CN116621980A - Anti-CEACAM6 single domain antibody and its fusion protein and application - Google Patents

Abstract

The invention discloses an anti-CEACAM6 single domain antibody and its fusion protein and application. The invention screens and obtains a group of anti-CEACAM6 single-domain antibodies, which have high activity, strong neutralization or binding ability, and can specifically bind CEACAM6. In the present invention, the single domain antibody is also humanized to obtain a humanized antibody with improved affinity. In the present invention, the single domain antibody or humanized single domain antibody is further fused with human IgG-Fc to obtain a fusion protein. The humanized single domain antibody and/or fusion protein of the present invention can be used for detecting or diagnosing CEACAM6 and for treating diseases related to abnormal expression of CEACAM6.

Description

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

This application is a divisional application of the patent application with application number "202011133599.8", application date "October 21, 2020", and invention name "Anti-CEACAM6 single-domain antibodies, humanized single-domain antibodies, fusion proteins and applications thereof".

Technical Field

The present invention relates to a single domain antibody, in particular to an anti-CEACAM6 single domain antibody and a fusion protein constructed by fusing the single domain antibody or a humanized single domain antibody with IgG1-Fc. The present invention further relates to their application in detecting CEACAM6 and treating diseases related to abnormal CEACAM6 expression, belonging to the field of anti-CEACAM6 single domain antibodies, humanized single domain antibodies and their application.

Background Art

Various carcinoembryonic antigen (CEA) related cell adhesion molecule (CEACAM) proteins belong to the family members of immunoglobulin (Ig) supergene, and the primary structure of the protein is composed of the extracellular region, transmembrane region and intracellular region (some members do not have an intracellular region). Common family members include CEACAM1, 3, 4, 5, 6, 7, 8, 16, 18, 19, 21, and the characteristics of the extracellular domain of the protein are: an N-terminal N domain, followed by no or 1-6 constant C2-like Ig domains (called A region or B region).

These extracellular domains of carcinoembryonic antigen-related cell adhesion molecules are necessary for CEACAM to display its functions as homophilic and heterophilic intercellular adhesion molecules or as receptors for human and rodent pathogens. CEACAM receptors can be oligomers or dimers that form multiple combinations 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 withinmammalian 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).

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), also known as non-specific cross-reacting antigen (NCA, NCA-50/90), CD66c is one of the important members of the carcinoembryonic antigen-related cell adhesion molecule protein family, and it has a high degree of homology with the molecules of family members CEACAM1/7/8. CEACAM6 is a glycosylphosphoinositol (GPI)-linked cell surface protein with an N domain and two C2-like domains. It mediates many possible cis- or trans-directed CEACAM interactions through its extracellular domain with various membrane receptors (some of which have been identified). Studies have reported that CEACAM6 is overexpressed in a variety of tumors, such as 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 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 overexpressed in many tumors, and many studies have reported that it is overexpressed in many 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: in all tumor types studied, CEACAM6 is expressed higher than CEA, and in cancers with overexpression of CEACAM6, tumors of different tissue types have different CEACAM6 levels. The expression abundance of EACAM6 is also different. For example, in breast tumors, the expression abundance of CEACAM6 is: papillary carcinoma > invasive ductal type > lobular > petiole type; in pancreatic cancer, the expression abundance of CEACAM6 is: moderately differentiated type > well differentiated type > poorly differentiated tumors; 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 tumor. There is no difference in the expression of CEACAM6 in prostate cancer tissue and its 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)).

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.

Single domain antibody (sdAb) or nanobody is a heavy chain antibody variable region fragment (VHH) found in alpaca blood that lacks light chain. It has a series of advantages such as simple structure, strong penetration, easy expression and purification, high affinity and stability, and low toxicity and side effects. The high-affinity anti-CEACAM6 single domain antibody screened by single domain antibody technology can be used for the detection and treatment of CEACAM6-overexpressing tumors, and can also provide new detection methods and treatment means for diseases related to CEACAM6-overexpressing tumors.

Summary of the invention

One of the purposes of the present invention is to provide a group of single-domain antibodies against CEACAM6 and their encoding genes;

The second purpose of the present invention is to humanize the single domain antibody against CEACAM6 to obtain a humanized single domain antibody;

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;

The 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;

The 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 to prepare a reagent for detecting CEACAM6 or to treat diseases related to abnormal CEACAM6 expression;

The above object of the present invention is achieved through the following technical solutions:

The present invention first provides a group of single-domain antibodies against CEACAM6, wherein the single-domain antibodies are composed of a framework region and three complementary determining regions, and the single-domain antibodies are selected from any one of NBC4, NBC5 or NBC6; wherein the amino acid sequences of the three complementary determining regions of the single-domain antibody NBC4 are respectively shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3; the amino acid sequences of the three complementary determining regions of the single-domain antibody NBC5 are respectively shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6; the amino acid sequences of the three complementary determining regions of the single-domain antibody NBC6 are respectively shown in SEQ ID No.7, SEQ ID No.8 and SEQID No.9;

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

Protein mutants obtained by deleting, replacing, 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 protection scope of the present invention; in addition, amino acid sequences that are at least 90% identical to any of the amino acid sequences shown above also fall within the protection scope of the present invention.

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 NBC4 is shown in SEQ ID No. 13, the nucleotide sequence of the coding gene of the single domain antibody NBC5 is shown in SEQ ID No. 14, and the nucleotide sequence of the coding gene of the single domain antibody NBC6 is shown in SEQ ID No. 15. Among them, the polynucleotide sequence that can hybridize with the complementary sequence of the polynucleotide sequence shown above under stringent hybridization conditions also belongs to the protection category of the present invention; in addition, the polynucleotide sequence that has at least 90% identity with any of the polynucleotide sequences shown above also belongs to the protection category of the present invention.

The present invention further provides a recombinant expression vector, which comprises one or more of the coding genes of the single-domain antibody; preferably, the recombinant expression vector can be a recombinant prokaryotic cell expression vector, a recombinant yeast expression vector, a recombinant eukaryotic cell expression vector or other recombinant cell expression vector.

The present invention also provides a recombinant host cell, which comprises the recombinant expression vector described above.

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.

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.

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 the 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, 4 subclasses, 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.) immunoglobulin.

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

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, and these conjugates can be used to detect CEACAM6 or treat various diseases related to abnormal CEACAM6 expression.

For example, anti-CEACAM6 humanized single domain antibody and Fc fusion protein are labeled with radioactive isotopes such as ⁶⁸ Ga, ⁸⁹ Zr, ⁶⁴ Cu, ¹⁸ F, ⁸⁶ Y, ⁹⁰ Y, ¹¹¹ In, ^99NV Tc, ¹²⁵ I, ¹²⁴ I, etc. to obtain labeled proteins for PET (positronemission tomography) or SPECT imaging detection. Alternatively, anti-CEACAM6 humanized single domain antibody and Fc fusion protein are labeled with radioactive isotopes such as 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 Bi or ⁶⁷ Ga to obtain labeled proteins for the treatment of diseases related to abnormal CEACAM6 expression.

The anti-CEACAM6 single domain antibody, humanized anti-CEACAM6 single domain antibody, or 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, radioactive isotope, fluorescent compound or chemiluminescent compound provided by the present invention mainly have the following uses:

(1) preparing drugs or reagents related to detecting CEACAM6;

(2) Application of the method for preparing a 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.

Definitions of terms used in this invention

The term "CEACAM6" used herein, carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), also known as non-specific cross-reacting antigen (NCA, NCA-50/90), CD66c is one of the important members of the carcinoembryonic antigen-related cell adhesion molecule protein family. CEACAM6 is a glycosylphosphoinositol (GPI)-linked cell surface protein with an N domain and two C2-like domains, which mediates many possible cis- or trans-directed CEACAM interactions through its extracellular domain with various membrane receptors (some of which have been identified). Studies have reported that CEACAM6 is overexpressed in a variety of tumors, such as 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, and CEACAM6 is a very attractive target for therapeutic intervention in cancer immunotherapy.

The new antibody against CEACAM6 and its Fc fusion protein are the research and development objects of this article, and ultimately the protection objects of this article. The scope of this article involves the obtained anti-CEACAM6 humanized single-domain antibody and its Fc fusion protein, materials (such as pharmaceutical compositions, kits, carriers, etc.) and applications (such as diagnostic applications, therapeutic applications, preparation applications, etc.) with the antibody as a component. However, those skilled in the art should understand that the protection objects of this article are not limited to these examples.

The term "single domain antibody (sdAb)" 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 with the mass of 150-160kDa of a complete antibody, a single domain antibody is much smaller, only about 12-17kDa. The first single domain antibody was artificially engineered from a camel's heavy chain antibody, called a "VHH segment."

The term "identity" of a sequence as used herein can be used interchangeably with "sameness" 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. A modified nucleotide sequence 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 substitution, etc.), the amino acid or nucleotide sequence shown in one or more of the sequences SEQ ID NO: 1-198 of the present invention is modified to obtain a sequence identity greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% 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.

The term "complementary" herein refers to two nucleotide sequences including antiparallel nucleotide sequences that can pair with each other after forming hydrogen bonds between the complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary chains are reverse complementary to each other when both sequences are viewed from the 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 do not necessarily have to be 100% completely complementary.

The term "amino acid sequence" refers to the order in which amino acids are linked to form a peptide chain (or polypeptide). The amino acid sequence can only be read in one direction. There are more than 100 different types of amino acids, of which 20 are commonly used. The present invention does not exclude the possibility that the amino acid chain is modified by other substances, such as sugars, lipids, etc., and the present invention is not limited to the 20 commonly used amino acids.

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 for which protection is sought in the present invention also cover RNA (rRNA, tRNA, mRNA) and their complementary sequences in addition to DNA sequences.

The substitution described in the present invention may be a conservative substitution, i.e., a specific amino acid residue is replaced by a residue having similar physical and chemical characteristics. Non-limiting examples of conservative substitutions include substitutions between amino acid residues containing aliphatic groups (e.g., mutual substitutions between Ile, Val, Leu or Ala), substitutions between polar residues (e.g., mutual 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 prepared by subjecting DNA encoding wild-type proteins to site-directed mutagenesis as a known technique (see, e.g., Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982, which is incorporated herein by reference in its entirety).

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

The term "framework region" refers to the skeleton region. There are about 110 amino acid sequences near the N-terminus of the H and L chains of immunoglobulins, which vary greatly, while the amino acid sequences of other parts are relatively constant. Based on this, the light chain and heavy chain can be divided into variable regions (V) and constant regions (C). The variable region contains the hypervariable region HVR (hypervariable region) or complementary determining region CDR (Complementarity-determining region) and the FR skeleton region.

The term "humanized" antibody refers to the Fr region of the variable region (VH or VHH) of the antibody, 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, 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 the present invention according to actual needs, which is within the scope of the present invention.

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 known to those skilled in the art.

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 a 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

FIG1 constructs a CEACAM6 gene library, and nested PCR amplifies the first round of PCR products. The fragments between 800 and 500 bp are heavy chain antibody gene fragments lacking light chains.

FIG2 : The VHH target gene was obtained by PCR amplification using VHH specific primers.

FIG3 is the SDS-PAGE electrophoresis result of part of the expressed anti-CEACAM6 anti-single domain antibody protein.

FIG. 4 is the SDS-PAGE electrophoresis result of the expressed part of CEACAM6-sdAB after purification by nickel column.

FIG. 5 shows the activity test results of the purified anti-CEACAM6 single domain antibody specifically binding to the human CEACAM6 antigen.

Figure 6 shows the results of SDS-PAGE reducing gel and non-reducing gel electrophoresis of three types of CEACAM6 humanized single domain antibodies after expression and purification; 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.

Fig. 7 Antibody modification and 89Zr labeling route.

Figure 8 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).

FIG9 is a bar graph showing the %ID/g value of radioactive material uptake in each tissue at each time point after administration of ⁸⁹ Zr-CEACAM637.2 antibody.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. 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, but these modifications and replacements all fall within the scope of protection of the present invention.

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

(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, and 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 the immunization was divided into 4-8 times. The absorption of the injection site was tracked to confirm the correct immunization. After the first immunization, the interval was 21 days, and the interval between the second injection 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.

(2) Isolation of alpaca peripheral blood lymphocytes and extraction of RNA: Isolate alpaca peripheral blood leukocytes and extract RNA using QIAGEN kit according to the instructions. RNA purification: RNA was purified using QIAGEN kit according to the instructions, and the obtained RNA concentration and OD260/280 ≥ 1.8 were measured.

(3) Heavy chain antibody variable region - VHH: Synthesize the first chain of 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 800bp in the first PCR amplification are ordinary heavy chain gene fragments, and the fragments between 800 and 500bp are heavy chain antibody gene fragments lacking light chains (Figure 1). The light chain and heavy chain antibody gene fragments lacking light chains were recovered by gel cutting. Using this as a template, VHH-specific primers were used to obtain the VHH target gene (~500bp) by PCR amplification. The gene amplification results are shown in Figure 2. Primers used:

First round PCR Fd5' primer: YF: CGC CAT CAA GGT ACC AGT TGA;

First round PCR Bd3’ primer: YBN: CAG CCG GCC ATG GCC SMK GTR CAG CTG GTG GAKTCT GGG GGA G;

Second round PCR primers:

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;

(4) Connection of VHH fragment and phage display vector and electroporation of TG1 competent cells: After SfI single restriction 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 connected by 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 electroporation bacterial solution was plated on LB/Amp plates, cultured overnight at 32°C, and the antibody connection efficiency was verified by colony PCR the next day. The connection efficiency of the phage antibody library was above 90%. The electroporation bacterial solution was plated on LB/Amp plates, cultured overnight at 32°C, washed with 2YT medium, added with 15% glycerol, and stored at -80°C. Phage library 1.8×10 ⁸ ; 30-50 clones were randomly selected, cloned by PCR, VHH gene insertion rate was 95%, and gene sequencing was performed, and the repetition rate of three CDR sequences in the VHH sequence was less than 2%.

(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.

Example 2 Screening of single domain antibodies against CEACAM6

(1) Screening of CEACAM6-specific single-domain antibodies

In the first round, the concentration of CEACAM6 protein was 50 μg/ml, and 0.5 ml was coated in an immunotube (Thermofisher) at 4°C overnight. In the second and third rounds, the concentrations of CEACAM6 protein were 20 μg/ml and 10 ug/ml, respectively, and 0.5 ml was coated in an immunotube at 4°C overnight. Blocking: 2% skim milk powder PBS, 37°C, incubate for 1.5 hours. Add phage, room temperature for 1 hour, wash 10 times with PBST and PBS respectively, elute the specifically bound phage with 0.5 ml of TEA, infect 2 ml of TG1 in the logarithmic growth phase, determine the titer, and culture and amplify the phage for a new round of screening.

Table 1 Screening results of CEACAM6-specific single domain antibodies

Screening times Add phage amount Phage recovery amount First round 1.1×10 ¹² 3.5×10 ⁵ Round 2 1.2×10 ¹² 4.3×10 ⁶ Round 3 5.0×10 ¹¹ 6.8×10 ⁷

(2) Phage ELISA method to select positive clones

From the colonies grown on the agar plates in the second and/or third rounds of screening, a single colony was randomly picked, inoculated in a 96-deep well culture plate containing 2YT liquid medium containing Amp, and superinfected with helper phage to induce the expression of phage antibodies. The expression supernatant was harvested, and ELISA was performed using CEACAM6 as an antigen, and CEACAM6-positive wells were selected. The gene sequence of the anti-single domain antibody clone was identified by DNA sequencing, and a series of single domain antibody gene sequences including the gene sequences shown in SEQ ID NO.13-15 were obtained for further expression and screening of specific and highly active single domain antibodies.

Example 3 Construction of specific CEACAM6 single domain antibody expression plasmid

The single domain antibody gene specific for CEACAM6 obtained in Example 2 was amplified by PCR to obtain a PCR product with restriction endonuclease 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, and the gene sequence was determined to determine the correctness of the sequence.

(1) Obtain the PCR amplification conditions of the VHH target gene of CEACAM6. Amplify with a 50 μl PCR system. The PCR reaction conditions are: 94° C. for 3 minutes, then 94° C. for 30 seconds; 72° C. for 45 seconds; 52° C. for 30 seconds; a total of 30 cycles; 72° C. for 7 minutes.

5’ primer—GAA GAAGAA GAC AA CAG GCC SAR GTG MAG CTG GWG GAK TCT;

3’ primer—gaagatctccggatccTGAGGAGACGGTGACCTGGGT;

(2) The target gene and vector are digested by enzymes, connected to the target gene and vector, transformed into TG1, PCR is used to identify clones containing the target fragment, and gene sequencing is performed to obtain a single-domain antibody expression plasmid with a correct gene sequence.

Example 4 Expression and purification of anti-single domain antibodies

Inoculate the strain containing plasmid sdAb-pSJF2 described in Example 3 on an LB culture plate containing ampicillin at 37°C overnight. Select a single colony and inoculate it in 15ml of LB culture medium containing ampicillin and culture it in a shaking incubator at 37°C overnight. Transfer 10ml of the overnight culture to 1L of 2YT culture medium containing ampicillin and culture it in a shaking incubator at 37°C at 240 rpm. When the OD value reaches 0.4-0.6, add 0.5-1.0mM IPTG and continue to culture overnight. Centrifuge and collect the bacteria. Add 25% hypertonic sucrose solution to extract the soluble expressed single domain antibody in the periplasm of the cells, centrifuge and collect the supernatant. Obtain a protein with a purity of more than 90% by Ni+ ion affinity chromatography. FIG. 3 is the SDS-PAGE electrophoresis result of part of the expressed CEACAM6 anti-single domain antibody protein, and FIG. 4 is the SDS-PAGE electrophoresis result of part of the expressed CEACAM6-sdAB after purification by nickel column.

Example 5 Binding test of purified CEACAM6 single domain antibody and CEACAM6 antigen (ELISA)

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).

2. Test methods

2.1 Coat with Human CEACAM6 Protein at a concentration of 2ug/ml, 100ul/well, and incubate at 4℃ overnight.

2.2 Add 2% skim milk PBS to block, 300ul/well. Incubate at 37℃ for 1.5h.

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

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

2.5 Add TMB colorimetric solution, 100ul/well, and react for 10 minutes in the dark.

2.6 Add 50ul/well 2M H2SO4 to terminate the reaction.

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

3. Test results

FIG. 5 shows the activity test results of the purified CEACAM6 single domain antibody specifically binding to the human CEACAM6 antigen.

Example 6 Anti-CEACAM6 single domain antibody affinity determination test

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);

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

2) Sample testing

The antigen to be tested was loaded through the SA sensor, and the antigen was diluted into 5 dilutions. The affinities of all single domain antibodies were 50nm, 20nm, 10nm, 1nm, 0.1nm, and 0.01nm. The affinities of some single domain antibodies are shown in Table 2, and their affinity ranges are shown in Table 2.

Table 2 Anti-CEACAM6 single domain antibody affinity test results

Example 7 Humanization of anti-CEACAM6 single domain antibody

The humanization method adopts the method of protein surface amino acid humanization (Resurfacing) and the universal antigen binding complementary region transplantation method (CDR grafting to a universal framework) of VHH humanization, and refers to the patent that has been applied for (Invention Name: Anti-EGFR Humanized Single Domain Antibody, Fc Fusion Protein, Heavy Chain Fab Protein and Its Application, Application Number: 2019113490209). The humanization steps are as follows: Homology modeling of anti-CEACAM6 single domain antibodies NBC4, 25 and 36, modeling software is Modeller9. Referring to the amino acid sequence of the well-soluble human antibody DP-47 and the homologous sequence NBBcII10 antibody, the anti-CEACAM6 single domain antibodies NBC4, 25 and 36 are humanized.

The humanization results are shown in Table 3.

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

*Note: X*: indicates the amino acid site that may undergo humanization changes. According to literature research reports, if it exceeds 80%, its immunogenicity is close to that of human antibodies.

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

(1) The first structure: sdAb1-Hinger-CH2-CH3 (IgG1-Fc). sdAb = NBC4HM2 or NBC25HM3 or NBC36HM2. (2) Construction steps: NBC4HM2 or NBC25HM3 or NBC36HM2 + human IgG1-Fc gene is fully synthesized, XhoI-EcoRI double enzyme digestion is added, the sdAb-Fc gene is connected to the p327.7 expression vector (patent publication number CN 104195173 A), and the corresponding restriction sites and stop codon are added, and XbaI-SalI double enzyme digestion is used to connect another sdAb-Fc gene to the p327.7 expression vector containing sdAb-Fc (XhoI-EcoRI double enzyme digestion and connection), so that one vector finally has two sdAb-Fc sequences.

The amino acid and gene sequence tables 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.

Table 4 Sequence list of anti-CEACAM6 humanized single domain antibody, Fc fusion protein, and heavy chain Fab protein

Example 9 Expression and purification of anti-CEACAM6 humanized single domain antibody Fc fusion protein

The expression vectors NBC4HM2-p327.7 or NBC25HM3-p327.7 or NBC36HM2-p327.7 were transfected into CHO/K1 cells respectively, and stable protein high-expressing cell lines were screened using MSX. A total of 3 stable expression cell lines were screened and the stable expression cell lines were cultured in 500 ml shake flasks for protein expression.

Protein purification: The above cell expression supernatant was purified by protein A affinity chromatography, and the purified protein was replaced with citric acid (0.05% Tween80, 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 reducing gel and non-reducing gel electrophoresis results of three CEACAM6 humanized single domain antibodies after expression and purification).

The theoretical value of the protein expressed by the above fusion protein expression vector is: containing 688, 688 and 682 amino acids respectively; molecular weight (MW), after Hinger disulfide bond connection molecular weight is 7.664KD, 7.704KD and 7.569KD respectively, isoelectric point (pI) is 7.88, 7.30 and 7.61 respectively, and the molecular weight after purification protein electrophoresis SDS-PAGE reduction is about 38KD, which is consistent with the theoretical value. The affinity determination test of the anti-CEACAM6 humanized single domain antibody fusion protein is the same as the above Example 6., and the affinity analysis results are shown in Table 5.

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

Example 10 Experiment on radioisotope-labeled CEACAM6 humanized single-domain antibody fusion protein

1. Test methods

(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 NaHCO ₃ /Na ₂ CO ₃ solution in the reaction bottle, measure the pH value to alkaline; stir and react at 37°C for 40 minutes. PD10 column purification. (2) Antibody labeling: Take a small amount of 89Zr, add 2M Na ₂ CO ₃ 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 front. The antibody modification and 89Zr labeling roadmap is shown in Figure 7.

2. Test results

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

Table 6 %ID values of radioactive material uptake in various tissues after administration of ⁸⁹ Zr-CEACAM6 (mean±SD, n=6)

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 (10)

1. Anti-CEACAM6 single-domain antibody NBC6, the single-domain antibody is composed of a framework region and three complementarity determining regions CDR1, CDR2 and CDR3, characterized in that the amino acids of the three complementarity determining regions CDR1, CDR2 and CDR3 The sequences are respectively shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 or one or more amino acids in any one of the amino acid sequences shown above are deleted, substituted, inserted and/or added. The protein mutant obtained has the same function as the protein before mutation; or an amino acid sequence having at least 90% identity with any of the amino acid sequences shown above. 2. The single domain antibody according to claim 1, characterized in that its amino acid sequence is shown in SEQ ID No. 12; or one or more amino acids in any of the amino acid sequences shown above are deleted, A protein mutant obtained by substitution, insertion and/or addition, the protein mutant has the same function as the protein before mutation; or an amino acid sequence having at least 90% identity with any of the amino acid sequences shown above. 3. The gene encoding the single domain antibody described in any one of claims 1 or 2. 4. according to the described encoding gene of claim 3, it is characterized in that, the nucleotide sequence of its described encoding gene is shown in SEQID No.15; Or a polynucleotide sequence capable of hybridizing to the complementary sequence of any of the above-mentioned polynucleotide sequences under stringent hybridization conditions; or having at least 90% identity with any of the above-mentioned polynucleotide sequences polynucleotide sequence. 5. A recombinant expression vector, characterized in that the recombinant expression vector comprises the coding gene of claim 3. 6. A fusion protein, characterized in that it is a fusion protein constructed by constructing the single domain antibody according to any one of claims 1 or 2 and IgG-Fc. 7. according to the described fusion protein of claim 6, it is characterized in that, described Fc gene sequence is derived from IgG, IgA, the Fc gene sequence of IgM or derives from IgG1, IgG2, IgG3 or IgG4; Described IgG is preferably Human IgG and its IgG1, 2, 3, 4 subclasses, human IgM, human IgA or Fc fragments of other animal immunoglobulins. 8. A conjugate, characterized in that the single domain antibody according to claim 1 or 2 is coupled with one or more of cytotoxic agents, enzyme phases, radioactive isotopes or chemiluminescent compounds to obtain conjugated compound. 9. The single domain antibody according to claim 1 or 2, the coding gene according to claim 3, the fusion protein according to claim 6 or 7 or the conjugate according to claim 8 in the preparation detection or diagnosis Use in medicines or reagents for diseases related to abnormal expression of CEACAM6. 10. The use according to claim 9, characterized in that the diseases related to the abnormal expression of CEACAM6 include non-small cell lung cancer, pancreatic cancer, breast cancer or ovarian cancer.