Patent Application
20230331862
The present invention relates to a monoclonal antibody. More specifically, the present application relates to a monoclonal antibody specifically binding to a glycosylated CEACAM5, preparation of a humanized antibody thereof, and use thereof.
In China, the gastrointestinal-related tumors including colorectal cancer, gastric cancer, and esophageal cancer are common, and their new case numbers are 520,000, 460,000, and 310,000 each year (WHO, 2018). Therefore, the gastrointestinal-related tumors have surpassed lung cancer and become the most common cancer in China. At present, the treatment methods for gastrointestinal tumors mainly include surgery, chemotherapy, targeted therapy and immunotherapy. Commonly used chemotherapy drugs include docetaxel, 5-fluorouracil, mitomycin C, platinum agents, etc.; targeted therapy drugs include VEGFR monoclonal antibody, Her2 monoclonal antibody, etc.; immunotherapy mainly includes PD1/PDL1 antibodies, etc.
The human carcinoembryonic antigen cell adhesion factor (CEACAM) family was discovered in the 1960s. The CEA family is highly expressed in a variety of gastrointestinal and lung cancer tumors, such as colorectal cancer, pancreatic cancer, lung cancer, gastric cancer, hepatocellular carcinoma, breast cancer, etc. CEACAM family consists of CEACAM subgroup and PSG family, and is characterized by IgV domains in series in the extracellular region which form a highly similar structure and are highly glycosylated (glycosylation moiety accounts for 50% of the molecular weight), and its extracellular region consists of A1-B1-A2-B2-A3-B3 structure in series (A1-3 or B1-3 is a highly homologous structure); the common CEA molecule is CEACAM5 (CD66e), which is coupled to the cell membrane through glycosylphosphatidylinositol (GPI), and can be released into the blood through enzymatic degradation of GPI (e.g., phospholipase C); CEACAM5 molecules are involved in cell adhesion (via a CEA family homo- or hetero-dimer, such as CEACAM6), intracellular signaling, tumor metastasis and generation of drug-resistance; at the same time, CEACAM5 is related to the adhesion of Escherichia coli in the gastrointestinal tract.
CEACAM5 is a highly glycosylated protein antigen, so the recombinantly expressed CEACAM5 (e.g., 293 system) antigen may have glycosylation different from the CEACAM5 protein expressed by tumor cells itself, so an antibody that can recognize the natural CEACAM5 antigen is needed in the art. In addition, CEACAM5 is expressed in a small amount in normal tissues such as the digestive tract, which is located on the apical surface of the digestive tract; in tumor cells, CEACAM5 is expressed on the apical and basolateral surfaces, and it is difficult for CEACAM5 antibody drugs to reach the tumor site. Therefore, it is needed in the art to address the problem of increasing the local concentration of CEACAM5 antibody drug.
One aspect of the present invention provides a monoclonal antibody or antigen-binding fragment thereof against a glycosylated CEACAM5, and the antibody or antigen-binding fragment thereof specifically binds to the domains A1-B1, A2-B2, and/or A3-B3 of the glycosylated CEACAM5.
In specific embodiments, the monoclonal antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:
In a specific embodiment, the monoclonal antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:
In one aspect, the present invention provides a humanized antibody or antigen-binding fragment thereof specifically binding to glycosylated CEACAM5, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein:
In a specific embodiment, in the humanized antibody of the present invention, the light chain variable region comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 68, 70, 72, 74, 76, 78, 80, 82, and the heavy chain variable region comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 69, 71, 73, 75, 77, 79, 81, 83.
In a preferred technical solution, the humanized antibody of the present invention comprises: a light chain variable region encoded by a nucleotide sequence selected from SEQ ID NOs: 52, 54, 56, 58, 60, 62, 64, 66, and a heavy chain variable region encoded by a nucleotide sequence selected from SEQ ID NOs: 53, 55, 57, 59, 61, 63, 65, 67.
In a preferred technical solution, the humanized antibody of the present invention comprises a light chain variable region selected from SEQ ID NOs: 68, 70, 72, 74, 76, 78, 80, 82 and a heavy chain variable region selected from SEQ ID NOs: 69, 71, 73, 75, 77, 79, 81, 83.
In a preferred technical solution, the humanized antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein:
In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof specifically binding to glycosylated CEACAM5.
In another aspect, the present invention relates to an expression vector comprising a nucleic acid molecule encoding the antibody specifically binding to glycosylated CEACAM5 as disclosed herein.
In another aspect, the present invention relates to a host cell comprising the expression vectors as disclosed herein.
In another aspect, the present invention relates to a pharmaceutical composition comprising at least one antibody specifically binding to glycosylated CEACAMS as disclosed herein and a pharmaceutically acceptable carrier.
In another aspect, the present invention relates to a method for preparing an antibody specifically binding to glycosylated CEACAM5, which comprises: expressing in a host cell a nucleic acid sequence encoding the antibody specifically binding to glycosylated CEACAM5 as disclosed herein, and separating the antibody specifically binding glycosylated CEACAM5 from the host cell.
In another aspect, the present invention provides a use of the antibody of the present invention in the manufacture of a medicament for treating a gastrointestinal tract-related tumor.
In another aspect, the present invention provides a method for treating a gastrointestinal tract-related tumor, comprising administering to a subject in need thereof the antibody of the present invention.
Monoclonal antibodies can be prepared as follows. Firstly, mice or other suitable host animals are immunized with an immunogen (added with an adjuvant if necessary). The immunogen or adjuvant is usually injected by subcutaneous multi-point injection or intraperitoneal injection. The immunogen can be pre-coupled to a certain known protein, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host. The adjuvant can be Freund's adjuvant or MPL-TDM, etc. After the animal is immunized, lymphocytes that secrete antibodies specifically binding to the immunogen will be produced in the body. In addition, lymphocytes can also be obtained by in vitro immunization. The target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent such as PEG to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). The preferred myeloma cells should have the characteristics of high fusion rate, stable antibody secretion ability, and sensitivity to HAT medium. The culture medium for growing hybridoma cells is used to detect the generation of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assay, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). For example, the affinity of monoclonal antibodies can be determined using the Scatchard assay described by Munson et al., Anal. Biochem. 107:220 (1980). After determining the specificity, affinity and reactivity of the antibody produced by the hybridoma, the target cell line can be subjected to subcloning by the standard limited dilution assay described by Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996. Suitable culture medium can be DMEM or RPMI-1640, etc. In addition, hybridoma cells can also be grown in animals in the form of ascitic tumors. Monoclonal antibodies secreted by subclonal cells can be isolated from cell culture, ascites, or serum using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, etc.
Monoclonal antibodies can also be obtained through genetic engineering and recombination techniques. The DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from hybridoma cells by using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody to carry out PCR amplification. The resulting DNA molecule is inserted into an expression vector, then transfected into a host cell (e.g., E. coli cell, COS cell, CHO cell, or other myeloma cell that does not produce immunoglobulin), and cultured under appropriate conditions, to obtain the antibody of interest as recombinantly expressed.
In the present invention, a tumor cell line (e.g., Lovo) with high expression of CEACAM5 is used to immunize mice to obtain antibodies that recognize natural CEACAM5 antigen.
Through CRISPR technology, sgRNA-guided Cas9 is used to cut the target genome, efficiently knock out gene expression, and construct a CEACAM5 knockout cell line for efficient screening of specific CEACAM5 antibodies. The antibody screened and obtained by the present invention binds to the three structural domains (A1-B1, A2-B2, A3-B3) at of CEACAM5 the same time, so it can increase the surface binding amount of tumor cells with high CEACAM5 expression, thereby increasing its local concentration and drug efficacy.
The humanization design and screening of the screened murine antibodies may reduce the HAMA (Human Anti Mouse Antibody) effect in clinical applications, thereby reducing the production of neutralizing antibodies in patients, and increasing the blood concentration of the drug to improve the efficacy.
A “humanized antibody” is an antibody comprising one or both of a humanized VH domain and a humanized VL domain. The one or more immunoglobulin constant regions need not be present, but if present, they are derived entirely or substantially from human immunoglobulin constant regions.
Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human “donor” antibody are grafted into human “recipient” antibody sequences (see, for example, Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No. 6,881,557). The recipient antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequence, a consensus sequence or germline sequence of human antibody sequence. The human recipient sequence can be chosen such that the variable region framework has a high degree of sequence identity to the donor sequence to match typical patterns and other criteria between the recipient and donor CDRs. Thus, a humanized antibody is an antibody in which CDRs are derived entirely or substantially from the donor antibody and variable region framework sequences, and constant regions (if present) are derived entirely or substantially from human antibody sequences. Similarly, a humanized heavy chain typically has all three CDRs derived substantially from donor antibody heavy chain and heavy chain variable region framework sequences, and heavy chain constant regions (if present) derived substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain typically has all three CDRs derived entirely or substantially from donor antibody light chain and light chain variable region framework sequences, and light chain constant regions (if present) derived substantially from human light chain variable region framework and constant region sequences. 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 system) are identical between the respective CDRs, or about 100% of the corresponding residues (as defined by Kabat numbering system) are identical, the CDRs in a humanized antibody are substantially derived from the corresponding CDRs 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 (variable regions are defined by Kabat numbering system and constant regions are defined by EU numbering system) are identical, or about 100% of the corresponding residues (variable regions are defined by Kabat numbering system and constant regions are defined by EU numbering system) are identical, the variable region framework sequences of the antibody chains or the constant regions of the antibody chains, respectively, are substantially derived from human variable region framework sequences or human constant regions.
While humanized antibodies often incorporate all six CDRs (preferably as defined by Kabat or IMGT) from a mouse antibody, the humanized antibodies can also be composed of fewer than all six CDRs (e.g., at least 3, 4 or 5 CDRs) from the mouse antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immuno1.36 :1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).
A CDR in a humanized antibody is “substantially derived from” the 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 by Kabat or IMGT) are identical between respective CDRs. In specific changes in which the CDRs are substantially derived from a humanized VH or VL domain of a non-human immunoglobulin, the CDRs of the humanized VH or VL domain span all three CDRs with respect to the corresponding non-human VH or VL CDRs, and have no more than six (e.g., no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1) amino acid substitutions (preferably conservative substitutions). 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 are defined by Kabat numbering system and constant regions are defined by EU numbering system) are identical, or about 100% of the corresponding residues (variable regions are defined by Kabat numbering system and constant regions are defined by EU numbering system) are identical, variable region framework sequences of antibody VH or VL domains or immunoglobulin constant region sequences (if present) are “substantially derived from” human VH or VL framework sequences or human constant regions, respectively. Thus, all portions of a humanized antibody (except CDRs) will usually be derived entirely or substantially from the corresponding portions of native human immunoglobulin sequences.
In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the laboratory operation steps of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all routine steps widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.
As used herein, the term “antibody” refers to an immunoglobulin molecule, usually composed of two pairs of polypeptide chains (each pair having a light chain and a heavy chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ϵ, and the antibody isotypes are respectively defined as IgM, IgD, IgG, IgA, and IgE. Within the light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, while the heavy chain also comprises a “D” region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain is composed 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 regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (Clq) of classical complement system. The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminal to carboxy-terminal. The variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site, respectively. Assignment of amino acids to regions or domains follows the definitions of the 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 to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be of a different isotype, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.
Herein, unless the context clearly dictates otherwise, when the term “antibody” is referred to, it includes not only entire antibody but also antigen-binding fragment of the antibody. As used herein, the term “antigen-binding fragment” of antibody refers to a polypeptide comprising a fragment of a full-length antibody, which 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 specifically binding to antigen, which is also referred to as an “antigen-binding moiety”. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibody can be obtained by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibody. In some cases, the antigen-binding fragments include Fab, Fab′, F(ab′)2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single chain antibody (e.g., scFv), chimeric antibody, diabody, and polypeptide comprising at least a portion of antibody sufficient to confer to the polypeptide the specific antigen-binding ability.
Antigen-binding fragments of antibody (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and can be used to screen antigen-binding fragment of antibody for specificity in the same manner as the intact antibody.
As used herein, the terms “mAb” and “monoclonal antibody” refer to an antibody or a fragment of antibody derived from a population of highly homogeneous antibody molecules, that is, a group of identical antibody molecules excluding natural mutations that may occur spontaneously. The mAbs are highly specific for a single epitope on an antigen. Compared with monoclonal antibodies, polyclonal antibodies usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on antigens. Monoclonal antibodies can usually be obtained by hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained by recombinant DNA technology (see, for example, U.S. Pat. No. 4,816,567).
For example, monoclonal antibodies can be prepared as follows. Mice or other suitable host animals are first immunized in injection manner with immunogen (added with an adjuvant if necessary). The immunogen or adjuvant is usually injected subcutaneously at multiple points or intraperitoneally. The immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host. The adjuvant can be Freund's adjuvant or MPL-TDM, etc. After the animal is immunized, lymphocytes that secrete antibodies that specifically bind to the immunogen will be produced in the body. In addition, lymphocytes can also be obtained by in vitro immunization. The lymphocytes of interest are collected and fused with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103, Academic Press, 1996). The hybridoma cells prepared above can be inoculated to grow in a suitable culture medium, which preferably contains one or more substances capable of inhibiting the growth of unfused, parental myeloma cells. For example, for parental myeloma cells lacking hypoxanthine guanine phosphotransferase (HGPRT or HPRT), adding substances such as hypoxanthine, aminopterin, and thymine (HAT medium) to the culture medium will inhibit the growth of HGPRT-defective cells. The preferred myeloma cells should have the characteristics of high fusion rate, stable antibody secretion ability, and sensitivity to HAT medium. Among them, the first choice for myeloma cells is murine myeloma, such as MOP-21 or MC-11 mouse tumor derivative strain (THE Salk Institute Cell Distribution Center, San Diego, Calif USA), and SP-2/0 or X63-Ag8-653 cell line (American Type Culture Collection, Rockville, Md. USA). In addition, there are also research reports, in which human myeloma and human mouse heteromyeloma cell lines are used to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp 51-63, Marcel Dekker, Inc., New York, 1987). The culture medium for growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assay, such as radioimmunoassay (MA), enzyme-linked immunosorbent assay (ELISA). For example, the affinity of mAbs can be determined using the Scatchard assay described by Munson et al., Anal. Biochem. 107:220 (1980). After determining the specificity, affinity and reactivity of the antibody produced by the hybridoma, the target cell line can be subjected to subcloning by the standard limited dilution assay described by Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996. A suitable culture medium can be DMEM or RPMI-1640, etc. In addition, hybridoma cells can also be grown in animals in the form of ascitic tumors. Monoclonal antibodies secreted by subcloned cells can be purified from cell culture fluid, ascites or serum by using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
Monoclonal antibodies can also be obtained through genetic engineering and recombination techniques. The DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from hybridoma cells by using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody to carry out PCR amplification. The resulting DNA molecule is inserted into an expression vector, then transfected into a host cell (e.g., E. coli cell, COS cell, CHO cell, or other myeloma cell that does not produce immunoglobulin), and cultured under appropriate conditions, to obtain the recombinantly expressed antibody of interest.
As used herein, the term “chimeric antibody” refers to an antibody whose light chain and/or heavy chain are partially derived from one antibody (which may be derived from a specific species or belong to a specific antibody class or subclass), and the other part 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 in any case, it still retains the binding activity to the target antigen (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
As used herein, the term “human antibody” refers to an antibody or antibody fragment obtained by replacing all or part of the CDR regions of a humanized antibody or human immunoglobulin (recipient antibody) by the CDR regions of a non-human antibody (donor antibody), in which the donor antibody may be a non-human (for example, mouse, rat or rabbit) antibody with desired specificity, affinity or reactivity. In addition, some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, so as to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, Jones et al., Nature, 321:522 525 (1986); Reichmann et al., Nature, 332:323 329 (1988); Presta, Curr. Op. Struct. Biol., 2:593 596 (1992); and Clark, Immunol. Today 21:397 402 (2000).
As used herein, the term “epitope” refers to a site on an antigen that is specifically bound by an immunoglobulin or an antibody. 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 comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation, which may be “linear” or “conformational”. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). In a linear epitope, all points of interaction between a protein and an interacting molecule (e.g., antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, 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 segment on an antigen that can serve as an epitope. In some cases, an epitope peptide alone is capable of being specifically recognized/bound by an antibody directed against the epitope. In other cases, it may be necessary 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 insert an epitope peptide at a specific position (e.g., internal, N-terminal or C-terminal of protein), so that the epitope peptide can be presented, so that the epitope peptide can be recognized by the antibody or the immune system. Such carrier proteins are well known to those skilled in the art and include, for example, HPV L1 protein (epitope peptide may be inserted between amino acids 130-131 or between amino acids 426-427 of the protein, see: Slupetzky, K. et al Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops[J]. J Gen Viro1,2001, 82:2799-2804; Varsani, A. et al Chimeric human papillomavirus type 16 (HPV-16) L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and HPV-16[J]. J Virol, 2003, 77:8386-8393), HBV core antigen (epitope peptide may be used to replace the amino acids 79-81 of the protein, see: Koletzki, D., et al. HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein[J]. Biol Chem,1999, 380:325-333), woodchuck hepatitis virus core protein (epitope peptide can be used to replace the amino acids 79-81 of the protein, see: Sabine Konig, Gertrud Beterams and Michael Nassal, J. Virol. 1998, 72(6):4997), CRM197 protein (epitope peptide can be linked to the N-terminus or C-terminus of the protein or fragment thereof). Optionally, a linker (e.g., flexible or rigid linker) can be used between the epitope peptide and the carrier protein to facilitate the folding thereof.
Antibodies can be competitively screened for binding to the same epitope using routine techniques known to those of skill 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., influenza virus hemagglutinin protein). A high-throughput method for obtaining antibodies binding to the same epitope based on their cross-competition is described in the International Patent Application WO03/48731. Thus, antibodies and antigen-binding fragments thereof (i.e., antigen-binding portions) that compete with the monoclonal antibodies of the present invention for binding to the same epitope on the influenza virus hemagglutinin protein can be obtained using routine 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 the reaction between an antibody and an antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody having specificity to an antigen) refers to an antibody that binds to the antigen with an affinity (KD) of less than about 10−5 M, such as less than about 10−6 M, 10−7 M, 10−8 M, 10−9 M or 10−10 M or less.
As used herein, the term “KD” 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 tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. It can be determined using various methods, for example in a BIACORE instrument using surface plasmon resonance (SPR).
As used herein, the terms “hybridoma” and “hybridoma cell line” are used interchangeably, and when the terms “hybridoma” and “hybridoma cell line” are referred to, they also include subclones and progeny cells of hybridomas. For example, when referring to hybridoma cell line 2F4, it also refers to subclones and progeny cells of hybridoma cell line 2F4.
Part of the sequence information involved in the present application is described in the table below, and the rest are described in the examples.
The embodiments of the present application will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present application, rather than limiting the scope of the present application. Various objects and advantages of the present application will become apparent to those skilled in the art from the following detailed description of the preferred embodiments.
In this example, a tumor cell line expressing CEACAM5 was used to immunize mice to prepare monoclonal antibodies.
The Lovo cell line ATCC CCL-229, which highly expressed CEACAM5, was cultured in RPMI1640 medium containing 10% FBS. After the Lovo cells were digested with TrypLE trypsin, they were resuspended in DPBS solution, and each SJL mouse was subcutaneously immunized at multiple points, 107 Lovo cells were immunized per time, once a week, 5 times in total. After the mice tested for serum titer were sacrificed, the spleen was taken, ground and sieved, and SP20 myeloma cells were fused according to the standard fusion procedure to obtain hybridoma cells.
The Lovo cells were labeled with anti-CEACAM5 antibody hMN14 (Immunomedics, Phase 2 drug), expressed through recombination, and the sequence was as follows
After labeling, anti-mouse Fc-PE fluorescence-labeled secondary antibody was added, sorted into 96-well plate by BD FACS Aria, after monoclonal culture was carried out, MN14 was used to detect the expression of monoclonal CEACAM5, and the results were as follows:
The above results showed that clone 6-1 had a higher purity than other clones, reaching 98.6%, and its CEACAM5 expression MFI was significantly higher than other clones, so that clone 6-1 was selected for CEACAM5 knockout.
The Lovo 6-1 clone was subjected to CEACAM5 gene knockout by the CRISPR method, and the Lovo CEACAM5 KO cell line (hereinafter referred to as the Lovo CEA KO cell line) was screened. The vector carrying CRISPR and sgRNA was packaged into a lentiviral vector (CEACAM5 KO1-3) and transduced into Lovo 6-1 cells. After transduction, CEACAM5 expression (MN14 antibody) was detected by FACS, and MN14 binding was detected by mouse Fc-APC secondary antibody. The results were shown in
The sequences of the three sgRNAs targeting CEACAM5 were as follows:
As shown in
Lovo cells and Lovo CEA KO cells were inoculated in 96-well plates, 104 per well, and cultured overnight, 1-10 μl of hybridoma supernatant was added to Lovo/Lovo CEA KO cell culture plates, incubated for 1 hour, the supernatant was discarded, anti-mouse Fc-FITC fluorescent secondary antibody was added, incubated for 1 hour, the supernatant was discarded, DPBS solution containing 2% BSA was added, the fluorescent signals and FITC staining areas were read and analyzed in Celigo.
Four clones of M19 (2F4), M7 (11B6), M17 (6A8) and M18 (7G1) were obtained by screening.
Four clones of M19 (2F4), M7 (11B6), M17 (6A8) and M18 (7G1) were obtained by screening. The selected hybridoma clones were sequenced according to the standard hybridoma sequencing method to obtain the heavy chain and light chain variable regions (VH and VL) of the selected clones. The VH and VL were synthesized through whole gene synthesis and ligated to human IgG1 and kappa chain constant regions, and the heavy chain and light chain sequences were ligated to pcDNA3.4 vector, subjected to transient expression in the 293 system and purified by protein A/G. The obtained chimeric recombinant antibody was subj ected to ultrafiltration for buffer replacement with PBS solution. The sequencing results were shown in the table below.
IHWYQQKPGKSPKPWIHGTSNLASG
YFMNWVRQAPGKALEWLGQMRNKVN
GDTTEYAESVEGRFTISRDISKNSL
YFDYWGQGTLVTVSS
TSSVSYMHWFQQKPGTSPKLWIYST
SNLASGVPARFSGSGSGTSYSLTIS
IDPENGDTEYASKFQGKATITADTS
YVNPHYYAMDYWGQGTSVTVSS
SSSVSYMHWFQQKPGTSPKLWIYTT
STLASGVPARFSGSGSGTSYFLTIS
IDPENGDTEYASKFQGKATITADTS
YYGSRGAMDYWGQGTSVTVSS
SQDINKFMAWYQHKPGKGPRLLIRY
TSTLQPGIPSRFSGSGSGRDYSFSI
The recombinant CEACAM5 antigen (Sinobiological, 11077-H08H) was diluted to 1 μg/ml with DPBS solution, added to a 96-well plate, 100 μl per well, and coated overnight at 2-8° C.; the coating solution was discarded, washing was performed twice with PBS solution, PBS solution containing 2% BSA was added, and blocked at room temperature for 2 hours; the blocking solution was discarded, the antibody diluted in concentration gradient was added, and incubated at 37° C. for 1 hour; the antibody solution was discarded, and washing was performed for 4 times with with PBS solution containing 0.05% Tween 20 (PBST solution); anti-human IgG Fc-HRP secondary antibody was added and incubated at 37° C. for 30 minutes; washing was performed for 4 times with PBST solution, TMB chromogenic substrate was added, color development was performed for 5-10 minutes, and the reaction was terminated with equal volume of 1M H2SO4; the absorbance at 450 nm was read on a microplate reader. The binding results of the above four antibodies M7, M17, M18, and M19 to CEACAM5 recombinant protein were shown in
The CEACAM5 molecule was split according to its extracellular domains (A1-B1-A2-B2-A3-B3), A1-B1-His Tag, A2-B2-His Tag, A3-B3-His Tag expression vectors were constructed, and purified using Ni column after being expressed in the 293 system, and their sequences were shown in the table below:
ATGCACAGCTCAGCACTGCTCTGTTGCCTG
GTCCTCCTGACTGGGGTGAGGGCCAAGCTC
ATGCACAGCTCAGCACTGCTCTGTTGCCTG
GTCCTCCTGACTGGGGTGAGGGCCGAGCCA
ATGCACAGCTCAGCACTGCTCTGTTGCCTG
GTCCTCCTGACTGGGGTGAGGGCCGAGCTG
The above CEACAM5 fragment was diluted to 1 μg/ml with DPBS solution, added to a 96-well plate, 100 μl per well, coated overnight at 2-8° C.; the coating solution was discarded, washing was performed twice with PBS solution, PBS solution containing 2% BSA was added, blocking was performed at room temperature for 2 hours; the blocking solution was discarded, antibody diluted in concentration gradient was added, and incubated at 37° C. for 1 hour; the antibody solution was discarded, and washing was performed for 4 times with PBS solution containing 0.05% Tween 20 (PBST solution); anti-human IgG Fc-HRP secondary antibody was added and incubated at 37° C. for 30 minutes; washing was performed for 4 times with PBST solution, TMB chromogenic substrate was added, color development was performed for 5-10 minutes, then the reaction was stopped with an equal volume of 1M H2SO4; the absorbance at 450 nm was read on a microplate reader. The results were shown in
The epitopes of the above four antibodies M7, M17, M18, and M19 binding to CEACAM5 molecule were as follows:
The above results showed that the M19 antibody could recognize and bind to all three CEACAMS domains, in which the domain binding EC50 to A2-B2 was the smallest (EC50 =0.003 μg/ml), which was much smaller than those of A1-B1 (EC50=0.95 μg/ml) and A3-B3 (EC50=3.23 μg/ml) domain binding; this indicated that the main binding position of the M19 antibody was located in the A2-B2 domain of CEACAM5 molecule, but it could also bind to the A1-B1 and A3-B3 domains, and possibly bind to B1-A2 and B2-A3 domains.
LS174T cells and KATO3 cells (high expression of CEACAMS; ATCC, CL-188) were cultured in RPMI1640 medium containing 10% FBS. After the cells were digested with TrypLE, they were centrifuged and resuspended in DPBS solution containing 2% BSA (FACS buffer solution, 4° C.), and added into a U-bottom 96-well plate, 5×105/100 μl/well, and antibody diluted in concentration gradient was added, incubated at 4° C. for 1 hour, the supernatant was discarded after centrifugation; 100 μl of solution containing anti-human IgG Fc-APC secondary antibody was added and incubated at 4° C. for 1 hour, washing was performed once with FACS buffer, resuspending was performed in 200 μl FACS buffer, and the fluorescence signal value was read on BD Cantoll. The results were shown in
Comparing the M19 antibody (m2F4) with the IMGT database, the human framework sequence with the highest homology to its VH/VL was selected, and subjected to CDR grafting, and computational chemical simulation was performed to maintain its binding to the antigen. The design of humanized antibody was shown in the following table.
KGIAYYFDYWGQGTLVTVSS
KGIAYYFDYWGQGTLVTVSS
KGIAYYFDYWGQGTLVTVSS
KGIAYYFDYWGQGTLVTVSS
KGIAYYFDYWGQGTLVTVSS
KGIAYYFDYWGQGTLVTVSS
KGIAYYFDYWGQGTLVTVSS
KGIAYYFDYWGQGTLVTVSS
KGIAYYFDYWGQGTLVTVSS
The VH and VL regions of the above antibodies were linked to the human IgG1 Fc region and the kappa constant region, and the heavy chain and light chain sequences of the antibody were inserted into the pcDNA3.4 vector, transiently expressed in 293 cells, and purified by protein A or G.
Recombinant CEACAM5 antigen (Sinobiological, 11077-H08H) was diluted to 1 μg/ml with DPB S solution, add into a 96-well plate, 100 μl per well, and coated overnight at 2-8° C.; the coating solution was discarded, washing was performed twice with PBS solution, PBS solution containing 2% BSA was added, and blocking was performed at room temperature for 2 hours; the blocking solution was discarded, the humanized antibody diluted in concentration gradient was added, and incubated at 37° C. for 1 hour; the antibody solution was discarded, and washing was performed for 4 times with PBS solution containing 0.05% Tween 20 (PBST solution); the anti-human IgG Fc-HRP secondary antibody was added and incubated at 37° C. for 30 minutes; washing was performed for 4 times with PBST solution, TMB chromogenic substrate was added, and color development was carried out for 5-10 minutes, then the reaction was terminated by adding equal volume of 1M H2SO4; the absorbance at 450 nm was read on a microplate reader. The binding results of the above humanized antibodies to CEACAM5 recombinant protein were shown in
KATO3 cells (high expression of CEACAM5) were cultured in RPMI1640 medium containing 10% FBS. After the cells were digested with TrypLE trypsin, they were centrifuged and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4° C.), and added to a U-bottom 96-well plate, 5×105/100 μl/well, the antibody diluted in concentration gradient was added, incubated at 4° C. for 1 hour, the supernatant was discarded after centrifugation; 100 μl of solution containing anti-human IgG Fc-APC secondary antibody was added to each well and incubated at 4° C. for 1 hour, then washing was performed with FACS buffer once, resuspending was performed in 200 μl of FACS buffer, and the fluorescence signal value was read on BD C6 plus. The results were shown in
The EC50 and Emax of humanized antibody binding to KATO3 cells were shown in the table below
The above results showed that hAb-005 lost its ability of binding to CEACAM5 protein and KATO3 cell line; in which hAb-009 humanized antibody had the strongest binding ability to KATO3 cells and maintained the maximum binding.
KATO3 cells (high expression of CEACAM5) were cultured in RPMI1640 medium containing 10% FBS. After the cells were digested with TrypLE trypsin, they were centrifuged and resuspended in DPB S solution containing 2% BSA (FACS buffer, 4° C.), and added a U-bottom 96-well plate, 5×105/100 μl/well, the mouse monoclonal antibody M19 antibody (m2F4) was added to 1 μg/ml and the humanized antibody diluted in gradient (50 μg/ml, 3-fold dilution) was added, then incubated at 4° C. for 1 hour, the supernatant was discarded after centrifugation; 100 μl of solution containing anti-mouse IgG Fc-APC secondary antibody was added to each well and incubated at 4° C. for 1 hour, washing was performed once with FACS buffer, resuspending was performed in 200 μl FACS buffer, and the fluorescence signal value was read on BD C6 plus. The results were shown in
The results of humanized antibodies blocking mouse monoclonal antibody M19 antibody (m2F4) binding were shown in the table below.
The above results showed that the hAb-003, hAb-006, hAb-009, hAb-010, hAb-013, hAb-016, hAb-017 antibodies could compete with the m2F4 antibody, indicating that they could bind to the same epitope on the CEACAM5 molecule.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/110514 | Aug 2020 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/070825 | 1/8/2021 | WO |
