CN1326881C - Trivalent bispecific antibody and its preparation process and use - Google Patents

Abstract

The invention discloses a trivalent bispecific antibody, its preparation method and application. The trivalent bispecific antibody of the present invention comprises the first constant region CH1 of the heavy chain of human IgG1, the CL domain of the constant region of the kappa chain, the variable region VH of the heavy chain and the variable region VL of the light chain of the A antibody, and the single chain of the B antibody Variable region scFv. The VH is connected to the N-terminal of CH1, the scFv is connected to the C-terminal of CH1, the VL is connected to the N-terminal of CL, and the scFv is connected to the C-terminal of CL. The preparation method of the bispecific antibody of the present invention includes the steps of constructing an expression vector, expressing and detecting the bispecific antibody, purifying the bispecific antibody, and analyzing the binding activity of the bispecific antibody. The trivalent bispecific antibody of the invention has the advantages of less side effects, good orientation and the like, and can be used to prepare tumor therapeutic drugs.

Description

A trivalent bispecific antibody, its preparation method and use

technical field

The present invention relates to a bispecific antibody, in particular to a trivalent bispecific antibody, as well as its preparation method and application.

Background technique

Bispecific antibody (bispecific antibody, BsAb) is an artificial antibody containing two specific antigen-binding sites, which can build a bridge between target cells and functional molecules (cells) to produce oriented effector functions. BsAb has broad application prospects in biomedicine, especially in tumor immunotherapy. Killing tumor cells through BsAb-mediated cytotoxicity is a hot topic in current immunotherapy application research. Its main feature is that BsAb can simultaneously bind to tumor-associated antigens and target molecules on effector cells, directly triggering the specific killing of tumor cells by effector cells . Currently, BsAbs can be prepared by methods such as chemical engineering, cell engineering, and genetic engineering. The biggest advantage of the genetic engineering method to construct BsAb is that it can be used to engineer the antibody, such as constructing humanized BsAb, small molecule BsAb, multivalent BsAb, etc. Bivalent BsAb can be designed and constructed by genetic engineering technology, and its construction mode includes bispecific miniantibody (Bispecific miniantibody), double chain antibody (Diabody), single chain bispecific antibody (Sc-BsAb) and so on. Bivalent BsAbs have the advantages of small molecular weight, good tumor tissue permeability, and easy construction and expression. However, because they only have one binding domain for each specific antigen, their affinity for target molecules is not high, and their guiding effect is poor. In order to improve the stability and therapeutic potential of BsAb, the construction mode of tetravalent BsAb has appeared in recent years, such as dimeric bispecific miniantibody (Dibi Miniantibody), tetravalent bispecific antibody Tandemdiabody (Tandab) , IgG-like bispecific antibodies, etc. In addition to their bispecific characteristics, they are also bivalent for each target antigen, so they have higher affinity and stronger effector functions for target cells. In human serum The stability has been enhanced, and the residence time in the circulation has also been significantly prolonged. However, since tetravalent BsAb is also bivalently bound to effector cells (T lymphocytes, NK cells, etc.), clinical application can cause cross-linking of receptors on the surface of effector cells in circulating blood, resulting in extensive activation of effector cells and the release of a large number of cytokines ( Such as TNFα, etc.), causing unacceptable toxic reactions. The results of animal and clinical trials in the past ten years have shown that BsAbs with greater clinical application value should have the following characteristics, (1) must be able to bind tumor-associated antigens with high selectivity and high affinity; (2) in the circulatory system Among them, the BsAb should have a relatively low affinity to effector cells or cytotoxic trigger molecules, so as to limit the side effects caused by systemic effector cell activation. Cell cytotoxicity; (3) BsAb should be a humanized antibody, thereby minimizing the human anti-mouse immune response, so that repeated administration is not limited; (4) BsAb molecules should be of moderate size, which can penetrate into tumor tissue Inside, it can also ensure proper cycle residence time. At present, bivalent bispecific or trivalent trispecific antibodies are constructed using the first constant region (CH1) of human IgG1 heavy chain and the constant region (CL) of kappa chain as the heterodimerization domain. The antigen has only one binding domain, which has a low affinity for the target molecule and poor guiding effect. Therefore, BsAbs with more reasonable structures and more powerful functional effects still need to be developed.

Contents of the invention

In order to solve the shortcomings of current bispecific antibodies, the present invention discloses a trivalent BsAb.

The present invention adopts the first constant region CH1 of human IgG1 heavy chain and the CL domain of the kappa chain constant region as heterodimerization domains, and designs a novel trivalent BsAb. The trivalent BsAb of the present invention comprises the first constant region CH1 of the human IgG1 heavy chain, the CL domain of the constant region of the kappa chain, the heavy chain variable region VH and the light chain variable region VL of the A antibody, and the single-chain variable region of the B antibody scFv. The heavy chain variable region VH of antibody A is connected to the N-terminus of CH1, and the single-chain variable region scFv of antibody B is connected to the C-terminus of CH1 (the structure is VH-VL or VL-VH), forming a VH-CH1-scFv structure ; The light chain variable region VL of antibody A is connected to the N-terminus of CL, and the scFv of antibody B is connected to the C-terminus of CL to form a VL-CL-scFv structure; natural dimerization between CH1 and CL forms a trivalent BsAb, and three See Figure 1 and Figure 2 for the arrangement sequence and structural diagram of each domain of the valent BsAb. In the trivalent BsAb, the A antibody recognizes trigger molecules on the surface of effector cells such as CD3, CD16, CD64, etc., and the B antibody recognizes target cell surface markers such as Her2/neu, CD20, CD30, EGFR, etc.

The invention also discloses a method for preparing the trivalent BsAb, the method comprising the following steps:

(1) Construction of expression vector An expression vector was selected to construct a trivalent bispecific antibody bicistronic expression vector. According to VL, CL, scFv, RBS (ribosome binding site, the sequence is aaggag), pelB (bacterial signal peptide sequence, as shown in sequence 15 in the sequence table), VH and CH1 gene sequences and multiple cloning sites in the vector Design primers. Wherein VL and CL, VH and CH1 are connected by overlapping extension PCR method, and RBS and pelB are fused to the 5' end of VH through primers. The PCR products were purified and recovered, and the VL-CL fragment, scFv fragment, and RBS-PelB-VH-CH1 fragment were digested respectively, and each fragment was inserted into the multiple cloning site in the vector in sequence to construct an expression vector.

(2) Expression and detection of recombinant protein BsAb in Escherichia coli The recombinant vector was transformed into Escherichia coli, a single colony was picked and cultured, induced by adding IPTG, samples were taken for SDS-PAGE and Western-blot to detect the expression of BsAb.

(3) Purification of BsAb The expressed culture medium was centrifuged to harvest the cells, resuspended in PBS, freeze-thawed, ultrasonicated, centrifuged, and the supernatant harvested. The supernatant was diluted with a binding buffer, passed through an affinity chromatography column, and eluted with an elution buffer, and the purified protein was detected by SDS-PAGE.

(4) FACS analysis to initially detect the binding activity of BsAb

① FACS analysis of the binding activity of BsAb to SK-BR-3 cells. Digest SK-BR-3 cells, wash with cold PBA by centrifugation, incubate with BsAb by shaking, wash by centrifugation, incubate with FITC-labeled goat anti-human IgG by shaking, wash by centrifugation, resuspend cells, and analyze.

② FACS analysis of the binding activity of BsAb to CD16 surface antigen-positive cells. Take peripheral blood from healthy people, separate mononuclear cells, wash by centrifugation, incubate with BsAb shaking, wash by centrifugation, incubate with phycoerythrinase-labeled mouse anti-human CD56, FITC-labeled goat anti-human IgG, wash by centrifugation, and resuspend cells Afterwards, two-color fluorescence analysis was performed.

The trivalent bispecific antibody of the present invention combines the advantages of the previous bivalent bispecific antibody and tetravalent bispecific antibody, and abandons its defects, and has the following advantages: (1) The trivalent BsAb binds to the effector cells in a monovalent manner. In the circulatory system, the affinity with effector cell triggering molecules is low, which can avoid or reduce the side effects caused by systemic effector cell activation; (2) trivalent BsAb binds to target cells with bivalent binding, which has higher The affinity of BsAb enhances the guiding function, and when it is used for in vivo treatment, it will be positioned at the tumor site more quickly, which can not only enhance the effector function, but also reduce the concentration of bispecific antibodies in circulation and reduce side effects; (3) The molecular weight of trivalent BsAb About 100kD, moderate molecular size, can not only penetrate into the tumor tissue, but also ensure proper circulation retention time.

The antibody prepared by the invention can be used to prepare anti-tumor therapeutic drugs, and has extremely important application value especially for removing postoperative residual foci and metastases of tumor patients.

Description of drawings

Figure 1 is a schematic diagram of the arrangement sequence of each structural domain of a trivalent BsAb.

Figure 2 is a schematic diagram of the structure of each domain of the trivalent BsAb

Fig. 3 is a schematic diagram of the bicistronic structure in the trivalent bispecific antibody expression vector. in

P/O is the T7 promoter/lac operator, RBS is the ribosome binding site,

pelB is the bacterial signal peptide sequence, VL is the anti-human CD16 light chain variable region sequence,

CL is the constant region sequence of human κ chain, scFv is the variable region sequence of anti-Her2/neu single chain antibody,

VH is anti-human CD16 heavy chain variable region sequence, CH1 is the first constant region of human IgG1 heavy chain.

Fig. 4 Electropherogram of PCR amplified VL, VH, CL and CH1 fragments. in

1 is VL; 2 is RBS-PelB-VH; 3 is DGL2000 DNA marker;

4 is CL; 5 is CH1

Fig. 5 is the electrophoresis diagram of PCR amplified scFv. in

1 is scFv (EcoRI, HindIII); 2 is DGL2000 DNA marker;

3 is scFv (NotI, XhoI)

Fig. 6 is the electrophoresis diagram of the VL-CL fragment amplified by the overlap extension PCR method. in

1 is DGL2000 DNA marker; 2 is VL-CL;

Fig. 7 is an electrophoresis diagram of the RBS-PelB-VH-CH1 fragment amplified by the overlap extension PCR method. in

1 is RBS-PelB-VH-CH1; 2 is DGL2000 DNA.

Figure 8 is a schematic diagram of the structure of the recombinant vector named pET22b(+)/BsAb.

Figure 9 is the detection of the expression (precipitation) of BsAb in BL21(DE3) by SDS-PAGE. in

1 is LMW protein marker;

2 is control (empty vector pET22b), not induced;

3 is control, IPTG 0.5mmol/L;

4 is control, IPTG 0.2mmol/L;

5 is pET22b/VLCL+seFv, not induced;

6 is VLCL+scFv, IPTG 0.5mmol/L;

7 is VLCL+scFv, IPTG 0.2mmol/L;

8 is pET22b/BsAb, not induced;

9 is BsAb, IPTG 0.5mmol/L;

10 is BsAb, IPTG 0.2mmol/L.

Fig. 10 detects the expression of BsAb in BL21 (DE3) for Western-blot, wherein

1 is LMW protein marker;

2 is no induced precipitation;

3 is uninduced supernatant;

4 is induced precipitation;

5 is the induction supernatant.

Fig. 11 is that SDS-PAGE detects purified BsAb, wherein

1 is LMW protein marker, 2 is purified BsAb.

Fig. 13 is the binding activity of FACS analysis BsAb and CD16 surface antigen positive cell, wherein

1 is negative control 2 is BsAb

Detailed ways

Example 1 Preparation of anti-Her2/neu×anti-CD16 trivalent BsAb

1. Materials

Mouse anti-human CD16 monoclonal antibody light chain, heavy chain variable region genes (VL, VH) were cloned from hybridoma cell B88-9, commercially available (refer to "Molecular Cloning" (Science Press, second edition) for experimental methods); The VL and VH genes of the mouse anti-P185 ^erbB2 monoclonal antibody were cloned from the hybridoma Her2 (commercially available), and the scFv was synthesized according to conventional methods. The amino acid sequence of the connecting peptide between VL and VH was (GGGGS) ₃ (see "Genetic Engineering Antibody", Dong Zhiwei, Wang Yan (editors); human IgG1 heavy chain first constant region CH1 and κ chain constant region CL genes provided by Dr. Tong Yigang, Academy of Military Medical Sciences (GenBank AF027159, X95747); Escherichia coli strain JM109, BL21(DE3), prokaryotic expression vector pET22b(+) is a standard strain, which is commercially available; human breast cancer cell line SK-BR-3, which highly expresses P185 ^erbB2 , is a standard strain (ATCC Number: HTB-30), which is purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Affinity chromatography column packing rProtein G Sepharose Fast Flow is a product of Pharmacia Biotech; T4 DNA ligase is a product of Gibco BRL; VentDNA polymerase and restriction endonuclease are products of Biolab; plasmid extraction kits, DNA fragment recovery reagents The box is the product of Beijing Dingguo Biotechnology Company; IPTG is the product of SIGMA Company; lymphocyte separation fluid is the product of Tianjin Chuanye Biochemical Products Co., Ltd.; goat anti-human IgG is made by our laboratory; horseradish peroxidase-labeled goat anti-human IgG , goat anti-mouse IgG (HRP-GAH, HRP-GAM), goat anti-human IgG labeled with fluorescein isothiocyanate (FITC-GAH) were purchased from Beijing Zhongshan Biotechnology Company; phycoerythrinase-labeled mouse anti-human CD56 (PE-MAH CD56) was purchased from Jingmei Bioengineering Co., Ltd.

2. Methods and results

1. Construction of expression vector

Select pET22b(+) as the expression vector to construct the bicistronic expression vector of the trivalent bispecific antibody. The bicistronic structure is shown in Figure 3. Primers were designed according to the VL, CL, scFv, ribosome binding site (RBS), bacterial signal peptide sequence (pelB), VH and CH1 gene sequences and the multiple cloning site in the pET22b(+) vector, see Table 1. Wherein VL and CL, VH and CH1 are connected by overlapping extension PCR method, and RBS and pelB are fused to the 5' end of VH through primers RBS/PelB1, PelB2, and PelB3/VH5.

Table 1 PCR primer sequences

Primer name (endonuclease) sequence Primer name (endonuclease) sequence VL5(NcoI) Sequence 1 in the sequence listing PelB2 Sequence 8 in the Sequence Listing VL3 Sequence 2 in the sequence listing PelB3/VH5 Sequence 9 in the Sequence Listing CL5 Sequence 3 in the sequence listing VH3 Sequence 10 in the Sequence Listing CL3(EcoRI) Sequence 4 in the sequence listing CH15 Sequence 11 in the Sequence Listing ScFv5(EcoRI) Sequence 5 in the sequence listing CH13(NotI) Sequence 12 in the Sequence Listing ScFv3(HindIII) Sequence 6 in the sequence listing ScFv5(NotI) Sequence 13 in the Sequence Listing RBS/PelB1(HindIII) Sequence 7 in the sequence listing ScFv3(XhoI) Sequence 14 in the Sequence Listing

The conditions for PCR amplification of VL, VH, CL and CH1 were 95°C for 2 min, and cycled 25 times according to the following parameters: denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 45 sec, and the last cycle of extension at 72°C for 10 min. The 5' end primer for amplifying VH is a mixture of RBS/PelB1, PelB2 and PelB3/VH5, and RBS and pelB are fused to the 5' end of VH, as shown in Figure 4.

The conditions for PCR amplification of scFv were 95°C for 2 min, and cycled 25 times according to the following parameters: denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 75 sec, and the last cycle of extension at 72°C for 10 min, as shown in Figure 5.

The condition for overlapping extension PCR to connect VL and CL is to use equal amounts of purified and recovered VL and CL (about 20 ng) as templates and primers for each other, and the rest of the reagents are the same as conventional PCR. Cycle 7 times according to the following parameters: denature at 94°C for 1 min, 53 Anneal for 1 min at ℃, extend for 40 min at 72°C; then add VL5`-end primer (VL5) and CL3`-end primer (CL3), and cycle 25 times according to the following parameters: denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 75 sec, and finally One cycle of extension at 72°C for 10 min, see Figure 6.

The conditions for overlapping extension PCR to connect RBS-PelB-VH and CH1 are to use equal amounts of purified and recovered VH and CH1 (about 20 ng) as templates and primers for each other, and the rest of the reagents are the same as conventional PCR. Cycle 7 times according to the following parameters: 94°C Denaturation for 1 min, annealing at 54°C for 1 min, extension at 72°C for 45 min; then add VH5`-end primer (RBS/PelB1) and CH13`-end primer (CH13), cycle 25 times according to the following parameters: denaturation at 94°C for 1 min, annealing at 56°C for 1 min, Extend at 72°C for 80 sec, and extend at 72°C for 10 min in the last cycle, see Figure 7.

DNA Fragment Recovery Kit Purification and recovery of PCR products, NcoI, EcoRI double enzyme digestion VL-CL fragment, EcoRI, HindIII double enzyme digestion scFv fragment, HindIII, NotI double enzyme digestion RBS-PelB-VH-CH1 fragment, NotI, XhoI double enzyme The scFv fragments were cut, and each restriction fragment was inserted into the multiple cloning site in the pET22b(+) vector in turn, and the recombinant vector was named pET22b(+)/BsAb, as shown in Figure 8.

2. Expression and detection of recombinant protein BsAb in Escherichia coli BL21(DE3) Transform the recombinant vector pET22b(+)/BsAb into Escherichia coli BL21(DE3), pick a single colony and inoculate it in LB culture containing 100mg/L ampicillin medium, shake culture at 37°C until OD ₆₀₀ is about 0.4, add IPTG to the final concentration of 0.2mmol/L and 0.5mmol/L, respectively, at room temperature, 130r/min, and continue to cultivate for 3h.

SDS-PAGE detection expression. Take 1ml of each sample, centrifuge at 10,000g for 15min to harvest bacterial cells, resuspend the cells in 200μl PBS, freeze and thaw three times, break the bacteria by ultrasonic, and centrifuge at 10,000g for 15min at 4°C, collect supernatant and precipitate respectively, and use 150μl 1× gel for precipitation Resuspend in loading buffer. The supernatant (10μl+10μl SDS gel loading buffer) and the precipitate were taken separately for 10% SDS-PAGE. The results showed that the bacteria transformed with the recombinant vector had a new protein band at a molecular weight of about 50 kD, as shown in FIG. 9 .

The expression of BsAb was detected by Western-blot. The proteins separated by SDS-PAGE were electroblotted to nitrocellulose membrane, and blocked overnight at 4°C with PBS containing 5% skimmed milk powder. TBS (NaCl 150mmol/L, Tris-HCl 50mmol/L, pH 7.5) washed three times, 10min each time, added HRP-GAH diluted in TBS, and incubated at 37°C for 1h. Wash with TBS three times, 10min each time, and develop color with DAB system. As a result, a specific protein band was developed at a molecular weight of about 50 kD, as shown in FIG. 10 .

3. Purification of BsAb

Frozen strains were activated overnight at 120r/min at 37°C. Inoculate 0.5ml of the activated strains into 120ml of LB medium (ambicillin 100mg/L), shake culture at 37°C and 250rpm until the _OD600 was 0.5, and add IPTG to the end. The concentration is 0.25mmol/L, 150r/min, and culture is continued at 30°C for 4h. Harvest the cells by centrifugation at 4000g for 10 min at 4°C, and resuspend the cells with 20% of the initial culture medium volume in PBS. The suspension was frozen and thawed once, and the bacteria were broken by ultrasonic waves. Centrifuge at 12000g for 20min at 4°C, and harvest the supernatant. The supernatant was diluted with 10 times volume of binding buffer (50mmol/L Tris-HCl, pH7.4, 150mmol/L NaCl) and passed over rProteinG Sepharose Fast Flow affinity chromatography column, 100mmol/L glycine, pH2.5 elution buffer liquid elution. The eluted protein was replaced with PBS buffer through YM-30 kD ultrafiltration membrane. The purified protein was detected by 10% SDS-PAGE, and the results are shown in FIG. 11 .

Example 2 FACS analysis to detect the activity detection of anti-Her2/neu×anti-CD16 trivalent BsAb

1. Materials

Same as embodiment one.

2. Methods and results

1. FACS analysis of the binding activity of BsAb to SK-BR-3 cells

Digest SK-BR-3 cells with 0.02% EDTA, centrifuge and wash twice with cold PBA (PBS, 2% BSA, 0.1% NaN ₃ ); incubate with BsAb (0.1 mg/L) at 0°C for 30 min with shaking; centrifuge with cold PBA Wash twice, and incubate with FITC-GAH at 0°C for 30 minutes; centrifuge and wash twice with cold PBA, resuspend cells in PBS, and analyze with Becton-Dickenson FACsort instrument. The results showed that the BsAb could specifically bind to intact SK-BR-3 cells, and the relative average fluorescence intensity was 92.08, which was 25.4 times that of the negative control, as shown in FIG. 12 .

2. FACS analysis of binding activity between BsAb and CD16 surface antigen-positive cells Take peripheral blood from healthy people, separate mononuclear cells with lymphocyte separation medium, and wash twice with cold PBA (PBS, 2% BSA, 0.1% NaN ₃ ) centrifugation ;Incubate with BsAb (0.1mg/L) at 0°C for 30min; Centrifuge and wash with cold PBA for 2 times, and incubate with PE-MAH CD56 and FITC-GAH for 30min at 0°C; Wash with cold PBA for 2 times, resuspend cells in PBS , two-color fluorescence analysis was performed with a Becton-Dickenson FACsort instrument. The results show that BsAb can specifically bind to NK cell subsets, as shown in FIG. 13 .

sequence listing

<110> Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Chinese People's Liberation Army

<120> A trivalent bispecific antibody, its preparation method and use

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Claims (3)

1. A trivalent bispecific antibody, characterized in that it comprises the first constant region CH1 of the human IgG1 heavy chain, the CL domain of the κ chain constant region, the heavy chain variable region VH and the light chain variable region VL of an anti-CD16 antibody , the single-chain variable region scFv of the anti-Her2/neu antibody; wherein the heavy chain variable region VH of the anti-CD16 antibody is connected to the N-terminal of CH1, and the single-chain variable region scFv of the anti-Her2/neu antibody is connected to the C-terminal of CH1 ; The VL of the light chain variable region of the anti-CD16 antibody is connected to the N-terminal of CL, the scFv of the anti-Her2/neu antibody is connected to the C-terminal of CL, and naturally dimerizes between CH1 and CL to form a trivalent BsAb. 2. The method for preparing the trivalent bispecific antibody of claim 1, comprising the following steps: (1) Construction of the expression vector: select the expression vector, design primers, connect the gene fragments by PCR method, and after digestion respectively, each restriction fragment is inserted into the multiple cloning site in the vector in sequence to construct the expression vector; (2) Expression and detection of the recombinant protein of the trivalent bispecific antibody: transform the recombinant vector into Escherichia coli, induce expression, and take samples to detect the expression of the trivalent bispecific antibody; (3) Purification of the trivalent bispecific antibody: collect the expressed bacteria, collect the supernatant after breaking the bacteria, and perform affinity chromatography purification; (4) Analyzing the binding activity of the trivalent bispecific antibody. 3. The use of the trivalent bispecific antibody according to claim 1 in the preparation of drugs for treating tumors.

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