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 bispecific antibody EpCAM multiplied by CD3

Technical Field

The present invention relates to the field of immunology. In particular to a construction and preparation method of a bispecific antibody.

Background

Bispecific antibodies (biabs) are artificial antibodies that contain two specific antigen binding sites and can bridge between target cells and functional molecules (cells) to produce targeted effector functions. BiAb has wide application prospect in biomedicine, especially in the immunotherapy of tumors. The method is mainly characterized in that the BiAb can be simultaneously combined with tumor-associated antigens and target molecules on immune effector cells to directly trigger the specific killing of the immune effector cells on the tumor cells. However, in the process of drug development of bispecific antibodies, there are various obstacles such as difficult expression, low yield, difficult purification, and poor stability, so it is very necessary to construct a novel bispecific antibody to overcome the above obstacles and establish a corresponding animal model for immune killing. The invention provides a novel dual-characteristic antibody construction, and describes a research method and a result of pharmacodynamics of the dual-characteristic antibody. The following is a description of some background art directed to the immune cell antigens and tumor cell antigens of interest, and related technology developments.

1.CD3

The CD3 molecule consists of 4 subunits: 18.9kDa, 23.1kDa, 20.5kDa and 18.7kDa, and 171, 207, 182 and 164 amino acid residues in length. Together, they form 6 peptides, often tightly associated with a T Cell Receptor (TCR) to form a TCR-CD3 complex containing 8 peptides, the structure of which is schematically shown in fig. 1. This complex has the function of T cell activation signaling, stabilizing the TCR structure. The CD3 cytoplasmic segment contains immunoreceptor tyrosine-based activation motif (ITAM), and TCR recognizes and binds to antigenic peptides presented by MHC (major histocompatibility complex) molecules, so that tyrosine residues of conserved sequences of ITAM of CD3 are phosphorylated by tyrosine protein kinase p56lck in T cells, and then other tyrosine protein kinases (such as ZAP-70) containing SH2(Scr homology 2) structural domains can be recruited. Phosphorylation of ITAM and binding to ZAP-70 are one of the important biochemical reactions in the early stages of the T cell activation signaling process. Thus, the function of the CD3 molecule is to transduce an activation signal generated by the TCR recognition antigen.

2.EpCAM

The epithelial cell adhesion molecule, EpCAM (CD 326), is a type I transmembrane glycoprotein encoded by the GA-733-2 gene and having a molecular weight of 40kDa and plays a role in epithelial carcinogenesis as a homophilic calcium-independent epithelial intercellular adhesion molecule. EpCAM is one of the first identified tumor-associated antigens using monoclonal antibody technology, and it is widely expressed on epithelial tissue surface in multimeric form, mediating the calcium-independent cell-cell homotypic adhesion function, and thus can be classified into adhesion molecule family. EpCAM also possesses other properties of the adhesion molecule family and is involved in a variety of processes including cell-matrix interaction, migration, cell differentiation, morphology, cell cycle regulation, signaling, metabolism, and the like. At the same time, EpCAM is overexpressed in tumors of various epithelial origins, suggesting that it is closely associated with tumors. In pathological cases, EpCAM is expressed to varying degrees in adenocarcinomas, including colorectal, gastric, breast, ovarian, lung, prostate, pancreatic, as well as hepatocellular and retinoblastoma. Several studies have demonstrated that EpCAM expression is associated with proliferation, cycle distribution, metastasis of breast and colon cancer cells (see table 1). Monospecific anti-EpCAM Monoclonal Antibodies (MAB), such as MAB 17-1A (glaxowell, Centocor), were the first approved adjuvant therapy for EpCAM-directed treatment of colorectal cancer in germany, however, extensive clinical dosing data show that such monospecific antibodies have no significantly more beneficial effects than chemotherapy. Currently, some other EpCAM-directed therapies, including bispecific antibodies, are being developed as the direction for cancer therapy, and bispecific antibodies MT110 and Catumaxomab, which have been approved by the european union in 2009 for the treatment of malignant cancerous ascites, are therapeutic diabody drugs against the tumor antigen EpCAM, with MT110 being in clinical studies. Apparently, EpCAM has become one of the hot targets for current tumor therapy studies.

TABLE 1EpCAM broad tumor distribution

Tumor(s)	EpCAM positive rate
		Ovarian cancer	88-100％
Stomach cancer	98％
		Cancer of colon	99％
Pancreatic cancer	96％
		Breast cancer	90％
Endometrial cancer	91-96％
		Lung cancer	87％
Prostate cancer	98％

3. Bispecific antibody technology development

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

The antibody medicine is a biological macromolecular medicine prepared by an antibody engineering technology taking a cell engineering technology and a genetic engineering technology as main bodies, and has the advantages of high specificity, uniform property, capability of being directionally prepared aiming at a specific target spot and the like. The monoclonal antibody is mainly applied to the following three aspects in clinic: tumor treatment, immune disease treatment, and anti-infection treatment. The tumor therapy is the most widely applied field of the monoclonal antibody at present, and the quantity of the monoclonal antibody used for tumor therapy is about 50 percent in the monoclonal antibody products which are clinically tested and marketed at present. The monoclonal antibody is an immunotherapy for killing target cells by combining specific targets of pathological cells through the monoclonal antibody, so that an immune system is stimulated, and in order to enhance the effect function of the antibody, particularly the effect of killing tumor cells, various methods are tried to modify antibody molecules, and the bispecific antibody is one of the development directions for improving the therapeutic effect of the antibody, and is now a hotspot in the field of antibody engineering research.

The bispecific antibody for immunotherapy is an artificial antibody containing 2 specific antigen binding sites, can bridge between target cells and functional molecules (cells), stimulates targeted immune response, and has wide application prospect in tumor immunotherapy.

4. Bispecific antibody preparation

Bispecific antibodies are available in a variety of ways, and are prepared mainly by: chemical coupling method, hybrid-hybridoma method and gene engineering antibody preparation method. The disadvantage of the chemical coupling method is obvious in that 2 different monoclonal antibodies are linked together by chemical coupling to prepare bispecific monoclonal antibodies, which is the earliest concept of bispecific monoclonal antibodies. The hybrid-hybridoma method is to produce bispecific monoclonal antibodies by means of cell hybridization or ternary hybridoma, which is obtained by fusion of established hybridomas or established hybridomas with lymphocytes obtained from mice, and which 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 genetic engineering bispecific antibodies appear, and the genetic engineering bispecific antibodies are mainly divided into four classes, namely bispecific miniantibodies, diabodies, single-chain diabodies and multivalent bispecific antibodies. At present, several genetic engineering bispecific antibody medicaments enter a clinical test stage internationally and show good application prospects.

5. Adoptive immunotherapy of tumors

The adoptive immunotherapy of tumor is to directly kill tumor cells and regulate and enhance the immune function of the body by inputting autologous or allogeneic immunocompetent cells into a patient after in vitro amplification, and mainly comprises immunotherapy of LAK cells, TIL cells, activated T lymphocytes and CIK cells. Immunotherapy can only eliminate a small amount 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 a large amount of tumor cells are cleaned by a conventional method, residual tumor cells are cleaned by an immunotherapy method, so that the comprehensive treatment effect of the tumor is improved. The adoptive immunotherapy is a new method in the comprehensive treatment of tumors, has been widely matched with conventional operative treatment, radiotherapy, chemotherapy and other cell and molecular therapies, and has shown wide application prospects in the treatment of various tumors. However, a more desirable way would be that one end of the bispecific antibody could bind to the surface antigen CD3 of cultured immune cells and be delivered into the body along with it, while the other end of the bispecific antibody binds well to the surface antigen of tumor cells; thus, the bispecific antibody can bridge between tumor cells and immune cells in vivo, so that the immune cells are concentrated around the tumor cells, and further kill the tumor cells. The method can effectively solve the problem of tumor cell metastasis and diffusion, and overcomes the defects of incomplete treatment, easy metastasis, great side effect and the like after three traditional treatment modes of operation, radiotherapy and chemotherapy.

Disclosure of Invention

Terms and abbreviations

BiAb bispecific antibody (bispecific antibody)

TA tumor antigen (tumor antigen)

VH heavy chain variable region (heavy chain variable region).

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

CL: a constant region of light chain (constant region of light chain).

CDR: the abbreviation is used for the English Complementary Determining Regions (CDRs), and refers to the antigen Complementarity determining regions of the antibody.

ScFv: single-chain variable region antibody fragments (also known as single-chain antibodies).

CLD cell line development (cell line)

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

Aiming at the defects of the conventional monoclonal antibody, the invention creates a new molecule-bispecific antibody by a genetic engineering and antibody engineering method, increases the immunotherapy of mediated T cells on the basis that the traditional monoclonal antibody kills tumor cells mainly by CDC, ADCC and apoptosis capacity, and greatly improves the efficiency of an immune system in killing the tumor cells.

Specifically, the invention provides the following technical scheme:

in one embodiment, there is provided a bispecific antibody, wherein the antibody comprises: (a) a monovalent unit that is a light-heavy chain pair selected from a T cell, an NK T cell, or a CIK cell for an immune cell; preferably, the light-heavy chain pair has specific binding capacity to the immune cell surface antigen CD 3; 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 capacity for a tumor cell surface antigen, preferably the tumor cell surface antigen is 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 a light chain variable region and a heavy chain variable region domain, both of which are targeted to 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 comprises an antibody anti-EpCAM against EpCAM, and the monovalent unit comprises an antibody anti-CD 3 against CD 3; preferably, the amino acid sequence of the heavy chain of anti-CD 3 is the amino acid sequence shown in sequence No. 1, the amino acid sequence of the light chain of anti-CD 3 is the amino acid sequence shown in sequence No. 3, and the amino acid sequence of the anti-EpCAM ScFv-Fc is the amino acid sequence shown in sequence No. 5; and the cysteine at position 222 of the anti-CD 3 heavy chain is disulfide-linked to the cysteine at position 213 of the light chain of anti-CD 3, the cysteine at positions 228 and 231 of the anti-CD 3 heavy chain is disulfide-linked to the cysteine at positions 263 and 266 of anti-EpCAM ScFv-Fc, respectively, the anti-CD 3 heavy chain is salt-bridged at positions 394 and 411 to positions 436 and 405 of anti-EpCAMScFv-Fc, and the anti-CD 3 heavy chain is protuberance-in-hole linked at position 368 to anti-EpCAMScFv-Fc at position 441.

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 humanized 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 fragment of the single chain unit are linked by a salt bridge and a knob-in-hole structure.

In one embodiment, a method of making a bispecific antibody is provided, the method comprising:

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

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

(3) separating the expression supernatant to obtain a purified bispecific antibody; preferably, the cells are CHO-S cells; or preferably, the separating step comprises: protein A affinity chromatographic column captures all antibodies with Fc structural domains from the expression supernatant, realizes the separation of the target bispecific antibody and byproducts by SP cation exchange chromatography, passes through a Q column, and finally concentrates and replaces buffer PBS.

In one embodiment, the first expression vector is pcho 1.0; the second expression vector was pCHO1.0-hygromycin.

In one embodiment, the monovalent unit is an anti-CD 3 antibody, primers for amplifying the light chain are Kozak (EcoR V) F, MK-leader (ecorv) F, L2K-vl (mk) F1 and higk (PacI) R, Kozak sequence, leader sequence, and cleavage sites EcoR V and PacI are introduced into the light chain by overlap PCR amplification; primers for amplifying the heavy chain are Kozak (Avr II) F, MK-leader (AvrII) F, L2K-VH (MK) F1 and hIgG1(sbfI) R, and Kozak sequence, leader sequence and cleavage sites AvrII and BstZl7I are introduced into the heavy chain through overlapping PCR amplification; carrying out homologous recombination on the amplified LC gene fragment and a pCHO1.0 expression vector which is cut by EcoRV and PacI enzyme to obtain an expression vector loaded with an anti-CD 3 light chain; then carrying out enzyme digestion on AvrII and BstZL7I, and then carrying out homologous recombination on the AvrII and BstZL7I and HC to obtain a pCHO1.0 expression vector of anti-CD 3, wherein the plasmid is named as pCHO1.0-anti-CD 3-HL-LDY;

the single-chain unit is an anti-EpCAM ScFv-Fc antibody, primers used for amplifying the anti-EpCAM ScFv-Fc antibody are Kozak (Avr II) F, MK-leader (AvrII) F, M701-VH F1 and hIgG1(sbfI) R, an anti-EpCAM ScFv-Fc domain is amplified through PCR, a Kozak sequence, a leader sequence, an enzyme cutting site AvrII and BstZL7I are introduced into ScFv-Fc, the amplified gene fragment and an enzyme-cut pCHO1.0-hygromycin expression vector are subjected to homologous recombination to obtain an expression vector filled with the anti-EpCAM ScFv-Fc, and the plasmid is named pCHO1.0-hygromycin-anti-EpCAM-ScFv-Fc-KKW.

In one embodiment, the use of a bispecific antibody of any one of the above or a bispecific antibody prepared according to any one of the above methods in the manufacture of a medicament for the treatment of a tumor or related disease caused by the expression of an EpCAM-specific antigen or for killing of EpCAM-expressing cells.

In one embodiment, the use of a bispecific antibody of any one of the above or a bispecific antibody prepared according to any one of the above methods in the manufacture of a medicament for screening for or evaluating the efficacy of a medicament for the treatment of a tumor cell associated disease expressing an EpCAM specific antigen in a human tumor cell line. The invention also provides the following technical scheme:

the invention provides a new method for preparing a bispecific antibody SMBODY (ScFv and monomerbispecific antibody) (shown in figure 2), which comprises two groups of heavy-light chain combinations, wherein one group specifically binds to an antigen, and the heavy chain Fc region is subjected to some modification, so that the bispecific antibody is not easy to form a dimer by itself compared with a wild type; while the other group specifically binds to the other antigen, and is also less prone to form dimers themselves by additional modifications in its heavy chain Fc region, whereas hybrid dimers are readily formed between the two heavy and light chains. And the antibody structure of one group is a monomeric Ab, and the other group is ScFv-Fc, so that the possibility of mismatching of the respective light chain and the heavy chain of the other party is avoided, and the bispecific antibody protein molecule with 125KD is formed. After Fc transformation, heavy chain and single chain of the monomeric Ab are naturally heterodimerized, and CL and CH1 are naturally dimerized at the same time, so that the SMBODY is finally formed, and the arrangement sequence and the structural schematic diagram of each structural domain of the SMBODY are shown in figure 2.

In the present invention, the bispecific antibody is prepared by the above method for preparing a bispecific antibody. In which the bispecific antibody targeting EpCAM and human CD3 was designated M702, as shown in fig. 2, the anti-CD 3 is in IgG format comprising heavy and light chains of anti-CD 3, and the anti-EpCAM is in ScFv-Fc format comprising VH, VL, Fc domains of anti-EpCAM. The bispecific antibody is constructed by an antibody genetic engineering method, a binary expression vector of a monomer Ab heavy chain and a monomer Ab light chain of the bispecific antibody SMBODY, and an ScFv-Fc expression vector. Primers were designed based on LC, HC, ScFv, Fc gene sequences and the multiple cloning sites in the vector. Wherein LC, HC, ScFv and Fc are respectively subjected to PCR amplification, gene fragments are obtained by a PCR method or an overlap extension PCR method, and then cloning is carried out by a homologous recombination method. The pCHO1.0 or pCHO1.0-hygromycin vector is enzyme-cut, then the PCR product and the enzyme-cut vector are purified and recovered, the LC fragment and the HC fragment are cloned on the pCHO1.0 vector through homologous recombination and the ScFv-Fc fragment is cloned on the pCHO1.0-hygromycin vector through homologous recombination in two steps, and the sequencing is carried out. The expression and detection of the recombinant protein SMBODY in mammalian cells, wherein plasmids respectively expressing a monovalent unit heavy chain, a monovalent unit light chain and a single chain unit are co-transfected into the mammalian cells by using a transfection reagent, and then the expression condition of the SMBODY is detected by collecting supernatant and carrying out SDS-PAGE and Western blotting. Centrifuging the supernatant of the culture solution after transfection expression, filtering, diluting with a binding buffer solution, passing through an affinity chromatography column, eluting with an elution buffer solution, and detecting and purifying the protein by SDS-PAGE.

The technical scheme of the invention has the beneficial technical effects that:

1. the present application provides a heterodimeric antibody comprising two distinct antigen-binding polypeptide units. The heterodimer has different molecular weight from the corresponding homodimer, and the purity of the bispecific antibody can be conveniently determined by using the molecular weight to distinguish the heterodimer from the homodimer. One of these two antigen-binding polypeptide units comprises a light chain-heavy chain pair similar to a wild-type antibody, which 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 a constant fragment (Fc) of an antibody. This fusion peptide is also referred to as a "single-chain unit" throughout the application.

2. The invention discloses establishment and application of a novel bispecific antibody SMBODY-mediated immune cell killing in-vitro efficacy experimental method. The invention comprises mediated immune cell killing in the process of bispecific antibody drug research, preparation of bispecific antibody, and establishment and detection of bispecific antibody in vitro efficacy model. The bispecific antibody SMBODY comprises a group of monovalent units (heavy-light chain combination) and a group of single-chain units (ScFv connecting Fc combination), wherein the single-chain units are specifically combined with a human tumor cell antigen, including a series of tumor cell membrane surface antigens such as EpCAM and the like, and a heavy chain Fc region is subjected to some transformation, so that the human tumor cell antigen is not easy to form a dimer by itself compared with a wild type; while another group of monovalent units specifically binds to another human T cell antigen, CD3, and also has some additional modifications in its heavy chain Fc region, it is not prone to self-dimer formation, and heterodimers are readily formed between these two groups of units. Meanwhile, the bispecific antibody can bridge between a target cell and a functional molecule (cell), stimulate a guided immune response, and has a wide application prospect in the immunotherapy of tumors.

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

In addition, because of the asymmetry, heterodimers have different isoelectric points than homodimers, which are 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 homodimers, greatly reducing difficulties in downstream process development that are prevalent with bispecific antibodies.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:

FIG. 1 is a molecular structure diagram of CD 3.

Figure 2. schematic of EpCAM x CD3SMBODY bispecific antibody molecule.

FIG. 3 is a diagram showing the detection of PCR products by electrophoresis; (A) m: DL1000 nucleic acid molecular marker; 1. an anti-CD 3 antibody light chain; (B) m: a DL2000 nucleic acid molecular marker; 1. an anti-CD 3 antibody heavy chain; 2. anti-EpCAM antibody ScFv-Fc.

FIG. 4 shows the purified double antibody electrophoresis and purity detection; (4A) SDS-PAGE electrophoresis, M: protein molecular weight markers; 1-2: a non-reducing EpCAM × CD3SMBODY diabody; 3-4: a reducing EpCAM × CD3SMBODY diabody; (4B) HPLC-SEC purity peak profile for EpCAM × CD3 SMBODY.

FIG. 5 is a graph showing the affinity of the EpCAM × CD3SMBODY diabody for HCT116 cells, as determined by flow cytometry; (●) an anti-EpCAM monoclonal antibody; (■) M702: EpCAM × CD3 SMBODY.

FIG. 6 is a graph showing the affinity of the EpCAM × CD3SMBODY diabody with Jurkat cells, as determined by flow cytometry; (●) anti-CD 3 monoclonal antibody L2K; (■) M702: EpCAM × CD3 SMBODY.

FIG. 7 is a graph of flow assays for EpCAM × CD3SMBODY diabody binding to both HCT116 and Jurkat cells; (●) M702: EpCAM × CD3SMBODY diabody; (■) anti-EpCAM mab; (. tangle-solidup.) anti-CD 3 monoclonal antibody L2K.

FIG. 8 is a graph showing the measurement of Tm value of EpCAM × CD3SMBODY diabody by differential scanning calorimeter scanning.

FIG. 9 is a graph showing the activity of the antibody after heat treatment; detection of binding activity to EpCAM, (●) anti-EpCAM monoclonal antibody; (. tangle-solidup.) M702: EpCAM × CD3SMBODY diabody; assay for binding activity to CD3, (●) anti-CD 3 monoclonal antibody L2K; (■) M702: EpCAM × CD3SMBODY diabody.

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

FIG. 11 is a graph showing the killing effect of CIK on target cells HCT116 (FIG. 11A) and NCI-N87 (FIG. 11B) by flow-assay in the presence of different concentrations of antibody; (■) M702: EpCAM × CD3SMBODY diabody, (. tangle-solidup.) Anti-EpCAM mab, (. xxx) Mco 101: control 4420X CD3 diabody, (. diamond-solid.) hIgG: human IgG.

FIG. 12 is a graph of flow measurements of killing of target cells HCT116 (FIG. 12A) and NCI-N87 (FIG. 12B) by effector PBMC in the presence of varying concentrations of antibody; (■) M702: EpCAM × CD3SMBODY diabody, (. tangle-solidup.) Mco 101: control 4420X CD3 diabody, (t) Anti-EpCAM mab, (. diamond-solid.) hIgG: human IgG.

FIG. 13 is a graph showing the results of an in vivo potency test of the diabody; (●) shows PBS, PBS was given only to the tail vein, (□) anti-EpCAM monoclonal antibody, (. DELTA.) 4420 × CD3 irrelevant control diabody, (. DELTA.) M702-1: EpCAM × CD3SMBODY diabody 4mg/kg concentration group (. smallcircle.) M702-2: EpCAM × CD3SMBODY diabody 2mg/kg concentration group.

Detailed Description

The invention is described in further detail below with reference to specific embodiments and with reference to the attached drawings. It should be understood that the examples in this specification are intended only to illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1: construction of expression vector for bispecific antibody (EpCAM X CD3SMBODY, M702)

1. Bispecific antibody sequence design

Bispecific antibodies targeting EpCAM and CD3 were designated M702(EpCAM × CD3 SMBODY), as in fig. 2, the anti-EpCAM side is in the form of ScFv-Fc, including anti-EpCAM VH, VL, Fc domains; anti-CD 3 is in the form of an IgG comprising an anti-CD 3 heavy and light chain, containing Fab and Fc domains. Wherein ScFv-Fc carries out KKW transformation on Fc, IgG form carries out LDY transformation on Fc, and the specific Fc transformation process is shown in PCT/CN2012/084982, so that the ScFv-Fc is not easy to form a homodimer and is easy to form a heterodimer, namely an EpCAM x CD3SMBODY bispecific antibody. Meanwhile, in order that the diabody can be expressed in CHO cells and secreted into the medium, a leader peptide sequence of the murine kappa chain was selected as a secretion signal peptide. The amino acid sequence and nucleic acid sequence of each domain and signal peptide are shown in SEQ ID NO 1-8. anti-CD 3 heavy chain amino acid sequence (SEQ ID NO: 1)

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

anti-CD 3 heavy chain nucleic acid sequence (SEQ ID NO: 2)

gatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcagcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgccgggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

anti-CD 3 light chain amino acid sequence (SEQ ID NO: 3)

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

anti-CD 3 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)

gaggtgcagctgctcgagcagtctggagctgagctggtaaggcctgggacttcagtgaagatatcctgcaaggcttctggatacgccttcactaactactggctaggttgggtaaagcagaggcctggacatggacttgagtggattggagatattttccctggaagtggtaatatccactacaatgagaagttcaagggcaaagccacactgactgcagacaaatcttcgagcacagcctatatgcagctcagtagcctgacatttgaggactctgctgtctatttctgtgcaagactgaggaactgggacgagcctatggactactggggccaagggaccacggtcaccgtctcctccggaggcggcggttcaggcggaggtggaagtggtggaggaggttctgagctcgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcactatgagctgcaagtccagtcagagtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcctcctaaactgttgatctactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggaacagatttcactctcaccatcagcagtgtgcaggctgaagacctggcagtttattactgtcagaatgattatagttatccgctcacgttcggtgctgggaccaagcttgagatcaaaggtgcggccgcagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgataccacgcctcccgtgctggactccgacggctccttcttcctctacagcgatctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

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

METDTLLLWVLLLWVPGSTG

Leader peptide sequence of mouse kappa chain nucleic acid sequence (SEQ ID NO: 8)

atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggt

2. Bispecific antibody gene cloning

pCHO1.0 was selected as an expression vector for cloning and expressing the anti-CD 3 heavy and light chain genes, and pCHO1.0-hygromycin expression vector was modified by replacing the puromycin gene in pCHO1.0 with the hygromycin resistance gene and was selected for cloning and expressing the anti-EpCAM ScFv-Fc fusion gene. The primers in Table 2 were designed according to the cloning protocol and sent to Sovium Kirgiz Biotechnology Inc. for synthesis. PCR amplification is carried out by using the primers in the table 1, the template is a gene plasmid synthesized by genes in an early experiment or subcloned to pCDNA3.1 or pUC57, the PCT/CN2012/084982 patent is described in detail, then anti-CD 3 heavy chain and anti-CD 3 light chain are respectively constructed on an expression vector of pCHO1.0, and anti-EpCAM ScFv-Fc is constructed on an 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, e.g., the light and heavy chains of the antibody of interest; 1 μ l of 10 μ M forward and reverse primers; 2.5. mu.l of 10 XPCR Buffer; 1 μ l of 10mM dNTP; 1 μ l of 2.5 units/. mu.l Pyrobest DNA polymerase (Takara, R005A); and distilled water to a total volume of 25 μ l were gently mixed in a microfuge tube and spun rapidly in a microfuge to collect the reaction mixture to the bottom of the tube. The PCR reaction was performed using GeneAmp PCR System 9700(Applied Biosystem) and the following settings: 5 minutes at 95 ℃; the following 25 cycles: at 95 ℃ for 30 seconds each time; 56 ℃ for 30 seconds; and 72 ℃ for 1 minute.

The Kozak sequence, leader sequence and cleavage sites EcoR V and PacI were introduced into the light chain by several rounds of overlapping PCR amplification (fig. 3); and corresponding primers introduce the Kozak sequence, leader sequence and cleavage sites AvrII and BstZL7I into the heavy chain (see FIG. 3). Firstly, carrying out homologous recombination on the amplified LC gene fragment and a pCHO1.0 expression vector which is cut by EcoR V and PacI enzyme to obtain an expression vector loaded with an anti-CD 3 light chain; then, the plasmid was cleaved with AvrII and BstZL7I, and then subjected to homologous recombination with HC to obtain an anti-CD 3 pCHO1.0 expression vector, which was named pCHO1.0-anti-CD 3-HL-LDY.

An anti-EpCAM ScFv-Fc domain is amplified through overlapping PCR, a Kozak sequence, a leader sequence, an enzyme cutting site AvrII and BstZL7I are introduced into ScFv-Fc, the amplified gene fragment (figure 3) and an enzyme-cut pCHO1.0-hygromycin expression vector are subjected to homologous recombination to obtain an expression vector loaded with the anti-EpCAM ScFv-Fc, and the plasmid is named pCHO1.0-hygromycin-anti-EpCAM-ScFv-Fc-KKW.

Example 2: bispecific antibody expression and purification

1. Expression of bispecific antibodies

Plasmid bulk extraction was performed using an endotoxin free bulk extraction kit (Qiagen, 12391), the specific procedures were performed according to the instructions provided by the manufacturer. CHO-S cell culture in CD CHO Medium (Gibco, 10743-029) at 37 ℃ with 5% CO according to the instructions provided by the manufacturer₂After culture in a cell incubator and cell preparation, the plasmid pCHO1.0-anti-CD 3-HL-LDY was co-transfected into CHO-S cells using a Maxcell STX electrotransfer according to the manufacturer' S instructions (Maxcell) together with pCHO1.0-hygromycin-anti-EpCAM-ScFv-Fc-KKW, both plasmids were designed to express bispecific antibodies to EpCAM × CD 3.

On day 2 post-transfection, the culture temperature was adjusted to 32 ℃ and supplemented with 3.5% feed a daily, and after 14 days of culture, expression supernatants were harvested by centrifugation at 800 × g.

2. Purification of bispecific antibodies

The expression supernatant was filtered through a 0.22uM filter, all the Fc domain-carrying antibodies were captured from the expression supernatant using a Mabselect Sure affinity column (available from GE, 18-1153-45,17-5438-01), and equilibration buffer (9.5mM NaH)₂PO₄+40.5mM Na₂HPO₄Ph7.0) and then passed through an affinity column and eluted with elution buffer (50mM citric acid +100mM arginine, ph 3.2). Separation of the target bispecific antibody from the byproducts was achieved by SP cation exchange chromatography, using equilibration buffer A (43.8mM NaH), a cation exchange column from GE (18-1153-44,17-1087-01)₂PO₄+6.2mM Na₂HPO₄pH 6.0) the column was equilibrated and the sample was diluted with impure water to a conductivity between 3.0 and 3.5ms and bound to the SP column using elution buffer B (43.8mM NaH)₂PO₄+6.2mM Na₂HPO₄+1M NaCl, pH 6.0)20 column volumes for linear elution; finally, the displacement buffer PBS was concentrated. The purified bispecific antibody was tested by SDS-PAGE and SEC and was more than 95% pure (see FIG. 4).

Example 3: determination of binding Activity of bispecific antibody to cells (FACS)

The bispecific antibodies of the invention bind to a target antigen on the corresponding cell. HCT116 (purchased from ATCC, CCL-247) was used as EpCAM-positive cells and Jurkat (Jurkat, TIB-152) was used as CD 3-positive cells, and the cell binding activity was measured using the diabody prepared according to the present invention.

1. Detection of binding Activity of bispecific antibodies to HCT116 cells Using flow assay

Sufficient HCT116 cells were cultured, digested with 0.25% trypsin, centrifuged to collect cells, bispecific antibody was simultaneously diluted at a concentration of 500nmol, 3-fold gradient diluted to obtain 12 concentration gradients for use, collected cells were washed twice with PBS + 1% FBS, and PBS + 1% FBS was added to resuspend the cells to 4 × 10⁶Cells/ml, cells were plated in 96-well plates at 50ul per well (2 × 10)⁵Individual cells), 50ul of diluted bispecific antibody was added and incubated at room temperature for 1 hour; is centrifuged to removeThe cell is washed twice by PBS, then the diluted PE-labeled anti-human IgG FC antibody (Biolegend, 409304) is used for resuspending the cell, the cell is incubated for 30 minutes at room temperature in a dark place, the PBS is washed twice, then the cell is resuspended by 100ul PBS, the detection is carried out on a computer, the average fluorescence intensity is used, and the KD value of the binding affinity of the diabody and the HCT116 is calculated by analyzing by using software GraphPad prism 5.0.

2. Detection of binding Activity of bispecific antibody with Jurkat cells by flow assay

Sufficient Jurkat suspension cells were cultured and harvested by centrifugation. The procedure of the following experiment was the same as in the above example, 100ul of PBS resuspended cells were tested on the machine and the binding affinity KD of the diabody to Jurkat cells was calculated by analysis with the software GraphPadprism5.0, with mean fluorescence intensity. The results showed that EpCAM × CD3SMBODY diabody had good binding activity to CD3 positive Jurkat cells, as shown in FIG. 6, with KD of only 50.90nM, with good cell binding activity.

3. Double antibody mediated co-binding activity assay

Cultured HCT116 and Jurkat cells were harvested by centrifugation and washed 2 times with PBS, stained with CFSE and PKH-26, respectively, while diluting bispecific antibody at a concentration of 10ug/ml, diluted in 3-fold gradients to obtain 12 concentration gradients for use, the stained HCT116 and Jurkat cells were centrifuged to remove supernatant, washed two times with PBS + 1% FBS, and resuspended in 4 × 10% PBS + 1% FBS⁶Each cell/ml, mixed well at 1:1, plated into 96-well plates at 50ul per well (2 × 10)⁵Individual cells) were added with 50ul of diluted bispecific antibody, incubated at room temperature for 1 hour, centrifuged to remove supernatant, washed twice with PBS, finally resuspended with 100ul of PBS, tested on the machine, analyzed for the rate of double positive cells, calculated by analysis with software GraphPad prism5.0, the results showed that the proportion of flow-detected bifluorescence was very low without M702 (fig. 7), and reached around 40% with the addition of EpCAM × CD3 diabody M702It was shown that M702 can bind to both EpCAM-positive HCT116 cells and CD 3-positive Jurkat cells, promoting co-aggregation of both cells, forming an immune-killing complex.

Example 4: thermal stability assay for bispecific antibodies

1. Determination of Tm value of bispecific antibody

The thermal stability of the bispecific antibody was determined by differential scanning calorimetry (MicroCal VP-DSC, GE Co.), the diabody samples were purified and replaced in PBS buffer, and calorimetric scanning data were obtained by scanning from 10 ℃ to 100 ℃ at a heating rate of 60 ℃/h, using PBS buffer as a control. The scanning results (FIG. 8) show that the Tm values of the bispecific antibodies are all above 70 ℃ and exhibit good thermal stability.

2. Thermal challenge experiments for bispecific antibodies

Single chain antibody fragment (ScFv) was formed by linking heavy chain variable region and light chain variable region via a linker peptide (Gly4Ser)3 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-promoter TRAIL-R2 and LTbeta R. MAbs.2009 Mar-Apr; 1(2): 128-41.) therefore, we diluted the antibody to 0.4mg/ml, 4 ℃, 37 ℃, 42 ℃, 47 ℃, 52 ℃, 57 ℃, 62 ℃, 67 ℃, 72 ℃, 77 ℃, 82 ℃, 1h with a PCR instrument, centrifuged the supernatant per tube 15 ul., flow-assayed according to the following steps, collected the suspension added to a 96 well plate, 3 × 10 single cells⁵Wells, addition of various treatment antibodies, and addition of a fluorescent secondary antibody, were tested on a flow machine, the results are shown in FIG. 9, FIG. 9A, which measures the thermostability of Anti-EpCAM and EpCAM × CD3SMBODY bispecific antibody in binding to EpCAM, and its T₅₀Values are 73.28 and 58.31, respectively, and figure 9B determines the thermostability of L2K and EpCAM × CD3 MSBODY bispecific antibodies in antibodies that bind CD3, their T₅₀The values were 69.33 and 61.30, respectively, both showing better thermal stability.

Example 5: dual antibody mediated in vitro cell killing assay

Isolation of PBMC cells and CIK cell culture

Taking fresh anticoagulated blood, centrifuging for 5min at 400g, and discarding the supernatant. Adding 10 times of cell volume of erythrocyte lysate, gently blowing and mixing, and lysing for 4-5 minutes at room temperature or on ice. During the lysis process, the red blood cells are preferably lysed by shaking the cells appropriately. Centrifuge at 400g for 5min at 4 ℃ and discard the red supernatant. If the red blood cells are not completely lysed, steps 2 and 3 are repeated once. Washing for 1-2 times. PBS of 5 times the cell pellet volume was added, the pellet was resuspended, centrifuged at 400g at 4 ℃ for 2-3 minutes, and the supernatant was discarded. The washing can be repeated 1 more time, and the washing is performed 1-2 times. According to the requirement of the experiment, after the cell sediment is re-suspended by PBS with proper 4 ℃, the subsequent experiments such as counting and the like can be carried out.

Culturing CIK cells, supplementing 30ml each cell with CIK cell activating culture medium (serum-free X-Vivo cell culture medium +750IU/ml IFN-gamma + -2% autologous plasma), adding into 75cm2 culture flask, placing in saturated humidity, 37 deg.C, and 5.0% CO₂Culturing in incubator for 24 hr, adding CIK cell stimulating factor mixture (serum-free X-Vivo cell culture solution +75ng/ml anti-human CD3, 750IU/ml IL-2, 0.6ng/ml IL-1 α), and further placing at saturated humidity, 37 deg.C, and 5.0% CO₂Culturing in an incubator. The next step is to determine the filling (serum-free X-Vivo culture solution +750IU/ml IL-2 + -2% autologous plasma) and bottle separation according to the growth of CIK cells, and basically to maintain the cells to grow at the concentration of 2X 10^ 6. Finally, the collected CIK cells were phenotypically examined by flow cytometer FC500, which included: CD3, CD56, CD4 and CD8, and the expression of the cell surface antigens in CIK cells is detected. The detection result is shown in a figure 10, and the phenotype result shows that the CIK cells have more than 15.9 percent of double positive CD3 and CD56, and the cultured cells have good NK T cell rate.

2. Double-antibody efficient mediated PBMC cell killing tumor cell detection

HCT116 or NCI-N87 cells were trypsinized to prepare single cell suspensions HCT116 or NCI-N87 were stained with CFSE at a final concentration of 5uM, and the cells were resuspended to 2 × 10 using 10% FBS-1640 from the cell culture after staining⁵Per ml, according to 2 × 10⁴Add to 96-well plate for overnight culture per well, i.e., 100 ul/well. Experimental design cultured CIK cells were added at 50 ul/well, control wells were set, and no addition was requiredThe wells of CIK cells are supplemented with culture medium with the same volume, corresponding antibodies are added according to the experimental design while the CIK cells are added, 50 ul/well is added, the wells without the addition of the antibodies are supplemented with the culture medium with the same volume, a 96-well plate is taken out after 48h, each well cell is digested by pancreatin to be single cell suspension, all supernatant and cell suspension in the process are correspondingly collected into a 1.5ml EP tube, 500g × min is centrifuged, the supernatant is discarded, 150ul 1% FBS-PBS is added into each well to be uniformly mixed and resuspended, each tube is added with PI (the final concentration is 1ug/ml) in the front 10-15min of a flow type machine to detect the proportion of CFSE and PI double positive cells in CFSE positive cells, namely the death rate of target cells HCT116 or NCI-N87, the result is shown in figure 11, and the result shows that EpSMBCCAM × CD3 ODY bispecific antibody mediates the killing of the CIK cells and the tumor cells shows good killing effect, the maximum killing efficiency and the death rate of Anti 539CAM 2-monoclonal antibody is obviously stronger than that of Epti-CAM.

3. Double-antibody efficient mediated PBMC cell killing tumor cell detection

HCT116 cells were trypsinized to prepare a single cell suspension HCT116 or NCI-N87 were stained with CFSE at a final concentration of 5uM, and the cells were resuspended to 2 × 10% cells in 10% FBS-1640 culture of the cells after staining⁵Per ml, according to 2 × 10⁴Adding cultured CIK cells into wells, namely 100 ul/well, culturing overnight, adding corresponding antibodies according to the experimental design, 50 ul/well, setting a control well, and supplementing the wells without adding CIK cells with the same volume of culture medium, adding corresponding antibodies into the wells according to the experimental design while adding CIK cells, 50 ul/well, supplementing the wells without adding antibodies with the same volume of culture medium, taking out 96-well plates after 48h, digesting the cells in each well with pancreatin to obtain single cell suspension, collecting all supernatants and cell suspensions in the process into 1.5ml EP tubes correspondingly, centrifuging 500g × min, abandoning the supernatants, adding 150ul 1% FBS-resuspended PBS into each well, mixing the cells uniformly, adding PI (final concentration of 1ug/ml) into each tube before the flow type machine, performing flow detection on CFSE, wherein the proportion of PI double positive cells in CFSE, the proportion of PI double positive cells in CFSE positive cells is the death rate of HCT116, the death rate of the monoclonal antibody of the CFSMB, the CD 6356 shows that the killing effect of the monoclonal antibody of the EpSMBCM CD 3683 and the monoclonal antibody mediated tumor cells is high in the highest in the tumor cell mediated killing effect of the EpSMCAM 35.

Example 6: drug effect detection of bispecific antibody killing subcutaneous transplantation tumor

The tumor xenograft model was established by mixing 5 × 10⁶SW480 and 5 × 10⁶CIK cells were pooled and grown by subcutaneous inoculation on the right dorsal side of female NOD/SCID mice (group N8). within 2 hours, mice were randomly grouped, antibody treated groups, started with 4, 2mg/kg of EpCAM × CD3SMBODY and were each injected intravenously via the tail vein.A control group comprised a group constructed with 4mg/kg of anti-EpCAM monoclonal antibody and a group of independently controlled SMBODY (4420 × CD 3). the control SMBODY was an anti-fluorescein antibody (4-4-20) sequence (Kranz DM, volume EW Jr.partial emulsification of anti-interferon in BALB/c micro: synthetic mutagenesis of secondary monoclonal antibodies in PBS: synthetic transfection efficiency of secondary monoclonal antibodies in molecular antigens. 1981; 18. vernier 8810) and continued to be injected with the same volume on day 3 as the control group (4-5929) and measured with the same volume per three days of intravenous injection of animals in the following equation 39 ×. the control group was continued with the same volume of dose on day 3. the same day of injection³)。

Evaluation of antitumor effects in EpCAM × CD3SMBODY was done by adoptive transfer graft tumor model. The gastric cancer tumor cell line SW480 was used to establish a transplantation tumor model on immunodeficient mouse NOD/SCID, and human CIK cells were obtained by stimulating culture after isolating peripheral blood mononuclear cells, and mixed inoculation was performed at a ratio of 1:1 before inoculation. As shown in FIG. 13, the PBS group, the Anti-EpCAM of the control antibody, and the 4420 XCD 3 antibody treatment all have no obvious inhibition on tumor growth, but the EpCAM XCD 3(2mg/kg, 4mg/kg) treatment group in the same experiment has no tumor growth, so that the EpCAM XCD 3 SMBODY-mediated immune tumor killing can obviously inhibit the tumor growth. Also as expected, the SMBODY molecule MCO101 lacking EpCAM specificity (4420 × CD3) did not show significant antitumor activity in vivo experiments even though the CD 3-specific molecule remained.

It is to be understood that the invention disclosed is not limited to the particular methodology, 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 present invention which will be 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 encompassed by the following claims.

Claims (12)

1. A bispecific antibody, characterized in that said antibody comprises: (a) a monovalent unit that is a light-chain-heavy-chain pair having specific binding capacity for the immune cell surface antigen CD 3; 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 capacity for a tumor cell surface antigen which is EpCAM;

the single chain unit comprises an antibody anti-EpCAM against EpCAM, the monovalent unit comprises an antibody anti-CD 3 against CD 3;

the amino acid sequence of the anti-CD 3 heavy chain is the amino acid sequence shown in sequence number 1, the amino acid sequence of the light chain of anti-CD 3 is the amino acid sequence shown in sequence number 3, and the amino acid sequence of the anti-EpCAM ScFv-Fc is the amino acid sequence shown in sequence number 5; and the cysteine at position 222 of the anti-CD 3 heavy chain is disulfide-linked to the cysteine at position 213 of the light chain of anti-CD 3, the cysteine at positions 228 and 231 of the anti-CD 3 heavy chain is disulfide-linked to the cysteine at positions 263 and 266 of anti-EpCAMScFv-Fc, respectively, the anti-CD 3 heavy chain is salt-bridged at positions 394 and 411 to positions 436 and 405 of anti-EpCAM ScFv-Fc, and the anti-CD 3 heavy chain is protuberance-into-hole-linked at position 368 to position 441 of anti-EpCAM ScFv-Fc.

2. The bispecific antibody according to claim 1, characterized in that: 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, characterized in that: the single-chain variable fragment consists 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 bound to the heavy chain by disulfide bonds; the heavy chain is bound to the fusion peptide by one or more disulfide bonds.

5. The bispecific antibody according to claim 1, characterized in that: the heavy chain in said monovalent unit comprises a human or humanized Fc fragment comprising a human IgG Fc fragment; the Fc fragment of the fusion peptide comprises a human or humanized Fc fragment comprising a human IgG Fc fragment.

6. The bispecific antibody according to claim 5, characterized in that: 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.

7. A method for the preparation of a bispecific antibody according to any one of claims 1 to 6, characterized in that said method comprises the steps of:

(1) respectively constructing heavy chains and light chains of the monovalent units on a first expression vector and constructing the single chain units on a 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 together into a cell, the cell being a CHO-S cell, culturing and taking the supernatant;

(3) separating the expression supernatant to obtain a purified bispecific antibody; the separating step comprises: protein A affinity chromatographic column captures all antibodies with Fc structural domains from the expression supernatant, realizes the separation of the target bispecific antibody and byproducts by SP cation exchange chromatography, passes through a Q column, and finally concentrates and replaces buffer PBS.

8. The method according to claim 7, characterized in that in step (1) of the method:

the monovalent unit is an anti-CD 3 antibody, primers for amplifying a light chain are Kozak (EcoRV) F, MK-leader (EcoRV) F, L2K-VL (MK) F1 and hIgK (PacI) R, and a Kozak sequence, a leader sequence, a restriction enzyme cutting site EcoRV and PacI are introduced into the light chain through overlapping PCR amplification; primers for amplifying the heavy chain are Kozak (Avr II) F, MK-leader (AvrII) F, L2K-VH (MK) F1 and hIgG1(sbfI) R, and Kozak sequence, leader sequence and cleavage sites AvrII and BstZl7I are introduced into the heavy chain through overlapping PCR amplification; carrying out homologous recombination on the amplified LC gene fragment and a pCHO1.0 expression vector which is cut by EcoRV and PacI enzyme to obtain an expression vector loaded with an anti-CD 3 light chain; then carrying out enzyme digestion on AvrII and BstZL7I, and then carrying out homologous recombination on the AvrII and BstZL7I and HC to obtain a pCHO1.0 expression vector of anti-CD 3, wherein the plasmid is named as pCHO1.0-anti-CD 3-HL-LDY;

the single-chain unit is an anti-EpCAM ScFv-Fc antibody, primers used for amplifying the anti-EpCAM ScFv-Fc antibody are Kozak (Avr II) F, MK-leader (AvrII) F, M701-VH F1 and hIgG1(sbfI) R, an anti-EpCAM ScFv-Fc domain is amplified through PCR, a Kozak sequence, a leader sequence, an enzyme cutting site AvrII and BstZL7I are introduced into ScFv-Fc, the amplified gene fragment and an enzyme-cut pCHO1.0-hygromycin expression vector are subjected to homologous recombination to obtain an expression vector filled with the anti-EpCAM ScFv-Fc, and the plasmid is named pCHO1.0-hygromycin-anti-EpCAM-ScFv-Fc-KKW.

9. Use of a bispecific antibody of any one of claims 1-6 in the manufacture of a medicament for the treatment of colon or gastric cancer caused by the expression of an antigen specific for EpCAM, or for killing colon or gastric cancer cells expressing EpCAM.

10. Use of a bispecific antibody prepared by the method of any one of claims 7-8 in the preparation of a medicament for the treatment of colon or gastric cancer caused by the expression of an antigen specific for EpCAM, or for killing colon or gastric cancer cells expressing EpCAM.

11. Use of a bispecific antibody of any one of claims 1-6 in the manufacture of a medicament for screening for or evaluating the efficacy of a medicament for treating colon or gastric cancer expressing an EpCAM-specific antigen in a tumor cell line.

12. Use of a bispecific antibody prepared by the method of any one of claims 7-8 in the preparation of a medicament for screening for or evaluating the efficacy of a medicament for treating colon or gastric cancer expressing an EpCAM-specific antigen in a tumor cell line.

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