CN115850483A - anti-CEACAM 6 single-domain antibody, humanized single-domain antibody, fc fusion protein and application thereof - Google Patents
anti-CEACAM 6 single-domain antibody, humanized single-domain antibody, fc fusion protein and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-CEACAM 6 single-domain antibody, a humanized single-domain antibody, an Fc fusion protein and application thereof. The invention obtains a group of CEACAM 6-resistant single-domain antibodies with stronger neutralizing or binding capacity by screening, and the antibodies can be specifically bound with CEACAM6. The invention also carries out humanization transformation on the single-domain antibody to obtain the humanized antibody with improved affinity. The single domain antibody or the humanized single domain antibody is further fused with human IgG-Fc to obtain the single domain antibody-Fc fusion protein, and the fusion protein can be well targeted to the in-vivo transplanted tumor of a mouse after being labeled by an isotope. The anti-CEACAM 6 single-domain antibody, the humanized single-domain antibody and the single-domain antibody-Fc fusion protein provided by the invention can be used for detecting or diagnosing CEACAM6 and treating diseases related to CEACAM6 abnormal expression.
Description
The application is a divisional application with the application number of "202011135295.5", the application date of "21/10/2020", the invention name of the invention is "anti-CEACAM 6 single domain antibody, humanized single domain antibody and Fc fusion protein and application thereof
Technical Field
The invention relates to a single domain antibody, in particular to a single domain antibody for resisting CEACAM6 and a fusion protein constructed by fusing the single domain antibody or a humanized single domain antibody with IgG1-Fc, and further relates to applications of the single domain antibody or the humanized single domain antibody in detecting CEACAM6 and treating CEACAM6 expression abnormality related diseases, belonging to the fields of the single domain antibody for resisting CEACAM6, the humanized single domain antibody and the application thereof.
Background
The proteins of various Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which constitute primary structures of extracellular, transmembrane and intracellular regions of cells (some of which have no intracellular region), belong to the family members of immunoglobulin (Ig) supergenes. Common family members are CEACAM1,3,4,5,6,7,8,16,18,19,21, whose extracellular domains of proteins are characterized by: there is an N-terminal N domain followed by either none or 1-6 constant C2-like Ig domains (referred to as a or B regions).
These extracellular domains of carcinoembryonic antigen-associated cell adhesion molecules are essential for CEACAM to display its function as homotropic and heterophilic cell adhesion molecules or as receptors for human and rodent pathogens. CEACAM receptors can be oligomers or dimers that form multiple combinations with other ligands in the cell membrane to modulate its important cellular functions. In addition to expression in human tissues, the CEACAM gene family is highly conserved among 27 other mammalian species (Robert kammer, wolf wing zimmermann. Coesolution of activating and inhibiting receptors with a macromolecular antibiotic family bmc biol.2010 Feb 4. The biological functions of CEACAM are to maintain Cell-to-Cell adhesion by their homotropic and heterotopic interactions, including the role in differentiation and formation of three-dimensional tissue structures, angiogenesis, apoptosis, tumor suppression and metastasis, among others (Kuespert K.et al CEACAMs: the role of the hair roller in physiology and pathophysiology. Current eye Cell biol.2006 Oct;18 (5): 565-71, athanasia Pavlopoulousu and Andrea Scorilas. A Comprehensive pharmacologic and Structural Analysis of the Carcinometric Analysis (CEA) Gene plane biol.2014Jun; 6 (6): 1314-1326).
Carcinoembryonic antigen-associated cell adhesion molecule 6 (CEACAM 6), also known as non-specific cross-reactive antigen (NCA, NCA-50/90)), CD66c is one of the important members of the carcinoembryonic antigen-associated cell adhesion molecule protein family, which has high homology with the molecule of family member CEACAM 1/7/8. CEACAM6 is a Glycosylphosphatidylinositol (GPI) -linked cell surface protein with one N domain and 2C 2-like domains, through which the extracellular domain with various membrane receptors (some of which have been identified) mediates many possible cis-or trans-directed CEACAM interactions. The CEACAM6 has been reported to be over-expressed in various tumors, such as non-small cell lung cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, gastric cancer, ovarian cancer and the like. The overexpression of CEACAM6 can cause metaplasia-like morphological change in epithelial tissues, tumor invasiveness and tolerance enhancement against chemical drugs, tumor metastasis, reduction of apoptosis and the like, and the reduction of gene expression of CEACAM6 by SiRNA and the inhibition of CEACAM6 protein function by monoclonal antibody can reverse the effects caused by CEACAM6 overexpression. Although CEACAM6 is also expressed in many normal tissues of human, such as the granulocytic cell line, it is overexpressed in many tumors, and many studies have been reported; the study comparing the expression levels of CEACAM6 and CEACAM5 (CEA) in lung, breast, prostate, colon, pancreatic and ovarian cancer tissues and their paraneoplastic and normal tissues shows that: CEACAM6 is expressed more than CEA in all tumor types studied, and CEACAM6 is expressed in different tissue types of tumors in cancers with over-expression of CEACAM6, as compared to breast tumor CEACAM 6: papillary carcinoma > invasive ductal pattern > lobular pattern > lobate petiolic pattern; the expression abundance of CEACAM6 in pancreatic cancer is: moderate differentiation type > full differentiation type > low differentiation type tumor; CEACAM6 expression is 3 times higher in mucinous ovarian adenocarcinoma than in serous ovarian adenocarcinoma; expression of CEACAM6 in non-small cell lung carcinoma is lung adenocarcinoma > lung squamous carcinoma; CEACAM6 expression in liver metastatic colon carcinoma > primary tumor > lymph node metastatic tumor. Expression of CEACAM6 in prostate cancer tissue is not different from that in paracancerous normal tissue (Nicode Beauchemin and dAzadehabazadeh. Carcino organizing anti-related cell adhesives (CEACAMs) in cancer progression and cancer. Rev.2013Dec.;32 (3-4): 643-71: rosalyn D Blumenthal et al.expression patches of ACACAM5 and CEACAMM 6 in primary and cancer. BMC cancer.2007.2 (1-15)).
As mentioned above, CEACEA6 may be a specific target antigen for these over-expressed tumors, CEACAM6 is a very attractive new target for therapeutic intervention in cancer immunotherapy.
The single domain antibody (sdAb) or nano antibody (nanobody) is a heavy chain antibody variable region fragment (VHH) which is found in alpaca blood and lacks a light chain, and has a series of advantages of simple structure, strong penetration, easy expression and purification, high affinity and stability, small toxic and side effects and the like.
Disclosure of Invention
One of the objects of the present invention is to provide a single domain antibody against CEACAM6 and a gene encoding the same;
the other purpose of the invention is to carry out humanized modification on the single domain antibody of the CEACAM6 to obtain a humanized single domain antibody;
the third purpose of the invention is to fuse the single-domain antibody or humanized single-domain antibody with human IgG1-Fc to obtain a fusion protein;
the fourth purpose of the invention is to couple the single-domain antibody or the humanized single-domain antibody with one or more of enzyme phase, radioactive isotope, fluorescent compound or chemiluminescent compound to obtain a conjugate;
the fourth purpose of the invention is to apply the anti-CEACAM 6 single-domain antibody, the anti-CEACAM 6 humanized single-domain antibody, the fusion protein and the conjugate to the preparation of a reagent for detecting CEACAM6 or the treatment of diseases related to CEACAM6 abnormal expression;
the above object of the present invention is achieved by the following technical solutions:
the invention firstly provides a single domain antibody against CEACAM6, which consists of a framework region and 3 complementarity determining regions, wherein the single domain antibody is NBC36; wherein, the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBC36 are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3;
the invention further provides an amino acid sequence of the single domain antibody, wherein the amino acid sequence of the single domain antibody NBC36 is shown as SEQ ID No. 4.
Protein mutants obtained by deleting, substituting, inserting and/or adding one or more amino acids in any one of the amino acid sequences shown above, wherein the protein mutants have the same functions as the protein before mutation, and the protein mutants belong to the protection scope of the invention; in addition, amino acid sequences having at least 90% identity to any of the above-described amino acid sequences are also within the scope of the present invention.
The invention further provides a coding gene sequence of the single domain antibody, wherein the nucleotide sequence of the coding gene of the single domain antibody NBC36 is shown as SEQ ID No. 5. Wherein, the polynucleotide sequence capable of hybridizing with the complementary sequence of the polynucleotide sequence under the strict hybridization condition also belongs to the protection scope of the invention; also, polynucleotide sequences having at least 90% identity to any of the polynucleotide sequences shown above are within the scope of the present invention.
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 can be a recombinant prokaryotic cell expression vector, a recombinant yeast expression vector, a recombinant eukaryotic cell expression vector or other recombinant cell expression vectors.
The present invention also provides a recombinant host cell comprising 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 recombinant yeast cell, and the recombinant prokaryotic expression cell is preferably escherichia coli.
The invention further carries out humanization transformation on the single domain antibody NBC36 to obtain 2 humanized antibodies NBC36HM1 and NBC36HM2, and the amino acid sequences of the humanized antibodies are respectively shown as SEQ ID No.6 and SEQ ID No. 7.
The invention further constructs a fusion protein by the anti-CEACAM 6 single-domain antibody or humanized single-domain antibody and IgG-Fc; wherein, the Fc gene sequence can be derived from IgG, igA and IgM or from IgG1, igG2, igG3 or IgG4. The IgG is preferably human IgG and subclasses of IgG1, 2, 3 and 4, and can also be Fc fragment gene and amino acid sequence of human IgM, human IgA or other animal (such as mouse, rabbit, monkey) immunoglobulin.
As a preferred embodiment of the invention, the humanized antibody NBC36HM2 is fused with a human IgG1-Fc gene to obtain a fusion protein with an amino acid sequence shown in SEQ ID No.8, and the nucleotide sequence of the coding gene is shown in SEQ ID No. 9.
The invention further couples the single-domain antibody or humanized single-domain antibody with one or more of enzyme phase (such as horseradish peroxidase, alkaline phosphatase, etc.), radioisotope, fluorescent compound or chemiluminescent compound (the chemiluminescent compound can be a fluorescent compound) to obtain conjugates, and the conjugates can be used for detecting CEACAM6 or treating various diseases related to abnormal expression of CEACAM6.
For example, anti-CEACAM 6 humanized single domain antibody, fc fusion protein 68 Ga, 89 Zr, 64 Cu, 18 F, 86 Y, 90 Y, 111 In, 99NV Tc, 125 I, 124 And I, labeling with radioactive isotopes to obtain labeled protein for imaging detection of PET (positron emission tomography) or SPECT (single photon emission computed tomography). Or anti-CEACAM 6 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 Bi or 67 The marked protein obtained by marking radioactive isotopes such as Ga is used for treating diseases related to CEACAM6 expression abnormality.
The single-domain antibody of anti-CEACAM 6, the humanized single-domain antibody of anti-CEACAM 6, or the fusion protein constructed by the humanized single-domain antibody and IgG-Fc, the conjugate obtained by coupling the single-domain antibody or the humanized single-domain antibody with an enzyme phase, a radioactive isotope, a fluorescent compound or a chemiluminescent compound mainly has the following uses:
(1) Preparing a medicine or reagent related to detecting CEACAM 6;
(2) The application of preparing the medicine for treating the diseases related to the CEACAM6 expression abnormality; preferably, the diseases related to abnormal CEACAM6 expression comprise tumor diseases such as non-small cell lung cancer, pancreatic cancer, breast cancer and ovarian cancer.
Definitions of terms to which the invention relates
The term "CEACAM6" as used herein, carcinoembryonic antigen-associated cell adhesion molecule 6 (CEACAM 6), also known as non-specific cross-reactive antigen (NCA, NCA-50/90), CD66c is one of the important members of the carcinoembryonic antigen-associated cell adhesion molecule protein family. CEACAM6 is a Glycosylphosphatidylinositol (GPI) -linked cell surface protein with one N domain and 2C 2-like domains, through which the extracellular domain with various membrane receptors (some of which have been identified) mediates many possible cis-or trans-directed CEACAM interactions. Research reports that CEACAM6 is overexpressed in various tumors, such as non-small cell lung cancer, pancreatic cancer, breast cancer, colorectal cancer, liver cancer, gastric cancer, ovarian cancer and the like, to different degrees. CEACEA6 may be a specific target antigen for these over-expressed tumors, CEACAM6 being a very attractive target for therapeutic intervention in cancer immunotherapy.
The scope of the present invention relates to substances (e.g., pharmaceutical compositions, kits, vectors, etc.), applications (e.g., diagnostic applications, therapeutic applications, preparative applications, etc.) and applications (e.g., diagnostic applications, etc.) of the obtained anti-CEACAM 6 humanized single domain antibody and Fc fusion protein thereof, which are components of the antibody, however, it should be understood by those skilled in the art that the subject of the present invention is not limited to those exemplified ones.
The term "single domain antibody (sdAb)" as used herein refers to a fragment comprising a single variable domain in an antibody, also known as a Nanobody. Like an intact antibody, it can selectively bind to a particular antigen. Single domain antibodies appear much smaller, approximately only 12-17kDa, compared to the 150-160kDa mass of intact antibodies. The first single domain antibody was artificially engineered from a camelid heavy chain antibody, referred to as a "VHH segment".
The term "identity" of sequences as used herein is used interchangeably with "identity" 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. The modified nucleotide sequence may be obtained by substitution, deletion and/or addition of one or several amino acids or bases to a known sequence. For example, by conventional means (e.g., conservative substitutions, etc.), the sequences of SEQ ID NOs: 1-198, and can have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% sequence identity thereto, and substantially the same properties, all within the scope of the present invention. Preferably, the present invention obtains sequence identity by conservative substitutions, but is not limited to conservative substitutions.
The term "complementary" as used herein refers to two nucleotide sequences comprising antiparallel nucleotide sequences capable of pairing with each other upon hydrogen bonding between 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 the sequences are viewed in both 5 'to 3' directions. It is also known in the art that two sequences that hybridize to each other under a given set of conditions do not necessarily have to be 100% perfectly complementary.
The term "amino acid sequence" refers to the order in which amino acids are linked to form a peptide chain (or polypeptide), and the amino acid sequence can only be read in one orientation. There are more than 100 different types of amino acids, 20 of which are commonly used, and the present invention does not exclude other substances such as saccharides, lipids, etc. from the amino acid chain, nor is the present invention limited to the amino acids commonly used in 20.
The term "nucleotide sequence" refers to the order of bases in DNA or RNA, i.e., A, T, G, C in DNA, or A, U, G, C in mRNA, and also includes rRNA, tRNA, mRNA. It is understood that the antibody genes claimed in the present invention also encompass RNA (rRNA, tRNA, mRNA) and their complementary sequences in addition to DNA sequences.
The substitutions described in the present invention may be conservative substitutions, i.e. the substitution of a specific amino acid residue for a residue 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), and the like. Mutants resulting from deletion, substitution, insertion and/or addition of amino acids can be prepared by subjecting DNA encoding a wild-type protein to, for example, site-directed mutagenesis as 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).
The term "Expression vectors" refers to vectors in which Expression elements (e.g., promoter, RBS, terminator, etc.) are added to the basic backbone of a cloning vector to enable the Expression of a desired gene. The four parts of the expression vector are as follows: target gene, promoter, terminator and marker gene. The present invention includes, but is not limited to, prokaryotic, eukaryotic, or other cellular expression vectors.
The term "Framework region", i.e., a Framework region, has a large variation of about 110 amino acid sequences near the N-terminus of H and L chains of an immunoglobulin, and the amino acid sequences of the other portions are relatively constant, whereby the light chain and the heavy chain can be distinguished into a variable region (V) and a constant region (C). The variable region includes the hypervariable region HVR (hypervariable region) or Complementarity determining region CDR (complementary-determining region) and FR framework regions.
The term "humanized" antibody refers to a portion of the Fr region of the variable region (VH or VHH), a portion of the constant region (i.e., the CH and CL regions) or all of the antibody encoded by human antibody genes. Humanized antibodies can greatly reduce the immune side effects of heterologous antibodies on the human body. Humanized antibodies include chimeric antibodies, modified antibodies, fully humanized antibodies, and the like. It will be appreciated that those skilled in the art will be able to prepare suitable humanized forms of the single domain antibodies of the invention as required and within the scope of the invention.
The terms "mutation" and "mutant" have their usual meanings herein, and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, which has the same meaning as is commonly known to those of skill 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 the recombinant host cell, e.g., direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.
Drawings
FIG. 1 is a CEACAM6 gene bank constructed, the first round PCR product is amplified by nested PCR, and the heavy chain antibody gene segment of the deletion light chain is between 800 and 500 bp.
FIG. 2 VHH target genes were obtained by PCR amplification using VHH specific primers.
FIG. 3 shows SDS-PAGE of the expressed partial anti-CEACAM 6 anti-single domain antibody protein.
FIG. 4 shows SDS-PAGE of the expressed fractions of CEACAM6-sdaB after nickel column purification.
FIG. 5 shows the results of activity assay of purified anti-CEACAM 6 single domain antibody specifically binding to human CEACAM6 antigen.
FIG. 6 shows the electrophoresis results of SDS-PAGE reducing gel and non-reducing gel after 3 CEACAM6 humanized single domain antibodies are expressed and purified; EG2M1-EG10M1-Fc-p327.7 expressed purified reducing protein band; EG2M1-Fc-EG10M1-p327.7 expression purified reducing protein band; the non-reducing protein band after EG2M1-EG10M1-Fc-p327.7 expression and purification; 4, expressing and purifying EG2M1-Fc-EG10M1-p327.7 to obtain a non-reducing protein band; 5. protein molecular weight standards (Marker); the molecular weight indicated by the arrow is 50kD.
FIG. 7 antibody modification and 89Zr labeling scheme.
FIG. 8 shows the distribution of 89Zr as the isotope labeled with Fc fusion protein in mouse tumor animal model (PET/CT scan).
Figure 9 administration 89 Histogram of% ID/g values of radioactive uptake by tissues at various time points after Zr-CEACAM637.2 antibody.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 construction of Single Domain antibody library specific for anti-CEACAM 6 antigen
(1) CEACAM6 antigen immunization alpaca: according to the conventional immunization method, adult healthy alpaca is selected from CEACAM6 antigen (Human CEACAM6 Protein, human, recombinant (His Tag)), the antigen is injected into the back of the neck at multiple points subcutaneously, the antigen and the equivalent volume of Freund's adjuvant are added, the immunization is divided into 4 to 8 times, and the absorption condition of the injection site block is followed to confirm the immunization correctness. After the first immunization, the interval is 21 days, the second needle immunization is started, the immunization interval is 7-15 days, after the 4 th immunization, blood is collected, the antigen immunization titer is determined, when the titer reaches about 5 ten thousand times (ELISA method), about 100ml of whole blood is collected, lymphocytes are separated, and the whole blood is stored at-80 ℃ for later use.
(2) Separation of alpaca peripheral blood lymphocytes and extraction of RNA: alpaca peripheral blood leukocytes were isolated and RNA was extracted using QIAGEN kit according to the instructions. RNA purification: RNA purification was performed using QIAGEN kit, and the concentration of the obtained RNA and OD260/280 were determined to be 1.8 or more according to the instructions.
(3) Heavy chain antibody variable region-VHH: first strand cDNA Synthesis: the procedure was followed using a cDNA synthesis Kit (MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0, TAKARA). Using this template, two sets of primers were used to perform PCR amplification of the heavy chain antibody VHH gene fragment. By adopting a nested PCR method, a common heavy chain gene fragment is larger than 800bp in the first PCR amplification, a heavy chain antibody gene fragment with a deleted light chain is between 800 and 500bp (figure 1), the gel is cut to recover the deleted light chain heavy chain antibody gene fragment, the gene fragment is used as a template, a VHH (VHH) target gene (-500 bp) is obtained by PCR amplification by using a VHH specific primer, and the gene amplification result is shown in figure 2. The primers used were:
the first round of PCRfd5' primer YF CGC CAT CAA GGT ACC AGT TGA;
the first round PCR Bd3' primer YBN CAG CCG GCC ATG GCC SMK GTR CAG CTG GTG GAK TCT 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) Ligation of VHH fragment and phage display vector and electrotransformation of TG1 competence: after SfI single digestion of VHH fragment and pHEN6 vector plasmid, VHH fragment and pHEN6 vector (Concrath, KEM other. Antisense Agents Chemothers (antisense Chemothery) 2001,45 (10) 2807-12.) were ligated with ligase (T.sub. 4 NEB company), were electroporated into TG1 competent cells, 10 were electroporated, plates were coated, and the antibody insertion rate was verified by colony PCR. Detecting the cloning efficiency of the recombinant gene: coating the electro-conversion bacterial liquid on an LB/Amp plate, culturing at 32 ℃ overnight, and verifying the connection efficiency of the antibody by a colony PCR method the next day, wherein the connection efficiency of a phage antibody library is more than 90%. The electrotransformation bacteria solution is spread on LB/Amp plate, cultured overnight at 32 ℃, washed with 2YT medium, added with 15% glycerol and stored at-80 ℃. Phage library 1.8X 10 8 (ii) a Randomly selecting 30-50 clones, cloning PCR, ensuring that the VHH gene insertion rate is 95%, carrying out gene sequencing, and ensuring that the repetition rate of three CDR sequences in the VHH sequence is less than 2%.
(5) Preparation of VHH phage antibody libraries: the antibody library was rescued by adding the helper phage M13K07 (Invitrogen): phage antibody libraries were prepared according to conventional methods and stored at-80 ℃ until use.
Example 2 screening of Single Domain antibodies against CEACAM6
(1) Screening for CEACAM 6-specific Single Domain antibodies
The first round CEACAM6 protein concentration was 50. Mu.g/ml, 0.5ml coated immune tubes (Thermofeisher Co.) and left overnight at 4 ℃. The second and third rounds were performed by coating the immunization tubes with CEACAM6 protein concentration of 20. Mu.g/ml, 10ug/ml,0.5ml, respectively, overnight at 4 ℃. And (3) sealing: 2% nonfat dry milk PBS,37 ℃, incubated for 1.5 hours. Phage was added, washed 10 times with PBST and PBS, each at room temperature for 1 hour, specifically bound phage was eluted with 0.5ml of TEA, 2ml of TG1 was infected at log phase, titer was determined, and amplified phage were cultured for a new round of screening.
TABLE 1 screening results for CEACAM 6-specific single-domain antibodies
| Number of screens | Adding phage amount | Amount of phage recovered by elution |
| First wheel | 1.1×10 12 | 3.5×10 5 |
| Second wheel | 1.2×10 12 | 4.3×10 6 |
| Third wheel | 5.0×10 11 | 6.8×10 7 |
(2) Selection of positive clones by phage ELISA
Colonies grown on agar plates were selected from round 2 and/or round 3, single colonies were randomly picked, inoculated in 96-well plates containing Amp 2YT broth, and superinfected with helper phage to induce expression of phage antibodies. And (3) harvesting expression supernatant, performing ELISA determination by taking CEACAM6 as an antigen, selecting CEACAM6 positive holes, and performing DNA sequencing to identify the gene sequence of the anti-single-domain antibody clone so as to obtain a series of single-domain antibody gene sequences including the gene sequences shown in SEQ ID NO.13-15 for further expressing and screening specific and high-activity single-domain antibodies.
Example 3 construction of expression plasmid for specific CEACAM6 Single Domain antibody
The single domain antibody gene of the specific CEACAM6 obtained in example 2 was amplified by PCR to obtain PCR products with restriction enzymes BbsI and BamHI sites, and the PCR products and the vector (pSJF 2 vector, kim is. Biosic biochem.2002,66 (5): 1148-51) were treated with restriction enzymes BbsI and BamHI, respectively, and subjected to T 4 Ligase is connected and recombined to obtain a plasmid sdAb-pSJF2 which can be efficiently expressed in Escherichia coli, and a gene sequence is measured to determine the correctness of the sequence.
(1) Obtaining PCR amplification conditions of the VHH target gene of CEACAM6, amplifying a 50-microliter PCR system, and carrying out PCR reaction conditions: firstly, 94 ℃ for 3 minutes, and then 94 ℃ for 30 seconds; 72 ℃,45 seconds, 52 ℃,30 seconds; 30 cycles in total; 72 ℃ for 7 minutes.
5' primer GAA GAAGAA GAC AA CAG GCC SAR GTG MAG CTG GWGGAK TCT;
3' primer-gaagatactcggatcttgatctgagacggtgaactgcctgggt;
(2) The target gene and the carrier are cut by enzyme, the target gene and the carrier are connected, TG1 is transformed, the clone containing the target segment is identified by PCR, the gene is sequenced, and the single domain antibody expression plasmid with correct gene sequence is obtained.
Example 4 expression and purification of anti-Single Domain antibodies
The plasmid sdAb-pSJF 2-containing strain described in example 3 was inoculated onto LB plates containing ampicillin overnight at 37 ℃. Individual colonies were selected and inoculated into 15ml of LB medium containing ampicillin and shake-cultured overnight at 37 ℃. Transferring 10ml of overnight culture into 1L of 2YT culture solution containing ampicillin, shake culturing at 37 ℃,240 rpm, adding 0.5-1.0 mM IPTG when OD value reaches 0.4-0.6, and continuing culturing overnight. And (4) centrifuging and collecting bacteria. Adding 25% hypertonic sucrose solution, extracting soluble expressed single domain antibody in periplasm of cells, centrifuging, and collecting supernatant. Obtaining the protein with the purity of more than 90 percent by Ni + ion affinity chromatography. FIG. 3 shows SDS-PAGE results of the expressed partial CEACAM6 anti-single domain antibody protein, and FIG. 4 shows SDS-PAGE results of the expressed partial CEACAM6-sdaB purified by nickel column.
EXAMPLE 5 binding assay (ELISA) of purified CEACAM6 Single Domain antibody to CEACAM6 antigen
1. Test materials: removable enzyme plate (Thermofish Co., ltd.), CEACAM6 antigen, anti-Myc tag antibody-HRP (Beijing Yinqiao Shenzhou Biotech Co., ltd.), TMB color solution (Beijing Meikoidde, cat: 1001), coating solution pH 9.6, BSA (Sigma Co., ltd.).
2. Test method
2.1 separately coated Human CEACAM6 Protein, concentration 2ug/ml,100 ul/well, incubated overnight at 4 ℃.
2.2 Add 2% skim milk PBS to block, 300 ul/well. Incubate at 37 ℃ for 1.5h.
2.3 dilution of different numbered CEACAM6 single domain antibodies to final concentrations of 10.0ug/ml and 1.0ug/ml,100 ul/well.
2.4 dilution Anti-Myc tag antibody (HRP) (1.
2.5 adding TMB color development liquid, 100 ul/hole, and reacting for 10min in dark.
2.6 stop the reaction by adding 50 ul/well of 2M H2SO 4.
2.7 OD measurement at 450 nm.
3. Test results
Fig. 5 is a result of an activity test of the purified CEACAM6 single domain antibody specifically binding to human CEACAM6 antigen.
Example 6 Single Domain antibody affinity assay for anti-CEACAM 6
1) Sample preparation of antigen: bio-CEACAM6 was diluted to 10. Mu.g/ml with 1 XPS buffer (1 XPBS with 0.05% Tween 20, 0.1% BSA, pH 7.2);
single domain antibodies: diluting with 1 × kinetic buffer solution sequentially to 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM;
2) Sample testing
The antigen to be detected is loaded through an SA sensor, the antigen is diluted by 5 dilutions, and the affinity of all single-domain antibodies is 50nm, 20nm, 10nm, 1nm, 0.1nm and 0.01nm. The partial single domain antibody affinities are shown in table 2, and the affinity ranges are shown in table 2.
TABLE 2 affinity assay results for anti-CEACAM 6 single domain antibodies
Example 7 humanization of anti-CEACAM 6 Single Domain antibodies
The humanization method is completed by adopting a protein surface amino acid humanization (Resurfacing) method and a VHH humanization general antigen binding complementary region transplantation method (CDR grafting to a universal framework), and refers to a patent already filed (anti-EGFR humanized single domain antibody, fc fusion protein, heavy chain Fab protein and application thereof, application number: 2019113490209).
The humanization procedure was as follows: anti-CEACAM 6 single domain antibodies NBC4, 25 and 36 were modeled homologously with the modeling software Modeller9. The anti-CEACAM 6 single domain antibodies NBC4, 25 and 36 were humanized with reference to the amino acid sequence of the well soluble human antibody DP-47 and the homologous sequence NBBcII10 antibody.
The results of humanization are shown in Table 3.
TABLE 3 NBC4, 25 and 36 Single Domain antibody humanization results
* Note that: x: indicating the possible sites of humanization changes for this amino acid. According to literature research reports, the immunogenicity of the antibody is close to that of a human antibody by more than 80 percent.
Example 8 vector construction of humanized Single Domain antibody Fc fusion protein against CEACAM6
(1) The first structure: sdAb 1-hinder-CH 2-CH3 (IgG 1-Fc). sdAb = NBC4HM2 or NBC25HM3 or NBC36HM2.
(2) The construction steps are as follows: NBC4HM2 or NBC25HM3 or NBC36HM2+ human IgG1-Fc gene was synthesized, xhoI-EcoRI double-digested was added, the sdAb-Fc gene was ligated to p327.7 expression vector (patent publication No. CN 104195173A), and the corresponding cleavage site and stop codon were added, xbaI-SalI double-digested was used, and the other sdAb-Fc gene was ligated to p327.7 expression vector already containing sdAb-Fc (already XhoI-EcoRI double-digested), and finally 2 sdAb-Fc sequences were added to one vector.
The sequence of amino acids and genes of the anti-CEACAM 6 humanized single domain antibody, fc fusion protein and heavy chain Fab protein provided by the invention is shown in 4.
TABLE 4 sequence Listing of anti-CEACAM 6 humanized single domain antibody, fc fusion protein, heavy chain Fab protein
Example 9 expression and purification of anti-CEACAM 6 humanized Single Domain antibody Fc fusion proteins
The expression vector NBC4HM2-p327.7 or NBC25HM3-p327.7 or NBC36HM2-p327.7 is transfected into CHO/K1 cell respectively, MSX is used to screen stable protein high expression cell strain, 3 stable expression cell strains are screened together, and the stable expression cell strains are cultured in 500ml shake flask for protein expression.
Protein purification: the cell expression supernatant was purified by affinity chromatography using protein A strain, and the purified protein was replaced with a citric acid (0.05% Tween80, pH 6.2) buffer. The protein expressed and purified by the anti-CEACAM 6 humanized single-domain antibody Fc fusion protein vector is shown in FIG. 6 (SDS-PAGE reduced gel and non-reduced gel electrophoresis results after 3 CEACAM6 humanized single-domain antibodies are expressed and purified).
The theoretical calculation value of the protein expressed by the fusion protein expression vector is as follows: 688 amino acids, 688 amino acids and 682 amino acids are contained respectively; the Molecular Weight (MW) is respectively 7.664KD, 7.704KD and 7.569KD through Hinger disulfide bond connection, the isoelectric point is respectively 7.88, 7.30 and 7.61, and the molecular weight after protein electrophoresis SDS-PAGE reduction after purification is about 38KD, which is consistent with the theoretical calculation value. The affinity assay for the anti-CEACAM 6 humanized single domain antibody fusion protein was the same as in example 6 above, and the results of the affinity assay are shown in table 5.
TABLE 5 affinity analysis results of anti-CEACAM 6 humanized single domain antibody fusion protein and human CEACAM6
Example 10 radioisotope-labeled CEACAM6 humanized Single Domain antibody fusion protein assay
1. Test method
(1) Antibody DFO modification: 1mL of antibody solution (2 mg/mL of one of the three fusion proteins) +1mL0.5M NaHCO is taken from a reaction bottle 3 /Na 2 CO 3 Measuring the pH value of the solution to be alkaline; the reaction was stirred at 37 ℃ for 40min. And (4) purifying by using a PD10 column. (2) antibody labeling: a little 89Zr was taken, and 2M Na was added 2 CO 3 Adjusting the pH of the solution to be neutral; (3) antibody quality control: glass fiber paper, developing agent; a sodium citrate system. Antibody label was at the origin, free 89Zr at the leading edge. Antibody modifications and 89Zr labeling scheme are shown in figure 7.
2. Test results
The distribution results of the single domain antibody-Fc fusion protein of the three antibody structures and the isotope 89Zr in the mouse tumor animal model in vivo important organs and tumor tissues are shown in Table 6, FIG. 8 and FIG. 9.
TABLE 6 administration of 89 % Radioactive substance uptake ID value of tissues after Zr-CEACAM6 (mean. + -. SD, n = 6)
Test results show that the single-domain antibody-Fc fusion isotope labeled can well target the transplanted tumors (non-small cell lung cancer, pancreatic cancer and the like) in the body of the mouse in a specific manner.
Claims (10)
1. The single domain antibody of anti-CEACAM 6, the single domain antibody is composed of a framework region and 3 complementarity determining regions, and is characterized in that the amino acid sequences of the 3 complementarity determining regions of the single domain antibody are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.
2. The single domain antibody of claim 1, wherein the amino acid sequence of said single domain antibody is set forth in SEQ ID No. 4.
3. A gene encoding the single domain antibody of any one of claims 1 or 2; preferably, the nucleotide sequence of the coding gene of the single domain antibody is shown as SEQ ID No. 5.
4. A recombinant expression vector comprising the coding gene of claim 3.
5. The humanized anti-CEACAM 6 single-domain antibody is characterized in that the single-domain antibody is humanized and transformed to obtain a 2-humanized antibody, and the amino acid sequences of the 2-humanized antibody are respectively shown as SEQ ID No.6 and SEQ ID No. 7.
6. A fusion protein constructed by the IgG-Fc of the single domain antibody of claim 1 or 2 or the humanized anti-CEACAM 6 single domain antibody of claim 5; preferably, the Fc gene sequence is an Fc gene sequence derived from IgG, igA, igM or from IgG1, igG2, igG3 or IgG4; the IgG is preferably human IgG and subclasses IgG1, 2, 3,4, human IgM, human IgA or Fc fragment of other animal immunoglobulin.
7. The fusion protein of claim 6, wherein the amino acid sequence is represented by SEQ ID No.8 and the nucleotide sequence of the gene encoding the fusion protein is represented by SEQ ID No. 9.
8. A conjugate obtained by coupling the single domain antibody of claim 1, 2 or 5 to one or more of a cytotoxic agent, an enzymatic phase, a radioisotope or a chemiluminescent compound.
9. Use of the single domain antibody of claim 1, 2 or 5, the encoding gene of claim 3, the fusion protein of claim 6 or 7 and the conjugate of claim 8 for the preparation of a medicament or reagent for the detection or diagnosis of CEACAM 6-related diseases.
10. Use of the single domain antibody of claim 1, 2 or 5, the encoding gene of claim 3, the fusion protein of claim 6 or 7, and the conjugate of claim 8 for the preparation of a medicament for the treatment of a disease associated with abnormal CEACAM6 expression; preferably, the diseases associated with abnormal CEACAM6 expression include non-small cell lung cancer, pancreatic cancer, breast cancer or ovarian cancer.
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