR1, CDR2 and CDR3 of the heavy and light chains), the constant regions and the complementarity determination The framework region sequence of the interval can be appropriately changed without substantially affecting the specificity and other properties of the antibody. The appropriate changes include conservative amino acid substitutions and the like.

The antibody of the invention can treat the following tumors, including but not limited to the following tumors, including primary, metastatic and refractory various solid tumors and non-solid tumors. Solid tumors include breast cancer, lung cancer, colon cancer, pancreatic cancer, glioma, head and neck cancer, prostate cancer, ovarian cancer, liver cancer, melanoma, stomach cancer, kidney cancer, skin cancer. Non-solid tumors include various leukemias, lymphomas, myelomas and other diseases.

The antibody of the invention can also treat the following non-neoplastic diseases, including but not limited to the following non-neoplastic diseases, including neovascular glaucoma, coronary artery disease, inflammatory diseases such as rheumatoid arthritis, ophthalmic diseases such as diabetic retinopathy and senile retina diseases such as macular degeneration.

The antibody of this invention can be used alone or in combination with other drugs, including various chemotherapy drugs, tumor suppressors such as small molecule compound inhibitors and monoclonal antibodies (such as monoclonal antibodies against EGFR, IGF1R, PDGFR).

The antibodies of the invention can also be conjugated to other compounds, such as cytotoxins, for the treatment of tumors.

The antibodies of the present invention provide new drugs for the treatment of diseases targeting angiogenesis.

Description of drawings

Figure 1: Phage Elisa values.

Figure 2: Identification of positive clones with blocked VEGF/VEGFR2 interaction.

Figure 3: Positive Cloned Fab Protein Blocks VEGF/VEGFR2 Interaction.

Figure 4: Positive clone Fab protein blocks VEGF-stimulated proliferation of human umbilical cord endothelial cells.

Figure 5: Positive Clone IgG1 Full Length Antibody Blocks VEGF/VEGFR2 Interaction.

Figure 6: The positive clone IgG1 full-length antibody blocks the proliferation of human umbilical cord endothelial cells stimulated by VEGF.

Detailed ways

The present invention will be further illustrated by the following non-limiting examples below. It is well known to those skilled in the art that many modifications can be made to the present invention without departing from the spirit of the present invention, and such modifications also fall within the scope of the present invention.

The following experimental methods are conventional methods unless otherwise specified, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified.

Example

Example 1. Construction of Human Monoclonal Antibody Phage Display Library

The present invention first constructs a scFv phage display library according to the literature report of Marks J et al ^. (J Mol Biol, 1991, 222, p581-597). XL1-Blue bacteria.

Example 2. Screening scFv phage library to obtain positive monoclonal anti-human VEGFR2

The screening steps are as follows:

1. Take 10 ml of XL1-Blue bacteria from the scFv library, inoculate them into 1 L of 2YTAG medium, culture them at 30°C to the logarithmic growth phase, add M13-K07 helper phage, and amplify overnight (30°C, 2YTAK culture base). Amplified phage were precipitated with 20% PEG8000, then resuspended in 100 ml PBS (20 mM phosphate pH 7.4) and phage titers were quantified.

2. Fix the antigen VEGFR2-Fc to the Maxisorp Star tube by conventional technical means, add milk to block for 1 hour (37°C), then add phage, wash with PBST (20mM phosphate 0.05%Tween-20pH7.4) for 15 times, wash with Wash with PBS 15 times. Bound phage were eluted with 0.1 M HCL, pH 2.2.

3. Infect XL1-Blue bacteria with the eluted phage, spread the infected bacteria on several 2YTAG plates, and incubate overnight at 37°C.

4. Randomly select 200 clones for phage Elisa.

5. Inoculate a single XL1-Blue clone into a 96-well plate, add M13K07 helper phage, and leave overnight. The amplified phage was blocked with milk, added to a 96-well plate coated with human VEGFR2-Fc, incubated for 1 hour, the plate was washed 4 times with PBST (same as above), and then rabbit anti-M13 phage-HRP conjugate was added . The plate was washed 4 times with PBST (same as above), added TMB peroxygen substrate, read at 450nm, the results are shown in Figure 1. Phage Elisa results showed that the Elisa readings of monoclonal 1D4B5, 1E10C3, 3D2G1, 3E10G9 and 3F1G10 were greater than 1.1 after 1:50 dilution of the phage supernatant, indicating that these clones had high affinity for VEGFR2 protein. Using a similar method, the monoclonal A8E2, 1H4D7, 1H6D8, 3E7G7, 1G2C10, 1F3C5, 1G4C11, 3C4F5 and 3D8G3 obtained in the present invention also have high affinity to VEGFR2 protein. The common feature of the fourteen antibodies obtained above is that their heavy chain sequences are the same, and the difference is only in the light chain sequences, but they all have high affinity for VEGFR2 protein.

Example 3. Screening for positive monoclonal antibodies that block VEGF/VEGFR2 interaction

Randomly select monoclonal 1D4B5, 1E10C3, 3D2G1, 3E10G9, 3F1G10 for VEGF/VEGFR2 interaction blocking screening test, the screening steps are briefly described as follows:

1. Inoculate the positive clone XL1-Blue bacteria that can bind to VEGFR2 into 2YTAG medium, add M13-K07 helper phage, and amplify overnight (30°C, 2YTAK medium). The resulting phages were mixed with VEGFR2 and incubated for 1 hour, and then transferred to VEGF-coated 96-well plates. After incubation for 1 hour, the plate was washed 4 times with PBST (same as above), and goat anti-human IgG-Fc-HRP conjugate was added. The plates were washed 4 times with PBST (same as above), and TMB peroxy substrate was added, and read at 450 nm.

2. Identify positive clones that can block VEGF/VEGFR2 interaction. Phage Elisa results show that monoclonal 1D4B5, 1E10C3, 3D2G1, 3E10G9, and 3F1G10 all have blocking effects. The results are shown in Figure 2. Similarly, the results of phage Elisa showed that monoclonal A8E2, 1H4D7, 1H6D8, 3E7G7, 1G2C10, 1F3C5, 1G4C11, 3C4F5 and 3D8G3 also had blocking effects.

3. Perform DNA sequencing on the positive clones that block the VEGF/VEGFR2 interaction, and obtain the heavy chain and light chain variable region sequences of the positive clones, wherein the heavy chain sequences of the antibodies are all shown in SEQ ID NO.2, which The nucleotide coding sequence is shown in SEQ ID NO.1, and the sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO.31-33, respectively. The light chain sequences of the above fourteen antibodies and the sequences of their complementarity determining regions are shown in Table 1.

Table 1

Example 4, Expression and Purification of Positive Monoclonal Antibody Fab Fragment

Randomly select the heavy chain and light chain variable regions of monoclonal 1D4B5 and 3E10G9 to prepare the corresponding monoclonal antibody Fab fragments, the steps are as follows:

1. The heavy chain and light chain variable region sequences of the above positive clones were recombined and cloned into the corresponding positions of the Fab expression plasmid pFab-1 (SfiI/NheI restriction site and ApaL1/BsiwI restriction site). The pFab-1 plasmid was constructed according to the literature report of Presta L et al. (Cancer Research, Presta L, 1997, 57, P4593-4599).

2. Induced expression

Transfect the obtained recombinant expression plasmid into bacteria HB2151, induce expression with IPTG, and select positive clones (simple high-expression clones by Western blotting); inoculate high-expression clones into 2YTAG medium at 30°C for logarithmic growth phase, and transfer into 2YTAI In the culture medium, 30°C was routinely induced overnight.

3. Purification

The induced bacterial pellet was collected by centrifugation, resuspended with TES buffer, placed at 4°C for 1 hour, and the supernatant was collected by centrifugation. The supernatant was purified with Ni column to obtain Fab protein.

Example 5, positive clone Fab protein blocks VEGF/VEGFR2 interaction

To verify whether the purified Fab protein obtained in Example 4 can block the interaction of VEGF/VEGFR2, the method is as follows:

1. Coat an effective amount of VEGF165 into the wells of a 96-well plate by conventional methods (200ng/well);

2. Mix different concentrations of positive Fab proteins (100nM to 0.8nM doubling dilution) with VEGFR2, incubate for 1 hour and add to the wells of a 96-well plate coated with VEGF165; wash the plate 4 times with PBST (same as above), Then add goat anti-human IgG-Fc-HRP conjugate.

3. After incubation for 1 hour, wash the plate with PBST (same as above) 4 times, add TMB peroxygen substrate, and read at 450nm with a BioTek microplate reader. The results show that the Fab proteins of monoclonal 1D4B5 and 3E10G9 have a strong blocking effect (image 3).

Example 6, Positive Clone Fab Protein Blocks VEGF to Stimulate the Proliferation of Human Umbilical Cord Endothelial Cells

In order to verify the physiological activity of the obtained Fab protein at the cellular level, the present invention uses the above-mentioned purified positive clone Fab protein to carry out an experiment of blocking VEGF to stimulate the proliferation of human umbilical cord endothelial cells, the method is as follows:

1. Obtain primary cells of human umbilical cord endothelial cells (HUVEC) from human umbilical cords using standard operating methods. In a preheated PBS culture dish, cut off the parts with clamp marks and hematoma, squeeze out the blood in the umbilical cord, trim the two sections with scissors, and divide them into 20cm sections; find out the umbilical veins (two with smaller lumens) The umbilical artery is the umbilical artery, and the one with a thicker lumen is the umbilical vein). Insert a syringe with a flat needle into the umbilical vein from one section, fix it with a blood vessel clamp, and rinse with preheated warm PBS for more than 3 times to completely wash away the blood. In order to prevent the residual blood from the umbilical artery from mixing, the artery can be separated a little and ligated with sterile thread, and the other end can be clamped with a hemostat, and 0.1% collagenase I (purchased from Sigma) can be injected from the flushed needle to make it full; take out the needle , clamp the injection end with a hemostatic forceps, move it into a sterile beaker, and incubate in a 37°C incubator for 15 minutes. Take it out, loosen the blood vessel clamp at the injection end, collect the digestive juice in the umbilical vein, and then inject PBS to rinse the lumen again , collect the digestion solution and washing solution together in a centrifuge tube, centrifuge at 1000r/min for 5 minutes, discard the supernatant. Resuspend the cells in 40ml PBS, centrifuge at 1000r/min for 5 minutes, discard the supernatant; Gently pipette the cells with a Pasteur pipette until uniform, inoculate 1×10 ⁶ cells/petri dish on a petri dish, place in a 5% CO ₂ incubator at 37°C, and replace the culture medium after 24 hours to remove unpasted cells. Then the medium was changed semi-quantitatively every 24 hours until the cells grew confluent.), and then the HUVEC primary cells were resuspended in EBM-2 (purchased from Clonetics), 2% fetal bovine serum, VEGF-free medium Inoculate in a 96-well culture plate, 5000×10 ³ cells/well, and culture in a 5% CO ₂ incubator at 37°C for 24 hours.

2. Add purified anti-VEGFR2 Fab protein at different concentrations (100nM to 3.125nM doubling dilution), add VEGF165 (concentration 50ng/ml) after 1 hour, and incubate in a 5% CO ₂ incubator at 37°C for 72 hours.

3. After adding cck-8 (purchased from Dojindo) and incubating for 4.5 hours, read (read at 450 nm with a BioTek microplate reader, the results show that the Fab proteins of monoclonal 1D4B5 and 3E10G9 can inhibit the proliferation of HUVEC, the results are shown in Figure 4).

Example 7. Expression and purification of positive clones in the form of full-length antibody IgG1

The present invention further randomly selects the heavy chain and light chain variable regions of monoclonal 1D4B5 and 3E10G9 to prepare the corresponding full-length monoclonal antibody (IgG1 format), and obtains a heavy monoclonal antibody containing the fully human-derived monoclonal antibody obtained in the present invention. Full-length antibodies (IgG1 format) to the variable regions of the chain and light chains, the method is briefly described as follows:

1. Recombinant expression vector: clone the variable regions of the light chain and heavy chain of positive clones into the corresponding regions of the mammalian cell expression vector pG1 (DraIII/BsiWI restriction site and MluI/NheI restriction site). pG1 was rebuilt from pCI-neo (Promega) (the rebuilding method is as follows: the heavy chain expression element (composed of CMV promoter, MluI/NheI polyclonal restriction site sequence, constant region of human antibody heavy chain gamma1 and SV40 tailed) Sequence composition) to replace the sequence of the BglII/XhoI restriction site of pCI-neo; the light chain expression element (composed of CMV promoter, DraIII/BsiWI polyclonal restriction site sequence, constant region of human antibody light chain Kappa and SV40 The sequence consisting of tailing sequence) replaces the sequence of the XhoI/XbaI restriction site of pCI-neo; the expression element of DHFR (dihydrofolatereductase) (composed of SV40 promoter, mouse DHFR expression gene and SV40 tailing sequence) replaces the sequence of pCI-neo Sequence of XbaI/NotI restriction site). pG1 contains the coding sequence of the light chain kappa constant region and the heavy chain gamma1 constant region, including CH1, hinge, CH2 and CH3 domains. The heavy chain and light chain coding sequences on pG1 are respectively induced by a CMV promoter.

2. Induced expression: The constructed mammalian cell expression plasmid pG1 was electroporatedly transfected into CHO cells, and the transfected cells were plated in six 96-well culture plates and cultured in a 5% CO ₂ incubator at 37°C. Two weeks later, single clones of cells were selected (only one germinal center under the microscope) for ELISA identification, and positive clones were obtained. The obtained positive clones were further screened, and MTX was added to the culture medium to induce antibody expression to obtain high-expression cell lines. The high-expression cell line was transferred to a serum-free medium (CD OptiCHO, Invitrogen) for amplification, and then transferred to a spinner bottle (500ml) for expression in a cell culture incubator at 37°C and 5% CO ₂ (add 50nM of MTX).

3. Purification: Collect the expressed cell culture supernatant and purify it with a Protein G column to obtain the protein in the form of full-length antibody IgG1.

Example 8, positive clone IgG1 full-length antibody blocks VEGF/VEGFR2 interaction

This example explores the influence of the full-length IgG1 antibody comprising the heavy chain and light chain variable regions of the fully human monoclonal antibody against VEGFR2 obtained in the present invention on the VEGF/VEGFR2 interaction. The method is briefly described as follows:

1. Coat an effective amount of VEGF165 into the wells of a 96-well plate by conventional methods (200ng/well);

2. Mix different concentrations of positive IgG1 full-length antibody (20nM to 0.04nM doubling dilution) with VEGFR2, incubate for 1 hour and add to the wells of a 96-well plate coated with VEGF165; wash the plate with PBST (same as above) for 4 The next time, goat anti-human IgG-Fc-HRP conjugate was added.

3. After incubation for 1 hour, wash the plate with PBST (same as above) 4 times, add TMB peroxygen substrate, and read at 450nm with a BioTek microplate reader. The results show that the full-length proteins of monoclonal 1D4B5 and 3E10G9 have strong blocking role (Figure 5).

Example 9, positive clone IgG1 full-length antibody blocks VEGF-stimulated proliferation of human umbilical cord endothelial cells

In order to verify the physiological activity of the obtained positive clone IgG1 full-length antibody at the cellular level, the present invention uses the above-mentioned purified positive clone IgG1 full-length antibody to carry out an experiment of blocking VEGF-stimulated human umbilical cord endothelial cell proliferation, the method is as follows:

1. Obtain primary cells of human umbilical cord endothelial cells (HUVEC, as above) from human umbilical cord using standard operating methods, and then resuspend the primary cells of HUVEC in EBM-2 (purchased from Clonetics), 2% fetal bovine serum, VEGF-free The culture medium was inoculated into 96-well culture plates, 5000×10 ³ cells/well, and cultured in a 5% CO ₂ incubator at 37°C for 24 hours.

2. Add different concentrations (20nM to 0.625nM multiple dilution) of purified IgG1 full-length antibody against VEGFR2, add VEGF165 (concentration 50ng/ml) after 1 hour, and incubate in a 5% CO ₂ incubator at 37°C for 72 hours .

3. After adding cck-8 and incubating for 4.5 hours, read (read at 450 nm with a BioTek microplate reader, the results show that both 1D4B5 and 3E10G9 can inhibit the proliferation of HUVEC, the results are shown in Figure 6).

Claims (16)

1. one kind comprises total man's resource monoclonal antibody, its function fragment or derivatives thereof of human VEGFR-3 2 for Mammals, complementary determining region CDR1, the CDR2 of its heavy chain and the sequence of CDR3 have at least 80% identity with the sequence shown in SEQ ID NO.31, SEQ ID NO.32 and SEQ ID NO.33 respectively, and complementary determining region CDR1, the CDR2 of its light chain and the sequence of CDR3 have respectively at least 80% identity with the sequence that is selected from following group:

Group 1:SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36;

Group 2:SEQ ID NO.37, SEQ ID NO.38 and SEQ ID NO.39;

Group 3:SEQ ID NO.40, SEQ ID NO.41 and SEQ ID NO.42;

Group 4:SEQ ID NO.43, SEQ ID NO.44 and SEQ ID NO.45;

Group 5:SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48;

Group 6:SEQ ID NO.49, SEQ ID NO.50 and SEQ ID NO.51;

Group 7:SEQ ID NO.52, SEQ ID NO.53 and SEQ ID NO.54;

Group 8:SEQ ID NO.55, SEQ ID NO.56 and SEQ ID NO.57;

Group 9:SEQ ID NO.58, SEQ ID NO.59 and SEQ ID NO.60;

Group 10:SEQ ID NO.61, SEQ ID NO.62 and SEQ ID NO.63;

Group 11:SEQ ID NO.64, SEQ ID NO.65 and SEQ ID NO.66;

Group 12:SEQ ID NO.67, SEQ ID NO.68 and SEQ ID NO.69;

Group 13:SEQ ID NO.70, SEQ ID NO.71 and SEQ ID NO.72; Or

Group 14:SEQ ID NO.73, SEQ ID NO.74 and SEQ ID NO.75.

2. total man's resource monoclonal antibody, its function fragment or derivatives thereof that comprises human VEGFR-3 2 for Mammals according to claim 1, it is characterized in that described identity is at least 85%, preferably at least 90%, more preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably, 100%.

3. the total man's resource monoclonal antibody that comprises human VEGFR-3 2 for Mammals according to claim 1 and 2, its function fragment or derivatives thereof, the CH that it is characterized in that described total man's resource monoclonal antibody is selected from α, δ, ε, γ or μ or and α, δ, ε, γ or μ have the sequence of at least 80% identity, constant region of light chain is selected from κ or λ or has the sequence of at least 80% identity with κ or λ, preferably, described identity is at least 85%, preferably at least 90%, more preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably, 100%.

4. according to comprising total man's resource monoclonal antibody, its function fragment or derivatives thereof of human VEGFR-3 2 for Mammals described in claims 1 to 3 any one, it is characterized in that its sequence of heavy chain and the identity of the sequence as shown in SEQ ID NO.2 are at least 80%, preferably, at least 85%, preferably at least 90%, more preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably, 100%.

5. according to comprising total man's resource monoclonal antibody of human VEGFR-3 2 for Mammals described in claim 1 to 4 any one, its function fragment or derivatives thereof, it is characterized in that its sequence of light chain with as the identity that is selected from the sequence of one of following sequence be 80%:SEQ ID NO.4 at least, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28 or SEQ ID NO.30, preferably, at least 85%, preferably at least 90%, more preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably, 100%.

6. according to comprising total man's resource monoclonal antibody, its function fragment or derivatives thereof of human VEGFR-3 2 for Mammals described in claim 1 to 5 any one, the type that it is characterized in that described antibody is IgA, IgD, IgE, IgG or IgM.

7. according to comprising total man's resource monoclonal antibody, its function fragment or derivatives thereof of human VEGFR-3 2 for Mammals described in claim 1 to 6 any one, the type that it is characterized in that described antibody is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

8. according to comprising total man's resource monoclonal antibody, its function fragment or derivatives thereof of human VEGFR-3 2 for Mammals described in claim 1 to 7 any one, it is characterized in that described function fragment or derivative are for (Fab ') 2, Fab, single-chain antibody, strand sv, multivalence single-chain antibody, bi-specific antibody or three-specific antibody.

9. a coding is according to comprising total man's resource monoclonal antibody of human VEGFR-3 2, the nucleotide sequence of its function fragment or derivatives thereof for Mammals described in claim 1 to 8 any one.

10. nucleotide sequence according to claim 9, it is characterized in that the encoding sequence of heavy chain and the identity of the sequence as shown in SEQID NO.1 are at least 80%, preferably, at least 85%, preferably at least 90%, more preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably, 100%; The sequence of light chain is at least 80%:SEQ IDNO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27 or SEQ ID NO.29 with the identity of the sequence that is selected from following sequence, preferably, at least 85%, preferably at least 90%, more preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably, 100%.

11. 1 kinds according to comprising total man's resource monoclonal antibody, its function fragment or derivatives thereof of human VEGFR-3 2 or being listed in the purposes of preparing in the interactional medicine of blocking VEGF/VEGFR2 according to the nucleotides sequence described in claim 9 or 10 for Mammals described in claim 1 to 8 any one.

Treat the purposes in the medicine of tumour and non-tumor disease according to comprising total man's resource monoclonal antibody, its function fragment or derivatives thereof of human VEGFR-3 2 or being listed in preparation according to the nucleotides sequence described in claim 9 or 10 for Mammals described in claim 1 to 8 any one for 12. 1 kinds.

13. purposes according to claim 12, it is characterized in that described tumour is idiopathic, metastatic and the various solid tumor of healing property of refractory and non-solid tumour, preferably, solid tumor is selected from mammary cancer, lung cancer, colorectal carcinoma, carcinoma of the pancreas, glioma, head and neck cancer, prostate cancer, ovarian cancer, liver cancer, melanoma, cancer of the stomach, kidney or skin carcinoma, and non-solid knurl is selected from various leukemia, lymphoma or myelomatosis; Preferably, described non-tumor disease is selected from neovascular glaucoma, coronary artery disease, the preferred rheumatic arthritis of diseases associated with inflammation, the preferred Diabetic retinopathy of ophthalmic diseases or the degeneration of senile macula retinae.

14. 1 kinds of main active ingredient are according to comprising total man's resource monoclonal antibody, its function fragment or derivatives thereof of human VEGFR-3 2 or according to the composition of the nucleotide sequence described in claim 9 or 10 for Mammals described in claim 1 to 8 any one.

15. compositions according to claim 14, is characterized in that it also contains chemotherapeutics, the preferred micromolecular compound inhibitor of tumor inhibitor or monoclonal antibody, the monoclonal antibody of preferred anti-EGFR, IGF1R or PDGFR.

16. 1 kinds according to comprising total man's resource monoclonal antibody, its function fragment or derivatives thereof of human VEGFR-3 2 for Mammals described in claim 1 to 8 any one, and its coupling has cytotoxin.