CN110872356B

CN110872356B - Bispecific antibodies and methods of use thereof

Info

Publication number: CN110872356B
Application number: CN201811028689.3A
Authority: CN
Inventors: 季匡华; 王愈善; 江欣倩; 黄逸君; 詹姆斯周
Original assignee: JOHNPRO BIOTECH Inc; SOURCING BIZ GUILIN Inc; Guangxi Hui Bao Source Health Industry Co ltd
Current assignee: JOHNPRO BIOTECH Inc; SOURCING BIZ GUILIN Inc; Guangxi Hui Bao Source Health Industry Co ltd
Priority date: 2018-09-03
Filing date: 2018-09-03
Publication date: 2023-06-13
Anticipated expiration: 2038-09-03
Also published as: WO2020047818A1; CN110872356A

Abstract

The present invention provides a bispecific antibody comprising a first peptide chain VL-CK-TM and a second peptide chain VH-CH1-VH '-VL', wherein VH is the heavy chain variable region of the first monoclonal antibody, VL is the light chain variable region of the first monoclonal antibody, CH1 is the heavy chain constant region 1 of the first monoclonal antibody, CK is the light chain constant region of the first monoclonal antibody, VH 'is the heavy chain variable region of the second monoclonal antibody, VL' is the light chain variable region of the second monoclonal antibody, and TM is the transmembrane protein domain. The bispecific antibody of the invention can particularly keep the affinity to T cells for a long time, and can be used for enhancing the killing effect to cancer cells.

Description

Bispecific antibodies and methods of use thereof

Technical Field

The invention belongs to the field of antibody engineering, and in particular relates to a bispecific antibody and application thereof, wherein the bispecific antibody has excellent effect, in particular to the effect of lasting maintenance of affinity to T cells.

Background

Antibodies are also known as immunoglobulins (Ig). Natural antibodies contain four heterologous polypeptide chains, of which the two chains of greater molecular weight are referred to as Heavy Chains (HC) and the two chains of lesser molecular weight are referred to as Light Chains (LC). The regions of the light and heavy chains that are greatly changed in amino acid sequence near the N-terminus are referred to as the variable regions, namely the light chain variable region (VL) and the heavy chain variable region (VH), which account for 1/4 and 1/2 of the heavy and light chains, respectively, the VL and VH each comprise three Complementarity Determining Regions (CDRs), namely VL-CDR1, VL-CDR2 and VL-CDR3 and VH-CDR1, VH-CDR2 and VH-CDR3; the region of relatively stable amino acid sequence near the C-terminus is referred to as the constant region, wherein the heavy chain constant region is divided into three segments, namely a heavy chain constant region 1 (CH 1), a heavy chain constant region 2 (CH 2) and a heavy chain constant region 3 (CH 3), in that order, CH1 and a light chain constant region (CL, also referred to as CK) are linked by disulfide bonds.

Natural monoclonal antibodies specifically bind to one antigen target, whereas bispecific antibodies (BsAb) are antibody fusions that specifically bind to two different antigen targets. Bispecific antibodies are currently studied in two more classes, tandem scFv and bispecific tetravalent antibodies (Tand-Abs), respectively.

For example, chinese patent application CN104892765a discloses a bispecific antibody against CD3 antigen and Her-2 antigen, comprising 2 identical light chain sequences and 2 different heavy chain sequences, which can recognize Her-2 antigen on the surface of breast cancer cells and CD3 antigen on the surface of T cells, respectively, and can significantly enhance the activity of antibody-mediated T cell killing tumor target cells;

chinese patent application CN106831996a provides a novel bispecific antibody comprising an antigen binding to human CD3E and/or an antigen binding to HER 2;

chinese patent application CN104829728A discloses a bispecific antibody HER2XCD3 consisting of a monovalent unit having specific binding capacity for the surface antigen CD3 of immune cells and a single chain unit having specific binding capacity for the tumor cell surface antigen HER2, and comprising a single chain variable fragment fused to an Fc fragment;

chinese patent application CN106632681a discloses anti-EGFR and anti-CD3 bispecific antibodies capable of specifically binding to tumor cell surface antigen epidermal growth factor receptor and immune cell surface antigen cluster 3, wherein the single chain antibody ScFv of the anti-CD3 antibody is located at the C-terminus of the constant region of the anti-EGFR antibody;

chinese patent application CN104774268A discloses a bispecific antibody egfr×cd3 consisting of a monovalent unit having specific binding capacity for the surface antigen CD3 of immune cells and a single chain unit having specific binding capacity for the tumor cell surface antigen EGFR, and comprising a single chain variable fragment fused to an Fc fragment;

chinese patent application CN104829729a discloses a bispecific antibody carrying anti Her2/CD3, which comprises a first antibody fragment and a second antibody fragment, wherein the first antibody fragment is an anti-tumor antigen antibody fragment, the second antibody fragment is an anti-human CD3 molecule antibody fragment, and the bispecific antibody is carried by adoptive transfer T cells and continuously expresses the antibody protein in vivo, so that the bispecific antibody exerts a killing effect in vivo, and simultaneously, the bispecific antibody accompanies sufficient T effector cells, thereby optimizing the efficiency of effector exertion.

However, the prior art does not suggest obtaining bispecific antibodies of the invention comprising specific tandem arrangements of transmembrane domains. Surprisingly, the bispecific antibody of the present invention designed by the present inventors is excellent in effect, particularly in effect of being able to permanently maintain adhesion to T cells, and is more advantageous for practical use.

Brief description of the invention

The present invention provides novel bispecific antibodies and intermediates of recombinant DNA techniques thereof, methods of preparation, therapeutic uses thereof, and the like.

In particular, in a first aspect, the present invention provides bispecific antibodies having the structure,

wherein VH is the heavy chain variable region of the first monoclonal antibody,

VL is the light chain variable region of the first monoclonal antibody,

CH1 is the heavy chain constant region 1 of the first monoclonal antibody,

CK is the light chain constant region of the first monoclonal antibody, where CH1 and CK are linked by disulfide bonds (|),

VH' is the heavy chain variable region of the second monoclonal antibody,

VL' is the light chain variable region of the second monoclonal antibody,

TM is the transmembrane protein domain, and,

-represent direct peptide bond linkage or via a linking peptide.

The antigen targets against which the first monoclonal antibody and the second monoclonal antibody are directed are different. Preferably, in the bispecific antibody of the first aspect of the present invention, one of the first monoclonal antibody and the second monoclonal antibody specifically binds to T cells. More preferably wherein the CD3 antigen specifically binds T cells.

It is also preferred that in the bispecific antibody of the first aspect of the present invention, the other of the first monoclonal antibody and the second monoclonal antibody specifically binds to a pathogen. More preferably, wherein the other of the first monoclonal antibody and the second monoclonal antibody specifically binds to a tumor or cancer cell, e.g., a tumor or cancer cell specific antigen or an over-expressed antigen, e.g., an epidermal growth factor receptor, carcinoembryonic antigen, or prostate specific membrane antigen.

Preferably in the bispecific antibody of the first aspect of the invention, the transmembrane protein domain is a transmembrane domain of a leukocyte differentiation antigen, for example a transmembrane domain which may be of cell adhesion molecule-1, CD48 or CD 80. More preferably, the amino acid sequence of the transmembrane protein domain is as set forth in SEQ ID NO: 1. 2 or 3.

Preferably in the bispecific antibody of the first aspect of the invention, VL or VL' comprises an amino acid sequence as set forth in SEQ ID NO: 4. 5, 6 or 11, and the light chain variable region or amino acid sequence in the fusion peptide shown in SEQ ID NO: 10. 13, 14 or 15, VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region in the fusion peptide.

It is also preferred that in the bispecific antibody of the first aspect of the present invention, VH or VH' comprises an amino acid sequence as set forth in SEQ ID NO: 7. 8, 9 or 12, and the heavy chain variable region or amino acid sequence in the fusion peptide shown in SEQ ID NO: 10. 13, 14 or 15, VH-CDR1, VH-CDR2 and VH-CDR3 in the heavy chain variable region of a fusion peptide.

In a specific embodiment of the invention, the amino acid sequence of VL-CK is as set forth in SEQ ID NO: 4. 5, 6 or 11, wherein the first three are VL-CK of a monoclonal antibody specifically binding to an EGF receptor, carcinoembryonic antigen and prostate specific membrane antigen, respectively; the latter is VL-CK of a monoclonal antibody that specifically binds the CD3 antigen of T cells.

In a specific embodiment of the invention, the amino acid sequence of VH-CH1 is as set forth in SEQ ID NO: 7. 8, 9 or 12, wherein the first three are VH-CH1 of a monoclonal antibody that specifically binds to an epidermal growth factor receptor, carcinoembryonic antigen and prostate specific membrane antigen, respectively; the latter is VH-CH1 of a monoclonal antibody that specifically binds to the CD3 antigen of T cells.

In a specific embodiment of the invention, the amino acid sequence of VH '-VL' is as set forth in SEQ ID NO: 10. 13, 14 or 15, wherein the former is VH '-VL' of a monoclonal antibody that specifically binds to CD3 antigen of T cells; the latter three are VH '-VL' of monoclonal antibodies that specifically bind to epidermal growth factor receptor, carcinoembryonic antigen and prostate specific membrane antigen, respectively.

In a specific embodiment of the present invention, the connecting peptide may be GGGSGGG, VEGGSGGSGGSGGSGGVD or (GGGGS) n, where n=1 to 5.

In a second aspect, the invention provides an isolated nucleic acid encoding a bispecific antibody of the first aspect of the invention.

In a third aspect, the invention provides a vector comprising a nucleic acid of the second aspect of the invention. Preferably the vector of the third aspect of the invention is a plasmid.

In a fourth aspect, the invention provides a host cell comprising or transfected with the vector of the third aspect of the invention. Preferably the host cell of the fourth aspect of the invention is a mammalian cell.

In a fifth aspect, the present invention provides a method of producing a bispecific antibody of the first aspect of the present invention comprising the steps of:

(a) Culturing the host cell of the fourth aspect of the invention; and, a method for producing the same.

(b) Collecting the bispecific antibody of the first aspect of the invention from the culture obtained in step (a).

In a sixth aspect, the invention provides a pharmaceutical composition comprising a bispecific antibody of the first aspect of the invention and a pharmaceutically acceptable adjuvant. The pharmaceutical composition of the sixth aspect of the invention is for use in the treatment of a disease, such as a tumour or cancer, treated with a first monoclonal antibody or a second monoclonal antibody.

In a seventh aspect, the present invention provides the use of a bispecific antibody of the first aspect of the present invention in the manufacture of a medicament for the treatment of a disease treated by a first monoclonal antibody or a second monoclonal antibody; accordingly, the present invention also provides a method of treating a disease treated by a first monoclonal antibody or a second monoclonal antibody comprising administering to an individual in need thereof an effective amount of a bispecific antibody of the first aspect of the present invention.

Preferably in the use or method of the seventh aspect of the invention, the disease treated by the first monoclonal antibody or the second monoclonal antibody is a tumour or cancer. More preferably wherein the tumor or cancer is selected from the group consisting of colorectal cancer, rectal cancer, throat cancer, head and neck cancer, lung cancer, stomach cancer, breast cancer, pancreatic cancer, cervical cancer, ovarian cancer, prostate cancer.

Drawings

FIG. 1 is a schematic diagram of the construct of bispecific antibody A of example 1.

FIG. 2 is a schematic structural diagram of bispecific antibody A of example 1.

FIG. 3 is a schematic diagram of the construct of bispecific antibody B of example 1.

FIG. 4 is a graph showing experimental results of T cell adhesion time test of example 1.

FIG. 5 is a schematic diagram of the construct of bispecific antibody C of example 2.

FIG. 6 is a schematic structural diagram of bispecific antibody C of example 2.

FIG. 7 is a schematic diagram of the construct of bispecific antibody D of example 2.

FIG. 8 is a graph showing experimental results of T cell adhesion time test of example 2.

Detailed Description

The bispecific antibodies of the invention consist of two peptide chains, comprising a first peptide chain and a second peptide chain, wherein the structure of the first peptide chain is shown as VL-CK-TM, the structure of the second peptide chain is shown as VH-CH1-VH '-VL', wherein VH is the heavy chain variable region of the first monoclonal antibody, VL is the light chain variable region of the first monoclonal antibody, CH1 is the heavy chain constant region 1 of the first monoclonal antibody, CK is the light chain constant region of the first monoclonal antibody, VH 'is the heavy chain variable region of the second monoclonal antibody, VL' is the light chain variable region of the second monoclonal antibody, TM is the transmembrane protein domain, and-represents a direct peptide bond linkage or via a linking peptide.

In this context, "first" and "second" when modifying the same product are intended to distinguish between the modified products, i.e., the first product and the second product are different, but are not limited to the structure and/or composition of the products themselves. For example, the first monoclonal antibody and the second monoclonal antibody are different monoclonal antibodies.

Herein, a monoclonal antibody is composed of a pair of light chains and a pair of heavy chains, which are linked to each other by disulfide bonds. Wherein each heavy chain typically comprises a heavy chain variable region and a heavy chain constant region. The heavy chain constant region typically comprises three domains, CH1, CH2, and CH3; each light chain typically comprises a light chain variable region and a light chain constant region. Each variable region typically comprises three complementarity determining regions, which are regions of formally highly variable sequence and/or structurally defined loops, separated by four Framework Regions (FR) that are more conserved, i.e., each variable region is arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

CDR sequences herein may be determined according to IMGT rules (Brochet x., nucleic Acids Research,2008, 36:w503-508 and Lefranc MP., nucleic Acids Research,1999, 27:209-212) or may be performed according to the methods described by Kabat et al (Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD.1991). The Kabat numbering of residues may be determined for a given antibody variable region.

Preferably in the bispecific antibody of the first aspect of the present invention, one of the first monoclonal antibody and the second monoclonal antibody specifically binds to a T cell, such as a CD3 antigen that specifically binds to a T cell; while the other of the first monoclonal antibody and the second monoclonal antibody specifically binds to a pathogen, preferably specifically to a tumor or cancer cell, e.g., a tumor or cancer cell specific antigen or an over-expressed antigen, e.g., epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), or Prostate Specific Membrane Antigen (PSMA). The medicinal use of the bispecific antibody of the first aspect of the present invention is mainly determined by a monoclonal antibody fragment specifically binding to a pathogen, and the other monoclonal antibody fragment specifically binding to T cells plays a role in permanently keeping adhesion with T cells, thereby promoting the corresponding medicinal effect.

In a specific embodiment of the invention, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 4. 5 or 6, wherein the light chain variable region or the amino acid sequence in the fusion peptide is shown in SEQ ID NO: 13. 14 or 15, and VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region of the fusion peptide depicted in SEQ ID NO:11, and the light chain variable region or amino acid sequence in the fusion peptide shown in SEQ ID NO:10, VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region of the fusion peptide shown in fig; vice versa, VL comprises an amino acid sequence as set forth in SEQ ID NO:11, and the light chain variable region or amino acid sequence in the fusion peptide shown in SEQ ID NO:10, VL' comprises the amino acid sequence of SEQ ID NO: 4. 5 or 6, wherein the light chain variable region or the amino acid sequence in the fusion peptide is shown in SEQ ID NO: 13. 14 or 15, VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region in the fusion peptide.

In a specific embodiment of the invention, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 7. 8 or 9, and the heavy chain variable region or the amino acid sequence in the fusion peptide shown in SEQ ID NO: 13. 14 or 15, VH' comprises an amino acid sequence as set forth in SEQ ID NO:12, the heavy chain variable region or amino acid sequence in the fusion peptide shown in SEQ ID NO:10, VH-CDR1, VH-CDR2, and VH-CDR3 in the heavy chain variable region of the fusion peptide shown in fig; vice versa, VH comprises an amino acid sequence as set forth in SEQ ID NO:12, the heavy chain variable region or amino acid sequence in the fusion peptide shown in SEQ ID NO:10, VH' comprises an amino acid sequence as set forth in SEQ ID NO: 7. 8 or 9, and the heavy chain variable region or the amino acid sequence in the fusion peptide shown in SEQ ID NO: 13. 14 or 15, VH-CDR1, VH-CDR2, and VH-CDR3 in the heavy chain variable region of a fusion peptide as set forth in seq id no.

In this context, a transmembrane protein domain or transmembrane domain refers to a region on a polypeptide or protein that is thermodynamically stable in the membrane and typically includes a single transmembrane alpha helix of the transmembrane protein, consisting essentially of hydrophobic amino acids. The preferred transmembrane protein domain of the invention is the transmembrane domain (CD) of a leukocyte differentiation antigen, which may be, for example, the transmembrane domain of cell adhesion molecule-1 (ICAM-1, also known as CD 54), CD48 or CD 80.

In this context, a linker peptide refers to one or more amino acid residues inserted between each variable, constant and transmembrane protein domain of a bispecific antibody that provide sufficient mobility for each region or domain to provide bispecific antibody binding function. The linker peptides typically comprise flexible amino acid residues, such as Gly, and in bispecific antibodies of the invention, each linker peptide is independent, may have the same sequence and/or length, or may be different from each other.

The nucleic acid of the second aspect of the invention is typically two, namely a first nucleic acid and a second nucleic acid, encoding a first peptide chain and a second peptide chain, respectively, in a bispecific antibody of the invention. The first nucleic acid and the second nucleic acid are preferably constructed on the same construct and expressed from the same vector. Of course, although not preferred, the first nucleic acid and the second nucleic acid may be constructed separately on separate vectors, expressed separately, and then combined together.

Herein, the vector is preferably an expression vector, including a derivative of SV40, a bacterial plasmid, phage DNA, baculovirus, yeast plasmid, a vector derived from a combination of plasmid and phage DNA, and a viral nucleic acid (RNA or DNA) vector, and the like. Vectors suitable for expression in bacterial cells include the already commercially available BlueScript (Stratagene), pIN vectors (Van Heeke, J Biol Chem,264,5503-5509 (1989)), pET vectors (Novagen, madison Wis.), and the like; vectors suitable for expression in yeast systems are vectors comprising constitutive or inducible promoters; preferred expression vectors are suitable for expression in mammalian cells, such as those employed in the embodiments of the present invention.

Expression vectors may contain or be associated with any suitable promoter, enhancer, and other elements that promote expression. Examples of such elements include a strong expression promoter (e.g., the human CMV IE promoter/enhancer and the RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), a polyA termination sequence, a replication initiation region for plasmid products in E.coli, an antibiotic resistance gene as a selectable marker, and/or a convenient cloning site.

The expression vector may be located in a host cell and/or the nucleic acid of the second aspect of the invention may be delivered (e.g. transfected) into a host cell via the vector. Examples of host cells include yeast, bacterial, and mammalian cells, such as CHO or HEK cells. In a specific embodiment of the invention, a nucleic acid encoding a bispecific antibody of the invention is transfected into an Expi293F cell.

The bispecific antibody of the first aspect of the present invention can be obtained by culturing the host cell of the fourth aspect of the present invention, and isolating and purifying the culture obtained by the culture. The separation and purification method comprises chromatography, magnetic bead separation or colloid filtration.

Pharmaceutically acceptable excipients in this context mean pharmaceutical excipients which do not cause side effects, allergies or other adverse reactions when used in mammals (in particular humans) as appropriate, including non-toxic solid or liquid fillers, diluents, encapsulating materials or formulation excipients and the like. Examples of suitable fillers, solvents, and/or excipients include one or more of water, amino acids, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. It is also preferred to include isotonic agents, for example, sugars, polyalcohols, or sodium chloride in the pharmaceutical compositions of the invention. It is further preferred that the pharmaceutical compositions of the present invention may further comprise an antioxidant (e.g., tryptamine) and a stabilizer (e.g., tween-20).

The form, route of administration, dosage and regimen of the pharmaceutical composition will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc. The pharmaceutical compositions of the present invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, and the like. Preferably the pharmaceutical composition of the invention is an injectable, such as an isotonic, sterile aqueous solution, or a dried (especially freeze-dried) composition thereof. The dried composition may be formulated into an injectable solution upon addition of sterile water or physiological saline.

The bispecific antibodies of the invention may be present in the pharmaceutical compositions of the invention in salt form. Pharmaceutically acceptable salts include acid addition salts, corresponding inorganic acids include hydrochloric acid or phosphoric acid, corresponding organic acids include acetic acid, oxalic acid, tartaric acid, and the like. Pharmaceutically acceptable salts may also include salts with free carboxyl groups on proteins, corresponding inorganic bases including sodium, potassium, ammonium, calcium or iron hydroxides, corresponding organic bases including isopropylamine, trimethylamine, glycine, histidine and the like.

In this context, individual refers to a human or non-human mammal, preferably a human, including men, women and children. The non-human mammal is preferably an animal for drug testing such as rat, mouse, rabbit, cat, dog, monkey, horse, etc.

Herein, treatment refers to reversing, alleviating, inhibiting the progression of one or more disorders or conditions of a given disease after administration to a subject having the disease. Thus, treatment refers not only to treatment that results in complete cure of the disease, but also to treatment that slows disease progression and/or prolongs survival of the subject.

An effective amount, as used herein, refers to the amount of drug required to achieve a therapeutic effect sufficient to treat the corresponding disease at a reasonable benefit/risk ratio. The total daily amount of bispecific antibody of the invention or pharmaceutical composition of the invention will be determined by the physician within the scope of sound medical judgment. The particular therapeutically effective amount for any particular patient will depend on a variety of factors, including the condition to be treated and the severity of the condition; the activity of the particular polypeptide employed; the specific composition employed; age, weight, general health, sex, and diet of the patient; the time of application; the route of administration and the rate of excretion of the polypeptide employed; duration of treatment; factors known in the medical arts, such as drugs used in combination or concurrently with the polypeptide employed.

The present invention refers to publications that are incorporated herein by reference in their entirety for the purpose of more clearly describing the invention as if they were repeatedly set forth herein in their entirety.

The present invention will be described in detail below with reference to specific embodiments and drawings for ease of understanding. It should be particularly pointed out that these descriptions are merely exemplary descriptions and do not constitute limitations on the scope of the invention. Many variations and modifications of the invention will be apparent to those skilled in the art in light of the teachings of this specification.

Detailed Description

The following examples further illustrate the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art and commercially available general instruments and reagents, and can be referred to in antibody engineering (second edition) (Beijing university medical press), molecular cloning experiment guidelines (3 rd edition) (scientific press), microbiological experiments (4 th edition) (higher education press), and manufacturer specifications of the corresponding instruments and reagents.

EXAMPLE 1 preparation of bispecific antibody A of the present invention and effect experiments thereof

Prepared according to conventional recombinant DNA techniques, briefly, a plasmid (pLNCX, available from Clontech) containing the construct shown in fig. 1 was first constructed, wherein: the light chain variable region and the light chain constant region (alpha-Tumor VL-CK, the amino acid sequences of which are shown as SEQ ID NO: 4), a connecting peptide (linker, amino acid sequence is GGGGSGGGGSGGGGS) and a cell adhesion molecule-1 (Intercellular Adhesion Molecule, ICAM-1) domain D1-D2-D3 (ICAM 1-D1-3, amino acid sequence of which is shown as SEQ ID NO: 1), a connecting peptide (scFv linker), a light chain variable region (alpha-CD 3 VL) of a CD3 antigen (anti-CD 3 VL) of an anti-T cell, and a DNA of a histidine tag (6 His) are inserted in sequence after the second Leader SEQ) carried by the plasmid, wherein the amino acid sequence is shown as SEQ ID NO:7, the connecting peptide (linker, amino acid sequence is GGGSGGG), the heavy chain variable region (alpha-CD 3 VH) of a CD3 antigen (anti-CD 3) of an anti-T cell, the light chain variable region (alpha-CD 3 VL) of an anti-T cell, the scFv-CD 3 antigen (anti-CD 3 VL) and the amino acid sequence of a-T cell are shown as SEQ ID NO: 7: shown at 10.

Next, positive plastids constructed as described above were transfected into an Expi293F cell (available from Thermo Fisher Scientific company) using a cell culture solution Expi293 ^TM Expression Medium (available from Thermo Fisher Scientific) was cultured in suspension at 37℃under a carbon dioxide concentration of 5%, and the number of the Expi293F cells in the Expi293F cell culture broth was maintained at 3 by subculture (subture)–5×10 ⁶ cells/ml. After 7 days of culture, cells were disrupted by sonication, cell supernatants were collected by centrifugation at 1000rpm for 10 minutes, and bispecific antibody A expressed by the Expi293F cells was purified from the above supernatants by nickel affinity chromatography (Nickel Affinity Chromatography).

The light chain constant region of alpha-Tumor VL-CK was identified to be covalently linked to CH1 of alpha-Tumor VH-CH1 by disulfide bonds to form an intact bispecific antibody. The structure of the bispecific antibody A after expression is shown in FIG. 2.

In addition, as shown in FIG. 3, a bispecific antibody B was constructed and prepared in substantially the same manner as described above, except that it did not have the domain D1-D2-D3 (ICAM 1-D1-3) of the cell adhesion molecule-1 (Intercellular Adhesion Molecule, ICAM-1) as compared to the bispecific antibody A.

Binding time tests for T cells were performed on bispecific antibodies A and B, specifically, 6 250mL cell culture flasks were first prepared, 20mL of serum-free cell culture medium AIM V (available from ThermoFisher Scientific Co.) was added to each cell culture flask, then, bispecific antibody A at a concentration of 5mg/mL was added to the cell culture medium in three of the cell culture flasks as a test group sample, and bispecific antibody B at a concentration of 5mg/mL was added to the cell culture medium in the other three cell culture flasks as a control group sample. The above 6 cell culture flasks were placed in a cell culture incubator, and incubated at 37℃and a carbon dioxide concentration of 5%, and at 4 time points of 0 hours after incubation, 24 hours after incubation, 48 hours after incubation, and 72 hours after incubation, the analysis samples were taken out of the cell fluid samples of the experimental group and the cell fluid samples of the control group, respectively, the analysis samples of the experimental group and the analysis samples of the control group were stained with fluorescent secondary antibody anti-human IgG fluorescent isothiocyanate (anti-human IgG-FITC), and the stained analysis samples were placed in a flow cytometer, and it was known whether the T cells in the experimental group and the control group were adhered to their corresponding bispecific antibody preparations by detecting T cells and the T cells labeled with anti-human IgG-FITC, because the anti-human IgG-FITC was bound to bispecific antibody A and bispecific antibody B.

As shown in fig. 4, the degrees of affinity of the bispecific antibody a (egfr_cd3+icam1) and the bispecific antibody B (egfr_cd3) to T cells decreased with time, but the degrees of affinity of the bispecific antibody a to T cells were significantly higher than the degrees of affinity of the bispecific antibody B to T cells at 3 time points of 24 hours, 48 hours, and 72 hours after the culture. As is clear from the experimental results of the above-described T cell adhesion time test of the bispecific antibody A of example 1, the domains D1-D2-D3 of ICAM-1 capable of stabilizing the immune synaptic structure contribute to the improvement of the affinity of the bispecific antibody A for T cells.

EXAMPLE 2 preparation of bispecific antibody C of the present invention and effect experiments thereof

Prepared according to conventional recombinant DNA techniques, briefly, a plasmid (pLNCX, available from Clontech) containing the construct shown in fig. 5 was first constructed, wherein: the light chain variable region and the light chain constant region (alpha-CD 3 VL-CK, the amino acid sequences of which are shown in SEQ ID NO: 11), a connecting peptide (linker, amino acid sequence is GGGGSGGGGSGGGGS) and a cell adhesion molecule-1 (Intercellular Adhesion Molecule, ICAM-1) domain D1-D2-D3 (ICAM 1-D1-3, amino acid sequence is shown as SEQ ID NO: 1), a connecting peptide (scFv linker), a light chain variable region (alpha-EGFR VL) of an anti-T cell CD3 antigen (anti-CD 3) and a DNA of CH1 (alpha-CD 3 VH-CH1, amino acid sequence is shown as SEQ ID NO: 12), a connecting peptide (linker, amino acid sequence is GGGSGGG), a heavy chain variable region of an anti-EGFR antibody (alpha-EGFR VH), a connecting peptide (scFv linker), a light chain variable region of an anti-EGFR antibody (alpha-EGFR VL) and a histidine tag (6 His) are inserted in sequence after a second Leader SEQ ID sequence (Leader SEQ), wherein the amino acid sequences of alpha-EGFR VH, VL and alpha-EGFR are shown as SEQ ID NO: shown at 13.

Next, bispecific antibody C was prepared by the same manner as in example 1 by the Expi293F cell and nickel affinity chromatography.

The light chain constant region of α -CD3 VL-CK was identified to be covalently linked to CH1 of α -CD3 VH-CH1 by disulfide bonds to form an intact bispecific antibody. The structure of the bispecific antibody C after expression is shown in FIG. 6.

In addition, as shown in FIG. 7, a bispecific antibody D was constructed and prepared in substantially the same manner as described above, except that it did not carry the domain D1-D2-D3 (ICAM 1-D1-3) of the cell adhesion molecule-1 (Intercellular Adhesion Molecule, ICAM-1) as compared to the bispecific antibody C.

Binding time assays for T cells on bispecific antibodies a and B were performed as described in example 1. As shown in fig. 8, the degrees of affinity of the bispecific antibody C (egfr_cd3+icam1) and the bispecific antibody D (egfr_cd3) to T cells decreased with time, but the degrees of affinity of the bispecific antibody C to T cells were significantly higher at 3 time points, 24 hours, 48 hours, and 72 hours after the culture, than the degrees of affinity of the bispecific antibody D to T cells. From the experimental results of the above-described bispecific antibody C-T cell binding time test of example 2 in combination with the results of example 1, it was found that the improvement of the affinity of bispecific antibodies having transmembrane proteins for T cells was not affected, regardless of whether VL-CK and VH-CH were allowed to bind to target cells and VH-VL was allowed to bind to T cells or VL-CK and VH-CH were allowed to bind to T cells.

<110> Guangxi Huibao Source health industry Co., ltd; jiang Pu raw technologies Co., ltd; gui Linshang plant products Co.Ltd

<120> bispecific antibodies and methods of use thereof

<130> CN,PCT

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 270

<212> PRT

<213> Artificial Sequence

<220>

<223> ICAM1 domain

<400> 1

Gln Thr Ser Val Ser Pro Ser Lys Val Ile Leu Pro Arg Gly Gly Ser

1 5 10 15

Val Leu Val Thr Cys Ser Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly

20 25 30

Ile Glu Thr Pro Leu Pro Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn

35 40 45

Arg Lys Val Tyr Glu Leu Ser Asn Val Gln Glu Asp Ser Gln Pro Met

50 55 60

Cys Tyr Ser Asn Cys Pro Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu

65 70 75 80

Thr Val Tyr Trp Thr Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Ser

85 90 95

Trp Gln Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly

100 105 110

Gly Ala Pro Arg Ala Asn Leu Thr Val Val Leu Leu Arg Gly Glu Lys

115 120 125

Glu Leu Lys Arg Glu Pro Ala Val Gly Glu Pro Ala Glu Val Thr Thr

130 135 140

Thr Val Leu Val Arg Arg Asp His His Gly Ala Asn Phe Ser Cys Arg

145 150 155 160

Thr Glu Leu Asp Leu Arg Pro Gln Gly Leu Glu Leu Phe Glu Asn Thr

165 170 175

Ser Ala Pro Tyr Gln Leu Gln Thr Phe Val Leu Pro Ala Thr Pro Pro

180 185 190

Gln Leu Val Ser Pro Arg Val Leu Glu Val Asp Thr Gln Gly Thr Val

195 200 205

Val Cys Ser Leu Asp Gly Leu Phe Pro Val Ser Glu Ala Gln Val His

210 215 220

Leu Ala Leu Gly Asp Gln Arg Leu Asn Pro Thr Val Thr Tyr Gly Asn

225 230 235 240

Asp Ser Phe Ser Ala Lys Ala Ser Val Ser Val Thr Ala Glu Asp Glu

245 250 255

Gly Thr Gln Arg Leu Thr Cys Ala Val Ile Leu Gly Asn Gln

260 265 270

<210> 2

<211> 252

<212> PRT

<213> Artificial Sequence

<220>

<223> CD48 domain

<400> 2

Met Cys Ser Arg Gly Trp Asp Ser Cys Leu Ala Leu Glu Leu Leu Leu

1 5 10 15

Leu Pro Leu Ser Leu Leu Val Thr Ser Ile Gln Gly His Leu Val His

20 25 30

Met Thr Val Val Ser Gly Ser Asn Val Thr Leu Asn Ile Ser Glu Ser

35 40 45

Leu Pro Glu Asn Tyr Lys Gln Leu Thr Trp Phe Tyr Thr Phe Asp Gln

50 55 60

Lys Ile Val Glu Trp Asp Ser Arg Lys Ser Lys Tyr Phe Glu Ser Lys

65 70 75 80

Phe Lys Gly Arg Val Arg Leu Asp Pro Gln Ser Gly Ala Leu Tyr Ile

85 90 95

Ser Lys Val Gln Lys Glu Asp Asn Ser Thr Tyr Ile Met Arg Val Leu

100 105 110

Lys Lys Thr Gly Asn Glu Gln Glu Trp Lys Ile Lys Leu Gln Val Leu

115 120 125

Asp Pro Val Pro Lys Pro Val Ile Lys Ile Glu Lys Ile Glu Asp Met

130 135 140

Asp Asp Asn Cys Tyr Leu Lys Leu Ser Cys Val Ile Pro Gly Glu Ser

145 150 155 160

Val Asn Tyr Thr Trp Tyr Gly Asp Lys Arg Pro Phe Pro Lys Glu Leu

165 170 175

Gln Asn Ser Val Leu Glu Thr Thr Leu Met Pro His Asn Tyr Ser Arg

180 185 190

Cys Tyr Thr Cys Gln Val Ser Asn Ser Val Ser Ser Lys Asn Gly Thr

195 200 205

Val Cys Leu Ser Pro Pro Cys Thr Leu Gly Lys Lys Asp Pro Trp Glu

210 215 220

Leu Arg Gly Ala Gln Gly Asn Trp Ser Cys Phe Glu Gln Arg Lys Ala

225 230 235 240

Gly Gly Pro Ile Gln Pro Pro Cys Thr Val Trp Trp

245 250

<210> 3

<211> 208

<212> PRT

<213> Artificial Sequence

<220>

<223> CD80 domain

<400> 3

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

20 25 30

Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

130 135 140

Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

145 150 155 160

Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

180 185 190

Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

195 200 205

<210> 4

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> anti EGFR_VL-CK

<400> 4

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210> 5

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> anti CEA_VL-CK

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Thr Val Ser Ala Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Ala Ser Tyr Arg Tyr Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 6

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> anti PSMA_VL-CK

<400> 6

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 7

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> anti EGFR_VH-CH1

<400> 7

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 8

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> anti CEA_VH-CH1

<400> 8

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Gly Met Asn Val Trp Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Ser Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Tyr Asp Tyr Val Asp Glu Ala Met Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 9

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> anti PSMA VH-CH1

<400> 9

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Lys Gln Ala Ser Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 10

<211> 226

<212> PRT

<213> Artificial Sequence

<220>

<223> anti CD3_VH-VL

<400> 10

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala

115 120 125

Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala

130 135 140

Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr

145 150 155 160

Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val

165 170 175

Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr

180 185 190

Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

195 200 205

Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

210 215 220

Lys Arg

225

<210> 11

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> anti CD3 VL-CK

<400> 11

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210> 12

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> anti CD3 VH-CH1

<400> 12

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 13

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> anti EGFR_VH-VL

<400> 13

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Asp Ile Leu Leu Thr Gln Ser Pro Val

115 120 125

Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala

130 135 140

Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln Arg Thr Asn

145 150 155 160

Gly Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly

165 170 175

Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

180 185 190

Ser Ile Asn Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln

195 200 205

Gln Asn Asn Asn Trp Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu

210 215 220

Leu Lys Arg

225

<210> 14

<211> 230

<212> PRT

<213> Artificial Sequence

<220>

<223> anti CEA_VH-VL

<400> 14

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Gly Met Asn Val Trp Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Ser Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Tyr Asp Tyr Val Asp Glu Ala Met Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Asp Ile Gln Met Thr Gln Ser

115 120 125

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

130 135 140

Lys Ala Ser Gln Thr Val Ser Ala Asn Val Ala Trp Tyr Gln Gln Lys

145 150 155 160

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Leu Ala Ser Tyr Arg Tyr

165 170 175

Arg Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

180 185 190

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

195 200 205

Cys His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr Phe Gly Gln Gly Thr

210 215 220

Lys Leu Glu Ile Lys Arg

225 230

<210> 15

<211> 223

<212> PRT

<213> Artificial Sequence

<220>

<223> anti PSMA_VH-VL

<400> 15

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Lys Gln Ala Ser Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala

115 120 125

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val

130 135 140

Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

145 150 155 160

Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg

165 170 175

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

180 185 190

Leu Gln Pro Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser

195 200 205

Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

210 215 220

Claims

1. A bispecific antibody against CD3 and EGFR having the following structure,

wherein VH is the heavy chain variable region of the first monoclonal antibody,

VL is the light chain variable region of the first monoclonal antibody,

CH1 is the heavy chain constant region 1 of the first monoclonal antibody,

CK is the light chain constant region of the first monoclonal antibody, wherein CH1 and CK are linked by disulfide bond,

VH' is the heavy chain variable region of the second monoclonal antibody,

VL' is the light chain variable region of the second monoclonal antibody,

TM is a transmembrane protein domain, and

representing a direct peptide bond linkage or via a linking peptide,

wherein,,

the amino acid sequence of VL-CK is shown in SEQ ID NO:4, the amino acid sequence of the VH-CH1 is shown as SEQ ID NO:7, the amino acid sequence of the transmembrane protein domain is shown as SEQ ID NO:1, and the amino acid sequence of the VH '-VL' is shown in SEQ ID NO:10 is shown in the figure; or,

the amino acid sequence of VL-CK is shown in SEQ ID NO:11, the amino acid sequence of the VH-CH1 is shown as SEQ ID NO:12, the amino acid sequence of the transmembrane protein domain is shown in SEQ ID NO:1, and the amino acid sequence of the VH '-VL' is shown in SEQ ID NO: shown at 13.

2. An isolated nucleic acid encoding the bispecific antibody of claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. The expression vector of claim 3, which is a plasmid.

5. A host cell comprising the expression vector of claim 3 or 4, or transfected with the vector of claim 3 or 4.

6. The host cell of claim 5 which is a mammalian cell.

7. A method of producing the bispecific antibody of claim 1, comprising the steps of:

(a) Culturing the host cell of claim 5 or 6; and, a step of, in the first embodiment,

(b) Collecting the bispecific antibody of claim 1 from the culture obtained in step (a).

8. A pharmaceutical composition comprising the bispecific antibody of claim 1 and a pharmaceutically acceptable adjuvant.

9. Use of the bispecific antibody of claim 1 in the manufacture of a medicament for the treatment of cancer.

10. The use of claim 9, wherein the cancer is selected from the group consisting of colorectal cancer, rectal cancer, throat cancer, head and neck cancer, lung cancer, gastric cancer, breast cancer, pancreatic cancer, cervical cancer, ovarian cancer, prostate cancer, and prostate cancer.