CN104592392B - The structure of bispecific antibody EpCAM × CD3 a kind of and application - Google Patents

Abstract

The present invention provides a kind of bispecific antibodies, the bispecific antibody of the application is made of strand unit and monovalent unit, wherein the strand unit for the surface antigen CD3 of immunocyte there is specific binding capacity, the unit price unit to have specific binding capacity for tumor cell surface antigen EpCAM；The strand unit includes the single chain variable fragment (scFv) with Fc segment compositions, which includes light chain and heavy chain pair.The application also provides the preparation method of bispecific antibody, the pharmaceutical use of these antibody.

Description

Construction and application of a bispecific antibody EpCAM×CD3

technical field

The present invention relates to the technical field of immunology. Specifically, it relates to a method for constructing and preparing a bispecific antibody, as well as a method for detecting the function and properties of the bispecific antibody.

Background technique

Bispecific antibody (bispecific antibody, BiAb) 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. BiAb has broad application prospects in biomedicine, especially in tumor immunotherapy. Killing tumor cells through BiAb-mediated cytotoxicity is a hot topic in current immunotherapy application research. Sexual killing. However, in the process of developing bispecific antibody drugs, there are many obstacles such as difficult expression, low yield, difficult purification, and poor stability. Therefore, it is necessary to construct new bispecific antibodies to overcome the above obstacles and establish corresponding The immune killing animal model is very necessary. The invention provides the construction of a novel bispecific antibody, and describes the research method and results of its pharmacodynamics. The following is an introduction to the background technology of the studied immune cell antigens and tumor cell antigens, as well as the development of related technologies.

1. CD3

The CD3 molecule consists of four subunits: δ, ε, γ, and ζ, with molecular masses of 18.9 kDa, 23.1 kDa, 20.5 kDa, and 18.7 kDa, and lengths of 171, 207, 182, and 164 amino acid residues, respectively. Together, they form 6 peptide chains, which are often tightly combined with T cell receptor (TCR) to form a TCR-CD3 complex containing 8 peptide chains. The schematic diagram of the structure is shown in Figure 1. This complex has the functions of T cell activation signal transduction and stable TCR structure. CD3 cytoplasmic segment contains immunoreceptor tyrosine-based activation motif (immunoreceptor tyrosine-based activation motif, ITAM), TCR recognizes and binds the antigenic peptide presented by MHC (major histo-compatibi lity complex) molecules, resulting in CD3 ITAM The tyrosine residues in the conserved sequence of T cells are phosphorylated by the tyrosine protein kinase p56lck in T cells, and then other tyrosine protein kinases (such as ZAP-70) containing the SH2 (Scr homology 2) domain can be recruited. Phosphorylation of ITAM and binding to ZAP-70 is one of the important biochemical reactions in the early stage of T cell activation signal transduction process. Therefore, the function of the CD3 molecule is to transduce the activation signal generated by the recognition of the antigen by the TCR.

2.EpCAM

EpCAM (CD326) is a type I transmembrane glycoprotein that acts as a specific cell adhesion molecule for epithelial cells. It is also involved in several other processes including cell migration, proliferation, differentiation, etc. EpCAM is one of the earliest tumor-associated antigens identified by monoclonal antibody technology. It is widely expressed on the surface of epithelial tissue in the form of multimers and mediates calcium-independent intercellular homotypic adhesion. family of molecules. EpCAM also has other characteristics of the adhesion molecule family, and participates in various processes including cell-matrix interaction, migration, cell differentiation, morphology, cell cycle regulation, signal transduction, and metabolism. At the same time, EpCAM was overexpressed in a variety of epithelial tumors, suggesting that it is closely related to tumors. Under pathological conditions, EpCAM is expressed in different degrees in adenocarcinoma, including colorectal cancer, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, hepatocellular carcinoma and retinoblastoma. A number of studies have confirmed that the expression of EpCAM is related to the proliferation, cycle distribution and metastasis of breast cancer and colon cancer cells (see Table 1). Monospecific anti-EpCAM monoclonal antibody (MAB), such as mAb 17-1A (glaxowellcome, Centocor), was the first approved adjuvant therapy for EpCAM-directed treatment of colorectal cancer in Germany, however, a large number of clinical drug data show that this There was no apparent more beneficial effect of this monospecific antibody compared with chemotherapy. At present, some other EpCAM-directed therapies, including bispecific antibodies, are being developed for cancer treatment. The bispecific antibody MT110 and Catumaxomab are both therapeutic double antibody drugs targeting the tumor antigen EpCAM. Catumaxomab was developed in 2009 by Approved by the European Union for the treatment of malignant ascites, MT110 is already in clinical research. Apparently, EpCAM has become one of the hot targets in current tumor therapy research.

Table 1 Wide tumor distribution of EpCAM

the tumor EpCAM positive rate ovarian cancer 88-100% stomach cancer 98% colon cancer 99% pancreatic cancer 96% breast cancer 90% endometrial cancer 91-96% lung cancer 87% prostate cancer 98%

3. Development of bispecific antibody technology

Bispecific antibodies, two antigen-binding sites in one antibody molecule can bind to two different antigen epitopes respectively.

Antibody drugs are biomacromolecular drugs prepared by antibody engineering technology based on cell engineering technology and genetic engineering technology. They have the advantages of high specificity, uniform properties, and directional preparation for specific targets. Monoclonal antibodies are mainly used clinically in the following three aspects: tumor treatment, immune disease treatment and anti-infection treatment. Among them, the treatment of tumors is currently the most widely used field of monoclonal antibodies. Among the monoclonal antibody products that have entered clinical trials and are on the market, the number of products used for tumor treatment accounts for about 50%. Monoclonal antibody treatment of tumors is an immunotherapy that stimulates the immune system to kill target cells by combining them with specific targets of diseased cells. In order to enhance the effector function of antibodies, especially the effect of killing tumor cells, people have tried various methods to transform Antibody molecules, bispecific antibodies are one of the development directions to improve the therapeutic effect of antibodies, and have become a hot spot in the field of antibody engineering research.

The bispecific antibody used for immunotherapy is an artificial antibody containing two specific antigen-binding sites, which can build a bridge between target cells and functional molecules (cells), stimulate a oriented immune response, and play an important role in the immune response of tumors. It has broad application prospects in treatment.

4. Bispecific antibody preparation

Bispecific antibodies can be obtained in a variety of ways, and their preparation methods mainly include: chemical coupling method, hybridization-hybridoma method and genetic engineering antibody preparation method. The chemical coupling method is to link two different monoclonal antibodies together by chemical coupling to prepare bispecific monoclonal antibodies. This is the earliest concept of bispecific monoclonal antibodies. The disadvantages of this preparation method are Obvious. Hybridization—The hybridoma method is to produce bispecific monoclonal antibodies by cell hybridization or ternary hybridomas. These cell hybridomas or ternary hybridomas are fused by established hybridomas, or established hybridomas and hybridomas derived from mice The fusion of obtained lymphocytes can only produce murine bispecific antibodies, and its application is greatly limited. With the rapid development of molecular biology technology, various construction modes of genetically engineered humanized bispecific antibodies have emerged, which are mainly divided into bispecific microantibodies, diabodies, single-chain diabodies, multivalent Four classes of bispecific antibodies. At present, several genetically engineered bispecific antibody drugs have entered the clinical trial stage in the world, and have shown good application prospects.

5. Adoptive immunotherapy of tumors

The adoptive immunotherapy of tumors is to inject autologous or allogeneic immunocompetent cells into the patient's body after in vitro expansion, directly kill tumor cells, regulate and enhance the immune function of the body, mainly including LAK cells, TIL cells, activated T lymphocytes and Immunotherapy of CIK cells. However, immunotherapy can only remove a small number of scattered tumor cells, and has limited efficacy for advanced solid tumors. Therefore, it is often used as an adjuvant therapy in combination with conventional methods such as surgery, chemotherapy, and radiotherapy. After removing a large number of tumor cells by conventional methods, immunotherapy is used to remove the remaining tumor cells to improve the effect of comprehensive tumor treatment. Among them, adoptive immunotherapy, as a new method in the comprehensive treatment of tumors, has been widely cooperated with conventional surgery, radiotherapy, chemotherapy and other cell and molecular therapies, and has shown broad application prospects in the treatment of various tumors. However, a more ideal way should be that one end of the bispecific antibody can bind to the surface antigen CD3 of the cultured immune cells, and then imported into the body together, while the other end of the bispecific antibody can bind tumor cells well In this way, the bispecific antibody can build a bridge between tumor cells and immune cells in vivo, so that immune cells can concentrate around tumor cells, and then kill tumor cells. This method can effectively solve the metastasis and spread of tumor cells, and overcome the disadvantages of "incomplete, easy to transfer, and large side effects" after the three traditional treatment methods of surgery, radiotherapy and chemotherapy.

Contents of the invention

Terms and Abbreviations

BiAb: bispecific antibody (bispecific antibody)

TA: tumor antigen (tumor antigen)

VH: heavy chain variable region.

VL: light chain variable region (light chain variable region).

CL: constant region of light chain.

CDR: It is the abbreviation of Complementarity determining regions (CDRs) in English, which refers to the complementarity determining regions of antibodies.

ScFv: single-chain variable region antibody fragment (single-chain variable fragment), also known as single-chain antibody.

CLD: cell line development

FACS: Fluorescence-activated cell sorting, also known as flow cytometry.

The present invention aims at the shortcomings of conventional monoclonal antibodies, and creates a new molecule-bispecific antibody through genetic engineering and antibody engineering methods. In traditional monoclonal antibodies, it mainly kills tumor cells through CDC, ADCC and apoptosis capabilities. On the basis of T cells, the immunotherapy that mediates T cells is added, which greatly improves the efficacy of the immune system in killing tumor cells.

Specifically, the present invention provides the following technical solutions:

In one embodiment, a bispecific antibody is provided, characterized in that the antibody comprises: (a) a monovalent unit, which is a light chain-heavy chain pair, and the light chain-heavy chain pair is directed against the tumor cell surface The antigen has specific binding ability, preferably the tumor cell surface antigen is EpCAM, CD20, CD30 and CD133, more preferably the tumor cell surface antigen is EpCAM; and (b) a single-chain unit is a fusion peptide, and the fusion peptide comprises Single-chain variable fragment ScFv and Fc fragment with hinge region, CH2 domain and CH3 domain, wherein the immune cells targeted by the fusion peptide are selected from T cells, NKT cells or CIK cells; preferably, the fusion peptide is effective on immune cells Surface antigen CD3 has specific binding ability.

In one embodiment, the CH2 domain of the single chain unit of the bispecific antibody is located between the scFv fragment and the CH3 domain; the single chain unit does not comprise a CH1 domain.

In one embodiment, the single chain variable fragment of the bispecific antibody consists of a light chain variable region and a heavy chain variable region domain, both of which target the epitope CD3.

In one embodiment, in the monovalent unit, both the light chain constant region domain and the light chain variable region domain of the light chain are targeted to the tumor antigen epitope EpCAM; the heavy chain constant structure of the heavy chain Domain CH1 and the heavy chain variable domain are also targeted to the tumor antigen epitope EpCAM; the light chain is bound to the heavy chain by a disulfide bond; the heavy chain is bound to the fusion peptide by one or more disulfide bonds.

In one embodiment, the single chain unit comprises the antibody anti-CD3 directed against CD3 and the monovalent unit comprises the antibody anti-EpCAM directed against EpCAM.

In one embodiment, the amino acid sequence of the heavy chain of the antibody anti-EpCAM is the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence of the light chain of the antibody anti-EpCAM is the amino acid sequence shown in SEQ ID NO: 3, and the The amino acid sequence of the anti-CD3ScFv-Fc is the amino acid sequence shown in SEQ ID NO: 5; and the cysteine at position 223 of the anti-EpCAM heavy chain is the same as the cysteine at position 220 of the light chain of anti-EpCAM The acids are connected in the form of disulfide bonds, and the cysteines at positions 229 and 232 of the anti-EpCAM heavy chain are connected with the cysteines at positions 255 and 258 of the anti-CD3ScFv-Fc with disulfide bonds, respectively. Ligated in the form of sulfur bonds, the anti-EpCAM heavy chain forms a salt bridge connection with the 428 and 397 positions of the anti-CD3ScFv-Fc at the 395 and 412 positions, and the anti-EpCAM heavy chain is at the 369 position The spot forms a bump-entry-hole junction with the 436 site of the anti-CD3ScFv-Fc.

In one embodiment, the heavy chain in the monovalent unit comprises a human or humanized Fc fragment, preferably, the Fc fragment of the heavy chain comprises a human IgG Fc fragment; the Fc fragment of the fusion peptide comprises a human or human Fc fragment. Fc fragment of Fc, preferably, the Fc fragment of the fusion peptide comprises a human IgG Fc fragment.

In one embodiment, the human IgG Fc segment of the monovalent unit and the IgG Fc of the single chain unit are connected by a salt bridge and a bump-in-hole structure.

In one embodiment, a method for preparing a bispecific antibody is provided, the method comprising:

(1) respectively constructing the heavy and light chains of the monovalent unit on the first expression vector, and constructing the single-chain unit on the second expression vector;

(2) co-transfecting the first and second expression vectors into the cells, culturing and taking the supernatant;

(3) Separating the expression supernatant to obtain the purified bispecific antibody; preferably, the cells are CHO-S cells; or preferably, the separation step includes: protein A affinity chromatography column from the expression All antibodies with Fc domains were captured in the supernatant, and the target bispecific antibody and by-products were separated by SP cation exchange chromatography, then passed through a Q column, and finally the replacement buffer PBS was concentrated.

In one embodiment, the first expression vector is pCHO1.0; the second expression vector is pCHO1.0-hygromycin.

In one embodiment, the monovalent unit is an anti-EpCAM antibody, and the primers used to amplify its light chain are Kozak (EcoR V) F, MK-leader sequence (EcoRV) F, M701-VL F1 and hIgK (PacI) R, through overlapping PCR amplification, the Kozak sequence, leader sequence and restriction sites EcoR V and PacI were introduced into the light chain; the primers used to amplify the heavy chain were Kozak (Avr II) F, MK-leader sequence (AvrII) F , M701-VH F1 and hIgG1 (sbfI) R, by overlapping PCR amplification, the Kozak sequence, leader sequence and restriction site AvrI I and BstZl7I are introduced into the heavy chain; the amplified LC gene fragment is combined with EcoR V and PacI The digested pCHO1.0 expression vector was subjected to homologous recombination to obtain the expression vector loaded with anti-EpCAM light chain; then digested with AvrI and BstZl7I and then subjected to homologous recombination with HC to obtain anti-EpCAM pCHO1. 0 expression vector, the plasmid was named pCHO1.0-anti-EpCAM-HL-KKW;

The single-chain unit is an anti-CD3ScFv-Fc antibody, and the primers used to amplify it are Kozak (Avr II) F, MK-leader sequence (AvrII) F, L2K-VH (MK) F1 and hIgG1 (sbfI) R, The anti-CD3 ScFv-Fc domain was amplified by overlapping PCR, and the Kozak sequence, leader sequence, and restriction sites AvrI I and BstZl7I were introduced into the ScFv-Fc, and the amplified gene fragment was combined with the enzyme-digested pCHO1.0- Homologous recombination was performed on the mycin expression vector to obtain the expression vector loaded with anti-CD3ScFv-Fc, and the plasmid was named pCHO1.0-hygromycin-L2K-ScFv-Fc-LDY.

In one embodiment, the use of any of the above-mentioned bispecific antibodies or bispecific antibodies prepared according to any of the above-mentioned methods in the preparation of medicines for the treatment of tumors or tumors caused by the expression of EpCAM-specific antigens related diseases, or to kill cells expressing EpCAM.

In one embodiment, the use of any of the above-mentioned bispecific antibodies or the bispecific antibodies prepared according to any of the above-mentioned methods in the preparation of drugs, the drugs are used for screening in tumor cell lines for the treatment of expression of EpCAM Double antibody drugs for tumor cell-related diseases of specific antigens or evaluation of the efficacy of double antibody drugs for the treatment of tumor cell-related diseases expressing EpCAM specific antigens. The present invention also provides the following technical solutions:

The present invention provides a new type of antibody called bispecific antibody, and establishes a method of using the immune system of the human body for immunotherapy and conducting research on the efficacy of the bispecific antibody. This bispecific antibody, as a new type of antibody and used as a drug effect model, introduces the specific cytotoxic effect of T cells on EpCAM and other tumor antigens.

The present invention provides a new method to prepare bispecific antibody MSBODY (monomer and ScFvbispecific antibody, as shown in Figure 2), the bispecific antibody includes two sets of heavy and light chain combinations, one of which specifically binds to one antigen, and Some modifications are made in the Fc region of its heavy chain, so that it is not easy to form dimers by itself compared with the wild type; while the other group specifically binds to another antigen, it also makes other modifications in the Fc region of its heavy chain, and it is not easy to form dimers by itself. dimers, and hybrid dimers are easily formed between these two groups of heavy and light chains. And the antibody structure of one group is monomeric Ab, and the other group is ScFv-Fc, which avoids the possibility of mismatching between the respective light chains and the heavy chains of the other party, thereby forming a 125KD bispecific antibody protein molecule. After Fc modification, the heavy chain and single chain of the monomeric Ab are naturally heterodimerized, and at the same time, CL and CH1 are naturally dimerized, and finally MSBODY is formed. The sequence and structural diagram of each domain of MSBODY are shown in Figure 2.

In the present invention, the above method for preparing bispecific antibodies is used to prepare bispecific antibodies. Among them, the bispecific antibody targeting EpCAM and CD3 is named EpCAMX CD3, as shown in Figure 2, the anti-EpCAM side is in IgG form, including anti-EpCAM heavy chain and light chain, and the anti-CD3 side is ScFv-Fc format, including anti-CD3 VH, VL, Fc domains. The above bispecific antibodies are constructed by antibody genetic engineering methods, the monomeric Ab heavy chain and monomeric Ab light chain binary expression vectors of the bispecific antibody MSBODY, and the ScFv-Fc expression vector. Primers were designed according to LC, HC, ScFv, Fc gene sequences and multiple cloning sites in the vector. Among them, LC, HC, ScFv and Fc are amplified by PCR respectively, and gene fragments are obtained by PCR or overlap extension PCR method, and then cloned by homologous recombination method. Digest the pCHO1.0 or pCHO1.0-hygromycin vector, then purify and recover the PCR product and the digested vector, clone the LC fragment and the HC fragment into the pCHO1.0 vector by homologous recombination in two steps, ScFv- The Fc fragment was cloned into pCHO1.0-hygromycin vector by homologous recombination and sequenced. Expression and detection of recombinant protein MSBODY in mammalian cells, using transfection reagents to co-transfect two plasmids expressing monomeric Ab heavy chain, monomeric Ab light chain and single chain (Single chain) respectively into mammals In the cells, the supernatant was collected for SDS-PAGE and Western blot to detect the expression of MSBODY. The culture supernatant after transfection and expression is centrifuged, filtered, diluted with binding buffer, passed through affinity chromatography column, eluted with elution buffer, and the purified protein is detected by SDS-PAGE.

The invention also provides methods for establishing tumor models and detecting drug effects. The establishment of the drug efficacy model and the drug efficacy test are mainly to use the stable human tumor cell line expressing human EpCAM and the isolated and cultured human CIK cells to form tumors efficiently in immunodeficient mice, and use the constructed double Specific antibody-mediated, which can simultaneously bind tumor cells, CD3-expressing T cells, and immune adjuvant cells that can bind to Fc, build a bridge between immune cells and tumor cells, and form an immune complex. The immune response of the immune system secretes a variety of cytokines to kill tumor cells, thereby inhibiting the growth of tumors. The model is carried out in a simulated immune system, using immune cells to kill tumors, and can effectively reflect the efficacy of bispecific antibodies in mediating immune cells to kill tumor cells. Bispecific antibody drug development provides a good method for drug efficacy evaluation.

The beneficial technical effect of technical scheme of the present invention has:

1. The present invention discloses the construction of a novel bispecific antibody MSBODY and the establishment and application of an animal model of immune cells killing tumor cells mediated by it. The invention includes immune cell killing mediated in the bispecific antibody drug research process, preparation of the bispecific antibody, and establishment and detection of a bispecific antibody drug effect model. The bispecific antibody MSBODY includes a combination of heavy and light chains, and another set of ScFv-linked Fc combinations, one of which specifically binds to a human tumor cell antigen, including a series of tumor cell membrane surface antigens such as EpCAM, and in its heavy Some modifications are made to the Fc region of the heavy chain, so that it is less likely to form dimers by itself than the wild type; while another group that specifically binds to another mouse T cell antigen CD3 also undergoes other modifications in the Fc region of the heavy chain, which is also not easy. Dimers are formed by themselves, and hybrid dimers are easily formed between these two sets of heavy and light chains. At the same time, the bispecific antibody can build a bridge between target cells and functional molecules (cells), and stimulate a directional immune response. With the participation of immune cells, the bispecific antibody of the present invention has a very strong effect on tumor cells Therefore, it has broad application prospects in tumor immunotherapy.

2. The application provides a heterodimeric antibody, which comprises two different antigen-binding polypeptide units. The heterodimer and its corresponding homodimer have different molecular weights, and the molecular weight can be used to distinguish the heterodimer and the homodimer, thereby effectively determining the purity of the bispecific antibody. One of these two antigen-binding polypeptide units comprises a light chain-heavy chain pair similar to a wild-type antibody, and is also referred to throughout the application as a "monovalent unit". Another antigen binding polypeptide unit comprises a single chain variable fragment (scFv). Such scFvs may be fused to the constant fragment (Fc) of an antibody. This fusion peptide is also referred to as a "single-chain unit" throughout this application.

Surprisingly, the present application demonstrates that such asymmetric antibodies are stable and have high antigen binding efficiency. This is surprising since homodimers of even single chain antibodies have been shown to be unstable under physiological conditions. For example, "scFv Antibody: Principles and Clinical Application," by Ahmad et al. (Clinical and Developmental Immunology, 2012:980250 (2012)), showed that scFv-based IgG class antibodies are unstable and require further engineering to reduce aggregation and improve stability .

In addition, because of their asymmetry, heterodimers have a different isoelectric point than homodimers composed of either antigen-binding polypeptide unit. Based on the difference in isoelectric points between heterodimers and homodimers, the required heterodimers can be easily separated from the homodimers, greatly reducing the downstream process development problems that are common in bispecific antibodies. difficulty.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the accompanying drawings that need to be used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments recorded in the present application , for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without paying creative work, wherein:

Figure 1. Schematic diagram of the molecular structure of CD3.

Figure 2. Molecular schematic diagram of EpCAM×CD3 bispecific antibody.

Figure 3. Electrophoresis detection of PCR products; M: DL10000 nucleic acid molecular marker; 1. Anti-CD3 antibody ScFv-Fc; 2. Anti-EpCAM antibody heavy chain; 3. Anti-EpCAM antibody light chain.

Figure 4. Electrophoresis and purity test results of purified double antibody; (4A) denatured SDS-PAGE electrophoresis, M: protein molecular weight marker; 1: EpCAM×CD3 double antibody; (4B) non-denatured SDS-PAGE electrophoresis, M: protein Molecular weight marker; 1: EpCAM×CD3 diabody; (4C) HPLC-SEC purity profile of EpCAM×CD3.

Figure 5. Affinity diagram of EpCAM×CD3 diabody and HCT116 cells determined based on flow cytometry method, (■) EpCAM×CD3 MSBODY; (●) Anti-EpCAM monoclonal antibody.

Figure 6. The affinity of EpCAM×CD3 diabody to Jurkat cells determined based on flow cytometry method, (■) EpCAM×CD3 MSBODY; (●) Anti-CD3 monoclonal antibody L2K.

Figure 7. Flow cytometric detection of the results of EpCAM×CD3 diabody binding to HCT116 and Jurkat cells at the same time, and pulling the two types of cells together; (●) EpCAM×CD3 diabody; (■) EpCAM monoantibody; (▲) Anti-CD3 monoclonal antibody L2K; (▼) control antibody MCO101.

Figure 8. The Tm value results of the differential scanning calorimeter scanning measurement of the EpCAM×CD3 MSBODY diabody.

Figure 9. Antibody activity test results after heat treatment, 9A. Binding activity detection with EpCAM, (●) anti-EpCAM monoclonal antibody; (▲) EpCAM×CD3 MSBODY double antibody; 9B. Binding activity detection with CD3, (●) Anti-CD3 monoclonal antibody L2K; (■) EpCAM×CD3 MSBODY diabody.

Figure 10. CIK phenotype detection results, CD3, CD56 double-positive NK cells in the upper right corner.

Figure 11. Flow cytometric detection of the killing effect of effector cell CIK on target cell HCT116 in the presence of different concentrations of antibodies; (■) EpCAM×CD3 MSBODY double antibody, (▲) Mco101: control 4420×CD3 double antibody, (▼ ) Anti-EpCAM: anti-EpCAM monoclonal antibody, (●) hIgG: human IgG. EpCAM×CD3

Figure 12. Flow cytometric detection of the killing effect of effector cell CIK on target cell NCI-N87 in the presence of different concentrations of antibodies; (■) EpCAM×CD3 MSBODY double antibody, (▲) Mco101: control 4420×CD3 double antibody, (▼) Anti-EpCAM: anti-EpCAM monoclonal antibody, (●) hIgG: human IgG.

Figure 13. Flow cytometric detection of the killing effect of effector cell PBMC on target cell HCT116 in the presence of different concentrations of antibodies; (■) EpCAM×CD3 MSBODY double antibody, (▲) Mco101: control 4420×CD3 double antibody, (▼ ) EpCAM: EpCAM monoclonal antibody, (•) hIgG: human IgG.

Figure 14. Flow cytometric detection of the killing effect of effector cell PBMC on target cell NCI-N87 in the presence of different concentrations of antibodies; (■) EpCAM×CD3 MSBODY diabody, (▲) Mco101: control 4420×CD3 diabody, (▼) EpCAM: EpCAM monoclonal antibody, (●) hIgG: human IgG.

Figure 15. In vivo drug efficacy test results of double antibodies, mice: NOD-SCID; inoculation (ih): mixed inoculation of SW480 (5X10 ⁶ ) and human CIK (5X10 ⁶ ); administration treatment (iv): EpCAM mAb: 4mg/ kg; Day(0,2,4), 4420×CD3:4mg/kg; Day(0,2,4), EpCAM×CD3(MSBODY)-1:4mg/kg; Day(0,2,4), EpCAM×CD3(MSBODY)-2: 2 mg/kg; Day (0,2,4). (●) indicates PBS, only PBS was given to the tail vein, (□) Anti-EpCAM monoclonal antibody, (△) 4420×CD3 irrelevant control double antibody, (▼) M701-1: EpCAM×CD3 SMBODY double antibody 4mg/kg concentration group (○) M701-2: EpCAM×CD3SMBODY diabody 2mg/kg concentration group.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that the embodiments in this specification are only for illustrating the present invention, but not limiting the scope of the present invention in any way.

Example 1: Construction of expression vectors for bispecific antibodies (EpCAM×CD3, M701)

1. Bispecific antibody sequence design

The bispecific antibody targeting EpCAM and CD3 is named M701, as shown in Figure 2, the anti-EpCAM side is in IgG form, including anti-EpCAM heavy chain and light chain, containing Fab and Fc domains; the anti-CD3 side It is in the form of ScFv-Fc, including anti-CD3 VH, VL, and Fc domains. Among them, the IgG form undergoes KKW transformation on one side of Fc, and the side of ScFv-Fc undergoes LDY transformation on Fc. For the specific Fc transformation process, refer to PCT/CN2012/084982, so that they are not easy to form homodimers, but easy to form hybrid dimers, namely EpCAM×CD3 bispecific antibody. At the same time, in order to express the diabody in CHO cells and secrete it into the culture medium, the leader peptide sequence of the mouse kappa chain was selected as the secretion signal peptide. The amino acid sequence and nucleic acid sequence of each structural domain and signal peptide are shown in the following sequence numbers: 1-8.

Anti-EpCAM heavy chain (amino acid sequence SEQ ID NO: 1)

EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-EpCAM heavy chain (nucleotide sequence number 2)

GAGGTGCAGCTGCTCGAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGATATCCTGCAAGGCTTCTGGATACGCCTTCACTAACTACTGGCTAGGTTGGGTAAAGCAGAGGCCTGGACATGGACTTGAGTGGATTGGAGATATTTTCCCTGGAAGTGGTAATATCCACTACAATGAGAAGTTCAAGGGCAAAGCCACACTGACTGCAGACAAATCTTCGAGCACAGCCTATATGCAGCTCAGTAGCCTGACATTTGAGGACTCTGCTGTCTATTTCTGTGCAAGACTGAGGAACTGGGACGAGCCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCCGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACGATACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCGATCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Anti-EpCAM light chain (amino acid sequence SEQ ID NO: 3)

ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFLTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKFSTYSLSSTLTLKGLNSKADYECSK

Anti-EpCAM light chain (nucleotide sequence number 4)

GAGCTCGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATAGTTATCCGCTCACGTTCGGTGCTGGGACCAAGCTTGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Anti-CD3ScFv-Fc (amino acid sequence number 5)

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-CD3ScFv-Fc (nucleotide sequence number 6)

CAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTCGTGCAGCCGGGCAGGTCCCTGAGACTGTCTTGTAAGGCTTCTGGATACACCTTCACTAGATACACAATGCACTGGGTCAGACAGGCTCCTGGAAAGGGACTCGAGTGGATTGGATACATTAATCCTAGCAGAGGTTATACTAACTACAATCAGAAGTTTAAGGACAGATTCACAATTTCTACTGACAAATCTAAGAGTACAGCCTTCCTGCAGATGGACTCACTCAGACCTGAGGATACCGGAGTCTATTTTTGTGCTAGATATTACGATGACCACTACTGTCTGGACTACTGGGGCCAAGGTACCCCGGTCACCGTGAGCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGAAGTGGTGGAGGAGGTTCTGATATTCAGATGACCCAGAGCCCGTCAAGCTTATCTGCTTCTGTCGGAGACAGAGTCACAATCACATGTTCTGCTTCTAGCTCTGTCTCTTACATGAACTGGTATCAGCAGACACCTGGAAAGGCTCCTAAGCGGTGGATCTACGACACATCTAAGCTCGCTTCTGGAGTCCCTTCTAGATTCTCTGGTTCTGGCTCTGGAACAGACTACACATTCACAATCTCTTCTCTCCAACCTGAGGACATCGCTACATACTACTGCCAACAGTGGTCTAGCAATCCTTTCACATTCGGACAGGGTACCAAACTGCAGATCACAAGAGGTGCGGCCGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

The leader peptide sequence of the mouse kappa chain (amino acid sequence sequence number 7)

METDTLLLWVLLLWVPGSTG

The leader peptide sequence of mouse kappa chain (nucleotide sequence number 8)

atggagacagaacacactcctgctatgggtactgctgctctgggttccaggttccactggt

2. Bispecific antibody gene cloning

pCHO1.0 was selected as the expression vector to clone and express the heavy and light chain genes resistant to EpCAM, and the pCHO1.0-hygromycin expression vector was obtained by replacing the puromycin gene in the pCHO1.0 vector with the hygromycin resistance gene Transformed and selected for cloning and expressing the anti-CD3 ScFv-Fc fusion gene. After the primers in Table 1 were designed according to the cloning scheme, they were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis. Perform PCR amplification with the primers in Table 1. The template is the gene plasmid that was synthesized or subcloned into pCDNA3.1 or pUC57 in the early experiments. The PCT/CN2012/084982 patent has detailed descriptions, and then anti-EpCAM heavy, The light chain was constructed on the expression vector of pCHO1.0, and the anti-CD3ScFv-Fc was constructed on the expression vector of pCHO1.0-hygromycin.

Table 1 Primers used in bispecific antibody gene cloning

Initial PCR amplification template DNA: 35ng of template DNA, such as the light and heavy chains of the target antibody; 1μl of 10μM forward primer and reverse primer; 2.5μl of 10×PCR Buffer buffer; 1μl of 10mM dNTP; 1μl 2.5 units/μl of Pyrobest DNA polymerase (Takara, R005A); and distilled water to a total volume of 25 μl were gently mixed in a microfuge tube and spun rapidly in a microcentrifuge to collect the reaction mixture to the bottom of the tube. PCR reactions were performed using a GeneAmp PCR System 9700 (Applied Biosystem) with the following settings: 95°C, 5 minutes; 25 cycles of: 95°C, 30 seconds each; 56°C, 30 seconds; and 72°C, 1 minute.

Through several rounds of overlapping PCR amplification, the Kozak sequence, leader sequence and restriction sites EcoR V and PacI were introduced into the light chain, as shown in Figure 3; and the corresponding primers introduced the Kozak sequence, leader sequence and restriction sites AvrII and BstZl7I See Figure 3 for the heavy chain. Homologous recombination was first performed between the amplified LC gene fragment and the pCHO1.0 expression vector digested with EcoR V and PacI to obtain an expression vector loaded with anti-EpCAM light chain; then digested with AvrII and BstZl7I and then The pCHO1.0 expression vector of anti-EpCAM was obtained by homologous recombination with HC, and the plasmid was named pCHO1.0-anti-EpCAM-HL-KKW.

The anti-CD3 ScFv-Fc domain was amplified by overlapping PCR, and the Kozak sequence, leader sequence, and restriction sites AvrII and BstZl7I were introduced into the ScFv-Fc, and the amplified gene fragment (Figure 3) was combined with the digested pCHO1. The 0-hygromycin expression vector was subjected to homologous recombination to obtain an expression vector loaded with anti-CD3ScFv-Fc, and the plasmid was named pCHO1.0-hygromycin-L2K-ScFv-Fc-LDY.

Example 2: Expression and purification of bispecific antibodies

1. Expression of bispecific antibodies

The endotoxin-free large-scale extraction kit (Qiagen, 12391) was used for large-scale extraction of plasmids, and the specific operation was performed according to the instructions provided by the manufacturer. CHO-S cells were cultured according to the instructions provided by the manufacturer in CD CHO medium (Gibco, 10743-029), placed in a 37°C, 5% _CO2 cell incubator for cultivation, after the cells were prepared, according to the manufacturer’s instructions (Maxcyte), the plasmid pCHO1.0-anti-EpCAM-HL-KKW and pCHO1.0-hygromycin-L2K-ScFv-Fc-LDY were co-transfected into CHO-S cells using a Maxcyte STX electroporator, and the design These two plasmids were co-transfected to express the bispecific antibody M701 to EpCAM×CD3.

On the second day after transfection, the culture temperature was lowered to 32° C., and 3.5% FeedA was added every day. After 14 days of culture, the expression supernatant was harvested by centrifugation at 800*g.

2. Purification of bispecific antibodies

The expression supernatant was filtered with a 0.22uM filter membrane, and all antibodies with Fc domains were captured from the expression supernatant by using Mabselect SuRe affinity chromatography column (purchased from GE Company, 18-1153-45, 17-5438-01), Equilibrate the chromatography column with equilibration buffer (9.5mM NaH ₂ PO ₄ +40.5mM Na ₂ HPO ₄ , pH7.0), pass through the affinity chromatography column, and use elution buffer (50mM citric acid + 100mM arginine, pH3.2) elution. Separation of the target bispecific antibody from by-products was achieved by SP cation exchange chromatography. The cation exchange columns were purchased from GE (18-1153-44, 17-1087-01), and the equilibration buffer A (43.8mM NaH ₂ PO ₄ +6.2mM Na ₂ HPO ₄ , pH 6.0) After equilibrating the chromatographic column, the sample was diluted with double pure water and the conductance was between 3.0-3.5ms. ₂ PO ₄ +6.2mM Na ₂ HPO ₄ +1M NaCl, pH 6.0) 20 column volumes for linear elution; finally concentrated and replaced with Buffer PBS. The purified bispecific antibody was detected by SDS-PAGE and SEC, and the purity was above 95%, as shown in Figure 3 .

Example 3: Determination of the binding activity of bispecific antibodies to cells (FACS)

The bispecific antibody of the invention binds to the target antigen on the corresponding cell. In the present invention, HCT116 (purchased from ATCC, CCL-247) is used as EpCAM-positive cells, Jurkat (Jurkat, TIB-152) is used as CD3-positive cells, and the cell binding activity is determined by the diabody prepared in the present invention.

1. Detection of binding activity of bispecific antibody to HCT116 cells by flow cytometry

Sufficient HCT116 cells were cultured, digested with 0.25% trypsin, and collected by centrifugation. Simultaneously dilute the bispecific antibody, the concentration starts from 10ug/ml, and 10-fold gradient dilution is obtained to obtain 12 concentration gradients for future use. Wash the collected cells twice with PBS + 1% FBS, then resuspend the cells in PBS + 1% FBS to 4×10 ⁶ cells/ml, plate the cells in 96-well plate, 50ul per well (2×10 ⁵ cells), add 50ul diluted bispecific antibody, and incubate at room temperature for 1 hour; centrifuge to remove the supernatant, wash the cells twice with PBS, and then reconstitute with diluted PE-labeled anti-human IgG FC antibody (Biolegend, 409304) Suspend the cells, incubate at room temperature in the dark for 30 minutes, wash twice with PBS, resuspend in 100ul PBS, detect on the machine, and calculate the binding affinity KD of the double antibody to HCT116 by analyzing the average fluorescence intensity with the software GraphPad Prism5.0 value. The results showed that the EpCAM×CD3 diabody had good binding activity to EpCAM-positive HCT116 cells, as shown in Figure 5, its KD value was 9.602nM, and the KD detection result of Anti-EpCAM was 1.661nM.

2. Detection of binding activity of bispecific antibody to Jurkat cells by flow cytometry

Sufficient Jurkat suspension cells were cultured and collected by centrifugation. The next experimental process was the same as the above example. The cells resuspended in 100ul PBS were detected on the computer, and then the average fluorescence intensity was analyzed by the software GraphPadPrism5.0 to calculate the binding affinity KD value of the double antibody to Jurkat cells. The results showed that the EpCAM×CD3 diabody had good binding activity to CD3-positive Jurkat cells, as shown in Figure 6, and its KD value was 14.27nM, showing good affinity.

3. Double-antibody-mediated co-binding experiment of immune cells and tumor cells

The cultured HCT116 and Jurkat cells were collected by centrifugation, washed twice with PBS, and stained with CFSE and PKH-26, respectively. Simultaneously dilute the bispecific antibody, the concentration starts from 10ug/ml, and 10-fold gradient dilution is obtained to obtain 12 concentration gradients for future use. Centrifuge the stained HCT116 and Jurkat cells to remove the supernatant, wash twice with PBS+1%FBS, then resuspend the cells in PBS+1%FBS to 4× ¹⁰⁶ cells/ml, mix evenly at 1:1, Cells were plated in a 96-well plate, 50ul per well (2× ¹⁰⁵ cells), 50ul of diluted bispecific antibody was added, and incubated at room temperature for 1 hour; the supernatant was removed by centrifugation, and the cells were washed twice with PBS, and finally washed with Resuspended in 100ulPBS, tested on the computer, analyzed the ratio of double-positive cells, and calculated by using the software GraphPad Prism5.0. The results show that in the absence of M701, the proportion of double fluorescence detected by flow cytometry is very low (Figure 7); in the case of adding EpCAM×CD3 diabody M701, the proportion of double fluorescence detected by flow cytometry reaches 27.5%, indicating that M701 can simultaneously Combine EpCAM-positive HCT116 cells and CD3-positive Jurkat cells to promote the co-aggregation of the two cells to form an immune killing complex.

Example 4: Determination of thermal stability of bispecific antibodies

1. Determination of Tm value of bispecific antibody

The thermal stability of the bispecific antibody was determined by a differential scanning calorimeter (MicroCal VP-DSC, GE Company). After the double antibody sample was purified, it was replaced in PBS buffer, and PBS buffer was used as a control. The calorimetric scanning data was expressed as A heating rate of 60°C/hour was obtained by scanning from 10°C to 100°C. The scanning results (Figure 8) show that the Tm values of the bispecific antibodies are all around 70°C, showing good thermal stability.

2. Thermal challenge experiment of bispecific antibody

The single-chain antibody fragment (scFv) is formed by linking the variable region of the heavy chain and the variable region of the light chain through a connecting peptide (Gly4Ser)3. However, it has been reported that the inherent instability of ScFv may affect the quality of antibody drugs [Michaelson JS1, etc., Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2and LTbetaR.MAbs.2009Mar-Apr; 1(2 ):128-41.]. Therefore, we diluted the antibody to 0.4mg/ml, respectively, 4°C, 37°C, 42°C, 47°C, 52°C, 57°C, 62°C, 67°C, 72°C, 77°C, 82°C, and PCR machine for 1h , 15ul per tube. Take the supernatant by centrifugation, and perform flow cytometry detection according to the following steps. Collect the single cell suspension and add it to a 96-well plate, 3×10 ⁵ /well, add various treatment antibodies, and add fluorescent secondary antibodies, and perform flow cytometry detection on the machine. Figure 9, Figure 9A measured the thermal stability of Anti-EpCAM and EpCAM×CD3 MSBODY bispecific antibodies in combination with EpCAM, and their T ₅₀ values were 73.28 and 61.01, respectively, and Figure 9B measured the bispecificity of L2K and EpCAM×CD3 MSBODY The T ₅₀ values of the antibody in the thermal stability of the CD3-binding antibody were 69.33 and 60.30, showing good thermal stability.

Example 5: Detection of double antibody-mediated cell killing in vitro

1. Isolation of PBMC cells and culture of CIK cells

Take fresh anticoagulated blood, centrifuge at 400g for 5min, and discard the supernatant. Add 10 times the volume of red blood cell lysate, mix gently by pipetting, and lyse at room temperature or on ice for 4-5 minutes. Shake properly during the lysis process to promote erythrocyte lysis. Centrifuge at 400g for 5min at 4°C, discard the red supernatant. If red blood cells are not completely lysed, repeat steps 2 and 3 once. Wash 1-2 times. Add PBS 5 times the volume of the cell pellet, resuspend the pellet, centrifuge at 400g for 2-3 minutes at 4°C, and discard the supernatant. It can be repeated 1 more times for a total of 1-2 washes. According to the needs of the experiment, the cell pellet was resuspended with appropriate 4°C PBS, and subsequent experiments such as counting could be carried out.

For the cultivation of CIK cells, use CIK cell starting culture medium (serum-free X-Vivo cell culture medium + 750IU/ml IFN-γ ± 2% autologous plasma) to fill up 30ml of each cell, add it to a 75cm ² culture flask, and place Cultivate in saturated humidity, 37°C, 5.0% CO ₂ incubator. After culturing for 24 hours, add 1ml of CIK cell stimulatory factor mixture (serum-free X-Vivo cell culture medium + 75ng/ml anti-human CD3ε, 750IU/ml IL-2, 0.6ng/ml IL-1α), and continue to place in saturated Humidity, 37 ° C, 5.0% CO ₂ incubator culture. The next step depends on the growth of CIK cells to decide on fluid replacement (serum-free X-Vivo culture medium + 750IU/ml IL-2±2% autologous plasma) and bottle splitting. Basically, the cells should be maintained at 2*10^6 concentration around the growth. Finally, the phenotype of collected CIK cells was detected by flow cytometer FC500, including: CD3, CD56, CD4, CD8, and the expression of these cell surface antigens in CIK cells was detected. The test results are shown in Figure 10, the phenotype results show that more than 35% of the CIK cells are double positive for CD3 and CD56, and the cultured cells have a good ratio of NK T cells.

2. Double antibody effectively mediates PBMC cell killing tumor cell detection

HCT116 or NCI-N87 cells were digested with trypsin to prepare single cell suspension. Stain HCT116 or NCI-N87 with CFSE at a final concentration of 5uM (see protocol-1 ^CFSE staining for the staining procedure). 10 ⁴ /well, that is, 100ul/well was added to a 96-well plate and cultured overnight. The experimental design added cultured CIK cells, 50ul/well, and set up control wells, and filled the wells without adding CIK cells with the same volume of medium. When adding CIK cells, add the corresponding antibody according to the experimental design, 50ul/well, and fill the wells that do not need to add the antibody with the same volume of medium. After 48 hours, the 96-well plate was taken out, and the cells in each well were digested with trypsin to form a single cell suspension. All supernatants and cell suspensions during this process were correspondingly collected into 1.5ml EP tubes, and centrifuged at 500g×5min. Discard the supernatant, add 150ul 1% FBS-PBS to each well to resuspend and mix the cells. Add PI (final concentration is 1ug/ml) to each tube 10-15 minutes before the flow cytometry machine to detect CFSE, and the ratio of PI double-positive cells to CFSE-positive cells is the death rate of the target cells HCT116 or NCI-N87. The results are shown in Figures 11 and 12. The cell killing results showed that the EpCAM×CD3 MSBODY bispecific antibody mediated CIK cells to kill tumor cells and showed a good killing effect, and its maximum killing efficiency and EC50 were significantly stronger than Anti-EpCAM monoclonal antibody.

3. Double antibody effectively mediates PBMC cell killing tumor cell detection

HCT116 or NCI-N87 cells were digested with trypsin to prepare single cell suspension. Stain HCT116 or NCI-N87 with CFSE at a final concentration of 5uM (see protocol-1 CFSE staining for the staining procedure), and resuspend the cells to 2×10^5/ml with 10% FBS-1640 cultured with the cells after staining, and follow the procedure of 2 ×10^4/well, that is, 100ul/well was added to a 96-well plate and cultured overnight. The experimental design added cultured CIK cells, 50ul/well, and set up control wells, and filled the wells without adding CIK cells with the same volume of medium. When adding CIK cells, add the corresponding antibody according to the experimental design, 50ul/well, and fill the wells that do not need to add the antibody with the same volume of medium. After 48 hours, the 96-well plate was taken out, and the cells in each well were digested with trypsin to form a single cell suspension. All supernatants and cell suspensions during this process were correspondingly collected into 1.5ml EP tubes, and centrifuged at 500g×5min. Discard the supernatant, add 150ul 1% FBS-PBS to each well to resuspend and mix the cells. Each tube was added PI (final concentration: 1ug/ml) 10-15 minutes before flow cytometry, and flow cytometry was used to detect CFSE, and the ratio of PI double-positive cells to CFSE-positive cells was the death rate of target cells HCT116 or NCI-N87. The results are shown in Figures 13 and 14. The cell killing results showed that the EpCAM×CD3 MSBODY bispecific antibody mediated PBMC to kill tumor cells and showed a good killing effect, and its maximum killing efficiency and EC50 were significantly stronger than Anti-EpCAM monoclonal antibody.

Example 6: Detection of drug efficacy of bispecific antibody in killing subcutaneous transplanted tumors

The tumor xenograft model was established by mixing 5×10 ⁶ SW480 and 5×10 ⁶ CIK cells and subcutaneously inoculating them on the right dorsal side of female NOD/SCID mice (N=8 groups). Within 2 hours, the mice were randomly divided into groups, and the antibody treatment group was given intravenous injection of 2, 1 and 0.5 mg/kg EpCAM×CD3 MSBODY respectively through the tail vein. The control group included a group with 2 mg/kg anti-EpCAM monoclonal antibody and a group with irrelevant control MSBODY (4420×CD3). The control MSBODY is constructed from an anti-fluorescein antibody (4-4-20) sequence (Kranz DM, Voss EW Jr., Partial elucidation of ananti-hapten repertoire in BALB/c mice: comparative characterization of several monoclonal antifluorescyl antibodies. Mol Immunol. 1981;18(10):889-898). And continue to administer the same dose on the 2nd and 4th day. Animals in the corresponding control group were intravenously injected with PBS. The tumor volume was measured with a digital vernier caliper every three days, and the result was calculated using the following formula: 1/2×length×width×width (mm ³ ).

The anti-tumor effect of EpCAM×CD3 MSBODY in vivo was evaluated by adoptive transfer xenograft model. Gastric cancer tumor cell line SW480 cells were used to establish xenograft tumor models on immunodeficient mice NOD/SCID. Human CIK cells were obtained by stimulating and culturing peripheral blood mononuclear cells, and mixed at a ratio of 1:1 before inoculation. Inoculate. As shown in Figure 15, the PBS group, the control antibody Anti-EpCAM, and the 4420×CD3 antibody treatment had no significant inhibition on tumor growth, but no tumor growth was found in the EpCAM×CD3 (2mg/kg, 4mg/kg) treatment group in the same experiment. Therefore, EpCAM×CD3 MSBODY-mediated immune tumor killing can significantly inhibit tumor growth. Also as expected, MCO101 (4420×CD3), which lacks the EpCAM-specific MSBODY molecule, did not show significant antitumor activity in vivo even though CD3-specific molecules were retained.

It is to be understood that the disclosed invention is not limited to the particular methodology, protocols and materials described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the appended claims.

Claims (9)

1. bispecific antibody, which is characterized in that the described antibody includes：(a) monovalent unit, is light-heavy chain pair, this is light Chain-heavy chain is EpCAM to being directed to tumour cell, and the amino acid sequence of anti-EpCAM heavy chains is amino acid sequence shown in sequence number 1 The amino acid sequence of row, the light chain of anti-EpCAM is amino acid sequence shown in sequence number 3；(b) strand unit, for fusion Peptide, the fusogenic peptide include single chain variable fragment ScFv and the Fc segments with hinge area, CH2 structural domains and CH3 structural domains, wherein It is amino shown in sequence number 5 that the fusogenic peptide has specific binding capacity, amino acid sequence to immune cell surface antigenic CD3 Acid sequence.

2. bispecific antibody according to claim 1, it is characterised in that：The CH2 structural domains of strand unit are located at scFv Between segment and CH3 structural domains；Not comprising CH1 structural domains.

3. bispecific antibody according to claim 1, it is characterised in that：Strand unit Build Order is " VH- connections Peptide-VL-Fc ".

4. bispecific antibody described in claim 1, it is characterised in that：Half Guang ammonia of the anti-EpCAM heavy chains on 223 sites Cysteine on sour 220 site of light chain with anti-EpCAM is connect in the form of disulfide bond, and the anti-EpCAM heavy chains exist Cysteine on 229 and 232 sites is with the cysteine on 255 and 258 sites of anti-CD3 ScFv-Fc respectively with two sulphur The form of key connects, the anti-EpCAM heavy chains on 395 and 412 sites with 428 and 397 sites of anti-CD3 ScFv-Fc Upper forming salt bridging connects, the anti-EpCAM heavy chains on 369 sites with formed on 436 sites of anti-CD3 ScFv-Fc it is grand Dash forward-enter-cave connection.

5. the method for preparing the bispecific antibody described in any one of claim 1-4, which is characterized in that the method packet Include step：

(1) weight of monovalent unit, light chain are building up to respectively on the first expression vector respectively, strand unit is building up to the second table Up on carrier；

(2) it by the first and second expression vectors together cotransfection to cell, cultivates and takes supernatant；

(3) the isolated bispecific antibody after purification of supernatant will be expressed；The cell is CHO-S cells；Point Include from step：Protein A affinity chromatography column captures the antibody of all band Fc structural domains from expression supernatant, is handed over by SP cations The separation that chromatography realizes target bispecific antibody and by-product is changed, after Q columns, buffer solution PBS is replaced in finally concentration.

6. according to the method described in claim 5, first expression vector is pCHO1.0；Second expression vector It is pCHO1.0- hygromycin.

7. according to the method described in claim 5, it is characterized in that, the step of the described method in (1)：

The unit price unit is anti-EpCAM antibody, and it is Kozak (EcoR V) F, MK- targeting sequencing to expand its light chain the primer (EcoRV) F, M701-VL F1 and hIgK (PacI) R, are expanded by over-lap PCR, by Kozak sequences, targeting sequencing and digestion position Point EcoR V and PacI introduces light chain；It is Kozak (Avr II) F, MK- targeting sequencing (AvrII) to expand its heavy chain the primer F, M701-VH F1 and hIgG1 (sbfI) R, are expanded by over-lap PCR, by Kozak sequences, targeting sequencing and restriction enzyme site AvrII and BstZl7I introduces heavy chain；The LC genetic fragments expanded are expressed with the pCHO1.0 of EcoR V and PacI digestions Carrier carries out homologous recombination, obtains the expression vector for being packed into anti-EpCAM light chains；Then it uses after AvrII and BstZl7I digestions again Homologous recombination is carried out with HC, obtains the pCHO1.0 expression vectors of anti-EpCAM, plasmid is named as the anti-EpCAM-HL- of pCHO1.0- KKW；

The strand unit is anti-CD3 ScFv-Fc antibody, and it is the leading sequence of Kozak (AvrII) F, MK- to expand its primer (AvrII) F, L2K-VH (MK) F1 and hIgG1 (sbfI) R are arranged, AntiCD3 McAb ScFv-Fc structural domains are expanded by over-lap PCR, and will Kozak sequences, targeting sequencing and restriction enzyme site AvrII and BstZl7I introduce ScFv-Fc, by the genetic fragment expanded and enzyme The pCHO1.0- hygromycin expression vectors cut through carry out homologous recombination, obtain the expression vector for being packed into AntiCD3 McAb ScFv-Fc, plasmid It is named as pCHO1.0- hygromycin-L2K-ScFv-Fc-LDY.

8. bispecific antibody described in any one of claim 1-4 is prepared according to any one of claim 5-7 The purposes of bispecific antibody prepared by the method for bispecific antibody in medicine preparation, the drug is for treating The expression of EpCAM specific antigens caused tumour or relevant disease, or for killing expression EpCAM cells.

9. any one of the claim 1-4 bispecific antibodies are prepared double according to any one of claim 5-7 The purposes of bispecific antibody prepared by the method for specific antibody in medicine preparation, the drug are used in tumor cell line The double antibody drug of tumour cell relevant disease of the middle screening for treating expression EpCAM specific antigens or evaluation are for treating Express the drug effect of the double antibody drug of the tumour cell relevant disease of EpCAM specific antigens.