Targeted human CEACAM5 full-human single-domain antibody drug conjugate and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to a fully humanized single-domain antibody drug conjugate targeting human CEACAM5 and application of the fully humanized single-domain antibody drug conjugate. The invention particularly relates to screening, expression and cysteine engineering modification of the fully humanized single-domain antibody and preparation and purification methods of an antibody drug conjugate. The invention relates to a fully-humanized single-domain antibody, which is characterized in that the 84th amino acid of the antibody is substituted by cysteine, and the substituted cysteine provides free sulfydryl which can be used for coupling, so that a cytotoxic agent, an enzyme or a detection marker and the like are coupled to the modified fully-humanized single-domain antibody, and the fully-humanized single-domain antibody is applied to cancer treatment or immunoassay.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a fully human single-domain antibody specifically binding to human CEACAM5 and an antibody-drug conjugate thereof, as well as methods for screening, expressing, engineering modification of cysteine ​​residues of the fully human single-domain antibody, and preparing and purifying the antibody-drug conjugate, a corresponding pharmaceutical composition and applications thereof. Background Art

[0002] Prior art discloses that antibody-based immunotherapy is very effective in treating a variety of cancers, and the development of successful immunotherapy depends mainly on the discovery of antibodies that target cell surface proteins that are preferentially expressed in tumor cells. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM5) is a cell surface glycoprotein that belongs to processes such as cell adhesion, signal transduction, angiogenesis, cancer occurrence and metastasis. CEACAM5 protein is highly expressed in a variety of tumors, including gastric cancer, colorectal cancer, lung cancer, pancreatic cancer, etc., but its expression level in normal tissues is extremely low. Therefore, it is widely used in the diagnosis, prognosis and monitoring of treatment effects of cancer, and is also an important target for tumor immunotherapy.

[0003] In recent years, researchers have discovered a class of antibodies containing only the heavy chain variable region in alpaca serum. It is the smallest antibody fragment that exists in nature and has the ability to bind to antigens, called nanobodies (VHH) or single-domain antibodies (sdAb) (Protein Engineering, 1994.7(9): p.1129-1135.). These nanobodies have high antigen affinity and specificity, similar to full-length IgG monoclonal antibodies. More importantly, these antibodies are easier to modify and can be expressed in prokaryotic systems, so the preparation cost is very low (Front Immunol, 2017.8: p.1802.). However, these small molecule antibodies are currently mainly derived from camels or sharks, and are prone to produce immune responses in the human body, thus limiting their clinical application. Although animal-derived nanobodies can be humanized through genetic engineering technology, the antibodies are prone to lose thermal stability and it is difficult to maintain antibody affinity during this process. Recently, it was found that a certain subfamily of human heavy chain variable regions also has excellent stability, solubility, low aggregation, excellent expression and purification yield, and characteristics suitable for phage display systems (J Mol Biol, 2008. 382 (3): p. 779-789.). Based on these findings, we used this heavy chain variable region subfamily as a framework and introduced the heavy chain CDR regions (CDR1, CDR2, CDR3) of all antibody subfamilies from healthy adults and newborns to construct a fully human single-domain antibody phage display library. This antibody library was used to screen for specific fully human single-domain antibodies targeting the human CEACAM5 target.

[0004] According to records, in the treatment of cancer, antibody conjugates (ADCs) are new anticancer drugs developed in recent years. Traditional antibody conjugates are generally prepared by reducing the disulfide bonds between antibody chains and coupling payloads to cysteine ​​or lysine. Lysine coupling will result in 0-8 drug molecules being coupled to each antibody. Peptide mapping experiments show that coupling sites generally occur on approximately 20 lysine residues in the heavy and light chains (there are approximately 40 lysines on an antibody). Therefore, this coupling method will produce millions of ADC drug molecules in different forms. In addition, the differences in drug molecular loading and coupling sites of ADC mixtures will also double the number of heterogeneous diversity. The diverse heterogeneous ADCs coupled at different sites also have different PK kinetic properties in vivo, and the difficulty of later industrial production will increase, and the consistency of samples between batches will face very large production challenges.

[0005] According to the data, site-specific conjugation technology can ensure that a certain number of drug molecules are site-specifically conjugated to specific sites of antibodies, which largely ensures drug homogeneity and batch production stability in production. In addition, the DAR value can also be precisely controlled. Site-specific ADCs technology not only makes it possible to study the effects of conjugation at different sites on the efficacy of ADCs, but can also be widely used in the development of conjugated drugs of other molecules such as nuclides, immunotoxins, proteins, precursor enzymes and antibodies, greatly improving the therapeutic effects and applications of these conjugated drugs. At present, site-specific ADC technology generally uses technologies such as engineering cysteine ​​sites, non-natural amino acids, selenocysteine ​​and enzyme (glutamine, sugar engineering, FGE) conjugation to specifically conjugate drug molecules to targeted antibodies. Summary of the invention

[0006] The purpose of the present invention is to solve the above technical problems and obtain a fully human single domain antibody that specifically binds to human CEACAM5 through a unique fully human single domain antibody research and development platform, and its amino acid sequence is shown in SEQ ID NO: 1. The present invention discloses a cysteine-engineered fully human single domain antibody that binds to human CEACAM5, which is mainly characterized in that the 84th amino acid of the antibody is replaced by cysteine, and the replaced cysteine ​​provides a free thiol group that can be used for coupling, thereby achieving the coupling of cytotoxic agents, enzymes or detection markers to the modified fully human single domain antibody, and applying it to cancer treatment or immunoassay.

[0007] Preferably, the fully human single-domain antibody that specifically binds to human CEACAM5 comprises the amino acid sequence shown in SEQ ID NO: 1 or a sequence that has at least 90% identity thereto.

[0008] Preferably, the single domain antibody is the heavy chain variable region (VH).

[0009] Preferably, the heavy chain variable region (VH) is fully human.

[0010] The present invention provides a cysteine-engineered fully human single-domain antibody that binds to human CEACAM5, characterized in that the sequence of the cysteine-engineered fully human single-domain antibody comprises an amino acid sequence after amino acid position 84 of SEQ ID NO: 1 is replaced by cysteine.

[0011] Preferably, the cysteine ​​engineered fully human single domain antibody of the present invention comprises at least one free thiol group.

[0012] Preferably, the free thiol groups of the present invention can be chemically coupled to cytotoxic agents, chemotherapeutic agents, radionuclides, nucleic acids or detectable labels.

[0013] The cytotoxic agents described in the present invention include any agents that are harmful to cells, including but not limited to cytochalasin B, gramicidin D, ethidium bromide, mitomycin, vincristine, vinblastine, colchicine, daunomycin, puromycin, cyclophosphamide, lidocaine, etc., and their analogs or homologs.

[0014] The chemotherapeutic agents of the present invention include, but are not limited to, paclitaxel, doxorubicin, carboplatin, melphalan, vinca alkaloids, methotrexate, mitomycin C and others.

[0015] The radioactive nuclides described in the present invention include, but are not limited to, zinc, sulfur, manganese, palladium, phosphorus, fluorine, cobalt, iodine, etc.; positron emitting metals using various positron emission tomography scans, and non-radioactive paramagnetic metal ions.

[0016] The nucleic acid of the present invention can be selected from DNA, RNA, short interfering RNA (siRNA), small molecule RNA, hairpin or nucleic acid mimetic such as peptide nucleic acid.

[0017] The detectable markers described in the present invention include but are not limited to various enzymes, including but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; prosthetic groups, including but not limited to avidin / biotin, antibiotic protein / biotin; fluorescent substances, including but not limited to 7-hydroxycoumarin, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, and phycoerythrin; luminescent substances, including but not limited to bioluminescent substances, including but not limited to luciferase, luciferin, and photoprotein.

[0018] The present invention provides nucleic acids encoding the cysteine ​​engineered fully human single domain antibodies that bind to human CEACAM5 antigen. The present invention provides plasmids comprising the nucleic acids, optionally operably linked to regulatory sequences, such as promoters, enhancers, and the like. The present invention provides host cells comprising the plasmids, and methods for producing and optionally recovering the cysteine ​​engineered fully human single domain antibodies that bind to human CEACAM5 antigen. The host cells of the present invention may be any prokaryotic or eukaryotic cells, including but not limited to bacterial cells (e.g., Escherichia coli, Bacillus subtilis), insect cells (e.g., using a baculovirus expression system), yeast or mammalian cells (e.g., CHO or BHK cell lines). Other suitable host cells are known to those skilled in the art.

[0019] The present invention provides an antibody conjugate of a fully human single-domain antibody that binds to a human CEACAM5 antigen, characterized in that it comprises the cysteine ​​engineered fully human single-domain antibody that binds to a human CEACAM5 antigen described in the present invention and a conjugate.

[0020] Preferably, the conjugate of the present invention is a cytotoxic agent, a chemotherapeutic agent, a radionuclide, a nucleic acid or a detectable marker.

[0021] The present invention provides a pharmaceutical composition, which is prepared by mixing an effective preventive or therapeutic dose of the cysteine ​​engineered fully human single domain antibody binding to human CEACAM5 antigen of the present invention or its nucleic acid molecule, plasmid or antibody conjugate of the cysteine ​​engineered fully human single domain antibody binding to human CEACAM5 antigen, and a physiologically or pharmaceutically acceptable carrier, excipient or stabilizer, and the composition includes but is not limited to a lyophilized dosage form, an aqueous solution dosage form, a liposome or a capsule dosage form, etc. The concentration of the cysteine ​​engineered fully human single domain antibody binding to human CEACAM5 antigen of the present invention or its nucleic acid molecule, plasmid or antibody conjugate of the cysteine ​​engineered fully human single domain antibody binding to human CEACAM5 antigen can be varied from about 0.1% to 100% (by weight).

[0022] further,

[0023] The present invention provides a method for preparing an antibody conjugate of a cysteine-engineered fully human single-domain antibody that binds to a human CEACAM5 antigen, the method comprising the following steps:

[0024] (1) expressing and purifying a cysteine-engineered fully human single-domain antibody that binds to the human CEACAM5 antigen;

[0025] (2) adding a conjugate that can chemically couple with the free thiol group to react and prepare an antibody conjugate;

[0026] (3) Filter the unreacted conjugate through molecular sieve.

[0027] Preferably, the conjugate in the method of the present invention includes, but is not limited to, a cytotoxic agent, a chemotherapeutic agent, a radionuclide, a nucleic acid or a detectable marker.

[0028] The present invention provides a detection kit, comprising the cysteine ​​engineered fully human single domain antibody, its nucleic acid molecule or plasmid, or its antibody conjugate according to the present invention. The detection kit is used to detect tumor cells, and the tumor cells include various benign tumor cells, malignant tumor cells (i.e., cancer cells), solid tumor cells, and hematogenous cancer cells.

[0029] The present invention provides a method for diagnosing, preventing or treating a disease, comprising administering to a subject the cysteine-engineered fully human single-domain antibody that binds to the human CEACAM5 antigen, the nucleic acid molecule, the plasmid, the antibody conjugate of the cysteine-engineered fully human single-domain antibody that binds to the human CEACAM5 antigen, and the pharmaceutical composition described in the present invention.

[0030] Disease of the present invention is cancer. Cancer of the present invention includes but is not limited to lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenoma, melanoma and non-leukemic leukemia or lymphoid malignancy. More specific examples of the above-mentioned cancer include squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, pancreatic cancer, malignant glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penis cancer, testicular cancer, esophageal cancer, bile duct tumor, head cancer, neck cancer, bone marrow stromal tumor, osteoclastoma, multiple myeloma, osteolytic bone cancers), central nervous system tumors, brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma, and retinoblastoma), nasopharyngeal carcinoma, basal cell carcinoma, bile duct carcinoma, Kaposi's sarcoma, primary liver or endometrial cancer, and tumors of the vascular system (angiosarcoma and hemagiopericytoma).

[0031] The cysteine ​​engineered fully human single domain antibody, nucleic acid molecule, plasmid, antibody conjugate of cysteine ​​engineered fully human single domain antibody and pharmaceutical composition binding to human CEACAM5 antigen of the present invention can be administered to human or animal subjects by various administration routes, which usually depend on the characteristics of the disease to be treated. In general, the method of the present invention can be implemented by any medically acceptable mode of administration, including oral, rectal, topical, intraocular, intracisternal, intraventricular, intratracheal, intranasal instillation, transdermal, subcutaneous, intrathecal, intramuscular, peritoneal, intraperitoneal, intracranial infusion or intravenous infusion.

[0032] The present invention performs cysteine ​​engineering modification on a fully human single domain antibody that can bind to the human CEACAM5 target, that is, the 84th amino acid of the human CEACAM5 fully human single domain antibody is replaced by cysteine, and the replaced cysteine ​​provides a free thiol group that can be used for coupling, thereby coupling cytotoxic agents, enzymes or detection markers to the modified fully human single domain antibody, and applying it to cancer treatment or immunoassay and detection. The site-specific coupling technology of the present invention can ensure that a certain number of drug molecules are site-specifically coupled to specific sites of the antibody, and to a large extent ensure drug homogeneity and batch production stability in production.

[0033] In order to more thoroughly understand the invention, some definitions are listed below. The above definitions are intended to include grammatical equivalents.

[0034] As used herein, "amino acid" means one of the 20 naturally occurring amino acids or any non-natural analogs, which may be located at a specified position. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chains may be in the (R) or (S) configuration. In a preferred embodiment, the amino acids are present in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substitutions may be used, for example to prevent or delay degradation in vivo.

[0035] The term "free thiol" as used herein refers to a functional group in cysteine ​​which is formed by a sulfur atom and a hydrogen atom connected together, and has no connecting group at its end.

[0036] "Antibody" herein means a protein consisting of one or more polypeptides encoded by all or part of a substantially recognized immunoglobulin gene. The recognized immunoglobulin genes, for example, in humans, include kappa (κ), lambda (λ) and heavy chain loci, which contain numerous variable region genes, and constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), alpha (α) encoding IgM, IgD, IgG, IgE and IgA isotypes, respectively. Antibodies herein are meant to include full-length antibodies and antibody fragments, as well as natural antibodies from any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic purposes or other purposes as further specified below. The term "antibody" includes antibody fragments, as known in the art, such as Fab, Fab', F(ab')2, Fv, scFv or other antigen-binding subsequences of antibodies, or antibody fragments produced by modifying intact antibodies or those synthesized de novo using recombinant DNA technology. The term "antibody" includes monoclonal and polyclonal antibodies. The antibody may be an antagonist, an agonist, a neutralizing antibody, or an inhibitory antibody, or a stimulatory antibody. The antibody of the present invention may be a non-human antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0037] As used herein, "antigen" means a compound, composition or substance that can stimulate antibody production or T cell response in an animal, including compositions injected or absorbed into an animal, which may be proteins, carbohydrates, lipids or other pathogens.

[0038] " Nucleic acid " used herein means the polymer consisting of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants and their synthesized non-naturally occurring analogs) by phosphodiester bonds. Therefore, the term includes nucleotide polymers, wherein nucleotides and the bonds therebetween include non-naturally occurring synthetic analogs, such as but not limited to thiophosphates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, peptide nucleic acids (PNA) etc. For example, these polynucleotides can be synthesized using an automatic DNA synthesizer. The term " oligonucleotide " generally refers to short polynucleotides, generally not more than about 50 nucleotides. It should be understood that when the nucleotide sequence is represented by a DNA sequence (i.e. A, T, G, C), this also includes RNA sequences (i.e. A, U, G, C) in which "U" replaces "T".

[0039] Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5' end; the left-hand direction of a double-stranded nucleotide sequence is the 5' direction. The direction in which 5' to 3' nucleotides are added to the nascent RNA transcript is called the transcription direction. The DNA strand with the same sequence as the mRNA is called the coding strand.

[0040] As used herein, "coding" means the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, used as a template for synthesizing other polymers and macromolecules in a biological process with a determined nucleotide sequence, or a determined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA produced by the gene produces protein in a cell or other biological system, the gene encodes a protein. The coding strand (its nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and the non-coding strand (used as a transcription template, gene or cDNA) can be referred to as a coded protein. Or other products of the gene or cDNA. Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The nucleotide sequences encoding proteins and RNA may include introns.

[0041] As used herein, "plasmid" means a plasmid artificially constructed on the basis of a natural plasmid to adapt to laboratory operations. A nucleic acid molecule can be introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication, and may also include one or more selectable marker genes and other genetic elements known in the art.

[0042] The "host cell" used herein is also called a recipient cell, and refers to a host cell that receives foreign genes during transformation and transduction (infection).

[0043] As used herein, "pharmaceutically acceptable carrier" means a conventional pharmaceutically acceptable carrier. Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, Pa., 15th edition (1975), describes compositions and formulations suitable for drug delivery of one or more therapeutic compounds or molecules (e.g., one or more antibodies), as well as additional agents.

[0044] As used herein, "diagnosing" a disease means determining a patient's condition and its progression after examination. "Preventing" a disease means inhibiting the full development of a disease. "Treatment" means therapeutic intervention to ameliorate the signs or symptoms of a disease or pathological condition after it has begun to develop.

[0045] "Administering" herein means selecting an appropriate route to introduce the substance into a subject. For example, if the selected route is intravenous, the composition is administered by introducing the substance into a vein of the subject.

[0046] Herein, "effective preventive / therapeutic dose" means a certain amount of a specific agent sufficient to achieve the desired effect in a subject treated with the agent. The exact dose will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques. The dosage range may be 0.01-100 mg / kg body weight or greater, such as 0.1, 1, 10 or 50 mg / kg body weight, preferably 1-10 mg / kg. As is known in the art, adjustments may be necessary for antibody or Fc fusion degradation, systemic or local drug delivery and new protease synthesis rates, as well as age, weight, general health status, sex, diet, administration time, drug interactions, and severity of the disease, and may be determined by those skilled in the art by conventional experimental methods. Such agents include monomeric Fc domain molecules described herein. In a non-limiting example, this may be the amount of HIV-specific monomeric Fc domains (or HIV-specific CH3 domain molecules) used to prevent, treat or improve HIV infection. Ideally, a therapeutically effective amount of an antibody is an amount sufficient to prevent, treat or ameliorate an infection or disease, such as caused by HIV infection in a subject without causing significant cytotoxic effects in the subject. The therapeutically effective amount of an agent used to prevent, ameliorate and / or treat a subject will depend on the subject being treated, the type and severity of the affliction, and the manner in which the therapeutic composition is administered.

[0047] "Cancer" herein is a solid tumor or hematogenous cancer. Solid tumors of the present invention are sarcomas or cancers, such as fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, or another sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, bladder cancer or central nervous system tumors (such as gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal glands, hemangioblastomas, acoustic neuromas, oligodendrogliomas, hemangiomas, melanomas, neuroblastomas or retinoblastomas). The blood-borne cancer described in the present invention is a leukemia, such as acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia or myelodysplasia.

[0048] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" include plural indicators. It should also be understood that all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weights or molecular weight values ​​are approximate and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprising" means "including". All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification (including term explanations) shall prevail. In addition, materials, methods and embodiments are illustrative only and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 .SDS-PAGE detection of B9 antibody.

[0050] Figure 2 .B9 antibody binding to CEACAM5 protein.

[0051] Figure 3 .ForteBio was used to detect the binding affinity of the fully human single-domain antibody B9 and the cysteine-modified B9 to the CEACAM5 antigen.

[0052] Figure 4 .Flow cytometry was used to detect the binding of fully human single-domain antibody B9 to various cell lines.

[0053] Figure 5 .SDS-PAGE detection of B9(S84C) and antibody conjugate B9(S84C)-vcMMAE.

[0054] Figure 6 .Binding of antibody conjugates to CEACAM5 protein.

[0055] Figure 7 .The cell-killing activity of antibody conjugates. DETAILED DESCRIPTION

[0056] The standard recombinant DNA technology and molecular cloning techniques used in the examples are well known in the art (Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience), and the materials and methods suitable for microbial growth are well known in the art. The main chemical and biological reagents were purchased from KAPABiosystems, New England Biolabs, TransGenBiotech, Thermo Fisher Scientific, OMEGAbio-tek, etc.

[0057] The present invention is described in detail below with reference to specific embodiments.

[0058] Example 1 Production of fully human single domain antibodies against CEACAM5

[0059] The preparation of the soluble expression product of the fully human single-domain antibody against CEACAM5 was basically carried out according to the literature (CellHostMicrobe.2017.22(4):471-483.e5.). Specifically, the recombinant plasmid of the fully human single-domain antibody B9 (antibody sequence is SEQID NO: 1) (see Chinese patent: 2016110409818) was transferred into HB2151 competent cells (from the plasmid vector strain cell gene preservation center), and a single colony was picked from an overnight grown ampicillin plate (16 g tryptone, 10 g yeast extract, 5 g NaCl, 2 g agar powder, distilled water to 1 L, autoclaved. 100 μg / ml Amp and 2% Glucose were added and stored at 4°C), and SB bacterial culture medium (30 g tryptone, 20 g yeast extract, 10 g 3-(N-morpholino)propanesulfonic acid (MOPS), distilled water to 1 L, and the pH was adjusted to 7.0, autoclaved, and stored at 4°C) was inoculated, and IPTG (23.8 g IPTG was dissolved in 100 mL of deionized water, filtered and sterilized with a 0.22 μm filter, and then dispensed into sterile 1.5 mL Eppendorf tubes, stored at -20°C, and expressed under induction conditions (using a concentration of 1 mM) for 12-14 hours. Ni-NTA resin (GE Healthcare, product number: 170531801) was equilibrated with an equilibrium buffer (PBS buffer containing 2.5 M NaCl), and the supernatant of the bacterial lysate containing the antibody was added to the Ni-NTA resin, and the flow rate was controlled at 1 mL / min. The impurities were eluted in sequence with 2 column volumes of equilibrium buffer and 20 mM imidazole (purchased from SigmaAldrich), and the bound antibodies were finally eluted with 250 mM imidazole. The eluted solution was transferred to an Amicon Ultra centrifugal ultrafiltration tube of 3 kD MWCO (purchased from Millipore), ultrafiltered at 4°C and 6500 g for 20 min, and the elution buffer was replaced with a PBS solution, and the ultrafiltration was repeated 3 to 4 times to collect the concentrated antibodies. Protein concentration was quantified by measuring the absorbance at 280 nm using a spectrophotometer (purchased from BioTek), and purity was determined by SDS-PAGE electrophoresis ( Figure 1 ).

[0060] Example 2 Binding of Antibody Conjugates to CEACAM5

[0061] The CEACAM5 protein was coated on an ELISA plate (purchased from Corning), and B9 with a starting concentration of 1000 nM and a 3-fold gradient dilution was added for incubation. Anti-FLAG tag antibody (purchased from SIGMA) was added to detect the binding ability of the antibody conjugate with the antigen CEACAM5 (for specific methods, see Cell Host Microbe. 2020. 27(6): 891-898.e5.). Figure 2 The ELISA results showed that B9 had a strong binding activity with CEACAM5 antigen.

[0062] Example 3 Binding kinetics of fully human single domain antibodies to CEACAM5

[0063] The binding activity of the fully human single-domain antibody B9 and the fully human single-domain antibody B9 (S84C) after cysteine ​​modification with CEACAM5 was determined using BLI technology (for specific methods, see Emerg Microbes Infect. 2017.6(10):e89.). CEACAM5 was fixed on the SA sensor (purchased from Pall Fortebio), and the antibody solution with gradient dilution was used as the analysis sample. The steps are as follows: CEACAM5 protein was diluted to 10 μg / mL with PBST solution (PBS solution containing 0.02% Tween-20), then immobilized on the SAbiosensor, and combined with a 3-fold gradient dilution of the antibody solution (concentration range of 3000-37.03 nM) in the running buffer (PBST: PBS solution containing 0.02% Tween-20), and no antibody was added to the blank control well. The program was set as: Association, 400 s; Dissociation, 400 s, and the temperature was set to 37°C. The data were analyzed using ForteBio Data analysis 10.1 software. The corresponding blank control wells were used as controls to subtract the background, and the curves of each concentration were fitted according to the 1:1 binding mode to obtain the kinetic analysis results. The report shows the equilibrium constant (K D ) value (see Figure 3 ). The results showed that after the cysteine ​​modification at position 84, the affinity of the B9 antibody was comparable to that before the modification.

[0064] Example 4 Flow cytometry detection of antibody-cell binding activity

[0065] Biotin (ThermoFisher, product number: A39257) and fully human single-domain antibody B9 (S84C) were incubated on ice for 1 hour to label B9 (S84C) with biotin. The cells were cultured to the logarithmic growth phase, digested with trypsin (Gibco, product number: 25200072), washed three times with PBS (Gibco, product number: 10010049), and fluorescent antibody B9 (S84C) (200nM) diluted with 2% FBS (Shanghai Yishen Biotechnology Co., Ltd., product number: 40130ES) was added and placed at room temperature in the dark for 1 hour. Wash three times with PBS, resuspend the cells in 0.3mL PBS solution, and then detect on a flow cytometer (BD Calibur). The results showed that the fully human single-domain antibody B9 (S84C) can bind to cells expressing CEACAM5, but not to FCHO cells that do not express CEACAM5 (see Figure 4 The human pancreatic cancer cells BxPC-3 were obtained from Fudan IBS Cell Resource Center (FDCC), the human gastric cancer cell line MKN45 was purchased from Wuhan Pronocell Life Science Co., Ltd., the human colon cancer cell line LS174T was donated by Professor Hu Guowei from the Affiliated Cancer Hospital of Fudan University, and the hamster ovary cells FCHO were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0066] Example 5 Preparation of anti-CEACAM5 single domain antibody and vcMMAE antibody conjugate

[0067] B9 (S84C), a fully humanized single-domain antibody against CEACAM5, was dissolved in a PBS solution containing 1 mM TCEP (purchased from SIGMA) to maintain its free thiol group and adjust the concentration to 2 mg / mL. A 5-fold molar concentration of vcMMAE (MedChemExpress, product number: HY-15575) was added and shaken continuously at room temperature for 1 hour. The reaction solution was removed, and a desalting column (GE Healthcare) was used to remove the residual small molecules in the reaction system and replace the buffer with a PBS solution. The molecular weight of B9 (S84C) coupled with vcMMAE increased as detected by SDS-PAGE (see Figure 5 ).

[0068] Example 6 Binding of Antibody Conjugates to CEACAM5

[0069] The CEACAM5 protein was coated on an ELISA plate (purchased from Corning), and B9(S84C)-vcMMAE with a starting concentration of 1000 nM and a 3-fold gradient dilution was added for incubation. Anti-FLAG tag antibody (purchased from SIGMA) was added to detect the binding ability of the antibody conjugate to the antigen CEACAM5 (for specific methods, see Cell Host Microbe. 2020. 27(6): 891-898.e5.). Figure 6 The ELISA results showed that B9(S84C) conjugated with vcMMAE retained the ability to bind to CEACAM5.

[0070] Example 7 Cytotoxicity of Antibody Conjugates to Cells

[0071] The cells were cultured to the logarithmic growth phase, and after digesting the cells with trypsin (purchased from Gibco), the cell density was adjusted to 10E6 cells / ml. The cells were inoculated in a 96-well culture plate at 5000 cells per well and cultured overnight at 37°C, 5% CO2. The culture medium was discarded, 200ul of antibody diluent (initial concentration was 1000nM) was added, and the culture was continued for 72h. The culture medium was discarded, and a culture medium containing 10% CCK8 (purchased from Biyuntian Biotechnology Co., Ltd.) was added and allowed to stand for 1-4h, and the absorbance value at 450nm was detected. Blank control wells and untreated wells were set, and the cell survival rate was calculated according to the formula: Cell survival rate (%) = (OD treatment group-OD blank control well) / (OD untreated well-OD blank control well)*100%. The results showed that B9(S84C)-vcMMAE linked to a single vcMMAE molecule had significant killing activity against tumor cell lines expressing CEACAM5, but had no killing activity against cell lines without CEACAM5 expression on their surface ( Figure 7 ).

[0072] Fudan University

[0073] Sequence Listing

[0074] SEQ ID NO:1(B9)

[0075] EVQLVESGGGLVQPGGSLRLSCAASGFTFGDYAMSWVRQAPRKGLEWIGEINHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTATYYCARVATAGYWGQGTLVIVSS

Claims

1. A fully human single domain antibody or antigen binding fragment against human CEACAM5, comprising: The heavy chain variable region of the antibody contains the CDR1 region at positions 26-33, the CDR2 region at positions 51-57, and the CDR3 region at positions 96-103 of SEQ ID NO: 1; The single domain antibody or antigen binding fragment specifically binds to CEACAM5.

2. The fully human single domain antibody or antigen-binding fragment of claim 1, in: The amino acid sequence of the heavy chain variable region is at least 90% identical to SEQ ID NO:

1.

3. The fully human single domain antibody or antigen binding fragment of any one of claims 1-2, wherein the fully human single domain antibody or antigen binding fragment is fully human.

4. The fully human single domain antibody or antigen binding fragment of any one of claims 1 to 3, wherein the fully human single domain antibody is VH, and the antigen binding fragment is VH, Fab, or scFv.

5. A cysteine-engineered fully human single-domain antibody, It is characterized in that The sequence of the cysteine-engineered fully human single-domain antibody comprises an amino acid sequence in which the amino acid at position 84 of SEQ ID NO: 1 is replaced by cysteine.

6. The cysteine ​​engineered fully human single domain antibody according to claim 5, It is characterized in that The cysteine ​​engineered fully human single domain antibody comprises at least one free thiol group.

7. The cysteine ​​engineered fully human single domain antibody according to claims 5-6, It is characterized in that The free thiol group can be chemically coupled to a cytotoxic agent, a chemotherapeutic agent, a radionuclide, a nucleic acid, or a detectable label.

8. A nucleic acid molecule, Features A nucleotide sequence encoding the fully human single domain antibody according to any one of claims 1 to 7.

9. A plasmid, Features Containing the nucleic acid molecule according to claim 8.

10. A host cell, Features Containing the plasmid according to claim 9.

11. A fully human single domain antibody-drug conjugate that binds to human CEACAM5 antigen, It is characterized in that The invention comprises any fully human single domain antibody and conjugate binding to human CEACAM5 antigen as claimed in claims 1 to 7.

12. The fully human single domain antibody-drug conjugate that binds to human CEACAM5 antigen according to claim 11, It is characterized in that The conjugate is a cytotoxic agent, a chemotherapeutic agent, a radionuclide, a nucleic acid, or a detectable label.

13. A method for preparing any one of claims 11-12 that binds to human CEACAM5 antigen and a fully human single domain antibody-drug conjugate, It is characterized in that The method comprises the following steps: (1) expressing and purifying any cysteine ​​engineered fully human single domain antibody that binds to human CEACAM5 antigen as described in any one of claims 5-6; (2) adding a conjugate that can chemically couple with the free thiol group to react and prepare an antibody conjugate; (3) Filter the unreacted conjugate through molecular sieve.

14. The method for preparing a fully human single domain antibody-drug conjugate binding to human CEACAM5 antigen according to claim 13, It is characterized in that The conjugate is a cytotoxic agent, a chemotherapeutic agent, a radionuclide, a nucleic acid, or a detectable label.

15. A pharmaceutical composition comprising an effective preventive or therapeutic dose of any fully human single domain antibody as described in claims 1 to 7 or a nucleic acid molecule as described in claim 8 or a plasmid as described in claim 9 or any fully human single domain antibody-drug conjugate that binds to human CEACAM5 antigen as described in claims 11 to 12, and a pharmaceutically acceptable carrier.

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