CD3-COL17A1 bispecific antibody

Abstract

The invention discloses a CD3-COL17A1 bispecific antibody and a preparation method thereof. The CD3-COL17A1 bispecific antibody provided by the invention can be combined with CD3 + T cells and COL17A1 + pancreatic cancer cells, can kill the pancreatic cancer cells at a lower dosage, provides a new thought for COL17A1 and / or CD3 positive tumor treatment, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a CD3-COL17A1 bispecific antibody. Background Art

[0002] Bispecific antibodies (BsAbs) or bispecific monoclonal antibodies (BsMAbs) are artificial proteins composed of two different antibody fragments. However, they face challenges on various fronts. First, they are difficult to produce on an industrial scale. More importantly, these artificial proteins are often unstable both in vitro and in vivo. When a protein contains two antigen-binding domains in close spatial proximity, whether these two antigen-binding domains retain their binding activity to each other is a major concern.

[0003] Therefore, providing BsAbs with higher stability and better therapeutic effects is an urgent problem to be solved in this field. Summary of the Invention

[0004] To overcome the deficiencies of the prior art, the present invention provides a CD3-COL17A1 bispecific antibody.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a bispecific antibody having the following configuration: the heavy chain and light chain of the CD3 antibody are located at the Fab end, and the scFv sequence of the COL17A1 antibody is located at the C-terminus of the constant region.

[0007] Furthermore, the heavy chain of the bispecific antibody comprises the heavy chain, constant region, and scFv of the CD3 antibody from N-terminus to C-terminus, and the light chain of the bispecific antibody consists of the light chain of the CD3 antibody.

[0008] Furthermore, the constant regions of the heavy chain and light chain of the bispecific antibody are of IgG1 type.

[0009] Further, through (G4S) n Linking the constant region to the scFv.

[0010] Furthermore, n is 3.

[0011] Furthermore, the COL17A1 antibody comprises heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 having at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3,

[0012] And light chain variable region complementary determining regions CDR1, CDR2, and CDR3 having at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 9, 10, and 11.

[0013] Furthermore, the amino acid sequences of the heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 of the COL17A1 antibody are shown in SEQ ID NOs: 1, 2, and 3, respectively.

[0014] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NOs: 9, 10, and 11, respectively.

[0015] Furthermore, the COL17A1 antibody further comprises heavy chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7.

[0016] And light chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOs: 12, 13, 14, and 15.

[0017] Furthermore, the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the COL17A1 antibody are shown in SEQ ID NOs: 4, 5, 6, and 7, respectively.

[0018] The amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

[0019] Furthermore, the heavy chain variable region of the COL17A1 antibody has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 8,

[0020] The light chain variable region has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0021] Furthermore, the amino acid sequence of the heavy chain variable region of the COL17A1 antibody is shown in SEQ ID NO: 8.

[0022] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

[0023] Furthermore, the variable region sequence of the COL17A1 antibody has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 17.

[0024] Furthermore, the variable region sequence of the COL17A1 antibody is shown in SEQ ID NO: 17.

[0025] Furthermore, the CD3 antibody comprises heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 having at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 18, 19, and 20,

[0026] And light chain variable region complementarity determining regions CDR1, CDR2, and CDR3 having at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 26, 27, and 28.

[0027] Furthermore, the amino acid sequences of the heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 of the CD3 antibody are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0028] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NOs: 26, 27, and 28, respectively.

[0029] Furthermore, the CD3 antibody further comprises heavy chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOs: 21, 22, 23, and 24,

[0030] And light chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOs: 29, 30, 31, and 32.

[0031] Furthermore, the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the CD3 antibody are shown in SEQ ID NOs: 21, 22, 23, and 24, respectively.

[0032] The amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NOs: 29, 30, 31, and 32, respectively.

[0033] Furthermore, the heavy chain variable region of the CD3 antibody has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 25,

[0034] The light chain variable region has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 33.

[0035] Furthermore, the amino acid sequence of the heavy chain variable region of the CD3 antibody is shown in SEQ ID NO: 25.

[0036] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33.

[0037] Furthermore, the variable region sequence of the CD3 antibody has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 34.

[0038] Furthermore, the variable region sequence of the CD3 antibody is shown in SEQ ID NO: 34.

[0039] Furthermore, the heavy chain variable region of the bispecific antibody has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 37,

[0040] The light chain variable region has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 38.

[0041] Furthermore, the amino acid sequence of the heavy chain variable region of the bispecific antibody is shown in SEQ ID NO: 37,

[0042] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38.

[0043] The second aspect of the present invention provides a nucleic acid encoding the bispecific antibody according to the first aspect of the present invention.

[0044] The third aspect of the present invention provides a vector comprising the nucleic acid described in the second aspect of the present invention.

[0045] Furthermore, the vector further comprises a promoter and / or an enhancer.

[0046] Furthermore, the vector also includes an operably linked nucleic acid molecule.

[0047] Furthermore, the operably linked nucleic acid molecule includes a tag.

[0048] Furthermore, the tags include epitope tags for positioning and tags for purification.

[0049] The fourth aspect of the present invention provides a host cell comprising the nucleic acid of the second aspect of the present invention or the vector of the third aspect of the present invention.

[0050] Furthermore, the host cells include prokaryotic cells and eukaryotic cells.

[0051] Furthermore, the eukaryotic cells include protist cells, animal cells or fungal cells.

[0052] Furthermore, the animal cells include mammalian cells, bird cells, and insect cells.

[0053] The fifth aspect of the present invention provides a derivative, wherein the derivative comprises the bispecific antibody according to the first aspect of the present invention or the nucleic acid according to the second aspect of the present invention linked to a detectable agent.

[0054] Furthermore, the detectable reagents include fluorescent dyes, radioactive labels, metal ions, enzymes, magnetic beads, and colorimetric labels.

[0055] The sixth aspect of the present invention provides a pharmaceutical composition, which comprises the bispecific antibody described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the derivative described in the fifth aspect of the present invention.

[0056] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0057] The seventh aspect of the present invention provides a product for detecting COL17A1 and / or CD3, wherein the product comprises the bispecific antibody according to the first aspect of the present invention or the nucleic acid according to the second aspect of the present invention.

[0058] Furthermore, the product includes a test kit and a test paper.

[0059] Furthermore, the kit further comprises a detectable label conjugated to the bispecific antibody.

[0060] Furthermore, the detectable label includes a fluorescent label, a radioisotope, a chemiluminescent molecule, a paramagnetic ion or a spin-trapping agent.

[0061] The eighth aspect of the present invention provides the use of the bispecific antibody described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the derivative described in the fifth aspect of the present invention in inhibiting tumor cell growth in vitro or in preparing a pharmaceutical composition for treating tumors.

[0062] Furthermore, the tumor is a COL17A1 and / or CD3 positive tumor.

[0063] Furthermore, the tumor is selected from pancreatic cancer.

[0064] Furthermore, the in vitro inhibition of tumor cell growth is inhibition of tumor cell growth for non-therapeutic purposes.

[0065] The ninth aspect of the present invention provides use of the bispecific antibody of the first aspect of the present invention, the nucleic acid of the second aspect of the present invention, the vector of the third aspect of the present invention, the host cell of the fourth aspect of the present invention, or the derivative of the fifth aspect of the present invention in detecting COL17A1 and / or CD3 or in preparing a product for detecting COL17A1 and / or CD3.

[0066] Furthermore, the detection of COL17A1 and / or CD3 is for non-diagnostic purposes.

[0067] Advantages and beneficial effects of the present invention:

[0068] The CD3-COL17A1 bispecific antibody provided in this application can bind to CD3 + T cells and COL17A1 + The combination of IL-17A1 and IL-6 can kill pancreatic cancer cells at a lower dose, providing a new idea for the treatment of COL17A1 and / or CD3 positive tumors and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Schematic diagrams of the structures of two configurations of COL17A1-CD3 bispecific antibodies, wherein 1A is a schematic diagram of the COL17A1-CD3 bispecific antibody structure, and 1B is a schematic diagram of the CD3-COL17A1 bispecific antibody structure;

[0070] Figure 2 Figures 2A and 2B show the expression, purification, and identification of COL17A1-CD3 and CD3-COL17A1 bispecific antibodies. Figure 2A shows the expression of COL17A1-CD3 bispecific antibodies detected by SDS-PAGE under non-reducing and reducing conditions, Figure 2B shows the expression and molecular weight of CD3-COL17A1 bispecific antibodies detected by SDS-PAGE under non-reducing and reducing conditions, and Figure 2C shows the purity and molecular weight of CD3-COL17A1 bispecific antibodies detected by SEC-HPLC.

[0071] Figure 3 Figure 3A shows the binding of the CD3-COL17A1 bispecific antibody to CD3+ T cells, Figure 3B shows the binding of the CD3-COL17A1 bispecific antibody to BxPC-3 cells expressing COL17A1, and Figure 3C shows the binding of the CD3-COL17A1 bispecific antibody to AsPC-1 cells expressing COL17A1.

[0072] Figure 4The diagram shows that CD3-COL17A1 dual antibody can kill COL17A1-positive pancreatic cancer cells. 4A shows the killing of AsPC-1 cells, and 4B shows the killing of BxPC-3 cells. DETAILED DESCRIPTION

[0073] The following provides definitions of some terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0074] The present invention provides a bispecific antibody having the following configuration: the heavy chain and light chain of the CD3 antibody are located at the Fab end, and the scFv sequence of the COL17A1 antibody is located at the C-terminus of the constant region.

[0075] In some embodiments, a bispecific antibody (BsAb) refers to an artificial antibody that can specifically bind to two different antigens or different antigen epitopes at the same time.

[0076] By (G4S) n Linking the constant region to the scFv.

[0077] In some embodiments, n is any natural number, such as 1-10, 1-15.

[0078] In a specific embodiment, n is 3.

[0079] The present invention provides nucleic acids encoding the above-mentioned bispecific antibodies.

[0080] In some embodiments, considering codon degeneracy, the gene encoding the antibody described herein may be modified within its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody. Those skilled in the art may also artificially synthesize and modify the gene based on the codon preference of the host cell expressing the antibody to improve the expression efficiency of the antibody.

[0081] The present invention provides a vector comprising the above nucleic acid.

[0082] In some embodiments, the vector also includes a transcriptional promoter and an optional enhancer, a translation signal, and a transcriptional and translational termination signal. Expression vectors for stable transformation typically have a selectable marker that allows selection and maintenance of transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cell. The vector can also include additional nucleotide sequences that are operably linked to the connected nucleic acid molecule, such as an epitope tag for localization, such as a 6-his tag or a myc tag, or a tag for purification, such as a GST fusion; and sequences for directing protein secretion and / or membrane association.

[0083] In some embodiments, the vector is a virus. The viral vector can be a retroviral vector or a lentiviral vector. The viral vector can also include a nucleic acid sequence encoding a transduction marker. Suitable viral vectors include RNA virus-based vectors, such as retroviral vectors, such as Moloney murine leukemia virus (MLV)-derived vectors, and more complex retroviral vectors, such as lentiviral vectors. HIV-1-derived vectors fall into this category.

[0084] Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses, including adenoviruses, herpes viruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus), and pox viruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, but are not limited to, norwalk viruses, togaviruses, flaviviruses, reoviruses, papillomaviruses, hepatitis viruses, and hepaciviruses. Examples of retroviruses include avian leukosarcoma virus, mammalian C, B, and D viruses, HTLV-BLV groups, lentiviruses, or foamy viruses.

[0085] In some embodiments, the vector is an expression vector. The expression vector according to the present application can direct the replication and expression of the nucleic acid of the present application in a host.

[0086] The present invention provides a host cell comprising the above nucleic acid or the above vector.

[0087] In some embodiments, host cells are cells used to receive, maintain, replicate, and amplify vectors. These include prokaryotic and eukaryotic cells. Prokaryotic cells include Gram-negative or Gram-positive organisms, such as Escherichia coli (DH5α, BL21DE3, BL21DE3pLysS, JM109, TOP10) or Bacilli. Eukaryotic cells include, but are not limited to, protist cells, animal cells, or fungal cells. Animal cells include mammalian cells, avian cells, and insect cells. Mammalian cells include, but are not limited to, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293 cells, 293T cells, and HEK 293F cells.

[0088] The present invention provides a derivative, wherein the derivative comprises a detectable agent or a therapeutic agent linked to the above-mentioned bispecific antibody or the above-mentioned nucleic acid.

[0089] In some embodiments, a detectable agent can be any substance having a detectable physical or chemical property. Such detectable agents are well developed in the field of immunoassays, and generally speaking, most any label useful in such methods can be applied to the provided methods. Thus, a label can be any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical methods. Detectable agents include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radiolabels (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), in particular, radiolabeled (e.g. 157 Gd, 55 Mn, 162 Dy, 52 Cr and 56 Fe), metal ions (e.g. 111 In, 97 Such as 67 Ga, 68 Ga, 72 As、 89 Zr and 201), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), electron transfer agents (including, for example, metal binding proteins and compounds), luminescent and chemiluminescent labels (e.g., luciferin and 2,3-dihydrophthalazines, e.g., luminol), magnetic beads (e.g., DYNABEADS™), and colorimetric labels, such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex).

[0090] The present invention provides a pharmaceutical composition comprising the above-mentioned bispecific antibody, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell or the above-mentioned derivative.

[0091] The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0092] In some embodiments, a pharmaceutically acceptable carrier refers to a non-toxic material that does not interact with the action of the active ingredient of the pharmaceutical composition. The pharmaceutically acceptable carrier refers to a natural or synthetic, organic or inorganic component that is used in conjunction with the active ingredient to facilitate application. According to the present application, a pharmaceutically acceptable carrier includes one or more compatible solid or liquid fillers, diluents, or encapsulating substances, and the pharmaceutically acceptable carrier is suitable for administration to a patient. The components of the pharmaceutical composition of the present application generally do not interact with each other in a manner that would significantly affect the desired therapeutic effect of the drug.

[0093] The present invention provides a product for detecting COL17A1 and / or CD3, wherein the product comprises the above-mentioned bispecific antibody or the above-mentioned nucleic acid.

[0094] The products include test kits and test strips.

[0095] In some embodiments, the kit is packaged in a suitable form. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit may optionally provide other components, such as a buffer. In some embodiments, the kit further comprises another pharmaceutical agent (e.g., the pharmaceutical agent described herein) and / or instructions for use. The instructions for use included with the kit in this application are typically written instructions on a label or package insert (e.g., a paper sheet included with the kit); machine-readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable.

[0096] The present invention provides use of the bispecific antibody, nucleic acid, vector, host cell or derivative for inhibiting tumor cell growth in vitro or in preparing a pharmaceutical composition for treating tumors.

[0097] The tumor is a COL17A1 and / or CD3 positive tumor.

[0098] In some embodiments, the tumor that is positive for COL17A1 and / or CD3 includes, but is not limited to, pancreatic cancer, breast cancer, liver cancer, kidney cancer, glioma, prostate cancer, bile duct cancer, esophageal cancer, mesothelioma, bladder cancer, testicular cancer, gastric cancer, colorectal cancer, endometrial cancer, ovarian cancer, cervical cancer, squamous cell carcinoma (squamous cell carcinoma, such as head and neck squamous cell carcinoma), melanoma, lung cancer, eye tumor, osteosarcoma, lymphoma, acute myeloid leukemia, and multiple myeloma.

[0099] In a specific embodiment, said tumor is selected from pancreatic cancer.

[0100] The present invention will be further described below with reference to specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0101] Example

[0102] 1. Experimental methods

[0103] 1.1 Construction of bispecific antibody expression vector

[0104] The target antibody gene sequence was obtained through gene synthesis and commissioned by Suzhou GeneWeiZhi Technology Co., Ltd. The amplified fragment was inserted into a mammalian expression vector (pcDNA3.1 backbone) via homologous recombination (using the HieffClone™ One-Step Cloning Kit, Yisheng Bio, Catalog No. 10905ES62). The ligation product was transformed into E. coli DH5α competent cells (TAKARA, Catalog No. 9057) for amplification. Positive clones were verified by sequencing (sequencing primers: pc3-L1, pcDNA3-R) to confirm the orientation and integrity of the insert.

[0105] 1.2 HEK293F cell culture and transient transfection of antibody expression plasmids

[0106] HEK293F cells were cultured in serum-free CD medium (Sino Biological, Cat. No. M293TI-1) at 37°C, 8% CO2, and a shaker at 150–175 rpm. Transient transfection procedures were as follows: the cell density was adjusted to 2 × 10 6 cells / mL. Plasmid DNA (heavy and light chain vectors) was mixed with transfection reagent TF1 (Sino Biological, Cat. No. STF02) at a 1:10 (w / v) ratio and added to the culture. Serum-free feed solution (Sino Biological, Cat. No. SMS-293-SUPI) was added on days 1, 3, and 5 post-transfection. Supernatant was collected after 6–7 days of culture for subsequent purification.

[0107] 1.3 Protein purification

[0108] Cell culture supernatants were centrifuged (Thermo ST40R centrifuge, 4,000 × g, 20 minutes) and filtered through a 0.45 μm membrane filter (Sartorius). The filtered supernatant was loaded onto a Protein A affinity chromatography column (GE Healthcare) pre-equilibrated in 50 mM Tris-HCl (pH 8.0). Contaminating proteins were removed by washing with the equilibration buffer. The target antibody was eluted with 100 mM glycine (pH 3.0) and immediately neutralized with 1 M Tris-HCl (pH 8.5). The eluate was then passed through a desalting column or dialyzed into PBS (pH 7.4).

[0109] 1.4 Molecular weight and purity identification of bispecific antibodies

[0110] (1) SDS-PAGE analysis: The purified protein was subjected to 12% SDS-PAGE electrophoresis under reducing (containing β-mercaptoethanol) and non-reducing conditions, and the purity was assessed by Coomassie Brilliant Blue staining (Sino Biological).

[0111] (2) Size exclusion chromatography (SEC-HPLC): Agilent 1290 system (TSKgel G3000SWxl column, 25°C) was used with a flow rate of 0.6 mL / min and a detection wavelength of 280 nm.

[0112] (3) Concentration determination: OD was measured by NanoDrop spectrophotometer (ThermoFisher) 280 .

[0113] 1.5 Flow cytometry detection of CD3-COL17A1 dual antibody and CD3 + T cells and COL17A1 + Binding of pancreatic cancer cells

[0114] (1) Cell preparation: ① CD3 + T cells: CD3 T cells were isolated from PBMC of healthy volunteers using a human T cell purification kit (STEMCELL, Cat. No. 17951). + T cells were activated using human T cell CD3 / CD28 activation magnetic beads (Gibco, 40203D) and the cells were harvested after 48 h for later use. + Pancreatic cancer cells: COL17A1 +Pancreatic cancer cells (Aspc-1 and Bxpc-3) were digested with trypsin (Biyuntian, C0201) and resuspended in RPMI1640 (VivaCell, C3010) medium supplemented with 10% fetal bovine serum (Biochannel, BC-SE-FBS01). They were washed once with PBS and used for further analysis. Aspc-1 and Bxpc-3 cells were purchased from ATCC and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum. All cells were authenticated by short tandem repeat (STR) analysis and confirmed to be free of mycoplasma contamination by polymerase chain reaction (PCR) before use.

[0115] (2) Antibody binding experiment: A two-step dilution method was used to prepare a 0.001–1000 nM concentration gradient: Step 1: dilute the original solution (3.9 μM) at a gradient of 1:10 (5 μL antibody + 45 μL PBS); Step 2: take 45 μL of the dilution solution and mix it with 135 μL PBS to achieve a 4-fold dilution. 5 Cells were incubated with 50 μL of bispecific antibodies at different concentrations on ice for 30 minutes. The cells were washed once with PBS (300 g, 5 minutes). 5% FBS·PBS containing 0.5 μL of YSFluor™ 488 Goat Anti-Rabbit IgG (H+L) fluorescent secondary antibody (YEASEN, 33106ES60) was added (50 μL / tube) and incubated on ice for 30 minutes in the dark. After washing, the cells were resuspended in 300 μL of PBS, filtered through a 40 μm nylon mesh, and analyzed by flow cytometry.

[0116] (3) Flow cytometry and data analysis: BD FACSCelesta flow cytometer was used to collect 10,000 cell events per sample; unstained cells and single-stained secondary antibody groups were used as controls. Median fluorescence intensity (MFI) was calculated using FlowJo v10; nonlinear regression fitting was performed using GraphPad Prism 9 to calculate EC 50 value.

[0117] 1.6 In vitro killing level of CD3-COL17A1 dual antibody based on luciferase fluorescence detection

[0118] (1) Tumor cell digestion, counting, and plating: Use a 96-well plate, add 8,000 luciferase-carrying AsPC-1 or 10,000 BxPC-3 tumor cells to each well in 100 µL of culture medium;

[0119] (2) On the second day, PBMCs were revived and cultured for 3-5 hours. After that, they were mixed with gradient-diluted bispecific antibodies at an effector-target ratio of 5:1 and added to the corresponding wells. 100 μL of the mixture was added to each well. 100 μL of tumor cell culture medium was added to the blank control wells. At the same time, the bispecific antibody mixture was prepared according to the gradient, and 100 μL of culture medium containing bispecific antibodies was added to each well.

[0120] (3) After 48 h of incubation, dilute the Luciferase substrate (Novozyme, DD1202) 1:1 with PBS, add 100 µL of the Luciferase substrate dilution solution to each well, place at room temperature for 10-15 min, then pipette 70 µL into a 96-well white bottom plate and detect using an enzyme-linked microplate reader.

[0121] 2. Experimental results

[0122] 2.1 Design of COL17A1-CD3 and CD3-COL17A1 bispecific antibodies

[0123] The bispecific antibody constructed in this study is a T cell-engaging bispecific antibody with an IgG-scFv fusion configuration. The Fab arm targets one target, retaining the complete IgG Fab structure; the scFv of the other target is fused to the C-terminus of human IgG1Fc through a flexible linker (G4S) 3 to form a dual-target binding capability; the Fc region retains the natural Fc structure of IgG1, aiming to achieve bispecific function in a modular manner while avoiding engineering modifications of the Fab arm. This application also designed two bispecific antibody configurations, one in which the heavy chain and light chain of anti-COL17A1 are located at the Fab end, and the scFv sequence of anti-CD3 is located at the C-terminus of human IgG1 Fc ( Figure 1 A). Another configuration is that the heavy and light chains of anti-CD3 are located at the Fab end, and the scFv sequence of anti-COL17A1 is located at the C-terminus of human IgG1 Fc ( Figure 1 B). Comparison of the ability of two configurations of CD3-COL17A1 bispecific antibodies to express and kill COL17A1-positive pancreatic cancer cells, as well as the differences between the two.

[0124] 2.2 Only the CD3-COL17A1 bispecific antibody could be successfully expressed and purified

[0125] Two bispecific antibodies with different configurations were expressed using 293F cells. However, the COL17A1-CD3 bispecific antibody failed to express successfully in HEK293F cells, and the target antibody could not be captured after purification by Protein A affinity chromatography. Figure 2A); The CD3-COL17A1 bispecific antibody was successfully expressed. Under reducing conditions, SDS-PAGE showed a single major band of approximately 200 kDa, which is consistent with the theoretical value ( Figure 2 B), the purity was 97.6%, indicating no significant degradation or impurities. The main peak of SEC-HPLC accounted for 96.6%, corresponding to the monomeric bispecific antibody with a molecular weight of approximately 200 kDa, which is consistent with the theoretical value ( Figure 2 C).

[0126] 2.3 CD3-COL17A1 dual antibody and CD3 + T cells and COL17A1 + Pancreatic cancer cell binding

[0127] Flow cytometry was used to detect the binding ability of CD3-COL17A1 bispecific antibody to T cells and pancreatic cancer cells BxPC-3 and AsPC-1 expressing COL17A1. + T cell binding, the concentration is close to saturation at around 10 nM; at the same time, CD3-COL17A1 can also bind to BxPC-3 and AsPC-1 cells expressing COL17A1, and the maximum binding concentrations of the two cells are different, which may be related to the difference in antigen density between the two cells.

[0128] 2.4 CD3-COL17A1 dual antibody can kill COL17A1-positive pancreatic cancer cells

[0129] The cytotoxicity of the CD3-COL17A1 dual antibody against COL17A1-positive pancreatic cancer cells, AsPC-1 and BxPC-3, was evaluated using human PBMCs as effector cells. Human PBMCs were co-cultured with BxPC-3 and AsPC-1 cells at effector-target ratios of 5:1 and 10:1 for 48 hours. Different concentrations of the CD3-COL17A1 dual antibody were then administered. Luciferase fluorescence activity assay was used to detect the cytotoxicity. Results showed that the CD3-COL17A1 dual antibody significantly cytotoxicized BxPC-3 and AsPC-1 cells in the presence of PBMCs, and the CD3-COL17A1 dual antibody was able to kill BxPC-3 and AsPC-1 cells at lower doses.

[0130] Amino acid sequences involved in this application

[0131] 1) Anti-COL17A1 heavy chain amino acid sequence

[0132] CDR1: GFTFSNYG (SEQ ID NO:1); CDR2: ISYDGSRK (SEQ ID NO:2); CDR3: AKGFYYDWGTYYDY (SEQ ID NO:3)

[0133] FR1: EVQLLEESGGGVVQPGRSLRLSCAAS (SEQ ID NO:4); FR2: MHWVRQAPGKGLEWVAA (SEQ ID NO:5); FR3: YYADSVKGRFTLSRDNSKNTLYLQMNSLIAEDTAVYYC (SEQ ID NO:6); FR4: WGQGTLVTVSS (SEQ ID NO:7)

[0134] Heavy chain (H chain): EVQLLEESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAAISYDGSRKYYADSVKGRFTLSRDNSKNTLYLQMNSLIAEDTAVYYCAKGFYYDWGTYYDYWGQGTLVTVSS (SEQ ID NO:8)

[0135] 2) Amino acid sequence of the light chain of anti-COL17A1

[0136] CDR1: SSNIGAGYD (SEQ ID NO:9); CDR2: ANS (SEQ ID NO:10); CDR3: QSYDSSLTGEV (SEQ ID NO:11)

[0137] FR1: ELTQPPSVSGAPGQRVTISCTGS (SEQ ID NO:12); FR2: VHWYQQLPGTAPKLLMY (SEQID NO:13); FR3: HRPSGVPDRFSGSKSGTSASLAIAGLQAEDEAHYYC (SEQ ID NO:14); FR4: FGGGTKLTVL (SEQ ID NO:15)

[0138] Light chain (L chain): ELTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLMYANSHRPSGVPDRFSGSKSGTSASLAIAGLQAEDEAHYYCQSYDSSLTGEVFGGGTKLTVLGQP (SEQ ID NO:16)

[0139] 3) Amino acid sequence of the scFv of anti-COL17A1

[0140] EVQLLEESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAAISYDGSRKYYADSVKGRFTLSRDNSKNTLYLQMNSLIAEDTAVYYCAKGFYYDWGTYYDYWGQGTLVTVSSGGGGSGGGGSGGGGSELTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLMYANSHRPSGVPDRFSGSKSGTSASLAIAGLQAEDEAHYYCQSYDSSLTGEVFGGGTKLTVLGQP (SEQ ID NO:17)

[0141] 4) Amino acid sequence of the heavy chain of anti-CD3

[0142] CDR1: GFTFNTYA (SEQ ID NO:18); CDR2: IRSKYNNYAT (SEQ ID NO:19); CDR3: ARHGNFGNSYVSWFAY (SEQ ID NO:20)

[0143] FR1: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:21); FR2: MNWVRQAPGKGLEWVAR (SEQ ID NO:22); FR3: YYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC (SEQ ID NO:23); FR4: WGQGTLVTVSS (SEQ ID NO:24)

[0144] Heavy chain (H chain): EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ IDNO:25)

[0145] 5) Amino acid sequence of the light chain of anti-CD3

[0146] CDR1: TGAVTTSNY (SEQ ID NO:26); CDR2: GTN (SEQ ID NO:27); CDR3: ALWYSNLWV (SEQ ID NO:28)

[0147] FR1: QTVVTQEPSLTVSPGGTVTLTCRSS (SEQ ID NO:29); FR2: ANWVQQKPGQAPRGLIG (SEQ ID NO:30); FR3: KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYC (SEQ ID NO:31); FR4: FGGGTKLTVL (SEQ ID NO:32)

[0148] Light chain (L chain): QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO:33)

[0149] 6) Amino acid sequence of scFv against anti-CD3

[0150] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO:34)

[0151] 7) Amino acid sequence of the heavy chain of the COL17A1-CD3 bispecific antibody

[0152] EVQLLEESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAAISYDGSRKYYADSVKGRFTLSRDNSKNTLYLQMNSLIAEDTAVYYCAKGFYYDWGTYYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO:35)

[0153] 8) Amino acid sequence of the light chain of the COL17A1-CD3 bispecific antibody

[0154] ELTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLMYANSHRPSGVPDRFSGSKSGTSASLAIAGLQAEDEAHYYCQSYDSSLTGEVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQID NO:36)

[0155] 9) Heavy chain amino acid sequence of CD3-COL17A1 bispecific antibody

[0156] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEVQLLEESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAAISYDGSRKYYADSVKGRFTLSRDNSKNTLYLQMNSLIAEDTAVYYCAKGFYYDWGTYYDYWGQGTLVTVSSGGGGSGGGGSGGGGSELTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLMYANSHRPSGVPDRFSGSKSGTSASLAIAGLQAEDEAHYYCQSYDSSLTGEVFGGGTKLTVLGQP (SEQ ID NO:37)

[0157] 10) Amino acid sequence of the light chain of the CD3-COL17A1 bispecific antibody

[0158] QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO:38)

[0159] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A bispecific antibody, characterized in that: The configuration of the bispecific antibody is as follows: the heavy chain and light chain of the CD3 antibody are located at the Fab end, and the scFv sequence of the COL17A1 antibody is located at the C-terminus of the constant region; Preferably, the heavy chain of the bispecific antibody comprises, from N-terminus to C-terminus, the heavy chain of the CD3 antibody, the constant region, and the scFv of the COL17A1 antibody, and the light chain of the bispecific antibody consists of the light chain of the CD3 antibody; Preferably, the constant regions of the heavy and light chains of the bispecific antibody are of IgG1 type; Preferably, through (G4S) n Linking the constant region to the scFv; Preferably, n is 3.

2. The bispecific antibody according to claim 1, characterized in that The COL17A1 antibody comprises heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 having at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, and light chain variable region complementarity determining regions CDR1, CDR2, and CDR3 having at least 95% sequence identity to the amino acid sequences shown in SEQ ID NOs: 9, 10, and 11; Preferably, the COL17A1 antibody further comprises heavy chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7. and light chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity to the amino acid sequences shown in SEQ ID NOs: 12, 13, 14, and 15; Preferably, the heavy chain variable region of the COL17A1 antibody has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:

8. The light chain variable region has at least 70% sequence identity to the amino acid sequence shown in SEQ ID NO: 16; Preferably, the variable region sequence of the COL17A1 antibody has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; Preferably, the CD3 antibody comprises heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 having at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 18, 19, and 20, and light chain variable region complementarity determining regions CDR1, CDR2, and CDR3 having at least 95% sequence identity to the amino acid sequences shown in SEQ ID NOs: 26, 27, and 28; Preferably, the CD3 antibody further comprises heavy chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOs: 21, 22, 23, and 24, and light chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity to the amino acid sequences shown in SEQ ID NOs: 29, 30, 31, and 32; Preferably, the heavy chain variable region of the CD3 antibody has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:

25. The light chain variable region has at least 70% sequence identity to the amino acid sequence shown in SEQ ID NO: 33; Preferably, the variable region sequence of the CD3 antibody has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 34; Preferably, the heavy chain variable region of the bispecific antibody has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO:

37. The light chain variable region has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO:

38.

3. A nucleic acid encoding the bispecific antibody according to claim 1 or 2.

4. A vector comprising the nucleic acid of claim 3; Preferably, the vector further comprises a promoter and / or an enhancer; Preferably, the vector further comprises an operably linked nucleic acid molecule; Preferably, the operably linked nucleic acid molecule comprises a tag; Preferably, the tag includes an epitope tag for localization and a tag for purification.

5. A host cell comprising the nucleic acid of claim 3 or the vector of claim 4; Preferably, the host cell includes a prokaryotic cell or a eukaryotic cell; Preferably, the eukaryotic cell comprises a protist cell, an animal cell or a fungal cell; Preferably, the animal cells include mammalian cells, avian cells, and insect cells.

6. A derivative, characterized in that The derivative comprises the bispecific antibody according to claim 1 or 2 or the nucleic acid according to claim 3 linked to a detectable agent; Preferably, the detectable reagent comprises a fluorescent dye, a radioactive label, a metal ion, an enzyme, a magnetic bead, or a colorimetric label.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the bispecific antibody of claim 1 or 2, the nucleic acid of claim 3, the vector of claim 4, the host cell of claim 5, or the derivative of claim 6; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8. A product for detecting COL17A1 and / or CD3, characterized in that: The product comprises the bispecific antibody of claim 1 or 2 or the nucleic acid of claim 3; Preferably, the product includes a test kit and a test paper; Preferably, the kit further comprises a detectable label conjugated to the bispecific antibody; Preferably, the detectable label comprises a fluorescent label, a radioisotope, a chemiluminescent molecule, a paramagnetic ion or a spin-trapping agent.

9. Use of the bispecific antibody of claim 1 or 2, the nucleic acid of claim 3, the vector of claim 4, the host cell of claim 5, or the derivative of claim 6 for inhibiting tumor cell growth in vitro or in preparing a pharmaceutical composition for treating tumors; Preferably, the tumor is a COL17A1 and / or CD3 positive tumor; Preferably, the tumor is selected from pancreatic cancer.

10. Use of the bispecific antibody according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4, the host cell according to claim 5, or the derivative according to claim 6 for detecting COL17A1 and / or CD3 or in the preparation of a product for detecting COL17A1 and / or CD3.

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