CN109836502B - A kind of bispecific antibody and its application - Google Patents

Abstract

The invention discloses a bispecific antibody. After the Fab domains of the two antibodies are respectively coupled to a first link arm and a second link arm, the azide linker between the two link arms is connected to an alkynyl group. The linker undergoes an azide-alkynyl cycloaddition reaction, thereby Fab coupling of the two antibodies to obtain a bispecific antibody. The preparation method of the bispecific antibody of the present invention has the advantages of high expression level, high coupling efficiency and high accuracy of assembly compared with the traditional preparation method of bispecific antibody, and the bispecific antibody in this form has higher affinity It can effectively activate lymphocytes and produce specific killing of CD20-positive tumor cells in vitro and in vivo. Moreover, due to the lack of the Fc segment of the antibody, it is not only easier to penetrate solid tumors, but also can avoid non-specificity caused by Fc. The toxic side effects of killing are more specific.

Description

Bispecific antibody and application thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a bispecific antibody and application thereof.

Background

Cancer is one of the biggest challenges facing human health in the 21 st century. The antibody drug plays an important role in the treatment of cancer, and with the continuous feedback of clinical data, people find that the clinical application of the monoclonal antibody drug is limited by the fact that the traditional monoclonal antibody drug is easy to cause drug resistance due to a single epitope or relapse due to poor drug effect of the antibody. Bispecific antibodies can simultaneously recognize and bind two different epitopes, and can be generally divided into two types depending on the way of action, one is to act by simultaneously blocking two different signaling pathways through synergistic action, and the other is to exert drug effect by activating effector cells (T cells, NK cells, etc.). Firstly, the drug resistance caused by single epitope of the monoclonal antibody can be solved by the bispecific antibody aiming at multiple epitopes; in addition, immunotherapy has been successful in treating tumors in recent years, and researchers have proposed that the use of the human immune system is the most effective way to treat tumors, and that the effector cell-dependent bispecific antibody can efficiently kill tumor cells using the human immune cells. Therefore, the bispecific antibody has a unique advantage and plays a significant role in the treatment of tumors.

The most widely studied antibody is now the T cell dependent bispecific antibody, one arm of which can bind to tumor associated antigens, while the other arm can bind to CD3 molecules on the surface of effector T cells to form an immunological synapse to activate T cells, which can release perforin and granzyme to kill tumor cells by lysis or apoptosis.

The primary methods for the early preparation of bispecific antibodies included the two-hybridoma cell method and chemical conjugation methods. The bispecific antibody produced by the double hybridoma cell method is generally a murine antibody, has stronger immunogenicity and is limited to be clinically applied. The chemical coupling method has poor reaction conditions and is easy to influence the activity of the antibody, so the application range is not wide. With the development of genetic engineering technology, more and more researchers try to prepare bispecific antibody by genetic engineering methods, such as the knob inteo hole technology developed by Tak, which is the most widely used gene, and the crosssmab technology developed by Roche on the basis of the knob inteo hole technology, and although the methods obviously improve the defects of the early methods, the defects of certain mismatching probability and low expression level of the antibodies still exist between two groups of antibodies.

The traditional bispecific antibody is in an IgG type, has a large molecular structure, and is difficult to permeate through the cortex and intercellular spaces in blood vessels to reach tumor cells at the deep part of a solid tumor, so that the therapeutic effect on the solid tumor is not good as that on blood tumors. Therefore, researchers have attempted to express miniaturized bispecific antibodies using genetic engineering techniquesAmong the most widely used BiTEs (bispecific T cell adaptors), the company blinatumomab (CD19 × CD3) was the FDA first approved for the marketing of relapsed refractory precursor B-cell acute lymphoblastic leukemia (Gerhard Zugmaier, Molecular Immunology, Clinical overview of anti-CD19, which is Philadelphia chromosome negative

and ex vivo data from anti-CD33

as examples for targeting T cells in biochemical magnegnanes 2015, (67) 58-66). BiTE is mainly composed of two single chain antibodies (scFv), with a molecular weight of about 50kDa, which has the advantage of easier penetration into tumor tissue than traditional IgG-type bispecific antibodies, but due to the smaller molecular weight and lack of Fc-fragment, the half-life of the antibody is very short (about 2h), requiring the deployment of continuous infusion devices in clinical use. Therefore, there is a great clinical need for small bispecific antibodies with suitable pharmacokinetic properties, and the most studied forms of antibodies currently include 2F (ab'), taFv-Fc, (scFv)₂HSA and the like.

The CD20 XCD 3bispecific antibody currently under clinical or preclinical investigation is mostly of IgG type using rituximab as a target antibody, such as FBTA-05 developed by Trion pharma in clinical stage II, CD20-TDB developed by Genentech in clinical stage I, and the like. Lu et al also tried fusion expression of IgG-CD20 with scFv-CD3 antibody via a flexible linker (Chia-Yen Lu, Biochemical and Biophysical Research Communications, genetic anti-CD20/CD3 biological antibody for the treatment of B cell lymphoma,2016, (473)808-813), and the prepared antibody had a molecular weight of about 200 kDa. At present, no research on the use of the antibody of Ofatumumab as a bispecific antibody targeting CD20 is available, and no miniaturized bispecific antibody form of CD20 × CD3 is reported, so that a miniaturized bispecific antibody of CD20 × CD3 with suitable pharmacokinetic properties is yet to be developed. In addition, for T cell-dependent bispecific antibodies, the off-target effect is a key factor responsible for their toxicity due to the strong immune cascade generated by the tumor cells after T cell activation. The bispecific antibody in the 2F (ab') form is lack of an Fc segment, so that not only is toxic and side effects caused by the interaction of the Fc segment and in-vivo FcRn expressing cells avoided, but also systemic immune side effects caused by the Fc segment are avoided, and the specificity of the antibody is further ensured. The bispecific antibody of the 2F (ab') form retains the property that the BiTE form is easy to penetrate the tumor, has better pharmacokinetic property, and is the first choice for expressing the bispecific antibody.

Disclosure of Invention

The invention provides a preparation method and application of a Bi-2F (ab') type bispecific antibody based on an antibody-based antigen binding fragment which takes CD20 as a target antigen and takes T cells as effector cells, aiming at the defects in the prior art.

A bispecific antibody is prepared by the following steps:

(1) fab domains of anti-CD20 and anti-CD 3 monoclonal antibodies are provided, respectively, and the C-terminal of the Fab domains carries LPXTG sequence, designated Fab-CD20 and Fab-CD3,

(2) respectively providing a first connecting arm and a second connecting arm, wherein both ends of the first connecting arm are respectively provided with an oligoglycine joint and an alkynyl joint, both ends of the second connecting arm are respectively provided with an oligoglycine joint and an azido joint,

(3) under the catalysis of Sortase A enzyme, the LPXTG sequence and the oligoglycine linker generate transpeptidation reaction, so that Fab-CD20 is connected with the first connecting arm, Fab-CD3 is connected with the second connecting arm, or Fab-CD20 is connected with the second connecting arm, Fab-CD3 is connected with the first connecting arm,

(4) and performing cycloaddition reaction on the azido linker and the alkynyl linker to connect the first connecting arm and the second connecting arm, so as to connect Fab-CD20 and Fab-CD3 to obtain the bispecific antibody.

The oligoglycine linker is generally 1-5 glycine residues, and the oligoglycine residue is coupled with the LPXTG sequence under the catalysis of Sortase A enzyme. After the Fab structural domains of the two antibodies are respectively coupled with the first connecting arm and the second connecting arm, the azide-alkyne cycloaddition reaction (SPAAC) is carried out between the azide linker and the alkyne linker between the two connecting arms, so that the bispecific antibody is obtained by the Fab coupling of the two antibodies. The SPAAC reaction is among the most classical reactions in click chemistry (click chemistry). The reaction can be carried out in aqueous solution, and has the advantages of simple operation, no need of exogenous catalyst, mild reaction condition and high reaction rate. The most outstanding advantages are high reaction yield, cost saving, and simultaneously, the production process reduces the toxic wastes which are easy to generate danger, thereby creating the possibility for realizing industrial large-scale production.

Preferably, both Fab domains carry the LPXTG sequence only at the C-terminus of the heavy chain.

The heavy chain amino acid sequence of the Fab-CD20 is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2.

The heavy chain amino acid sequence of the Fab-CD3 is shown as SEQ ID No.5, and the light chain amino acid sequence is shown as SEQ ID No. 6.

The LPXTG sequence is LPETG, Fab-CD20 and Fab-CD3, and a flexible linker is arranged between the LPXTG sequence and the LPXTG sequence, and the flexible linker is composed of a plurality of glycine residues and/or serine residues.

Preferably, the flexible joint is GGGGS (GGGGS)₂Or (GGGGS)₃。

The ends of the LPXTG sequences of the Fab-CD20 and the Fab-CD3 provided in the step (1) are also connected with a histidine tag for protein purification. The histidine tag is used for purifying the antibody obtained by expression, and the histidine tag can be removed when the histidine tag is coupled with the connecting arm in the first step, so that the structure of the subsequent bispecific antibody is not influenced.

The first connecting arm is GGG- (PEG)_m-DBCO, the second linker arm being GGG- (PEG)_n-N₃Wherein m is less than or equal to 4 and n is less than or equal to 4. PEG is polyethylene glycol, and DBCO is diphenyl cyclooctyne linker.

The invention also provides application of the bispecific antibody in preparation of antitumor drugs.

Preferably, the tumor type is a CD20 positive B lymphoma.

Compared with the traditional preparation method of the bispecific antibody, the preparation method of the bispecific antibody has the advantages of high expression level, high coupling efficiency and high assembly accuracy, the bispecific antibody in the form has higher affinity and specificity, can effectively activate lymphocytes in vitro and in vivo and generate specific killing on CD20 positive tumor cells, is easier to penetrate solid tumors due to lack of Fc sections of the antibody, and has higher specificity by avoiding the toxic and side effects of nonspecific killing caused by Fc.

Drawings

FIG. 1 is a linker arm small molecule GGG- (PEG)₃-N₃And GGG- (PEG)₄Schematic chemical structural formula of-DBCO.

FIG. 2 is a schematic diagram of the preparation of Bi-2F (ab') -CD20-CD 3bispecific antibodies by enzymatic-chemical coupling.

FIG. 3 is a graph showing the results of molecular sieve purification of the click chemistry reaction product Bi-2F (ab') -CD20-CD 3.

FIG. 4 is a graph showing the results of SDS-PAGE analysis of the molecular sieve purified conjugate product Bi-2F (ab ') -CD20-CD3, wherein lanes 1 and 2 are in the reduced state, lanes 3 and 4 are in the non-reduced state, lanes 1 and 3 are samples of the conjugate product 2F (ab') -CD20-CD3, and lanes 2 and 4 are samples of the unconjugated product Fab-CD20-N₃And Fab-CD 3-DBCO.

FIG. 5 is a graph showing the results of HPLC analysis of the coupled product Bi-2F (ab') -CD20-CD 3.

FIG. 6 is a graph showing the results of affinity assay of Bi-2F (ab') -CD20-CD3 in Ramos cell assay, wherein the cases of antibody addition in panels A to E are respectively as follows: negative control, Fab-CD 201. mu.g/ml, Fab-CD 205. mu.g/ml, Bi-2F (ab ') -CD20-CD 31. mu.g/ml, Bi-2F (ab') -CD20-CD 35. mu.g/ml.

FIG. 7 is a graph showing the result of affinity assay for Bi-2F (ab') -CD20-CD3 in PBMC, wherein the graphs A-E show the following results when antibodies are added: negative control, Fab-CD 31. mu.g/ml, Fab-CD 35. mu.g/ml, Bi-2F (ab ') -CD20-CD 31. mu.g/ml, Bi-2F (ab') -CD20-CD 35. mu.g/ml.

FIG. 8 is a graph showing the results of Bi-2F (ab') -CD20-CD3 in vitro killing experiments.

FIG. 9 is a diagram showing the results of specific verification of Bi-2F (ab') -CD20-CD3 and IgG-CD20-CD 3.

FIG. 10 is a flow chart of flow analysis of early activation marker CD69 specific to PBMC by Bi-2F (ab ') -CD20-CD3, wherein, panel A is a negative control, panel B is CD3 antibody at 2000ng/ml, and panels C-G are Bi-2F (ab') -CD20-CD3 antibody at concentrations of 16, 80, 400, 2000, 10000ng/ml, respectively.

FIG. 11 is a flow chart of flow analysis of specific late activation marker CD25 of Bi-2F (ab ') -CD20-CD3 on PBMC, wherein, panel A is a negative control, panel B is CD3 antibody at 2000ng/ml, and panels C-G are Bi-2F (ab') -CD20-CD3 antibody with concentration gradient of 16, 80, 400, 2000 and 10000ng/ml respectively.

FIG. 12 is a graph showing the proliferation of T cells after flow analysis of Bi-2F (ab') -CD20-CD3 on PBMC incubated with K562 CD 20-negative cells.

FIG. 13 is a graph of flow analysis of T cell proliferation after incubation of PBMC with CD20 positive cells Ramos for Bi-2F (ab ') -CD20-CD3, wherein A-H are PBMC blank, PBMC-CFSE blank, 0, 16, 80, 400, 2000, 10000ng/ml Bi-2F (ab') -CD20-CD3 antibody, respectively.

FIG. 14 is a graph showing the experimental results of in vitro anti-tumor activity of Bi-2F (ab') -CD20-CD 3.

Detailed Description

Volunteer fresh peripheral blood samples: obtained by cooperating with the school hospital of Zhejiang university, approved by the medical ethics committee of the college of medicine of Zhejiang university, and numbered: (2018) lon. Lon (003).

Example 1

Preparation of Fab-CD20 or Fab-CD 3.

Fab represents an antigen binding fragment and comprises a heavy chain (H) and a light chain (L), wherein the heavy chain amino acid sequence of the Fab-CD20 is shown as SEQ ID No.1, the light chain amino acid sequence is shown as SEQ ID No.2, the heavy chain coding gene sequence is shown as SEQ ID No.3, and the light chain coding gene sequence is shown as SEQ ID No. 4. The heavy chain amino acid sequence of Fab-CD3 is shown as SEQ ID No.5, the light chain amino acid sequence is shown as SEQ ID No.6, the heavy chain coding gene sequence is shown as SEQ ID No.7, and the light chain coding gene sequence is shown as SEQ ID No. 8. In the present invention, Fab-CD20 was also prepared on the basis of antigen-binding fragment of the original OFA monoclonal antibodyModification for coupling with drug, and modification of Fab-CD3 based on antigen binding fragment of original OKT3 monoclonal antibody for coupling with drug, wherein the modification mode is that the end (C end) of CH1 of heavy chain is provided with (GGGGS)₃-LPETGGHHHHHH (GGGGS)₃For flexible linker peptide, LPETG is the SortaseA enzyme recognition sequence, 6 × His tag for protein purification.

The light chain and heavy chain coding gene sequences of the antigen binding fragments of the OFA monoclonal antibody and the OKT3 monoclonal antibody are synthesized by the generation of Biotechnology engineering (Shanghai) GmbH, and then the modification is completed by a PCR method. Then, the pMH3 expression vectors were ligated to human embryonic kidney cells (293F) and the antibodies were purified using His Trap HP affinity column to obtain Fab-CD20 or Fab-CD3 monoclonal antibodies.

Example 2

The bispecific antibody Bi-2F (ab') -CD20-CD3 was prepared according to the scheme shown in FIG. 2.

1、Fab-CD20-N₃Preparation of Fab-CD3-DBCO conjugate

(1) In a 10ml reaction system, 6. mu.M Fab mab (in 50mM Tris-HCl, 150mM NaCl, pH7.4), 150. mu.M GGG-PEG dissolved in buffer (50mM Tris-HCl, 150mM NaCl, pH7.4) were added₄-DBCO/GGG-PEG₃-N₃(FIG. 1, synthesized by Nanjing Lining Biotech Ltd.), 50 μm sortase A, 5mM CaCl₂And reacting in water bath at 37 ℃ for 12 h.

(2) The reaction solution was purified using protein L (1ml, GE) column, and the eluate was eluted to give an antibody conjugate, which was adjusted to pH 7.0 with 1M Tris, concentrated by ultrafiltration using a 10kDa millipore ultrafiltration tube and replaced with PBS buffer, and stored at-20 ℃ for further use. Fab-CD20 with GGG-PEG₃-N₃Reaction to obtain Fab-CD20-N₃Fab-CD3 with GGG-PEG₄The DBCO reaction to obtain Fab-CD 3-DBCO.

2、Fab-CD20-N₃Fab-CD3-DBCO click chemistry coupling reaction and purification

(1) Fab-CD20-N in a 1ml reaction (50mM Tris-HCl, 150mM NaCl, pH7.4)₃And Fab-CD3-DBCO were reacted with shaking at 4 ℃ overnight in a molar ratio of 1: 1.

(2) Purifying the reaction solution with molecular sieve (Superdex 200Increase 10/300GL, GE) column, collecting the effluent of the first peak as shown in FIG. 3, which is the coupled product of the two and is named Bi-2F (ab') -CD20-CD3, and the second peak is unconjugated Fab-CD20-N₃And Fab-CD3-DBCO (molecular weights close to each other), based on the charge (3mg Fab-CD 20-N)₃And 3mg Fab-CD20-N₃) And the amount of protein recovered (. about.2.7 mg) was calculated to give a recovery of about 45%. Due to the large steric hindrance between protein molecules, the coupling efficiency cannot reach 100%. The desired product was concentrated by ultrafiltration using a 30kDa millipore ultrafiltration tube and replaced with PBS buffer, and stored at-20 ℃ until use.

(3) SDS-PAGE analysis of the purified product As shown in FIG. 4, there was a band of about 50kDa (two heavy chains click chemically linked together) and a band of 25kDa light chain (two light chains of similar size and difficult to separate) under reducing conditions; under non-reducing conditions, a band of about 100kDa corresponds to the theoretical value.

(4) The purity of the purified product (under non-reducing conditions) was analyzed by HPLC, and as shown in fig. 5, the protein purity was about 95%.

Example 3

Flow affinity assay.

(1) Collecting 1X 10⁶Individual Ramos cells were incubated with Fab-CD20, Bi-2F (ab') -CD20-CD3 (1. mu.g/ml and 5. mu.g/ml), respectively, for 30min at 4 ℃; same collection 1X 10⁶Incubating PBMC with Fab-CD3 and Bi-2F (ab') -CD20-CD3 (1. mu.g/ml and 5. mu.g/ml) for 30min at 4 ℃;

(2) after three PBS washes, 200. mu.l of anti-human murine monoclonal antibody Fab (1: 1500 dilution) was added to each tube and incubated at 4 ℃ for 30 min;

(3) after three PBS washes, 200. mu.l of FITC-labeled goat anti-mouse IgG (H + L) (1: 250 dilution) was added to each tube, and after incubation at 4 ℃ for 30min, the tubes were washed 3 times with PBS and examined by flow cytometry.

(4) The affinity of the flow cytometer detection antibodies to CD20/CD3 positive cells is mainly indicated by the mean fluorescence intensity of FITC after secondary antibody labeling.

(5) The enzymatic-chemical coupling product Bi-2F (ab ') -CD20-CD3 showed significantly higher affinity than Fab-CD20 and Fab-CD3 alone as shown in FIGS. 6 and 7, indicating that the bivalent 2F (ab') bispecific antibody has higher affinity and stability than the monovalent Fab antibody.

Example 4

In vitro Activity assay

(1) The CD20 positive Ramos, Daudi, Raji cells and CD20 negative K562 cells were collected, counted and resuspended in RPMI-1640 (10% FBS) medium to a cell density of 1X 10⁶Cell suspensions, plated in 12-well plates at 100 μ l/well, respectively;

(2) fresh peripheral blood of volunteers was collected, and PBMC of lymphocytes were separated from the collected human lymphocyte fraction, washed twice with PBS buffer, counted, and resuspended in 1X 10 with PBS⁷The cells were suspended and appropriate amounts of CFSE were added to a final concentration of 5. mu.M. After being washed twice with PBS and counted in a dark water bath at 37 ℃ for 15min, the cells were resuspended in RPMI-1640 (10% FBS) medium to a cell density of 1.25X 10⁶Cell suspensions, plated in 12-well plates in (1) at 400. mu.l/well, respectively;

(3) adding Bi-2F (ab') -CD20-CD3 antibodies with different concentrations (10000, 2000, 400, 200, 80, 16 and 8ng/ml), incubating for 48h, collecting cells, washing the cells with PBS, then resuspending the cells with 100 ul of 5 ul of Annexin V and 5 ul of PI, incubating for 15min at room temperature, supplementing 400 ul of binding solution, and detecting the apoptosis percentage by a flow cytometer;

(4) as can be seen from the results in FIG. 8, the Bi-2F (ab') -CD20-CD3 antibody has no obvious killing effect on CD20 negative K562 cells, but has concentration gradient-dependent specific killing effect on CD20 positive tumor cells Ramos, Daudi and Raji, and can kill the cells at a concentration of 8ng/ml, so that the in vitro tumor killing effect is very obvious.

Example 5

In vitro specificity verification experiment

(1) FcRn receptor-expressing K562 cells were selected as the study subjects, counted after collection, and resuspended at a cell density of 1X 10 in RPMI-1640 (10% FBS) medium⁶Cell suspensions, plated in 12-well plates at 100 μ l/well, respectively;

(2) fresh peripheral blood of volunteers was collected, and PBMC of lymphocytes were separated from the collected human lymphocyte fraction, washed twice with PBS buffer, counted, and resuspended in 1X 10 with PBS⁷The cells were suspended and appropriate amounts of CFSE were added to a final concentration of 5. mu.M. After being washed twice with PBS and counted in a dark water bath at 37 ℃ for 15min, the cells were resuspended in RPMI-1640 (10% FBS) medium to a cell density of 1.25X 10⁶Cell suspensions, plated in 12-well plates in (1) at 400. mu.l/well, respectively;

(3) adding Bi-2F (ab') -CD20-CD3 antibody and IgG-CD20-CD3 (IgG form antibody which is constructed by the applicant according to the knob int hole and crosssmab technology and has one arm of anti-CD20 antibody and the other arm of anti-CD 3 antibody) with different concentrations (10000, 100 and 10ng/ml) respectively, incubating for 48h, collecting cells, washing the cells with PBS, resuspending with 100 ul of antibody containing 5 ul of Annexin V and 5 ul of PI, incubating for 15min at room temperature, supplementing 400 ul of binding solution, and detecting the apoptosis percentage by a flow cytometer;

(4) as shown in FIG. 9, the Bi-2F (ab ') -CD20-CD3 antibody had no significant killing effect on FcRn-expressing K562 at three concentrations, while IgG-CD20-CD3 had significant killing effect on FcRn-expressing K562 at 1000ng/ml, indicating that the Bi-2F (ab') -CD20-CD3 antibody lacking the Fc fragment had better specificity.

Example 6

PBMC specific activation assay

(1) CD20 positive Ramos cells were collected, counted and resuspended in RPMI-1640 (10% FBS) medium to a cell density of 1X 10⁶Cell suspensions, plated in 12-well plates at 100 μ l/well, respectively;

(2) fresh peripheral blood of volunteers was collected, and after separating lymphocytes PBMC from the human lymphocyte isolate, the cells were washed twice with PBS buffer and counted, and then resuspended in RPMI-1640 (10% FBS) medium to a cell density of 1.25X 10⁶Cell suspensions, plated in 12-well plates in (1) at 400. mu.l/well, respectively;

(3) Bi-2F (ab') -CD20-CD3 antibodies were added at different concentrations (10000, 2000, 400, 80, 16ng/ml), a control group of 2000ng/ml CD3 antibody was selected for incubation for 48h, the collected cells were washed with PBS, and then an appropriate concentration of anti-CD 4-FITC, CD8-FITC, CD69-PE, CD25-APC antibody (antibody purchased from BD company, USA) was added and incubated at 4 ℃ for 30 min. Flow assays were performed after two washes with PBS.

(4) The results are shown in FIGS. 10 and 11, with CD25 and CD69 being surface markers of T cell activation, and Fab-CD3 antibody alone at a concentration of 2000ng/ml for T (CD 4)⁺CD8⁺) The cells had weak activation (lower CD 4)⁺CD8⁺CD69⁺Or CD4⁺CD8⁺CD25⁺Positive ratio) but significantly lower than the ability of Bi-2F (ab') -CD20-CD3 antibody at the same concentration to activate T cells. The Bi-2F (ab') -CD20-CD3 antibody can specifically activate T cells, and the activation ratio is in positive correlation with the administration concentration.

Example 7

PBMC specific proliferation assay

(1) CD20 positive Ramos cells were collected, counted and resuspended in RPMI-1640 (10% FBS) medium to a cell density of 1X 10⁶Cell suspensions, plated in 12-well plates at 100 μ l/well, respectively;

(2) fresh peripheral blood of volunteers was collected, and PBMC of lymphocytes were separated from the collected human lymphocyte fraction, washed twice with PBS buffer, counted, and resuspended in 1X 10 with PBS⁷The cells were suspended and appropriate amounts of CFSE were added to a final concentration of 5. mu.M. After being washed twice with PBS and counted in a dark water bath at 37 ℃ for 15min, the cells were resuspended in RPMI-1640 (10% FBS) medium to a cell density of 1.25X 10⁶Cell suspensions, plated in 12-well plates in (1) at 400. mu.l/well, respectively;

(3) adding Bi-2F (ab') -CD20-CD3 antibodies with different concentrations (10000, 2000, 400, 200, 80, 16, 8ng/ml), incubating for 48h, collecting cells, washing with PBS and resuspending, and detecting cell proliferation by a flow cytometer;

(4) as shown in FIGS. 12 and 13, the Bi-2F (ab') -CD20-CD3 antibody did not induce PBMC proliferation when co-incubated with the CD20 negative cell line K562, but Ramos cells positive for CD20 specifically induced PBMC proliferation in proportion to the administered concentration.

Example 8

In vivo antitumor Activity test

(1) Fresh peripheral blood of volunteers was collected, and PBMC of lymphocytes were separated from the collected human lymphocyte fraction, washed twice with PBS buffer and counted, and then resuspended to 2X 10 with PBS⁸A cell suspension;

(2) culturing in vitro CD20 positive cells Ramos, centrifuging, collecting, washing twice with PBS, counting, resuspending with a certain volume of PBS to 5 × 10⁷cell/ml cell suspension;

(3) the effector PBMC suspension and the Target Ramos suspension were mixed at a ratio of 1: 1(v/v) (Effect: Target 4: 1) and inoculated in the right underarm of SCID Beige mice. And mice were randomly divided into four groups: a saline control group, a CD3 antibody control group (3mg/kg), a Bi-2F (ab ') -CD20-CD3 low dose group (1mg/kg), and a Bi-2F (ab') -CD20-CD3 high dose group (3 mg/kg). 24h after tumor inoculation, administration was started once every two days for a total of five times.

(4) As shown in FIG. 14, the mice in the Saline control group and the CD3 antibody control group showed gradual growth of tumors, all of the tumors disappeared in the Bi-2F (ab ') -CD20-CD3 high dose group (3mg/kg), and all of the tumors disappeared in the Bi-2F (ab') -CD20-CD3 low dose group (1mg/kg), with only one tumor growing slowly. The experimental result shows that Bi-2F (ab') -CD20-CD3 can well inhibit the growth of tumors under the condition of low-concentration administration dose and has stronger anti-tumor activity.

Sequence listing

<110> Zhejiang university

<120> bispecific antibody and application thereof

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Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

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Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

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Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

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Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

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gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa gtccctgtat 240

ctgcaaatga acagtctgag agctgaggac acggccttgt attactgtgc aaaagatata 300

cagtacggca actactacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360

tcctcagcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420

tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480

gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600

cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 660

gagcccaaat cttgtgacaa aactcacaca ggcggtggag gctctggagg cgggggttcc 720

ggtggcggag gttcactgcc ggagactggt ggtcatcacc accatcacca ttaa 774

<210> 4

<211> 645

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg cagtttatta ctgtcagcag cgtagcaact ggccgatcac cttcggccaa 300

gggacacgac tggagattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645

<210> 5

<211> 254

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Leu Pro Glu Thr Gly Gly His His His His His His

245 250

<210> 6

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 7

<211> 765

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

caggtccagc tgcagcagtc tggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaagg cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcagct 360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac aggcggtgga ggctctggag gcgggggttc cggtggcgga 720

ggttcactgc cggagactgg tggtcatcac caccatcacc attaa 765

<210> 8

<211> 645

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gtgccagctc aagtgtaagt tacatgaact ggtaccagca gaagtcaggc 120

acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcac 180

ttcaggggca gtgggtctgg gacctcttac tctctcacaa tcagcggcat ggaggctgaa 240

gatgctgcca cttattactg ccagcagtgg agtagtaacc cattcacgtt cggctcgggg 300

acaaagttgg aaataaaccg gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645

<210> 9

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Leu Pro Glu Thr Gly

1 5

<210> 10

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Gly Gly Gly Ser

1 5

<210> 11

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 12

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 13

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gly Gly Gly

1

<210> 14

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu

1 5 10 15

Pro Glu Thr Gly Gly His His His His His His

20 25

Claims (7)

1. a bispecific antibody, is characterized in that, preparation method comprises the following steps: (1) The Fab domains of monoclonal antibodies against CD20 and anti-CD3 are provided respectively, and the C-termini of the two Fab domains have LPXTG sequences, named Fab-CD20 and Fab-CD3, respectively, (2) respectively providing a first linking arm and a second linking arm, the two ends of the first linking arm are respectively provided with an oligoglycine linker and an alkynyl linker, and the two ends of the second linking arm are respectively provided with an oligoglycine linker and a stack nitrogen-based linker, (3) Under the catalysis of Sortase A enzyme, the LPXTG sequence and the oligoglycine linker undergo a transpeptidation reaction, so that Fab-CD20 is connected to the first linker, Fab-CD3 is linked to the second linker, or Fab-CD20 is linked to the second linker, Fab-CD3 is attached to the first tether, (4) a cycloaddition reaction occurs between the azido linker and the alkynyl linker to connect the first linker with the second linker, thereby linking Fab-CD20 and Fab-CD3 to obtain the bispecific antibody, Both Fab domains only carry LPXTG sequence at the C-terminus of the heavy chain, The heavy chain amino acid sequence of the Fab-CD20 is shown in SEQ ID No.1, and the light chain amino acid sequence is shown in SEQ ID No.2, The heavy chain amino acid sequence of the Fab-CD3 is shown in SEQ ID No.5, and the light chain amino acid sequence is shown in SEQ ID No.6. 2. The bispecific antibody of claim 1, wherein the LPXTG sequence is LPETG, and there is a flexible linker between Fab-CD20 and Fab-CD3 and the LPXTG sequence, and the flexible linker is composed of several glycine residues and/or serine residues. 3. The bispecific antibody of claim 2, wherein the flexible linker is GGGGS, (GGGGS) ₂ or (GGGGS) ₃ . 4 . The bispecific antibody of claim 1 , wherein the LPXTG sequences of Fab-CD20 and Fab-CD3 provided in step (1) are further connected with a histidine tag for protein purification. 5 . 5. The bispecific antibody of claim 1, wherein the first linker is GGG-(PEG) _m -DBCO, and the second linker is GGG-(PEG) _{n -} N3, wherein m≤4, n≤4. 6. The application of the bispecific antibody according to claims 1 to 5 in the preparation of antitumor drugs. 7. The use according to claim 6, wherein the tumor type is CD20 positive B lymphoma.

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