CN104592391B - Construction and application of bispecific antibody EpCAM multiplied by CD3 - Google Patents

Abstract

The invention provides a bispecific antibody, which consists of a single chain unit and a monovalent unit, wherein the monovalent unit has specific binding capacity for a surface antigen CD3 of an immune cell, and the single chain unit has specific binding capacity for a tumor cell surface antigen EpCAM; the single chain unit comprises a single chain variable fragment (ScFv) fused to an Fc fragment, and the monovalent unit comprises a light chain and a heavy chain pair. The present application also provides methods for the preparation of bispecific antibodies, pharmaceutical uses of these antibodies.

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 the construction and preparation of bispecific antibodies.

Background technique

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. BiAbs have broad application prospects in biomedicine, especially in tumor immunotherapy. Killing tumor cells through BiAb-mediated cytotoxicity is a hot spot in current immunotherapy application research. Its main feature is that BiAb can simultaneously bind tumor-associated antigens and target molecules on immune effector cells, directly triggering the specificity of immune effector cells to tumor cells. Sexual Killing. However, in the development of bispecific antibody drugs, there are many obstacles such as difficulty in expression, low yield, difficult purification, and poor stability. Therefore, new bispecific antibodies are constructed to overcome the above obstacles and establish corresponding An animal model of immune killing is very necessary. The present invention provides the construction of a novel bispecific antibody, and describes the research methods and results of its pharmacodynamics. The following is an introduction to some background technologies for the studied immune cell antigens and tumor cell antigens, as well as the development of related technologies.

1. CD3

The CD3 molecule consists of 4 subunits: δ, ε, γ, ζ, its molecular mass is 18.9kDa, 23.1kDa, 20.5kDa, 18.7kDa, and its length is 171, 207, 182, 164 amino acid residues, respectively. Together they form 6 peptide chains, which are often tightly combined with T cell receptors (TCR) to form a TCR-CD3 complex containing 8 peptide chains. The schematic diagram is shown in Figure 1. This complex has the functions of T cell activation signal transduction and stabilization of TCR structure. The CD3 cytoplasmic segment contains an immunoreceptor tyrosine-based activation motif (ITAM), and TCR recognizes and binds to antigenic peptides presented by MHC (major histo-compatibility complex) molecules, resulting in the activation of CD3 ITAM. Conserved sequence tyrosine residues are phosphorylated by the tyrosine protein kinase p56lck in T cells, which can then recruit other SH2 (Scr homology 2) domain-containing tyrosine protein kinases (eg, ZAP-70). Phosphorylation of ITAM and binding to ZAP-70 is one of the important biochemical reactions in the early stage of T cell activation signaling process. Therefore, the function of the CD3 molecule is to transduce the activation signal generated by TCR recognition of the antigen.

2. EpCAM

Epithelial cell adhesion molecule EpCAM (epithelial cell adhesion molecule, CD326), is a type I transmembrane glycoprotein with a molecular weight of 40kDa encoded by the GA-733-2 gene, as a homophilic calcium-independent epithelial cell Mesenchymal adhesion molecules play a role in epithelial carcinogenesis. 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. molecular family. EpCAM also possesses other properties of the adhesion molecule family and is involved 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-derived tumors, suggesting that it is closely related to tumors. Pathologically, EpCAM is expressed to varying degrees in adenocarcinomas, including colorectal cancer, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, as well as hepatocellular carcinoma and retinoblastoma. Several studies have confirmed that the expression of EpCAM is related to the proliferation, cycle distribution and metastasis of breast and colon cancer cells (see Table 1). Monospecific anti-EpCAM monoclonal antibodies (MABs), such as mAb 17-1A (glaxowellcome, Centocor), were the first adjuvant therapy approved for EpCAM-directed treatment of colorectal cancer in Germany. A monospecific antibody did not have a significantly more beneficial effect than chemotherapy. At present, some other EpCAM-directed therapies, including bispecific antibodies, are being developed in the direction of cancer treatment. Bispecific antibodies MT110 and Catumaxomab are both therapeutic diabody drugs against the tumor antigen EpCAM, of which Catumaxomab was approved by EpCAM in 2009. Approved in the European Union for the treatment of malignant cancerous ascites, MT110 is already in clinical studies. Obviously, EpCAM has become one of the hot targets in current tumor therapy research.

Table 1 EpCAM broad tumor distribution

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 are antibodies in which the two antigen-binding sites in an antibody molecule can respectively bind to two different antigenic epitopes.

Antibody drugs are biological macromolecular drugs prepared by antibody engineering technology with cell engineering technology and genetic engineering technology as the main body. Monoclonal antibodies are mainly used in the following three clinical aspects: tumor treatment, immune disease treatment and anti-infection treatment. Among them, the treatment of tumors is the most widely used field of monoclonal antibodies. Among the monoclonal antibody products that have entered clinical trials and are currently 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 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 and bispecific antibodies are one of the development directions to improve the therapeutic effect of antibodies, and have now become a hot spot in the field of antibody engineering research.

Bispecific antibodies used in immunotherapy are artificial antibodies containing two specific antigen binding sites, which can build a bridge between target cells and functional molecules (cells), stimulate a directional immune response, and play a role in tumor immunity. It has broad application prospects in treatment.

4. Bispecific Antibody Preparation

Bispecific antibodies can be obtained in various ways, and their preparation methods mainly include: chemical coupling method, hybrid-hybridoma method and genetic engineering antibody preparation method. The chemical coupling method is to connect two different monoclonal antibodies together by chemical coupling to prepare bispecific monoclonal antibodies. This is the earliest concept of bispecific monoclonal antibodies. The disadvantage of this preparation method is that Obvious. The hybrid-hybridoma method is the production of bispecific monoclonal antibodies by cell hybridization or ternary hybridomas that are fused by established hybridomas, or established hybridomas and hybridomas derived from mice. The obtained lymphocyte fusion can only produce bispecific antibodies of murine origin, and its application is greatly limited. With the rapid development of molecular biology technology, various construction modes of genetically engineered bispecific antibodies have emerged, which are mainly divided into bispecific microantibodies, diabodies, single-chain diabodies, and multivalent bispecific antibodies. Four classes of 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

Adoptive immunotherapy of tumors is to infuse autologous or allogeneic immunocompetent cells into patients after in vitro expansion to 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. First, a large number of tumor cells are removed by conventional methods, and then the remaining tumor cells are removed by immunotherapy to improve the effect of comprehensive tumor therapy. Among them, adoptive immunotherapy, as a new method in comprehensive tumor therapy, has been widely cooperated with conventional surgery, radiotherapy, chemotherapy and other cell and molecular therapy, and has shown a wide range of 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 enter the body together, while the other end of the bispecific antibody can well bind to tumor cells In this way, the bispecific antibody can build a bridge between tumor cells and immune cells in the body, so that the immune cells can concentrate around the tumor cells, and then kill the tumor cells. This method can effectively solve the metastasis and spread of tumor cells, and overcome the disadvantages of "incompleteness, easy metastasis, and large side effects" after the three traditional treatment methods of surgery, radiotherapy and chemotherapy.

SUMMARY OF THE INVENTION

Terms and Abbreviations

BiAb: bispecific antibody

TA: tumor antigen

VH: heavy chain variable region.

VL: light chain variable region.

CL: constant region of light chain.

CDR: is the abbreviation of English Complementarity determining regions (CDRs), which refers to the antigenic 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.

Aiming at the shortcomings of conventional monoclonal antibodies, the present invention creates a new molecule-bispecific antibody through genetic engineering and antibody engineering. Traditional monoclonal antibodies mainly kill tumor cells through CDC, ADCC and apoptosis. On the basis of T-cell-mediated immunotherapy, the efficacy of the immune system in killing tumor cells is greatly improved.

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 that is directed against immune cell selection. from T cells, NK T cells or CIK cells; preferably, the light chain-heavy chain pair has specific binding ability to the immune cell surface antigen CD3; and (b) a single chain unit, which is a fusion peptide comprising a single chain Chain variable fragment ScFv and Fc fragment with hinge region, CH2 domain and CH3 domain, wherein the fusion peptide has specific binding ability to tumor cell surface antigens, preferably the tumor cell surface antigens are EpCAM, CD20, CD30 and CD133, more preferably the tumor cell surface antigen is EpCAM.

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.

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

In one embodiment, in a monovalent unit, 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 includes an antibody against EpCAM, anti-EpCAM, and the monovalent unit includes an antibody against CD3, anti-CD3; preferably, the amino acid sequence of the anti-CD3 heavy chain is shown in SEQ ID NO: 1 The amino acid sequence, the amino acid sequence of the anti-CD3 light chain is the amino acid sequence shown in SEQ ID NO: 3, and the amino acid sequence of the anti-EpCAM ScFv-Fc is the amino acid sequence shown in SEQ ID NO: 5; and the anti-CD3 heavy chain The cysteine at position 222 is disulfide bonded to the cysteine at position 213 of the anti-CD3 light chain, and the anti-CD3 heavy chain at positions 228 and 231 Cysteine and anti-EpCAM ScFv-Fc at positions 263 and 266 of the cysteines are linked by disulfide bonds, respectively, and the anti-CD3 heavy chain at positions 394 and 411 with anti- - A salt bridge is formed at positions 436 and 405 of EpCAMScFv-Fc, and the anti-CD3 heavy chain at position 368 forms a bump-in-hole junction at position 441 of anti-EpCAMScFv-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-derived Fc fragment Preferably, the Fc fragment of the fusion peptide comprises a human IgG Fc fragment.

In one embodiment, the human IgG Fc fragment of the monovalent unit and the IgG Fc of the single chain unit are linked by a salt bridge and a knob-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 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 supernatant All antibodies with Fc domains were captured in the medium, and the target bispecific antibody and by-products were separated by SP cation exchange chromatography, then passed through the 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-CD3 antibody, and the primers used to amplify its light chain are Kozak (EcoRV) F, MK-Leader (EcoRV) F, L2K-VL (MK) F1 and hIgK ( PacI) R, by overlapping PCR amplification, the Kozak sequence, leader sequence and restriction site 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 (AvrII) F, L2K-VH(MK)F1 and hIgG1(sbfI)R were amplified by overlapping PCR, and Kozak sequence, leader sequence and restriction site AvrII and BstZ17I were introduced into the heavy chain; EcoRV was subjected to homologous recombination with the pCHO1.0 expression vector digested with PacI to obtain an expression vector loaded with anti-CD3 light chain; then digested with AvrII and BstZ17I, and then subjected to homologous recombination with HC to obtain anti-CD3 pCHO1.0 expression vector, the plasmid is named pCHO1.0-anti-CD3-HL-LDY;

The single-chain unit is an anti-EpCAM ScFv-Fc antibody, and the primers used to amplify it are Kozak (Avr II) F, MK-Leader (AvrII) F, M701-VH F1 and hIgG1 (sbfI) R, amplified by PCR Anti-EpCAM ScFv-Fc domain, the Kozak sequence, leader sequence and restriction site AvrII and BstZ17I were introduced into ScFv-Fc, and the amplified gene fragment was homogenized with the digested pCHO1.0-hygromycin expression vector. The source was recombined to obtain an expression vector loaded with anti-EpCAM ScFv-Fc, and the plasmid was named pCHO1.0-hygromycin-anti-EpCAM-ScFv-Fc-KKW.

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 medicaments for treating tumors or related diseases caused by the expression of EpCAM-specific antigens disease, or to kill EpCAM-expressing cells.

In one embodiment, the use of any of the above bispecific antibodies or bispecific antibodies prepared according to any of the above methods in the preparation of a medicament for screening human tumor cell lines for therapeutic expression Drugs for tumor cell-related diseases expressing EpCAM-specific antigens or evaluating the efficacy of drugs for treating tumor-cell-related diseases expressing EpCAM-specific antigens. The present invention also provides the following technical solutions:

The present invention provides a novel method for preparing a bispecific antibody SMBODY (ScFv and monomerbispecific antibody) (as shown in Figure 2 ). And some modifications are made in the Fc region of its heavy chain to make it relatively wild-type, and it is not easy to form dimers by itself; while another group specifically binds to another antigen, and also undergoes other modifications in the Fc region of its heavy chain, and it is not easy to form itself. dimers are formed, and hybrid dimers are readily formed between these two sets of heavy and light chains. And the antibody structure of one group is a monomer Ab, and the other group is ScFv-Fc, which avoids the possibility of mismatch between each light chain and each other's heavy chain, thus forming a 125KD bispecific antibody protein molecule. After Fc transformation, the heavy chain and single chain of the monomer Ab are naturally heterodimerized, and at the same time, CL and CH1 are naturally dimerized, and finally SMBODY is formed.

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 human CD3 is named M702, as shown in Figure 2, the anti-CD3 side is in the form of IgG, including the anti-CD3 heavy chain and light chain, and the anti-EpCAM side In ScFv-Fc format, including anti-EpCAM VH, VL, Fc domains. The above bispecific antibodies are constructed by antibody genetic engineering methods, the bispecific antibody SMBODY monomer Ab heavy chain and monomer Ab light chain binary expression vector, and 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. The pCHO1.0 or pCHO1.0-hygromycin vector was digested with enzymes, and then the PCR product and the digested vector were purified and recovered. The LC fragment and the HC fragment were cloned into the pCHO1.0 vector by homologous recombination in two steps, and the ScFv- The Fc fragment was cloned into pCHO1.0-hygromycin vector by homologous recombination and sequenced. Expression and detection of recombinant protein SMBODY in mammalian cells, using transfection reagents to co-transfect plasmids expressing monovalent unit heavy chain, monovalent unit light chain and single chain unit into mammalian cells, and then collect the supernatant for SDS - The expression of SMBODY was detected by PAGE and Western blotting. The supernatant of the transfected and expressed culture medium was centrifuged, filtered, diluted with binding buffer, passed through an affinity chromatography column, eluted with elution buffer, and the purified protein was detected by SDS-PAGE.

The beneficial technical effects of the technical solution of the present invention include:

1. The present application provides a heterodimeric antibody comprising 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, so that the purity of the bispecific antibody can be easily determined. One of the two antigen-binding polypeptide units comprises a light chain-heavy chain pair similar to a wild-type antibody, and this unit is also referred to as a "monovalent unit" throughout this application. Another antigen-binding polypeptide unit comprises a single-chain variable fragment (scFv). Such scFv can 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.

2. The present invention discloses the establishment and application of a novel bispecific antibody SMBODY-mediated in vitro pharmacodynamic test method for killing immune cells. The invention includes the killing of immune cells mediated in the research process of bispecific antibody drugs, the preparation of bispecific antibodies, and the establishment and detection of in vitro pharmacodynamic models of bispecific antibodies. The bispecific antibody SMBODY includes a set of monovalent units (heavy and light chain combination) and a single chain unit (ScFv linked to Fc combination), wherein the single-chain unit specifically binds a human tumor cell antigen, including EpCAM and other A series of tumor cell membrane surface antigens, and some modifications are made in the Fc region of its heavy chain to make it less likely to form dimers by itself compared to the wild type; while another group of monovalent units specifically binds another human T cell antigen CD3, also in the The Fc region of its heavy chain undergoes some other modifications, and it is not easy to form a dimer by itself, and it is easy to form a heterodimer between these two groups of units. At the same time, bispecific antibodies can build a bridge between target cells and functional molecules (cells), stimulate a directional immune response, and have broad application prospects in tumor immunotherapy.

Surprisingly, the present application demonstrates that such asymmetric antibodies are stable and have high antigen binding efficiency. This is surprising since even single chain antibody homodimers have been shown to be unstable under physiological conditions. For example, Ahmad et al., "ScFv Antibody: Principles and Clinical Application," 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 the asymmetry, heterodimers have different isoelectric points than homodimers composed of any of the antigen-binding polypeptide units. Based on the isoelectric point difference between heterodimers and homodimers, the desired heterodimers can be easily separated from the homodimers, greatly reducing the ubiquitous downstream process development of bispecific antibodies. difficulty.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present application more clearly, the following briefly introduces the accompanying drawings that need to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments described in the present application. , for those of ordinary skill in the art, under the premise of no creative work, other drawings can also be obtained from these drawings, wherein:

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

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

Figure 3. Electrophoresis detection of PCR products; (A) M: DL1000 nucleic acid molecular marker; 1. Anti-CD3 antibody light chain; (B) M: DL2000 nucleic acid molecular marker; 1. Anti-CD3 antibody heavy chain; 2. Anti-EpCAM Antibody ScFv-Fc.

Figure 4. The electrophoresis and purity detection of purified double antibody; (4A) SDS-PAGE electrophoresis, M: protein molecular weight marker; 1-2: non-reduced EpCAM×CD3 SMBODY double antibody; 3-4: reduced EpCAM× CD3 SMBODY diabody; (4B) HPLC-SEC purity peak profile of EpCAM x CD3 SMBODY.

Figure 5. Affinity of EpCAM×CD3 SMBODY diabody to HCT116 cells determined by flow cytometry; (•) anti-EpCAM monoclonal antibody; (■) M702: EpCAM×CD3 SMBODY.

Figure 6. Affinity of EpCAM×CD3 SMBODY diabody to Jurkat cells determined by flow cytometry; (•) anti-CD3 monoclonal antibody L2K; (■) M702: EpCAM×CD3 SMBODY.

Figure 7. Flow cytometry detection of EpCAM×CD3 SMBODY double antibody binding to HCT116 and Jurkat cells simultaneously; (●) M702: EpCAM×CD3 SMBODY double antibody; (■) Anti-EpCAM mAb; (▲) Anti-CD3 mAb L2K .

Figure 8. Differential scanning calorimeter scanning measurement of Tm values of EpCAM x CD3 SMBODY diabody.

Figure 9. Antibody activity detection after heat treatment; 9A. Detection of binding activity to EpCAM, (●) anti-EpCAM monoclonal antibody; (▲) M702: EpCAM×CD3 SMBODY double antibody; 9B. Detection of binding activity to CD3, (•) anti-CD3 monoclonal antibody L2K; (■) M702: EpCAM x CD3 SMBODY diabody.

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

Figure 11. Flow cytometry detection of the killing effect of effector cell CIK on target cells HCT116 (Figure 11A) and NCI-N87 (Figure 11B) in the presence of different concentrations of antibodies; (■) M702: EpCAM×CD3 SMBODY double antibody, (▲) Anti-EpCAM monoclonal antibody, (▼) Mco101: control 4420X CD3 diabody, (◆) hIgG: human IgG.

Figure 12. Flow cytometry detection of the killing effect of effector cell PBMC on target cells HCT116 (Figure 12A) and NCI-N87 (Figure 12B) in the presence of different concentrations of antibodies; (■) M702: EpCAM×CD3 SMBODY double antibody, (▲) Mco101: control 4420X CD3 diabody, (▼) Anti-EpCAM monoclonal antibody, (◆) hIgG: human IgG.

Figure 13. The results of the in vivo efficacy experiment of diabodies; (●) represents PBS, only PBS was administered to the tail vein, (□) anti-EpCAM monoclonal antibody, (△) 4420×CD3 irrelevant control diabody, (▼) M702- 1: EpCAM×CD3 SMBODY diabody 4 mg/kg concentration group (○) M702-2: EpCAM×CD3 SMBODY diabody 2 mg/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 the present specification are only for illustrating the present invention, rather than limiting the scope of the present invention in any way.

Example 1: Construction of expression vector for bispecific antibody (EpCAM x CD3 SMBODY, M702)

1. Bispecific antibody sequence design

The bispecific antibody targeting EpCAM and CD3 was named M702 (EpCAM×CD3 SMBODY), as shown in Figure 2, the anti-EpCAM side is in the form of ScFv-Fc, including anti-EpCAM VH, VL, and Fc domains; CD3 here is in IgG form, including anti-CD3 heavy and light chains, containing Fab and Fc domains. Wherein ScFv-Fc undergoes KKW transformation on one side of Fc, and IgG format on the other side of Fc undergoes LDY transformation. For the specific Fc transformation process, please refer to PCT/CN2012/084982, so that each of them is not easy to form homodimers, but is easy to form heterodimers, namely EpCAM x CD3 SMBODY bispecific antibody. At the same time, in order to express the diabody in CHO cells and secrete it into the medium, the leader peptide sequence of the mouse kappa chain was selected as the secretion signal peptide. The amino acid and nucleic acid sequences of each domain and signal peptide are shown in SEQ ID NOs: 1-8 below. Anti-CD3 heavy chain amino acid sequence (SEQ ID NO: 1)

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-CD3 heavy chain nucleic acid sequence (SEQ ID NO: 2)

gatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcagcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccccca tcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgccgggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

Anti-CD3 light chain amino acid sequence (SEQ ID NO: 3)

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Anti-CD3 light chain nucleic acid sequence (SEQ ID NO: 4)

gacattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag

Anti-EpCAM ScFv-Fc amino acid sequence (SEQ ID NO: 5)

EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSGGGGSGGGGSGGGGSELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIKGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-EpCAM ScFv-Fc nucleic acid sequence (SEQ ID NO: 6)

gaggtgcagctgctcgagcagtctggagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggatacgccttcactaactactggctaggttgggtaaagcagaggcctggacatggacttgagtggattggagatattttccctggaagtggtaatatccactacaatgagaagttcaagggcaaagccacactgactgcagacaaatcttcgagcacagcctatatgcagctcagtagcctgacatttgaggactctgctgtctatttctgtgcaagactgaggaactgggacgagcctatggactactggggccaagggaccacggtcaccgtctcctccggaggcggcggttcaggcggaggtggaagtggtggaggaggttctgagctcgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcactatgagctgcaagtccagtcagagtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcctcctaaactgttgatctactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcactctcaccatcagcagtgtgcaggctgaagacctggcagtttattactgtcagaatgattatagttatccgctcacgttcggtgctgggaccaagcttgagatcaaaggtgcggccgcagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgataccacgcctcccgtgctggactccgacggctccttcttcctctacagcgatctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

Leader peptide sequence amino acid sequence of mouse kappa chain (SEQ ID NO: 7)

METDTLLLWVLLLWVPGSTG

Nucleic acid sequence of leader peptide sequence of mouse kappa chain (SEQ ID NO: 8)

atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggt

2. Bispecific antibody gene cloning

Select pCHO1.0 as the expression vector to clone and express the anti-CD3 heavy and light chain genes, the pCHO1.0-hygromycin expression vector is by replacing the puromycin gene in the pCHO1.0 vector with the hygromycin resistance gene was engineered and selected for cloning and expression of an anti-EpCAM ScFv-Fc fusion gene. After the primers in Table 2 were designed according to the cloning scheme, they were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis. Carry out PCR amplification with the primers in table 1, the template is the gene plasmid of gene synthesis or subcloning on pCDNA3.1 or pUC57 in the early experiment, PCT/CN2012/084982 patent has detailed description, and then the anti-CD3 heavy, The light chain was constructed on the expression vector of pCHO1.0, and the anti-EpCAM ScFv-Fc was constructed on the expression vector of pCHO1.0-hygromycin.

Table 2. Primers used in bispecific antibody gene cloning

Initial PCR amplification of template DNA: 35ng of template DNA, eg, light and heavy chains of target antibody; 1 μl of 10 μM forward and reverse primers; 2.5 μl of 10x PCR Buffer; 1 μl of 10 mM dNTPs; 1 μl of 2.5 units/μl Pyrobest DNA polymerase (Takara, R005A); and distilled water to a total volume of 25 μl were mixed gently in a microfuge tube and spun quickly 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, Kozak sequence, leader sequence and restriction sites EcoR V and PacI were introduced into the light chain (Fig. 3); and corresponding primers introduced Kozak sequence, leader sequence and restriction sites AvrII and BstZl7I heavy chain (see Figure 3). First, homologously recombine the amplified LC gene fragment with the pCHO1.0 expression vector digested with EcoR V and PacI to obtain an expression vector loaded with anti-CD3 light chain; then digest with AvrII and BstZ17I, and then Homologous recombination was performed with HC to obtain an anti-CD3 pCHO1.0 expression vector, and the plasmid was named pCHO1.0-anti-CD3-HL-LDY.

The anti-EpCAM ScFv-Fc domain was amplified by overlapping PCR, and the Kozak sequence, leader sequence and restriction sites AvrII and BstZ17I were introduced into ScFv-Fc, and the amplified gene fragment (Fig. 3) was mixed with the digested pCHO1 The .0-hygromycin expression vector was subjected to homologous recombination to obtain an expression vector loaded with anti-EpCAM ScFv-Fc, and the plasmid was named pCHO1.0-hygromycin-anti-EpCAM-ScFv-Fc-KKW.

Example 2: Bispecific antibody expression and purification

1. Expression of Bispecific Antibodies

Plasmids were amplified using an endotoxin-free amplification kit (Qiagen, 12391), and the specific operations were carried out according to the instructions provided by the manufacturer. CHO-S cells were cultured in CD CHO medium (Gibco, 10743-029) according to the manufacturer's instructions in a 37°C, 5% _CO2 cell incubator, after preparing the cells, according to the manufacturer's instructions (Maxcyte), the plasmid pCHO1.0-anti-CD3-HL-LDY was co-transfected into CHO-S cells together with pCHO1.0-hygromycin-anti-EpCAM-ScFv-Fc-KKW using a Maxcyte STX electroporator , co-transfection of these two plasmids was designed to express bispecific antibodies to EpCAM × CD3.

On the 2nd day after transfection, the culture temperature was lowered to 32°C, and 3.5% FeedA was supplemented 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, and all antibodies with Fc domains were captured from the expression supernatant using a Mabselect SuRe affinity chromatography column (purchased from GE, 18-1153-45, 17-5438-01). After equilibrating the column with equilibration buffer (9.5mM NaH ₂ PO ₄ + 40.5 mM Na ₂ HPO ₄ , pH 7.0), the column was subjected to affinity chromatography, followed by elution buffer (50 mM citric acid + 100 mM arginine, pH3.2) elution. The separation of the target bispecific antibody and by-products was achieved by SP cation exchange chromatography. The cation exchange column was purchased from GE (18-1153-44, 17-1087-01), and the equilibration buffer A (43.8mM NaH ₂ was used) After the column was equilibrated with PO ₄ +6.2mM Na ₂ HPO ₄ , pH 6.0), the sample was diluted with double pure water and the conductance was between 3.0-3.5ms. _2PO4 + 6.2 mM _{Na2HPO4 + 1} M NaCl, pH 6.0) 20 column volumes linear elution; finally concentrated displacement buffer PBS. The purified bispecific antibody was detected by SDS-PAGE and SEC, and the purity was above 95% (see Figure 4).

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

The bispecific antibodies of the present invention bind to the target antigen on the corresponding cell. The present invention uses HCT116 (purchased from ATCC, CCL-247) as EpCAM-positive cells and Jurkat (Jurkat, TIB-152) as CD3-positive cells, and uses the double antibody prepared in the present invention to measure its cell-binding activity.

1. Detection of the binding activity of bispecific antibodies to HCT116 cells by flow analysis

Sufficient HCT116 cells were cultured, digested with 0.25% trypsin, and collected by centrifugation. At the same time, dilute the bispecific antibody, starting from 500 nmol, with a 3-fold gradient dilution to obtain 12 concentration gradients for use. The collected cells were washed twice with PBS+1% FBS, and then resuspended to 4×10 ⁶ cells/ml with PBS+1% FBS, and the cells were plated in a 96-well plate with 50ul (2×10 ⁵ ) per well. cells), add 50ul of the diluted bispecific antibody, and incubate at room temperature for 1 hour; centrifuge to remove the supernatant, wash the cells twice with PBS, and resuspend the cells with the diluted PE-labeled anti-human IgG FC antibody (Biolegend, 409304). Suspend the cells, incubate in the dark at room temperature for 30 minutes, wash twice with PBS, resuspend in 100ul PBS, detect on the computer, and calculate the binding affinity KD of the diabody to HCT116 by analyzing the average fluorescence intensity with the software GraphPad Prism5.0 value. The results show that the EpCAM×CD3SMBODY diabody has good binding activity to EpCAM-positive HCT116 cells, as shown in Figure 5, and its KD value is only 69.82nM, which has good cell binding activity.

2. Detection of the binding activity of bispecific antibodies to Jurkat cells by flow analysis

Sufficient Jurkat suspension cells were cultured and collected by centrifugation. The following experimental process is the same as the above-mentioned example. The cells resuspended in 100ul PBS were detected on the machine, and then the average fluorescence intensity was used to analyze and calculate the binding affinity KD value of the diabody to Jurkat cells by analyzing with the software GraphPad Prism 5.0. The results showed that the EpCAM×CD3SMBODY diabody had good binding activity to CD3-positive Jurkat cells, as shown in Figure 6, and its KD value was only 50.90nM, showing good cell binding activity.

3. Double antibody-mediated co-binding activity assay

The cultured HCT116 and Jurkat cells were collected by centrifugation, washed twice with PBS, and stained with CFSE and PKH-26, respectively. At the same time, dilute the bispecific antibody, the concentration starts from 10ug/ml, and the 3-fold gradient dilution is used to obtain 12 concentration gradients for use. Centrifuge the stained HCT116 and Jurkat cells to remove the supernatant, wash twice with PBS+1%FBS, add PBS+1%FBS to resuspend the cells to 4×10 ⁶ cells/ml, and mix evenly at 1:1. The cells were plated in a 96-well plate, 50ul per well (2×10 ⁵ cells), 50ul of the diluted bispecific antibody was added, and incubated at room temperature for 1 hour; centrifuged to remove the supernatant, and washed the cells twice with PBS. 100ul of PBS was resuspended, and the ratio of double-positive cells was analyzed on the computer for analysis and calculation by using the software GraphPad Prism5.0. The results showed that in the absence of M702, the proportion of double fluorescence detected by flow cytometry was very low (Fig. 7); in the case of adding EpCAM × CD3 diabody M702, the proportion of double fluorescence detected by flow cytometry reached about 40%, indicating that M702 can Simultaneously combined with 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: Thermal Stability Assay of Bispecific Antibodies

1. Determination of Tm value of bispecific antibodies

The thermal stability of the bispecific antibody was measured by a differential scanning calorimeter (MicroCal VP-DSC, GE Company). The double antibody sample was purified and replaced with PBS buffer. The PBS buffer was used as a control. A heating rate of 60°C/hour was obtained by scanning from 10°C to 100°C. The scanning results (Fig. 8) showed that the Tm values of the bispecific antibodies were all above 70°C, showing good thermal stability.

2. Thermal Challenge Experiments with Bispecific Antibodies

Single-chain antibody fragments (ScFv) are formed by linking the variable regions of the heavy and light chains by a linking 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-likebispecific antibodies targeting TRAIL-R2 and LTbetaR. MAbs. 2009 Mar-Apr; 1 ( 2): 128-41). Therefore, we diluted the antibody to 0.4mg/ml, 4°C, 37°C, 42°C, 47°C, 52°C, 57°C, 62°C, 67°C, 72°C, 77°C, 82°C, respectively, and the PCR machine was processed for 1h. , 15ul per tube. Centrifuge to take the supernatant, carry out flow detection according to the following steps, collect single cell suspension and add it to 96-well plate, 3×10 ⁵ /well, add various processing antibodies, and add fluorescent secondary antibodies, flow on the machine for detection, see the results. Figure 9, Figure 9A determined the thermal stability of Anti-EpCAM and EpCAM × CD3 _SMBODY bispecific antibodies in binding to EpCAM, with T50 values of 73.28 and 58.31, respectively, and Figure 9B determined the L2K and EpCAM × CD3 MSBODY bispecifics The thermal stability of the antibody in binding to CD3, the _T50 values of which were 69.33 and 61.30, respectively, showed good thermal stability.

Example 5: Double antibody-mediated in vitro cell killing assay

1. Isolation of PBMC cells and culture of CIK cells

Take fresh anticoagulant, centrifuge at 400g for 5min, and discard the supernatant. Add 10 times the cell volume of red blood cell lysate, mix by gently pipetting, and lyse for 4-5 minutes at room temperature or on ice. Shake appropriately during the lysis process to facilitate lysis of red blood cells. Centrifuge at 400g for 5 min at 4°C and discard the red supernatant. If red blood cell lysis is incomplete, repeat steps 2 and 3 once. Wash 1-2 times. Add 5 times the cell pellet volume of PBS, resuspend the pellet, centrifuge at 400g for 2-3 minutes at 4°C, and discard the supernatant. It can be repeated 1 more time for a total of 1-2 washes. Follow-up experiments such as counting can be performed after resuspending the cell pellet with appropriate 4 ℃ PBS according to the experimental needs.

For the cultivation of CIK cells, each cell was filled with 30ml of CIK cell starter medium (serum-free X-Vivo cell culture medium + 750IU/ml IFN-γ±2% autologous plasma), added to a 75cm2 culture flask, and placed in a saturated Humidity, 37°C, 5.0% CO ₂ incubator. After 24 hours of culture, add 1 ml of CIK cell stimulating factor mixture (serum-free X-Vivo cell culture medium + 75ng/ml anti-human CD3ε, 750IU/ml IL-2, 0.6ng/ml IL-1α), continue to place in Culture in a saturated humidity, 37°C, 5.0% CO ₂ incubator. The next steps are based on the growth of CIK cells to determine the supplementation (serum-free X-Vivo medium + 750IU/ml IL-2±2% autologous plasma) and bottle separation, basically maintaining the cells at 2*10^6 concentration around growth. Finally, flow cytometry FC500 was used to detect the phenotype of the collected CIK cells, including: CD3, CD56, CD4, CD8, to detect the expression of these cell surface antigens in CIK cells. The test results are shown in Figure 10. The phenotype results show that the CIK cells are double positive for CD3 and CD56 over 15.9%, and the cultured cells have a good ratio of NK T cells.

2. Double antibody effectively mediates PBMC cells to kill tumor cells

Single-cell suspensions were prepared by trypsinizing HCT116 or NCI-N87 cells. Stain HCT116 or NCI-N87 with CFSE at a final concentration of 5uM. After staining, resuspend the cells to 2×10 ⁵ /ml with 10% FBS-1640 of the cell culture, according to 2×10 ⁴ /well, that is, 100ul/well Add to 96-well plate and culture overnight. The experimental design added cultured CIK cells, 50ul/well, and set up control wells. The wells that did not need to add CIK cells were supplemented with the same volume of medium. When adding CIK cells, the corresponding antibodies were added according to the experimental design, 50ul/well, and the same volume of culture medium was supplemented to the wells that did not need to add antibodies. 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 collected into 1.5ml EP tubes, and centrifuged at 500g × 5min. The supernatant was discarded, and 150ul of 1% FBS-PBS was added to each well to resuspend the cells. PI (final concentration 1ug/ml) was added to each tube 10-15min before the flow-through machine to detect the proportion of CFSE and PI double-positive cells to CFSE-positive cells, which was the death rate of target cells HCT116 or NCI-N87. The results are shown in Figure 11. The cell killing results show that the EpCAM×CD3 SMBODY bispecific antibody mediated the killing of tumor cells by CIK 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 the killing of tumor cells by PBMC cells

HCT116 cells were trypsinized to prepare a single-cell suspension. Stain HCT116 or NCI-N87 with CFSE at a final concentration of 5uM. After staining, resuspend the cells to 2×10 ⁵ /ml with 10% FBS-1640 of the cell culture, according to 2×10 ⁴ /well, that is, 100ul/well Add to 96-well plate and culture overnight. The experimental design added cultured CIK cells, 50ul/well, and set up control wells. The wells that did not need to add CIK cells were supplemented with the same volume of medium. When adding CIK cells, the corresponding antibodies were added according to the experimental design, 50ul/well, and the same volume of culture medium was supplemented to the wells that did not need to add antibodies. 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 collected into 1.5ml EP tubes, and centrifuged at 500g × 5min. The supernatant was discarded, and 150ul of 1% FBS-PBS was added to each well to resuspend the cells. Each tube was added with PI (final concentration of 1ug/ml) 10-15min before the flow-through machine to detect CFSE and PI double-positive cells accounted for the proportion of CFSE-positive cells, which was the death rate of the target cell HCT116. The results are shown in Figure 12. The cell killing results show that the EpCAM×CD3 SMBODY bispecific antibody mediated the killing of tumor cells by CIK cells and showed a good killing effect, and its maximum killing efficiency and EC50 were significantly stronger than Anti-EpCAM monoclonal antibody.

Example 6: Pharmacodynamic detection of bispecific antibodies in killing subcutaneously transplanted tumors

The tumor xenograft model was established by mixing 5×10 ⁶ SW480 and 5×10 ⁶ CIK cells and inoculated subcutaneously on the right dorsal side of female NOD/SCID mice (N=8 groups). Within 2 hours, mice were randomly divided into antibody-treated groups, and 4, 2 mg/kg EpCAM×CD3 SMBODY was started by intravenous injection through the tail vein, respectively. The control group consisted of a group with 4 mg/kg of anti-EpCAM mAb and a group of unrelated control SMBODY (4420×CD3). The control SMBODY was constructed from the sequence of an anti-fluorescein antibody (4-4-20) (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 days 2 and 4. PBS was intravenously injected into the corresponding control animals. Tumor volume was measured with a digital vernier caliper every three days, and the results were calculated using the following formula: 1/2 x length x width x width (mm ³ ).

The evaluation of the antitumor effect of EpCAM×CD3SMBODY in vivo was accomplished by adoptive transfer xenograft model. The gastric cancer tumor cell line SW480 was used to establish a xenograft model in immunodeficient mice NOD/SCID. Human CIK cells were obtained by isolating peripheral blood mononuclear cells and stimulating culture, and inoculated in a 1:1 ratio before inoculation . As shown in Figure 13, PBS group, control antibody Anti-EpCAM, 4420×CD3 antibody treatment did not significantly inhibit tumor growth, but in the same experiment, EpCAM×CD3 (2mg/kg, 4mg/kg) treatment group did not find tumor growth, Therefore, EpCAM × CD3 SMBODY-mediated immune tumor killing can significantly inhibit tumor growth. Also as expected, even if the CD3-specific molecule was retained, the SMBODY molecule MCO101 (4420×CD3) lacking EpCAM-specificity did not show significant antitumor activity in vivo.

It is to be understood that the invention disclosed is not limited to the particular methods, protocols and materials described, as these may vary. It is also to 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 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 included in the appended claims.

Claims (12)

1. bispecific antibody, it is characterized in that, described antibody comprises: (a) monovalent unit, is light chain-heavy chain pair, and this light chain-heavy chain pair has specific binding ability to immune cell surface antigen CD3; And (b) a single-chain unit, which is a fusion peptide comprising a single-chain variable fragment ScFv and an Fc fragment having a hinge region, a CH2 domain and a CH3 domain, wherein the fusion peptide has specific binding to tumor cell surface antigens ability, the tumor cell surface antigen is EpCAM; The single-chain unit includes an antibody against EpCAM, anti-EpCAM, and the monovalent unit includes an antibody against CD3, anti-CD3; The amino acid sequence of the anti-CD3 heavy chain is the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence of the anti-CD3 light chain is the amino acid sequence shown in SEQ ID NO: 3, and the amino acid sequence of the anti-EpCAM ScFv-Fc is the amino acid sequence shown in SEQ ID NO: 5; and the cysteine at position 222 of the anti-CD3 heavy chain is connected with the cysteine at position 213 of the light chain of anti-CD3 in the form of a disulfide bond, The cysteines at positions 228 and 231 of the anti-CD3 heavy chain are connected with the cysteines at positions 263 and 266 of the anti-EpCAMScFv-Fc in the form of disulfide bonds, respectively. The anti-CD3 heavy chain forms a salt bridge at positions 394 and 411 with the anti-EpCAM ScFv-Fc at positions 436 and 405, and the anti-CD3 heavy chain binds to the anti-EpCAM ScFv at position 368. - Knock-in-hole junction is formed at position 441 of Fc. 2. The bispecific antibody according to claim 1, wherein the CH2 domain of the single-chain unit is located between the ScFv fragment and the CH3 domain. 3. The bispecific antibody according to claim 1, wherein the single-chain variable fragment is composed of a light chain variable region and a heavy chain variable region domain, both of which are targeted to the epitope EpCAM . 4. The bispecific antibody according to claim 1, characterized in that: in the monovalent unit, the light chain is combined with the heavy chain through a disulfide bond; the heavy chain is connected to the heavy chain through one or more disulfide bonds. to the fusion peptide. 5. The bispecific antibody according to claim 1, wherein the heavy chain in the monovalent unit comprises a human or humanized Fc fragment, and the Fc fragment of the heavy chain comprises a human IgG Fc fragment; the The Fc fragment of the fusion peptide comprises a human or humanized Fc fragment, and the Fc fragment of the fusion peptide comprises a human IgG Fc fragment. 6. The bispecific antibody according to claim 5, wherein the human IgG Fc fragment 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. 7. The method for preparing the bispecific antibody of any one of claims 1-6, wherein the method comprises the steps of: (1) The heavy and light chains of the monovalent unit are respectively constructed on the first expression vector, and the single-chain unit is constructed on the second expression vector; the first expression vector is pCHO1.0, and the second expression vector is pCHO1.0-hygromycin; (2) co-transfecting the first and second expression vectors into cells, culturing and taking the supernatant, the cells are CHO-S cells; (3) Separating the expression supernatant to obtain the purified bispecific antibody; the separation step includes: a protein A affinity chromatography column captures all antibodies with Fc domains from the expression supernatant, and passes SP cation exchange chromatography To achieve the separation of the target bispecific antibody and by-products, pass through the Q column, and finally concentrate the replacement buffer PBS. 8. The method according to claim 7, wherein in the step (1) of the method: The monovalent unit is an anti-CD3 antibody, and the primers used to amplify its light chain are Kozak (EcoRV) F, MK-Leader (EcoRV) F, L2K-VL (MK) F1 and hIgK (PacI) R, amplified by overlapping PCR. Increase, introduce Kozak sequence, leader sequence and restriction enzyme site EcoRV and PacI into light chain; The primers used for amplifying its heavy chain are Kozak(Avr II)F, MK-Leader(AvrII)F, L2K-VH(MK)F1 and hIgG1(sbfI)R, amplified by overlapping PCR, Kozak sequence, leader sequence and restriction site AvrII and BstZ17I were introduced into the heavy chain; the amplified LC gene fragment and pCHO1. 0 The expression vector was subjected to homologous recombination to obtain an expression vector loaded with anti-CD3 light chain; then it was digested with AvrII and BstZ17I and then subjected to homologous recombination with HC to obtain an anti-CD3 pCHO1.0 expression vector, and the plasmid was named as pCHO1.0-anti-CD3-HL-LDY; The single-chain unit is an anti-EpCAM ScFv-Fc antibody, and the primers used to amplify it are Kozak (Avr II) F, MK-Leader (AvrII) F, M701-VH F1 and hIgG1 (sbfI) R, amplified by PCR Anti-EpCAM ScFv-Fc domain, the Kozak sequence, leader sequence and restriction site AvrII and BstZ17I were introduced into ScFv-Fc, and the amplified gene fragment was homogenized with the digested pCHO1.0-hygromycin expression vector. The source was recombined to obtain an expression vector loaded with anti-EpCAM ScFv-Fc, and the plasmid was named pCHO1.0-hygromycin-anti-EpCAM-ScFv-Fc-KKW. 9. Use of the bispecific antibody according to any one of claims 1-6 in the preparation of a medicament for treating colon cancer or gastric cancer caused by the expression of EpCAM-specific antigens, or for killing EpCAM-expressing Colon or gastric cancer cells. 10. Use of the bispecific antibody prepared by the method of any one of claims 7-8 in the preparation of a medicament for treating colon cancer or gastric cancer caused by the expression of EpCAM-specific antigen, or for killing the expression of Colon or gastric cancer cells of EpCAM. 11. Use of the bispecific antibody according to any one of claims 1 to 6 in the preparation of a medicament for screening a medicament in a tumor cell line for the treatment of colon cancer or gastric cancer expressing an EpCAM specific antigen or To evaluate the efficacy of drugs for the treatment of colon or gastric cancers expressing EpCAM-specific antigens. 12. Use of the bispecific antibody prepared by the method of any one of claims 7-8 in the preparation of a medicament for screening a tumor cell line for the treatment of colon cancer or gastric cancer expressing an EpCAM specific antigen Drugs or to evaluate the efficacy of drugs for the treatment of colon cancer or gastric cancer expressing EpCAM-specific antigens.

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