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CN115505043A - Antibodies specifically binding glycosylated CEACAM5 - Google Patents

Antibodies specifically binding glycosylated CEACAM5 Download PDF

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Publication number
CN115505043A
CN115505043A CN202110697514.7A CN202110697514A CN115505043A CN 115505043 A CN115505043 A CN 115505043A CN 202110697514 A CN202110697514 A CN 202110697514A CN 115505043 A CN115505043 A CN 115505043A
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antibody
seq
antigen
ser
substitution
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牟男
于跃
杜靓
袁纪军
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Shanghai Genbase Biotechnology Co Ltd
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Shanghai Genbase Biotechnology Co Ltd
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Priority to CN202110697514.7A priority Critical patent/CN115505043A/en
Priority to PCT/CN2022/098746 priority patent/WO2022267936A1/en
Publication of CN115505043A publication Critical patent/CN115505043A/en
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    • A61K47/6851—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 determinant of a tumour cell
    • A61K47/6853—Carcino-embryonic antigens
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Abstract

The present application provides antibodies that specifically bind CEACAM5, preparation and uses of humanized antibodies thereof.

Description

Antibodies specifically binding glycosylated CEACAM5
Technical Field
The present invention relates to antibodies. More specifically, the present application relates to monoclonal antibodies that specifically bind to glycosylated CEACAM5, the preparation and use of humanized antibodies thereof.
Background
In 1960s, gold and Freedman first found CEA antigen (CEACAM 5) in human colon cancer tissue extracts and subsequently detected free CEA antigen in the peripheral blood of cancer patients such as colorectal cancer. CEA antigens are widely involved in cell adhesion, cell differentiation, cell proliferation, and cell survival. CEA antigen is widely and highly expressed in digestive tract cancers, such as colorectal cancer, esophageal cancer, gastric cancer and the like, and has been used as a tumor marker for decades.
CEACAM5 (CD 66e, carcinoembrynic antigen related cell adhesion molecule 5), a Carcinoembryonic antigen, belongs to the CEA family. The CEA family contains 12 genes, all located on chromosome 19q13; CEACAM5 is the gene with the largest molecular weight in the CEA family, is expressed in a cell membrane (GPI anchoring) and can fall off to enter peripheral blood to become free CEA; it consists of 702 amino acids (N-A1-B1-A2-B2-A3-B3 domains are connected in series), and the molecular weight is about 77KDa; the molecular weight of the CEACAM5 protein, which is recombinantly expressed or purified from ascites fluid of colorectal cancer patients, is about 180kDa, indicating that CEACAM5 is highly glycosylated in the cells.
CEA family antigen exists in three forms of transmembrane, GPI anchoring and secretion, and the extracellular region of the antigen consists of immunoglobulin structural domain. The extracellular region comprises an N-terminal structural domain, an A1-3 structural domain and a B1-3 structural domain which are connected in series. CEACAM1, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM18, CEACAM20, CEACAM21 contain N-terminal and AB domains; CEACAM3, CEACAM4, CEACAM19 comprise only N-terminal domains; CEACAM16 is a secreted protein. CEACAM family proteins, such as CEACAM1, CEACAM3, CEACAM5, CEACAM6, CEACAM8, have high homology and different tissue distribution, such as CEACAM1 mainly distributed in urinary tract epithelium and intestinal tract epithelium; CEACAM6 is mainly distributed in alveoli and bile ducts, CEACAM8 is mainly distributed in bone marrow and hematopoietic system.
CEACAM5 is widely and highly expressed in a plurality of cancers, such as digestive tract cancer (colorectal cancer, gastric cancer, pancreatic cancer, esophageal cancer), lung cancer, breast cancer and the like, and is an ideal Tumor Associated Antigen Target (TAA). The development of the CEACAM5 targeting drug is mainly focused on the field of antibody discovery, and the CEA family sequence has higher homology, so that the screening of the CEACAM5 targeting antibody can reduce the side effect in clinical use, thereby improving the treatment window and the drug effect.
Disclosure of Invention
The invention provides a humanized antibody specifically binding CEACAM5, which can be applied to the treatment of CEACAM5 high-expression tumors.
One aspect of the present invention provides a monoclonal antibody or antigen-binding fragment thereof directed against CEACAM5, which antibody or antigen-binding fragment thereof specifically binds to the N-A1-B1 domain and not to the A2-B2 and A3-B3 domains of CEACAM 5.
In a specific embodiment, the monoclonal antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein:
a. the heavy chain variable region comprises: a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) to or to CDR-H1 shown by SEQ ID NO. 1, a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) to or to CDR-H2 shown by SEQ ID NO. 2, and a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) to or to CDR-H3 shown by SEQ ID NO. 3; and the light chain variable region comprises: a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) of CDR-L1 shown by SEQ ID NO. 4 or compared thereto, a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) of CDR-L2 shown by SEQ ID NO. 5 or compared thereto, and a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) of CDR-L3 shown by SEQ ID NO. 6 or compared thereto; or
b. The heavy chain variable region comprises: a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) to or from CDR-H1 shown by SEQ ID NO. 7, a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) to or from CDR-H2 shown by SEQ ID NO. 8, and a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) to or from CDR-H3 shown by SEQ ID NO. 9; and the light chain variable region comprises: a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) to or from CDR-L1 shown by SEQ ID NO:10, a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) to or from CDR-L2 shown by SEQ ID NO:11, and a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) to or from CDR-L3 shown by SEQ ID NO: 12.
In one embodiment, the monoclonal antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein:
a) The heavy chain variable region comprises the polypeptide shown in SEQ ID NO. 13 or a sequence having one or more amino acid substitutions, deletions or additions (such as 1,2 or 3 amino acid substitutions, deletions or additions) compared with the polypeptide, while the light chain variable region comprises the polypeptide shown in SEQ ID NO. 14 or a sequence having one or more amino acid substitutions, deletions or additions (such as 1,2 or 3 amino acid substitutions, deletions or additions) compared with the polypeptide; or
b) The heavy chain variable region comprises the polypeptide shown in SEQ ID NO. 15 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) compared thereto, while the light chain variable region comprises the polypeptide shown in SEQ ID NO. 16 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) compared thereto.
Preferably, the substitution of any one of SEQ ID NOs 1-12 is a conservative substitution;
preferably, the CDRs of any of SEQ ID NOs 1-12 are defined according to the Kabat numbering system.
In one aspect, the antibody of the invention is a humanized antibody. In a specific embodiment, the present invention provides a humanized antibody or an antigen-binding fragment thereof that specifically binds CEACAM5, wherein the humanized antibody comprises a heavy chain variable region and a light chain variable region, wherein:
a) The heavy chain variable region comprises the polypeptide shown in SEQ ID NO. 17 or a sequence having one or more amino acid substitutions, deletions or additions (such as 1,2 or 3 amino acid substitutions, deletions or additions) compared thereto, while the light chain variable region comprises the polypeptide shown in SEQ ID NO. 18 or a sequence having one or more amino acid substitutions, deletions or additions (such as 1,2 or 3 amino acid substitutions, deletions or additions) compared thereto; or
b) The heavy chain variable region comprises the polypeptide shown in SEQ ID NO. 19 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) compared thereto, while the light chain variable region comprises the polypeptide shown in SEQ ID NO. 20 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) compared thereto.
Preferably, the substitution of any one of SEQ ID NOs 17-20 is a conservative substitution;
preferably, the CDRs of any of SEQ ID NOs 17-20 are defined according to the Kabat numbering system.
The present invention provides a humanized antibody or an antigen-binding fragment thereof that specifically binds CEACAM5, wherein said antibody or antigen-binding fragment thereof comprises a constant region derived from a human immunoglobulin or a variant thereof;
preferably, the antibody or antigen-binding fragment thereof comprises:
(a) A heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., substitutions, deletions or additions of 1,2, 3,4, or 5 amino acids or any combination thereof) compared to the sequence from which it is derived; and/or
(b) A light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., substitutions, deletions or additions of 1,2, 3,4, or 5 amino acids or any combination thereof) compared to the sequence from which it is derived;
preferably, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, igG2, igG3, or IgG4 heavy chain constant region;
preferably, the antibody or antigen-binding fragment thereof comprises the heavy chain variable region (VH) set forth in SEQ ID NO:17 or SEQ ID NO: 19;
preferably, the light chain constant region is a kappa light chain constant region;
preferably, the antibody or antigen-binding fragment thereof comprises the light chain variable region (VL) set forth in SEQ ID NO:18 or SEQ ID NO: 20.
The present invention provides a humanized antibody or an antigen binding fragment thereof that specifically binds CEACAM5, wherein the antigen binding fragment is selected from the group consisting of Fab, fab ', (Fab ') 2, fv, disulfide linked Fv, bsFv, dsFv, (dsFv) 2, dsFv-dsFv ', scFv dimer, camelized single domain antibody (camelized single chain domain antibody), diabody (diabody), ds diabody (ds diabody), nanobody, single domain antibody (sdAb), diabody; and/or, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.
Wherein the antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds CEACAM 5.
In another aspect, the invention relates to an expression vector comprising a nucleic acid molecule encoding an antibody that specifically binds CEACAM5 as disclosed herein.
In another aspect, the invention relates to a host cell comprising an expression vector disclosed herein.
In another aspect, the present invention relates to a pharmaceutical composition comprising at least one antibody that specifically binds CEACAM5 disclosed herein and a pharmaceutically acceptable carrier.
Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent;
preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, mitotic inhibitor, anti-tumor antibiotic, anti-metabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide, radiosensitizer, anti-angiogenic agent, cytokine, molecularly targeted drug, immune checkpoint inhibitor or oncolytic virus;
preferably, the antibody or antigen binding fragment thereof, bispecific or multispecific molecule or immunoconjugate and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.
In another aspect, the invention provides a chimeric antigen receptor comprising an antigen binding domain of said humanized antibody or antigen binding fragment thereof;
preferably, the antigen binding domain comprises the heavy chain variable region and the light chain variable region of the humanized antibody or antigen binding fragment thereof;
preferably, the antigen binding domain is a scFv;
preferably, the antigen binding receptor comprises the humanized antibody or antigen binding fragment thereof;
preferably, the antigen binding receptor is expressed by an immune effector cell (e.g., a T cell).
The invention provides an isolated nucleic acid molecule encoding the chimeric antigen receptor.
The present invention provides a vector comprising the isolated nucleic acid molecule; preferably, it is used for the preparation of chimeric antigen receptor T cells.
The present invention provides a host cell comprising said isolated nucleic acid molecule or said vector;
preferably, the host cell is an immune effector cell (e.g., a T cell or NK cell);
preferably, the host cell is a chimeric antigen receptor T cell (CAR-T).
The present invention provides a method for reducing the expression level of CEACAM5 on the surface of a cell, comprising contacting said cell with said humanized antibody or antigen-binding fragment thereof, or said pharmaceutical composition, or said chimeric antigen receptor, or said host cell, such that the expression level of CEACAM5 on the surface of said cell is reduced; wherein the cell expresses CEACAM5 on its surface;
preferably, the cell is a CEACAM5 expressing tumor cell.
The present invention provides a method of inhibiting the growth and/or killing a CEACAM5 expressing tumor cell comprising contacting said tumor cell with an effective amount of said humanized antibody or antigen binding fragment thereof, or said pharmaceutical composition, or said chimeric antigen receptor, or said host cell.
In another aspect, the present invention relates to a method for preparing an antibody specifically binding CEACAM5 comprising expressing in a host cell a nucleic acid sequence encoding an antibody specifically binding CEACAM5 as disclosed herein and isolating the antibody specifically binding CEACAM5 from the host cell.
In another aspect, the present invention provides a use of the antibody of the present invention for the preparation of a medicament for treating CEACAM 5-highly expressing cancer, wherein the CEACAM 5-highly expressing cancer is selected from colorectal, gastric, pancreatic, esophageal, lung or breast cancer.
In another aspect, the present invention provides a method for treating a CEACAM 5-highly expressing cancer comprising administering the antibody of the present invention to a subject in need thereof, wherein the CEACAM 5-highly expressing cancer is selected from colorectal, gastric, pancreatic, esophageal, lung, or breast cancer.
Preferably, the subject is a mammal, such as a human;
preferably, the method further comprises administering an additional agent with anti-tumor activity, such as an alkylating agent, mitotic inhibitor, anti-tumor antibiotic, anti-metabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide, radiosensitizer, antiangiogenic agent, cytokine, molecularly targeted drug, immune checkpoint inhibitor, or oncolytic virus;
preferably, the method further comprises administering an additional anti-tumor therapy, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy or palliative therapy.
The present invention also provides an antibody-drug conjugate comprising:
(a) The antibody as described above; and
(b) A drug conjugated to the antibody, wherein the drug is a peptide,
preferably, the drug is a toxin, preferably monomethyl auristatin (monomethylauristatin), calicheamicin, a maytansinoid, or a combination thereof; more preferably selected from: monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), or combinations thereof.
The invention provides application of the antibody drug conjugate in preparing drugs.
The present invention provides a bispecific antibody comprising a domain that specifically binds CEACAM5 and a domain that specifically binds CD3, CD28 or 41BB,
wherein the domain that specifically binds CEACAM5 comprises: CDR-H1 shown in SEQ ID NO. 1; CDR-H2 shown in SEQ ID NO. 2, CDR-H3 shown in SEQ ID NO. 3, CDR-L1 shown in SEQ ID NO. 4, CDR-L2 shown in SEQ ID NO. 5, and CDR-L3 shown in SEQ ID NO. 6; or
CDR-H1 shown in SEQ ID NO. 7, CDR-H2 shown in SEQ ID NO. 8, CDR-H3 shown in SEQ ID NO. 9, CDR-L1 shown in SEQ ID NO. 10, CDR-L2 shown in SEQ ID NO. 11, and CDR-L3 shown in SEQ ID NO. 12.
The invention also provides the use of the bispecific antibody in the preparation of a medicament.
Preparation and screening of monoclonal antibodies
Monoclonal antibodies can be prepared as follows. Mice or other suitable host animals are first immunized with the immunogen (with adjuvant added if necessary). The mode of injection of the immunogen or adjuvant is usually subcutaneous multi-site injection or intraperitoneal injection. The immunogen may be pre-coupled to certain known proteins, such as serum albumin or pancreatin inhibitor, to enhance the immunogenicity of the antigen in the host. The adjuvant may be Freund's adjuvant or MPL-TDM, etc. After the animal is immunized, lymphocytes that secrete antibodies that specifically bind the immunogen will be produced in vivo. Alternatively, lymphocytes may be obtained by in vitro immunization. The lymphocytes of interest are collected and fused with myeloma cells using a suitable fusing agent such as PEG to obtain hybridoma cells (Goding, monoclonal Antibodies: principles and Practice, pp.59-103, academic Press, 1996). Preferred myeloma cells should have high fusion rate, stable antibody secretion ability, sensitivity to HAT culture medium, and the like. The culture medium of the growing hybridoma cells was used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of a monoclonal antibody produced by a hybridoma cell include, for example, immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA). For example, the affinity of a monoclonal antibody can be determined using the Scatchard assay described by Munson et al, anal. Biochem.107:220 (1980). After the specificity, affinity and reactivity of the Antibodies produced by the hybridomas have been determined, the cell lines of interest may be subcloned by standard limiting dilution methods as described in Goding, monoclonal Antibodies: principles and Practice, pp.59-103, academic Press, 1996. Suitable culture medium may be DMEM or RPMI-1640 or the like. In addition, hybridoma cells can also be grown in animals as ascites tumors. The monoclonal antibodies secreted by the subcloned cells can be isolated from the cell culture fluid, ascites fluid, or serum using conventional immunoglobulin purification methods, such as protein a sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
Monoclonal antibodies can also be obtained by genetic engineering recombination techniques. The DNA molecules encoding the monoclonal antibody heavy chain and light chain genes can be isolated from the hybridoma cells by PCR amplification using nucleic acid primers that specifically bind to the monoclonal antibody heavy chain and light chain genes. The resulting DNA molecule is inserted into an expression vector, and then host cells (e.g., E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin) are transfected and cultured under appropriate conditions to obtain a recombinantly expressed antibody of interest.
The invention adopts CEACAM5 high-expression tumor cell line (such as Lovo) to immunize mice so as to obtain the antibody for recognizing natural CEACAM5 antigen.
Humanized antibodies
A "humanized antibody" is an antibody that comprises one or both of a humanized VH domain and a humanized VL domain. One or more immunoglobulin constant regions need not be present, but if present, are derived entirely or substantially from a human immunoglobulin constant region.
Humanized antibodies are genetically engineered antibodies in which the CDRs from a non-human "donor" antibody are grafted into human "acceptor" antibody sequences (see, e.g., queen, US 5,530,101 and 5,585,089 winter, US 5,225,539 carter, US 6,407,213 adair, US 5,859,205; and Foote, US 6,881,557. The recipient antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a reproductive region sequence. The human acceptor sequence may be selected such that the variable region framework has a high degree of sequence identity with the donor sequence to match the canonical form and other criteria between the acceptor CDR and the donor CDR. Thus, a humanized antibody is an antibody in which the CDRs are derived completely or substantially from the donor antibody and variable region framework sequences and the constant regions (if present) are derived completely or substantially from human antibody sequences. Similarly, humanized heavy chains typically have all three CDRs entirely or substantially from the donor antibody heavy and heavy chain variable region framework sequences and a heavy chain constant region (if present) substantially from the human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain typically has all three CDRs entirely or substantially from the donor antibody light and light chain variable region framework sequences and a light chain constant region (if present) substantially from the human light chain variable region framework and constant region sequences. A CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the corresponding residues (as defined by Kabat numbering) between the respective CDRs are identical or wherein about 100% of the corresponding residues (as defined by Kabat numbering) are identical. A variable region framework sequence of an antibody chain or a constant region of an antibody chain is substantially derived from a human variable region framework sequence or a human constant region, respectively, when at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the corresponding residues (variable regions defined by Kabat numbering and constant regions defined by EU numbering) are the same or about 100% of the corresponding residues (variable regions defined by Kabat numbering and constant regions defined by EU numbering) are the same.
Although humanized antibodies often incorporate all six CDRs from a mouse antibody (preferably as defined by Kabat or IMGT), humanized antibodies may also be composed of fewer than all six CDRs (e.g., at least 3,4, or 5) from a mouse antibody (e.g., pascales et al, J.Immunol.169:3076,2002 Vajdos et al, journal of Molecular Biology, 320-428, 2002 Iwahashi et al, mol.Immunol.36:1079-1091,1999, tamura et al, journal of Immunology, 164-1432-1441, 2000.
A CDR in a humanized antibody is "substantially from" a corresponding CDR in a non-human antibody when at least 60%, at least 85%, at least 90%, at least 95%, or 100% of the corresponding residues (as defined in Kabat or IMGT) between the individual CDRs are identical. In particular variations in which the CDRs are substantially from a humanized VH or VL domain of a non-human immunoglobulin, the CDRs of the humanized VH or VL domain have no more than six (e.g., preferably conservative substitutions) amino acid substitutions relative to the corresponding non-human VH or VL CDRs across all three CDRs. A variable region framework sequence of an antibody VH or VL domain or a sequence of an immunoglobulin constant region (if present) is "substantially derived from" a human VH or VL framework sequence or a human constant region, respectively, when at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the corresponding residues (variable regions defined by Kabat numbering and constant regions defined by EU numbering) are identical or about 100% of the corresponding residues (variable regions defined by Kabat numbering and constant regions defined by EU numbering) are identical. Thus, all portions of a humanized antibody (except the CDRs) are typically derived entirely or substantially from the corresponding portion of a natural human immunoglobulin sequence.
Definition of general terms
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are all conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain and one heavy chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, igD, igG, igA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to the various regions or domains follows either Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia & Lesk (1987) J.mol.biol.196:901-917; chothia et al (1989) Nature 342, 878-883. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be of different isotypes, e.g., igG (e.g., igG1, igG2, igG3, or IgG4 subtypes), igA1, igA2, igD, igE, or IgM antibodies.
Herein, when the term "antibody" is referred to, it includes not only intact antibodies, but also antigen-binding fragments of antibodies, unless the context clearly indicates otherwise. As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion. In some cases, antigen-binding fragments include Fab, fab ', F (ab') 2, fd, fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabodies), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding capability on the polypeptide.
Antigen-binding fragments of antibodies (e.g., antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation), and the antigen-binding fragments of antibodies are specifically screened for in the same manner as for intact antibodies.
As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. The monoclonal antibody has high specificity to a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using hybridoma technology first reported by Kohler et al (Nature, 256, 495, 1975), but can also be obtained using recombinant DNA technology (see, e.g., U.S. P4, 816, 567).
For example, monoclonal antibodies can be prepared as follows. Mice or other suitable host animals are first immunized with the immunogen (adjuvant added if necessary). The mode of injection of the immunogen or adjuvant is usually subcutaneous multi-site injection or intraperitoneal injection. Immunogens can be pre-conjugated to certain known proteins, such as serum albumin or soybean pancreatin inhibitors, to enhance the immunogenicity of the antigen in the host. The adjuvant may be Freund's adjuvant or MPL-TDM, etc. After an animal is immunized, it will produce lymphocytes that secrete antibodies that specifically bind the immunogen in vivo. Alternatively, lymphocytes may be obtained by in vitro immunization. The lymphocytes of interest are collected and fused with myeloma cells using a suitable fusing agent such as PEG to obtain hybridoma cells (Goding, monoclonal Antibodies: principles and Practice, pp.59-103, academic Press, 1996). The hybridoma cells prepared as described above may be grown by inoculating into a suitable culture medium, preferably containing one or more substances capable of inhibiting the growth of unfused, maternal myeloma cells. For example, for parental myeloma cells that lack hypoxanthine guanine phosphotransferase (HGPRT or HPRT), the addition of hypoxanthine, aminopterin, and thymidine (HAT medium) to the culture medium will inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells should have high fusion rate, stable antibody secretion ability, sensitivity to HAT culture medium, and the like. Among them, THE myeloma cells are preferably derived from murine myelomas such as MOP-21 or MC-11 mouse tumor-derived strains (THE salt Institute Cell Distribution Center, san Diego, calif. USA), and SP-2/0 or X63-Ag8-653 Cell strains (American Type Culture Collection, rockville, md. USA). In addition, there have been studies reported that human Monoclonal antibodies (Kozbor, J.Immunol.,133 (1984); brodeur et al, monoclonal Antibody Production Techniques and Applications, pp.51-63, marcel Dekker, inc., new York, 1987) were prepared using human myeloma and human murine allogeneic myeloma cell lines. The culture medium of the growing hybridoma cells was used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of a monoclonal antibody produced by a hybridoma cell include, for example, immunoprecipitation or in vitro binding assays, such as Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). For example, the affinity of a mAb can be determined using the Scatchard assay described by Munson et al, anal. Biochem.107:220 (1980). After the specificity, affinity and reactivity of the Antibodies produced by the hybridomas are determined, the cell lines of interest can be subcloned by standard limiting dilution methods as described by (Goding, monoclonal Antibodies: principles and Practice, pp.59-103, academic Press, 1996). Suitable culture medium may be DMEM or RPMI-1640 or the like. In addition, hybridoma cells can also be grown in animals as ascites tumors. The monoclonal antibodies secreted by the subcloned cells can be isolated from the cell culture fluid, ascites fluid, or serum using conventional immunoglobulin purification methods, such as protein a sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
Monoclonal antibodies can also be obtained by genetic engineering recombination techniques. The DNA molecules encoding the monoclonal antibody heavy chain and light chain genes can be isolated from the hybridoma cells by PCR amplification using nucleic acid primers that specifically bind to the monoclonal antibody heavy chain and light chain genes. The resulting DNA molecule is inserted into an expression vector, and then host cells (e.g., E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin protein) are transfected and cultured under appropriate conditions to obtain a recombinantly expressed antibody of interest.
As used herein, the term "chimeric antibody" refers to an antibody of which a portion of the light chain or/and heavy chain is derived from one antibody (which may be derived from a certain species or belong to a certain antibody class or subclass) and another portion of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which nevertheless retains binding activity to a target antigen (u.s.p 4,816,567to Cabilly et al; morrison et al, proc.natl.acad.sci.usa,81 6851 6855 (1984)).
As used herein, the term "human antibody" refers to a humanized antibody, an antibody or antibody fragment obtained by replacing all or a portion of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. In addition, some amino acid residues of the Framework Region (FR) of the acceptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further refine or optimize the performance of the antibody. For more details on humanized antibodies, see, e.g., jones et al, nature, 321; reichmann et al, nature, 332; presta, curr, op, struct, biol., 2; and Clark, immunol. Today 21 (2000).
As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to in the art as an "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrates or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically includes at least 3,4,5,6,7,8,9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, ed. (1996). In a linear epitope, the points of all interactions between a protein and an interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the protein. In conformational epitopes, the points of interaction exist across protein amino acid residues that are separated from each other.
As used herein, the term "epitope peptide" refers to a peptide fragment on an antigen that can serve as an epitope. In some cases, the epitope peptide alone is capable of being specifically recognized/bound by an antibody directed against the epitope. In other cases, it may be desirable to fuse the epitope peptide to a carrier protein so that the epitope peptide can be recognized by a specific antibody. As used herein, the term "carrier protein" refers to a protein that can act as a carrier for an epitope peptide, i.e., it can be inserted into the epitope peptide at a specific position (e.g., inside the protein, N-terminal or C-terminal) so that the epitope peptide can be presented so that it can be recognized by an antibody or immune system. Such carrier proteins are well known to those skilled in the art and include, for example, the HPV L1 protein (epitope peptides can be inserted between amino acids 130-131 or between amino acids 426-427 of the protein, see Slupetzky, K. Et al, molecular plasmid expression a for infection epitope on peptide surface loops [ J]J Gen Virol,2001, 82; varsani, A. Et al, chimeric human papillomavir type 16 (HPV-16) L1 particles presenting the common neutral hepatitis for the L2 minor capsid protein of HPV-6and HPV-16[ J ], []J Virol,2003,77 8386-8393), HBV core antigen (amino acids 79-81 of the protein may be replaced by epitope peptides, see Koletzki, D., et al, HBV core particles alone the infection and surface exposure of the organic pore protective region of cancer modifying nuclear protein [ J Virol].Biol Chem1999,380, 325-333), the woodchuck hepatitis virus core protein (amino acids 79-81 of the protein can be replaced by an epitope peptide, see Sabine
Figure BDA0003128428790000161
Gertrud Beterams and Michael Nassal, J.Virol.1998,72 (6): 4997), CRM197 protein (epitope peptides can be attached to the N-terminus or C-terminus of the protein or fragment thereof). Optionally, a linker (e.g., a flexible or rigid linker) may be used between the epitope peptide and the carrier protein to facilitate folding of each.
Antibodies can be screened for binding competition with the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to an antigen (e.g., an influenza virus hemagglutinin protein). A high throughput method for obtaining antibodies binding to the same epitope based on their cross-competition is described in International patent application WO 03/48731. Accordingly, antibodies and antigen-binding fragments (i.e., antigen-binding portions) thereof that compete with the monoclonal antibodies of the invention for binding to the same epitope on the influenza virus hemagglutinin protein can be obtained using conventional techniques known to those skilled in the art.
As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody is present in an amount less than about 10 -5 M, e.g. less than about 10 -6 M、10 -7 M、10 -8 M、10 -9 M or 10 -10 M or less affinity (K) D ) Binding the antigen.
As used herein, the term "K D "refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Can be determined using a variety of methods, e.g., using surface plasmonsDaughter resonance Spectroscopy (SPR) was measured in a BIACORE instrument.
Drawings
FIG. 1 shows the results of the binding of the recombinant chimeric antibody to the CEACAM5 recombinant protein.
FIG. 2 shows the binding of the recombinant chimeric antibody to the CEACAM5 domain.
FIG. 3 shows the binding of recombinant chimeric antibodies to LS174T cells and KATO3 cells highly expressing CEACAM 5.
Figure 4 shows the binding of humanized antibodies to recombinant CEACAM5 antigen.
Figure 5 shows the binding of humanized antibodies to LS174T cells highly expressing CEACAM 5.
Sequence information
The partial sequence information referred to in the present application is described in the following table, and the rest is described in the examples.
Figure BDA0003128428790000171
Figure BDA0003128428790000181
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and do not limit the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description of the preferred embodiments.
EXAMPLE 1 preparation of monoclonal antibodies
This example uses a CEACAM 5-expressing tumor cell line to immunize mice to prepare monoclonal antibodies.
1.SJL mouse immunization/hybridoma fusion
Lovo cell line ATCC CCL-229 highly expressing CEACAM5 was cultured in RPMI1640 medium containing 10% FBS, and Lovo cells were digested with TrypLE trypsin and then resuspended in DPBSIn solution, each SJL mouse is immunized in subcutaneous multiple points, and each immunization is 10 times 7 Lovo cells, immunized once a week for a total of 5 times. After the mice which are detected by serum titer are killed, spleen is taken, ground and sieved, and SP20 myeloma cells are fused according to a standard fusion process to obtain hybridoma cells.
Construction and screening of Lovo CEACAM5 KO cell line
2.1 Screening of Lovo CEACAM5 high expression monoclonal cell line
The sequence of the recombinant expression of the Lovo cells by using anti-CEACAM 5 antibody hMN14 (immunolodics, phase2 drug) is as follows
>hMN14 VH
EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
>hMN14 VL
<xnotran> DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) , Fc-PE , BD FACS Aria 96 , , hMN14 CEACAM5 , : </xnotran>
Figure BDA0003128428790000201
The results show that the purity of the clone No. 6-1 is higher than that of other clones, and reaches 98.6%, and the CEACAM5 expression MFI is obviously higher than that of other clones, so the clone No. 6-1 is selected for CEACAM5 knockout.
2.2 Lovo CEACAM5 KO cell line selection
The clone Lovo 6-1 was subjected to CEACAM5 gene knockout by CRISPR method, and Lovo CEACAM5 KO cell line (hereinafter referred to as Lovo CEA KO cell line) was selected. The vector carrying CRISPR and sgRNA was packaged as a lentiviral vector (CEACAM 5 KO 1-3), and transformed into Lovo 6-1 cells, after which CEACAM5 expression (MN 14 antibody) was detected by FACS, and MN14 binding was detected by a murine Fc-APC secondary antibody. The result shows that CEACAM5 KO1-3 can knock out CEACAM5, wherein the knockout efficiency of CEACAM5 KO3 carrier is higher, and the CEACAM5 negative population is more clear. Sequences targeting CEACAM5 three sgrnas are as follows:
No sgRNA
CEACAM5 KO1 GATCTGACTTTATGACGTGT
CEACAM5 KO2 GATGACTGAATCACTGCGCC
CEACAM5 KO3 CAGGGGATGCACCATCTGTG
3. hybridoma cloning and screening
Lovo cells and Lovo CEA KO cells were plated at 10 per well 4 After overnight incubation in 96-well plates, hybridoma supernatants 1-10 μ l were added to Lovo/Lovo CEA KO cell culture plates, incubated for 1 hour, the supernatants were discarded, anti-mouse Fc-FITC fluorescent secondary antibody was added, after incubation for 1 hour, the supernatants were discarded, a DPBS solution containing 2% BSA was added, and the fluorescent signals and the FITC staining area were read and analyzed in Celigo.
Two clones, M22 (26A 10) and M25 (28C 11), were selected.
Figure BDA0003128428790000211
The above results indicate that clones M22 (26 a 10) and M25 (28C 11) bind specifically to Lovo and not to Lovo CEA KO cells.
4. Hybridoma clone screening and chimeric antibody expression
The heavy and light chain variable regions (VH and VL) of the picked clones were obtained by sequencing the hybridomas of the picked clones according to standard hybridoma sequencing methods.
VH and VL were synthesized by total gene synthesis and the human IgG1 and kappa chain constant regions were ligated, the heavy and light chain sequences were ligated into pcDNA3.4 vector and transiently expressed in 293 system and protein A/G purified. The obtained chimeric recombinant antibody was ultrafiltered and buffer-replaced with a PBS solution. The sequencing results are shown in the table below.
Figure BDA0003128428790000221
Example 2 antigen binding ELISA assay
Diluting recombinant CEACAM5 antigen (Sinobiological, 11077-H08H) with DPBS solution to 1 μ g/ml, adding to 96-well plate according to 100 μ l per well, and coating overnight at 2-8 deg.C; the coating solution was discarded and washed 2 times with PBS solution, and 2% BSA in PBS solution was added and blocked at room temperature for 2 hours; removing the blocking solution, adding the antibody with the concentration gradient dilution, and incubating at 37 ℃ for 1 hour; discarding the antibody solution and washing 4 times with a PBS solution (PBST solution) containing 0.05% Tween 20; anti-human IgG Fc-HRP secondary antibody was added and incubated at 37 ℃ for 30 minutes; washing with PBST solution for 4 times, adding TMB chromogenic substrate, developing for 5-10 min, and adding 1 MH 2 Stopping the reaction by SO 4; the absorbance at 450nm was read on a microplate reader. The binding results for CEACAM5 recombinant protein are shown in fig. 1. The results show that: both chimeric antibodies cM22hIgG1 and cM25hIgG1 bound CEACAM5 recombinant protein.
Example 3 antibody Epitope binding assay
The CEACAM5 molecule is split according to the extracellular domain (A1-B1-A2-B2-A3-B3) thereof, expression vectors of A1-B1-His Tag, A2-B2-His Tag and A3-B3-His Tag are respectively constructed, and after expression in 293 system, the expression vectors are purified by using Ni column, and the sequence is shown in the following table:
Figure BDA0003128428790000231
Figure BDA0003128428790000241
diluting the CEACAM5 fragment with DPBS solution to 1 μ g/ml, adding to 96-well plate at a volume of 100 μ l per well, and coating at 2-8 deg.C overnight; the coating solution was discarded and washed 2 times with PBS solution, and 2% BSA in PBS solution was added and blocked at room temperature for 2 hours; removing the blocking solution, adding the antibody with the concentration gradient dilution, and incubating at 37 ℃ for 1 hour; discarding the antibody solution and washing 4 times with a PBS solution (PBST solution) containing 0.05% Tween 20; add anti-human IgG Fc-HRP secondary antibody and incubate for 30 min at 37 ℃; washing with PBST solution for 4 times, adding TMB chromogenic substrate, developing for 5-10 min, and performing equal volume of 1 MH 2 Stopping the reaction by SO 4; the absorbance at 450nm was read on a microplate reader. The results are shown in FIG. 2.
The results indicate that the M22 and M25 antibodies bind to the N-A1-B1 domain of CEACAM5 and not to the A2-B2 and A3-B3 domains.
Example 4 CEACAMM 5 Positive tumor cell binding assay
LS174T cells and KATO3 cells were cultured in 10-vol% FBS-containing RPMI1640 medium, the cells were digested with trypsin, and then centrifuged and resuspended in 2-vol% BSA-containing DPBS solution (FACS buffer, 4 ℃ C.) at 5X 10 5 Adding 100 mul/well into a U-shaped bottom 96-well plate, adding the antibody diluted by concentration gradient, incubating for 1 hour at 4 ℃, centrifuging and then discarding the supernatant; after adding 100. Mu.l of Fc-PE secondary antibody containing anti-human IgG to each well and incubating at 4 ℃ for 1 hour, the cells were washed 1 time with FACS buffer and resuspended in 200. Mu.l of FACS bufferIn solution, the fluorescence signal values were read in BD CantoII. The results are shown in FIG. 3. The results show that: both antibodies bound LS174T cells and KATO3 cells.
Example 5 antibody CEA family protein selectivity assay
The CEA family protein, following the following Table mRNA, was inserted into a lentiviral vector (puro-resistant) and packaged as a lentivirus. RKO cells (CEA family expression negative) were transduced with CEACAM1-8 lentiviruses and screened using puromycin to construct stably expressing CEACAM1-8 cell lines.
CEA family Gene ID mRNA sequences
CEACAM1 634 NM_001712.5
CEACAM3 1084 NM_001815.5
CEACAM5 1048 NM_004363.6
CEACAM6 4680 NM_002483.7
CEACAM8 1088 NM_001816.4
The cM22hIgG1 and cM25hIgG1 antibodies were added to the above cells, i.e., RKO-CEA1, RKO-CEA3, RKO-CEA5, RKO-CEA6, RKO-CEA8 at a final concentration of 5ug/ml for cell binding FACS determination, the percentage of positive binding of the antibodies to the corresponding cell lines was as follows:
Figure BDA0003128428790000251
the above results indicate that cM22hIgG1 and cM25hIgG1 highly specifically bind CEACAM5 and not other CEA proteins; while control hAb796hIgG1 binds to CEACAM5 but also to a different extent to CEA family proteins, such as CEACAM8.
Example 6 humanized antibody design and expression
Comparing the M22 antibody and M25 with an IMGT database, selecting a humanized Framework sequence with the highest VH/VL homology, carrying out CDR graft, and carrying out computational chemistry simulation to maintain the binding of the humanized antibody and the antigen, wherein the design of the humanized antibody is shown in the following table.
Figure BDA0003128428790000261
Figure BDA0003128428790000271
The VH and VL regions of the antibody are linked with the Fc region of human IgG1 and the kappa constant region, and the heavy chain and light chain sequences of the antibody are inserted into pcDNA3.4 vector, transiently expressed in 293 cells, and purified by protein A or G.
Example 7 humanized antibody ELISA binding assay
Diluting recombinant CEACAM5 antigen (Sinobiological, 11077-H08H) with DPBS solution to 1 μ g/ml, adding to 96-well plate according to 100 μ l per well, and coating overnight at 2-8 deg.C; coating liquidDiscarded and washed 2 times with PBS solution, added with 2% BSA in PBS solution, blocked for 2 hours at room temperature; removing the blocking solution, adding the humanized antibody diluted by concentration gradient, and incubating at 37 ℃ for 1 hour; discarding the antibody solution and washing 4 times with a PBS solution (PBST solution) containing 0.05% Tween 20; anti-human IgG Fc-HRP secondary antibody was added and incubated at 37 ℃ for 30 minutes; washing with PBST solution for 4 times, adding TMB chromogenic substrate, developing for 5-10 min, and adding 1 MH 2 Stopping the reaction by SO 4; the absorbance at 450nm was read on a microplate reader. The binding results of the above humanized antibodies to CEACAM5 recombinant protein are shown in fig. 4. The results show that: both humanized antibodies hM22.9hIgG1 and hM25.6hIgG1 can bind to CEACAM5-His antigen.
Example 7 cell binding assay for humanized antibodies
LS174T cells (CEACAM 5 high expression; ATCC, CL-188) were cultured in 10% FBS RPMI1640 medium, trypsinized, centrifuged, resuspended in 2% BSA in DPBS solution (FACS buffer, 4 ℃) at 5X 10 5 Adding 100 mul/well into a U-shaped bottom 96-well plate, adding the antibody diluted by concentration gradient, incubating for 1 hour at 4 ℃, centrifuging and then discarding the supernatant; after adding 100. Mu.l of a secondary antibody containing anti-human IgG Fc-APC to each well and incubating at 4 ℃ for 1 hour, the wells were washed 1 time with FACS buffer, resuspended in 200. Mu.l of FACS buffer, and the fluorescence signal values were read on BD CantoII. The results are shown in FIG. 5.
Example 8 CEACAMM 5-specific antibody endocytosis assay
hM22.9hIgG1, hM25.6hIgG1, hAb796hIgG1, hPR1A3hIgG1 and hIgG Ctrl are dye-labeled with pHAb amino-coupled dyes (labeled antibodies are endocytosed in a low pH environment, and excitation fluorescence is detected at 560nm at 532nm excitation light). Adding the antibody into MKN45 cells to a final concentration of 10 mu g/ml, culturing and incubating for 24 hours at 37 ℃, washing the cells, reading a fluorescence value of a PE channel, and detecting the endocytosis of the labeled antibody.
Antibodies Endocytosis% (PE +%)
hM22.9hIgG1 96.7％
hM25.6hIgG1 96.9％
hAb796hIgG1 95.9％
hPR1A3hIgG1 13.4％
hIgG Ctrl 1.3％
The above results indicate that the hM22.9hIgG1 and hM25.6hIgG1 antibodies can produce endocytosis on MKN45 tumor cells.
Sequence listing
<110> Ji-fold biotech GmbH in Shanghai
<120> antibody specifically binding to glycosylated CEACAM5
<130> IDC210182
<160> 28
<170> PatentIn version 3.5
<210> 1
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> M22 heavy chain variable region CDR1
<400> 1
Asp Tyr Tyr Met His
1 5
<210> 2
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> M22 heavy chain variable region CDR2
<400> 2
Tyr Ile Tyr Pro Asn Asn Gly Gly Asn Gly Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 3
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> M22 heavy chain variable region CDR3
<400> 3
Asp Gly Gly Leu Arg Arg Gly Phe Asp Tyr
1 5 10
<210> 4
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> M22 light chain variable region CDR1
<400> 4
Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Ala
1 5 10
<210> 5
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> M22 light chain variable region CDR2
<400> 5
Ala Ala Thr Ser Leu Ala Asp
1 5
<210> 6
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> M22 light chain variable region CDR3
<400> 6
Gln Gln Leu Tyr Ser Ala Pro Trp Thr
1 5
<210> 7
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> M25 heavy chain variable region CDR1
<400> 7
Ser Tyr Trp Met His
1 5
<210> 8
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> M25 heavy chain variable region CDR2
<400> 8
Arg Ile His Pro Ser Asp Ser Asp Thr Asn Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 9
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> M25 heavy chain variable region CDR3
<400> 9
Val Glu Trp Met Phe Asp Tyr
1 5
<210> 10
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> M25 light chain variable region CDR1
<400> 10
Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala
1 5 10
<210> 11
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> M25 light chain variable region CDR2
<400> 11
Ser Ala Ser Tyr Arg Tyr Ser
1 5
<210> 12
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> M25 light chain variable region CDR3
<400> 12
Gln Gln Tyr Tyr Ser Tyr Phe Thr
1 5
<210> 13
<211> 119
<212> PRT
<213> Artificial sequence
<220>
<223> M22 heavy chain variable region
<400> 13
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Asn Asn Gly Gly Asn Gly Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Thr Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gly Gly Leu Arg Arg Gly Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 14
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> M22 light chain variable region
<400> 14
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly
1 5 10 15
Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Phe Leu Ile
35 40 45
Tyr Ala Ala Thr Ser Leu Ala Asp Gly Ala Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala
65 70 75 80
Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Ser Ala Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 15
<211> 116
<212> PRT
<213> Artificial sequence
<220>
<223> M25 heavy chain variable region
<400> 15
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile His Pro Ser Asp Ser Asp Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Val Glu Trp Met Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 16
<211> 106
<212> PRT
<213> Artificial sequence
<220>
<223> M25 light chain variable region
<400> 16
Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Tyr Ser Tyr Phe Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 17
<211> 119
<212> PRT
<213> Artificial sequence
<220>
<223> hM22.9 heavy chain variable region
<400> 17
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Asn Asn Gly Gly Asn Gly Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gly Gly Leu Arg Arg Gly Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 18
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> hM22.9 light chain variable region
<400> 18
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile
35 40 45
Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Tyr Ser Ala Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 19
<211> 116
<212> PRT
<213> Artificial sequence
<220>
<223> hM25.6 heavy chain variable region
<400> 19
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile His Pro Ser Asp Ser Asp Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Val Glu Trp Met Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 20
<211> 106
<212> PRT
<213> Artificial sequence
<220>
<223> hM25.6 light chain variable region
<400> 20
Ala Ile Arg Met Thr Gln Ser Pro Ser Ser Phe Ser Ala Ser Thr Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Tyr Phe Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 21
<211> 292
<212> PRT
<213> Artificial sequence
<220>
<223> N-A1-B1 Domain
<400> 21
Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys Glu
1 5 10 15
Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly Tyr Ser
20 25 30
Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile Gly Tyr
35 40 45
Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly Arg
50 55 60
Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile Ile Gln
65 70 75 80
Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp Leu Val
85 90 95
Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu Pro Lys
100 105 110
Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys Asp Ala
115 120 125
Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr Leu Trp
130 135 140
Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser
145 150 155 160
Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn Asp Thr
165 170 175
Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg Arg Ser
180 185 190
Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro Thr Ile
195 200 205
Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn Leu Ser
210 215 220
Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe Val Asn
225 230 235 240
Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr
245 250 255
Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser Asp Thr
260 265 270
Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala His His
275 280 285
His His His His
290
<210> 22
<211> 184
<212> PRT
<213> Artificial sequence
<220>
<223> A2-B2 Domain
<400> 22
Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu
1 5 10 15
Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr
20 25 30
Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg
35 40 45
Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr
50 55 60
Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Glu Leu Ser
65 70 75 80
Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp
85 90 95
Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn
100 105 110
Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser
115 120 125
Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile
130 135 140
Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn
145 150 155 160
Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val
165 170 175
Ser Ala His His His His His His
180
<210> 23
<211> 193
<212> PRT
<213> Artificial sequence
<220>
<223> A3-B3 Domain
<400> 23
Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu
1 5 10 15
Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln Asn Thr
20 25 30
Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg
35 40 45
Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr
50 55 60
Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser Val Ser
65 70 75 80
Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly Pro Asp
85 90 95
Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn
100 105 110
Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln Tyr Ser
115 120 125
Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu Phe Ile
130 135 140
Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe Val Ser
145 150 155 160
Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile Thr Val
165 170 175
Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala His His His His His
180 185 190
His
<210> 24
<211> 449
<212> PRT
<213> Artificial sequence
<220>
<223> antibody hMN14 heavy chain variable region
<400> 24
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys
85 90 95
Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Pro Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 25
<211> 213
<212> PRT
<213> Artificial sequence
<220>
<223> antibody hMN14 light chain variable region
<400> 25
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> CEACAM5 KO1 sgRNA sequence
<400> 26
gatctgactt tatgacgtgt 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> CEACAM5 KO2 sgRNA sequence
<400> 27
gatgactgaa tcactgcgcc 20
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> CEACAM5 KO3 sgRNA sequence
<400> 28
caggggatgc accatctgtg 20

Claims (23)

1. A humanized antibody or antigen-binding fragment thereof that specifically binds CEACAM5, wherein said antibody comprises a heavy chain variable region and a light chain variable region, wherein:
(i) The heavy chain variable region comprises: 1, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) thereto; 2, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) thereto; and CDR-H3 shown in SEQ ID NO. 3, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) compared thereto; and the light chain variable region comprises CDR-L1 shown in SEQ ID NO. 4, or a sequence having substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1,2 or 3 amino acids) compared thereto; CDR-L2 of SEQ ID NO. 5, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) compared thereto; and CDR-L3 shown in SEQ ID NO. 6, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) compared thereto; or
(ii) The heavy chain variable region comprises: CDR-H1 of SEQ ID NO. 7, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) compared thereto; CDR-H2 of SEQ ID NO. 8, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) compared thereto; and CDR-H3 shown by SEQ ID NO. 9, or a sequence having substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1,2 or 3 amino acids) thereto; and the light chain variable region comprises CDR-L1 shown in SEQ ID NO. 10, or a sequence having substitution, deletion or addition of one or more amino acids (for example, substitution, deletion or addition of 1,2 or 3 amino acids) compared thereto; CDR-L2 of SEQ ID NO. 11, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1,2 or 3 amino acids) compared thereto; and CDR-L3 shown by SEQ ID NO. 12, or a sequence having substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1,2 or 3 amino acids) thereto,
preferably, the substitution of any one of SEQ ID NOs 1 to 12 is a conservative substitution; preferably, the CDRs of any of SEQ ID NOs 1 to 12 are defined according to the Kabat numbering system.
2. The humanized antibody or antigen-binding fragment thereof of claim 1, wherein:
a) The heavy chain variable region comprises the polypeptide shown in SEQ ID NO. 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) compared thereto, while the light chain variable region comprises the polypeptide shown in SEQ ID NO. 18, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) compared thereto; or
b) The heavy chain variable region comprises the polypeptide shown in SEQ ID NO:19, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) compared thereto, while the light chain variable region comprises the polypeptide shown in SEQ ID NO:20, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) compared thereto,
preferably, the substitution of any one of SEQ ID NO 17-20 is a conservative substitution;
preferably, the CDRs of any of SEQ ID NOs 17-20 are defined according to the Kabat numbering system.
3. The humanized antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a constant region derived from a human immunoglobulin or a variant thereof;
preferably, the antibody or antigen-binding fragment thereof comprises:
(a) A heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., substitutions, deletions or additions of 1,2, 3,4, or 5 amino acids or any combination thereof) compared to the sequence from which it is derived; and/or
(b) A light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., substitutions, deletions or additions of 1,2, 3,4, or 5 amino acids or any combination thereof) compared to the sequence from which it is derived;
preferably, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, igG2, igG3, or IgG4 heavy chain constant region;
preferably, the antibody or antigen-binding fragment thereof comprises the heavy chain variable region (VH) set forth in SEQ ID NO:17 or SEQ ID NO: 19;
preferably, the light chain constant region is a kappa light chain constant region;
preferably, the antibody or antigen-binding fragment thereof comprises the light chain variable region (VL) set forth in SEQ ID NO:18 or SEQ ID NO: 20.
4. The humanized antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', (Fab ') 2, fv, disulfide-linked Fv, bsFv, dsFv, (dsFv) 2, dsFv-dsFv ', scFv dimer, camelized single domain antibody (camelized single chain domain antibody), diabody (diabody), ds diabody (ds diabody), nanobody, single domain antibody (sdAb), diabody; and/or, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.
5. The humanized antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.
6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the humanized antibody or antigen-binding fragment thereof of any one of claims 1-5.
7. A vector comprising the nucleic acid molecule of claim 6.
8. A host cell comprising the vector of claim 7.
9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5 and a pharmaceutically acceptable carrier,
preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent;
preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, mitotic inhibitor, anti-tumor antibiotic, anti-metabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide, radiosensitizer, anti-angiogenic agent, cytokine, molecularly targeted drug, immune checkpoint inhibitor or oncolytic virus;
preferably, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate is provided as a separate component or as a component of the same composition with the additional pharmaceutically active agent.
10. A chimeric antigen receptor comprising the antigen binding domain of the humanized antibody or antigen binding fragment thereof of any one of claims 1-5;
preferably, the antigen binding domain comprises the heavy chain variable region and the light chain variable region of the humanized antibody or antigen binding fragment thereof of any one of claims 1-5;
preferably, the antigen binding domain is a scFv;
preferably, the antigen binding receptor comprises the humanized antibody or antigen binding fragment thereof of any one of claims 1-5;
preferably, the antigen binding receptor is expressed by an immune effector cell (e.g., a T cell).
11. An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 10.
12. A vector comprising the isolated nucleic acid molecule of claim 11; preferably, it is used for the preparation of chimeric antigen receptor T cells.
13. A host cell comprising the isolated nucleic acid molecule of claim 11 or the vector of claim 12;
preferably, the host cell is an immune effector cell (e.g., a T cell or NK cell);
preferably, the host cell is a chimeric antigen receptor T cell (CAR-T).
14. A method for reducing the expression level of CEACAM5 on the surface of a cell comprising contacting said cell with the humanized antibody or antigen-binding fragment thereof of any one of claims 1-5, or the pharmaceutical composition of claim 9, or the chimeric antigen receptor of claim 10, or the host cell of claim 13, such that the expression level of CEACAM5 on the surface of said cell is reduced; wherein the cell expresses CEACAM5 on its surface;
preferably, the cell is a CEACAM5 expressing tumor cell.
15. A method of inhibiting growth and/or killing a CEACAM 5-expressing tumor cell comprising contacting the tumor cell with an effective amount of the humanized antibody or antigen-binding fragment thereof of any one of claims 1-5, or the pharmaceutical composition of claim 9, or the chimeric antigen receptor of claim 10, or the host cell of claim 13.
16. A method for preparing an antibody specifically binding CEACAM5 comprising expressing the nucleic acid molecule of claim 11 in a host cell and isolating the antibody specifically binding CEACAM5 from the host cell.
17. Use of an antibody of any one of claims 1-2 in the manufacture of a medicament for the treatment of a cancer highly expressing CEACAM 5.
18. The use of claim 17, wherein the CEACAM 5-highly expressing cancer is selected from colorectal, gastric, pancreatic, esophageal, lung, or breast cancer.
19. A method for treating a CEACAM 5-highly expressing cancer comprising administering to a subject in need thereof the antibody of any one of claims 1-2 or the pharmaceutical composition of claim 6, wherein the CEACAM 5-highly expressing cancer is selected from colorectal, gastric, pancreatic, esophageal, lung or breast cancer,
preferably, the subject is a mammal, such as a human;
preferably, the method further comprises administering an additional agent with anti-tumor activity, such as an alkylating agent, mitotic inhibitor, anti-tumor antibiotic, anti-metabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide, radiosensitizer, antiangiogenic agent, cytokine, molecularly targeted drug, immune checkpoint inhibitor, or oncolytic virus;
preferably, the method further comprises administering an additional anti-tumor therapy, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy or palliative therapy.
20. An antibody-drug conjugate, comprising:
(a) The antibody of claim 1 or 2; and
(b) A drug conjugated to the antibody, wherein the drug is selected from the group consisting of,
preferably, the drug is a toxin, preferably monomethyl auristatin (monomethylauristatin), calicheamicin, maytansinoids, or combinations thereof; more preferably selected from: monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), or combinations thereof.
21. Use of an antibody drug conjugate according to claim 20 in the manufacture of a medicament.
22. A bispecific antibody comprising a domain that specifically binds CEACAM5 and a domain that specifically binds CD3, CD28 or 41BB,
wherein the domain that specifically binds CEACAM5 comprises: CDR-H1 shown in SEQ ID NO. 1; CDR-H2 shown in SEQ ID NO. 2, CDR-H3 shown in SEQ ID NO. 3, CDR-L1 shown in SEQ ID NO. 4, CDR-L2 shown in SEQ ID NO. 5, and CDR-L3 shown in SEQ ID NO. 6; or
CDR-H1 shown in SEQ ID NO. 7, CDR-H2 shown in SEQ ID NO. 8, CDR-H3 shown in SEQ ID NO. 9, CDR-L1 shown in SEQ ID NO. 10, CDR-L2 shown in SEQ ID NO. 11, and CDR-L3 shown in SEQ ID NO. 12.
23. Use of a bispecific antibody of claim 22 in the manufacture of a medicament.
CN202110697514.7A 2021-06-23 2021-06-23 Antibodies specifically binding glycosylated CEACAM5 Pending CN115505043A (en)

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CN116178556A (en) * 2023-01-09 2023-05-30 南方医科大学第三附属医院（广东省骨科研究院） CEACAM 5-targeted nano antibody and preparation method and application thereof

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WO2024165403A1 (en) * 2023-02-06 2024-08-15 Philogen S.P.A. Anti-cea antibodies

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BR112021024544A2 (en) * 2019-06-04 2022-02-08 Biotheus Inc Anti-ceacam5 monoclonal antibody and method of preparation and use of same

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116178556A (en) * 2023-01-09 2023-05-30 南方医科大学第三附属医院（广东省骨科研究院） CEACAM 5-targeted nano antibody and preparation method and application thereof
CN116178556B (en) * 2023-01-09 2024-01-09 南方医科大学第三附属医院（广东省骨科研究院） CEACAM 5-targeted nano antibody and preparation method and application thereof

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