CN106810610A - Anti-EpCAM and the double targeting antibodies of CD3 specificity and its preparation method and application, the minicircle dna containing this pair of targeting antibodies expression cassette and application - Google Patents

Abstract

The invention provides a specific dual targeting antibody and its method and application. The specific dual-targeting antibody has a first antigen-binding domain that specifically binds to human CD3 and a second antigen-binding domain that specifically binds to the EpCAM antigen, and can specifically recognize and bind to tumor cells that overexpress the EpCAM antigen , including liver cancer and other EpCAM-positive tumor diseases, etc., and can also bind T cell receptors; and mediate T cells to play a killing role, thereby eliminating tumor cells, which has great application value in the diagnosis, treatment and detection of EpCAM-positive tumor diseases . The present invention also provides a microcircle DNA having the nucleotide sequence encoding the specific dual-targeting antibody. The specific dual targeting antibody and microcircle DNA provided by the invention can be used to prepare antibody pharmaceutical composition or EpCAM positive tumor cell diagnostic reagent.

Description

Anti-EpCAM and CD3 specific dual targeting antibody and its preparation method and application, Microcircle DNA containing the double-targeting antibody expression cassette and its application

technical field

The invention relates to the fields of biomedicine and diagnosis and treatment, in particular to an anti-EpCAM and CD3 specific dual-targeting antibody, a preparation method and application thereof, and a microcircle DNA containing the expression cassette of the dual-targeting antibody and its application.

Background technique

EpCAM (Epithelial cell adhesion molecule), also known as TACSTD1 (tumor-associated calcium signal transducer1), CD326 (cluster of differentiation), 17-A, ESA, EGP40, EPCAM is a kind of GA-733-2 The gene encodes a transmembrane glycoprotein with a molecular weight of 40kDa, which acts as a homophilic calcium-independent adhesion molecule between epithelial cells in the process of epithelial carcinogenesis.

EpCAM was first discovered as a tumor-associated antigen, and has attracted special attention because of its high-level expression in tumor tissues of various types of diseases. At present, a large number of experiments have confirmed that EpCAM can be used as a marker protein for diagnosis and treatment of tumors. Many human primary liver cancer cell lines isolated and established from primary liver tumors contain the expression of EpCAM, and the expressed tumor cells have Some typical characteristics of cancer stem cells play a decisive role in the occurrence and development of tumors. In addition, after the discovery of colorectal cancer stem cells with the phenotype of EpCAM+/CD44+, the characteristics of stem cell function have been confirmed in many aspects. Recent studies have shown that EpCAM protein is highly expressed in embryonic stem cells. In epithelial tumor cells, its multidirectional differentiation potential is realized by inhibiting the expression of p53 protein and improving the differentiation ability of mouse embryonic fibroblasts, indicating that EpCAM transmembrane signaling Reprogramming is achieved by inhibiting the p53-p21 signaling pathway. Therefore EpCAM is considered as a marker of tumor stem cells.

Edrecolomab (ED) is a mouse-derived monoclonal IgG2 antibody against EpCAM that recognizes human tumor-associated antigen 17-1A. This monoclonal antibody has been marketed in Germany and has been used in breast cancer and colorectal cancer. It has just been reported that whether it is used alone or combined with other adjuvant chemotherapy, it has a significant advantage in terms of recurrence-free and overall survival. However, in the subsequent four large prospective randomized clinical trials, whether used alone or combined with chemotherapy, ED was ineffective for postoperative stage II and III colorectal cancer.

In the case of poor results in clinical trials of mouse-derived antibodies, the advent of humanized anti-EpCAM monoclonal IgG1 antibody Adecatumumab (MT201) has brought new hope to the clinical immune-directed therapy of tumors. In vitro experiments have a stronger antibody-dependent cytotoxicity than ED. At present, it has shown certain effects in the treatment of breast cancer, uterine serous papillary carcinoma, ovarian cancer, and prostate cancer. It is currently being studied in the treatment of breast cancer. more. In their phase II clinical study, Schmidt et al. found that patients who received high doses and high expression of EpCAM had significantly reduced tumor progression ability, and had lower or mild adverse reactions related to treatment. The results showed that the single dose of MT201 in the treatment of advanced breast cancer, although it could not objectively cause the tumor to regress, could slow down the progress of the tumor, which provided theoretical support for the next step in the treatment of MT201 in other tumors that overexpress EpCAM and have low tumor burden. At present, the phase II and phase III clinical studies of other tumors are still in progress, and the prospects are impressive, but there are no reports on gastrointestinal tumors, so its status in the treatment needs to be further studied.

In recent years, in the field of tumor immunotherapy, a new therapy using bispecific antibody (BsAb) to mediate T cells to kill cancer cells has emerged. In the human immune system, cytotoxic T lymphocytes (Cytotoxic T Lymphocytes, CTLs) play an important role. They have T cell receptors (T cell receptors, TCRs), which can specifically recognize and bind cells expressing target antigens, and secrete Perforin and granzymes kill target cells. Due to the lack of immune adhesion molecules and co-stimulatory molecules in tumor cells, the body's immunity cannot be effectively recognized and activated, so that CTL cannot clear cancer cells. BsAb can bind to specific antigens of T cells and tumor cells at the same time, can form an immune synapse between T cells and tumor cells, and play the same function as T cell receptor (TCR), thereby activating the immune mechanism of T cells , which mediates the killing of target cells by T cells.

Catumaxomab (Catumaxomab) is a trifunctional bispecific antibody that can specifically bind to EpCAM molecule, CD3 antigen and FC receptor. It is the first marketed BsAb antibody reported so far. It was approved in the European Union in 2009 for the treatment of patients with malignant ascites with EPCAM-positive tumors, and achieved good results.

Strhlein et al. studied the phase I clinical effect of Catumaxomab perfusion in malignant ascites, and adopted dose-escalating (10-200 μg) intraperitoneal treatment of malignant ascites from the gastrointestinal tract. After 2 years of follow-up, they found that Catumaxomab treatment could remove tumor cells in peritoneal lavage samples, delay Disease progression, prolonged survival. Parsons et al. studied the phase II and III clinical effects of Catumaxomab infusion in cancerous ascites, and observed 258 cases of EpCAM-positive malignant ascites of epithelial origin. The initial observation endpoint was puncture-free survival time, and the control group was allowed to receive Catumaxomab infusion by peritoneal puncture after the observation endpoint. The results showed that there was a statistically significant difference in puncture-free survival time, the symptoms and signs of ascites in the two groups were significantly relieved, the median overall survival time was prolonged, and survival benefited. Treatment after the end point in the control group prolonged the puncture-free time. Overall treatment adverse reactions were usually mild or infrequent and completely reversible. Therefore, intraperitoneal infusion of Catumaxomab in cancerous ascites can prolong puncture-free survival time and puncture-free time, and can significantly reduce the symptoms and signs of ascites.

However, Catumaxomab, a BsAb against CD3 and epithelial cell adhesion molecule (EpCAM), was obtained from the hybridization of rat and mouse antibody sequences and was approved in the European Union in 2009 for use in Treatment of patients with malignant ascites with EpCAM-positive tumors. Another BsAb antibody in phase II clinical research: Solitomab, a mouse bispecific antibody against CD3 and epithelial cell adhesion molecule (EpCAM), is currently under observation. In summary, the antigen-binding region sequences of these two BsAbs are all derived from murine monoclonal antibodies, and long-term application will cause partial graft-versus-host reaction (graft versus host reaction, GVHR). At present, there is no report of a dual-targeting antibody whose antigen-binding region sequence is derived from EpCAM of a human monoclonal antibody. The present invention aims to provide a high-efficiency, low-cost anti-EpCAM and CD3-specific dual-targeting antibody derived from human sources and its preparation method and application.

Contents of the invention

In a first aspect, the present invention provides a specific dual-targeting antibody, which has a first antigen-binding domain that specifically binds to human CD3 and a second antigen that specifically binds to an EpCAM antigen binding domain.

The specific dual-targeting antibody (Bispecific antibody, BsAb) provided by the present invention can simultaneously bind to CD3 and EpCAM antigens; specifically recognize and bind to EpCAM antigen-positive cells, and also bind to T cells expressing CD3, and can be used on T cells Form an immune synapse (immune synapse) with tumor cells, and play the same function as T cell receptor (TCR), thereby activating the immune mechanism of T cells and mediating T cells to play the role of killing EpCAM-positive tumor cells. Thereby eradicating EpCAM-positive tumor cells.

The "dual targeting" in the present invention means "capable of binding to CD3 and EpCAM antigens at the same time".

In one embodiment of the present invention, the specific dual-targeting antibody provided by the present invention can bind to CD3 and EpCAM; the specific dual-targeting antibody is also referred to herein as "anti-CD3/anti-EpCAM specific dual targeting antibodies". The anti-EpCAM portion of the “anti-CD3/anti-EpCAM specific dual targeting antibody” can be used to target EpCAM expressing cells, such as the HepG2 liver cancer cell line, while the anti-CD3 portion of the specific dual targeting antibody molecule can be used to Activate T cells. Promotes directed killing (cytolysis) of targeted EpCAM-positive cells by activated T cells through simultaneous binding to cMET on EpCAM-positive cells and CD3 on T cells.

The anti-CD3/anti-EpCAM specific dual-targeting antibody of the present invention is especially used for diagnosing and treating EpCAM-positive tumor cells, as well as for treating, preventing and/or controlling the abnormal expression of EpCAM and/or the abnormality caused by EpCAM Methods and compositions for expression-induced physiological disturbances. Such disorders include, but are not limited to: cancer, non-cancerous hyperproliferative cellular disorders.

Exemplary specific dual targeting antibodies of the invention are listed in Tables 1 and 2 herein. In the specific dual-targeting antibody provided by the present invention, both the first antigen-binding domain and the second antigen-binding domain include a heavy chain variable region (HCVR) and a light chain variable region (LCVR). Table 1 and Table 2 list the amino acid sequences of HCVR and LCVR of the specific dual-targeting antibody provided by the present invention, and their sequence numbers in the sequence listing. Table 2 lists the nucleotide sequences corresponding to the amino acid sequences encoding HCVR and LCVR described in the present invention, and their sequence numbers in the sequence listing.

The amino acid sequence of the specific dual-targeting antibody provided by the present invention includes: at least one HCVR amino acid sequence of the first antigen-binding domain, at least one LCVR amino acid sequence of the first antigen-binding domain, at least one second antigen-binding domain HCVR amino acid sequence, and at least one LCVR amino acid sequence of the second antigen-binding domain; the specific HCVR and LCVR are preferably any amino acid sequence selected from Table 1.

The nucleotide sequence encoding the specific dual-targeting antibody provided by the present invention includes: at least one HCVR nucleotide sequence of the first antigen-binding domain, at least one LCVR nucleotide sequence of the first antigen-binding domain, at least one The HCVR nucleotide sequence of the second antigen-binding domain, and at least one LCVR nucleotide sequence of the second antigen-binding domain; the specific HCVR and LCVR are preferably any nucleotide sequence selected from Table 2.

Table 1 Amino Acid Sequences in Sequence Listing

In an embodiment of the present invention, three anti-CD3/anti-EpCAM specific dual-targeting antibodies with different sequences are numbered BsAb-1, BsAb-2, and BsAb-3 in Table 1, wherein BsAb-1 It is the specific dual-targeting antibody with the best effect after screening.

In some embodiments of the present invention, each sequence in Table 1 is specifically:

Specifically, among the sequences in Table 1, the hypervariable region is the underlined sequence as follows:

In SEQUENCE NO.1,

CD3.HCVR

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS;

In SEQUENCE NO.2,

CD3.LCVR

DIQLTQSPAIMSASPGKVTMTCRASSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK;

In SEQUENCE NO.3,

EPCAM-HCVR

EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSS;

In SEQUENCE NO.4,

EPCAM-LCVR

DIELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIK;

In SEQUENCE NO.5,

EPCAM-HCVR

EVQLLEQSGAELVRPGTSVRLSCKASGYAFTNYWLGWVKQRPGHGIEWLGDIFPGSGNIRYNEKFKGKATITADKSSSTAYMQLSSLTFEDSAVYFCARIKNWDEPMDYWGQGTTVTVSS;

In SEQUENCE NO.6,

EPCAM-LCVR

DIELVMTQSPSSLTVTMGERVTMSCKSSQSLLNSGNQKNYITWYQQKPGQPPKLLIYWASTRESGVPDKFTGSSSGTDFLTTISSVQAEDLAVYYCQNDYSQPITFGAGTKLEIK;

In SEQUENCE NO.7,

EPCAM-HCVR

EVQLLEQSGAELVKPGSSVKISCKASGYAFTNYWLGWVKQRPGKGIEWIGDIFPGSGNIHYNEKFKGKATLTADKSGSTAYMGLSSLTFEDSAVYFCARLKNWDEPSDYWGQGTTVTVSS;

In SEQUENCE NO.8,

EPCAM-LCVR

DIELVMTQSPSSLTVTAGERVTMSCKGSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDKFTGSGSGTDFTIISSVQAEDLAVYTCQNDTGYPLTFGAGTKLEIK;

Table 2 Nucleotide sequences in the sequence listing

In some embodiments of the present invention, each sequence in Table 2 is specifically:

SEQUENCE NO.9,

CD3.HCVR

GACATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACACCTTTACTAGGTACACCATGCACTGGGTCAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCAGAGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGCGCAAGATATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA；

SEQUENCE NO.10,

CD3.LCVR

GACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTCAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTCATACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA；

In SEQUENCE NO.11,

EPCAM-HCVR

GAGGTGCAGCTGCTCGAGCAGTCTGGAGCTGAGCTGGTGAGGCCTGGGACTTCAGTGAAGATCTCCTGCAAGGCTTCTGGATACGCCTTCACTAACTACTGGCTTGGTTGGGTCAAGCAGAGGCCTGGACATGGACTTGAGTGGATTGGAGATATTTTCCCTGGAAGTGGTAATATCCACTACAATGAGAAGTTCAAGGGCAAAGCCACACTGACTGCAGACAAATCTTCCAGCACAGCCTATATGCAGCTCAGTAGCCTGACATTTGAGGACTCTGCTGTCTATTTCTGCGCAAGACTGAGGAACTGGGACGAGCCTATGGACTACTGGGGCCAAGGGACCACAGTCACCGTCTCCTCC；

In SEQUENCE NO.12,

EPCAM-LCVR

GAGCTCGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGCTTAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCCAGAATGATTATAGTTATCCTCTCACCTTCGGTGCTGGGACCAAACTTGAGATCAAA；

In SEQUENCE NO.13,

GAGGTGCAGCTGCTGGAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCACCAGCGTGCGCCTGAGCTGCAAGGCCAGCGGCTACGCCTTCACCAACTACTGGCTGGGCTGGGTGAAGCAGCGCCCCGGCCACGGCATCGAGTGGCTGGGCGACATCTTCCCCGGCAGCGGCAACATCCGCTACAACGAGAAGTTCAAGGGCAAGGCCACCATCACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCTTCGAGGACAGCGCCGTGTACTTCTGCGCCCGCATCAAGAACTGGGACGAGCCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC；

In SEQUENCE NO.14,

GACATCGAGCTGGTGATGACCCAGAGCCCCAGCAGCCTGACCGTGACCATGGGCGAGCGCGTGACCATGAGCTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACATCACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCAGCACCCGCGAGAGCGGCGTGCCCGACAAGTTCACCGGCAGCAGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCGTGCAGGCCGAGGACCTGGCCGTGTACTACTGCCAGAACGACTACAGCCAGCCCATCACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG；

In SEQUENCE NO.15,

GAGGTGCAGCTGCTGGAGCAGAGCGGCGCCGAGCTGGTGAAGCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCACCAACTACTGGCTGGGCTGGGTGAAGCAGCGCCCCGGCAAGGGCATCGAGTGGATCGGCGACATCTTCCCCGGCAGCGGCAACATCCACTACAACGAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCGGCAGCACCGCCTACATGGGCCTGAGCAGCCTGACCTTCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCTGAAGAACTGGGACGAGCCCAGCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC；

In SEQUENCE NO.16,

GACATCGAGCTGGTGATGACCCAGAGCCCCAGCAGCCTGACCGTGACCGCCGGCGAGCGCGTGACCATGAGCTGCAAGGGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCAGCACCCGCGAGAGCGGCGTGCCCGACAAGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCATCACCATCAGCAGCGTGCAGGCCGAGGACCTGGCCGTGTACACCTGCCAGAACGACACCGGCTACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG；

Specifically, SEQUENCE NO.9-16 encodes the amino acid sequences of SEQUENCE NO.1-8, respectively.

In view of sequence homology, those skilled in the art can understand that the following embodiments should be included in the protection scope of the present invention:

In some embodiments of the present invention, the HCVR of the first antigen-binding domain has at least 90%, at least 95%, or at least 98% of the amino acid sequence of any HCVR in the first antigen-binding domain shown in Table 1. Amino acid sequences that are % or at least 99% homologous.

In some embodiments of the present invention, the LCVR of the first antigen-binding domain is at least 90%, at least 95%, at least 98% identical to any LCVR amino acid sequence in the first antigen-binding domain shown in Table 1. Amino acid sequences that are % or at least 99% homologous.

In some embodiments of the present invention, the HCVR of the second antigen-binding domain has at least 90%, at least 95%, or at least 98% of the amino acid sequence of any HCVR in the first antigen-binding domain shown in Table 1. Amino acid sequences that are % or at least 99% homologous.

In some embodiments of the present invention, the LCVR of the second antigen-binding domain is at least 90%, at least 95%, at least 98% identical to any LCVR amino acid sequence in the first antigen-binding domain shown in Table 1. Amino acid sequences that are % or at least 99% homologous.

In some embodiments of the present invention, the sequence encoding the first antigen-binding domain HCVR is at least 90%, at least 95% identical to any HCVR nucleotide sequence in the first antigen-binding domain shown in Table 2. %, at least 98% or at least 99% homologous nucleotide sequences.

In some embodiments of the present invention, the sequence encoding the first antigen-binding domain LCVR has at least 90%, at least 95% of any LCVR nucleotide sequence in the first antigen-binding domain shown in Table 2. %, at least 98% or at least 99% homologous nucleotide sequences.

In some embodiments of the present invention, the sequence encoding the second antigen-binding domain HCVR is at least 90%, at least 95% identical to any HCVR nucleotide sequence in the first antigen-binding domain shown in Table 2. %, at least 98% or at least 99% homologous nucleotide sequences.

In some embodiments of the present invention, the sequence encoding the second antigen-binding domain LCVR is at least 90%, at least 95% identical to any LCVR nucleotide sequence in the first antigen-binding domain shown in Table 2. %, at least 98% or at least 99% homologous nucleotide sequences.

It is understandable that due to base degeneracy and mutation in the "nucleotide sequence", ordinary technicians in the industry can adjust the types of certain bases. Although the base changes lead to changes in codons, but Amino acid changes that do not cause codon translation, very common, leucine codons have multiple codons, such as: UUA, UUG, CUU.

In addition, the amino acid sequence of the EpCAM antibody described in the present invention is derived from the amino acid sequence of a human antibody, which can reduce host versus graft reaction (HVGR) in human applications. The nucleotide sequence is codon-optimized for expression in mammalian cells.

In some embodiments of the present invention, the amino acid sequence of the specific dual-targeting antibody is: optionally selected from the sequences in Table 1, obtained by a combination of (a) to (h) in the following connection form (" V _L CD3" indicates the LCVR of the first antigen-binding domain of the specific dual-targeting antibody; "V _H CD3" indicates the HCVR of the first antigen-binding domain of the specific dual-targeting antibody; "V _L EpCAM" indicates the specific LCVR of the second antigen-binding domain of a specific dual-targeting antibody; "V _H EpCAM" indicates the HCVR of the second antigen-binding domain of a specific dual-targeting antibody):

(a) _VL EpCAM- _linker1 - _VH EpCAM-linker2-VLCD3- _linker1 -VHCD3,

(b) V _H EpCAM-linker1-V _L EpCAM-linker2-V _L CD3-linker1-V _H CD3,

(c) _VL EpCAM-linker1- _VH EpCAM-linker2- _VHCD3 - _linker1 -VLCD3,

(d) V _H EpCAM-linker1-V _L EpCAM-linker2-V _H CD3-linker1-V _L CD3,

(e) V _L CD3-linker1-V _H CD3-linker2-V _L EpCAM-linker1-V _H EpCAM,

(f) V _L CD3-linker1-V _H CD3-linker2-V _H EpCAM-linker1-V _L EpCAM,

(g) VHCD3- _linker1 -VLCD3-linker2- _VLEpCAM - _linker1 - _VHEpCAM ,

( _h ) VHCD3-linker1-VLCD3-linker2- _VHEpCAM - _linker1 - _VLEpCAM ,

Wherein, the horizontal bar "-" represents the sequence arrangement of each amino acid sequence.

In one embodiment of the present invention, the amino acid sequence of Linker (Linker1) between HCVR (V _H ) and LCVR (V _L ) of the first antigen-binding domain or the second antigen-binding domain is (GGGGS) _n (n =1-4), (GGS) ₄ or (Gly)n (n=6-8), but not limited thereto.

In one embodiment of the present invention, the amino acid sequence of the Linker (Linker2) between the antigen-binding domains is (GGGGS)n (n=1-4), but not limited thereto.

In a preferred embodiment of the present invention, the amino acid sequence of the bispecific single-chain antibody has no less than 90%, 95%, 98% or 99% homology with any of the sequences shown in (a) to (h). amino acid sequence.

In some embodiments of the present invention, the HCVR (V _H ) and LCVR (V _L ) of each antigen-binding domain, and the antigen-binding domains are connected by a linker, and the Linker is a connecting peptide, and the connecting peptide mainly adopts A polypeptide sequence mainly composed of glycine and serine, among which, glycine is the amino acid with the smallest molecular weight and the shortest side chain, which can increase the flexibility of the side chain; serine is the most hydrophilic amino acid, which can increase the hydrophilicity of the peptide chain .

In some embodiments of the present invention, the N segment of the specific dual targeting antibody has the amino acid sequence of the signal peptide of the immunoglobulin κ chain.

The N-terminal of the specific dual-targeting antibody provided by the present invention is added with the secretion signal peptide of the light chain κ chain of immunoglobulin, so as to ensure the expression and secretion of the antibody outside the host cell.

In the second aspect, the present invention also provides a method for preparing the specific dual-targeting antibody described in the first aspect, the method comprising: using the sequence of the specific dual-targeting antibody described in the first aspect to construct an expression The recombinant expression vector of the specific dual-targeting antibody; introduced into host cells; under the condition of expression, the host cells are cultured, expressed, separated and purified to obtain the specific dual-targeted antibody.

In the third aspect, the present invention also provides the application of the specific dual-targeting antibody as described in the first aspect by one or more of the following methods:

(1) applying the specific dual-targeting antibody alone;

(2) Applying the specific dual-targeting antibody in combination with one or more of chemotherapy, radiotherapy, surgery, biological therapy, and immunotherapy;

(3) using in vivo delivery to directly deliver the specific dual-targeting antibody to the patient for treatment;

(4) First, the specific dual-targeting antibody is mixed with the immune effector cells by in vitro transfection technology, and then the immune effector cells mixed with the specific dual-targeting antibody are infused back into the patient for treatment.

In some embodiments of the present invention, in the uses (3) and (4) of the third aspect, the antibody as described above is administered to the patient, and the patient may be administered more than once.

In some embodiments of the present invention, in the application (3) as described in the third aspect, the delivery method is targeted delivery, including but not limited to liposome liposomes (or polysaccharides thereof) that can be delivered in a targeted manner. Polymer) and other industries commonly used carriers for delivery.

By administering the antibody provided in the first aspect of the present invention or the pharmaceutical composition provided in the fourth aspect to mammals including humans, liver cancer or other EpCAM-related diseases, such as diseases related to abnormal or disordered EpCAM expression, can be prevented or treated.

In the fourth aspect, the present invention provides a pharmaceutical composition, including at least one specific dual-targeting antibody as described in the first aspect, and a pharmaceutically acceptable carrier, excipient or diluent.

The pharmaceutical composition provided by the invention can be made into pharmaceutical preparations according to conventional methods. During formulation, the antibody is preferably mixed with or diluted with a carrier, or packed into a carrier in the form of a container. When the carrier acts as a diluent, it can be solid, semi-solid or liquid, used as a vesicle, excipient or medium for the antibody. Thus, the preparations can be tablets, pills, powders, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile solutions for injection, sterile Powder and other forms. Examples of suitable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, hydroxybenzene Methyl formate, propyl paraben, talc, magnesium stearate and mineral oil. The formulations may also include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like.

In some embodiments of the present invention, the pharmaceutical composition further includes a second therapeutic agent, and the second therapeutic agent is any active agent that is advantageously combined with the specific dual-targeting antibody. Including but not limited to other active agents that bind and/or activate CD3 signaling (including other antibodies or antigen-binding fragments thereof, etc.), and/or active agents that do not directly bind to CD3 but activate or stimulate immune cell activation.

In some embodiments of the present invention, the second therapeutic agent includes interfering agents that act synergistically with antibodies, anti-EpCAM monoclonal antibodies, anti-EpCAM polyclonal antibodies, nucleoside analogs, DNA polymerase inhibitors, siRNA preparations, Therapeutic vaccines, cytokines, toxins, chemotherapeutics, radiotherapeutics, hormones, antibody Fc fragments, TLR agonists, CpG containing molecules or immune co-stimulatory molecules used as antiviral agents.

In the fifth aspect, the present invention provides a diagnostic reagent, including at least one specific dual-targeting antibody as described in the first aspect, and a diagnostic agent, including but not limited to fluorophores, chromophores , dyes, radioisotopes, chemiluminescent molecules, paramagnetic ions, or spin-trapping reagents.

In the sixth aspect, the present invention provides an antibody drug, which is any specific dual-targeting antibody as described in the first aspect, which binds to human CD3 and induces the proliferation of human T cells in vitro.

In the seventh aspect, the present invention provides an antibody drug, which is any specific dual-targeting antibody as described in the first aspect, which binds to human CD3 and induces T cell-mediated killing of EpCAM-positive cells in vitro.

In the eighth aspect, the present invention provides a nucleic acid molecule encoding the amino acid sequence of the specific dual-targeting antibody described in the first aspect; in some embodiments, the amino acid sequence encoded by the nucleic acid molecule is the same as The amino acid sequence of the specific dual targeting antibody according to the first aspect has an amino acid sequence of at least 90%, at least 95%, at least 98% or at least 99% homology.

In the ninth aspect, the present invention provides a recombinant vector expressing the specific dual-targeting antibody as described in the first aspect; in some embodiments, the recombinant vector has any of the Nucleic acid molecule sequence.

In some embodiments of the present invention, the vector is a minicircle DNA recombination master plasmid or a minicircle DNA.

In some embodiments of the present invention, the minicircle DNA recombination master plasmid is formed by inserting any nucleic acid molecular sequence provided in the eighth aspect of the present invention into the multiple cloning site of the minicircle DNA empty plasmid. The specific insertion method can be conventional gene cloning or seamless cloning.

In some embodiments of the present invention, the empty minicircle DNA plasmid is preferably p2ФC31 plasmid or pMC.BESPX plasmid. Specifically, the construction method of the empty plasmid p2ФC31 refers to Chen ZY et al., Molecular Therapy, 8(3), 495-500 (2003), Chen ZY et al., HumanGene Therapy, 16(1), 126-131 (2005) and the US Patent US7897380 B2. Specifically, the construction method of the empty plasmid pMC.BESPX and the complete gene sequence refer to Chen ZY et al., Nature Biotechnology, 28, (12), 1289-1291 (2010).

The p2ФC31 plasmid and pMC.BESPX plasmid used in the typical embodiments of the present invention are respectively obtained from the p2ФC31 recombinant mother plasmid and pMC.BESPX recombinant mother plasmid. nucleotide sequence, but there is no nucleotide sequence encoding ФC31 recombinase and I-Sce1 endonuclease on the pMC.BESPX vector, so the minicircle DNA master plasmid prepared by pMC.BESPX vector is more high-quality, greatly reducing the number of recombinase and I-Sce1 endonuclease Contamination of the nucleotide sequence of the endonuclease; but pMC.BESPX needs to be matched with E. coli ZYCY10P3S2T engineering bacteria, ZYCY10P3S2T engineering bacteria have the function of encoding ФC31 recombinase and I-Sce1 endonuclease, without ФC31 recombinase ( That is, the pMC.BESPX recombination mother plasmid of phiC31 group enzyme) and I-Sce1 endonuclease nucleotide coding sequence needs to be matched with ZYCY10P3S2T engineering bacteria to produce site-specific recombination in vivo (and E. coli TOP 10 does not have this function ) and finally produce microcircle DNA; correspondingly, the p2ФC31 recombination parent plasmid can be used in conjunction with TOP 10 to produce site-specific recombination in vivo, and finally produce microcircle DNA.

In some embodiments of the present invention, the minicircle DNA is produced by site-specific recombination of specific recombination sites using the minicircle DNA recombination master plasmid provided by the present invention. The microcircle DNA recombination master plasmid provided by the present invention has specific recombination sites, and the "minicircle DNA recombination master plasmid" generates microcircle DNA and backbone DNA sequences through site-specific recombination of specific recombination sites. The method of producing minicircle DNA from the p2ФC31 recombinant mother plasmid refers to Chen ZY et al., Molecular Therapy, 8(3), 495-500 (2003), Chen ZY et al., Human Gene Therapy, 16(1), 126-131(2005) and US Patent US7897380 B2. The method of producing minicircle DNA from pMC.BESPX recombinant mother plasmid refers to Chen ZY et al., Nature Biotechnology, 28, (12), 1289-1291 (2010).

As used herein, "backbone DNA sequence" has DNA sequences in standard plasmids that are responsible for the replication of bacterial plasmids, or selection of hosts containing plasmids, such as bacterial replication sequences, resistance genes, unmethylated CpG motifs, and the like.

In some embodiments of the present invention, the recombinant vector also includes a gene expression system that expresses all the necessary elements required for the expression of the target polypeptide (specific dual-targeting antibody in the present invention), which generally includes the following elements: promoter, A gene sequence encoding a polypeptide, a terminator; in addition, it may optionally include a signal peptide coding sequence, etc.; these elements are operably linked.

As used herein, the "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, thus, the promoter region is "operably linked" to the nucleic acid sequence. Preferably, the signal peptide is an immunoglobulin κ chain signal peptide. Preferably, the tag is at least one of His tag, GST tag, c-myc tag and Flag tag. The above types of signal peptides and tags are preferred by the inventors, and those skilled in the art can select appropriate signal peptides and tags according to specific needs.

In the tenth aspect, the present invention provides a host cell (preferably a eukaryotic cell or a mammalian cell including a human) having the vector as described in the ninth aspect; it also provides the introduction of the vector as described in the ninth aspect A method in a host cell; also provided is a method by culturing a host cell with the vector under conditions allowing antibody production, and isolating the antibody produced.

In the eleventh aspect, the present invention provides a detection kit, comprising a solid detection support, and the solid detection support comprises at least one specific dual-targeting antibody as described in the first aspect. In some embodiments, the solid support is a solid surface that can attach macromolecules such as antibodies, proteins, polypeptides, peptides, polynucleotides, such as magnetic beads, latex beads, microtiter plate wells, glass plates, nylon, agar Sugar, polyacrylamide, silica particles, nitrocellulose membrane, etc.

In a twelfth aspect, the present invention provides a method for detecting abnormal cells in a sample to be tested, which comprises contacting the sample with at least one specific dual-targeting antibody as described in the first aspect. In some embodiments, the sample is from the liver or a resected tumor bed.

As used herein, an "abnormal cell" is any cell that has characteristics that are atypical for that cell type, including atypical growth, typical growth at an atypical location, or typical effects on atypical targets. Such cells include cancer cells, benign hyperplastic or dysplastic cells, inflammatory cells or autoimmune cells.

In some embodiments of the present invention, the donated tissues or organs before transplantation are screened by the method as described in the twelfth aspect, so as to provide tissues or organs substantially free of EpCAM.

In some embodiments of the present invention, the blood supply is screened using the method described in the twelfth aspect to provide a blood supply substantially free of EpCAM.

In the thirteenth aspect, the application of the specific dual-targeting antibody as described in the first aspect or the recombinant vector as described in the ninth aspect in the preparation of reagents or drugs for diagnosis, prevention, and treatment of EpCAM-related diseases.

As described in the present invention, the "EpCAM disease" includes but not limited to diseases related to EpCAM abnormal expression or disorder, for example, EpCAM antigen-positive liver cancer.

The technical solution provided by the present invention is to design a group of specific dual-targeting antibodies, and insert the antibody expression cassette (DNA sequence) into a minicircle DNA vector (preferably Kay MA*, He CY and Chen ZY* (*corresponding author) .A Robust system for production of minicircleDNA vectors. Nat Biotechnology, 28:1287, 2010). The antibody expressed by the microcircle DNA has the following characteristics: 1) specifically binding to the EpCAM positive antigen; 2) specifically binding to the T cell; 3) mediating the killing effect of the T cell on the EpCAM antigen positive cell. In addition, in some embodiments, since the antibody expression cassette has a coding sequence for a secretion signal peptide, the antibody can be secreted extracellularly and maintain biological activity after expression.

Description of drawings

Fig. 1 is the plasmid map of the minicircle master plasmid prepared in Example 1 of the present invention;

Fig. 2 is the plasmid map of the microcircle DNA prepared in Example 2 of the present invention;

Fig. 3 is the result of the expression level of BsAb antibody in the medium supernatant detected by Western Blot provided by Example 3 of the present invention;

Figures 4-5 are the results of flow cytometry detection of the EpCAM×CD3 BsAb cell binding experiment provided in Example 4 of the present invention;

Fig. 6 The results of the EpCAM×CD3 BsAb combined with DCIK cell killing experiment provided in Example 5 of the present invention.

detailed description

The following description is a preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also considered Be the protection scope of the present invention.

Unless otherwise specified in the examples of the present invention, all reagents and consumables used are commercially available.

Example 1

The construction of the minicircle parent plasmid with the nucleotide sequence encoding EpCAM×CD3 BsAb antibody comprises the following steps:

1) Design BsAb antibody gene expression cassette

In addition to the nucleotide sequence encoding BsAb, the BsAb antibody gene expression cassette of Example 1 of the present invention also includes a coding sequence for a promoter, a secretion signal peptide, a protein purification tag, and a polyA tailing signal.

In a specific embodiment, the BsAb antibody gene expression cassette of Example 1 of the present invention includes sequentially connected:

Nucleotide sequence encoding immunoglobulin κ chain signal peptide (Murine Ig kapa-chain signal peptide, SEQ ID NO: 17)-gene sequence of BsAb antibody (V _L EpCAM-linker1-V _H EpCAM-linker2-V _H CD3 -linker1-V _L CD3; Concrete nucleotide sequence corresponds to the nucleotide sequence (SEQ ID NO: 18) - double stop codon (TAGTGA); wherein,

The nucleotide sequence encoding the signal peptide of the immunoglobulin κ chain is shown in SEQ ID NO:17.

The nucleotide sequence encoding the histidine 6-His tag is shown in SEQ ID NO:18.

The BsAb antibody gene expression cassette of Example 1 of the present invention also includes the nucleotide sequence of the cytomegalovirus CMV promoter, which is located in the N segment of the Signalpeptide secretion signal peptide; the sequence is SEQ ID NO:19.

The BsAb antibody gene expression cassette of Example 1 of the present invention also includes the nucleotide sequence of bovine growth hormone polynucleotide poly A (BGH poly A), which is located at the C-terminus of the stop codon (TGATAG); the sequence is SEQ ID NO :20.

In order to enhance the expression efficiency of the microcircle DNA vector, an element chimeric intron (between the CMV promoter and the Signal peptide secretion signal peptide; reported in the literature, can be enhanced by 10-20 times on various cells) was added, and the sequence is SEQ ID NO: 21.

In the BsAb antibody gene expression cassette of Example 1 of the present invention, linker1 is (GGGGS) ₃ , and the nucleotide sequence is the nucleotide sequence shown in SEQUENCE NO.22.

Linker2 is GGGGS, and its nucleotide sequence is the nucleotide sequence shown in SEQUENCE NO.23.

Linker3 is (GGGGS) ₃ , and its nucleotide sequence is the nucleotide sequence shown in SEQUENCE NO.22.

Specifically, the sequences are as follows:

In SEQUENCE NO.17,

Murine Ig kapa-chain signal peptide

ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT;

In SEQUENCE NO.18,

6-His

CATCATCACCATCATCAT;

In SEQUENCE NO.19,

CMV promoter

GTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGGTCGTTTAGTGAACCGTCAGATCACTAGTAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCT；

In SEQUENCE NO.20,

BGH poly A

GGTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGTGGGGCAGGACAGCAAGGGGGAGGACTGGGAAGAGGAATAGCAGGCATGCTGGG

In SEQUENCE NO.21,

chimeric intron

AACGCAGTCAGTGCTCGACTGATCACAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGGCCAATAGAAACTGGGCTTGTCGAGACAGAGAAGATTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGGGTACCGAAGCCGCTAGCGCTACCGGTC;

In SEQUENCE NO.22,

Linker1

GGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCT;

In SEQUENCE NO.23,

Linker2

GGAGGTGGTGGATCC;

2) Insert the BsAb antibody gene expression cassette between the attB and attP sites of p2ФC31 empty vector or pMC.BESXP empty vector, and the specific steps include any one of A) or B):

A)

For specific steps, refer to the description of the PCT application (application number: PCT/CN2014/083741) submitted by the inventor in 2014:

Example 1 "A method for constructing a minicircle DNA recombination parent plasmid p2ФC31.Bab containing a genetically engineered antibody gene expression cassette", to obtain the p2ФC31 microcircle with the nucleotide sequence encoding the EpCAM×CD3 BsAb antibody in Example 2 of the present invention ring parent plasmid; and

Steps of Example 6 "A Method for Constructing a Minicircular DNA Recombination Mother Plasmid pMC.Bab Containing a Genetic Engineering Antibody Gene Expression Cassette"; to obtain the nucleotide sequence encoding the EpCAM×CD3 BsAb antibody in Example 2 of the present invention pMC.BESXP minicircle master plasmid.

The only difference between step 1-2) of the present invention and the above PCT application is that step 1-2) of embodiment 2 of the present invention replaces the "gene sequence of BiTE" in embodiment 1 or 6 of the above PCT application with the embodiment of the present invention "Gene sequence of EpCAM×CD3 BsAb" of 2.

B)

Directly adopt the method of enzyme digestion-ligation, design two restriction sites of SmaI-ApaI at both ends of the BsAb antibody gene expression cassette and pMC.BESXP empty vector or p2ФC31 empty vector, and synthesize the BsAb antibody gene expression cassette by enzyme digestion- Insert between the attB and attP sites of the p2ФC31 empty vector or the pMC.BESXP empty vector by ligation; respectively obtain the p2ФC31 minicircle master plasmid or pMC with the nucleotide sequence encoding the EpCAM×CD3 BsAb antibody in Example 1 of the present invention. BESXP minicircle parent plasmid.

The plasmid map of the pMC.BESXP minicircle master plasmid prepared in Example 1 of the present invention is shown in Figure 1 (named p-EpCAM×CD3).

Example 2

Construction of Microcircle DNA with Nucleotide Sequence Encoding EpCAM×CD3 BsAb Antibody

Referring to the description of the PCT application (application number: PCT/CN2014/083741) submitted by the inventor in 2014:

Example 2 and Example 7 "A method for preparing a microcircle DNA containing a genetically engineered antibody gene expression cassette", respectively to obtain the microcircle DNA having the nucleotide sequence encoding the EpCAM×CD3 BsAb antibody in Example 2 of the present invention .

The difference between the present invention and the above-mentioned PCT application example 2 and example 7 is only that, in the present invention example 3, the minicircle master plasmid in the above-mentioned PCT application example 2 or 7 is replaced by the minicircle provided by the present invention example 1 parent plasmid.

The microring pattern prepared in Example 2 of the present invention is shown in FIG. 2 .

Example 3

The expression and purification of BsAb antibody comprises the following steps:

(1) Expression of EpCAM×CD3 BsAb antibody in HEK 293T cells

X-treme GENEHP DNA Transfection Reagent Plasmid Transfection Kit (Roche Company) was used to transfect HEK 293T cells with the microcircle DNA of Example 3 (the microcircle DNA prepared from the microcircle master plasmid shown in Figure 2), and the HEK 293T cells were transfected with After 72 hours of transfection with the EpCAM BsAb microcircle carrier, Western Blot was used to detect the expression level of BsAb antibody in the culture supernatant and cytoplasm. As shown in Figure 3, both BsAb-1 and BsAb-2 were expressed in the cytoplasm, but only BsAb-1 was Culture supernatant (secretory expression).

(2) Purification of EpCAM BsAb antibody

The medium-scale transfection of the secreted and expressed antibody BsAb-1 into HEK 293T cells was performed, and the culture supernatant was collected for low-temperature ultracentrifugation. The supernatant was collected and then purified by His-Tag affinity resin (Omplete His-Tag Purification Resin, Roche ), the purified antibody was tested for protein concentration by ELISA method; the purified protein concentration reached 0.6mg/ml, and stored at -80°C for long-term storage for use in flow cytometry detection.

Example 4

The binding activity of EpCAM×CD3 BsAb-1 antibody to K562, HepG2, and Jurkat cells was detected by flow cytometry, including the following steps:

a) Cell culture: K562 (DMEM medium+10% FBS culture; suspension growth, no EpCAM expression cell line; negative control), HepG2 (DMEM medium+10% FBS culture; EpCAM positive liver cancer cell line), Jurkat cells (1640medium +10% FBS culture; suspension growth, CD3 positive cell line);

b) Digest HepG2 with trypsin, add serum-containing medium for neutralization, and then centrifuge and discard the supernatant to obtain cells; Jurkat is for suspension cells and directly centrifuges to collect cells;

c) Wash the tears twice with pre-cooled PBS, centrifuge at 200g for 5min, collect the cells, and count them respectively;

d) Evenly distribute 1×10 ⁵ samples per experimental group, grouped as follows:

K562, Group 2: blank group (Blank, supernatant after pMC.BESXP empty vector transfection of HEK 293T cells; pre-prepared); EpCAM BsAb group (purified EpCAM BsAb antibody obtained in Example 3; pre-prepared)

HepG2, 2 groups: grouping as above, blank group & EpCAM BsAb group (the purified EpCAM BsAb antibody obtained in Example 3; pre-prepared)

Jurkat, 2 groups: grouping as above, blank group & EpCAM BsAb group (the purified EpCAM BsAb antibody obtained in Example 3; pre-prepared)

e) Add 100 μl of the above supernatant/group and incubate on ice for 30 minutes;

f) Add 1ml of pre-cooled PBS to wash, centrifuge at 200g for 5min, collect the cells, add 100μl of pre-cooled and diluted APC-mouse anti-6His antibody in PBS (1:1000 dilution), incubate on ice for 30min,

g) Add 1 ml of pre-cooled PBS to wash, centrifuge at 200 g for 5 min, collect cells, and resuspend with 200 μl of PBS. Detect with a flow cytometer (BDC6) to observe the binding situation.

Figure 4 is the result of flow cytometry detection of BsAb binding to CD3 positive cells (Jurkat); Figure 5 is the result of flow cytometry detection of BsAb binding to EpCAM antigen positive cells (HepG2). In Figure 4 and Figure 5, curve C is the blank group; curve B is the BsAb experimental group. It can be seen from the figure that BsAb-1 has the function of binding to the relevant antigen on the cell surface.

Example 5

Method of EpCAM×CD3 BsAb-1 Antibody Combined with DCIK Cells to Kill HepG2 Cells

In this example, the target cell (T) is HepG2 cell; the effector cell (E) is DCIK cell (dendritic cell activated and cytokine induced killer cell)

step:

a) Seed the target cells (HepG2) in a 96-well plate one day in advance, and the inoculum amount of the cells is 2×10 ⁴ /well; set up groups at the same time (Table 3 below; 3 replicate wells for each group):

Table 3 Different groups

b) Replace with fresh opti-DMEM medium the next day, 100 μL/well;

c) Count DCIK (E) effector cells and resuspend in opti-DMEM medium. According to the ratio of T:E=1:8, add corresponding amount of DCIK (1.6×10 ⁵ /well, 100μL/well) according to the grouping in a). );

d) According to the grouping, add 10 μl/well of purified BsAb antibody (concentrations are 0.02, 0.04, 0.06 μg/μL) to the corresponding well, and mix well;

e) Incubate at 37°C for 6 hours in a cell incubator;

f) Add CCK-8 (Dojindo, Japan) reagent (20 μl/well), incubate at 37° C. for 2 hours in a cell incubator, and measure the absorbance at 450 nm;

g) According to the instructions of the CCK-8 kit (Dojindo, Japan), count the number of living cells and calculate the killing efficiency:

Kill rate = [((T+E)-T&E)/T]×100%

Among them, T represents the number of living target cells; E represents the number of living effector cells; then T+E is equal to the total number of living target cells and effector cells; T&E represents the number of living cells after the target cells are killed by effector cells.

The results of EpCAM×CD3 BsAb combined with DCIK cell killing experiment are shown in FIG. 6 .

In Fig. 6, the vertical axis represents the relative value of target cell death rate; the horizontal axis represents different groups.

It can be seen from Figure 6 that BsAb 0.02, BsAb 0.04, and BsAb 0.06 correspond to the settings of BsAb antibody concentrations of 0.02, 0.04, and 0.06 μg/μl in Table 3-Group 4 respectively; DCIK is the result of Table 3-Group 3; thus It can be seen that simply adding effector cells DCIK has a poor killing effect on target cells, which is only about 28%; while adding DCIK and BsAb at the same time in group 4, the killing effects on target cells can reach 62%, 85% and 91%, respectively, and with time The higher the concentration of BsAb antibody, the better the killing effect.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (10)

1. A specific dual-targeting antibody, characterized in that, the specific dual-targeting antibody has a first antigen-binding domain that specifically binds to human CD3 and a second antigen-binding structure that specifically binds to an EpCAM antigen area. 2. The specific dual-targeting antibody according to claim 1, wherein the specific dual-targeting antibody comprises at least one HCVR amino acid sequence of the first antigen-binding domain, at least one first antigen-binding domain LCVR amino acid sequence, at least one HCVR amino acid sequence of the second antigen-binding domain, and at least one LCVR amino acid sequence of the second antigen-binding domain; wherein, the HCVR and LCVR are optional HCVR and LCVR amino acid sequence. 3. A specific dual targeting antibody is applied according to one or more of the following methods: (1) applying the specific dual-targeting antibody alone; (2) Applying the specific dual-targeting antibody in combination with one or more of chemotherapy, radiotherapy, surgery, biological therapy, and immunotherapy; (3) using in vivo delivery to directly deliver the specific dual-targeting antibody to the patient for treatment; (4) First, the specific dual-targeting antibody is mixed with the immune effector cells by in vitro transfection technology, and then the immune effector cells mixed with the specific dual-targeting antibody are infused back into the patient for treatment. 4. A pharmaceutical composition, comprising at least one specific dual-targeting antibody according to claim 1, and a diagnostic agent, a pharmaceutically acceptable carrier, excipient or diluent. 5. A nucleic acid molecule, characterized in that it encodes the amino acid sequence of the specific dual-targeting antibody according to claim 1. 6. A recombinant vector, characterized in that it has any nucleic acid molecule sequence as claimed in claim 7. 7. The recombinant vector according to claim 6, characterized in that, the recombinant vector is an expression vector or a minicircle DNA. 8. A host cell, comprising the recombinant vector according to claim 6. 9. An antibody drug, characterized in that it is any specific dual-targeting antibody as claimed in claim 1, which binds to human CD3 and induces T cell-mediated killing of EpCAM-positive cells in vitro. 10. The application of the specific dual targeting antibody as claimed in claim 1 or the recombinant vector as claimed in claim 6 in the preparation of reagents or medicines for diagnosis, prevention and treatment of EpCAM diseases.