CN116940599A

CN116940599A - Antibody fusion proteins and uses thereof

Info

Publication number: CN116940599A
Application number: CN202280018156.5A
Authority: CN
Inventors: 金磊; 王思勤; 王涛; 郭洪瑞; 闫宏博; 刘宁; 刘爽
Original assignee: Changchun Genescience Pharmaceutical Co Ltd
Current assignee: Changchun Genescience Pharmaceutical Co Ltd
Priority date: 2021-03-01
Filing date: 2022-02-28
Publication date: 2023-10-24
Also published as: WO2022184015A1; CN112961250A; CN112961250B

Abstract

The application provides antibody fusion proteins and uses thereof. The antibody fusion protein has high expression level, high assembly rate, high affinity, convenient purification and good cytological activity and in vivo pharmacodynamic activity.

Description

Antibody fusion proteins and uses thereof

Technical Field

The application relates to the field of medicines, in particular to an antibody fusion protein and application thereof.

Background

Vascular Endothelial Growth Factor (VEGF) proteins and their receptors (VEGFR) play an important role in angiogenesis (the development of embryonic vasculature from early differentiated endothelial cells), angiogenesis (the process of forming new blood vessels from existing vessels), and lymphangiogenesis (the process of forming new lymphatic vessels). Platelet Derived Growth Factor (PDGF) proteins and their receptors (PDGFR) are involved in regulating cell proliferation, survival and migration of several cell types.

Dysfunction of the endothelial cell modulating system is a key feature of cancer and various diseases associated with abnormal angiogenesis, vasculogenesis and lymphangiogenesis. Angiogenesis occurs in embryonic development and normal tissue growth, repair and regeneration, female reproductive cycle, establishment and maintenance of pregnancy, repair of wounds and fractures. In addition to angiogenesis occurring in healthy individuals, angiogenic events are also involved in many pathological processes, especially tumor growth and metastasis, and other conditions in which vascular proliferation (especially of the microvasculature) is increased, such as diabetic retinopathy, psoriasis and arthrosis. Inhibition of angiogenesis may be used to prevent or reduce these pathological processes or to slow their progression.

The etiology of neovascular ophthalmic diseases is complex and the mechanism is not completely clear. The Anti-VEGF drugs have remarkable curative effects in clinical treatment, and replace laser Photocoagulation (PDT) to become a first-line therapy. Both the international raw ranibizumab and Ai Liya are approved by CFDA and marketed in China, and Kangbai cilipp of Cheng Doukang Hongzhu pharmaceutical industry is marketed in 2013, and the 3 medicines can obviously improve the vision of more than about half of people who are treated initially, but about 15% of patients do not respond at all and about 15% -20% of patients are desensitized after anti-VEGF treatment, and the curative effect is lost. The compensatory increase in VEGF-C after neutralization of VEGF-A was found to be an important factor in desensitizing anti-VEGF treatment in wAMD patients.

Bispecific antibodies (bispecific monoclonal antibody, bsAb) are a special antibody that has been engineered to bind two different antigens simultaneously. Bispecific antibodies do not exist in nature and can only be prepared manually. Bispecific or multispecific antibodies in the art can bind to more than 2 antigens and can be produced using cell fusion, chemical conjugation, or recombinant DNA techniques. A wide variety of recombinant bispecific antibody formats have recently been developed, for example by fusing tetravalent bispecific antibodies, such as IgG antibody formats, with single chain domains (see, e.g., co1oma, M.J., et al, nature Biotech.15 (1997) 159-163; WO2001077342; and Morrison, S.L., nature Biotech.25 (2007) 1233-1234). In addition, many other novel forms capable of binding more than 2 antigens have been developed in which the antibody central structure (IgA, igD, igE, igG or IgM) no longer holds as diabodies, triabodies or tetrabodies and several single chain forms (scFv, bis-scFv) (Holliger, p., et al, nature biotech.23 (2005) 1126-1136; fischer, n., and leger, o., pathio 1ogy (2007) 3-14; shen, j., et al, journal 1 of immunology 1 Methods 318 (2007) 65-74; wu, c., et al, nature biotech.25 (2007) 1290-1297).

However, the bispecific antibody proteins reported in the prior art have low expression levels, and have undesirable affinity and assembly rate.

Disclosure of Invention

In view of the above, the application provides an antibody fusion Protein and application thereof, wherein the antibody fusion Protein has the advantages of high expression level, high assembly rate and higher affinity, and simultaneously has the advantage of convenient subsequent purification, and the purification purity of more than 95% can be achieved by using Protein A or Protein L in one step.

The antibody fusion protein provided by the application comprises:

a) An antibody that specifically binds VEGFC;

b) A protein that specifically binds VEGFA; and

c) A flexible peptide linking a) and b);

the antibody specifically binding to VEGFC is an intact antibody, a single chain antibody or a VEGFC antigen binding fragment specifically binding to VEGFC.

In some embodiments, the antibody that specifically binds VEGFC comprises one or more of a light chain variable region, a light chain constant region, a heavy chain variable region, a heavy chain constant region 1, a heavy chain constant region 2, and a heavy chain constant region 3.

In some embodiments, the antibody that specifically binds to VEGFC is an intact antibody that specifically binds to VEGFC, i.e., the P01-H1L4 antibody obtained by panning a natural humanized library according to the present application.

In some embodiments, the antibody that specifically binds VEGFC comprises a light chain variable region and a light chain constant region, the protein that specifically binds VEGFA is a Trap protein, and the antibody fusion protein is VL-CL-linker-Trap, such as antibody fusion protein C031 shown in fig. 2.

In some embodiments, the antibody that specifically binds VEGFC comprises a heavy chain variable region and a heavy chain constant region 1, a heavy chain constant region 2, and a heavy chain constant region 3, the protein that specifically binds VEGFA is a Trap protein, and the antibody fusion protein is a VH-CH1-CH2-CH3-linker-Trap, such as the antibody fusion protein C003 shown in fig. 1.

In some embodiments, the antibody fusion proteins of the application are shown in FIG. 2, comprising: a) An antibody that specifically binds VEGFC; b) A protein that specifically binds VEGFA; and c) a flexible peptide linking a) and b); the antibody specifically binding to VEGFC is an intact antibody, comprising a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), a heavy chain constant region 1 (CH 1), a heavy chain constant region 2 (CH 2) and a heavy chain constant region 3 (CH 3), wherein the protein specifically binding to VEGFA is Trap protein, and the flexible peptide is linker; the light chain of the antibody fusion protein sequentially comprises a light chain variable region (VL), a light chain constant region (CL), a linker and a Trap protein (namely VL-CL-linker-Trap), and the heavy chain of the antibody fusion protein sequentially comprises a heavy chain variable region (VH), a heavy chain constant region 1 (CH 1), a heavy chain constant region 2 (CH 2) and a heavy chain constant region 3 (CH 3) (namely VH-CH1-CH2-CH 3).

In some embodiments, the antibody fusion proteins of the application are shown in FIG. 1, comprising: a) An antibody that specifically binds VEGFC; b) A protein that specifically binds VEGFA; and c) a flexible peptide linking a) and b); the antibody specifically binding to VEGFC is an intact antibody, comprising a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), a heavy chain constant region 1 (CH 1), a heavy chain constant region 2 (CH 2) and a heavy chain constant region 3 (CH 3), wherein the protein specifically binding to VEGFA is Trap protein, and the flexible peptide is linker; the light chain of the antibody fusion protein sequentially comprises a light chain variable region (VL) and a light chain constant region (CL) (namely VL-CL), and the heavy chain of the antibody fusion protein sequentially comprises a heavy chain variable region (VH), a heavy chain constant region 1 (CH 1), a heavy chain constant region 2 (CH 2), a heavy chain constant region 3 (CH 3), a linker and a Trap protein (namely VH-CH1-CH2-CH 3-linker-Trap).

In some embodiments, the flexible peptide is (G4S) n, wherein n is an integer greater than 0; preferably, n is 1, 2,3, 4 or 5; more preferably, n is 3.

In some embodiments, the amino acid sequences of light chain CDR1, CDR2, and CDR3 in the antibody that specifically binds VEGFC are as set forth in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15; the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: shown at 18.

In some embodiments, the light chain variable region has an amino acid sequence as set forth in SEQ ID NO:2, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 3.

In some specific embodiments, the light chain is Kappa-type or Lambda-type.

In some embodiments of the present application, in some embodiments,

(I) The amino acid sequence of the heavy chain is shown as SEQ ID NO:4 or SEQ ID NO:5 is shown in the figure;

the amino acid sequence of the light chain is shown as SEQ ID NO:6 or SEQ ID NO: shown in figure 7;

or (II) an amino acid sequence having the same or similar function as the amino acid sequence shown in (I) obtained by substituting, deleting or adding one or more amino acids in the sequence shown in (I);

(III) an amino acid sequence having a homology of 90% or more with the sequence of (I).

In some embodiments, the heavy chain has an amino acid sequence as set forth in SEQ ID NO:4, the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 6.

In some embodiments, the heavy chain has an amino acid sequence as set forth in SEQ ID NO:5, the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 7.

In some embodiments, the heavy chain has an amino acid sequence as set forth in SEQ ID NO:5 is shown in the figure; the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 8.

The application also provides nucleic acids encoding the antibody fusion proteins, having:

(IV) encoding the amino acid sequence set forth in SEQ ID NO:2, and a light chain variable region; encoding the sequence as shown in SEQ ID NO:3, and a heavy chain variable region; or (b)

(V) a nucleotide sequence having a homology of 90% or more with the sequence of (IV).

In some embodiments, the nucleic acids encoding the antibody fusion proteins of the application have:

(VI), encoding a polypeptide as set forth in SEQ ID NO:4, and a heavy chain; encoding the sequence as shown in SEQ ID NO:6, the nucleotide sequence of the light chain shown in FIG. 6; or (b)

(VII) a nucleotide sequence having a homology of 90% or more with the sequence of (VI).

(VIII) encodes an amino acid sequence as set forth in SEQ ID NO:5, and a nucleotide sequence of the heavy chain shown in seq id no; encoding the sequence as shown in SEQ ID NO:7, a nucleotide sequence of a light chain shown in seq id no; or (b)

(IX) a nucleotide sequence having a homology of 90% or more with the sequence of (VIII).

(X) encodes an amino acid sequence as set forth in SEQ ID NO:5, and a nucleotide sequence of the heavy chain shown in seq id no; encoding the sequence as shown in SEQ ID NO:8, and a light chain nucleotide sequence shown in seq id no; or (b)

(XI) a nucleotide sequence having a homology of 90% or more with the sequence of (X).

The application also provides a conjugate which is formed by connecting the antibody fusion protein of the application with isotopes, immunotoxins and chemical drugs.

The application also provides a conjugate, which is formed by coupling the antibody fusion protein of the application with a solid medium or a semisolid medium, or by coupling the conjugate of the application with a solid medium or a semisolid medium.

The application also provides application of the antibody fusion protein or the conjugate in preparing medicines for treating or diagnosing diseases.

The disease is breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, non-hodgkin's lymphoma, chronic lymphoma leukemia, multiple myeloma, acute myelogenous leukemia, acute lymphoblastic leukemia, diseased glioma, melanoma, age-related macular degeneration, retinal vein occlusion, diabetic macular edema, polypoidal choroidal vasculopathy, diabetic retinopathy, choroidal neovascularization secondary to pathological myopia, central serous chorioretinopathy, neonatal neovascular disease, neovascular glaucoma, idiopathic sub-macular neovascular disease, outer exudative retinopathy or vitreoretinal preoperative adjuvant.

The application also provides a pharmaceutical composition comprising the antibody fusion protein of the application, or the conjugate.

The application also provides a kit comprising the antibody fusion protein, or the conjugate of the application.

The present application also provides an antibody that specifically binds VEGFC, comprising CDRs 1-3 of the light chain variable region and CDRs 1-3 of the heavy chain variable region, wherein the amino acid sequences of light chain CDR1, CDR2 and CDR3 are as set forth in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15; the amino acid sequences of heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: shown at 18.

In certain embodiments, the light chain variable region of an antibody of the application that specifically binds VEGFC has an amino acid sequence as set forth in SEQ ID NO:2, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 3.

In certain embodiments, the antibodies of the application that specifically bind to VEGFC are intact antibodies, single chain antibodies, or VEGFC antigen binding fragments that specifically bind to VEGFC.

In certain embodiments, antibodies of the application that specifically bind VEGFC comprise one or more of a light chain variable region, a light chain constant region, a heavy chain variable region, a heavy chain constant region 1, a heavy chain constant region 2, and a heavy chain constant region 3.

In certain preferred embodiments, the antibodies of the application that specifically bind to VEGFC are intact antibodies that specifically bind to VEGFC, i.e., the P01-H1L4 antibodies obtained by panning a natural human repertoire of the application.

The antibody fusion protein provided by the application has high expression quantity, and the transient transfection expression quantity in the mammalian cell 293E is 100-150mg/L; the preferred bispecific fusion protein of the application has a structure which has high assembly rate and more than 95% of correct assembly rate; the bispecific fusion protein obtained by the application has high affinity, and the binding KD value of the unilateral antibody/fusion protein and the antigen is equivalent to that of the positive control monoclonal antibody/fusion protein and the antigen; the bispecific fusion Protein disclosed by the application also brings the advantage of convenient subsequent purification of the Protein due to the structural characteristics, and the purification purity of the Protein A or Protein L can reach more than 95% by one step. The bispecific fusion protein also has good cytological activity and in-vivo pharmacodynamic activity, and the cytological activity and the in-vivo pharmacodynamic activity are superior to those of the contrast marketed drugs.

Drawings

FIG. 1 shows a schematic representation of a C003 bispecific antibody fusion protein;

FIG. 2 shows a schematic representation of a C031 bispecific antibody fusion protein;

FIG. 3 shows SDS-PAGE detection of transient expression supernatants of antibody fusion proteins: m is Maker; lanes 1, 2,3, 4 are C003, C031, C002 and C004, respectively;

FIG. 4 shows SDS-PAGE detection after purification of the antibody fusion protein: m is Maker; lanes 1, 2,3, 4 are C003, C031, C002 and C004, respectively;

FIG. 5 shows the results of an antibody fusion protein binding hVEGFA Elisa assay;

FIG. 6 shows the results of an antibody fusion protein binding hVEGFC Elisa assay;

FIG. 7 shows the results of detection of binding of antibody fusion proteins to hVEGFA and hVEGFC sandwich Elisa;

FIG. 8 shows the results of antibody fusion protein cytologically blocking hVEGFA activity reporter gene assay;

FIG. 9 shows the results of antibody fusion protein cytologically blocking hVEGFC activity reporter gene assay;

FIG. 10 shows the results of antibody fusion protein cytologically blocking hVEGFA and hVEGFC activity reporter gene assays;

FIG. 11 shows the results of an antibody fusion protein blocking hVEGFA from inhibiting HUVEC cell proliferation assay;

FIG. 12 shows the results of an antibody fusion protein blocking hVEGFC inhibiting HUVEC cell proliferation assay;

FIG. 13 shows the results of assays for blocking hVEGFA and hVEGFC by antibody fusion proteins to inhibit HUVEC cell proliferation.

Detailed Description

The application provides antibody fusion proteins and uses thereof, and those skilled in the art can, in light of the disclosure herein, suitably modify the process parameters to achieve. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present application. While the methods and applications of this application have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the application can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the application.

The test materials adopted by the application are all common commercial products and can be purchased in the market. Wherein Eylea is an anti-hVEGFA drug marketed by the regeneration company;

the application is further illustrated by the following examples:

example 1: bispecific antibody fusion protein preparation

1. hVEGFC antibody P01-H1L4 panning of Natural human Single chain antibody display library

Sterile extraction of fresh peripheral blood from healthy volunteers, separation of lymphocytes from the peripheral blood by lymphocyte separation (GE) using Invitrogen corporation(15596-026) extraction of 100×10 ⁶ Total RNA of individual cells. pCANTAB5E was selected as a phage display vector. For a stock building scheme see patent CN 106928354a.

Phage display and screening of a human antibody library. 100 times of stock capacity of the human VH and VL single-chain antibody stock solution is inoculated with 750 ml of 2YT-AG culture medium (containing 100 mu g/ml ampicillin and 2% (w/v) glucose), cultured at 37 ℃ and 200rpm until OD600 = 0.5-0.6, helper phage with 100 times of cell density is added, infected for 1.5h, the cells are collected by centrifugation, 400ml of 2YT-AK culture medium (containing 100 mu g/ml ampicillin and 75 mu g/ml kanamycin) is used for resuspension of the cells, and cultured at 30 ℃ and 200rpm overnight. Centrifuging 10000g of the culture obtained in the last step at 4 ℃ for 20min, collecting supernatant, adding 1/4 volume of PEG/NaCl, mixing well, and standing on ice for 1h; centrifuging at 4deg.C for 25min at 12000g, discarding supernatant, and reversely fastening the centrifuge tube on flat paper to remove liquid; phage pellet was resuspended in 2ml pre-chilled 1 XPBS, centrifuged at 12000g for 10min at 4 ℃. The supernatant was transferred to a new 15ml centrifuge tube and BSA was added at a final concentration of 3% (w/v) to obtain the first round of starting phage. Coating an immune tube by taking hVEGFC-His as an antigen, and blocking by adopting 2% (w/v) M-PBS; then add 10 ¹³ Antibody antigen binding was performed on the first round of starting phage of (A), unbound phage was washed off with PBST, and the antibody was used to wash off the bound phage1ml Glycine-HCl (pH 2.2) eluted phage, the eluted phage was re-infected with TG1, the eluted product was amplified, and PEG/NaCl precipitated purified phage was used for the next round of screening. And 4 rounds of enrichment screening of phage libraries are carried out, the antigen amount is reduced in sequence, the washing strength is enhanced in sequence, and each round of elution products are subjected to titer measurement.

Limiting dilution of bacterial liquid after 1 st to 4 th rounds of panning, coating the bacterial liquid on a flat plate, and culturing overnight; selecting a monoclonal to be cultured on a 96-well deep-hole plate which is subpackaged with 0.5 ml/well 2YT-AG culture medium for overnight; the overnight culture was then transferred 1:10 into a 96-well deep well plate containing 0.5 ml/well 2YT-AG medium, cultured until OD600 = 0.5-0.6, the cells were collected by centrifugation at 3000g, the cells were resuspended in 2YT-AI medium (containing 100. Mu.g/ml ampicillin and 1mM IPTG), induced overnight at 30℃and the supernatant was transferred by centrifugation the next day to a clean 96-well deep well plate, and BSA was added at a final concentration of 3% (w/v) to obtain a monoclonal phage sample. Coating a 96-well ELISA plate by taking hVEGFC-His as an antigen, adding 50 μl of a monoclonal phage sample into each well after sealing, and incubating for 1h at 25 ℃; then 200 μl of PBST is added into each well, the solution is discarded and repeated for 3-5 times after 5-10S of shaking; then 50 μl of anti-M13-HRP antibody PBS diluent was added to each well and incubated for 1h at 25deg.C; then 200 μl of PBST is added to each well, the solution is discarded and repeated 5 times after shaking for 5-10S; mu.l of TMB developing solution was added to each well and developed for 3 to 10 minutes (the specific development time was determined depending on the development speed), and then 50. Mu.l of 1M H was added to each well ₂ SO ₄ Terminating the color development; OD450 values were determined using a microplate reader. 860 ELISA positive samples were selected based on the monoclonal phage ELISA data, competitive ELISA screening was performed, the positive control antibodies were diluted to 200. Mu.g/ml, 50. Mu.l of the dilution was mixed with 50. Mu.l of the monoclonal phage samples, ELISA was performed as above, and the results showed that 18 of the monoclonal antibodies were epitope-competitive with the positive control antibodies, and the clones were selected for sequencing.

Construction of expression vectors by transient transfection of full length antibodies and transfection, expression and detection in mammalian cells 293E. Based on the expression level and ELISA detection values of binding of antibodies to VEGFC, P01-H1L4 was determined by selecting relatively low EC50 values and relatively high ELISA Rmax values. The P01-H1L4 light chain variable region and the CDR1, CDR2 and CDR3 sequences are SEQ ID No.2, SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15 respectively. The P01-H1L4 heavy chain variable region and the CDR1, CDR2 and CDR3 sequences are SEQ ID No.3, SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18 respectively.

As shown in Table 5, the affinity of the P01-H1L4 antibody with VEGFC was tested to reach 7.04E-10M; as shown in fig. 6, the P01-H1L4 antibody was able to bind VEGFC (EC 50 is 0.5011); as shown in FIG. 9, the reporter gene assay for the P01-H1L4 antibody was able to block the VEGFC signaling pathway (IC 50 is 1.32 nM); as shown in FIG. 12, the inhibition of cell proliferation activity assay for the P01-H1L4 antibody was able to block the VEGFC signaling pathway (IC 50 is 10.37 nM).

2. Construction of bispecific antibody fusion protein transient transfection expression vector

Material

The Trap (SEQ ID No. 1) sequence combined with human VEGFA comes from the Eylea of the regeneration company, which is a drug on the market (the sequence is < 210 & gt 6 in the sequence table of Chinese patent CN 103349781B); the monoclonal antibodies against human VEGFC were derived from the sequences of VL (SEQ ID No. 2) and VH (SEQ ID No. 3) of P01-H1L 4.

Method

pGS003 was selected to construct the expression vectors for the heavy and light chains of bispecific antibody fusion proteins C003 and C031 (see FIG. 1 and FIG. 2 for schematic structure), respectively. Primers were designed based on the coding nucleotides of VEGFR1Domain2 and VEGFR2Domain3, the coding nucleotide of anti-human VEGFC monoclonal antibody P01-H1L4, the coding nucleotide of the IgG1 heavy chain constant region, and the nucleotide sequence of the Lambda chain constant region and the multiple cloning sites in the vector. After PCR amplification, 2 heavy chain coding sequences and 2 light chain coding sequences were cloned into pGS003 using in vitro recombination (nanjing gold, cloneEZ PCR Cloning Kit) as shown in table 3. After sequencing and identifying that the target gene is correctly inserted, the recombinant expression vector is transformed into escherichia coli TOP10F', single colony is selected and inoculated into LB culture medium containing 100 mug/mL ampicillin, and the recombinant expression vector is cultured for 16 hours at 37 ℃ in a shaking way. Plasmids were extracted using the enzyme Research's large endotoxin removal kit, and finally the plasmids were dissolved in 1mL of ultrapure water, and the plasmid concentration and OD260/280 were determined using a spectrophotometer. OD260/280 is 1.8-1.9, which is plasmid DNA with higher purity.

TABLE 3 transient transfection expression vector lists for heavy and light chains

3. Transfection, expression and detection in mammalian cells 293E

Transient transfection expression assessment of 2ml of the 293E system was performed after combining the 2 heavy chain expression vectors and 2 light chain expression vectors described above. The expression level and the correct assembly rate of the antibody were measured, and the results are shown in FIG. 3 and Table 4. The expression of C003 and C031 is 100-150mg/L, and the assembly rate is more than 95%. C002 and C004 are antibody fusion proteins of two other structures.

Amplified transient transfection expression of C003 and C031 was performed using 293E in Freestyle medium. 24 hours prior to transfection, 0.5X10 cells were inoculated in 1L cell culture flasks ⁶ Cell/ml 293E cell 300ml,37℃5% CO ₂ Shaking culture was performed at 120rpm in an incubator. In transfection, 300. Mu.l of 293fectin is firstly added into 5.7ml of OPtiMEM, and after being fully and evenly mixed, the mixture is incubated for 2 minutes at room temperature; at the same time, the total amount of expression plasmids used in C003 and C031 was diluted to 6ml using OPtiMEM at 300. Mu.g each. Mixing the diluted transfection reagent and plasmid thoroughly, incubating at room temperature for 15 min, adding the mixture into cells, mixing well, and mixing with 5% CO at 37deg.C ₂ Shaking culture in incubator at 120rpm for 7 days.

Table 4, C003 and C031 transient transfection expression levels

Antibody name	C003	C031
Expression level (mg/L)	100	150

Example 2: purification and detection of bispecific antibody fusion proteins

The cell culture broth was centrifuged at 2000g for 20min, the supernatant was collected, filtered through a 0.22 μm filter, subjected to Mabselect Sure (GE) chromatography, eluted with 20mM sodium citrate, pH3.0, and pH adjusted to neutral with 1M Tris base. Purified samples were subjected to SDS-PAGE using 4-20% gradient gel (Kirschner Biotechnology Co., ltd.) to detect purified proteins, the results of which are shown in FIG. 4.

Example 3: ELISA detection of bispecific antibody fusion protein binding to human VEGF-A and human VEGFC

ELISA detection of bispecific antibody fusion protein binding to human VEGFA

1. Antigen coating: human VEGFA-His (gold self-construct, SEQ ID No. 10) was diluted to 1. Mu.g/ml with PBS and added to 96-well ELISA plates and incubated overnight at 4℃at 50. Mu.l per well.

2. Closing: after three plate washes, the plates were blocked with 3% (w/v) BSA, 250 μl per well, and incubated for 2 hours at 37 ℃.

3. Adding a candidate antibody: after washing the plate 3 times, 12 concentration gradient candidate antibody samples or positive or negative controls were added at an initial concentration of 10mg/ml, 2-fold dilution. Mu.l per well, incubated at 25℃for 1 hour.

4. Adding a secondary antibody: after washing the plates 3 times, HRP-labeled streptavidin (1:10000) was added and incubated for 1 hour at 25℃in 50. Mu.l per well.

5. Color development: after washing the plate 4 times, 50. Mu.l of TMB developing solution was added to each well, and developed for 10 minutes at room temperature in the dark.

6. And (3) terminating: the reaction was terminated by directly adding 50. Mu.l of a stop solution per well.

7. And (3) detection: immediately after the reaction is terminated, the ELISA plate is placed into an ELISA apparatus, OD value is measured at 450nm, and original data are stored for finishing. The results are shown in FIG. 5. The results showed that C003 and C031 were comparable to the positive control drug Eylea activity.

ELISA detection of (two) bispecific antibody fusion proteins binding to human VEGFC

1. Antigen coating: human VEGFC-His (gold self-construct, SEQ ID No. 11) was diluted to 1. Mu.g/ml with PBS and added to 96-well ELISA plates and incubated overnight at 4℃at 50. Mu.l per well.

7. And (3) detection: immediately after the reaction is terminated, the ELISA plate is placed into an ELISA apparatus, OD value is measured at 450nm, and original data are stored for finishing. The results are shown in FIG. 6. The results showed that C003 and C031 were comparable to the positive control antibody P01-H1L4 in activity, and superior to C002 and C004.

ELISA detection of (III) bispecific antibody fusion proteins binding to human VEGFA and human VEGFC

1. Coating a first antigen: human VEGFA-His (gold self-construct, SEQ ID No. 10) was diluted to 1. Mu.g/ml with PBS and added to 96-well ELISA plates and incubated overnight at 4℃at 50. Mu.l per well.

4. Adding a second antigen: after washing the plates three times, human VEGFC-mFc (gold self-construct, SEQ ID NO: 12) was diluted to 10. Mu.g/ml with PBS and added to 96-well ELISA plates and incubated at 25℃for 1h at 50. Mu.l per well.

5. Adding a secondary antibody: after washing the plates 3 times, HRP-labeled streptavidin (1:10000) was added and incubated for 1 hour at 25℃in 50. Mu.l per well.

6. Color development: after washing the plate 4 times, 50. Mu.l of TMB developing solution was added to each well, and developed for 10 minutes at room temperature in the dark.

7. And (3) terminating: the reaction was terminated by directly adding 50. Mu.l of a stop solution per well.

8. And (3) detection: immediately after the reaction is terminated, the ELISA plate is placed into an ELISA apparatus, OD value is measured at 450nm, and original data are stored for finishing. The results are shown in FIG. 7.

The results show that both C003 and C031 have activity to bind to the double antigen and that the activity is superior to that of C002 and C004, whereas the control drug eyea does not have activity to bind to the double antigen.

Example 4: bispecific antibody fusion protein affinity assays

The affinity of each antibody fusion protein was detected using a Biacore T200 instrument. The specific method comprises the following steps: human VEGFC-His (gold self-organization, SEQ ID No. 11) and human VEGFA-His (gold self-organization, SEQ ID No. 10) were coupled to a CM5 biosensing chip (GE), antibodies of different concentrations were flowed through the chip at a flow rate of 30. Mu.l/min, antigen was combined with candidate antibody for 120s, and dissociation time was 300s. Kinetic fitting was performed with BIAevaluation software (GE) to obtain affinity constant results for affinities of 5.73E-10M and 3.27E-10M for C003 and C031, respectively, as shown in Table 5, table 6, below; the affinities of C003 and C031 for VEGFA were 3.74E-10M and 5.62E-10M, respectively.

Table 5, C003 and C031 affinity measurements with VEGFC

Table 6, C003 and C031 affinity measurements with VEGFA

Example 5: bispecific antibody fusion protein blocking cell VEGFA/VEGFC signaling pathway Activity assay

Reporter gene method

NFAT-RE-luc 2P-KDR-HEK293 cells (gold self-constructs, KDR expressed on HEK293 cell surface) 30000/well were inoculated in 96-well plates and incubated in incubator. The initial concentration of BA006 samples was 1000nM, 3-fold diluted, 10 concentration spots and incubated for 1h. Adding VEGFA or VEGFC or VEGFA+VEGFC (after the VEGFA/VEGFC recognizes that KDR receptor starts a signal path, the generated NFAT can start the expression of Luciferase, chemical light emission is generated after adding a substrate, and the anti-VEGFA/C or KDR antibody can reduce the expression and the corresponding activity of Luciferase by blocking the combination of VEGFA/C and receptor KDR), incubating for 4 hours, and adding a Luciferase detection solution after cleavage, and detecting. As a result, see fig. 8-10, the blocking activity of C003 and C031 on VEGFA signal was comparable to that of the marketed control drug eyread, the blocking activity of C003 and C031 on VEGFC signal was comparable to that of control antibody P01-H1L4, and C003 and C031 could block VEGFA and VEGFC pathway simultaneously, while eyread could only partially reduce fluorescence intensity, showing that it only blocks VEGFA pathway, but not VEGFA and VEGFC pathway simultaneously.

Method for inhibiting cell proliferation activity

HUVEC cells 30000/well were inoculated in 96-well plates and placed in an incubator for 3.5h. BA006 samples, initial 1.5 μm, 4-fold dilution, 10 concentration spots, were incubated for 1.5h. The incubation was performed for 92h at either VEGFA concentration of 0.1 μg/ml or VEGFC concentration of 0.75 μg/ml or VEGFA+VEGFC concentration of 23+300 ng/ml. CCK8 was added, incubated for 3.5h, and detected. The results are shown in FIGS. 11-13, where the blocking activity of C003 and C031 on VEGFA signal is equivalent to that of Eylea, a marketed control drug, and the blocking activity of C003 and C031 on VEGFC signal is equivalent to that of control antibody P01-H1L4, and C003 and C031 can block VEGFA and VEGFC channels at the same time, and Eylea can only partially inhibit cell proliferation activity, showing that it only blocks VEGFA channels, but not VEGFA and VEGFC channels at the same time.

Example 6: animal efficacy determination of bispecific antibody fusion proteins

And (3) model: MC38 (colorectal cancer cell), 2×10 ⁵ cells/mouse, C57BL/6 mice were inoculated subcutaneously.

Administration: subcutaneous tumor volume of about 100-150mm ³ The mice were randomly grouped, 6 mice per group were given intraperitoneally, 2 times per week, for a total of 3 weeks; eylea,2mg/kg; combination 1, P01-H1L4+Eylea,3mg/kg+2mg/kg, is administered in the morning and evening; combination 2, antibody VC4.5 (see GENENTECH patent US9102720B 2) +Eylea,3mg/kg+2mg/kg, administered in the morning and evening; c003,3.8mg/kg; c031,3.8mg/kg; c002,3.8mg/kg.

As shown in Table 7, the tumor inhibition rate of the single Eylea drug as the marketed control drug was 24.58%, the tumor inhibition rate of the combination of P01-H1L4 and Eylea drug was 13.9%, the tumor inhibition rate of the combination of VC4.5 and Eylea drug was 10.87%, the tumor inhibition rates of the bispecific antibody fusion proteins C003 and C031 were 37.81% and 34.82%, respectively, and the activity was superior to that of C002. Thus, the activity of P01-H1L4 is superior to that of VC4.5, and the activity of the bispecific fusion proteins C003 and C031 of the application is obviously superior to that of single medicine and other combined medicine groups.

TABLE 7 tumor inhibition rates of C003 and C031

Medicament	TGI(％)
Eylea(2mg/kg)	24.58
Eylea+anti-P01-H1L4(2.0+3.0mg/kg)	13.90
Eylea+anti-VC4.5(2.0+3.0mg/kg)	10.87
C003(3.8mg/kg)	37.81
C031(3.8mg/kg)	34.82
C002(3.8mg/kg)	0

Example 7: bispecific antibody fusion protein monkey eye CNV model pharmacodynamic assay

And (3) model: the cynomolgus monkey is subjected to modeling on the first day (D1). The laser-induced fundus Choroidal Neovascularization (CNV) model of the ocular fundus of the cynomolgus monkey has the laser firing number of 6-8 for each eye. When bubbles are generated at the position of the photocoagulation spot, if bubbles are not generated, additional photocoagulation is performed at the position of the laser photocoagulation spot.

Administration: and on the 16 th day (D16), animals with 4-level fluorescent leakage light spots are selected to enter groups, and grouping is carried out according to average light spot leakage distribution, so that no obvious difference of average light spot leakage distribution of each group of animals is ensured during grouping. D16, eylea (5 mg/ml 50. Mu.l) and C003 (10 mg/ml 50. Mu.l) were administered in equimolar single glass doses with adjuvant solution without drug as negative control.

As shown in Table 8, the average improvement rate of the light spot leakage of the Eylea of the marketed control drug was 71.81%, the average improvement rate of the light spot leakage of the bispecific antibody fusion protein C003 was 76.09%, and the activity was slightly superior to that of Eylea.

Table 8, C003 fluorescence spot leakage improvement rate

Medicament	Improvement rate (%)
Auxiliary materials	27.59
Eylea(5mg/ml 50μl)	71.81
C003(10mg/ml 50μl)	76.09

The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.

Claims

An antibody fusion protein comprising:

a) An antibody that specifically binds VEGFC;

b) A protein that specifically binds VEGFA; and

c) A flexible peptide linking a) and b);

the antibody specifically binding to VEGFC is an intact antibody, a single chain antibody or a VEGFC antigen binding fragment specifically binding to VEGFC.
The antibody fusion protein of claim 1, wherein the antibody that specifically binds VEGFC comprises one or more of a light chain variable region, a light chain constant region, a heavy chain variable region, a heavy chain constant region 1, a heavy chain constant region 2, and a heavy chain constant region 3.
The antibody fusion protein of claim 1, wherein the antibody that specifically binds VEGFC comprises a light chain variable region and a light chain constant region, wherein the protein that specifically binds VEGFA is a Trap protein, and wherein the antibody fusion protein is a VL-CL-linker-Trap.
The antibody fusion protein of claim 1, wherein the antibody that specifically binds VEGFA comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), a heavy chain constant region 1 (CH 1), a heavy chain constant region 2 (CH 2), and a heavy chain constant region 3 (CH 3), wherein the protein that specifically binds VEGFA is a Trap protein, wherein the flexible peptide is a linker, wherein the light chain of the antibody fusion protein is a VL-CL-linker-Trap, and wherein the heavy chain of the antibody fusion protein is a VH-CH1-CH2-CH3.
The antibody fusion protein of claim 1, wherein the antibody that specifically binds VEGFC comprises a heavy chain variable region and a heavy chain constant region 1, a heavy chain constant region 2, and a heavy chain constant region 3, wherein the protein that specifically binds VEGFA is a Trap protein, and wherein the antibody fusion protein is a VH-CH1-CH2-CH3-linker-Trap.
The antibody fusion protein of claim 1, wherein the antibody that specifically binds VEGFA comprises a heavy chain variable region (VH), a heavy chain constant region 1 (CH 1), a heavy chain constant region 2 (CH 2), a heavy chain constant region 3 (CH 3), a light chain variable region (VL), and a light chain constant region (CL), wherein the protein that specifically binds VEGFA is a Trap protein, wherein the flexible peptide is a linker, wherein the heavy chain of the antibody fusion protein is VH-CH1-CH2-CH3-linker-Trap, and wherein the light chain of the antibody fusion protein is VL-CL.
The antibody fusion protein of claim 1, wherein the flexible peptide is (G4S) n, wherein n is an integer greater than 0; preferably, n is 1, 2,3, 4 or 5; more preferably, n is 3.
The antibody fusion protein of any one of claims 1-7, wherein the amino acid sequences of light chain CDR1, CDR2, and CDR3 in the antibody that specifically binds VEGFC are as set forth in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15; the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: shown at 18.
The antibody fusion protein of claim 8, wherein the variable region of the light chain has an amino acid sequence as set forth in SEQ ID NO:2, the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 3.
The antibody fusion protein of any one of claim 1 to 9, wherein,

(I) The amino acid sequence of the heavy chain is shown as SEQ ID NO:4 or SEQ ID NO:5 is shown in the figure;

the amino acid sequence of the light chain is shown as SEQ ID NO:6 or SEQ ID NO: shown in figure 7;

or (II) an amino acid sequence having the same or similar function as the amino acid sequence shown in (I) obtained by substituting, deleting or adding one or more amino acids in the sequence shown in (I);

(III) an amino acid sequence having a homology of 90% or more with the sequence of (I).
The antibody fusion protein of claim 10, wherein the heavy chain has an amino acid sequence set forth in SEQ ID NO:4, the amino acid sequence of the light chain is shown as SEQ ID NO:6 is shown in the figure; or (b)

The amino acid sequence of the heavy chain is shown in SEQ ID NO:5, the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 7.
A nucleic acid encoding the antibody fusion protein of any one of claims 1 to 11, comprising:

(IV) encoding the amino acid sequence set forth in SEQ ID NO:2, and a light chain variable region; encoding the sequence as shown in SEQ ID NO:3, and a heavy chain variable region; or (b)

(V) a nucleotide sequence having a homology of 90% or more with the sequence of (IV).
A conjugate comprising the antibody fusion protein of any one of claims 1-11 linked to an isotope, immunotoxin, or chemical.
A conjugate comprising the antibody fusion protein of any one of claims 1-11 or the conjugate of claim 13 coupled to a solid or semi-solid medium.
Use of an antibody fusion protein according to any one of claims 1 to 11 or a conjugate according to claim 13 or a conjugate according to claim 14 in the manufacture of a medicament for the treatment or diagnosis of a disease;

the disease is breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, non-hodgkin's lymphoma, chronic lymphoma leukemia, multiple myeloma, acute myelogenous leukemia, acute lymphoblastic leukemia, diseased glioma, melanoma, age-related macular degeneration, retinal vein occlusion, diabetic macular edema, polypoidal choroidal vasculopathy, diabetic retinopathy, choroidal neovascularization secondary to pathological myopia, central serous chorioretinopathy, neonatal neovascular disease, neovascular glaucoma, idiopathic sub-macular neovascular disease, outer exudative retinopathy or vitreoretinal preoperative adjuvant.
A pharmaceutical composition comprising the antibody fusion protein of any one of claims 1-11, or the conjugate of claim 13, or the conjugate of claim 14.
A kit comprising the antibody fusion protein of any one of claims 1-11, or the conjugate of claim 13, or the conjugate of claim 14.
An antibody that specifically binds VEGFC, characterized in that:

the amino acid sequences of the light chain CDR1, CDR2 and CDR3 in the antibody specifically binding to VEGFC are respectively shown in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15; the amino acid sequences of heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: shown at 18.
The antibody of claim 18, wherein the light chain variable region of the antibody has an amino acid sequence as set forth in SEQ ID NO:2, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 3.