CN103965359B - Anti-epithelial cell adhesion molecule and the bi-specific antibody of T cell antigen - Google Patents

Abstract

A bispecific antibody comprising (1) a first functional domain binding to EpCAM, (2) a second functional domain binding to CD3, and (3) a linker connecting the first and second functional domains, the The first functional domain comprises at least one light chain variable region and at least one heavy chain variable region, the light chain variable region comprising the following three complementarity determining regions: CDR-L1: RASQDISNYLN; CDR-L2: YTSRLHS; CDR-L1: L3: QQGSTLPYT, the heavy chain variable region includes the following three complementarity determining regions: CDR-H1: GYTFTYYGMN; CDR-H2: WINTYTGEPTYGDDFKG; CDR-H3: TGRATSFDY.

Description

Bispecific antibodies against epithelial cell adhesion molecules and T cell antigens

technical field

The present invention relates to antibodies related to tumor treatment, more specifically, the field of genetically engineered antibodies.

Background technique

Current studies have known that epithelial cell adhesion molecule (EpCAM, Epithelialcelladhesionmolecule; CD326) is a glycosylated type I transmembrane glycoprotein with a molecular weight of 30 to 40kD, and its structure includes an epidermal growth factor-like, a thyroglobulin-like The extracellular region, a transmembrane region and a 26 amino acid cytoplasmic region. EpCAM is mainly localized between epithelial cells and functions in a directed and highly ordered manner to adhere epithelial cells. Once the malignant transformation of epithelial cells, the rapid growth of tumor cells will abandon the high order of epithelial cells. As a result, the surface distribution of EpCAM becomes less constrained and the molecule is more exposed on tumor cells. Most tumor cells express EpCAM on their surface due to their epithelial origin [7]. Therefore, EpCAM can be the target site for antibody immunotherapy on tumor cells and tumor stem cells expressing this molecule on the surface.

At present, a large number of immunotherapy studies targeting EpCAM have been carried out, such as Adecatumumab, Catumaxomab, and an anti-EpCAM/CD3 trifunctional antibody have entered the clinical trial research stage. Among them, the effect of Catumaxomab antibody in the treatment of colon cancer/gastric cancer/or pancreatic cancer, the therapeutic effect of Adecatumumab antibody against prostate cancer, and the therapeutic effect of anti-EpCAM/CD3 trifunctional antibody against ovarian cancer have been reported in the literature [12-14]. recorded.

Anti-EpCAM/CD3 bispecific antibodies purportedly shown to treat pancreatic cancer, colorectal cancer, and ovarian cancer have also been published in research literature [15-18], respectively. In addition, Chinese patent application CN1812999A also reported a drug comprising an anti-EpCAM×CD3 bispecific single-chain antibody construct, which is expected to be used for the treatment, alleviation and/or prevention of epithelial cancer or minimal residual cancer.

However, no EpCAM antibody that has expected and ideal therapeutic effect on liver cancer has been reported so far. Therefore, there is still a need in this field to develop anti-EpCAM antibodies with excellent properties including but not limited to effective targeting and treatment of liver cancer, so as to develop targeted drugs with more significant tumor immunotherapy effects.

Contents of the invention

The present invention provides a bispecific antibody molecule with EpCAM specific binding functional domain, and the molecule also has the ability to trigger T cell targeting cytotoxicity through binding with CD3 molecule to kill tumor cells. The bispecific antibody of the present invention shows excellent specific killing effect on various liver cancer tumor cell lines and tumors in tumor mice, especially specific killing effect on liver cancer cells.

The present invention relates to a bispecific antibody comprising (1) a first functional domain specifically binding to EpCAM (2) a second functional domain specifically binding to CD3 and (3) connecting the first and second functional domains The linker of said first functional domain comprises at least one light chain variable region and at least one heavy chain variable region, said light chain variable region comprises three complementarity determining regions as follows:

CDR-L1: RASQDISNYLN (SEQ ID NO: 7)

CDR-L2: YTSRLHS (SEQ ID NO: 8)

CDR-L3: QQGSTLPYT (SEQ ID NO: 9)

The heavy chain variable region comprises the following three complementarity determining regions:

CDR-H1: GYTFTYYGMN (SEQ ID NO: 10)

CDR-H2: WINTYTGEPTYGDDFKG (SEQ ID NO: 11)

CDR-H3: TGRATSFDY (SEQ ID NO: 12).

"Functional domain" refers to a three-dimensional structure capable of specifically recognizing and/or binding to an epitope, such as an antibody or antibody fragment, including native intact antibody, single chain antibody (scFV), Fd fragment, Fab fragment, F(ab ') ₂ fragments, single domain antibody fragments, isolated CDR fragments, and derivatives thereof. "Single-stranded" here means that the first and second functional domains are covalently linked, preferably in the form of a colinear amino acid sequence that can be encoded by one nucleic acid molecule.

Preferably or alternatively, the first and/or second functional domains in the above-mentioned bispecific antibody of the present invention are selected from monoclonal antibodies, intact antibodies, single-chain antibodies (scFV), Fab fragments, Fd fragments, and Fv fragments , F(ab')2 fragments and functional derivatives thereof. It is worth noting that the binding sites of the functional domains of the present invention may not only come from antibodies, but also from other proteins that bind to EpCAM and/or CD3, such as naturally occurring surface receptors or ligands.

A "whole antibody" is composed of two identical heavy and light chains, each chain comprising a variable region (V region) and one or more constant regions (C regions), respectively. The variable region is responsible for binding to the antigen, while the constant region is primarily responsible for binding effector molecules. Within each variable domain are three flexible loops of high diversity, called complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The rest of the variable domains contain rigid beta sheets and support the so-called framework regions (FRs). CDRs and FRs are arranged alternately to form a sandwich structure.

"Single-chain antibody (scFV) fragment" refers to an antibody fragment constructed by genetic engineering, which is a recombinant protein with a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker , the linker brings these two domains into association to form an antigen-binding site. The size of ScFv is generally 1/6 of a whole antibody. A single-chain antibody is preferably a sequence of one amino acid chain encoded by one nucleotide chain.

"Fd fragment" refers to an antibody fragment composed of heavy chain VH and CH1.

"Fab fragment" refers to the heterodimer formed by the Fd fragment (consisting of heavy chain VH and CH1) and the entire light chain via interchain disulfide bonds. A "Fab antibody" is 1/3 the size of a full antibody and contains only one antigen-binding site.

"F(ab') ₂ fragment" refers to a bivalent fragment comprising two linked Fab fragments.

A "single domain antibody" consists of either a heavy chain variable region or a light chain variable region. The antibody fragment is so named because it consists of only one domain. The size of this fragment is 1/12 of a whole antibody.

"Derivatives of antibodies" include, for example, when the derivatives of the antibodies are obtained by phage display technology, surface plasmon resonance technology as used in BIAcore system can be used to increase the efficiency of phage antibody binding to GPC3 or CD3 epitope ( Schier, Human Antibody Hybridoma 7 (1996), 97-105; Malmborg, J Immunol Methods 183 (1995), 7-13). Also included are methods for the production of chimeric antibodies as described, for example, in WO89/09622, methods for the production of humanized antibodies as described in EP-A10239400 and WO90/07861, as described in WO91/10741, WO94/02602 and WO96/33735 Method for generating heterologous antibodies, such as human antibodies in mice.

The antibodies or fragments thereof used in the present invention can be further modified individually or in combination using conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and/or recombination and/or other modification methods. Methods for introducing such modifications into the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art; see, eg, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. The modifications referred to are preferably carried out at the nucleic acid level.

"Binding" defines the affinity interaction between a specific epitope on an antigen and its corresponding antibody, and is generally understood as "specific recognition". "Specific recognition" means that the bispecific antibody of the present invention does not or substantially does not cross-react with any polypeptide other than the target antigen. And the degree of specificity can be judged by immunological techniques, including but not limited to western blotting, immunoaffinity chromatography, flow cytometry and so on. In the present invention, the specific recognition is preferably determined by flow cytometry, and in specific cases, the standard of specific recognition can be judged by those of ordinary skill in the art based on their common knowledge in the field.

"CD3" refers to the antigen expressed on T cells as part of the T cell receptor complex, which consists of three distinct chains CD3ε, CD3δ and CD3γ. Clustering of CD3 on T cells by, for example, their immobilization by anti-CD3 antibodies results in T cell activation similar to T cell receptor mediated activation, but independent of the specificity of the TCR clone. The vast majority of anti-CD3 antibodies recognize the CD3ε chain.

The second functional domain that specifically recognizes the T cell surface receptor CD3 of the present invention is not specifically limited, as long as it can specifically recognize CD3. For example but not limited to CD3 antibodies mentioned in the following patents: US7,994,289, US6,750,325; US6,706,265; US5,968,509; US8,076,459; US7,728,114; US20100183615.

In one embodiment of the present invention, the first functional domain of the bispecific antibody is a single-chain antibody comprising a light chain having the amino acid sequence described in SEQ ID NO: 1 and a light chain having the amino acid sequence described in SEQ ID NO: 3. heavy chain.

In one embodiment of the present invention, the bispecific antibody has the amino acid sequence set forth in SEQ ID NO:5.

As mentioned above, the bispecific antibody of the present invention also includes a linker linking the first and second functional domains. The linker sequence can be a short peptide chain, such as but not limited to (Gly ₄ Ser)n, where n is from 1 to 5, and n is an integer. In a particular embodiment, n is preferably 3. Characterization of such linkers comprising a lack of secondary structure promotion is known in the art and described eg in Dall's Acqua et al. (Biochem. 1998, 37, 9266-9273). Contemplated linkers having fewer than 5 amino acids may comprise 4, 3, 2, or 1 amino acid. A preferred single amino acid is Gly. Interlinkage of said first and second functional domains is provided by, for example, a genetically engineered linker, as described in the Examples below. Methods for making fused and operably linked bispecific antibodies and expressing antibodies in mammalian cells or bacteria are well known in the art.

In one embodiment of the invention, said bispecific antibody is a chimeric antibody or a humanized antibody.

In one embodiment of the present invention, the bispecific antibody is also linked to other drugs or labels.

The present invention also relates to a nucleic acid encoding the above-mentioned bispecific antibody, or a nucleic acid that hybridizes to said nucleic acid under stringent conditions, or a nucleic acid composed of a degenerate sequence of said nucleic acid. The term "hybridization" is official in the art, ie the person skilled in the art knows what hybridization conditions have to be used. According to the stringent hybridization conditions of the present invention, it is meant to contain, for example, 50% formamide, 5*SSC (750mM sodium chloride, 75mM sodium citrate), 50mM sodium phosphate, 5*Denhardt's solution and 10% dextran sulfate and 20 μg /ml of denatured sheared salmon sperm DNA was incubated overnight at 42°C, and then the filter was washed with 0.1×SSC at approximately 65°C. Also contemplated are nucleic acid molecules that hybridize to the nucleic acids of the invention under less stringent conditions. Variations in the stringency of hybridization and signal detection can be achieved by controlling formamide concentration (lower concentrations of formamide result in lower stringency), salt concentration or temperature. In addition, to achieve lower stringency, washes after stringent hybridization can be performed at higher salt concentrations (eg, 5×SSC). In addition, variations in the stringency conditions described above may actually be achieved by including and/or replacing blocks used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, heparin, denatured salmon sperm DNA and other commercially available preparations. The nucleic acid molecule can be, for example, DNA, cDNA, RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of these nucleic acids, alone or in combination.

In one embodiment of the present invention, the nucleic acid encoding the above-mentioned bispecific antibody comprises (1) the nucleic acid sequence SEQ ID NO: 2 encoding the amino acid sequence of SEQ ID NO: 1, and (2) the nucleic acid sequence SEQ ID NO: 4 encoding the amino acid sequence of SEQ ID NO: 2 .

In one embodiment of the present invention, the nucleic acid encoding the above-mentioned bispecific antibody has the nucleic acid sequence SEQ ID NO:6 encoding the amino acid sequence described in SEQ ID NO:5.

The invention also relates to a vector comprising said nucleic acid. Many suitable vectors are known to those skilled in the art, the choice of which depends on the intended function, including cloning and expression vectors. Vectors include plasmids, cosmids, viruses, bacteriophages and other vectors routinely used in genetic engineering. In addition, nucleic acids and vectors of the invention can be reconstituted into liposomes for delivery to target cells.

The expression vector may be a eukaryotic cell vector or a prokaryotic cell vector. Preferably, the expression vector further includes a regulatory sequence operatively linked to the nucleic acid. Regulatory sequences refer to DNA sequences required to affect the expression of coding sequences to which they are linked. Operative linkage means that the regulatory sequences of the coding sequences are linked in a manner compatible therewith to achieve the expression of the coding sequences. Such regulatory sequences include, for example: (a) an origin of replication sequence that enables the vector to replicate in the host cell, (b) a gene sequence that encodes a selectable marker that encodes a protein that is activated by the host cell Necessary for survival and growth in specific selective media, etc. In case the host cell is not transfected or transformed with a vector containing the gene, the host cell cannot survive in the specific selection medium. The proteins encoded by typical selection marker genes include proteins that are resistant to antibiotics or toxins, such as ampicillin, kanamycin, tetracycline, neomycin, hygromycin, methotrexate, etc.; Related proteins that compensate for auxotrophs and supply key nutrients that are not present in the medium, such as the gene encoding D-alanine racemase. An example of using resistance selection includes transfecting a foreign vector containing a neomycin resistance gene so that the host cells can continue to survive and grow in the medium containing the drug neomycin or G418. Another example is the use of the dihydrofolate reductase (DHFR) selection marker in mammalian cells such as Chinese hamster ovary cells (CHO). A mammalian cell host cell is a DHFR-deficient cell that does not contain the dihydrofolate reductase gene. , cannot synthesize nucleic acid and must grow in a medium containing HT. When using the vector to transfect host cells, the positive clones of the exogenous vector containing both the target gene and the DHFR gene can be obtained through selection and screening of the above-mentioned medium conditions. (c) its coding sequence contains the sequence of the promoter, (d) the expression vector may also include other constituent sequences, including signal peptide sequence, transcription termination sequence, enhancer sequence, etc., preferably, the vector of the present invention is a eukaryotic cell vector . Preferably, the vector of the present invention is a pH vector used for antibody eukaryotic expression, which contains CMV promoter, internal ribosome entry site sequence (Internal ribosome entry site, IRES), DHFR screening marker and other elements. Methotrexate (MTX) is an inhibitor of DHFR that blocks its action. When the cell culture medium contains MTX, DHFR is inhibited, and through feedback regulation, the gene self-amplifies, and its upstream and downstream genes are also amplified, so that the target gene is also amplified, and the expression of the target protein can be increased. Useful regulatory sequences are known to those skilled in the art and are commercially available.

The present invention also relates to an expression system containing the above-mentioned vector, that is, to a host cell containing the vector for expressing the desired multifunctional antibody polypeptide. According to the vector used, the host cell of the present invention can be any prokaryotic host cell or eukaryotic host cell. Depending on the host used, the protein encoded by the nucleic acid of the invention may be glycosylated or non-glycosylated. It is to fuse the nucleic acid sequence of the present invention with a C-terminal His tag. Eukaryotic host cells, including yeast, insect cells, plant cells, mammalian cells, etc. may be preferred, because eukaryotic cells have complex post-translational modifications (such as glycosylation) of the protein of interest, and are increasingly used for large-scale cultivation. Commonly used host cell lines include monkey kidney cells (COS-7ATCCCRL1651), human embryonic kidney cells 293 and its subclone cell lines, baby hamster kidney cells (BHK, ATCCCCL10), Chinese hamster ovary cells (CHO), etc. Preferably, the eukaryotic host cell of the present invention is a Chinese hamster ovary cell.

The present invention also relates to the application of the anti-EpCAM/CD3 bispecific antibody in the preparation of drugs for treating, preventing or alleviating tumors. The tumors include but not limited to liver cancer, pancreatic cancer, colorectal cancer, ovarian cancer, lung cancer, breast cancer, head and neck tumors. Preferably, the tumor is liver cancer.

The present invention also relates to a pharmaceutical composition, which comprises a safe and effective amount of anti-EpCAM/CD3 bispecific antibody and a pharmaceutically acceptable carrier.

Description of drawings

Figure 1 is a schematic structural view of the recombinant plasmid pIH-anti-EpCAM/CD3scFv comprising the nucleic acid sequence encoding the bispecific antibody EpCAM/CD3 of the present invention.

Figure 2 is the gel denatured protein electrophoresis (SDS-PAGE) results of the bispecific antibody 1H8/CD3 of the present invention recombinantly expressed in a genetic engineering manner, and the left column is a nucleic acid molecular weight marker (purchased from: Shanghai Shengzheng Biotechnology Co., Ltd. , trade name: low molecular weight standard protein marker).

Fig. 3 is a flow cytometric analysis graph (FACS) of the binding of the bispecific antibody 1H8/CD3 of the present invention to peripheral blood lymphocytes (PBMC).

Figure 4 is a flow cytometric analysis of the binding of the bispecific antibody 1H8/CD3 of the present invention to EpCAM-positive tumor cells Huh-7 and SMMC-7721 and to EpCAM-negative tumor cells PLC/PRF/5 and SK-Hep-1 respectively picture.

Fig. 5 is a bar graph showing the numerical percentage (%) of PBMC-mediated cytotoxicity against EpCAM-positive tumor cells Hep3B and Huh-7 induced by the bispecific antibody 1H8/CD3 of the present invention.

Fig. 6 is a bar graph showing the numerical percentage (%) of the PBMC-mediated cytotoxic effect on the EpCAM-negative tumor cell SK-Hep-1 induced by the bispecific antibody 1H8/CD3 of the present invention.

Fig. 7 is a microscopic enlarged photo showing that the bispecific antibody 1H8/CD3 of the present invention recruits PBMCs to the EpCAM-positive tumor cell Huh-7. The red arrows indicate Huh-7 cells, the green arrows indicate PBMCs, and the black arrows indicate PBMCs gathered around Huh-7 cells.

Fig. 8 shows enlarged micrographs showing that the bispecific antibody 1H8/CD3 of the present invention does not recruit PBMCs to EpCAM-negative tumor cell SK-Hep-1. The red arrows in Figure 8 indicate SK-Hep-1 cells, and the green arrows indicate PBMCs.

Fig. 9 is a line graph showing the volume change of implanted tumors in Huh-7 tumor-bearing mice treated with different doses of the bispecific antibody 1H8/CD3 of the present invention and control medium,

Fig. 10 is a line graph showing the changes in the volume of implanted tumors in vivo after treatment of Hep3B tumor-bearing mice with different doses of the bispecific antibody 1H8/CD3 of the present invention and a control medium.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. In the following examples, as the experimental method of specific conditions is not indicated, then according to conventional conditions such as Sambrook etc., the conditions described in "Molecular Cloning: Experimental Handbook (NewYork: Cold Spring Harbor Laboratory Press, 1989)", and clearly stated in the examples that there are When using the instructions from the reagent company, follow the conditions suggested in the instructions.

Example 1. Antigen immunization and hybridoma screening

1.1 Antigen immunization

(1) Recombinant protein immunization

EpCAM extracellular region recombinant protein (purchased from Shanghai Ruijin Biotechnology Co., Ltd., product number: RO720) was fully emulsified and mixed with an equal volume of complete Freund's adjuvant (purchased from Sigma, product number: 5881) to obtain a total volume of 200 μL, which contained EpCAM antigen 100μg, subcutaneous injection to immunize six-week-old BALB/c mice. After 4 weeks, the recombinant EpCAM antigen was emulsified and mixed with an equal volume of incomplete Freund's adjuvant (purchased from Sigma, product number: 5506) to obtain a total volume of 200 μL, which contained 50 μg of EpCAM antigen, and intraperitoneally injected to immunize mice. At intervals of 2 weeks, intraperitoneal booster immunization was repeated once. Three weeks after the completion of the last booster immunization, the mice were immunized intrasplenicly with 20 μg of EpCAM antigen.

1.2 Establishment and screening of hybridoma cell lines

Four days after intrasplenic immunization of the mice, the spleen was taken under aseptic conditions, the lymphocytes were separated with a 100-mesh filter, fused with the myeloma SP2/0 cell line, and treated with hypoxanthine, aminopterin and thymidine containing 10% fetal bovine serum (purchased from: PAA Company, product number: A15-151), DMEM (purchased from Gibco Company, product number: 12800-082) selective medium for culture, add xanthine and thymidine after 3 days to continue the cell Cultivate for a week.

Coating with recombinant protein antigen of EpCAM extracellular region, positive clones were screened by enzyme-linked immunosorbent assay (ELISA), and positive clones were subcloned three times by limiting dilution method, cultured continuously for 2 months, and finally stable Positive clone, the hybridoma cell line was named 1H8.

Example 2. Preparation of anti-EpCAM bispecific antibody

2.1 Genetic engineering preparation of anti-EpCAM single-chain antibody

Use Trizol (Invitrogene Company, product number: 15596-026) to extract the total RNA of the hybridoma cell line 1H8 in the above-mentioned Example 1, and then use a reverse transcription RT-PCR kit (Promega Company, product number: A3800) to operate according to the instructions of the kit The step is to perform reverse transcription on the total RNA extracted above to obtain a complementary DNA sequence.

Use the 5'-FullRace kit (Takara, product number: D315) to amplify the antibody heavy chain or light chain variable region and its upstream and downstream sequences using the complementary DNA sequence obtained above as a template according to the steps described in the kit. A DNA sequence in which:

The upstream primers are provided by the kit,

The downstream outer primer is: CCAGAGTTCCAGGTCACTGTCACT (SEQ ID NO: 13),

The downstream inner primer is: CCAGGGTCACCATGGAGTTAGTTT (SEQ ID NO: 14),

Then clone the antibody heavy chain and light chain variable regions amplified by PCR into T-easy vectors (Promega, Cat. No.: A1360), transform competent Escherichia coli Top10, and screen for resistance culture to pick out monoclonal strains , were sequenced separately.

Then the sequencing results were entered into the PubmedBlastIgBlast database for comparison. According to the comparison results, the database showed FWR1-CDR1-FWR2-CDR2-FWR3 of the antibody, and according to the characteristic amino acids at the end of the mouse antibody CDR3, that is, the heavy chain: Val-Ser-Ala or Val- Ser-Ser; light chain: Ile-Lys-Arg, accurately identify the starting sites of the heavy chain and light chain variable regions of the hybridoma 1H8 monoclonal cell line. The determined amino acid sequence of the variable region of the light chain is SEQ ID NO: 1, and the nucleic acid sequence encoding it is SEQ ID NO: 2; the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 3, and the nucleic acid sequence encoding it is SEQ ID NO: 4.

SEQ ID NO: 1 (1H8V _L )

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDFSLTISNLDQEDIATYFCQQGSTLPYTFGGGTKLEIKR

SEQ ID NO: 2 (1H8V _L )

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTTCCAATTATTTAAACTGGTATCAACAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTTCTCTCACCATTAGCAACCTGGACCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAGTACGCTTCCGTACACGTTCGGGGGGGGGACCAAGCTGGAAATAAAACGG

SEQ ID NO: 3 (1H8V _H )

QIQLVQSGPELKKPGETVKISCKASGYTFTYYGMNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASASASLQINNLKNEDTATYFCARTGRATSFDYWGQGTTLKVSS

SEQ ID NO: 4 (1H8V _H )

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACATATTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGGTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCGCTGCCTCTTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAACAGGTCGGGCTACGTCCTTTGACTACTGGGGCCAAGGCACCACTCTCAAAGTCTCCTCA

Then add (Gly ₃ Ser) ₃ short peptide coding DNA sequences at the primer end of the downstream of the light chain variable region and the upstream end of the heavy chain variable region, and then pass the nucleic acid sequences of the heavy chain and the light chain variable region through the linker (Gly ) by OverlapPCR. 3Ser _{) 3 short} peptide encoding DNA linkage. The specific implementation method is as follows:

The 1H8vL fragment was amplified by 5'-1H8vL/3'-1H8vL primers, and the linker (Gly ₃ Ser) ₃ short peptide encoding DNA was added downstream of 1H8vL.

1H8vH was amplified by 5'-1H8vH/3'-1H8vH primers, and (Gly ₃ Ser) ₃ short peptide coding DNA was added upstream.

The above two stages of PCR product gel recovery. Then, 20 ng of the products recovered from the gel were taken for PCR reaction according to the following method.

reaction system:

Reaction conditions: annealing temperature 50°C, 10 cycles

After the reaction was completed, 5 μL was taken as a template and 5'-1H8vL/3'-1H8vH as primers for 28 cycles of PCR reaction. The reaction product is 1H8scFv, which is recovered and purified by gel.

The primer sequences are as follows:

5'-1H8vL: GATATCCAGATGACACAGAC (SEQ ID NO: 15)

3'-1H8vL:

CTGCGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCTCTCCACCCCGTTTTATTTTCCAGCTTG (SEQ ID NO: 16)

5'-1H8vH:

GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGATCCAGTTGGTGCAGTCTG (SEQ ID NO: 17)

3'-1H8vH: TGAGGAGACTTTGAGAGTGG (SEQ ID NO: 18)

1H8scFv (SEQ ID NO: 19)

GSTLPYTFGGGTKLEIKRGGGGSGGGGSGGGGSQIQLVQSGPELKKPGETVKISCKASGYTFTYYGMNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASAASLQINNLKNEDTATYFCARTGRATSFDYWGQGTTLKVSS

1H8scFv (SEQ ID NO: 20)

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTTCCAATTATTTAAACTGGTATCAACAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTTCTCTCACCATTAGCAACCTGGACCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAGTACGCTTCCGTACACGTTCGGGGGGGGGACCAAGCTGGAAATAAAACGGGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACATATTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGGTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCGCTGCCTCTTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAACAGGTCGGGCTACGTCCTTTGACTACTGGGGCCAAGGCACCACTCTCAAAGTCTCCTCA

2.2 Preparation of CD3 single-chain antibody nucleic acid sequence

The V _LCD3 and V _HCD3 gene fragments are respectively obtained according to the 857-1585th nucleotide sequence of SEQ ID NO: 9 published in the patent document US7112324B1, wherein the nucleotide sequence encoding the CD3 single-chain antibody fragment is obtained by the VLCD3 and VHCD3 nucleotides A nucleotide sequence encoding the amino acid sequence VE(GGS) ₄ GG linker is introduced between the fragments.

SEQ ID NO: 21 (CD3scFv)

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

SEQ ID NO: 22 (CD3scFv)

GATATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGTCGAAGGTGGAAGTGGAGGTTCTGGTGGAAGTGGAGGTTCAGGTGGAGTCGACGACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTCATACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

2.3 Construction of 1H8/CD3 bispecific antibody coding sequence nucleic acid

The anti-EpCAM single-chain antibody prepared in the above steps and the anti-CD3 scFv were connected by the PCR method with the DNA encoding the linker Gly ₃ Ser short peptide to prepare the open reading frame sequence of the anti-EpCAM/CD3 bispecific antibody.

2.4 Construction of expression vector

The 1H8scFv fragment was amplified by 5'-81/3'-1H8scFv primers, the 1H8scFv was added with a signal peptide upstream, and a Gly ₄ Ser linker linker peptide was added downstream. The CD3 single-chain antibody was amplified by 5'-CD3/3'-CD3-Histag primers, the Gly ₄ Ser linker was added upstream, and the 6×Histag coding sequence was introduced downstream. The above two stages of PCR product gel recovery. Then, 20 ng of the products recovered from the gel were taken for PCR reaction according to the following method.

reaction system:

Reaction conditions: annealing temperature 50°C, 10 cycles

After the reaction was completed, 5 μL was used as a template, and 5'-81/3'-CD3-Histag was used as a primer for 28 cycles of PCR reaction. The reaction product was recovered and purified by gel. The nucleic acid sequence of the reaction product is 1H8/CD3 bispecific antibody is SEQ ID NO: 6, and the amino acid sequence encoded by it is SEQ ID NO: 5. Wherein the reaction product introduces the NheI restriction site due to the 5'-81 primer, and contains the NotI restriction site due to the introduction of the 3'-CD3-Histag primer.

Digested with NheI and NdeI, ligated with T4 ligase, and transformed into Top10 competent Escherichia coli. Pick positive clones for sequencing identification. (NheI, NdeI, and T4 enzymes were purchased from NEB Company, and the reaction system and conditions refer to the instruction manual)

The primer sequences are as follows:

5'-81:

TAGCTAGCCACCATGGTGTCCACAGCTCAGTTCCTTGCATTCTTGTTGCTTTGGTTTCCAGGTGCAAGATGTGATATCCAGATGACACAGAC (SEQ ID NO: 23)

3'-1H8scFV:

CTGACTGCTGCAGTTTGATATCGGATCCACCACTCCTGAGGAGACTTTG (SEQ ID NO: 24)

5'-CD3:

CAAAGTCTCTCTCAGGAGGTGGTGGATCCGATATCAAACTGCAGCAGTCAG (SEQ ID NO: 25)

3'-CD3-Histag:

TATGCGGCCGCCTAATGATGATGGTGATGATGTTTCAGTCCAGCTTGGTCC (SEQ ID NO: 26)

Amino acid sequence of 1H8/CD3 bispecific antibody

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDFSLTISNLDQEDIATYFCQQGSTLPYTFGGGTKLEIKRGGGGSGGGGSGGGGSQIQLVQSGPELKKPGETVKISCKASGYTFTYYGMNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASAASLQINNLKNEDTATYFCARTGRATSFDYWGQGTTLKVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKHHHHHH(SEQIDNO：5)

1H8/CD3 bispecific antibody nucleic acid sequence

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTTCCAATTATTTAAACTGGTATCAACAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTTCTCTCACCATTAGCAACCTGGACCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAGTACGCTTCCGTACACGTTCGGGGGGGGGACCAAGCTGGAAATAAAACGGGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACATATTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGGTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCGCTGCCTCTTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAACAGGTCGGGCTACGTCCTTTGACTACTGGGGCCAAGGCACCACTCTCAAAGTCTCCTCAGGAGGTGGTGGATCCGATATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACAT CTGAGGACTCTGCAGTCTATTACTGTGCAAGATATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGTCGAAGGTGGAAGTGGAGGTTCTGGTGGAAGTGGAGGTTCAGGTGGAGTCGACGACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTCATACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACATCATCACCATCATCAT(SEQIDNO：6)

2.5 Screening of stable transformed host cells

The pIH plasmid pIH-1H8/CD3 containing the nucleic acid sequence encoding the bispecific antibody 1H8/CD3 was digested overnight with Pvui (purchased from NEB Company) enzyme according to the instructions. Add 70% isopropanol to the reaction system to precipitate on ice for 30 minutes, centrifuge at 12,000 g for 10 minutes at 4°C, discard the supernatant; wash twice with 70% ice ethanol, dry at room temperature for 10 minutes, add sterilized supernatant Dissolve in pure water, quantify with a spectrophotometer, and then freeze at -80°C overnight.

The linearized plasmid was used to transform CHO-DG44 cells (according to the instructions of Invitrogen Company, Cat. No. A10999-01). After 48 hours of transfection, the cells were counted, and 500 cells per well were planted in a 96-well plate, and a total of 10 plates were planted. After 14 days, the recombinant EpCAM protein was coated on the ELISA plate at 30 ng per well, and coated overnight at 4°C. In the next morning, the liquid in the ELISA plate was discarded and blocked with 5% milk prepared in PBS at 37°C for 2 hours. Discard the liquid in the plate, wash 3 times with 0.5% PBST, and dry the liquid in the plate. Then pipette 50 μL of CHO-DG44 cell culture supernatant transformed with pIH-1H8/CD3 plasmid as the primary antibody, add it to the blocked ELISA plate, incubate at 37°C for 2 hours, discard the liquid in the plate, and wash 3 times with 0.5% PBST , Buckle the liquid in the dry plate. Dilute the mouse anti-6×His antibody (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) at 1:1000 (PBS) as a secondary antibody, add 50 μL to each well, incubate at 37°C for 2 hours, discard the plate The liquid in the plate was washed 3 times with 0.5% PBST, and the liquid in the plate was dried. Dilute FITC-labeled goat anti-mouse antibody (purchased from Shanghai Kangcheng Bioengineering Co., Ltd.) at 1:1000 (PBS) as the third antibody, add 50 μL to each well, incubate at 37°C for 2 hours, discard the liquid in the plate , washed 3 times with 0.5% PBST, dried the liquid in the plate, added ABTS (2,2'-azino-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) containing H ₂ O ₂ )) 100μL (Preparation of ABTS working solution: Weigh 14.7g trisodium citrate, dissolve in dH ₂ O. Adjust the pH to 4.0, weigh 0.22g ABTS, mix well, dilute to 1L, and store at 4° away from light) (add 26mL ABTS 31 μL H ₂ O ₂ ), and detect the absorbance at a wavelength of 450 nm. According to the results, single clones with high antibody expression were selected for culture.

2.6 Expression and purification of 1H8/CD3 bispecific antibody

The CHO-DG44 cell culture conditions were cultivated according to the method recommended by Invitrogen (Product No. A10999-01). The cell culture supernatant was collected, centrifuged at 9000 rpm at 4°C for 30 minutes, and then filtered with a 0.45 micron filter membrane (purchased from Millex, model: SLHV033RB) to remove solid impurities. Metal-affinity chromatography column NiSepharose ^TM 6FastFlow (purchased from GE Healthcare Bio-Science AB company) was used to carry out the purification of diabody heterosexual antibody, and the elution condition was to contain 20mM, 50mM, 100mM, 250mM, phosphate buffer (pH 8 .0, Na ₂ HPO ₄ 50Mm, NaCl 300mM) for elution in sequence, and the elution volume of each imidazole concentration group was 5 times the volume of the metal affinity chromatography column NiSepharose ^TM 6FastFlow. The eluted product was subjected to SDA-PAGE gel electrophoresis, and the size of the eluted product was determined by means of small molecular weight markers. Carry out Coomassie Brilliant Blue staining gel then, decolorization solution decolorization, identify the elution product (specific process strictly according to: Molecular cloning: the condition described in the experimental handbook (NewYork: Cold Spring Harbor laboratory Press, 1989)) that is specifically eluted solution, and the specific eluted product was dialyzed in 1× ice PBS, the dialyzed volume was 1:1000. After the dialysis, the antibody was concentrated using CentrifugalFilterUnits (purchased from Amicon, product number: UFC801096).

Example 3 Detection of Binding Ability of Bispecific Antibody

3.11H8/CD3 bispecific antibody FACS analysis

A.Material:

1) cells:

b. steps

Carry out similar operations as follows for each cell line in the above table:

Take the cells listed in Table 1 in the logarithmic growth phase and inoculate them into 6cm plates, cultivate them in a 5% _CO2 incubator at 37°C, and use them for flow cytometry when the cells cover 90% of the plates.

1) Use 10mM EDTA to digest the cells, and centrifuge at 5000rpm×5min to collect the cells in each well of the plate into several 2mL Eppendorf tubes. The cells were washed twice with 1× phosphate buffered saline (PBS) containing 1% NCS (Neonatal Calf Serum), and the supernatant was discarded after centrifugation.

2) Add the bispecific antibody 1H8/CD3 of the present invention in 100 μL 1×PBS with a final concentration of 10 μg/mL in a tube of a test group as the primary antibody, add the same volume of PBS in a tube of a control group to resuspend the cells in the cell pellet, and place on ice Incubate the cells for 45min. Then centrifuge, discard the supernatant, wash the cells twice with PBS containing 1% NCS, centrifuge, discard the supernatant.

3) Add 100 μL of 1:50 diluted mouse-derived anti-6×His antibody (Sangon Bioengineering (Shanghai) Co., Ltd.) as the secondary antibody to resuspend the cells, incubate the cells in ice bath for 45 min, centrifuge, discard the supernatant, and wash with 1 Wash the cells 2 times in PBS with % NCS, centrifuge and discard the supernatant.

4) Add 100 μL 1:50 diluted goat anti-mouse FITC-labeled antibody (Shanghai Kangcheng Bioengineering Co., Ltd.) as the third antibody to resuspend the cells, incubate the cells in ice bath for 45 min, centrifuge, discard the supernatant, and wash the cells with PBS containing 1% NCS 2 times, centrifuge and discard the supernatant.

5) The cell pellets in each tube were resuspended with 400 μL of PBS, and then transferred to flow cytometry tubes respectively, and the fluorescence intensity of the cell surface produced by the antibody bound to the cells was monitored by flow cytometry.

6) The flow cytometer data analysis software WinMDI2.9 was used to analyze the data. At least 10,000 cells per sample were analyzed by flow cytometry.

C. Results

As shown in Figure 3, PBMC cells showed a fluorescence intensity spectrum significantly higher than that of the PBS control, indicating that the experimental 1H8/CD3 bispecific antibody of the present invention can specifically bind to CD3-positive PBMC cells.

As shown in Figure 4, EpCAM-positive cells SMMC-7721 and Huh-7 showed significantly higher fluorescence intensity spectra than the PBS control, in which the peak fluorescence intensity of SMMC-7721 was about 4-5 times that of the PBS control, and that of Huh-7 The peak fluorescence intensity was about 10 times that of the PBS control. Also as shown in Fig. 4, the EpCAM-negative cells PLC/PRF/5 and SK-Hep-1 showed a fluorescence intensity spectrum and a fluorescence intensity peak that almost coincided with those of the PBS control.

The results in Figure 4 show that the bispecific antibody 1H8/CD3 of the present invention can specifically bind to EpCAM positive cells but not to EpCAM negative cells.

Based on the above figures 3 and 4, the bispecific antibody 1H8/CD3 of the present invention has the ability to specifically bind to CD3-positive PBMC cells and EpCAM-positive tumor cells.

Example 4 In vitro cytotoxicity analysis of anti-EpCAM bispecific antibody

step:

Peripheral blood mononuclear cells (PBMC) were isolated from blood of healthy human donors using Ficoll (from Biochrom) density gradient centrifugation method following standard procedures. After centrifugation, the cells were washed with 0.1M phosphate buffered saline (PBS) and then resuspended in RPMI1640 complete medium (Gibco) to adjust the cell concentration to 7×10 ⁵ /mL. PBMCs were used as effector cells in cytotoxicity experiments. Different tumor cells were used as target cells, including the EpCAM-positive human liver cancer cell line Hep3B (liver cancer cell line, ATCC: HB-8064 ^TM ), the aforementioned Huh-7 and the EpCAM-negative SK-Hep-1.

Adjust the target cell concentration to 7×10 ⁴ /mL with RPMI1640 complete medium. Equal volumes of target cells and effector cells were mixed so that the effector:target (E:T) ratio was 10:1.

The mixed cell suspension was added to a 96-well plate at a volume of 75 μL/well. Then, 25 μL of 1H8/CD3 single-chain bifunctional antibody serially diluted ten-fold from 30 ng/mL to 0.03 ng/mL was added to each well.

After incubation for 48 hours at 37°C in a 5% _CO2 incubator, according to the manufacturer's instructions, use The non-radioactive cytotoxicity assay kit (Non-Radioactive Cytotoxicity Assaykit, from Promega) detects the cytotoxicity of the antibody.

The non-radioactive cytotoxicity assay is a colorimetric-based assay that can replace the ⁵¹ Cr release method. The assay quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytosolic enzyme that is released upon cell lysis in much the same way as ⁵¹ Cr is released in radioactive assays. The released LDH medium supernatant can be detected by a 30-minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) into red formazan. The amount of red product produced is directly proportional to the number of cells lysed.

The tumor cell killing rate (i.e., % cytotoxicity) was determined according to The non-radioactive cytotoxicity assay G1780 product manual provides the following formula for calculation:

in:

"Experiment" refers to the LDH release value produced by the experimental wells added with antibody/effector cells/target cells,

"Spontaneous effector cell" refers to spontaneous LDH release from control wells containing only effector cells. In the present invention, it refers to the control well of effector cells

"Target cell spontaneous" refers to LDH release from control wells containing only target cells when the target cells were not treated with other factors.

"Target cell maximum" is the LDH release produced by the target cells after treatment with 0.8% TritonX-100 to completely lyse the control wells,

"Target cell maximum - target cell spontaneous" represents the release of LDH produced by the complete lysis of target cells after external treatment.

3.3 Experimental results:

The results of cytotoxicity of bispecific antibody 1H8/CD3 on various tumors at different concentrations are as follows.

From the above table and the results shown in Figures 5 and 6, it can be seen that the bispecific antibody 1H8/CD3 can reach about 30% in the lower dose group, such as the 0.3ng/ml group, on the EpCAM positive tumor cell Hep3B, and in the higher dose group. Dose group, such as 30ng/ml group, is as high as 86%. Similarly, the effect of the bispecific antibody 1H8/CD3 on EpCAM-positive tumor cells Huh-7 reached about 71% in the lower dose group, such as the 0.3ng/ml group, while in the higher dose group, such as the 30ng/ml group , it is as high as 102%. This shows that the recombinant bispecific antibody 1H8/CD3 of the present invention has a specific killing effect on EpCAM positive tumor cells. In contrast, the bispecific antibody 1H8/CD3 of the present invention has almost no killing effect on the EpCAM negative tumor cell SK-Hep-1, except in the case of a very high dose (100ng/ml), showing about 14.1% cell killing toxicity. This may be due to the activation of T cells by large doses of CD3 antibodies, leading to non-specific killing of tumors.

Example 5

In vivo antitumor activity of bifunctional antibody in tumor-bearing mice

Immunodeficient NOD/SCID mice aged 6-10 weeks (provided by the Animal Experiment Center of Shanghai Medical College, Fudan University) were used to construct xenograft models of human EpCAM-related tumors, whose genetic characteristics are lack of T cells, B cells, NK cell and macrophage function.

Two treatment groups (6 mice in each group) were inoculated subcutaneously on the right side with a mixed cell suspension consisting of 2×10 ⁶ Huh-7 or 4×10 ⁶ Hep3B tumor cells and a cell concentration of 1× 10 ⁶ unstimulated PBMCs were prepared by mixing 1:1 (the ratio of cell numbers). One hour after the inoculation, the mice of each group were administered 0.25 mg/kg/d and 0.5 mg/kg/d of 1H8/CD3 through the tail vein, respectively, and the administration was repeated for 10 consecutive days.

Control groups included (1) Control 1, which was inoculated with tumor cells not mixed with an equal volume of PBMCs, as a background control for assessing the effect of PBMCs on tumor growth, and (2) Control 2, which was inoculated with tumor cells mixed with PBMCs After the suspension, only PBS without antibody was administered to the group to evaluate the non-specific killing effect induced by PBMC effector cells. The administration method and administration time of the control group were parallel to those of the treatment group. The control group consisted of 6 mice per group.

The size of the tumor in each group of mice was measured with a caliper, and the tumor volume was calculated according to the following formula:

As shown in Figures 9 and 10, two groups of mice were treated with different concentrations of the bispecific antibody 1H8/CD3 of the present invention, and the tumor volumes in their bodies were significantly smaller than those of mice in the control group 1 and control group 2 in this experiment Tumor volume in vivo.

Specifically, as shown in Figure 9, for example, on the 27th day after tumor cell inoculation, the tumor volumes of the Huh-7PBS group, that is, the control group 1 and the Huh-7PBMCPBS group, that is, the control group 2 mice were all above 2000 mm ³ , while the two The tumor volumes of the mice treated with different doses of the bispecific antibody of the present invention were significantly less than 2000mm ³ , and the average tumor volume of the mice in the 0.25mg/kg dose group was as low as about 463mm ³ , while the mice in the 0.5mg/kg dose group No tumor grew. As shown in Figure 10, from the 26th day to the 42nd day after tumor inoculation, the tumor volume of the mice in the control group increased rapidly, from less than 100 mm ³ to about 700 mm ³ and about 2000 mm ³ respectively, while the two experimental mice There was no tumor growth in the mice in the dose group no matter whether it was the 0.25 mg/kg dose group or the 0.5 mg/kg dose group.

Figures 9 and 10 show that the bispecific antibody 1H8/CD3 of the present invention has significant specific tumor-killing therapeutic effects on Huh-7 tumor-bearing mice and Hep-3B tumor-bearing mice.

The contents of all publications listed in this specification are incorporated herein. In addition, those skilled in the art can understand that various modifications and changes are possible in the present invention without departing from the technical scope and spirit described in the claims. The present invention also includes the above-mentioned modifications and changes.

references:

1. Baeuerle PA, Gires O. EpCAM (CD326) finding its role in cancer. BrJ Cancer 2007; 96: 417-423.

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3. Visvader JE, Lindeman GJ. Cancer stem cells in solid tumors: accumulating evidence and unresolved questions. Nat Rev Cancer 2008; 8: 755-768.

4.GiresO, KleinCA, BaeuerlePA. OntheabundanceofEpCAMoncancerstemcells.NatRevCancer2009;9:143;authorreply143.

5. MunzM, BaeuerlePA, GiresO.

6. SchmelzerE, ReidLM. EpCAM expressioninnormal, non-pathologicaltissues. FrontBiosci2008; 13: 3096-3100.

7. McLaughlinPM, HarmsenMC, DokterWH, KroesenBJ, van derMolenH, BrinkerMG, HollemaH, et al. Theepithelialglycoprotein2 (EGP-2) promoter-drivenepithelial-specificexpressionofEGP-2intransgenicmice: anewmodeltostudycarcinoma-directedimmunotherapy.CancerRes2: 501-401-401;

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11. DalerbaP, DyllaSJ, ParkIK, LiuR, WangX, ChoRW, HoeyT, etal.

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Claims (12)

1. the bi-specific antibody suppressing hepatoma carcinoma cell, it comprises (1) and combines first functional domain of EpCAM, (2) second functional domain of CD3 is combined, and (3) connect the joint of described first and second functional domains, described first functional domain comprises a variable region of light chain and a variable region of heavy chain, described variable region of light chain comprises following three complementary determining region: SEQIDNO:7, SEQIDNO:8, SEQIDNO:9；Described variable region of heavy chain comprises following three complementary determining region: SEQIDNO:10, SEQIDNO:11, SEQIDNO:12；

Further, described first functional domain is single-chain antibody, its comprise aminoacid sequence as described in SEQIDNO:1 variable region of light chain and as described in SEQIDNO:3 the variable region of heavy chain of aminoacid sequence.

2. the bi-specific antibody of claim 1, its aminoacid sequence is as described in SEQIDNO:5.

3. the bi-specific antibody of one of claim 1-2, it is also connected with label.

4. the nucleic acid of the bi-specific antibody of one of coding the claims.

5. the nucleic acid of claim 4, its comprise (1) coding the aminoacid sequence shown in SEQIDNO:1 nucleic acid sequence SEQ ID NO: 2, and (2) coding the aminoacid sequence shown in SEQIDNO:3 nucleic acid sequence SEQ ID NO: 4.

6. the nucleic acid of claim 4, its nucleotide sequence is as shown in SEQIDNO:6.

7. contain the carrier of the nucleic acid of one of claim 4-6.

8. the carrier of claim 7, it is expression vector.

9. the carrier of claim 7 or 8, it also comprises the regulation sequence being effectively connected to described nucleic acid.

10. comprise the host cell of the carrier of one of claim 7-9.

The application in the medicine of tumor is treated, prevented or alleviate to the bi-specific antibody of one of 11. claim 1-3 in preparation；Wherein said tumor is hepatocarcinoma.

12. 1 kinds of pharmaceutical compositions, the bi-specific antibody of one of its claim 1-3 comprising safe and effective amount and pharmaceutically acceptable carrier.