Anti-CEACAM6 single-domain antibodies, humanized single-domain antibodies, Fc fusion proteins thereof, and applications thereof

Abstract

The present invention discloses an anti-CEACAM6 single-domain antibody, a humanized single-domain antibody, an Fc fusion protein thereof, and applications thereof. The present invention screens and obtains a group of anti-CEACAM6 single-domain antibodies with strong neutralizing or binding capabilities, which can specifically bind to CEACAM6. The present invention also humanizes the single-domain antibody to obtain a humanized antibody with improved affinity. The present invention further fuses the single-domain antibody or the humanized single-domain antibody with human IgG-Fc to obtain a single-domain antibody-Fc fusion protein, which can specifically target transplanted tumors in mice after being labeled with an isotope. The anti-CEACAM6 single-domain antibody, humanized single-domain antibody, and single-domain antibody-Fc fusion protein provided by the present invention can be used to detect or diagnose CEACAM6 and to treat diseases related to abnormal CEACAM6 expression.

Description

[0001] This application is a divisional application with application number "202011135295.5", filing date "October 21, 2020", and invention name "Anti-CEACAM6 single-domain antibody, humanized single-domain antibody and Fc fusion protein and application thereof" Technical Field

[0002] The present invention relates to single-domain antibodies, in particular to anti-CEACAM6 single-domain antibodies and fusion proteins constructed by fusing the single-domain antibodies or humanized single-domain antibodies with IgG1-Fc. The present invention further relates to their use in detecting CEACAM6 and treating diseases associated with abnormal CEACAM6 expression, belonging to the field of anti-CEACAM6 single-domain antibodies, humanized single-domain antibodies and their applications. Background Art

[0003] Various carcinoembryonic antigen (CEA)-related cell adhesion molecule (CEACAM) proteins belong to the immunoglobulin (Ig) supergene family. Their primary structure consists of an extracellular region, a transmembrane region, and an intracellular region (some members lack an intracellular region). Common family members include CEACAM1, 3, 4, 5, 6, 7, 8, 16, 18, 19, and 21. Their extracellular domains are characterized by an N-terminal N domain followed by one or six constant C2-like Ig domains (referred to as the A region or B region).

[0004] These extracellular domains of carcinoembryonic antigen-related cell adhesion molecules are essential for CEACAM to function as homophilic and heterophilic intercellular adhesion molecules or as receptors for human and rodent pathogens. CEACAM receptors can form oligomers or dimers, forming multiple associations with other ligands on the cell membrane to regulate their important cellular functions. In addition to expression in human tissues, the CEACAM gene family is highly conserved in 27 other mammalian species (Robert Kammerer, Wolfgang Zimmermann. Coevolution of activating and inhibitory receptors within mammalian carcinoembryonic antigen families. BMC Biol. 2010 Feb 4;8:12). The biological function of CEACAM is to maintain cell-cell adhesion through its homophilic and heterophilic interactions, including its role in the differentiation and formation of three-dimensional tissue structures, angiogenesis, apoptosis, tumor suppression and metastasis (Kuespert K. et al. CEACAMs: their role in physiology and pathophysiology. Curr Opin Cell Biol. 2006 Oct; 18(5): 565-71; Athanasia Pavlopoulou and Andreas Scorilas. A Comprehensive Phylogenetic and Structural Analysis of the Carcinoembryonic Antigen (CEA) Gene Family. Genome Biol Evol. 2014 Jun; 6(6): 1314–1326).

[0005] Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), also known as non-specific cross-reacting antigen (NCA, NCA-50 / 90), and CD66c, is a key member of the CEACAM protein family. It shares high homology with family members CEACAM1 / 7 / 8. CEACAM6 is a glycosylphosphoinositol (GPI)-linked cell surface protein with an N domain and two C2-like domains. Its extracellular domain, which harbors various membrane receptors (some of which have been identified), mediates numerous possible cis- and trans-directed CEACAM interactions. Studies have reported that CEACAM6 is overexpressed in various tumors, including non-small cell lung cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, gastric cancer, and ovarian cancer. Overexpression of CEACAM6 can lead to mesenchymal-like morphological changes in epithelial tissues, increased tumor invasiveness and resistance to chemotherapy drugs, tumor metastasis, and reduced cell apoptosis. Reducing CEACAM6 gene expression with siRNA and inhibiting CEACAM6 protein function with monoclonal antibodies can reverse these effects of CEACAM6 overexpression. Although CEACAM6 is also expressed in many normal human tissues, such as granulocytes, it has been reported in many studies to be overexpressed in various tumors. Studies comparing the expression of CEACAM6 and CEACAM5 (CEA) in lung cancer, breast cancer, prostate cancer, colon cancer, pancreatic cancer, and ovarian cancer tissues, as well as in tissues adjacent to tumor tissues and normal tissues have shown that CEACAM6 is expressed at a higher level than CEA in all studied tumor types. Moreover, in cancers with overexpression of CEACAM6, tumors of different tissue types have different CEACAM6 expression patterns. The expression abundance of EACAM6 also varies. For example, in breast tumors, the expression abundance of CEACAM6 is as follows: papillary carcinoma > invasive ductal type > lobular type > lobular petiole type; in pancreatic cancer, the expression abundance of CEACAM6 is as follows: moderately differentiated type > well differentiated type > poorly differentiated tumor; the expression of CEACAM6 in mucinous ovarian adenocarcinoma is 3 times higher than that in serous ovarian adenocarcinoma; in non-small cell lung cancer, the expression of CEACAM6 is lung adenocarcinoma > lung squamous cell carcinoma; in liver metastasis of colon cancer, the expression of CEACAM6 is > primary tumor > lymph node metastasis.The expression of CEACAM6 in prostate cancer tissue is no different from that in adjacent normal tissue (Nicode Beauchemin and Azadeh Arabzadeh. Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis. Cancer Metastasis Rev. 2013 Dec.; 32(3-4): 643-71; Rosalyn D Blumenthal et al. Expression patterns of CEACAM5 and CEACAM6 in primary and metastatic cancers. BMC Cancer. 2007 7: 2(1-15)).

[0006] As mentioned above, CEACEA6 may be a specific target antigen for these overexpressing tumors, and CEACAM6 is a very attractive new target for therapeutic intervention in cancer immunotherapy.

[0007] Single-domain antibodies (sdAbs), also known as nanobodies, are heavy-chain antibody variable region fragments (VHHs) found in alpaca blood that lack light chains. They possess a number of advantages, including simple structure, strong penetrating power, ease of expression and purification, high affinity and stability, and minimal toxicity. High-affinity anti-CEACAM6 sdAbs screened using sdAb technology can be used for the detection and treatment of CEACAM6-overexpressing tumors and may also provide new detection methods and treatments for diseases associated with CEACAM6-overexpressing tumors. Summary of the Invention

[0008] One of the objectives of the present invention is to provide a single-domain antibody against CEACAM6 and a gene encoding the same;

[0009] The second purpose of the present invention is to humanize an anti-CEACAM6 single domain antibody to obtain a humanized single domain antibody;

[0010] The third object of the present invention is to fuse the single domain antibody or humanized single domain antibody with human IgG1-Fc to obtain a fusion protein;

[0011] A fourth object of the present invention is to couple the single domain antibody or humanized single domain antibody with one or more of an enzyme, a radioisotope, a fluorescent compound or a chemiluminescent compound to obtain a conjugate;

[0012] A fourth object of the present invention is to use the anti-CEACAM6 single domain antibody, anti-CEACAM6 humanized single domain antibody, fusion protein, and conjugate described above for preparing reagents for detecting CEACAM6 or treating diseases associated with abnormal CEACAM6 expression;

[0013] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0014] The present invention first provides an anti-CEACAM6 single-domain antibody, wherein the single-domain antibody comprises a framework region and three complementary determining regions, and the single-domain antibody is NBC36; wherein the amino acid sequences of the three complementary determining regions of the single-domain antibody NBC36 are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively;

[0015] The present invention further provides the amino acid sequence of the single-domain antibody, wherein the amino acid sequence of the single-domain antibody NBC36 is shown in SEQ ID No.4.

[0016] Protein mutants obtained by deleting, substituting, inserting and / or adding one or more amino acids in any of the amino acid sequences shown above have the same function as the protein before mutation, and these protein mutants all fall within the scope of protection of the present invention; in addition, amino acid sequences that have at least 90% identity with any of the amino acid sequences shown above also fall within the scope of protection of the present invention.

[0017] The present invention further provides the coding gene sequence of the single-domain antibody, wherein the nucleotide sequence of the coding gene of the single-domain antibody NBC36 is shown in SEQ ID No. 5. Among them, polynucleotide sequences that can hybridize with the complementary sequence of the polynucleotide sequence shown above under stringent hybridization conditions also fall within the scope of protection of the present invention; in addition, polynucleotide sequences that have at least 90% identity with any of the polynucleotide sequences shown above also fall within the scope of protection of the present invention.

[0018] The present invention further provides a recombinant expression vector comprising one or more of the genes encoding the single-domain antibody; preferably, the recombinant expression vector may be a recombinant prokaryotic cell expression vector, a recombinant yeast expression vector, a recombinant eukaryotic cell expression vector or other recombinant cell expression vector.

[0019] The present invention also provides a recombinant host cell comprising the recombinant expression vector described above.

[0020] Preferably, the recombinant host cell is a recombinant prokaryotic expression cell, a recombinant eukaryotic expression cell, a recombinant fungal cell or a recombinant yeast cell, and the recombinant prokaryotic expression cell is preferably Escherichia coli.

[0021] The present invention further humanized the single-domain antibody NBC36 to obtain two humanized antibodies NBC36HM1 and NBC36HM2, whose amino acid sequences are shown in SEQ ID No. 6 and SEQ ID No. 7, respectively.

[0022] The present invention further constructs a fusion protein by combining the anti-CEACAM6 single-domain antibody or humanized single-domain antibody with IgG-Fc; wherein the Fc gene sequence can be derived from an Fc gene sequence of IgG, IgA, IgM, or from IgG1, IgG2, IgG3, or IgG4. The IgG is preferably human IgG and its IgG1, 2, 3, and 4 subclasses, and can also be the Fc fragment gene and amino acid sequence of human IgM, human IgA, or immunoglobulins from other animals (e.g., mouse, rabbit, monkey, etc.).

[0023] As a preferred embodiment of the present invention, the humanized antibody NBC36HM2 is fused with the human IgG1-Fc gene to obtain a fusion protein having an amino acid sequence as shown in SEQ ID No. 8, and the nucleotide sequence of the encoding gene is shown in SEQ ID No. 9.

[0024] The present invention further couples the single-domain antibody or humanized single-domain antibody with one or more of an enzyme (such as horseradish peroxidase, alkaline phosphatase, etc.), a radioactive isotope, a fluorescent compound, or a chemiluminescent compound (the chemiluminescent compound can be a fluorescent compound) to obtain a conjugate. These conjugates can be used to detect CEACAM6 or treat various diseases associated with abnormal CEACAM6 expression.

[0025] For example, humanized single-domain antibodies and Fc fusion proteins against CEACAM6 were used 68 Ga, 89 Zr, 64 Cu, 18 F, 86 Y, 90 Y, 111 In, 99NV Tc, 125 I, 124 I and other radioisotopes to obtain labeled proteins for PET (positronemission tomography) or SPECT imaging detection. Alternatively, anti-CEACAM6 humanized single domain antibody, Fc fusion protein 90 Y, 177 Lu, 125 I, 131 I, 211 At, 111 In,152 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 225 Ac, 213 Bi, 212 Bior 67 The labeled protein is labeled with radioactive isotopes such as Ga and is used to treat diseases related to abnormal CEACAM6 expression.

[0026] The anti-CEACAM6 single-domain antibody, humanized anti-CEACAM6 single-domain antibody, or fusion protein constructed from a humanized single-domain antibody and IgG-Fc, and conjugates obtained by coupling a single-domain antibody or a humanized single-domain antibody with an enzyme, a radioisotope, a fluorescent compound, or a chemiluminescent compound provided by the present invention have the following uses:

[0027] (1) preparing drugs or reagents related to the detection of CEACAM6;

[0028] (2) Application of the drug for treating diseases associated with abnormal CEACAM6 expression; preferably, the diseases associated with abnormal CEACAM6 expression include tumor diseases such as non-small cell lung cancer, pancreatic cancer, breast cancer and ovarian cancer.

[0029] Definitions of terms used in this invention

[0030] The term "CEACAM6," as used herein, refers to carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), also known as non-specific cross-reacting antigen (NCA, NCA-50 / 90), and CD66c, a key member of the CEACAM protein family. CEACAM6 is a glycosylphosphoinositol (GPI)-linked cell surface protein with an N domain and two C2-like domains. Its extracellular domain, which harbors various membrane receptors (some of which have been identified), mediates numerous possible cis- and trans-directed CEACAM interactions. Studies have reported that CEACAM6 is overexpressed in various tumors, including non-small cell lung cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, gastric cancer, and ovarian cancer. CEACAM6 may be a specific target antigen for these overexpressing tumors, making it an attractive target for therapeutic intervention in cancer immunotherapy.

[0031] The novel antibodies targeting CEACAM6 and their Fc fusion proteins are the research and development objects of this article and are ultimately the objects of protection of this article. The scope of this article covers the obtained anti-CEACAM6 humanized single-domain antibodies and their Fc fusion proteins, as well as materials (e.g., pharmaceutical compositions, kits, vectors, etc.) and applications (e.g., diagnostic applications, therapeutic applications, preparation applications, etc.) containing the antibodies as components. However, those skilled in the art should understand that the objects of protection of this article are not limited to these examples.

[0032] The term "single-domain antibody (sdAb)" as used herein refers to a fragment containing a single variable domain of an antibody, also known as a nanobody. Like a full antibody, it can selectively bind to a specific antigen. Compared to the 150-160 kDa mass of a full antibody, a sdAb is significantly smaller, approximately 12-17 kDa. The first sdAb was engineered from the heavy chain antibody of a camel, known as the "VHH segment."

[0033] As used herein, the term "identity" of a sequence can be used interchangeably with "homogeneity" and refers to the degree of similarity between sequences as determined by sequence alignment software such as BLAST. Methods and software for sequence alignment are well known to those skilled in the art. Modified nucleotide sequences can be obtained by substituting, deleting and / or adding one or more amino acids or bases to a known sequence. For example, by conventional means (such as conservative substitutions, etc.), the amino acid or nucleotide sequence shown in one or more of the sequences SEQ ID NOs: 1-198 of the present invention is modified to obtain sequences having greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% sequence identity with these sequences, and having substantially the same properties, which are all within the scope of protection of the present invention. Preferably, the present invention obtains sequence identity by conservative substitution, but is not limited to conservative substitution.

[0034] The term "complementary" herein refers to two nucleotide sequences comprising antiparallel nucleotide sequences that can pair with each other after hydrogen bonding between the complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary strands are reverse complementary to each other when both sequences are viewed in a 5' to 3' direction. It is also known in the art that two sequences that can hybridize to each other under a given set of conditions are not necessarily 100% completely complementary.

[0035] The term "amino acid sequence" refers to the order in which amino acids are linked together to form a peptide chain (or polypeptide). An amino acid sequence can only be read in one direction. There are over 100 different types of amino acids, 20 of which are commonly used. This invention does not exclude modifications of the amino acid chain with other substances, such as carbohydrates and lipids, and is not limited to the 20 commonly used amino acids.

[0036] The term "nucleotide sequence" refers to the order of bases in DNA or RNA, i.e., the order of A, T, G, C in DNA, or the order of A, U, G, C in mRNA, and also includes the order of bases in rRNA, tRNA, and mRNA. It should be understood that the antibody genes claimed in the present invention encompass not only DNA sequences but also RNA (rRNA, tRNA, mRNA) and their complementary sequences.

[0037] The substitutions described herein may be conservative substitutions, i.e., replacement of specific amino acid residues with residues having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include substitutions between amino acid residues containing aliphatic groups (e.g., substitutions between Ile, Val, Leu, or Ala), substitutions between polar residues (e.g., substitutions between Lys and Arg, Glu and Asp, Gln and Asn), etc. Mutants resulting from deletions, substitutions, insertions, and / or additions of amino acids can be prepared by subjecting DNA encoding the wild-type protein to, for example, site-directed mutagenesis, a well-known technique (see, for example, Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982, which is incorporated herein by reference in its entirety).

[0038] The term "expression vector" refers to a vector that adds expression elements (such as a promoter, RBS, and terminator) to the basic cloning vector framework to enable the expression of a target gene. An expression vector consists of four parts: the target gene, promoter, terminator, and marker gene. The present invention includes, but is not limited to, prokaryotic expression vectors, eukaryotic expression vectors, and other cell expression vectors.

[0039] The term "framework region" refers to the framework region, which contains approximately 110 amino acids near the N-terminus of the immunoglobulin H and L chains, with significant variation in sequence. The remaining amino acid sequences are relatively constant, and are therefore used to distinguish the light and heavy chains into the variable (V) and constant (C) regions. The variable region comprises the hypervariable region (HVR), also known as the complementarity-determining region (CDR), and the framework region (FR).

[0040] The term "humanized" antibody refers to an antibody in which the Fr region of the variable region (VH or VHH), the constant region (i.e., CH and CL regions), or all of the antibody is encoded by human antibody genes. Humanized antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Humanized antibodies include chimeric antibodies, remodeled antibodies, and fully humanized antibodies. It should be understood that those skilled in the art can prepare suitable humanized forms of the single-domain antibodies of the present invention according to actual needs, which is within the scope of the present invention.

[0041] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those generally understood by those skilled in the art.

[0042] The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector, such as a plasmid, or may be integrated into the host genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 A CEACAM6 gene library was constructed, and the first-round PCR products were amplified by nested PCR. The fragments between 800 and 500 bp were heavy chain antibody gene fragments lacking light chains.

[0044] Figure 2 The VHH target gene was amplified by PCR using VHH-specific primers.

[0045] Figure 3 The figure shows the SDS-PAGE electrophoresis results of some expressed anti-CEACAM6 single-domain antibody proteins.

[0046] Figure 4 Figure 3 is the SDS-PAGE electrophoresis result of the expressed CEACAM6-sdAB after purification by nickel column.

[0047] Figure 5 The results are activity tests of the purified anti-CEACAM6 single domain antibody specifically binding to human CEACAM6 antigen.

[0048] Figure 6The following are the results of SDS-PAGE reducing and non-reducing gel electrophoresis of three humanized CEACAM6 single-domain antibodies after expression and purification; 1. Reducing protein band of EG2M1-EG10M1-Fc-p327.7 after expression and purification; 2. Reducing protein band of EG2M1-Fc-EG10M1-p327.7 after expression and purification; 3. Non-reducing protein band of EG2M1-EG10M1-Fc-p327.7 after expression and purification; 4. Non-reducing protein band of EG2M1-Fc-EG10M1-p327.7 after expression and purification; 5. Protein molecular weight marker; the arrow indicates a molecular weight of 50KD.

[0049] Figure 7 Antibody modification and 89Zr labeling roadmap.

[0050] Figure 8 The distribution results of single-domain antibody-Fc fusion protein labeled with isotope 89Zr in important organs and tumor site tissues in mouse tumor animal model (PET / CT scan image).

[0051] Figure 9 give 89 Bar graph of %ID / g values ​​of radioactive substance uptake in each tissue at different time points after Zr-CEACAM637.2 antibody administration. DETAILED DESCRIPTION

[0052] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0053] Example 1 Construction of anti-CEACAM6 antigen-specific single domain antibody library

[0054] (1) Immunization of alpacas with CEACAM6 antigen: According to the conventional immunization method, the purchased CEACAM6 antigen (HumanCEACAM6 Protein, Human, Recombinant (His Tag)) was used to select healthy adult alpacas. The antigen was injected subcutaneously at multiple points on the back of the neck. The antigen and an equal volume of Freund's adjuvant were added and immunized 4-8 times. The absorption of the injection site was tracked to confirm the correct immunization. After the first immunization, the second immunization was carried out 21 days later. The immunization interval was 7-15 days. After the fourth immunization, serum was collected to determine the antigen immune titer. When the titer reached about 50,000 times (ELISA method), about 100 ml of whole blood was collected, lymphocytes were separated, and stored at -80℃ for later use.

[0055] (2) Isolation of alpaca peripheral blood lymphocytes and RNA extraction: Isolate alpaca peripheral blood leukocytes and extract RNA using the QIAGEN kit according to the manufacturer's instructions. RNA purification: RNA was purified using the QIAGEN kit according to the manufacturer's instructions. The resulting RNA concentration and OD260 / 280 were determined to be ≥1.8.

[0056] (3) Heavy chain antibody variable region - VHH: Synthesis of the first chain cDNA: Use a cDNA synthesis kit (MiniBESTAgaroseGel DNA Extraction Kit Ver.4.0, TAKARA) according to the instructions. Using this template, two sets of primers were used to PCR amplify the heavy chain antibody VHH gene fragment. Using the nested PCR method, the fragments larger than 800 bp in the first PCR amplification were normal heavy chain gene fragments, and the fragments between 800 and 500 bp were heavy chain antibody gene fragments lacking light chains ( Figure 1 ), the missing light chain and heavy chain antibody gene fragments were recovered by gel excision, and the VHH target gene (~500bp) was amplified by PCR using VHH specific primers as a template. The gene amplification results are shown in Figure 2 Primers used:

[0057] First round PCRFd5' primer: YF: CGC CAT CAA GGT ACC AGT TGA;

[0058] First-round PCR Bd3′ primer: YBN: CAG CCG GCC ATG GCC SMK GTR CAG CTG GTG GAKTCT GGG GGA G;

[0059] Second round PCR primers:

[0060] YV-BACK: CAT GTG CATGGCCTA GAC TCG CGG CCCAGC CGG CCA TGG CC; YV-FOR: CAT GTG TAG ATT CCT GGC CGG CCT GGC CTG AGG AGA CGG TGA CCT GG;

[0061] (4) Connection of VHH fragment and phage display vector and electroporation into TG1 competent cells: After SfI single enzyme digestion of VHH fragment and pHEN6 vector plasmid, the VHH fragment and pHEN6 vector (Conrath, KEM other. Antimicrob Agents Chemother (Antimicrobial Chemotherapy) 2001, 45: (10) 2807-12.) were ligated with ligase (T4, NEB) and electroporated into TG1 competent cells. Ten electroporations were performed, plated, and the antibody insertion rate was verified by colony PCR. Recombinant gene cloning efficiency detection: The electroporated bacterial solution was plated onto LB / Amp plates and cultured overnight at 32°C. The next day, the antibody ligation efficiency was verified by colony PCR. The ligation efficiency of the phage antibody library was above 90%. The electroporated bacterial solution was plated onto LB / Amp plates and cultured overnight at 32°C. The solution was washed with 2YT medium, added with 15% glycerol, and stored at -80°C. Phage library 1.8×10 8 ; Randomly select 30-50 clones, clone PCR, VHH gene insertion rate 95%, and perform gene sequencing. The repetition rate of the three CDR sequences in the VHH sequence is less than 2%.

[0062] (5) Preparation of VHH phage antibody library: Helper phage M13K07 (Invitrogen) was added to the antibody library for rescue: The phage antibody library was prepared according to conventional methods and stored at -80°C for future use.

[0063] Example 2 Screening of single-domain antibodies against CEACAM6

[0064] (1) Screening of CEACAM6-specific single-domain antibodies

[0065] In the first round, 0.5 ml of CEACAM6 protein was coated onto immunotubes (Thermofisher) at a concentration of 50 μg / ml and incubated overnight at 4°C. In the second and third rounds, 0.5 ml of CEACAM6 protein was coated onto immunotubes at concentrations of 20 μg / ml and 10 μg / ml, respectively, and incubated overnight at 4°C. Blocking was performed with 2% skim milk powder in PBS at 37°C for 1.5 hours. Phage was added and incubated at room temperature for 1 hour. The cells were washed 10 times with PBST and 10 times with PBS. Specifically bound phage were eluted with 0.5 ml of TEA and infected into 2 ml of TG1 cells in the logarithmic growth phase. The titer was determined and the phage was cultured and amplified for a new round of screening.

[0066] Table 1 Screening results of CEACAM6-specific single domain antibodies

[0067] Number of screenings Add phage amount Phage recovery amount First round <![CDATA[1.1×10 12 ]]> <![CDATA[3.5×10 5 ]]> Second round <![CDATA[1.2×10 12 ]]> <![CDATA[4.3×10 6 ]]> Round 3 <![CDATA[5.0×10 11 ]]> <![CDATA[6.8×10 7 ]]>

[0068] (2) Phage ELISA method to select positive clones

[0069] From the second and / or third rounds of screening, colonies growing on agar plates were randomly selected and inoculated into 96-well plates in 2YT liquid medium containing Amp. Phage antibody expression was induced by superinfection with helper phage. The expression supernatant was harvested and assayed by ELISA using CEACAM6 as the antigen. CEACAM6-positive wells were selected and DNA sequenced to identify the gene sequences of anti-single-domain antibody clones. A series of single-domain antibody gene sequences, including those shown in SEQ ID NOs. 13-15, were obtained for further expression and screening of specific, highly active single-domain antibodies.

[0070] Example 3 Construction of a specific CEACAM6 single-domain antibody expression plasmid

[0071] The CEACAM6-specific single-domain antibody gene obtained in Example 2 was amplified by PCR to obtain a PCR product containing restriction endonucleases BbsI and BamHI sites. The PCR product and vector (pSJF2 vector, Kim Is. Biosic Biochem. 2002, 66(5):1148-51) were treated with restriction endonucleases BbsI and BamHI, respectively, and recombined by ligation with T4 ligase to obtain a plasmid sdAb-pSJF2 that can be efficiently expressed in Escherichia coli. The gene sequence was then determined to confirm the correctness of the sequence.

[0072] (1) The PCR amplification conditions for the target gene of CEACAM6 VHH were obtained. A 50 μl PCR system was used for amplification. The PCR reaction conditions were: 94°C for 3 minutes, followed by 94°C for 30 seconds, 72°C for 45 seconds, and 52°C for 30 seconds; a total of 30 cycles; and 72°C for 7 minutes.

[0073] 5' primer—GAA GAAGAA GAC AA CAG GCC SAR GTG MAG CTG GWG GAK TCT;

[0074] 3′ primer—gaagatctccggatccTGAGGAGACGGTGACCTGGGT;

[0075] (2) The target gene and vector were digested by enzymes, connected to the target gene and vector, transformed into TG1, and clones containing the target fragment were identified by PCR. The gene was sequenced to obtain a single-domain antibody expression plasmid with the correct gene sequence.

[0076] Example 4 Expression and purification of anti-single domain antibodies

[0077] The strain containing the plasmid sdAb-pSJF2 described in Example 3 was inoculated onto an LB plate containing ampicillin and incubated overnight at 37°C. A single colony was selected and inoculated into 15 ml of LB medium containing ampicillin and incubated overnight at 37°C on a shaker. 10 ml of the overnight culture was transferred to 1 L of 2YT medium containing ampicillin and incubated at 37°C on a shaker at 240 rpm. When the OD reached 0.4-0.6, 0.5-1.0 mM IPTG was added and the culture continued overnight. The cells were harvested by centrifugation. A 25% hypertonic sucrose solution was added to extract the soluble expressed single-domain antibody from the periplasm, followed by centrifugation and collection of the supernatant. Protein with a purity exceeding 90% was obtained by Ni+ ion affinity chromatography. Figure 3 The results of SDS-PAGE electrophoresis of the expressed CEACAM6 anti-single domain antibody protein are shown in Figure 2. Figure 4 Figure 3 is the SDS-PAGE electrophoresis result of the expressed CEACAM6-sdAB after purification by nickel column.

[0078] Example 5 Binding Assay of Purified CEACAM6 Single Domain Antibody to CEACAM6 Antigen (ELISA)

[0079] 1. Test materials: detachable ELISA plate (Thermofisher), CEACAM6 antigen, Anti-Myc tagantibody-HRP (Beijing Sino Biological Technology Co., Ltd.), TMB colorimetric solution (Beijing Meikewande, Cat: 1001), coating solution pH 9.6, BSA (Sigma).

[0080] 2. Test methods

[0081] 2.1 Coat the cells with Human CEACAM6 Protein at a concentration of 2 μg / ml, 100 μl / well, and incubate at 4°C overnight.

[0082] 2.2 Block the plate by adding 2% skim milk in PBS (300 μl / well). Incubate at 37°C for 1.5 h.

[0083] 2.3 Dilute CEACAM6 single domain antibodies of different numbers to a final concentration of 10.0 ug / ml and 1.0 ug / ml, 100 ul / well.

[0084] 2.4 Dilute Anti-Myc tag antibody (HRP) (1:5000), 100ul / well, incubate at 37℃ for 1h.

[0085] 2.5 Add TMB colorimetric solution, 100 μl / well, and react for 10 minutes in the dark.

[0086] 2.6 Add 50 μl / well of 2M H2SO4 to terminate the reaction.

[0087] 2.7 Measure the OD value at a wavelength of 450nm.

[0088] 3. Test results

[0089] Figure 5 The results of the activity test of the purified CEACAM6 single domain antibody specifically binding to the human CEACAM6 antigen.

[0090] Example 6 Anti-CEACAM6 Single Domain Antibody Affinity Determination Test

[0091] 1) Sample Preparation Antigen: Bio-CEACAM6 was diluted to 10 μg / ml in 1× dynamic buffer (1× PBS, containing 0.05% Tween 20, 0.1% BSA, pH 7.2);

[0092] Single domain antibody: diluted in 1× kinetic buffer to 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM;

[0093] 2) Sample testing

[0094] The antigen to be tested was loaded onto the SA sensor and diluted to five different dilutions. The affinities of all single-domain antibodies were 50 nM, 20 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM. The affinities of some single-domain antibodies are shown in Table 2, along with their affinity ranges.

[0095] Table 2 Affinity determination results of anti-CEACAM6 single domain antibodies

[0096]

[0097] Example 7 Humanization of anti-CEACAM6 single domain antibody

[0098] The humanization method uses the protein surface amino acid humanization (Resurfacing) method and the universal antigen-binding complementary region transplantation method (CDR grafting to a universal framework) of VHH humanization to complete, and refers to the already applied patent (anti-EGFR humanized single-domain antibody, Fc fusion protein, heavy chain Fab protein and its application, application number: 2019113490209).

[0099] The humanization steps were as follows: Homology modeling of the anti-CEACAM6 single-domain antibodies NBC4, 25, and 36 was performed using Modeller 9. The anti-CEACAM6 single-domain antibodies NBC4, 25, and 36 were humanized based on the amino acid sequences of the highly soluble human antibody DP-47 and the homologous sequence NBBcII10 antibody.

[0100] The humanization results are shown in Table 3.

[0101] Table 3 Humanization results of NBC4, 25 and 36 single domain antibodies

[0102]

[0103]

[0104] *Note: X* indicates the amino acid site that may be altered for humanization. According to literature research, if the amino acid change exceeds 80%, the immunogenicity is close to that of human antibodies.

[0105] Example 8 Vector Construction of Anti-CEACAM6 Humanized Single Domain Antibody Fc Fusion Protein

[0106] (1) First structure: sdAb1-Hinger-CH2-CH3 (IgG1-Fc). sdAb = NBC4HM2 or NBC25HM3 or NBC36HM2.

[0107] (2) Construction steps: NBC4HM2 or NBC25HM3 or NBC36HM2+human IgG1-Fc gene was fully synthesized, and XhoI-EcoRI double enzyme digestion was added. The sdAb-Fc gene was ligated to the p327.7 expression vector (Patent Publication No. CN 104195173 A), and the corresponding restriction sites and stop codon were added. After XbaI-SalI double enzyme digestion, another sdAb-Fc gene was ligated to the p327.7 expression vector containing sdAb-Fc (which had been double enzyme digested and ligated with XhoI-EcoRI), so that one vector finally had two sdAb-Fc sequences.

[0108] The amino acid and gene sequence of the anti-CEACAM6 humanized single domain antibody, Fc fusion protein and heavy chain Fab protein provided by the present invention are shown in Table 4.

[0109] Table 4 Sequence Listing of Anti-CEACAM6 Humanized Single Domain Antibody, Fc Fusion Protein, and Heavy Chain Fab Protein

[0110]

[0111]

[0112] Example 9 Expression and Purification of Anti-CEACAM6 Humanized Single Domain Antibody Fc Fusion Protein

[0113] The expression vectors NBC4HM2-p327.7, NBC25HM3-p327.7, or NBC36HM2-p327.7 were transfected into CHO / K1 cells, respectively. Stable protein high-expressing cell lines were screened using MSX. A total of three stable expression cell lines were screened. The stable expression cell lines were cultured in 500 ml shake flasks for protein expression.

[0114] Protein purification: The cell expression supernatant was purified by protein A affinity chromatography, and the purified protein was replaced with citric acid (0.05% Tween 80, pH 6.2) buffer. The purified protein expressed by the anti-CEACAM6 humanized single domain antibody Fc fusion protein vector is shown in Figure 6 (SDS-PAGE electrophoresis results of reducing gel and non-reducing gel after expression and purification of three humanized CEACAM6 single-domain antibodies).

[0115] The theoretically estimated values ​​for the proteins expressed by the above-mentioned fusion protein expression vectors are: 688, 688, and 682 amino acids, respectively; molecular weights (MW) of 7.664 kD, 7.704 kD, and 7.569 kD, respectively, linked by Hinger disulfide bonds; and isoelectric points (pIs) of 7.88, 7.30, and 7.61, respectively. After purification, the molecular weight after SDS-PAGE reduction was approximately 38 kD, consistent with the theoretically estimated value. Affinity determination of the anti-CEACAM6 humanized single-domain antibody fusion protein was performed as described in Example 6 above. The affinity analysis results are shown in Table 5.

[0116] Table 5 Affinity analysis results of anti-CEACAM6 humanized single domain antibody fusion protein and human CEACAM6

[0117]

[0118] Example 10 Radioisotope-labeled CEACAM6 humanized single-domain antibody fusion protein assay

[0119] 1. Test methods

[0120] (1) Antibody DFO modification: Take 1 mL of antibody solution (2 mg / ml of one of the three fusion proteins mentioned above) and add 1 mL of 0.5 M NaHCO3 / Na2CO3 solution to the reaction bottle, and measure the pH value until it is alkaline; stir and react at 37°C for 40 minutes. Purify with PD10 column. (2) Antibody labeling: Take a small amount of 89Zr, add 2 M Na2CO3 solution, and adjust the pH to neutral; (3) Antibody quality control: glass fiber paper, developing agent; sodium citrate system. The antibody marker is at the origin and the free 89Zr is at the forefront. See the antibody modification and 89Zr labeling roadmap for details. Figure 7 .

[0121] 2. Test results

[0122] The single-domain antibody-Fc fusion proteins of three antibody structures were labeled with the isotope 89Zr. The distribution results in important organs and tumor tissues in mouse tumor animal models are shown in Table 6. Figure 8 and Figure 9 .

[0123] Table 6 gives 89 %ID values ​​of radioactive substance uptake in various tissues after Zr-CEACAM6 (mean±SD, n=6)

[0124]

[0125] The experimental results show that the single-domain antibody-Fc fusion can specifically target transplanted tumors (non-small cell lung cancer, pancreatic cancer, etc.) in mice after isotope labeling.

Claims

1. A single-domain antibody against CEACAM6, comprising a framework region and three complementarity-determining regions, characterized in that: The amino acid sequences of the three complementary determining regions CDR1-3 of the single-domain antibody are shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively.

2. The single domain antibody according to claim 1, characterized in that The amino acid sequence of the single-domain antibody is shown in SEQ ID No.

4.

3. A gene encoding the single domain antibody according to any one of claims 1 or 2.

4. The coding gene according to claim 3, characterized in that The nucleotide sequence of the gene encoding the single-domain antibody is shown in SEQ ID No.

5.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the coding gene according to claim 3 or 4.

6. A humanized single-domain antibody against CEACAM6, characterized in that: The single-domain antibodies were humanized to obtain two humanized antibodies, whose amino acid sequences are shown in SEQ ID No. 6 and SEQ ID No. 7, respectively.

7. A fusion protein, characterized in that A fusion protein is obtained by constructing the single-domain antibody according to any one of claims 1 or 2 or the humanized anti-CEACAM6 single-domain antibody according to claim 6 with IgG-Fc.

8. The fusion protein according to claim 7, characterized in that The amino acid sequence is shown in SEQ ID No.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID No.

9.

9. A conjugate, characterized in that The single domain antibody according to claim 1, 2 or 6 is coupled to one or more of an enzyme, a radioisotope or a chemiluminescent compound to obtain a conjugate.

10. Use of the single domain antibody of claim 1, 2 or 6, the encoding gene of claim 3 or 4, the fusion protein of claim 7 or 8, and the conjugate of claim 9 in the preparation of a drug or reagent for detecting or diagnosing a CEACAM6-overexpressing tumor; the CEACAM6-overexpressing tumor is non-small cell lung cancer, pancreatic cancer, breast cancer or ovarian cancer.

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