CN116621980A - anti-CEACAM 6 single domain antibody, fusion protein and application thereof - Google Patents
anti-CEACAM 6 single domain antibody, fusion protein and application thereof Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K2317/52—Constant or Fc region; Isotype
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
The application discloses an anti-CEACAM 6 single domain antibody, fusion protein and application thereof. The application screens and obtains a group of anti-CEACAM 6 single domain antibodies which have high activity and stronger neutralization or binding capacity and can specifically bind to CEACAM6. The application also discloses humanized antibody with improved affinity obtained by humanized modification of the single-domain antibody. The application further fuses the single domain antibody or the humanized single domain antibody with human IgG-Fc to obtain fusion proteins. The humanized single domain antibodies and/or fusion proteins of the application are useful for detecting or diagnosing CEACAM6 and for treating CEACAM6 expression abnormality related diseases.
Description
The application is a divisional application of patent application with the application number of 202011133599.8, the application date of 2020, 10 months and 21 days, and the application name of anti-CEACAM 6 single domain antibody, humanized single domain antibody, fusion protein and application.
Technical Field
The application relates to a single domain antibody, in particular to a single domain antibody of anti-CEACAM 6 and a fusion protein constructed by fusing the single domain antibody or a humanized single domain antibody with IgG1-Fc, and further relates to application of the single domain antibody or the humanized single domain antibody in detecting CEACAM6 and treating diseases related to CEACAM6 abnormal expression, belonging to the single domain antibody of anti-CEACAM 6, the humanized single domain antibody and application fields thereof.
Background
Proteins of a variety of carcinoembryonic antigen-related cell adhesion molecules (Carcinoembryonic antigen (CEA) related cell adhesion molecule (CEACAM)) belong to family members of the immunoglobulin (Ig) supergene whose primary structure is the extracellular, transmembrane and intracellular regions of the cell (some members do not have intracellular regions). Common family members are CEACAM1,3,4,5,6,7,8,16,18,19,21, the extracellular domain of which is characterized by: an N domain with an N-terminal followed by none 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 essential for CEACAM to display its function as homophilic and heterophilic intercellular adhesion molecules or as human and rodent pathogen receptors. CEACAM receptors can be oligomers or dimers that form multiple combinations with other ligands at the cell membrane to modulate their cell important functions. In addition to expression in human tissues, the CEACAM gene family is highly conserved among 27 other mammalian species (Robert Kammerer, wolfgang zimmermann. Cooling of activating and inhibitory receptors within mammalian carcinoembryonic antigen family.bmc biol.2010feb 4; 8:12). The biological function of CEACAM is to maintain Cell-to-Cell adhesion by its homophilic and heterophilic interactions, including roles in differentiation and formation of three-dimensional tissue structures, angiogenesis, apoptosis, tumor suppression and metastasis, and the like (Kuespert k.et al CEACAM: their role in physiology and pathhysiology.curr Opin Cell biol.2006oct;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.2014jun;6 (6): 1314-1326).
Carcinoembryonic antigen-related 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-related cell adhesion molecule protein family, which has a high degree of homology with the molecules of family members CEACAM 1/7/8. CEACAM6 is a Glycosyl Phosphatidylinositol (GPI) -linked cell surface protein having one N domain and 2C 2-like domains, through which extracellular domains with various membrane receptors (some of which have been identified) mediate many possible cis-or trans-directed CEACAM interactions. CEACAM6 has been reported to be 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 varying degrees. Overexpression of CEACAM6 can result in altered morphology of leaf matrix in epithelial tissue, increased tumor invasiveness and resistance to chemical agents, metastasis of tumors, and reduced apoptosis, and the reduction of CEACAM6 gene expression with SiRNA and inhibition of CEACAM6 protein function with monoclonal antibodies can reverse these effects resulting from CEACAM6 overexpression. Although CEACAM6 is also expressed in many normal tissues of humans, such as granulocyte lines, it is overexpressed in a variety of tumors, and many studies have been reported; a study comparing the expression levels of CEACAM6 and CEACAM5 (CEA) in lung, breast, prostate, colon, pancreas and ovary cancer tissues and tissues beside and in normal tissues of the tissues showed that: in all tumor types studied, CEACAM6 was expressed higher than CEA, and in CEACAM6 overexpressing cancers, the abundance of CEACAM6 expressed was also different in tumors of different tissue types, e.g., in breast tumors CEACAM6 expressed: mastoid carcinoma > invasive ductal > lobular > petiole; the abundance of CEACAM6 expression in pancreatic cancer is: moderate differentiation type > fully differentiation type > poorly differentiated type tumors; the expression of CEACAM6 of mucinous ovarian adenocarcinoma is 3 times higher than that of serous ovarian adenocarcinoma; the expression in non-small cell lung cancer CEACAM6 is lung adenocarcinoma > lung squamous carcinoma; CEACAM6 expression > primary tumor > lymph node metastasis tumor in liver metastasis colon cancer. The expression of CEACAM6 in prostate cancer tissue is not different from that in its paracancerous normal tissue (Nicode Beauchemin and zadehabazadeh. Carcinoebryonic anti-related cell adhesion molecules (CEACAMs) in cancer progression and metatasis. Cancer metatasis Rev.2013Dec.;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, CEACAM6 may be a specific target antigen for these overexpressed tumors, CEACAM6 being a very attractive new target for therapeutic intervention in cancer immunotherapy.
The single domain antibody (single domain antibody (sdAb) or nano antibody (nanobody) is a heavy chain antibody variable region fragment (VHH) of a missing light chain found in alpaca blood, has a series of advantages of simple structure, strong penetrability, easy expression and purification, high affinity and stability, small toxic and side effects and the like, can be used for detecting and treating and researching CEACAM6 over-expression tumors, and can provide a novel detection method and a novel treatment means for related diseases of the CEACAM6 over-expression tumors by screening the anti-CEACAM 6 single domain antibody with high affinity by utilizing a single domain antibody technology.
Disclosure of Invention
It is an object of the present application to provide a set of single domain antibodies against CEACAM6 and genes encoding the same;
the second purpose of the application is to humanize the anti-CEACAM 6 single domain antibody to obtain a humanized single domain antibody;
the third object of the application is to fuse the single domain antibody or the humanized single domain antibody with human IgG1-Fc to obtain fusion protein;
the fourth object of the present application is to couple the single domain antibody or the humanized single domain antibody with one or more of an enzyme phase, a radioisotope, a fluorescent compound or a chemiluminescent compound to obtain a conjugate;
the fourth object of the present application is to apply the single domain antibody of anti-CEACAM 6, the humanized single domain antibody of anti-CEACAM 6, the fusion protein and the conjugate to the preparation of a reagent for detecting CEACAM6 or the treatment of CEACAM6 expression abnormality related diseases;
the above object of the present application is achieved by the following technical solutions:
the present application first provides a set of single domain antibodies against CEACAM6, each consisting of a framework region and 3 complementarity determining regions, selected from any one of NBC4, NBC5 or NBC 6; wherein the amino acid sequences of the 3 complementarity determining regions of the single domain antibody NBC4 are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; the amino acid sequences of the 3 complementarity determining regions of the single domain antibody NBC5 are shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 respectively; the amino acid sequences of the 3 complementarity determining regions of the single domain antibody NBC6 are shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, respectively;
the application further provides an amino acid sequence of the single-domain antibody, wherein the amino acid sequence of the single-domain antibody NBC4 is shown as SEQ ID No.10, the amino acid sequence of the single-domain antibody NBC5 is shown as SEQ ID No.11, and the amino acid sequence of the single-domain antibody NBC6 is shown as SEQ ID No. 12.
Protein mutants obtained by deleting, substituting, inserting and/or adding one or more amino acids in any one of the above-mentioned amino acid sequences, which have the same function as the protein before mutation, are within the scope of the present application; in addition, amino acid sequences having at least 90% identity to any of the amino acid sequences set forth above are also within the scope of the present application.
The application further provides a coding gene sequence of the single domain antibody, wherein the nucleotide sequence of the coding gene of the single domain antibody NBC4 is shown as SEQ ID No.13, the nucleotide sequence of the coding gene of the single domain antibody NBC5 is shown as SEQ ID No.14, and the nucleotide sequence of the coding gene of the single domain antibody NBC6 is shown as SEQ ID No. 15. Among them, polynucleotide sequences which hybridize under stringent hybridization conditions to the complementary sequences of the polynucleotide sequences shown above are also within the scope of the present application; in addition, polynucleotide sequences having at least 90% identity to any of the polynucleotide sequences described above are also within the scope of the present application.
The application further provides a recombinant expression vector comprising one or more of the genes encoding the single domain antibody; preferably, the recombinant expression vector may be a recombinant prokaryotic cell expression vector, a recombinant yeast expression vector, a recombinant eukaryotic cell expression vector or other recombinant cell expression vector.
The application 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 yeast recombinant parent cell, and the recombinant prokaryotic expression cell is preferably escherichia coli.
The application further carries out humanized transformation on the single domain antibody NBC4 to obtain 5 humanized antibodies NBC4HM1, NBC4HM2, NBC4HM3, NBC4HM4 and NBC4HM5, and the amino acid sequences of the humanized antibodies are respectively shown as SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 and SEQ ID No. 20.
The application further constructs fusion protein of the anti-CEACAM 6 single domain antibody or the humanized single domain antibody and IgG-Fc; wherein the Fc gene sequence may be an Fc gene sequence derived from IgG, igA, igM or derived 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 genes and amino acid sequences of human IgM, human IgA or immunoglobulin of other animals (such as mice, rabbits, monkeys, etc.).
As a preferred embodiment of the application, the humanized antibody NBC4HM2 and the human IgG1-Fc gene are fused to obtain a fusion protein with the amino acid sequence shown as SEQ ID No.21, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 22.
The present application further couples the single domain antibody or humanized single domain antibody to one or more of an enzyme phase (e.g., horseradish peroxidase, alkaline phosphatase, etc.), a radioisotope, a fluorescent compound, or a chemiluminescent compound (which may be a fluorescent compound) to provide conjugates that can be used to detect CEACAM6 or to treat a variety of diseases associated with CEACAM6 expression abnormalities.
For example, anti-CEACAM 6 humanized single domain antibodies and Fc fusion proteins 68 Ga, 89 Zr, 64 Cu, 18 F, 86 Y, 90 Y, 111 In, 99NV Tc, 125 I, 124 And (3) labeling the radioisotope such as I to obtain a labeled protein for imaging detection of PET (positron emission tomography) or SPECT. Or humanized anti-CEACAM 6 single domain antibody and 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 Bi 67 The radioactive isotopes such as Ga are labeled to obtain a labeled protein which is used for treating diseases related to abnormal expression of CEACAM6.
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 single domain antibody or the humanized single domain antibody is coupled with an enzyme phase, a radioactive isotope, a fluorescent compound or a chemiluminescent compound to obtain a conjugate, which mainly has the following purposes:
(1) Preparing a drug or reagent for detecting CEACAM 6;
(2) The application of the medicine for treating diseases related to abnormal expression of CEACAM6 is provided. Preferably, the CEACAM6 expression abnormality related disease includes tumor diseases such as non-small cell lung cancer, pancreatic cancer, breast cancer, ovarian cancer and the like.
The application is characterized in thatDefinition of terms involved
The term "CEACAM6", carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM 6), also known as non-specific cross-reactive antigen (NCA, NCA-50/90)), CD66c, as used herein, is one of the important members of the carcinoembryonic antigen-related cell adhesion molecule protein family. CEACAM6 is a Glycosyl Phosphatidylinositol (GPI) -linked cell surface protein having one N domain and 2C 2-like domains, through which extracellular domains with various membrane receptors (some of which have been identified) mediate many possible cis-or trans-directed CEACAM interactions. CEACAM6 has been reported to be 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 varying degrees. CEACEA6 may be a specific target antigen for these overexpressed tumors, CEACAM6 being a very attractive target for therapeutic intervention in cancer immunotherapy.
The novel antibodies against CEACAM6 and Fc fusion proteins thereof are subject of development herein, and ultimately of protection herein, and the scope of the present disclosure relates to the resulting anti-CEACAM 6 humanized single domain antibodies and the substances (e.g., pharmaceutical compositions, kits, vectors, etc.) of which the Fc fusion proteins are a component, uses (e.g., diagnostic applications, therapeutic applications, preparative applications, etc.), however, it will be understood by those skilled in the art that the subject of protection herein is not limited to such examples.
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 (Nanobody). As with intact antibodies, it can bind selectively to specific antigens. Single domain antibodies appear much smaller than the 150-160kDa mass of intact antibodies, approximately only 12-17kDa. The first single domain antibody was engineered from a heavy chain antibody of a camel and is referred to as the "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 engineered 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 the application SEQ ID NO:1-198, and having substantially the same properties as those having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% sequence identity thereto, and which are within the scope of the present application. Preferably, the present application achieves sequence identity through 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 after 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 complementary to each other in reverse when the sequences are all seen in the 5 'to 3' direction. It is also known in the art that two sequences which 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 one another to form a peptide chain (or polypeptide), and the amino acid sequence can be read in only one direction. There are 100 different types of amino acids, 20 of which are commonly used, and the present application does not exclude other substances on the amino acid chain, such as sugar, lipid, etc., and the present application is not limited to the commonly used amino acids in 20.
The term "nucleotide sequence" refers to the arrangement of bases in DNA or RNA, i.e., A, T, G, C in DNA, or A, U, G, C in mRNA, including rRNA, tRNA, mRNA. It should be understood that the antibody genes claimed in the present application encompass RNA (rRNA, tRNA, mRNA) and their complements in addition to DNA sequences.
The substitutions described in the present application may be conservative substitutions, i.e. the substitution of a particular amino acid residue for one having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include those between aliphatic group-containing amino acid residues (e.g., ile, val, leu or inter-substitution between Ala), those between polar residues (e.g., between Lys and Arg, glu and Asp, gln and Asn), and the like. Mutants resulting from the deletion, substitution, insertion and/or addition of amino acids can be made by subjecting the DNA encoding the wild-type protein to site-directed mutagenesis as known in the art (see, for example, nucleic Acid Research, vol.10, no.20, p.6487-6500, 1982, incorporated herein by reference in its entirety).
The term "expression vector (Expression vectors)" refers to a vector in which expression elements (e.g., promoter, RBS, terminator, etc.) are added to the basic skeleton of a cloning vector so that a desired gene can be expressed. Expression vector four parts: a target gene, a promoter, a terminator and a marker gene. The application includes, but is not limited to, prokaryotic expression vectors, eukaryotic expression vectors, or other cellular expression vectors.
The term "Framework region", i.e., the Framework region, varies widely about 110 amino acid sequences near the N-terminus of the H and L chains of an immunoglobulin, and the amino acid sequences of the other parts are relatively constant, thereby distinguishing the light and heavy chains into variable (V) and constant (C) regions. The variable region comprises a hypervariable region HVR (hypervariable region), or Complementarity determining region CDR (determining region), and an FR framework region.
The term "humanized" antibody refers to the antibody in which the variable region (VH or VHH) Fr region portion, the constant region portion (i.e., CH and CL regions) or all of the antibody is encoded by a human antibody gene. Humanized antibodies can greatly reduce the immune side effects of heterologous antibodies on the human body. Humanized antibodies include chimeric antibodies, diabodies, and fully humanized antibodies. It will be appreciated that it is within the scope of the present application for the person skilled in the art to be able to prepare suitable humanized forms of the single domain antibodies of the application as required in practice.
The terms "mutation" and "mutant" have their usual meaning herein, referring to genetic, naturally occurring or introduced changes in a nucleic acid or polypeptide sequence, which are in the same sense as commonly known to those skilled in the art.
The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the application, regardless of the method used to insert to produce a recombinant host cell, such as 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 construction of CEACAM6 gene library, nested PCR amplification of the first round of PCR products, heavy chain antibody gene fragments between 800 and 500bp with missing light chain.
FIG. 2 PCR amplification of VHH-specific primers to obtain the VHH gene of interest.
FIG. 3 shows the result of SDS-PAGE of expressed partial anti-CEACAM 6 anti-single domain antibody protein.
FIG. 4 shows the result of SDS-PAGE of the expressed partial CEACAM6-sdAB after nickel column purification.
FIG. 5 shows the results of activity assays for specific binding of purified anti-CEACAM 6 single domain antibodies to human CEACAM6 antigen.
FIG. 6 shows the results of SDS-PAGE of reducing and non-reducing gel electrophoresis after expression purification of 3 CEACAM6 humanized single domain antibodies; expression of EG2M1-EG10M1-Fc-p327.7 purified reductive protein bands; expression of EG2M1-Fc-EG10M1-p327.7 purified reductive protein bands; expression of the purified non-reducing protein band in EG2M1-EG10M1-Fc-p 327.7; expression of the purified non-reducing protein band in EG2M1-Fc-EG10M1-p 327.7; 5. protein molecular weight standard (Marker); the molecular weight indicated by the arrow is 50KD.
FIG. 7 is a diagram showing the route of antibody modification and 89Zr labeling.
FIG. 8 distribution results (PET/CT scan) of the single domain antibody-Fc fusion protein labeled isotope 89Zr in vital organs and tumor site tissues in a mouse tumor animal model.
FIG. 9 administration 89 Histogram of% ID/g uptake of radioactive material by each tissue at various time points after Zr-CEACAM637.2 antibody.
Detailed Description
The application will be further described with reference to specific embodiments, and advantages and features of the application will become apparent from the description. These examples are merely exemplary and do not limit the scope of the application in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the application without departing from the spirit and scope of the application, but these modifications and substitutions are intended to be within the scope of the application.
EXAMPLE 1 construction of Single-Domain antibody library specific for the anti-CEACAM 6 antigen
(1) CEACAM6 antigen immunized alpaca: the immunization was performed according to the conventional immunization method using the purchased CEACAM6 antigen (Human CEACAM6 Protein, human, recombinant (His Tag)), selecting adult healthy alpaca, subcutaneously injecting the antigen into the nape, adding the antigen and the equal volume of the freund adjuvant, performing immunization for 4-8 times, and tracking and observing the absorption of the injection site to confirm the immunization. After the first immunization, the interval is 21 days, the second needle immunization is started, the interval time is 7-15 days, after the 4 th immunization, blood is collected, antigen immune titer is measured, when the titer reaches about 5 ten thousand times (ELISA method), about 100ml of whole blood is collected, lymphocytes are separated, and the blood is preserved at-80 ℃ for standby.
(2) Separation of alpaca peripheral blood lymphocytes and extraction of RNA: the alpaca peripheral blood leucocytes were isolated and RNA was extracted using QIAGEN kit according to instructions. And (3) RNA purification: RNA purification is carried out by using a QIAGEN kit, and the concentration of the obtained RNA and OD260/280 are measured to be more than or equal to 1.8 according to the specification.
(3) Heavy chain antibody variable region-VHH: first strand cDNA Synthesis: the cDNA synthesis was carried out using a cDNA synthesis kit (MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0, TAKARA Co.) according to the instructions. With this template, PCR amplification of heavy chain antibody VHH gene fragments was performed using two sets of primers, respectively. The nested PCR method is adopted, the heavy chain gene fragment with the length of more than 800bp is common heavy chain gene fragment in the first PCR amplification, the heavy chain antibody gene fragment with the length of between 800 and 500bp is the light chain deletion (figure 1), the light chain deletion heavy chain antibody gene fragment is recovered by cutting glue, the VHH target gene (500 bp) is obtained by using VHH specific primers as templates through PCR amplification, and the gene amplification result is shown in figure 2. The primers used were:
first round PCRFd5' 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 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 fragments and phage display vectors and electrotransformation TG1 competence: sfI after cleavage of the VHH fragment and pHEN6 vector plasmid, the VHH fragment and pHEN6 vector (establishment, KEM other. Antimicrob Agents Chemoter (Antimicrobial Chemotherapy) 2001,45 (10) 2807-12.) were digested with the enzyme ligase (T) 4 NEB company), were electrotransformed into TG1 competent cells, 10 electrotransformed, plated, and colony PCR was used to verify antibody insertion rate. Recombinant gene cloning efficiency detection: the electrotransformation bacterial liquid is coated on an LB/Amp plate, cultured overnight at 32 ℃, and the connection efficiency of the antibody is verified by a colony PCR method the next day, and the connection efficiency of a phage antibody library is more than 90%. The electrotransformation bacteria liquid is coated on LB/Amp plates, cultures are washed off with 2YT medium at 32 ℃ overnight, 15% glycerol is added, and the culture is preserved at-80 ℃. Phage library 1.8X10 8 The method comprises the steps of carrying out a first treatment on the surface of the 30-50 clones were randomly selected, cloned PCR was performed, the VHH gene insertion rate was 95%, and gene sequencing was performed, with a VHH sequence repetition rate of less than 2% for three CDRs.
(5) Preparation of a VHH phage antibody library: the antibody pool was rescued by adding helper phage M13K07 (Invitrogen): phage antibody libraries were prepared according to conventional methods and stored at-80℃for use.
EXAMPLE 2 screening of Single-Domain antibodies against CEACAM6
(1) Screening for CEACAM 6-specific single domain antibodies
The first round of CEACAM6 protein concentration was 50. Mu.g/ml, 0.5ml coated immune tubes (thermo fisher Co.) overnight at 4 ℃. The second and third rounds of coating the immune tubes with CEACAM6 protein at 20. Mu.g/ml, 10. Mu.g/ml, 0.5ml, respectively, overnight at 4 ℃. Closing: 2% nonfat dry milk PBS,37℃and incubated for 1.5 hours. Phage were added, washed 10 times with PBST and PBS each at room temperature for 1 hour, the specifically bound phage eluted with 0.5ml TEA, 2ml TG1 in logarithmic growth phase was infected, titers were 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 screening | Amount of phage added | Elution and recovery of phage quantity |
| 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) Phage ELISA method for selecting positive clones
Colonies grown on agar plates were selected from rounds 2 and/or 3, individual colonies were randomly picked, inoculated in 96 deep well plates containing Amp in 2YT liquid medium and cultured, and phage antibodies were induced by superinfection with helper phage. The expression supernatant is harvested, ELISA determination is carried out by taking CEACAM6 as an antigen, CEACAM6 positive holes are selected, and DNA sequencing is carried out to identify the gene sequence of anti-single domain antibody clone, so that a series of single domain antibody gene sequences including the gene sequences shown in SEQ ID NO.13-15 are obtained and are used for further expressing and screening specific and high-activity single domain antibodies.
EXAMPLE 3 construction of expression plasmid for specific CEACAM6 Single-domain antibody
PCR amplification of the specific CEACAM6 single domain antibody Gene obtained in example 2 to obtain PCR products with restriction enzymes BbsI and BamHI sites, the PCR products and vectors were treated with restriction enzymes BbsI and BamHI, respectively (pSJF 2 vector, kim is. Biosic biochem.2002,66 (5): 1148-51), T-passed 4 The ligase is connected and recombined to obtain plasmid sdAb-pSJF2 which can be efficiently expressed in escherichia coli, and gene sequence determination is carried out to determine the sequence correctness.
(1) PCR amplification conditions for obtaining the VHH target gene of CEACAM6, 50. Mu.l PCR amplification system, PCR reaction conditions: 94℃for 3 minutes, then 94℃for 30 seconds; 72 ℃,45 seconds, 52 ℃ and 30 seconds; 30 cycles total; 72℃for 7 min.
5' primer-GAA GAAGAA GAC AA CAG GCC SAR GTG MAG CTG GWG GAK TCT;
3' primer-gaagatctccggatccTGAGGAGACGGTGACCTGGGT;
(2) And (3) carrying out enzyme digestion on the target gene and the vector, connecting the target gene and the vector, transforming TG1, identifying clone containing the target fragment by PCR, and carrying out gene sequencing to obtain the single domain antibody expression plasmid with correct gene sequence.
EXAMPLE 4 expression and purification of anti-Single Domain antibodies
The strain containing plasmid sdAb-pSJF2 described in example 3 was inoculated on an ampicillin-containing LB plate overnight at 37 ℃. Individual colonies were selected and inoculated in 15ml of LB medium containing ampicillin, and shake cultured overnight at 37 ℃. Transferring 10ml of overnight culture into 1L 2YT culture solution containing ampicillin, shaking at 37 ℃ for 240 revolutions per minute, adding 0.5-1.0 mM IPTG when the OD value reaches 0.4-0.6, and continuing to culture overnight. And (5) centrifuging and collecting bacteria. Adding 25% high-permeability sucrose solution, extracting soluble expressed single domain antibody in periplasm of cell, centrifuging, and collecting supernatant. Obtaining the protein with purity reaching more than 90% through Ni+ ion affinity chromatography. FIG. 3 shows the result of SDS-PAGE of the expressed partial CEACAM6 anti-single domain antibody protein, and FIG. 4 shows the result of SDS-PAGE after purification of the expressed partial CEACAM6-sdaB by a nickel column.
EXAMPLE 5 binding assay (ELISA) of purified CEACAM6 Single-domain antibodies to CEACAM6 antigen
1. Test materials: removable ELISA plate (Thermofiser Co.), CEACAM6 antigen, anti-Myc tag Anti-HRP (Beijing Yiqiao Shenzhou Biotechnology Co., ltd.), TMB color development solution (Beijing Mei Kemo De, cat: 1001), coating solution pH 9.6, BSA (Sigma Co.).
2. Test method
2.1 separately coated Human CEACAM6 Protein, concentration 2ug/ml,100 ul/well, incubated overnight at 4 ℃.
2.2 blocking with 2% skim milk PBS, 300 ul/well. Incubate at 37℃for 1.5h.
2.3 dilution of differently 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 Anti-ibody (HRP) (1:5000), 100 ul/well, incubation at 37℃for 1h.
2.5 adding TMB color development liquid, 100 ul/hole, and reacting for 10min in dark.
2.6 the reaction was stopped by adding 50 ul/well 2M H2SO 4.
2.7 OD measured at 450 nm.
3. Test results
FIG. 5 shows the results of activity assays for specific binding of purified CEACAM6 single domain antibodies to human CEACAM6 antigen.
Example 6 anti-CEACAM 6 Single-domain antibody affinity assay
1) Sample preparation antigen: bio-CEACAM6 was diluted to 10. Mu.g/ml with 1 Xdynamic buffer (1 XPBS, 0.05% Tween 20, 0.1% BSA, pH 7.2);
single domain antibodies: sequentially diluted with 1 Xkinetic buffer to 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM;
2) Sample testing
The antigen to be tested is loaded by SA sensor, the antigen is diluted for 5 dilutions, and the affinity of all single domain antibodies is 50nm, 20nm, 10nm, 1nm, 0.1nm and 0.01nm. The affinity of the partial single domain antibodies is shown in Table 2, and the affinity ranges are shown in Table 2.
TABLE 2 assay results for affinity assay of 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 humanized universal antigen binding complementary region transplantation method (CDR grafting to a universal framework), and reference is made to the filed patent (the name of the application: anti-EGFR humanized single domain antibody, fc fusion protein, heavy chain Fab protein and application thereof, application number: 2019113490209). The humanization steps were as follows: homology modeling against CEACAM6 single domain antibodies NBC4, 25 and 36 was performed with modeling software model 9. anti-CEACAM 6 single domain antibodies NBC4, 25 and 36 were humanized with reference to the amino acid sequences of human antibody DP-47 and the homologous sequence NBBcII10 antibody, which were well soluble.
The humanization results are shown in Table 3.
TABLE 3 humanized results of NBC4, 25 and 36 Single Domain antibodies
* Annotation: x: indicating that the amino acid may be subjected to a humanized variant site. According to literature studies, more than 80% of the antibodies are more immunogenic than human antibodies.
EXAMPLE 8 vector construction of anti-CEACAM 6 humanized Single-domain antibody Fc fusion protein
(1) A first structure: sdAb 1-ringer-CH 2-CH3 (IgG 1-Fc). sdab=nbc4hm2 or nbc25hm3 or nbc36hm2. (2) construction: the NBC4HM2 or NBC25HM3 or NBC36HM2+ human IgG1-Fc gene was synthesized in full, double XhoI-EcoRI cleavage was added, the sdAb-Fc gene was ligated into the p327.7 expression vector (patent publication No. CN 104195173A), the corresponding cleavage site and stop codon were added, double XbaI-SalI cleavage was used, the other sdAb-Fc gene was ligated into the p327.7 expression vector already containing sdAb-Fc (double XhoI-EcoRI cleavage ligation) and finally one vector had 2 sdAb-Fc sequences.
The sequence table of amino acid and gene of the anti-CEACAM 6 humanized single domain antibody, fc fusion protein and heavy chain Fab protein provided by the application is shown in 4.
TABLE 4 sequence listing of anti-CEACAM 6 humanized Single-domain antibodies, fc fusion proteins, heavy chain Fab proteins
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EXAMPLE 9 expression and purification of anti-CEACAM 6 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-expression cell lines were selected by MSX, 3 stable expression cell lines were co-selected, and protein expression was performed by culturing the stable expression cell lines in 500ml shake flasks.
Protein purification: the cell expression supernatant was purified by affinity chromatography using protein A strain, and the purified protein was replaced with a buffer of citric acid (0.05% Tween80, pH 6.2). The purified proteins expressed by the anti-CEACAM 6 humanized single domain antibody Fc fusion protein vector are shown in figure 6 (SDS-PAGE reducing gel and non-reducing gel electrophoresis results after purification of 3 CEACAM6 humanized single domain antibodies).
The theoretical estimated value of the protein expressed by the fusion protein expression vector is as follows: 688, 688 and 682 amino acids, respectively; molecular Weight (MW), through ringer disulfide bond connection molecular weight 7.664KD, 7.704KD and 7.569KD, isoelectric point (pI) 7.88, 7.30 and 7.61, respectively, and molecular weight about 38KD after purification and electrophoresis SDS-PAGE reduction is consistent with theoretical estimated value. Affinity assay for anti-CEACAM 6 humanized single domain antibody fusion proteins the results of affinity assays are set forth in table 5 above in example 6.
TABLE 5 affinity analysis results of anti-CEACAM 6 humanized Single-domain antibody fusion proteins with 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) +1mL of 0.5M NaHCO was taken from the reaction flask 3 /Na 2 CO 3 Measuring the pH value of the solution to be alkaline; the reaction was stirred at 37℃for 40min. And (5) purifying the PD10 column. (2) antibody labeling: taking a little 89Zr, adding 2MNA 2 CO 3 A solution, wherein the pH is adjusted to be neutral; (3) antibody quality control: glass fiber paper, developing agent; and a sodium citrate system. The antibody label is at the origin and free 89Zr is at the leading edge. The antibody modification and 89 Zr-labeled roadmap is shown in FIG. 7.
2. Test results
The distribution results of the single domain antibody-Fc fusion protein labeled isotope 89Zr of the three antibody structures in the important organs and tumor part tissues in the mouse tumor animal model are shown in Table 6, FIG. 8 and FIG. 9.
Table 6 administration 89 The% uptake ID value (mean+ -SD, n=6) of radioactive substance for each tissue after Zr-CEACAM6
The test result shows that the single-domain antibody-Fc fusion isotope labeled can target mice to transplanted tumors (non-small cell lung cancer, pancreatic cancer and the like) in vivo with good specificity.
Claims (10)
1. A single domain antibody NBC6 against CEACAM6, said single domain antibody comprising a framework region and 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequences of said 3 complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, respectively, or a protein mutant obtained by deletion, substitution, insertion and/or addition of one or more amino acids of any one of the amino acid sequences shown above, said protein mutant having the same function as the protein prior to mutation; or an amino acid sequence having at least 90% identity to any one of the amino acid sequences shown above.
2. The single domain antibody according to claim 1, wherein the amino acid sequence is shown in SEQ ID No. 12; or a protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in any one of the above-mentioned amino acid sequences, which has the same function as the protein before mutation; or an amino acid sequence having at least 90% identity to any one of the amino acid sequences shown above.
3. A gene encoding the single domain antibody of any one of claims 1 or 2.
4. A coding gene according to claim 3, wherein the nucleotide sequence of the coding gene is shown in SEQ ID No. 15;
or a polynucleotide sequence capable of hybridizing under stringent hybridization conditions to a complement of any one of the polynucleotide sequences shown above; or a polynucleotide sequence having at least 90% identity to any one of the polynucleotide sequences shown above.
5. A recombinant expression vector comprising the coding gene of claim 3.
6. A fusion protein constructed by combining the single domain antibody of any one of claims 1 or 2 with IgG-Fc.
7. The fusion protein of claim 6, wherein the Fc gene sequence is an Fc gene sequence derived from IgG, igA, igM or an Fc gene sequence derived from IgG1, igG2, igG3 or IgG4; the IgG is preferably human IgG and subclasses of IgG1, 2, 3,4, human IgM, human IgA or Fc fragments of other animal immunoglobulins.
8. A conjugate, characterized in that the single domain antibody of claim 1 or 2 is conjugated to one or more of a cytotoxic agent, an enzyme phase, a radioisotope or a chemiluminescent compound to give the conjugate.
9. Use of the single domain antibody of claim 1 or 2, the encoding gene of claim 3, the fusion protein of claim 6 or 7, or the conjugate of claim 8 in the manufacture of a medicament or agent for detecting or diagnosing a disease associated with abnormal CEACAM6 expression.
10. The use according to claim 9, wherein the CEACAM6 expression abnormality related disease comprises non-small cell lung cancer, pancreatic cancer, breast cancer or ovarian cancer.
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