CN107556388A - The double targeting antibodies of anti-CD44v6 and CD3 specificity, the minicircle dna containing this pair of targeting antibodies expression cassette and application - Google Patents

Abstract

The invention provides the double targeting antibodies of a species specificity and its methods and applications.The first antigen-binding domains and specificity and the second antigen-binding domains of CD44v6 antigen bindings that there is the double targeting antibodies of specificity specificity to be combined with mankind CD3, the tumour cell that can specifically identify and be overexpressed with reference to CD44v6 antigens and CD44v6, including positive tumor disease of liver cancer and other CD44v6 etc., while it can also combine φt cell receptor；And mediate T cell plays lethal effect, so as to remove CD44v6 infection, there is very big application value in diagnosis and treatment, prevention, detection CD44v6 relevant diseases.Present invention also offers a kind of minicircle dna for the nucleotide sequence for having and encoding the double targeting antibodies of the specificity.The invention provides the double targeting antibodies of specificity and minicircle dna can be used for preparing antibody pharmaceutical compositions or CD44v6 positive tumor cell diagnostic reagents.

Description

Anti-CD44v6 and CD3 specific dual targeting antibody, containing the dual targeting antibody expression cassette Microcircle DNA and its application

technical field

The invention relates to the fields of biomedicine and diagnosis and treatment, in particular to an anti-CD44v6 and CD3 specific dual-targeting antibody, a microcircle DNA containing the expression cassette of the dual-targeting antibody, and applications.

Background technique

CD44 molecule is an integral membrane protein on the cell surface and belongs to the adhesion molecule family. Its normal function is to act as a receptor to recognize hyaluronic acid (HA) and collagen I and IV, and is mainly involved in immune recognition, lymphocyte aggregation and cell-cell, cell - Specific adhesion process between substrates. It can be divided into standard type (CD44s) and splice variant type (CD44v). Currently, it is believed that CD44 splicing variant 6 (CD44v6) is closely related to the degree of tumor progression, invasion, and metastasis. It is mainly involved in the heterogeneous adhesion of tumor cells to host cells and host matrix, enabling tumor cells to infiltrate or metastasize from the adhesion to surrounding cells, and to promote the invasion and metastasis of tumor cells, that is, to directly participate in tumor progression and transfer process. In addition, by strengthening the activation of vascular signaling pathways such as c-Met and VEGFR-2 in tumor cells, it can promote the formation of new blood vessels in tumor tissue, thereby promoting the growth of tumor tissue. However, its expression level in normal tissues is very low, and compared with EGFR and other tumor-associated antigens, the average amount of CD44v6 protein on the surface of tumor cells is more. Therefore, it is an ideal molecular target for tumor diagnosis and targeted therapy.

In recent years, in the field of tumor immunotherapy, the use of bispecific antibodies (BsAb) to mediate the important role of T cells has emerged, with T cell receptors (T cell receptors, TCR), which can specifically recognize and bind to expressed Target antigen cells, and secrete perforin and granzymes to 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.

A CD44v6 antibody, namely cMAb U36, is currently reported, which was developed by the Free University Hospital in the Netherlands. It is a human-mouse chimeric antibody and is currently used in combination with ¹²⁵ I and other radioactive isotope labeling for the treatment of metastatic squamous cell carcinoma of the head and neck ( head and neck squamous cell carcinoma, HNSCC), currently in pre-clinical trials. The antibody can only bind to CD44v6 antigen and CD44v6-positive cells, but cannot bind to T cells and cannot mediate T cell killing; it can be targeted and killed by ADCC (antibody-dependent cell-mediated cytotoxicity) or CDC (complement-dependent cytotoxicity) CD44v6 antigen-positive tumor cells can kill tumors through labeled ¹²⁵ I and other radioactive isotopes, because its mouse origin has not been completely eliminated, and there will be host-versus-graft reactions when used for human treatment. The main related papers (British Journal of Cancer (1997) 75, 1049–1060.).

Another CD44v6 antibody, namely AbD15179, was developed by Uppsala University in Sweden. It is a fully human Fab fragment obtained through phage library technology screening. It is currently used in combination with ¹²⁵ I and other radioactive isotope labeling to treat metastatic head and neck Squamous cell carcinoma (head and neck squamous cell carcinoma, HNSCC) is currently in preclinical trials. The antibody can only bind to CD44v6 antigen and CD44v6-positive cells, but cannot bind to T cells, and cannot mediate T cell killing; it cannot yet be targeted by ADCC (antibody-dependent cell-mediated cytotoxicity) or CDC (complement-dependent cytotoxicity). CD44v6 antigen-positive tumor cells can only be killed by labeled radioactive isotopes such as ¹²⁵ I. Main related paper (Haylock et al. EJNMMI Research 2014, 4:11).

Another CD44v6 antibody, the humanized monoclonal antibody Bivatuzumab (also known as BIWA 4), was developed by Boehringer-Ingelheim and has been humanized to reduce its immunogenicity. It is currently used to treat metastatic head and neck squamous cell carcinoma (HNSCC), in phase I clinical trials. The antibody can only bind to CD44v6 antigen and CD44v6-positive cells, it cannot bind to T cells, and cannot mediate T cell killing; it can only be targeted and killed by ADCC (antibody-dependent cell-mediated cytotoxicity) or CDC (complement-dependent cytotoxicity) CD44v6 antigen-positive tumor cells, main related papers (Clin Cancer Res. 2006 Oct 15; 12(20Pt 1): 6064-72.).

In addition, there is no approved CD44v6×CD3 dual-targeting antibody on the market. The present invention aims to provide a high-efficiency, low-cost, anti-CD44v6 and CD3 specific dual-targeting antibody and its preparation method and application.

Contents of the invention

In the first aspect, the present invention provides a specific dual-targeting antibody, the specific dual-targeting antibody has a first antigen-binding domain that specifically binds to human CD3 and a second antigen that specifically binds to the CD44v6 antigen binding domain.

The specific dual-targeting antibody (Bispecific antibody, BsAb) provided by the present invention can simultaneously bind to CD3 and CD44v6 antigens; specifically recognize and bind to CD44v6 antigen-positive cells, and also bind to T cells expressing CD3, and can be used on T cells Form immune synapse (immune synapse) with tumor cells, play the same function as T cell receptor (TCR), thereby activating T cell immune mechanism, mediating T cells to play the role of killing CD44v6 positive tumor cells, thereby Eliminate CD44v6 positive tumor cells.

"Dual targeting" in the present invention means "capable of binding to CD3 and CD44v6 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 CD44v6; the specific dual-targeting antibody is also referred to herein as "anti-CD3/anti-CD44v6 specific dual targeting antibodies". The anti-CD44v6 part of the "anti-CD3/anti-CD44v6 specific dual targeting antibody" can be used to target CD44v6 expressing cells, such as the SMMC-7721 liver cancer cell line, while the anti-CD3 part of the specific dual targeting antibody molecule Can be used to activate T cells. Promotes directed killing (cytolysis) of targeted CD44v6-positive cells by activated T cells through simultaneous binding to CD44v6 on CD44v6-positive cells and CD3 on T cells.

The anti-CD3/anti-CD44v6 specific dual targeting antibody of the present invention is especially used for diagnosis and treatment of CD44v6 positive tumor cells, as well as treatment, prevention and/or control of abnormal expression of CD44v6 and/or abnormality caused by CD44v6 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 lists 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.

Amino Acid Sequences in Table 1 Sequence Listing

In an embodiment of the present invention, three anti-CD3×anti-CD44v6 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:

In SEQUENCE NO.1,

CD3.HCVR

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGRVNPNRRGTTYNQKFEGRVTMTTDTSTSTAYMELRRSLRSDDTAVYYCARANWLDYWGQGTTVTVSS;

In SEQUENCE NO.2,

CD3.LCVR

DIQMTQSPSSLSASVGDRVTITCSVSSSVSSIYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQVYSGYPLTFGGGTKVEIK;

In SEQUENCE NO.3,

CD44V6-HCVR

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSS;

In SEQUENCE NO.4,

CD44V6-LCVR

EIVLTQSPATLSSLSPGERATLSCGSASGSINYIYWLQQKPGQAPRILIYLTSNLASGVPAKFSGSGSGTDFLTIGSLEPEDFAVYYCLQWSGNPLTFGSGTKVEIK;

In SEQUENCE NO.5,

CD44V6-HCVR

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSS;

In SEQUENCE NO.6,

CD44V6-LCVR

EIVLTQSPATLSSLSPGERATLSCGSASGSINYIYWLQQKPGQAPRILIYLTSNLASGVPAKFSGSGSGTDFLTIGSLEPEDFAVYYCLQWSGNPLTFGSGTKVEIK;

In SEQUENCE NO.7,

CD44V6-HCVR

EVQLVESGSGLVKPGGSLKLSCAASGFTFSSYDMSWVRQAPGKSLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNGLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSS;

In SEQUENCE NO.8,

CD44V6-LCVR

EIVLTQGPATLGISPGEKATLSCGGASGSINYIYWLQQKPGQAPRILIYLTSNLASSVPARFSGSGSGTDFTLTLGSLEPEDFAVYYCLQWGSNPLTFGSGTKVEIK;

Nucleotide sequences in the sequence listing of Table 2

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

SEQUENCE NO.9,

CD3.HCVR

CAGGTTCAGCTGGTGCAGTCTGGTGCTGAGGTGAAGAAGCCTGGTGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACATTCACTGACTACTACATGCACTGGGTGAGACAGGCCCCTGGTCAAGGTCTTGAGTGGATGGGTAGAGTTAATCCTAACCGGAGGGGTACTACCTACAACCAGAAATTCGAGGGCCGTGTCACCATGACCACAGACACATCCACCAGCACAGCCTACATGGAGCTGCGTAGCCTGCGTTCTGACGACACCGCCGTGTATTACTGCGCCCGTGCTAACTGGCTTGACTACTGGGGCCAGGGCACCACCGTCACCGTCTCCTCC；

SEQUENCE NO.10,

CD3.LCVR

CAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCAGTGTCAGCTCAAGTGTTTCCTCCATTTACTTGCACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGCACATCCAACTTGGCTTCTGGAGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGCCAAGTCTACAGTGGTTACCCTCTCACCTTCGGCGGAGGGACCAAGGTGGAGATCAAA；

In SEQUENCE NO.11,

CD44V6-HCVR

GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCACCATCAGCAGCGGCGGCAGCTACACCTACTACCTGGACAGCATCAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCCAGGGCCTGGACTACTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGC；

In SEQUENCE NO.12,

CD44V6-LCVR

GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCAGCAGCGCCAGCAGCAGCATCAACTACATCTACTGGCTGCAGCAGAAGCCCGGCCAGGCCCCCCGCATCCTGATCTACCTGACCAGCAACCTGGCCAGCGGCGTGCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCTGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAG；

In SEQUENCE NO.13,

CD44V6-HCVR

GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCACCATCAGCAGCGGCGGCAGCTACACCTACTACCTGGACAGCATCAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCCAGGGCCTGGACTACTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGC；

In SEQUENCE NO.14,

CD44V6-LCVR

GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCGGCAGCGCCAGCGGCAGCATCAACTACATCTACTGGCTGCAGCAGAAGCCCGGCCAGGCCCCCCGCATCCTGATCTACCTGACCAGCAACCTGGCCAGCGGCGTGCCCGCCAAGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCGGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCTGCAGTGGAGCGGCAACCCCCTGACCTTCGGCAGCGGCACCAAGGTGGAGATCAAG；

In SEQUENCE NO.15,

CD44V6-HCVR

GAGGTGCAGCTGGTGGAGAGCGGCAGCGGCCTGGTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGCGCCAGGCCCCCGGCAAGAGCCTGGAGTGGGTGAGCACCATCAGCAGCGGCGGCAGCTACACCTACTACCTGGACAGCATCAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACGGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCCAGGGCCTGGACTACTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGC；

In SEQUENCE NO.16,

CD44V6-LCVR

GAGATCGTGCTGACCCAGGGCCCCGCCACCCTGGGCATCAGCCCCGGCGAGAAGGCCACCCTGAGCTGCGGCGGCGCCAGCGGCAGCATCAACTACATCTACTGGCTGCAGCAGAAGCCCGGCCAGGCCCCCCGCATCCTGATCTACCTGACCAGCAACCTGGCCAGCAGCGTGCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCCTGGGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCTGCAGTGGGGCAGCAACCCCCTGACCTTCGGCAGCGGCACCAAGGTGGAGATCAAG；

Specifically, SEQUENCE NO.9-16 correspond to the amino acid sequences encoding 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 CD44V6 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 future 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 CD44v6" indicates the specific LCVR of the second antigen-binding domain of the specific dual-targeting antibody; "V _H CD44v6" indicates the HCVR of the second antigen-binding domain of the specific dual-targeting antibody):

(a) V _L CD44v6-linker1-V _H CD44v6-linker2-V _L CD3-linker3-V _H CD3,

(b) _VHCD44v6 - _linker1 - _VLCD44v6 -linker2-VLCD3- _linker3 -VHCD3,

(c) V _L CD44v6-linker1-V _H CD44v6-linker2-V _H CD3-linker3-V _L CD3,

(d) V _H CD44v6-linker1-V _L CD44v6-linker2-V _H CD3-linker3-V _L CD3,

(e) V _L CD3-linker1-V _H CD3-linker2-V _L CD44v6-linker3-V _H CD44v6,

(f) V _L CD3-linker1-V _H CD3-linker2-V _H CD44v6-linker3-V _L CD44v6,

(g) VHCD3- _linker1 -VLCD3-linker2- _VLCD44v6 - _linker3 - _VHCD44v6 ,

(h) V _H CD3-linker1-V _L CD3-linker2-V _H CD44v6-linker3-V _L CD44v6,

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, Linker3) 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 CD44v6-related diseases, such as diseases related to abnormal or disordered expression of CD44v6, 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-CD44v6 monoclonal antibodies, anti-CD44v6 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 CD44v6 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., Human Gene Therapy, 16(1), 126-131 (2005) and US Patent US7897380B2. 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 mother plasmid provided by the present invention has a specific recombination site, and the "minicircle DNA recombination mother plasmid" generates a microcircle DNA and a backbone DNA sequence through site-specific recombination of the specific recombination site. 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 the United States Patent US7897380B2. The method for 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 using the method as described in the twelfth aspect, so as to provide tissues or organs substantially free of CD44v6.

In some embodiments of the present invention, blood supplies are screened using the method described in the twelfth aspect to provide blood supplies substantially free of CD44v6.

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 CD44v6-related diseases.

As described in the present invention, the "CD44v6 disease" includes but not limited to diseases related to abnormal or disordered expression of CD44v6, for example, CD44v6 antigen-positive liver cancer.

In the fourteenth aspect, the present invention also provides a method for treating cancer or tumor using the specific dual-targeting antibody as described in the first aspect, including any one of the following methods:

(1) using the genetically engineered antibody alone for treating cancer or tumor;

(2) Combining the genetically engineered antibody with one or more of chemotherapy, radiotherapy, surgery, biological therapy, and immunotherapy for the treatment of cancer or tumors;

(3) Directly delivering the genetically engineered antibody into the patient's body by way of in vivo targeted delivery for cancer or tumor treatment;

(4) Firstly, the genetically engineered antibody is mixed with the immune effector cells by in vitro transfection technology, and then the immune effector cells mixed with the genetically engineered antibody are infused back into the patient's body for cancer or tumor treatment.

In some embodiments of the present invention, in the method (4), the immune effector cells are CIK cells or D-CIK cells.

In some embodiments of the present invention, in the method (4), the mixing ratio of the specific dual-targeting antibody of the first aspect and immune effector cells is: 0.001-1ug/ul specific dual-targeting antibody Mix with 1×10 ⁴ -1.6×10 ⁵ immune effector cells.

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 Robustsystem for production of minicircle DNA vectors. Nat Biotechnology, 28:1287, 2010). The antibody expressed by the microcircle DNA has the following characteristics: 1) specifically binding to CD44v6 positive antigen; 2) specifically binding to T cells; 3) mediating T cells to play a killing effect on CD44v6 antigen positive cells. 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;

Figure 3 is the result of Western Blot detection of the expression level of BsAb antibody in the medium supernatant provided by Example 3 of the present invention; wherein, a-c respectively represent the results of BsAb-1, BsAb-2, and BsAb-3; B is the result of BsAb, C is the control (control);

Figures 4-5 are the flow cytometry detection results of the CD44v6×CD3BsAb cell binding experiment provided in Example 4 of the present invention;

6-7 The results of the CD44v6×CD3BsAb 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 the CD44v6×CD3BsAb 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 CD44v6-linker1-V _H CD44v6-linker2-V _H CD3- linker3-V _L CD3; the specific nucleotide sequence corresponds to the CD44v6LCVR-linker1-CD44v6HCVR-linker2-CD3HCVR-linker3-CD3LCVR in Table 2)-the nucleotide sequence encoding histidine 6-His tag (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 Signal peptide 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 (BGHpoly 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 microcircular 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 SEQID NO :twenty one.

In the BsAb antibody gene expression cassette of Example 1 of the present invention, the amino acid sequences of linker1 and linker2 are the same, both are (GGGGS) ₃ , and the nucleotide sequences are different, which are the cores shown in SEQUENCE NO.22 and SEQUENCE NO.23 respectively. nucleotide sequence.

The amino acid sequence of Linker3 is VEGGSGGSGGSGGSGGVD, and the nucleotide sequence is the nucleotide sequence shown in SEQUENCE NO.24.

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

GGCGGCGGAGGCAGCGGCGGCGGAGGCAGCGGCGGAGGCGGATCC;

In SEQUENCE NO.24,

Linker3

GTCGAAGGTGGAAGTGGAGGTTCTGGTGGAAGTGGAGGTTCAGGTGGAGTTGAC;

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 microcircle DNA recombination parent plasmid p2ФC31.Bab containing a genetically engineered antibody gene expression cassette", to obtain the p2ФC31 microcircle with the nucleotide sequence encoding CD44v6×CD3BsAb antibody in Example 2 of the present invention parent plasmid; and

Steps of Example 6 "A Method for Constructing a Microcircular DNA Recombination Mother Plasmid pMC.Bab Containing Genetic Engineering Antibody Gene Expression Cassette"; to obtain the pMC with the nucleotide sequence encoding CD44v6×CD3BsAb antibody in Example 2 of the present invention .BESXP minicircle parent 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 CD44v6×CD3BsAb" 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 the attB and attP sites of the p2ФC31 empty vector or pMC.BESXP empty vector by ligation; respectively obtain the p2ФC31 minicircle master plasmid or pMC.BESXP minicircle with the nucleotide sequence encoding the BsAb antibody in Example 1 of the present invention 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-CD44v6×CD3).

Example 2

Construction of microcircle DNA with nucleotide sequence encoding CD44v6×CD3BsAb 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 Preparation Method of Microcircle DNA Containing Gene Expression Cassette of Genetic Engineering Antibody", respectively to obtain the microcircle DNA with the nucleotide sequence encoding CD44v6×CD3BsAb 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 CD44v6×CD3BsAb antibody in HEK 293T cells

Using the X-treme GENEHP DNA Transfection Reagent Plasmid Transfection Kit (Roche Company), the microcircle DNA of Example 3 (the microcircle DNA prepared from the microcircle master plasmid shown in Figure 2) was transfected into HEK293T cells, and the HEK293T cells were transfected with CD44v6BsAb After 72 hours of microcircle carrier, the expression level of CD44v6 BsAb antibody in the medium supernatant and cytoplasm was detected by Western Blot. As shown in Figure 3, the three BsAbs were all expressed in the medium supernatant and cytoplasm.

(2) Purification of CD44v6×CD3BsAb antibody

Transfect HEK 293T cells with medium-scale secreted antibody BsAb, collect the culture supernatant, and then use His-Tag affinity resin to purify (Omplete His-Tag Purification Resin, Roche). The purified antibody is detected by ELISA method Concentration: After purification, the protein concentration reaches 1.0mg/ml, and it is stored at -80°C for a long time, and it will be used for flow cytometry detection and in vitro tumor killing experiments.

Example 4

The binding activity of CD44v6×CD3BsAb-1 and CD44v6×CD3BsAb-2 antibodies to Jurkat and SMMC-7721 cells was detected by flow cytometry, including the following steps:

a) Cell culture: Jurkat cells (1640medium; suspension growth, cell line expressing CD3); SMMC-7721 (DMEM medium; liver cancer cell line expressing CD44v6),

b) Digest SMMC-7721 with trypsin, add serum-containing medium to neutralize, then centrifuge and discard the supernatant to obtain cells; Jurkat is directly centrifuged to collect cells for suspension 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:

Jurkat cells, 2 groups: blank group (Blank, supernatant of HEK 293T cells transfected with pMC.BESXP empty vector; pre-prepared); CD44v6×CD3BsAb group (purified antibody obtained in Example 3; pre-prepared)

SMMC-7721 cells, 2 groups: grouping as above, blank group (same as above) and CD44v6BsAb group (purified antibody obtained in Example 3; pre-prepared) Jurkat, 2 groups: grouping as above, blank group (same as above) and CD44v6×CD3BsAb group (purified 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 for washing, centrifuge at 200 g for 5 min, collect the cells, and resuspend with 200 μl of PBS. The combination was observed on a flow cytometer (BD Accuri C6).

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 CD44v6 antigen positive cells (SMMC-7721). In each of Fig. 4 and Fig. 5, the left curve is the blank group; the right curve is the BsAb experimental group. Experimental results Figure 4 shows that only BsAb-1 has a strong binding function to the CD3 antigen, BsAb-2 has a weak binding function, and BsAb-3 has almost no binding function. Experimental Results Figure 5 shows that only BsAb-1 has weak specific binding to tumor cells, while BsAb-2 and BsAb-3 have no binding ability. .

Example 5

Method for killing SMMC-7721 cells by CD44v6×CD3BsAb-1 antibody combined with DCIK cells

In this example, the target cells (T) are SMMC-7721 cells; the effector cells (E) are DCIK cells (dendritic cell activated and cytokine induced killer cells) Kit CytoTox96Non-Radioactive Cytotoxicity Assay (Promega, cat.G1780)

Refer to the kit instructions for the steps, as follows:

a) The cultured target cells (SMMC-7721) were collected by trypsinization, counted, resuspended in DMEM medium containing 5% FBS, grouped according to 2×10 ⁴ /well and shown in Figure 6, and inoculated to 96 in the orifice plate.

b) The effector cells DCIK (E) were collected by centrifugation and counted, resuspended in DMEM medium containing 5% FBS, respectively according to the ratio of E:T=4:1 and E:T=8:1, according to the middle figure Add the 6 groups to the corresponding wells.

c) According to Figure 6, add each BsAb (dilute the purified solution to 1 ug/ml, that is, 1 ng/ul), the addition amount is 10 ng/well, 10 μl, set a group of no BsAb control (DCIK group), and each group has 4 duplicate wells ;

d) Incubate at 37°C for 3 hours and 15 minutes in a cell incubator;

e) Add 10 μl of lysate to the target cell maximum release lactate dehydrogenase (LDH) well, and mix well;

f) After continuing to incubate at 37°C for 45 minutes, centrifuge 96 wells at 250 g for 4 minutes, transfer 10 μl of supernatant from each well to a new 96-well plate, add 50 μl of LDH substrate solution to each well, and incubate at room temperature for 30 minutes;

g) Add 50 μl of stop solution to each well, and measure the absorbance at 490 nm.

h) According to the absorbance value, calculate the tumor killing efficiency according to the following formula:

Tumor killing efficiency (%)=[(E _BsAb -E _spon -T _spon )/(T _max -T _spon )]×100

Among them, E _BsAb represents the release of lactate dehydrogenase (LDH) in each experimental group; E _spon represents the spontaneous release of LDH by effector cells; T _spon represents the spontaneous release of LDH by target cells; T _max represents the maximum release of LDH by target cells. (According to the actual instructions, the culture medium background control was subtracted from the data of all wells before processing, and the volume correction value was subtracted from the experimental group).

The results of the combined DCIK cell killing SMMC-7721 cell experiment are shown in FIG. 7 .

In Figure 7, the vertical axis represents the relative value of the tumor killing rate; the horizontal axis represents different groups and the effect-to-target ratio (E:T).

It can be seen from Figure 7 that, compared with the DCIK group alone (no BsAb group), the addition of EGFRvIII×CD3BsAb increased the tumor killing rate, among which EGFRvIII×CD3BsAb-1 had the best tumor killing effect, and the effective target ratio (E:T) was At 4:1 and 8:1, they can reach 42.69% and 71.96%, respectively, indicating that EGFRvIII×CD3BsAb can specifically mediate the killing of target cells by DCIK (main T cells).

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 having a first antigen binding domain that specifically binds human CD3 and a second antigen binding domain that specifically binds CD44v6 antigen.

2. The specific dual-targeting antibody of claim 1, wherein said specific dual-targeting antibody comprises at least one HCVR amino acid sequence of a first antigen-binding domain, at least one LCVR amino acid sequence of a first antigen-binding domain, at least one HCVR amino acid sequence of a second antigen-binding domain, and at least one LCVR amino acid sequence of a second antigen-binding domain; wherein the HCVR and LCVR are optional HCVR and LCVR amino acid sequences, respectively, in Table 1.

3. A specific dual-targeting antibody is applied by one or more of the following methods:

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

(2) the specific double-targeting antibody is combined with one or more of chemotherapy, radiotherapy, surgery, biotherapy and immunotherapy for application;

(3) delivering the specific dual-targeted antibody directly into a patient for treatment by in vivo delivery;

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

4. A pharmaceutical composition comprising at least one specific dual-targeting antibody of claim 1, and a diagnostic agent, a pharmaceutically acceptable carrier, excipient or diluent.

5. A nucleic acid molecule encoding the amino acid sequence of the specific dual-targeting antibody of claim 1.

6. A recombinant vector having the sequence of any one of the nucleic acid molecules of claim 7.

7. The recombinant vector of claim 6, wherein the recombinant vector is an expression vector or a minicircle DNA.

8. A host cell comprising the recombinant vector of claim 6.

9. An antibody drug, which is the specific dual-targeting antibody of claim 1, which binds to human CD3 and induces T-cell mediated killing of CD44v6 positive cells in vitro.

10. Use of the specific dual-targeting antibody of claim 1 or the recombinant vector of claim 6 for the preparation of a reagent or a medicament for the diagnosis, prevention, treatment of CD44v6 disease.