CN110872356A

CN110872356A - Bispecific antibodies and methods of use thereof

Info

Publication number: CN110872356A
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; Guangxi Hui Bao Source Health Industry Co Ltd
Current assignee: JOHNPRO BIOTECH Inc; Guangxi Hui Bao Source Health Industry Co Ltd
Priority date: 2018-09-03
Filing date: 2018-09-03
Publication date: 2020-03-10
Anticipated expiration: 2038-09-03
Also published as: WO2020047818A1; CN110872356B

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 a 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 a second monoclonal antibody, VL' is the light chain variable region of the second monoclonal antibody, and TM is a transmembrane protein domain. The bispecific antibody of the present invention can maintain the affinity for T cell and can be used to enhance the killing effect on cancer cell.

Description

Bispecific antibodies and methods of use thereof

Technical Field

The present invention belongs to the field of antibody engineering, and more specifically, relates to a bispecific antibody having an excellent effect, particularly an excellent effect of maintaining the affinity for T cells for a long period, and to the use thereof.

Background

Antibodies are also known as immunoglobulins (Ig). Natural antibodies contain four heterologous polypeptide chains, of which the two chains with the larger molecular weight are called Heavy Chains (HC) and the two chains with the smaller molecular weight are called Light Chains (LC). The regions of the light and heavy chains that vary greatly in amino acid sequence near the N-terminus are called variable regions, light chain variable regions (VL) and heavy chain variable regions (VH), 1/4 and 1/2 of the heavy and light chains, respectively, and VL and VH each include three Complementarity Determining Regions (CDRs), namely VL-CDR1, VL-CDR2 and VL-CDR3 and VH-CDR1, VH-CDR2 and VH-CDR 3; the region in which the amino acid sequence near the C-terminus is relatively stable is referred to as a constant region, in which the heavy chain constant region is divided into three segments, which are, in turn, heavy chain constant region 1(CH1), heavy chain constant region 2(CH2), and heavy chain constant region 3(CH3), and CH1 is linked to the light chain constant region (CL, also referred to as CK) by a disulfide bond.

Natural monoclonal antibodies specifically bind to one antigen target, while bispecific antibodies (BsAb) are antibody fusions that can specifically bind to two different antigen targets. Two types of bispecific antibodies are currently being studied, namely tandem scFv and bispecific tetravalent antibodies (Tang-Abs).

For example, chinese patent application CN104892765A discloses a bispecific antibody against CD3 antigen and Her-2 antigen, which comprises 2 identical light chain sequences and 2 different heavy chain sequences, can respectively recognize Her-2 antigen on the surface of breast cancer cell and CD3 antigen on the surface of T cell, and can significantly enhance the activity of T cell mediated killing tumor target cells by antibody;

chinese patent application CN106831996A provides a novel bispecific antibody comprising an antigen binding portion against human CD3E and/or an antigen binding portion against HER 2;

chinese patent application CN104829728A discloses a bispecific antibody HER2XCD3, consisting of a monovalent unit with specific binding capacity against the surface antigen CD3 of immune cells and a single chain unit with specific binding capacity against the tumor cell surface antigen HER2, and the single chain unit comprises 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 of differentiation 3, wherein the single chain antibody ScFv of anti-CD3 antibody is located at the C-terminus of the anti-EGFR antibody constant region;

chinese patent application CN104774268A discloses a bispecific antibody EGFR x CD3, consisting of a monovalent unit with specific binding capacity against the surface antigen CD3 of immune cells and a single chain unit with specific binding capacity against 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-Her 2/CD3, which comprises a first antibody segment and a second antibody segment, wherein the first antibody segment is an anti-tumor antigen antibody segment, the second antibody segment is an anti-human CD3 molecular antibody segment, and the antibody protein is carried by the T cells which are transmitted by the relay and is continuously expressed in vivo, so that the bispecific antibody can exert killing effect in vivo and simultaneously, the efficiency of exerting effect is optimized along with sufficient T effector cells.

However, the prior art does not suggest to obtain bispecific antibodies of the present invention comprising specific tandem connections of transmembrane domains. Surprisingly, the bispecific antibody of the present invention designed by the present inventors is excellent in effect, and particularly, can maintain the effect of adhesion to T cells for a long time, which is more advantageous for practical use.

Brief description of the invention

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

In particular, in a first aspect, the invention provides a bispecific antibody 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 a 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,

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

The first monoclonal antibody and the second monoclonal antibody are directed against different antigenic targets. Preferably in the bispecific antibody of the first aspect of the invention, one of the first monoclonal antibody and the second monoclonal antibody specifically binds to a T cell. More preferably, among these, the CD3 antigen that specifically binds T cells.

Also preferred in the bispecific antibody of the first aspect of the 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 overexpressed antigen, e.g., 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, which may be, for example, the transmembrane domain of cell adhesion molecule-1, CD48 or CD 80. More preferably, wherein 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, wherein the light chain variable region or amino acid sequence of the fusion peptide is as shown in SEQ ID NO: 10. 13, 14 or 15, VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region.

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

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 that specifically binds to epidermal growth factor receptor, carcinoembryonic antigen and prostate specific membrane antigen, respectively; the latter is VL-CK of a monoclonal antibody that specifically binds to the CD3 antigen of T cells.

In a particular 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 epidermal growth factor receptor, carcinoembryonic antigen and prostate specific membrane antigen, respectively; the latter is VH-CH1 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 '-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 epidermal growth factor receptor, carcinoembryonic antigen and prostate specific membrane antigen, respectively.

In a specific embodiment of the invention, the linker peptide may be GGGSGGG, vegggsggsggsggsggvd or (GGGGS) n, wherein n is 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 present invention provides a vector comprising a nucleic acid according to the second aspect of the invention. Preferably the vector of the third aspect of the invention is a plasmid.

In a fourth aspect, the present invention provides a host cell comprising, or transfected with, a 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 invention, comprising the steps of:

(a) culturing the host cell of the fourth aspect of the invention; and (c).

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

In a sixth aspect, the present invention provides a pharmaceutical composition comprising a bispecific antibody of the first aspect of the invention and a pharmaceutically acceptable excipient. The pharmaceutical composition of the sixth aspect of the invention is used for treating a disease, such as a tumor or cancer, treated by the first monoclonal antibody or the second monoclonal antibody.

In a seventh aspect, the invention provides the use of a bispecific antibody of the first aspect of the 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 invention.

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

Drawings

Figure 1 is a schematic of the construct for bispecific antibody a of example 1.

Figure 2 is a schematic diagram of the structure of bispecific antibody a of example 1.

Figure 3 is a schematic of the construct for bispecific antibody B of example 1.

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

Figure 5 is a schematic of the construct for bispecific antibody C of example 2.

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

Figure 7 is a schematic of the construct for bispecific antibody D of example 2.

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

Detailed Description

The bispecific antibody of the present invention is composed of two peptide chains, including a first peptide chain and a second peptide chain, wherein the first peptide chain has a structure shown as VL-CK-TM, and the second peptide chain has a structure 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-means direct peptide bond linkage or linkage through a linking peptide.

In this context, "first" and "second" when referring to the same product are intended to distinguish the modified product, i.e., the first product and the second product are different, but do not limit the structure and/or composition of the product itself. For example, the first monoclonal antibody and the second monoclonal antibody are different monoclonal antibodies.

In this context, a monoclonal antibody consists of a pair of light chains and a pair of heavy chains, which are interconnected 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, namely CH1, CH2, and CH 3; 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 formally highly variable regions of a sequence and/or structurally defined loop, separated by four more conserved Framework Regions (FR), i.e., each variable region is arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4.

CDR Sequences herein may be determined according to the IMGT rules (Brochet X., Nucleic Acids Research, 2008, 36: W503-508 and Lefranc MP., Nucleic Acids Research, 1999, 27:209-212), or 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 can be determined for a given antibody variable region.

Preferably in the bispecific antibody of the first aspect of the invention, one of the first monoclonal antibody and the second monoclonal antibody specifically binds to a T cell, such as the CD3 antigen which specifically binds to a T cell; and the other of the first monoclonal antibody and the second monoclonal antibody specifically binds to a pathogen, preferably a specific antigen or an overexpressed antigen, such as Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), or Prostate Specific Membrane Antigen (PSMA), of a tumor or cancer cell, such as a tumor or cancer cell. The medicinal use of the bispecific antibody of the first aspect of the invention is mainly determined by the monoclonal antibody fragment specifically binding to the pathogen, and the other monoclonal antibody fragment specifically binding to the T cell has the effect of maintaining the adhesion to the T cell for a long time, thereby promoting the corresponding medicinal effect.

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

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

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 comprises a single transmembrane α helix of a transmembrane protein, composed primarily of hydrophobic amino acids.

Herein, a linker peptide refers to one or more amino acid residues inserted between each variable region, constant region and transmembrane protein domain of the bispecific antibody that provide each region or domain with sufficient mobility to provide the binding function of the bispecific antibody. The linking peptide typically comprises flexible amino acid residues, such as Gly, and in the bispecific antibodies of the invention, each linking peptide is independent, and 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, i.e. 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 different vectors, expressed separately, and then combined together.

In this context, the vector is preferably an expression vector, including derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors, and the like. Vectors suitable for expression in bacterial cells include the already commercially available BlueScript (Stratagene), pIN vector (VanHeeke, J Biol Chem, 264,5503-5509(1989)), and pET vector (Novagen, Madison Wis.), among others; 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 used in the embodiments of the present invention.

The expression vector may comprise or be associated with any suitable promoter, enhancer, and other elements that facilitate expression. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter/enhancer and RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), polyA termination sequences, origins of replication for plasmid products in e.coli, antibiotic resistance genes as selectable markers, and/or convenient cloning sites.

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

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.

Herein, pharmaceutically acceptable excipients refer to pharmaceutical excipients that do not produce side effects, allergies or other adverse reactions when administered to mammals (particularly humans) as appropriate, including non-toxic solid or liquid fillers, diluents, encapsulating materials or formulation excipients and the like. Examples of suitable bulking agents, solvents, and/or excipients include one or more of water, amino acids, saline, phosphate buffered saline, glucose, glycerol, ethanol, and the like, as well as combinations thereof. It is also preferred that isotonic agents, such as sugars, polyalcohols or sodium chloride, are included in the pharmaceutical compositions of the present invention. It is also preferred that the pharmaceutical composition of the present invention further comprises 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, intraocular administration, or the like. Preferably, the pharmaceutical compositions of the present invention are injectable formulations, such as isotonic, sterile aqueous solutions, or dried (especially freeze-dried) compositions thereof. The dried composition may constitute 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 the form of a salt. Pharmaceutically acceptable salts include acid addition salts and the corresponding inorganic acids include hydrochloric or phosphoric acids and the corresponding organic acids include acetic, oxalic, tartaric acids and the like. Pharmaceutically acceptable salts may also include salts formed with free carboxyl groups on proteins, corresponding inorganic bases include sodium, potassium, ammonium, calcium or iron hydroxides, and corresponding organic bases include isopropylamine, trimethylamine, glycine, histidine and the like.

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

As used 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 not only directs treatment that results in a complete cure for the disease, but also treatment that slows disease progression and/or prolongs the survival of the subject.

In this context, an effective amount refers to an amount of a drug that is required to achieve a treatment with a reasonable benefit/risk ratio sufficient to treat the corresponding disease. The total daily amount of the bispecific antibody of the invention or the pharmaceutical composition of the invention will be determined by a physician within the scope of safe medical judgment. The specific therapeutically effective amount for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular polypeptide employed; the particular composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration; the route of administration and the rate of excretion of the polypeptide employed; the duration of treatment; a drug used in combination or concomitant use with the polypeptide to be used, and the like.

The present invention incorporates publications which are intended to describe the invention more clearly and which are incorporated herein by reference in their entirety as if reproduced in their entirety.

For the purpose of facilitating understanding, the present invention will be described in detail below with reference to specific embodiments and the accompanying drawings. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in light of the teachings of this specification.

Detailed Description

The present invention is further illustrated by the following examples. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art and commercially available instruments and reagents, and can be referred to in "antibody engineering (second edition) (beijing university medical press)", "molecular cloning experimental guidelines (3 rd edition) (scientific press)", "microbiological experiments (4 th edition) (advanced education press), and the manufacturer's specifications of the corresponding instruments and reagents.

Example 1 preparation of bispecific antibody A of the present invention and experiment of the Effect thereof

Briefly, a plasmid (pLNCX, available from Clontech) containing the construct shown in FIG. 1 was constructed by inserting a light chain variable region and a light chain constant region (α -Tumor VL-CK) encoding an anti-epidermal growth factor receptor antibody between a Leader sequence (Leader SEQ) carried by the plasmid and an Internal Ribosome Entry Site (IRES) in this order, a linker (linker, whose amino acid sequence is shown in SEQ ID NO: 4) having a GGGGSGGGGSGGGGGS) and a cell adhesion Molecule-1 (Intercellularadhesion Molecule 1, ICAM-1) domain D1-D2-D3(ICAM1-D1-3, whose amino acid sequence is shown in SEQ ID NO: 1) having a linker peptide, a DNA encoding an anti-epidermal growth factor receptor antibody and a heavy chain variable region (CD 6326-VH antigen of CD linker) having a Leader sequence (CD 6326-VL-CD 9) linked to a heavy chain variable region (CD antigen), CD linker peptide (CD 9-VL-GCV) having a Leader sequence of GCGGGGSGGGGSGGGGGGGGGGGGGGGGGGGGS and a cell adhesion Molecule-1 (IntercelluladsAGGRGG-1) having an amino acid sequence of D1-D2-D3(ICAM 1-11) having an amino acid sequence shown in this linker amino acid sequence, CD linker peptide sequence shown in SEQ ID NO: 1, CD linker peptide sequence, CD 6326-CD linker peptide linked to CD linker peptide sequence, CD 9-CD linker peptide (CD linker peptide linker) having a CD 9-CD 9, CD 9-CD 9, CD linker peptide linked to CD.

Subsequently, the positive plasmids constructed as described above were transfected into Expi293F cells (available from Thermo Fisher scientific Co., Ltd.) in cell culture medium Expi293^TMExpi Medium (available from Thermo Fisher scientific Co., Ltd.) was cultured in suspension at 37 ℃ and a carbon dioxide concentration of 5%, and the number of Expi293F cells in the Expi293F cell culture solution was maintained at 3-5X 10 by subculture (subculture)⁶cells/ml. After culturing for 7 days, the cells were disrupted by sonication, the cell supernatant was collected by centrifugation at 1000rpm for 10 minutes, and bispecific antibody A expressed by Expi293F cells was purified from the supernatant by Nickel Affinity Chromatography (Nickel Affinity Chromatography).

The light chain constant region of α -Tumor VL-CK and CH1 of α -Tumor VH-CH1 are covalently linked by disulfide bond to form the complete bispecific antibody, the structure of which after expression is shown in FIG. 2.

In addition, as shown in FIG. 3, bispecific antibody B, which was constructed and prepared as a control in substantially the same manner as described above, was different from bispecific antibody A only in that it did not carry cell adhesion Molecule-1 (intercellular adhesion Molecule 1, ICAM-1) domain D1-D2-D3(ICAM 1-D1-3).

The binding time test for T cells was performed for bispecific antibodies a and B, specifically, first, 6 250mL cell culture flasks were prepared, and 20mL of serum-free cell culture medium AIM V (available from thermo fisher Scientific) was added to each of the cell culture flasks, and then bispecific antibody a was added to the cell culture medium in three of the cell culture flasks at a concentration of 5mg/mL as an experimental group sample, and bispecific antibody B was added to the cell culture medium in the other three cell culture flasks at a concentration of 5mg/mL as a control group sample. The 6 cell culture flasks were placed in a cell culture chamber and cultured at 37 ℃ and 5% carbon dioxide, and at 4 time points of 0 hour after the culture, 24 hours after the culture, 48 hours after the culture and 72 hours after the culture, respectively taking out the analysis samples from the cell sap sample of the experimental group and the cell sap sample of the control group, staining the analysis samples of the experimental group and the analysis samples of the control group by fluorescent secondary antibody anti-human IgG fluorescent isothiocyanate (anti-human IgG-FITC), and putting the stained analysis samples into a flow cytometer, because anti-human IgG-FITC binds to bispecific antibody A and bispecific antibody B, therefore, by detecting T cells and T cells marked by anti-human IgG-FITC, it is possible to know whether or not the T cells in the experimental group and the control group are adhered to the corresponding bispecific antibody.

As shown in fig. 4, the affinity of bispecific antibody a (EGFR _ CD3+ ICAM1) and bispecific antibody B (EGFR _ CD3) to T cells decreased with time, but at 3 time points 24 hours, 48 hours, and 72 hours after culture, the affinity of bispecific antibody a to T cells was significantly higher than that of bispecific antibody B to T cells. From the experimental results of the T cell adhesion time test of the bispecific antibody A of example 1, it can be seen that the domains D1-D2-D3 of ICAM-1, which are capable of stabilizing the immune synapse 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 test thereof

Briefly, a plasmid (pLNCX, available from Clontech) containing the construct shown in FIG. 5 was constructed by inserting a light chain variable region and a light chain constant region (α -CD3 VL-CK), whose amino acid sequences are shown in SEQ ID NO: 11), a linker (linker, whose amino acid sequences are GGGGSGGGGSGGGGS) and a cell adhesion Molecule-1 (interstitial addition Molecule 1, ICAM-1) domain D1-D2-D3(ICAM1-D1-3, whose amino acid sequences are shown in SEQ ID NO: 1), a DNA, whose amino acid sequences are shown in SEQ ID NO: 11, a heavy chain variable region (anti-CD3 antigen (anti-CD3), a heavy chain constant region (α -CD3 VL-CK), a linker (heavy chain constant region) and an anti-T cell growth peptide-IgG-11), a heavy chain variable region and an anti-T cell growth peptide (EGFR-VH peptide-VL-IgG 1), a heavy chain variable region (anti-CD-VL-scFv) antibody, whose amino acid sequences are shown in sequence of GGGGGGGGSGGGGGGGGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGGGGGGGGGSGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGS) and a-1, and a heavy cell-GSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSS, and a heavy chain constant region-1, and a heavy chain constant region-IgG-binding peptide-IgG-.

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

The light chain constant region of α -CD3 VL-CK and CH1 of α -CD3 VH-CH1 are covalently linked by disulfide bonds to form the complete bispecific antibody, the structure of which after C expression is shown in FIG. 6.

In addition, as shown in FIG. 7, bispecific antibody D, which was constructed and prepared as a control in substantially the same manner as described above, was different from bispecific antibody C only in that it did not carry cell adhesion Molecule-1 (ICAM-1) domain D1-D2-D3(ICAM 1-D1-3).

The bispecific antibodies a and B were tested for binding time against T cells according to the test method described in example 1. As shown in fig. 8, the affinity of bispecific antibody C (EGFR _ CD3+ ICAM1) and bispecific antibody D (EGFR _ CD3) for T cells decreased with time, but at 3 time points 24 hours, 48 hours, and 72 hours after culture, the affinity of bispecific antibody C for T cells was significantly higher than that of bispecific antibody D for T cells. From the experimental results of the test for the binding time of the bispecific antibody C to T cells in example 2 and the results of example 1, it is clear that neither the improvement in the affinity of the bispecific antibody having a transmembrane protein for T cells nor the improvement in the affinity of the bispecific antibody having a transmembrane protein for T cells is affected by the binding specificity of VL-CK and VH-CH to target cells and the binding specificity of VH-VL to T cells.

Claims

1. A bispecific antibody having the structure of the formula,

VL-CK-TM

|

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,

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

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

2. The bispecific antibody of claim 1, wherein one of the first monoclonal antibody and the second monoclonal antibody specifically binds to a T cell, preferably to the CD3 antigen of a T cell; the other of the first and second monoclonal antibodies specifically binds to a pathogen, preferably to a tumor or cancer cell, more preferably to epidermal growth factor receptor, carcinoembryonic antigen or prostate specific membrane antigen.

3. The bispecific antibody of claim 1, wherein the transmembrane protein domain is a transmembrane domain of a leukocyte differentiation antigen, preferably of cell adhesion molecule-1, CD48 or CD80, more preferably the amino acid sequence of the transmembrane protein domain is as set forth in SEQ ID NO: 1. 2 or 3.

4. The bispecific antibody of claim 1, wherein the amino acid sequence of VL-CK is as set forth in SEQ ID NO: 4. 5, 6 or 11.

5. The bispecific antibody of claim 1, wherein the amino acid sequence of VH-CH1 is as set forth in SEQ ID NO: 7. 8, 9 or 12.

6. The bispecific antibody of claim 1, wherein the amino acid sequence of VH '-VL' is as set forth in SEQ ID NO: 10. 13, 14 or 15.

7. An intermediate product of recombinant DNA technology, which is:

(1) an isolated nucleic acid encoding the bispecific antibody of any one of claims 1-6;

(2) a vector (preferably a plasmid) comprising the nucleic acid according to (1) above; or the like, or, alternatively,

(3) a host cell (preferably a mammalian cell) comprising the vector of (2) above or transfected with the vector of (2) above.

8. A method of producing a bispecific antibody of any one of claims 1-6, comprising the steps of:

(a) culturing the host cell of claim 7; and the combination of (a) and (b),

(b) collecting the bispecific antibody of any one of claims 1-6 from the culture obtained in step (a).

9. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-6 and a pharmaceutically acceptable excipient.

10. Use of a bispecific antibody of any one of claims 1-6 in the manufacture of a medicament for the treatment of a disease treated by the first monoclonal antibody or the second monoclonal antibody, preferably wherein the disease treated by the first monoclonal antibody or the second monoclonal antibody is a tumor 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, gastric cancer, breast cancer, pancreatic cancer, cervical cancer, ovarian cancer, prostate cancer, and prostate cancer.