Anti-CEACAM6 single-domain antibody and its fusion protein and application

Abstract

This invention discloses anti-CEACAM6 single-domain antibodies, their fusion proteins, and their applications. This invention screened and obtained a group of anti-CEACAM6 single-domain antibodies with high activity, strong neutralizing or binding ability, and the ability to specifically bind to CEACAM6. This invention also humanizes the single-domain antibodies to obtain humanized antibodies with improved affinity. This invention further fuses the single-domain antibodies or humanized single-domain antibodies with human IgG-Fc to obtain fusion proteins. The humanized single-domain antibodies and / or fusion proteins of this invention can be used for the detection or diagnosis of CEACAM6 and for the treatment of diseases related to abnormal CEACAM6 expression.

Description

[0001] This application is a divisional application of patent application No. 202011133599.8, filed on October 21, 2020, entitled "Anti-CEACAM6 Single-Domain Antibody, Humanized Single-Domain Antibody and its Fusion Protein and Application". Technical Field

[0002] This invention relates to single-domain antibodies, and more particularly to single-domain antibodies against CEACAM6 and fusion proteins constructed by fusing the single-domain antibody or a humanized single-domain antibody with IgG1-Fc. The invention further relates to their application in detecting CEACAM6 and treating diseases related to abnormal CEACAM6 expression, and belongs to the field of single-domain antibodies against CEACAM6, humanized single-domain antibodies and their applications. Background Technology

[0003] Multiple 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. The extracellular domains of these proteins are characterized by an N-terminal N-domain, followed by one or 1-6 constant C2-like Ig domains (referred to as the A or B region).

[0004] These extracellular domains of carcinoembryonic antigen-associated cell adhesion molecules are essential for CEACAM to perform its function as both isotropic and heterotropic cell adhesion molecules or as receptors for human and rodent pathogens. CEACAM receptors can form multiple combinations with other ligands at the cell membrane, either as oligomers or dimers, thereby regulating their vital 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 functions of CEACAMs are to maintain cell-cell adhesion through their isophilic and heterophilic interactions, including their roles 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-associated cell adhesion molecule 6 (CEACAM6), also known as non-specific cross-reacting antigen (NCA, NCA-50 / 90) or CD66c, is an important member of the CEACAM family of proteins, sharing high homology with family members CEACAM1 / 7 / 8. CEACAM6 is a glycosylphosphoinositol (GPI)-linked cell surface protein with one N-domain and two C2-like domains. It mediates numerous possible cis- or trans-guided CEACAM interactions through its extracellular domains containing various membrane receptors (some of which have been identified). Studies have reported overexpression of CEACAM6 in various tumors, including non-small cell lung cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, gastric cancer, and ovarian cancer, to varying degrees. Overexpression of CEACAM6 can lead to mesenchymal-like morphological changes in epithelial tissues, increased tumor invasiveness and resistance to chemotherapy, tumor metastasis, and reduced 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 expressed in many normal human tissues, such as granulocytes, its overexpression in various tumors has been reported in numerous studies. Studies comparing the expression levels of CEACAM6 and CEACAM5 (CEA) in lung cancer, breast cancer, prostate cancer, colon cancer, pancreatic cancer, and ovarian cancer tissues, as well as adjacent tissues and normal tissues, have shown that CEACAM6 expression is higher than CEA in all tumor types studied. Moreover, in cancers with CEACAM6 overexpression, different tissue types show that CEACAM6 expression is higher than CEA expression. The expression abundance of CEACAM6 also varies. For example, in breast tumors, the expression abundance of CEACAM6 is: papillary carcinoma > invasive ductal type > lobular type > foliate stalk type; in pancreatic cancer, the expression abundance of CEACAM6 is: moderately differentiated type > well differentiated type > poorly differentiated type; 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 metastases of colon cancer, the expression of CEACAM6 is: primary tumor > lymph node metastases.The expression of CEACAM6 in prostate cancer tissues was not different from that in adjacent normal tissues (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 variable region fragments (VHHs) of heavy chain antibodies lacking the light chain, discovered in alpaca blood. They possess a range of advantages, including simple structure, strong penetrability, ease of expression and purification, high affinity and stability, and low toxicity. Screening for high-affinity anti-CEACAM6 single-domain antibodies using single-domain antibody technology can be used for the detection and treatment of CEACAM6-overexpressing tumors, and can also provide new detection methods and treatments for related diseases. Summary of the Invention

[0008] One objective of this invention is to provide a set of single-domain antibodies against CEACAM6 and their encoding genes;

[0009] The second objective of this invention is to humanize anti-CEACAM6 single-domain antibodies to obtain humanized single-domain antibodies.

[0010] A third objective of this invention is to fuse the aforementioned single-domain antibody or humanized single-domain antibody with human IgG1-Fc to obtain a fusion protein;

[0011] The fourth objective of this invention is to couple the single-domain antibody or humanized single-domain antibody with one or more of the following phases: enzyme phase, radioactive isotope, fluorescent compound, or chemiluminescent compound to obtain a conjugate.

[0012] The fourth objective of this invention is to apply the aforementioned anti-CEACAM6 single-domain antibody, anti-CEACAM6 humanized single-domain antibody, fusion protein, and conjugate to the preparation of reagents for detecting CEACAM6 or for the treatment of diseases related to abnormal CEACAM6 expression.

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

[0014] This invention first provides a group of single-domain antibodies against CEACAM6, each single-domain antibody consisting of a frame region and three complementarity-determining regions (CDRs). The single-domain antibodies are selected from any one of NBC4, NBC5, or NBC6. Specifically, the amino acid sequences of the three CDRs of single-domain antibody NBC4 are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively; the amino acid sequences of the three CDRs of single-domain antibody NBC5 are shown in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively; and the amino acid sequences of the three CDRs of single-domain antibody NBC6 are shown in SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9, respectively.

[0015] The present invention further provides the amino acid sequences of the single-domain antibodies, wherein the amino acid sequence of single-domain antibody NBC4 is shown in SEQ ID No. 10, the amino acid sequence of single-domain antibody NBC5 is shown in SEQ ID No. 11, and the amino acid sequence of single-domain antibody NBC6 is shown in SEQ ID No. 12.

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

[0017] The present invention further provides the coding gene sequences of the single-domain antibodies, wherein the nucleotide sequence of the coding gene for single-domain antibody NBC4 is shown in SEQ ID No. 13, the nucleotide sequence of the coding gene for single-domain antibody NBC5 is shown in SEQ ID No. 14, and the nucleotide sequence of the coding gene for single-domain antibody NBC6 is shown in SEQ ID No. 15. Polynucleotide sequences whose complementary sequences to the above-described polynucleotide sequences can hybridize under stringent hybridization conditions are also within the scope of protection of the present invention; in addition, polynucleotide sequences that have at least 90% identity with any of the above-described polynucleotide sequences are also within the scope of protection of the present invention.

[0018] The present invention further provides a recombinant expression vector, wherein the recombinant expression vector contains one or more of the encoding genes of 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 yeast recombinant mother cell, and the recombinant prokaryotic expression cell is preferably Escherichia coli.

[0021] The present invention further humanized the single-domain antibody NBC4 to obtain five humanized antibodies NBC4HM1, NBC4HM2, NBC4HM3, NBC4HM4 and NBC4HM5, whose amino acid sequences are shown in SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 and SEQ ID No.20, respectively.

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

[0023] In a preferred embodiment of the present invention, the humanized antibody NBC4HM2 is fused with the human IgG1-Fc gene to obtain a fusion protein with the amino acid sequence shown in SEQ ID No. 21, and the nucleotide sequence of the encoding gene is shown in SEQ ID No. 22.

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

[0025] For example, using humanized single-domain antibodies against CEACAM6 and Fc fusion proteins...68 Ga, 89 Zr, 64 Cu, 18 F, 86 Y, 90 Y, 111 In, 99NV Tc, 125 I, 124 Labeled proteins obtained by labeling with radioactive isotopes such as I-1 can be used for imaging detection in PET (positron emission tomography) or SPECT. Alternatively, humanized single-domain antibodies against CEACAM6 or Fc fusion proteins can be used... 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 Labeled proteins obtained by labeling with radioactive isotopes such as Ga are used to treat diseases related to abnormal CEACAM6 expression.

[0026] The single-domain antibody against CEACAM6, the humanized single-domain antibody against CEACAM6, or the fusion protein constructed by humanized single-domain antibody and IgG-Fc, and the conjugate obtained by coupling single-domain antibody or humanized single-domain antibody with enzyme phase, radioactive isotope, fluorescent compound or chemiluminescent compound provided by the present invention mainly have the following applications:

[0027] (1) Prepare drugs or reagents for detecting CEACAM6;

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

[0029] Definition of terms involved in this invention

[0030] The term "CEACAM6" used in this article refers to carcinoembryonic antigen-associated cell adhesion molecule 6 (CEACAM6), also known as non-specific cross-reacting antigen (NCA, NCA-50 / 90) or CD66c, and is an important member of the carcinoembryonic antigen-associated cell adhesion molecule protein family. CEACAM6 is a glycosylphosphoinositol (GPI)-linked cell surface protein with one N domain and two C2-like domains. It mediates many possible cis- or trans-guided CEACAM interactions through its extracellular domains containing various membrane receptors (some of which have been identified). 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 to varying degrees. CEACAM6 may be a specific target antigen for these overexpressing tumors, making it a very attractive target for therapeutic interventions in cancer immunotherapy.

[0031] The novel antibody against CEACAM6 and its Fc fusion protein are the research and development objects of this paper, and ultimately also the objects of protection of this paper. The scope of this paper covers the obtained humanized single-domain antibody against CEACAM6 and its Fc fusion protein, as well as substances (e.g., pharmaceutical compositions, kits, vectors, etc.) and applications (e.g., diagnostic applications, therapeutic applications, preparative applications, etc.) with the antibody as a component. However, those skilled in the art should understand that the objects of protection of this paper are not limited to the examples mentioned above.

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

[0033] The term "identity" used herein is interchangeable with "similarity" and refers to the degree of similarity between sequences as determined by sequence alignment software such as BLAST. Sequence alignment methods and software 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 modifying one or more amino acid or nucleotide sequences shown in SEQ ID NO: 1-198 of the present invention using conventional means (e.g., conservative substitution), sequences with greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% identity and substantially the same properties can be obtained, all within the scope of this invention. Preferably, the present invention obtains sequence identity through conservative substitution, but is not limited to conservative substitution.

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

[0035] The term "amino acid sequence" refers to the order in which amino acids link 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, of which 20 are commonly used. This invention does not exclude the possibility of other substances, such as carbohydrates or lipids, modifying the amino acid chain, nor is it limited to the 20 commonly used amino acids.

[0036] The term "nucleotide sequence" refers to the arrangement of bases in DNA or RNA, specifically the A, T, G, C sequence in DNA, or the A, U, G, C sequence in mRNA, and also includes the base sequences in rRNA, tRNA, and mRNA. It should be understood that the antibody gene for which protection is sought in this invention covers not only DNA sequences but also RNA (rRNA, tRNA, mRNA) and their complementary sequences.

[0037] The substitutions described in this invention can be conservative substitutions, which involve replacing 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 the deletion, substitution, insertion, and / or addition of amino acids can be created by performing site-directed mutagenesis on the DNA encoding the wild-type protein, for example, using well-known techniques (see, for example, Nucleic Acid Research, Vol. 10, No. 20, pp. 6487-6500, 1982, incorporated herein by reference in its entirety).

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

[0039] The term "framework region" refers to the backbone region. In immunoglobulins, approximately 110 amino acid sequences near the N-terminus of the H and L chains exhibit significant variation, while the amino acid sequences of the remaining parts are relatively constant. Based on this, the light and heavy chains can be divided into variable (V) and constant (C) regions. The variable region contains 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 whose variable region (VH or VHH) is encoded entirely by a human antibody gene, including the Fr region, constant region (i.e., CH and CL regions), or all of the antibody's variable region. Humanized antibodies can significantly reduce the immune side effects caused by heterologous antibodies in the human body. Humanized antibodies include several types, such as chimeric antibodies, modified 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 this invention according to actual needs, which is within the scope of this invention.

[0041] The terms “mutation” and “mutant” have their common meanings here, referring to genetic, naturally occurring, or introduced changes in a nucleic acid or polypeptide sequence, and their meanings are the same as those commonly known to those skilled in the art.

[0042] The terms "host cell" or "recombinant host cell" refer to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotides may remain as, for example, non-integrating vectors of plasmids or may be integrated into the host genome. Attached Figure Description

[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 that lacked the light chain.

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

[0045] Figure 3 The results of SDS-PAGE electrophoresis of partially expressed anti-CEACAM6 anti-single-domain antibody protein.

[0046] Figure 4 The results of SDS-PAGE electrophoresis of the CEACAM6-sdAB fraction after purification by nickel column were obtained.

[0047] Figure 5 The results are from an activity assay showing the specific binding of the purified anti-CEACAM6 single-domain antibody to the human CEACAM6 antigen.

[0048] Figure 6 The following are the SDS-PAGE results of three humanized CEACAM6 single-domain antibodies after expression and purification: reducing and non-reducing gel electrophoresis; 1. Reduced protein band after expression and purification of EG2M1-EG10M1-Fc-p327.7; 2. Reduced protein band after expression and purification of EG2M1-Fc-EG10M1-p327.7; 3. Non-reduced protein band after expression and purification of EG2M1-EG10M1-Fc-p327.7; 4. Non-reduced protein band after expression and purification of EG2M1-Fc-EG10M1-p327.7; 5. Protein molecular weight standard (Marker); the molecular weight indicated by the arrow is 50KD.

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

[0050] Figure 8 Distribution of single-domain antibody-Fc fusion protein-labeled isotope 89Zr in important organs and tumor sites in a mouse tumor animal model (PET / CT scan).

[0051] Figure 9 give 89Bar chart of radioactive uptake %ID / g values ​​in each tissue at different time points after Zr-CEACAM637.2 antibody treatment. Detailed Implementation

[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 a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

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

[0054] (1) Immunization of alpacas with CEACAM6 antigen: The immunization was carried out according to the conventional method. Using purchased CEACAM6 antigen (Human CEACAM6 Protein, Human, Recombinant (His Tag)), healthy adult alpacas were selected. Multiple subcutaneous injections of the antigen were made into the neck and back of the alpacas, along with an equal volume of Freund's adjuvant. Immunization was administered in 4-8 doses, and the absorption of the injection site lumps was monitored to confirm correct immunization. After the first immunization, a 21-day interval was observed before the second immunization. The immunization interval was 7-15 days. After the fourth immunization, serum was collected to determine the antigen titer. When the titer reached approximately 50,000 times (ELISA method), approximately 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 extraction of RNA: Alpaca peripheral blood leukocytes were isolated, and RNA was extracted using the QIAGEN kit according to the instructions. RNA purification: RNA was purified using the QIAGEN kit according to the instructions, and the concentration of the obtained RNA and OD260 / 280 ≥ 1.8 were measured.

[0056] (3) Heavy chain antibody variable region - VHH: First-strand cDNA synthesis: The cDNA synthesis kit (MiniBESTAgaroseGel DNA Extraction Kit Ver. 4.0, TAKARA) was used according to the manufacturer's instructions. Using this template, two sets of primers were used to amplify the heavy chain antibody VHH gene fragment by PCR. Nested PCR was employed. In the first PCR amplification, fragments larger than 800 bp were considered normal heavy chain gene fragments, while those between 800 and 500 bp were heavy chain antibody gene fragments lacking the light chain. Figure 1The gene fragment lacking the light and heavy chains of the antibody was recovered by gel extraction. Using this fragment as a template, the VHH target gene (~500 bp) was amplified by PCR using VHH-specific primers. The gene amplification results are shown below. Figure 2 Primers used:

[0057] First-round PCR Fd5' primers: YF: CGC CAT CAA GGT ACC AGT TGA;

[0058] First round PCR primers: 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) Ligation of VHH fragment and phage display vector and electroporation of TG1 competent cells: After digesting VHH fragment and pHEN6 vector plasmid with SfI, 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, and the cells were plated. The antibody insertion rate was verified by colony PCR. Recombinant gene cloning efficiency detection: The electroporated bacterial culture was plated on LB / Amp plates and cultured overnight at 32°C. The antibody ligation efficiency was verified by colony PCR the next day. The ligation efficiency of the phage antibody library was above 90%. The electroporated bacterial culture was plated on LB / Amp plates and cultured overnight at 32°C. The culture was washed with 2YT medium, 15% glycerol was added, and the culture was stored at -80°C. Phage library 1.8 × 10 8 30-50 clones were randomly selected, cloned by PCR, with a VHH gene insertion rate of 95%, and gene sequencing was performed. The repetition rate of the three CDR sequences in the VHH sequence was less than 2%.

[0062] (5) Preparation of VHH phage antibody library: The antibody library was rescued by adding helper phage M13K07 (Invitrogen): The phage antibody library was prepared according to the conventional method and stored at -80℃ for later use.

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

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

[0065] The first round used CEACAM6 protein at a concentration of 50 μg / ml, coated in 0.5 ml immunotubes (Thermofisher), and incubated overnight at 4°C. The second and third rounds used CEACAM6 protein concentrations of 20 μg / ml and 10 μg / ml, respectively, coated in 0.5 ml immunotubes and incubated overnight at 4°C. Blocking was performed using 2% skim milk PBS at 37°C for 1.5 hours. Phages were added, incubated at room temperature for 1 hour, washed 10 times each with PBST and PBS, and eluted with 0.5 ml of TEA to remove specifically bound phages. The phages were then infected with 2 ml of TG1 in logarithmic growth phase, the titer was measured, and the phages were cultured and amplified for a new round of screening.

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

[0067] Number of filters Added phage amount Elution and recovery of phage volume Round 1 <![CDATA[1.1×10 12 ]]> <![CDATA[3.5×10 5 ]]> Second round <![CDATA[1.2×10 12 ]]> <![CDATA[4.3×10 6 ]]> Third round <![CDATA[5.0×10 11 ]]> <![CDATA[6.8×10 7 ]]>

[0068] (2) Phage ELISA method for selecting positive clones

[0069] From the second and / or third round of screening of colonies grown on agar plates, single colonies were randomly selected and inoculated into 96-well deep-well plates containing 2YT liquid medium with Amp. Phage antibody expression was induced by helper phage superinfection. The expression supernatant was harvested and ELISA was performed using CEACAM6 as the antigen. CEACAM6-positive wells were selected, and DNA sequencing was performed to identify the gene sequences of the anti-single-domain antibody clones, yielding a series of single-domain antibody gene sequences, including those shown in SEQ ID NO. 13-15, 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 specific CEACAM6 single-domain antibody gene obtained in Example 2 was amplified by PCR to obtain PCR products with restriction endonucleases BbsI and BamHI sites. The PCR products and the vector (pSJF2 vector, kim Is. Biosic Biochem. 2002, 66(5):1148-51) were treated with restriction endonucleases BbsI and BamHI, respectively. The vectors were then ligated and recombined with T4 ligase to obtain plasmid sdAb-pSJF2, which can be efficiently expressed in Escherichia coli. The gene sequence was determined to confirm the correctness of the sequence.

[0072] (1) Obtain the PCR amplification conditions for the VHH target gene of CEACAM6. Amplify the PCR system in 50 μl. PCR reaction conditions: first 94℃ for 3 minutes, then 94℃ for 30 seconds; 72℃ for 45 seconds, 52℃ for 30 seconds; for a total of 30 cycles; 72℃ 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 are digested with enzymes, the target gene and vector are ligated, TG1 is transformed, clones containing the target fragment are identified by PCR, gene sequencing is performed, and single-domain antibody expression plasmids with correct gene sequences are obtained.

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

[0077] The bacterial strain containing plasmid sdAb-pSJF2 described in Example 3 was inoculated onto an LB agar plate containing ampicillin and incubated overnight at 37°C. A single colony was selected and inoculated into 15 ml of LB agar containing ampicillin, and cultured overnight at 37°C on a shaker. 10 ml of the overnight culture was transferred to 1 L of 2YT agar containing ampicillin and cultured at 37°C on a shaker at 240 rpm until the OD value reached 0.4–0.6. Then, 0.5–1.0 mM IPTG was added, and the culture was continued overnight. The cells were centrifuged and collected. Soluble single-domain antibodies expressed in the periplasm were extracted by adding 25% hypertonic sucrose solution, centrifuged, and the supernatant was collected. Protein with a purity of over 90% was obtained by Ni+ affinity chromatography. Figure 3 The results of SDS-PAGE electrophoresis of the partially expressed CEACAM6 anti-single-domain antibody protein are shown. Figure 4 The results of SDS-PAGE electrophoresis of the CEACAM6-sdAB fraction after purification by nickel column were obtained.

[0078] Example 5: Binding assay (ELISA) of purified CEACAM6 single-domain antibody to CEACAM6 antigen.

[0079] 1. Experimental materials: removable ELISA plate (Thermofisher), CEACAM6 antigen, Anti-Myc tagantibody-HRP (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), TMB chromogenic solution (Beijing Meikewande, Cat: 1001), coating solution pH 9.6, BSA (Sigma).

[0080] 2. Test Methods

[0081] 2.1 Coat each well with Human CEACAM6 Protein at a concentration of 2ug / ml, 100ul / well, and incubate overnight at 4°C.

[0082] 2.2 Add 2% skim milk PBS for blocking, 300 μL / well. Incubate at 37°C for 1.5 h.

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

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

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

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

[0087] 2.7 OD value was measured at a wavelength of 450 nm.

[0088] 3. Test Results

[0089] Figure 5 The results are from an activity assay showing the specific binding of purified CEACAM6 single-domain antibody to human CEACAM6 antigen.

[0090] Example 6: Affinity Assay for Anti-CEACAM6 Single-Domain Antibody

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

[0092] Single-domain antibodies: Dilute sequentially with 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 using an SA sensor. The antigen was diluted five times, and the affinities of all single-domain antibodies were at 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, and their affinity ranges are also shown in Table 2.

[0095] Table 2 Results of Affinity Assay for Anti-CEACAM6 Single-Domain Antibodies

[0096]

[0097] Example 7: Humanization of Anti-CEACAM6 Single-Domain Antibody

[0098] The humanization method employed protein surface amino acid resurfacing and the universally applicable antigen-binding complementary region grafting (CDR grafting to a universal framework) technique, referencing a previously filed patent (Invention Title: Anti-EGFR Humanized Single-Domain Antibody, Fc Fusion Protein, Heavy Chain Fab Protein and Its Applications, Application No.: 2019113490209). The humanization steps were as follows: Homologous modeling of the anti-CEACAM6 single-domain antibodies NBC4, 25, and 36 was performed using Modeller9 software. The amino acid sequences of the highly soluble human antibody DP-47 and the homologous sequence NBBcII10 antibody were used to humanize the anti-CEACAM6 single-domain antibodies NBC4, 25, and 36.

[0099] The results of humanization are shown in Table 3.

[0100] Table 3. Humanization results of NBC4, 25, and 36 single-domain antibodies

[0101]

[0102]

[0103] *Note: X*: Indicates a site where the amino acid may undergo humanization. According to literature reports, over 80% of these antibodies have immunogenicity close to that of human antibodies.

[0104] Example 8: Construction of a vector for the anti-CEACAM6 humanized single-domain antibody Fc fusion protein

[0105] (1) First structure: sdAb1-Hinger-CH2-CH3(IgG1-Fc). sdAb = NBC4HM2 or NBC25HM3 or NBC36HM2. (2) Construction steps: The NBC4HM2 or NBC25HM3 or NBC36HM2 + human IgG1-Fc gene is fully synthesized, and XhoI-EcoRI is added for double digestion. The sdAb-Fc gene is ligated into the p327.7 expression vector (patent publication number CN 104195173 A), and the corresponding restriction sites and stop codons are added. The gene is then double-digested with XbaI-SalI, and another sdAb-Fc gene is ligated into the p327.7 expression vector that already contains sdAb-Fc (which has been double-digested and ligated with XhoI-EcoRI), so that one vector has two sdAb-Fc sequences.

[0106] The amino acid and gene sequence listings of the anti-CEACAM6 humanized single-domain antibody, Fc fusion protein, and heavy chain Fab protein provided by this invention are shown in Figure 4.

[0107] Table 4. Sequence listings of anti-CEACAM6 humanized single-domain antibody, Fc fusion protein, and heavy chain Fab protein.

[0108]

[0109]

[0110]

[0111] Example 9: Expression and purification of the anti-CEACAM6 humanized single-domain antibody Fc fusion protein

[0112] The expression vectors NBC4HM2-p327.7, NBC25HM3-p327.7, or NBC36HM2-p327.7 were transfected into CHO / K1 cells, respectively. Stable high-expression cell lines were screened using MSX, and a total of 3 stable expression cell lines were screened. Protein expression was performed by culturing the stable expression cell lines in 500 ml shake flasks.

[0113] Protein purification: The cell expression supernatant was purified by protein A affinity chromatography. The purified protein was then replaced with citrate (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 below. Figure 6 (SDS-PAGE electrophoresis results of reducing and non-reducing gels after expression and purification of three humanized CEACAM6 single-domain antibodies).

[0114] The theoretically calculated values ​​of the proteins expressed by the above fusion protein expression vectors are: 688, 688, and 682 amino acids, respectively; molecular weights (MW), linked by Hinge disulfide bonds, are 7.664 KD, 7.704 KD, and 7.569 KD, respectively; isoelectric points (pI) are 7.88, 7.30, and 7.61, respectively; and the molecular weight after purification and SDS-PAGE reduction is approximately 38 KD, consistent with the theoretically calculated values. The affinity assay for the anti-CEACAM6 humanized single-domain antibody fusion protein was performed as described in Example 6 above, and the affinity analysis results are shown in Table 5.

[0115] Table 5. Affinity analysis results of anti-CEACAM6 humanized single-domain antibody fusion protein and human CEACAM6.

[0116]

[0117] Example 10: Assay for Radioactively Labeled CEACAM6 Humanized Single-Domain Antibody Fusion Protein

[0118] 1. Test Methods

[0119] (1) Antibody DFO modification: Take 1 mL of antibody solution (2 mg / mL of one of the three fusion proteins mentioned above) + 1 mL of 0.5 M NaHCO3 / Na2CO3 solution in the reaction flask, and measure the pH value to alkaline; stir the reaction at 37℃ for 40 min. Purify by 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 solvent; sodium citrate system. The antibody label is at the origin, and the free 89Zr is at the leading edge. See the antibody modification and 89Zr labeling route diagram. Figure 7 .

[0120] 2. Test Results

[0121] Table 6 shows the distribution of single-domain antibody-Fc fusion proteins labeled with the isotope 89Zr in important organs and tumor tissues of a mouse tumor model. Figure 8 and Figure 9 .

[0122] Table 6 gives 89 Zr-CEACAM6 radioactive uptake percentage ID values ​​(mean ± SD, n = 6) in various tissues

[0123]

[0124] Experimental results show that the single-domain antibody-Fc fusion isotope labeling can specifically target transplanted tumors (non-small cell lung cancer, pancreatic cancer, etc.) in mice.

Claims

1. A single-domain antibody against CEACAM6, wherein each single-domain antibody comprises a framework region and three complementarity-determining regions (CDR1, CDR2, and CDR3), characterized in that, The amino acid sequences of the three complementarity-determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9, respectively.

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

12.

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

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

15.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the coding gene as described in claim 3.

6. A fusion protein, characterized in that, The fusion protein is obtained by constructing a single-domain antibody of any one of claims 1 or 2 with IgG-Fc.

7. A conjugate, characterized in that, The single-domain antibody of claim 1 or 2 is coupled with one or more phases of an enzyme phase, a radioisotope, or a chemiluminescent compound to obtain a conjugate.

8. Use of the single-domain antibody of claim 1 or 2, the encoding gene of claim 3, the fusion protein of claim 6, or the conjugate of claim 7 in the preparation of a medicament or reagent for detecting or diagnosing diseases associated with abnormal CEACAM6 expression; wherein the diseases associated with abnormal CEACAM6 expression are non-small cell lung cancer, pancreatic cancer, breast cancer, or ovarian cancer.

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