CN118184785A - CEA antibodies and uses thereof - Google Patents
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- CN118184785A CN118184785A CN202410307425.0A CN202410307425A CN118184785A CN 118184785 A CN118184785 A CN 118184785A CN 202410307425 A CN202410307425 A CN 202410307425A CN 118184785 A CN118184785 A CN 118184785A
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Abstract
The invention provides a CEA antibody and application thereof. In particular, nucleic acids encoding the antibodies, compositions comprising the antibodies, methods of making the antibodies, and methods of using the antibodies to treat or prevent diseases, such as cancer and/or inflammatory diseases, are also described.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a CEA antibody and application thereof.
Background
Gastrointestinal tract tumor is a great threat to human life, and although the treatment means such as operation treatment, radiotherapy and chemotherapy, interventional treatment and the like have certain curative effects on the tumor, the survival rate of patients is not improved obviously. At present, therapeutic antibodies of great interest and CAR-T cell therapy techniques are expected to be therapeutic breaches.
Carcinoembryonic antigen (carcinoembryonic antigen, CEA) is an acidic glycoprotein, is initially found in colon cancer and fetal intestinal tissues, is proved to be widely present in digestive system tumors of endocotyl origin, such as gastric cancer, liver cancer, pancreatic cancer (over 75%), colorectal cancer (60%), can be simultaneously present in serum of various tumor patients, belongs to a tumor marker which is very commonly used clinically, and is mainly used for assisting diagnosis, prognosis, monitoring curative effect, tumor recurrence and the like of malignant tumors clinically.
Notably, CEA is hardly expressed in other normal cells except for low expression in intestinal epithelial cells. Therefore, it is considered as a target point of great therapeutic potential for solid tumors.
Thus, there is a need in the art to develop therapeutic antibodies against CEA with greater anticancer potential, lower toxicity, lower dosage, and thus effectively treat a variety of solid tumors.
Disclosure of Invention
The invention provides a CEA antibody and application thereof
In a first aspect of the present invention there is provided a CEA antibody or antigen-binding fragment thereof having three complementarity determining region CDRs (HCDRs) of the heavy chain variable region and three complementarity determining region CDRs (LCDRs) of the light chain variable region selected from the group consisting of:
(1) HCDR1 shown in SEQ ID NO. 2,
HCDR2 shown in SEQ ID NO. 3,
HCDR3 shown in SEQ ID NO. 4,
LCDR1 shown in SEQ ID NO. 6,
LCDR2 shown in SEQ ID NO. 7,
LCDR3 shown in SEQ ID NO. 8;
(2) HCDR1 shown in SEQ ID NO. 12,
HCDR2 shown in SEQ ID NO. 3,
HCDR3 shown in SEQ ID NO. 13,
LCDR1 shown in SEQ ID NO. 15,
LCDR2 as shown in SEQ ID NO. 16,
LCDR3 shown in SEQ ID NO. 8;
(3) HCDR1 shown in SEQ ID NO. 20,
HCDR2 shown in SEQ ID NO. 21,
HCDR3 shown in SEQ ID NO. 22,
LCDR1 shown in SEQ ID NO. 24,
LCDR2 as shown in SEQ ID NO. 25,
LCDR3 as shown in SEQ ID NO. 26.
In another preferred embodiment, the antibody or antigen binding fragment thereof has a sequence selected from the group consisting of seq id no:
(1) The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.1, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 5;
(2) The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 11, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 14;
(3) The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 19, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 23; .
In another preferred embodiment, the heavy chain of the antibody or antigen binding fragment thereof further comprises a heavy chain constant region; the light chain of the antibody or antigen binding fragment thereof further comprises a light chain constant region.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the heavy chain constant region is of human or murine origin.
In another preferred embodiment, the light chain constant region is of human or murine origin.
In another preferred embodiment, the antibody is an antibody full-length protein, or an antigen-binding fragment.
In another preferred embodiment, the antibody is a monoclonal antibody.
In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.
In another preferred embodiment, the antibody further comprises a connecting peptide between the heavy chain variable region and the light chain variable region.
In a second aspect of the present invention, there is provided a recombinant protein having:
(i) An antibody or antigen-binding fragment thereof according to the first aspect of the invention; and
(Ii) Optionally a tag sequence that facilitates expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a third aspect of the invention there is provided a polynucleotide molecule encoding an antibody or antigen binding fragment thereof according to the first aspect of the invention.
In another preferred embodiment, the polynucleotide has the sequence shown in SEQ ID NO. 9, 10, 17, 18, 27, or 28.
In a fourth aspect of the invention there is provided a vector comprising a polynucleotide molecule according to the third aspect of the invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In another preferred embodiment, the vector is a eukaryotic expression vector.
In a fifth aspect of the invention there is provided an engineered cell comprising a vector or genome according to the fourth aspect of the invention incorporating a polynucleotide molecule according to the third aspect of the invention, or expressing a CEA antibody or antigen binding fragment thereof according to the first aspect of the invention.
In another preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell.
In another preferred embodiment, the cells are immune cells and have a chimeric antigen receptor expressed on their surface.
In another preferred embodiment, the immune cells are T cells, NK cells, or a combination thereof.
In another preferred embodiment, the immune cell is a chimeric antigen receptor T cell (CAR-T cell).
In another preferred embodiment, the chimeric antigen receptor is selected from the group consisting of: CEA, mesothelin, GPC3, NKG2D, MUC1, or a combination thereof.
In a sixth aspect of the invention, there is provided an immunoconjugate comprising:
(a) An antibody or antigen-binding fragment thereof according to the first aspect of the invention; and
(B) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.
In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) An antibody or antigen-binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, or an immunoconjugate according to the sixth aspect of the invention; and
(Ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is in the form of an injection.
In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating a tumor selected from the group consisting of: gastric cancer, esophageal cancer, cholangiocarcinoma, pancreatic cancer, lung cancer, bladder cancer, ovarian cancer, cervical cancer, endometrial cancer, colon cancer, prostate cancer, oral cancer, laryngeal cancer, tonsil cancer, or a combination thereof.
In an eighth aspect of the invention there is provided the use of an antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, or an immunoconjugate according to the sixth aspect of the invention, for the preparation of a medicament, reagent, assay plate or kit;
The reagent, assay plate or kit is for: detecting CEA protein in the sample;
The agent is used for treating or preventing tumors expressing CEA protein.
In another preferred embodiment, the reagent comprises a chip, an immune microparticle coated with an antibody.
In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating a tumor selected from the group consisting of: gastric cancer, esophageal cancer, cholangiocarcinoma, pancreatic cancer, lung cancer, bladder cancer, ovarian cancer, cervical cancer, endometrial cancer, colon cancer, prostate cancer, oral cancer, laryngeal cancer, tonsil cancer, or a combination thereof.
In a ninth aspect of the present invention, there is provided a method of detecting a CEA protein in a sample, the method comprising the steps of:
(1) Contacting a sample with an antibody or antigen-binding fragment thereof according to the first aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of a CEA protein in the sample.
In another preferred embodiment, the sample comprises: human or animal tissue samples, tumor resection samples, and exfoliated cell samples.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In another preferred embodiment, the method is an in vitro method.
In another preferred embodiment, the method further comprises the step of (3) analyzing the affinity of the antibody for the antigen.
In a tenth aspect of the invention there is provided a test plate comprising a substrate (support plate) and a test strip comprising an antibody or antigen binding fragment thereof according to the first aspect of the invention, or an immunoconjugate according to the sixth aspect of the invention.
In another preferred embodiment, the test strip further comprises an antigen spotting region.
In another preferred embodiment, the test strip is formed by sequentially overlapping a sample filtering paper, a chromatographic material, a nitrocellulose membrane and a water absorbing paper.
In an eleventh aspect of the present invention, there is provided a kit comprising:
(1) A first container comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention; and/or
(2) A second container comprising a second antibody against the antibody or antigen-binding fragment thereof according to the first aspect of the invention; and/or
(3) A third container containing a cell lysis reagent therein;
Or alternatively
The kit comprises a detection plate according to the tenth aspect of the invention.
In another preferred embodiment, the antibodies in the first container are detectably labeled.
In another preferred embodiment, the antibodies in the second container are detectably labeled.
In a twelfth aspect of the invention there is provided a method of preparing a PD-1 antibody or antigen-binding fragment thereof according to the first aspect of the invention, comprising culturing a cell comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof under conditions suitable for production of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment from the cell or culture.
In a thirteenth aspect of the invention there is provided a method of preparing an engineered cell according to the fifth aspect of the invention comprising the steps of: transduction of a polynucleotide molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention into a cell, thereby obtaining the engineered cell.
In another preferred embodiment, the cells are immune cells.
In another preferred embodiment, the immune cells are T cells, NK cells, or a combination thereof.
In another preferred embodiment, the immune cell is a chimeric antigen receptor T cell (CAR-T cell in a fourteenth aspect of the invention, there is provided a method of producing a recombinant polypeptide, the method comprising:
(a) Culturing a cell according to the fifth aspect of the invention under conditions suitable for expression;
(b) Isolating from the culture a recombinant polypeptide which is an antibody or antigen-binding fragment thereof according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention.
In a fifteenth aspect of the present invention, there is provided a method of preventing and/or treating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a CEA antibody or antigen-binding fragment thereof of the first aspect of the present invention, an engineered cell of the fifth aspect of the present invention, or a pharmaceutical composition of the seventh aspect of the present invention.
In another preferred embodiment, the disease is cancer or tumor.
In another preferred embodiment, the cancer or tumor is selected from the group consisting of: hematological tumors, lymphomas, solid tumors, or combinations thereof.
In another preferred embodiment, the hematological neoplasm is selected from the group consisting of: acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.
In another preferred embodiment, the lymphoma is selected from the group consisting of: hodgkin Lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), follicular Lymphoma (FL), chronic Lymphocytic Leukemia (CLL), small Lymphocytic Lymphoma (SLL), marginal Zone Lymphoma (MZL), mantle Cell Lymphoma (MCL), burkitt Lymphoma (BL), and other complex B-cell non-hodgkin lymphomas.
In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or a combination thereof.
In a sixteenth aspect of the invention there is provided an immunodetection site inhibitor comprising an antibody or antigen binding fragment thereof according to the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows binding of post-three-immune mouse serum to CHO-CEA cells as detected by FACS.
FIG. 2 shows binding of post-four-immune mouse serum to CHO-CEA cells as detected by FACS.
FIG. 3 shows TMB color development of mice serum after four-phase detection by ELISA.
FIG. 4 shows wells with better screening positive results detected by FACS.
FIG. 5 shows the results of CEA-10-1E6 positive subclone detection by FACS.
FIG. 6 shows the results of CEA-10-2D10 positive subclone detection by FACS.
FIG. 7 shows the results of CEA-10-2E7 positive subclone detection by FACS.
FIG. 8 shows the results of CEA-10-4G4 positive subclone detection by FACS.
FIG. 9 shows the results of CEA-10-5C7 positive subclone detection by FACS.
FIG. 10 shows the results of CEA-10-5H2 positive subclone detection by FACS.
FIG. 11 shows the results of expression identification of CEA-10-1E6, CEA-10-2D10 and CEA-10-5H2 recombinant antibodies by flow analysis.
FIG. 12 shows the results of expression identification of CEA-10-2E7 and CEA-10-5H2 recombinant antibodies by flow analysis.
Detailed Description
The present inventors have conducted extensive and intensive studies and, as a result of extensive screening, have unexpectedly found a class of anti-CEA monoclonal antibodies, specifically including CEA-10-1E6, CEA-10-2D10 and CEA-10-2E7. Experimental results show that the monoclonal antibody provided by the invention has high specificity and strong affinity, and can be specifically combined with CEA. The present invention has been completed on the basis of this finding.
CEA
Carcinoembryonic antigen (CEA) is a non-specific serum biomarker, originally found in colon cancer and fetal intestinal tissue, and later proved to be widely present in digestive system tumors of endocotyl origin, such as gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, and medullary thyroid cancer, breast cancer, mucinous ovarian cancer, lung cancer, and the like.
CEA is a glycoprotein of molecular weight 200kDa, typically derived from fetal embryonic endodermal epithelium, under the control of fetal oncogenes. It generally disappears from serum after birth; however, small amounts of CEA may remain in the colon tissue. CEA and related genes constitute the human CEA family and accumulate on chromosome 19q13.2.
Currently, CEA is a clinically common broad-spectrum tumor marker, widely used for differential diagnosis, disease monitoring and prognosis evaluation of multiple tumors such as colon cancer, rectal cancer, gastric cancer, lung cancer and the like, and because it is related to various types of malignant and non-malignant diseases, elevated serum CEA is not a definite marker of a specific site of origin of cancer. Therefore, it is often screened in combination with other indicators, such as CEA in combination with CA199, where an increase in the indicators suggests a higher risk of pancreatic cancer, gall bladder cancer, etc.
CEA is one of the carcinoembryonic antigen-related cell adhesion molecule (CEACAMs) family members, CEACAMs is a family of cell surface glycoproteins, comprising a total of 12 family members that are differentially expressed in normal and tumor tissues. Among them, CEA glycoprotein is most closely related to tumor development, and plays an important role in tumor progression, metastasis, angiogenesis, and inflammatory processes.
The CEA molecule belongs to an immunoglobulin superfamily adhesion molecule, and the N end of the protein molecule is provided with an immunoglobulin variable region (IgV) like structural domain composed of 108 amino acids and 0-6 immunoglobulin constant region (IgC) 2 type like structural domains. CEA is linked to the cell membrane by Glycosyl Phosphatidylinositol (GPI). CEACAMs are involved in a number of pathophysiological processes such as phagocytosis, proliferation, signal transduction, cell adhesion, and tumor suppression. CEA is currently being studied as a target for the targeted treatment of various cancers.
Terminology
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to an antibody that specifically binds to a CEA protein, such as a protein or polypeptide having a heavy chain variable region (e.g., the amino acid sequence of SEQ ID NO:1, 11, or 19) and/or a light chain variable region (e.g., the amino acid sequence of SEQ ID NO:5, 14, or 23). They may or may not contain an initiating methionine.
Preferably, the antibody numbering and corresponding sequence numbering of the invention is shown in table 1 below.
TABLE 1
| Antibody numbering | VH | HCDR1 | HCDR2 | HCDR3 | VL | LCDR1 | LCDR2 | LCDR3 |
| CEA-10-1E6-1C1 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| CEA-10-2D10-1E7 | 11 | 12 | 3 | 13 | 14 | 15 | 16 | 8 |
| CEA-10-2E7-1B6 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 |
Note that: each numerical value in the table indicates a sequence number, i.e. "1" indicates "SEQ ID NO:1", and the sequence numbers of VH, HCDR1, HCDR2, HCDR3, VL, LCDR1, LCDR2, LCDR3 shown in the table are the numbers of the amino acid sequences thereof.
Preferably, the CEA-10-1E6-1C1 antibody of the present invention has a heavy chain sequence as shown in SEQ ID NO.1 and a light chain sequence as shown in SEQ ID NO. 5; the CEA-10-2D10-1E7 antibody has a heavy chain sequence shown as SEQ ID NO. 11 and a light chain sequence shown as SEQ ID NO. 14; the CEA-10-2E7-1B6 antibody has a heavy chain sequence shown as SEQ ID NO. 19 and a light chain sequence shown as SEQ ID NO. 23.
In another preferred embodiment, the antibody is a murine or human chimeric monoclonal antibody to the CEA protein, the heavy and/or light chain constant regions of which may be humanized heavy or light chain constant regions. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgG1, igG2, or the like.
The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain and a light chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy chain and light chain of the antibodies of the invention.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.
The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to an antigen. Thus, the invention includes those molecules having monoclonal antibody light and heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.
The invention includes not only intact monoclonal antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
As used herein, the term "heavy chain variable region" is used interchangeably with "VH".
As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".
As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
The antibody of the present invention refers to a polypeptide having CEA protein binding activity and comprising the above CDR regions. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as fusion proteins comprising a human antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table 2.
TABLE 2
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to or degenerate as set forth in SEQ ID NO. 9, 10, 17, 18, 27 and/or 28. As used herein, a "degenerate variant" refers in the present invention to a nucleic acid sequence encoding a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but differing from the coding region sequences set forth in SEQ ID No. 9, 10, 17, 18, 27 and/or 28.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID No. 1-16, 19-26.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl 2 method using procedures well known in the art. Another approach is to use MgCl 2. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.
Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, cancer metastasis reviews (CANCER METASTASIS REVIEWS) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, nature 339, 394; epel et al, 2002, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. cytokines such as IL-2 et al (Gillies et al, 1992, proc. Natl. Acad. Sci. USA (PNAS) 89, 1428; card et al, 2004, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 53, 345; halin et al, 2003, cancer research (CANCER RESEARCH) 63, 3202); 4. gold nanoparticles/nanorods (Lapotko et al, 2005, cancer communication (CANCER LETTERS) 239, 36; huang et al, 2006, journal of the american Society of chemistry (Journal of THE AMERICAN CHEMICAL Society) 128, 2115); 5. viral particles (Peng et al, 2004, gene therapy (GENE THERAPY) 11, 1234); 6. liposomes (Mamot et al, 2005, cancer research (CANCER RESEARCH) 65, 11631); 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticle, and the like.
Antibodies to
As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or different types of immunoglobulins, i.e., igM, igD, igG, igA and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses again due to differences in chemical structure and biological function: igG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by the difference in constant regions. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 FR Regions (FR) that are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in sequence from amino-to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain, namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3; the 3 CDR regions of the heavy chain, namely heavy chain hypervariable regions (HCDR), refer to HCDR1, HCDR2 and HCDR3. The number and positions of CDR amino acid residues in the LCVR and HCVR regions of an antibody or antigen binding fragment according to the invention are in accordance with the known Kabat numbering convention or in accordance with the Chothia and IMGT numbering convention. The four FR regions in the natural heavy and light chain variable regions are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and form together with the CDRs of the other chain an antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody. The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
As used herein, the term "antigen binding fragment" refers to a Fab fragment, fab 'fragment, F (ab') 2 fragment, or a single Fv fragment having antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) A F (ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragments; (v) an scFv molecule; (vi) a dAb fragment; and (vii) a minimal recognition unit consisting of amino acid residues mimicking an antibody hypervariable region (e.g., an independent Complementarity Determining Region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. As used herein, the expression "antigen binding fragment" also encompasses within other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small Modular Immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.
The invention also includes monoclonal antibodies having the corresponding amino acid sequences of the anti-CEA protein monoclonal antibodies, monoclonal antibodies having the variable region chains of the anti-CEA protein monoclonal antibodies, and other proteins or protein conjugates and fusion expression products having these chains. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain comprising a hypervariable region (complementarity determining region, CDR), provided that the hypervariable region is identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light chain and heavy chain of the invention.
Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules in combination with said anti-CEA protein monoclonal antibodies or fragments thereof. The invention also includes cell surface markers or antigens that bind to the anti-CEA protein monoclonal antibodies or fragments thereof.
The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') 2 fragments; antibody heavy chain; an antibody light chain.
As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell by splicing the V region gene of a murine antibody to the C region gene of a human antibody into a chimeric gene, followed by insertion into a vector. The high specificity and affinity of the parent mouse antibody are maintained, and the human Fc segment of the parent mouse antibody can effectively mediate biological effect functions.
As used herein, the term "humanized antibody", a variable region engineered version of the murine antibody of the present invention, has CDR regions derived (or substantially derived) from a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; i.e., grafting murine-resistant CDR region sequences onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing expression vectors.
In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding CAR-T cell as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The antibodies of the invention may also be used for cellular therapy where the nucleotide sequence is expressed intracellularly, e.g., for chimeric antigen receptor T cell immunotherapy (CAR-T), etc.
The pharmaceutical compositions of the present invention can be used directly to bind CEA protein molecules and thus can be used to prevent and treat CEA related diseases. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the monoclonal antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
Where a pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Abbreviations
CDR: complementarity determining regions
FR: amino acid residues other than CDR residues in the variable region of an antibody
VH: antibody heavy chain variable region
VL: antibody light chain variable regions
IgG: immunoglobulin G
Kabat: immunoglobulin alignment and numbering System as proposed by Elvin A.Kabat
Chothia: immunoglobulin numbering system as proposed by Chothia et al
IMGT: numbering system based on the international immunogenetics information system initiated by Lefranc et al
ELISA: ELISA adsorption
FACS: flow cytometry
And (2) PCR: polymerase chain reaction
LS174t cells: is a human colon adenocarcinoma cell, is a trypsinized variant of the LS180 colon adenocarcinoma cell line. It is easier to passage than the parent cell and can produce large amounts of carcinoembryonic antigen (CEA).
Hybridoma cell strain
The invention also provides a hybridoma cell strain capable of producing the CEA protein monoclonal antibody; preferably, the present invention provides hybridoma cell lines with high titers against CEA protein monoclonal antibodies.
After obtaining the hybridoma producing the CEA protein monoclonal antibody of the invention, the skilled artisan can conveniently use the hybridoma cell line to prepare the antibody. Furthermore, the structure of the antibodies of the invention (e.g., the heavy and light chain variable regions of the antibodies) can be readily known to those skilled in the art, and then the monoclonal antibodies of the invention can be prepared by recombinant methods.
Preparation of monoclonal antibodies
Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, the antigens of the invention may be administered to animals to induce monoclonal antibody production. For monoclonal antibodies, hybridoma technology can be used to prepare (see Kohler et al, nature 256;495,1975; kohler et al, eur. J. Immunol.6:511,1976; kohler et al, eur. J. Immunol.6:292,1976; hammerling et al, in Monoclonal Antibodies and T Cell Hybridomas, elsevier, N.Y., 1981) or can be prepared using recombinant DNA methods (U.S. Pat. No. 4,816,567).
Representative myeloma cells are those that fuse efficiently, support stable high levels of antibody production by the antibody-producing cell of choice, and are sensitive to the medium (HAT medium matrix), including myeloma cell lines, e.g., murine myeloma cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, san diego, california, usa) and SP-2, NZ0 or X63-Ag8-653 cells (available from AMERICAN TYPE Culture Collection, rocyvale, maryland, usa). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [ Kozbor, j.immunol.,133:3001 (1984); brodeur et al, monoclonal antibody production techniques and uses (Monoclonal Antibodies Production Techniques and Applications), pages 51-63 (MARCEL DEKKER, inc., new york, 1987).
The culture medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies having the desired specificity, such as by an in vitro binding assay, e.g., an enzyme-linked immunosorbent assay (ELISA) or a Radioimmunoassay (RIA). The location of cells expressing the antibody can be detected by FACS. The hybridoma clones can then be subcloned by limiting dilution steps (subcloned) and grown by standard methods (Goding, monoclonal antibody (Monoclonal Antibodies): principles and practices (PRINCIPLES AND PRACTICE), ACADEMIC PRESS (1986) pages 59-103). Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can grow as ascites tumors in animals.
Monoclonal antibodies secreted by the subclones are suitably isolated from culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
The invention provides a monoclonal antibody aiming at CEA protein, in particular to a monoclonal antibody aiming at CEA protein. In a preferred embodiment of the invention, the monoclonal antibodies are prepared by culturing hybridoma cells. Taking supernatant of hybridoma cell culture, roughly extracting IgG by a saturated ammonium sulfate precipitation method, and purifying the roughly extracted antibody by an affinity chromatography column (Protein G-Sephrose).
In a preferred embodiment of the present invention, the monoclonal antibody is prepared by the method of producing a monoclonal antibody by using Balb/C mouse ascites. Approximately hybridoma cells were inoculated into the abdominal cavity of sensitized mice, and obvious abdominal distension was seen around 10 days. Extracting ascites, coarse extracting with saturated ammonium sulfate precipitation, and purifying the coarse extracted antibody with affinity chromatographic column (Protein G-Sephrose).
Labeled immunoglobulins
In a preferred embodiment of the invention, the immunoglobulin is provided with a detectable label. More preferably, the marker is selected from the group consisting of: colloidal gold labels, colored labels or fluorescent labels.
Colloidal gold labelling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the present invention, the monoclonal antibody to CEA protein is labeled with colloidal gold to obtain a colloidal gold-labeled monoclonal antibody.
The CEA protein monoclonal antibody has good specificity and high titer.
Methods and samples
The present invention relates to a method for detecting tumors in a sample lysed in cells and/or tissues. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving a sample in a medium; detecting the level of CEA protein in the solubilized sample. The sample used in the method of the invention may be any sample comprising cells present in a cell preservation fluid, as used in liquid-based cell assays.
Kit for detecting a substance in a sample
The invention also provides a kit comprising the antibody (or fragment thereof) of the invention or the detection plate of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.
The invention further relates to a detection kit for detecting CEA level, which comprises an antibody for recognizing CEA protein, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates and the like. The detection kit may be an in vitro diagnostic device.
The main advantages of the invention include:
(1) The antibody of the invention has high specificity and strong affinity, can be prepared in large quantity, and has easy control of monoclonal antibody quality.
(2) The antibody of the invention can be used for targeting drugs, antibody drug conjugates or multifunctional antibodies which specifically target CEA positive tumor cells.
(3) The antibodies of the invention can be used for preparing reagents for diagnosing tumors or for preparing chimeric antigen receptor immune cells.
(4) The antibody of the invention can be used as a secretory antibody which is expressed by chimeric antigen receptor immune cells together and acts on local tumor.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Example 1
Anti-CEA antibody preparation
The main technical scheme of the embodiment is as follows:
1. Mice were immunized with four DNA immunizations +1 cell impact, two weeks apart. Wherein, the DNA refers to human CEA DNA, and the cell used is CHO-CEA (i.e. CHO cell expressing CEA).
2. FACS screening was performed using CHO-CEA (CEA expressing CHO cells).
The method comprises the following experimental steps:
1. Immunized mice
Experimental mice were immunized according to the protocol of table 3:
TABLE 3 time protocol for immunization of mice
| Mouse ID | Exempt from | Two-way valve | Three-free | Four-free | Impact immunity |
| CEA-A-01 | 2020/09/08 | 2020/09/22 | 2020/10/10 | 2020/10/23 | 2020/12/04 |
| CEA-A-02 | 2020/09/08 | 2020/09/22 | 2020/10/10 | 2020/10/23 | |
| CEA-A-03 | 2020/09/08 | 2020/09/22 | 2020/10/10 | 2020/10/23 | 2020/12/04 |
| CEA-A-04 | 2020/09/08 | 2020/09/22 | 2020/10/10 | 2020/10/23 | |
| CEA-A-05 | 2020/09/08 | 2020/09/22 | 2020/10/10 | 2020/10/23 | |
| CEA-A-06 | 2020/09/08 | 2020/09/22 | 2020/10/10 | 2020/10/23 | |
| CEA-A-07 | 2020/09/08 | 2020/09/22 | 2020/10/10 | 2020/10/23 | |
| CEA-A-08 | 2020/09/08 | 2020/09/22 | 2020/10/10 | 2020/10/23 | 2020/12/04 |
| CEA-A-09 | 2020/09/08 | 2020/09/22 | 2020/10/10 | 2020/10/23 | |
| CEA-A-10 | 2020/09/08 | 2020/09/22 | 2020/10/10 | 2020/10/23 | 2020/12/04 |
Wherein, one exempts from: CEA-A-01-10 each mouse was DNA immunized (60. Mu.g DNA, intramuscular injection); and (2) avoiding: CEA-A-01-10 each mouse was DNA immunized; three-way: CEA-A-01-10 each mouse was DNA immunized; four-exemption: CEA-A-01-10 each mouse was DNA immunized; impact immunization: CEA-A-01, 03 mice impact an immune protein antigen.
2. Serum detection
(1) And (3) flow detection:
1) Appropriate amounts of CHO-CEA (or LS174 t) cells were dispensed into 1.5mL Ep tubes, 4000rpm,5min, and the supernatant was pipetted off.
2) 50. Mu.L of serum diluted with PBS1:100 resuspended the above cells and stood at 4℃for 15min.
3) 4000Rpm,5min, the supernatant was pipetted off.
4) Mu.L of Goat anti-mouseIgGFc-FITC diluted with PBS1:500 resuspended the above cells and stood at 4℃for 15min.
5) And 3, the same step as the step.
6) Cells were resuspended in 200 μl PBS and FACS analyzed.
The binding of mouse serum after the three-phase immunization to CHO-CEA was detected by FACS as shown in FIG. 1. The results showed that no significant immune response was detected in 10 mice after three-way, four-way, and ELISA was used for detection.
The binding of mouse serum after the four-immunization to CHO-CEA was detected by FACS as shown in FIG. 2. The results show that no obvious immune response was detected in serum after detection of the four-way immune response using CHO-CEA cells, and that better immune response was detected using LS174t cells.
(2) ELISA detection
Serum from mice after the four-immunization was assayed by ELISA, and experimental materials and conditions are shown in Table 3.
TABLE 3 Table 3
The TMB color development results are shown in FIG. 3, and the OD450 readings are shown in Table 4.
TABLE 4 Table 4
As is clear from the results, the detection results using the protein antigen were good, and CEA-A-01, 03 mice were selected for impact immune fusion.
3. Fusion of
After two weeks of impact immunization, B lymphocytes of the mice were fused with myeloma cells, respectively, to obtain corresponding hybridoma cells.
4. Post-fusion screening
(1) FACS detection screening: each mouse was plated with 5 96-well plates (12×8 wells, 1-8 for row number, A-H for column number, as in Table 1) and numbered 1,2, 3, 4,5, respectively, for FACS detection.
Table 5 preliminary screening results
As shown in FIG. 4 and Table 5, CEA-10-1E6, CEA-10-2D10, CEA-10-2E7, CEA-10-4G4, CEA-10-5C7, and CEA-10-5H2 were selected for subcloning based on the results.
(2) Subclone screening
The results are shown in FIGS. 5-10, as determined by FACS. The results show that CEA-10-1E6, CEA-10-2D10, CEA-10-2E7, CEA-10-4G4, CEA-10-5C7, CEA-10-5H2 were subcloned successfully.
5. Subcloning strain
Preferred positive subclones are summarized in Table 6 as determined by FACS as described above.
TABLE 6 preferred Positive subclone summary results
| Clone number | Subcloning number | Subtype type |
| CEA-10-1E6 | 1C1 | IgG1 |
| CEA-10-2D10 | 1E7、1A2、1E1、1G11 | IgG1 |
| CEA-10-2E7 | 1D9、1B6、1G10 | IgG2b |
| CEA-10-4G4 | 1A2、1C1、1C7 | IgG1 |
| CEA-10-5C7 | 1C10、1A5、1A11、1C3 | IgG1 |
| CEA-10-5H2 | 1A12、1A2、1A1、1C11 | IgG1 |
Example 2
2.1 Construction of recombinant CEA antibodies
The preferred positive subclone antibodies selected from example 1 were: CEA-10-1E6-1C1, CEA-10-2D10-1E7, CEA-10-5H2-1A12, CEA-10-2E7-1B6, which were subcloned based on the flow positive results of the hybridoma cell supernatants on LS174t cells after fusion.
And subjected to antibody sequence and subtype analysis.
First, the above subclones (CEA-10-2E 7-1B6, etc.) were determined for antibody sequence and subtype analysis.
The specific experimental steps are as follows:
(1) LS174t cells were aliquoted into 1.5mL EP tubes.
(2) Centrifuge at 4000rpm for 5min and discard supernatant.
(3) 50. Mu.L of hybridoma supernatant was taken and resuspended, and left to stand at 4℃for 15min.
(4) Centrifuge at 4000rpm for 5min and discard supernatant.
(5) The cells were resuspended in 50. Mu.L of Goat anti-mouse IgG1-FITC, goat anti-mouse IgG2a-FITC and Goat anti-mouse IgG2b-FITC diluted 1:200 and allowed to stand at 4℃for 15min.
(6) Centrifuge at 4000rpm for 5min and discard supernatant.
(7) Cells were resuspended in 300 μl PBS and flow analyzed.
The results of the flow analysis are shown in Table 7. The results show that: the CEA-10-2E7-1B6 antibody subtype is IgG2B.
Antibody gene sequencing was performed using the Sanger method of capillary electrophoresis.
Details of the CEA-10-1E6-1C1, CEA-10-2D10-1E7, CEA-10-5H2-1A12, and CEA-10-2E7-1B6 antibodies are summarized in Table 7.
TABLE 7 subtype and gene sequence of antibodies
| Clone number | Subtype type | Heavy chain | Light chain |
| CEA-10-1E6-1C1 | IgG1 | IGHV14-4 | IGKV4-57 |
| CEA-10-2D10-1E7 | IgG1 | IGHV14-4 | IGKV4-57 |
| CEA-10-5H2-1A12 | IgG1 | IGHV3-6 | IGKV3-2 |
| CEA-10-2E7-1B6 | IgG2b | IGHV2-9 | IGKV10-96 |
The heavy chain IGHV14-4 sequence of CEA-10-1E6-1C1 is shown as SEQ ID NO 9 (DNA) and SEQ ID NO 1 (amino acid).
The light chain IGKV4-57 sequence of CEA-10-1E6-1C1 is shown as SEQ ID NO. 10 (DNA) and SEQ ID NO. 5 (amino acid).
The heavy chain IGHV14-4 sequence of CEA-10-2D10-1E7 is shown in SEQ ID NO:17 (DNA) and SEQ ID NO:11 (amino acid).
The light chain IGKV4-57 sequence of CEA-10-2D10-1E7 is shown as SEQ ID NO. 18 (DNA) and SEQ ID NO. 14 (amino acid).
The heavy chain IGHV2-9 sequence of CEA-10-2E7-1B6 is shown in SEQ ID NO 27 (DNA) and SEQ ID NO 19 (amino acid).
The light chain IGKV10-96 sequence of CEA-10-2E7-1B6 is shown as SEQ ID NO. 28 (DNA) and SEQ ID NO. 23 (amino acid).
2.2 Expression verification of recombinant antibodies
The method comprises the following specific steps: the nucleotide sequences of the heavy chain variable region and the light chain variable region of the CEA-10-1E6-1C1, CEA-10-2D10-1E7 and CEA-10-2E7-1B6 antibody are cloned into pCAG eukaryotic expression vectors respectively to obtain recombinant expression vectors for expressing the heavy chain variable region and the light chain variable region of the antibody respectively.
10 Μg of light and heavy chain expression vector was co-transferred into 293T cells in 10cm dishes using the calcium phosphate method, and the supernatant was collected three days later and FACS verified in LS174T cells.
The FACS validation experiment includes the following steps:
(1) LS174t cells were aliquoted into 1.5mL EP tubes.
(2) Centrifuge at 4000rpm for 5min and discard supernatant.
(3) 50. Mu.L of 293T cells were transfected into the supernatant and resuspended, and allowed to stand at 4℃for 15min.
(4) Centrifuge at 4000rpm for 5min and discard supernatant.
(5) The cells were resuspended in 50. Mu.L of Goat anti-mouse IgG Fc-FITC diluted 1:200 and allowed to stand at 4℃for 15min.
(6) Centrifuge at 4000rpm for 5min and discard supernatant.
(7) Cells were resuspended in 300 μl PBS and flow analyzed.
The results of the FACS validation are shown in FIGS. 11-12. The results show that CEA-10-1E6-1C1, CEA-10-2D10-1E7 and CEA-10-2E7-1B6 transfected supernatant had better results and correct sequences. The CEA-10-5H2-1A12 signal was slightly worse, and after this transfection verification, the signal was still worse.
The sequence information of the present invention is shown in Table 8.
TABLE 8
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All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (10)
1. A CEA antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof has three complementarity determining region CDRs (HCDR) of a heavy chain variable region and three complementarity determining region CDRs (LCDR) of a light chain variable region selected from the group consisting of:
(1) HCDR1 shown in SEQ ID NO. 2,
HCDR2 shown in SEQ ID NO. 3,
HCDR3 shown in SEQ ID NO. 4,
LCDR1 shown in SEQ ID NO. 6,
LCDR2 shown in SEQ ID NO. 7,
LCDR3 shown in SEQ ID NO. 8;
(2) HCDR1 shown in SEQ ID NO. 12,
HCDR2 shown in SEQ ID NO. 3,
HCDR3 shown in SEQ ID NO. 13,
LCDR1 shown in SEQ ID NO. 15,
LCDR2 as shown in SEQ ID NO. 16,
LCDR3 shown in SEQ ID NO. 8;
(3) HCDR1 shown in SEQ ID NO. 20,
HCDR2 shown in SEQ ID NO. 21,
HCDR3 shown in SEQ ID NO. 22,
LCDR1 shown in SEQ ID NO. 24,
LCDR2 as shown in SEQ ID NO. 25,
LCDR3 as shown in SEQ ID NO. 26.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof has a sequence selected from the group consisting of seq id no:
(1) The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.1, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 5;
(2) The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 11, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 14;
(3) The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 19, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 23; .
3. A recombinant protein, wherein the recombinant protein has:
(i) The antibody or antigen-binding fragment thereof of claim 1; and
(Ii) Optionally a tag sequence that facilitates expression and/or purification.
4. A polynucleotide molecule encoding the antibody or antigen-binding fragment thereof of claim 1.
5.A vector comprising the polynucleotide molecule of claim 4.
6. An engineered cell comprising the vector or genome of claim 5 integrated with the polynucleotide molecule of claim 4 or expressing the CEA antibody or antigen-binding fragment thereof of claim 1.
7. An immunoconjugate, characterized in that the immunoconjugate comprises:
(a) The antibody or antigen-binding fragment thereof of claim 1; and
(B) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
8. A pharmaceutical composition, comprising:
(i) The antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 3, or the immunoconjugate of claim 7; and
(Ii) A pharmaceutically acceptable carrier.
9. Use of the antibody or antigen binding fragment thereof of claim 1, the recombinant protein of claim 3, or the immunoconjugate of claim 7, for the preparation of a medicament, reagent, assay plate, or kit;
The reagent, assay plate or kit is for: detecting CEA protein in the sample;
The agent is used for treating or preventing tumors expressing CEA protein.
10. A method of detecting CEA protein in a sample, the method comprising the steps of:
(1) Contacting a sample with the antibody or antigen-binding fragment thereof of claim 1;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of a CEA protein in the sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410307425.0A CN118184785A (en) | 2024-03-18 | 2024-03-18 | CEA antibodies and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410307425.0A CN118184785A (en) | 2024-03-18 | 2024-03-18 | CEA antibodies and uses thereof |
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| Publication Number | Publication Date |
|---|---|
| CN118184785A true CN118184785A (en) | 2024-06-14 |
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ID=91414736
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| Application Number | Title | Priority Date | Filing Date |
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| CN202410307425.0A Pending CN118184785A (en) | 2024-03-18 | 2024-03-18 | CEA antibodies and uses thereof |
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| Country | Link |
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| CN (1) | CN118184785A (en) |
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