CN117430708B - anti-Claudin18.2 antibody - Google Patents
anti-Claudin18.2 antibody Download PDFInfo
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- CN117430708B CN117430708B CN202311764560.XA CN202311764560A CN117430708B CN 117430708 B CN117430708 B CN 117430708B CN 202311764560 A CN202311764560 A CN 202311764560A CN 117430708 B CN117430708 B CN 117430708B
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Abstract
The invention provides an anti-Claudin 18.2 antibody. Specifically, the anti-Claudin 18.2 antibody specifically binds Claudin18.2, has a higher internalization rate and can mediate higher ADCC action than the positive control antibody Zolbetuximab.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to an anti-Claudin 18.2 antibody.
Background
Claudins belong to the family of Tight Junction (TJ) proteins, which can join cells and form cellular barriers, are predominantly distributed in gastric, pancreatic and pulmonary tissues, and are useful in disease diagnosis and treatment. Although the various claudens are co-expressed in the same tissue, some members of the claudens family are expressed in a specific manner in the tissue. Claudin18 is a typical example. The Claudin18 protein is encoded by the Claudin18 gene, and the first exon is divided into two types, encoding two different tight junction proteins, with different cell lineages, claudin18.1 and Claudin18.2, due to the presence of different restriction sites on the Claudin18 site. Claudin18.1 is mainly expressed in lung tissue, whereas Claudin18.2 is mainly distributed in stomach tissue, these two proteins differ only by 8 amino acid residues in the first exon sequence of 51 amino acids. Since claudin18.2 is buried in the gastric mucosa in normal tissues, it cannot be contacted by monoclonal antibodies. However, cancer cells can disrupt tight junctions, exposing claudin18.2, and thus are widely expressed in a variety of primary malignancies, including but not limited to pancreatic, gastric, biliary, genitourinary/small intestine and colorectal cancers, as specific targets. Therefore, it is a very potential tumor therapeutic target.
Thus, in view of the role and function of claudin18.2 in various related diseases, there is a need in the art to develop anti-claudin 18.2 antibodies suitable for treating patients.
Disclosure of Invention
The invention aims at providing an anti-Claudin 18.2 antibody with high affinity and high biological activity.
In a first aspect of the invention there is provided an antibody against claudin18.2, said antibody comprising a heavy chain and a light chain, and wherein the heavy chain variable region of said heavy chain comprises the following three heavy chain CDRs:
HCDR1 as shown in SEQ ID No.:2,
HCDR2 as shown in SEQ ID No.:3, and
HCDR3 as shown in SEQ ID No.: 4; and
the light chain variable region of the light chain comprises the following three light chain CDRs:
LCDR1 as shown in SEQ ID No.:6,
LCDR2 with sequence WAS, and
LCDR3 as shown in SEQ ID No. 7.
In another preferred embodiment, the heavy chain of the antibody comprises the three heavy chain CDRs and a heavy chain framework region for connecting the heavy chain CDRs; and the light chain of the antibody comprises the three light chain CDRs and a light chain framework region for connecting the light chain CDRs.
In another preferred embodiment, the heavy chain variable region is a heavy chain variable region having the sequence set forth in SEQ ID NO. 1.
In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.
In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin, preferably of human origin.
In another preferred embodiment, the light chain variable region is a light chain variable region having the sequence set forth in SEQ ID NO. 5.
In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.
In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin, preferably of human origin.
In another preferred embodiment, the antibody is a humanized antibody.
In another preferred embodiment, the antibody specifically binds claudin18.2.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the antibody is a monoclonal antibody.
In another preferred embodiment, the antibody comprises a monospecific, bispecific, or trispecific antibody.
In a second aspect of the present invention, there is provided a recombinant protein having:
(i) Light chain and/or heavy chain, or an antibody against Claudin18.2 formed by said light chain and said heavy chain,
the heavy chain variable region of the heavy chain comprises the following three heavy chain CDRs:
HCDR1 as shown in SEQ ID No.:2,
HCDR2 as shown in SEQ ID No.:3, and
HCDR3 as shown in SEQ ID No.: 4; and
the light chain variable region of the light chain comprises the following three light chain CDRs:
LCDR1 as shown in SEQ ID No.:6,
LCDR2 with sequence WAS, and
LCDR3 as shown in SEQ ID NO. 7.
And (ii) optionally a tag sequence to aid expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In another preferred embodiment, the recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused to said element (i).
In a third aspect of the invention, there is provided an antibody preparation comprising:
(a) An antibody according to the first aspect of the invention; and
(b) A carrier, said carrier comprising: buffering agents, sterile water, optionally surfactants.
In a fourth aspect of the invention there is provided a kit comprising an antibody preparation according to the third aspect of the invention, and a container for containing the antibody preparation.
In a fifth aspect of the invention, there is provided a CAR construct, the scFv segment of the antigen binding region of which is a binding region that specifically binds to claudin18.2, and the scFv segment has a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the heavy chain comprises the following three heavy chain CDRs:
HCDR1 as shown in SEQ ID No.:2,
HCDR2 as shown in SEQ ID No.:3, and
HCDR3 as shown in SEQ ID No.: 4; and
the light chain variable region of the light chain comprises the following three light chain CDRs:
LCDR1 as shown in SEQ ID No.:6,
LCDR2 with sequence WAS, and
LCDR3 as shown in SEQ ID No. 7.
In a sixth aspect of the invention there is provided a recombinant immune cell expressing an exogenous CAR construct according to the fifth aspect of the invention.
In another preferred embodiment, the immune cells are selected from the group consisting of: NK cells, T cells.
In another preferred embodiment, the immune cells are derived from a human or non-human mammal (e.g., a mouse).
In a seventh aspect of the present invention, there is provided an antibody drug conjugate comprising:
(a) An antibody moiety selected from the group consisting of: the antibody of the first aspect of the invention, or the recombinant protein of the second aspect of the invention, or a combination thereof; and
(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.
In an eighth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, a CAR construct according to the fifth aspect of the invention, an immune cell according to the sixth aspect of the invention, an antibody drug conjugate according to the seventh aspect of the invention, or a combination thereof, the active ingredients being for:
(a) Preparing a detection reagent or a kit;
(b) Preparing a medicament or preparation for preventing and/or treating Claudin18.2 related diseases; and/or
(c) Preparing medicine or preparation for preventing and/or treating cancer or tumor.
In another preferred embodiment, the cancer or tumor is selected from the group consisting of: lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, leukemia.
In a ninth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) An active ingredient selected from the group consisting of: an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, a CAR construct according to the fifth aspect of the invention, an immune cell according to the sixth aspect of the invention, an antibody drug conjugate according to the seventh aspect of the invention, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a liquid formulation.
In another preferred embodiment, the pharmaceutical composition is an injection.
In another preferred embodiment, the pharmaceutical composition is for the treatment of tumors.
In another preferred embodiment, the tumor is a tumor that highly expresses claudin 18.2.
In a tenth aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:
(1) An antibody according to the first aspect of the invention; or (b)
(2) A recombinant protein according to the second aspect of the invention;
(3) A CAR construct according to the fifth aspect of the invention.
In an eleventh aspect of the invention there is provided a vector comprising a polynucleotide according to the tenth aspect of the invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a twelfth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the eleventh aspect of the invention having incorporated therein a polynucleotide according to the tenth aspect of the invention.
In a thirteenth aspect of the invention, there is provided a method for the in vitro non-diagnostic detection (including diagnostic or non-diagnostic) of claudin18.2 protein in a sample, said method comprising the steps of:
(1) Contacting the sample with an antibody according to the first aspect of the invention in vitro;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of Claudin18.2 protein in the sample.
In a fourteenth aspect of the invention, there is provided a method for detecting Claudin18.2 protein in an in vitro sample (including diagnostic or non-diagnostic), said method comprising the steps of:
(1) Contacting the sample with an antibody according to the first aspect of the invention in vitro;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of Claudin18.2 protein in the sample.
In a fifteenth aspect of the present invention, there is provided a method of treating a claudin 18.2-associated disease, the method comprising:
administering to a subject in need thereof an antibody according to the first aspect of the invention, a drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows cell affinity diagrams of murine Claudin18.2 antibody and 293-Claudin 18.2 cell line
FIG. 2 shows cell affinity diagrams of murine Claudin18.2 antibody and 293-Claudin 18.1 cell line
FIG. 3 shows a graph of C102X02 antibody mediated internalization of XCC08 cell Claudin18.2
FIG. 4 shows a graph of C102X02 mediated ADCC results
Detailed Description
The present inventors have conducted extensive and intensive studies and as a result, unexpectedly obtained an anti-Claudin 18.2 antibody having excellent affinity. The Claudin18.2 antibody specifically binds Claudin18.2, has higher internalization rate and can mediate higher ADCC action compared with the positive control antibody Zolbetuximab. The present invention has been completed on the basis of this finding.
Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The three-letter and one-letter codes for amino acids used in the present invention are as described in J.biol. Chem,243, p3558 (1968).
As used herein, the terms "administering" and "treating" refer to the application of an exogenous drug, therapeutic, diagnostic, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contacting a reagent with the cell, contacting a reagent with a fluid, and contacting a fluid with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, study and diagnosis; comprising contacting an anti-Claudin18.2 antibody with a human or animal, subject, cell, tissue, physiological compartment or physiological fluid.
As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including any one of the anti-claudin 18.2 antibodies of the invention, and compositions thereof, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) effective to alleviate one or more symptoms of the disease.
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that there may be, but need not be, 1, 2, or 3 antibody heavy chain variable regions of a particular sequence.
"sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, or deletions of mutations. The sequence identity between the sequences described in the present invention and sequences with which it has identity may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.
Antibodies to
As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or different types of immunoglobulins, namely IgM, igD, igG, igA and IgE, the heavy chain constant regions corresponding to the different classes of immunoglobulins being referred to as a, d, e, g, and m, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses again due to differences in chemical structure and biological function: igG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by the difference in constant regions. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 FR Regions (FR) that are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions (framework regions) arranged in sequence from amino-to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain, namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3; the 3 CDR regions of the heavy chain, namely heavy chain hypervariable regions (HCDR), refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in numbers and positions that meet the known Kabat numbering convention (LCDR 1-3, HCDR 2-3), or that meet the numbering convention of Kabat and chothia (HCDR 1). The four FR regions in the natural heavy and light chain variable regions are generally in a b-folded configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of a b-folded structure. The CDRs in each chain are held closely together by the FR regions and form together with the CDRs of the other chain an antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody. The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
As used herein, the term "antigen binding fragment" refers to a Fab fragment, fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding.
As used herein, the term "epitope" refers to a discrete, three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using DNA recombination techniques well known in the art.
As used herein, the term "monoclonal antibody" refers to an antibody secreted from a clone derived from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cells may be eukaryotic, prokaryotic or phage clonal cell lines.
As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell by splicing the V region gene of a murine antibody to the C region gene of a human antibody into a chimeric gene, followed by insertion into a vector. The high specificity and affinity of the parent mouse antibody are maintained, and the human Fc segment of the parent mouse antibody can effectively mediate biological effect functions.
As used herein, the term "humanized antibody", a variable region engineered version of the murine antibody of the present invention, has CDR regions derived (or substantially derived) from a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; i.e., grafting murine-resistant CDR region sequences onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing expression vectors.
In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table a.
anti-Claudin18.2 antibodies
As used herein, the term "claudin18.2" refers generally to natural or recombinant human claudin18.2, as well as non-human homologs of human claudin 18.2.
The present invention provides a high specificity and high affinity antibody to claudin18.2 comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.
Preferably, the heavy chain variable region comprises the following three heavy chain CDRs:
HCDR1 as shown in SEQ ID No.:2,
HCDR2 as shown in SEQ ID No.:3, and
HCDR3 as shown in SEQ ID No.: 4;
the light chain variable region comprises the following three light chain CDRs:
LCDR1 as shown in SEQ ID No.:6,
LCDR2 as shown in WAS, and
LCDR3 as shown in SEQ ID No. 7.
Wherein any one of the above amino acid sequences further comprises a derivative sequence having Claudin18.2 binding affinity with at least one (e.g., 1-5, 1-3, preferably 1-2, more preferably 1) amino acid(s) added, deleted, modified and/or substituted.
In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence having a homology of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.
The antibody of the present invention may be a double-or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.
The antibody derivatives of the invention may be single chain antibodies, and/or antibody fragments, such as: fab, fab ', (Fab') 2 or other antibody derivatives known in the art, and the like, as well as IgA, igD, igE, igG and any one or more of IgM antibodies or antibodies of other subtypes.
Wherein the animal is preferably a mammal, such as a mouse.
The antibodies of the invention may be murine antibodies, chimeric antibodies, humanized antibodies, CDR grafted and/or modified antibodies that target human claudin 18.2.
In a preferred embodiment of the invention, any one or several of the sequences SEQ ID NO. 2, 3, 4 described above, or a sequence thereof having Claudin18.2 binding affinity with addition, deletion, modification and/or substitution of at least one amino acid, is located in the CDR region of the heavy chain variable region (VH).
In a preferred embodiment of the invention, any one or more of the sequences SEQ ID NO. 6, 7, or WAS described above, or a sequence thereof having Claudin18.2 binding affinity with at least one amino acid added, deleted, modified and/or substituted thereto, is located in the CDR region of the light chain variable region (VL).
In a more preferred embodiment of the invention, each of H CDR1, H CDR2, H CDR3 is independently selected from any one or several of SEQ ID NO. 2, 3, 4 or a sequence having Claudin18.2 binding affinity with addition, deletion, modification and/or substitution of at least one amino acid; LCDR1, LCDR2, LCDR3 are each independently selected from any one or more of SEQ ID No. 6, 7, or WAS, or a sequence thereof having claudin18.2 binding affinity that has been added, deleted, modified and/or substituted for at least one amino acid.
In the above-described aspect of the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids in the original amino acid sequence.
In the present invention, the number of the added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, most preferably 1.
Humanized anti-Claudin 18.2 antibodies
Jones et al first transplanted murine monoclonal antibody heavy chain CDRs into human antibody heavy chain framework regions, then assembled the murine monoclonal antibody heavy chain into complete antibodies with murine monoclonal antibody light chains and maintained affinity similar to the original murine monoclonal antibodies, providing ideas for the development of antibody humanization technology. Queen et al in 1989 successfully constructed anti-CD 25 humanized antibodies by CDR grafting using humanized Eu framework regions of human antibodies, with murine amino acids retained at partial sites of the framework regions to maintain affinity. Presta et al, 1992, reported a successful construction of humanization by CDR grafting using a human antibody subgroup consensus sequence (consensus sequence) as a template. The humanization of antibodies by the method of surface remodeling (resurfacing) was reported by Pedersen et al 1994. Hsiao et al, 1994, reported a method of humanizing CDR grafting with the framework region of the Germline sequence of a human antibody. The humanization method was successfully constructed by Jeppers et al, 1994, using phage library (shuffling library).
The invention also provides a humanized antibody with high specificity and high affinity against Claudin 18.2. The sequence formed is preferably an amino acid sequence having a homology of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%. The antibodies of the invention may be double-or single-chain antibodies, and may preferably be fully humanized antibodies.
The antibody derivatives of the invention may be single chain antibodies, and/or antibody fragments, such as: fab, fab ', (Fab') 2 Or other antibody derivatives known in the art, and IgA, igD, igE, igG and any of IgM antibodies or other subclasses of antibodiesOr several.
The antibodies of the invention may be humanized antibodies, CDR grafted and/or modified antibodies targeting claudin 18.2.
In the above-described aspect of the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids in the original amino acid sequence.
Preparation of antibodies
Any suitable method for producing monoclonal antibodies can be used to produce the Claudin18.2 antibodies of the invention. For example, animals may be immunized with the linked or naturally occurring Claudin18.2 protein or fragment thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.
Any suitable form of claudin18.2 can be used as an immunogen (antigen) for generating non-human antibodies specific for claudin18.2, and screening the antibodies for biological activity. The immunogens may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. The immunogen may be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic (e.g., cDNA) in origin. DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, baculoviral vectors, plasmids, and non-viral vectors.
The humanized antibody may be selected from any class of immunoglobulins, including IgM, igD, igG, igA and IgE. Also, any type of light chain may be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the present invention.
An exemplary method for preparing the Claudin18.2 antibodies of the invention is described in example 1.
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.
The term "nucleic acid molecule" refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The term "host cell" refers to a cell into which an expression vector has been introduced. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant or animal cells (e.g., mammalian cells).
The steps described herein for transforming a host cell with recombinant DNA may be performed using techniques well known in the art. The transformant obtained can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultivated in conventional medium under suitable conditions.
Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
Antibody-drug conjugates (ADC)
The invention also provides an antibody-conjugated drug (ADC) based on the antibody.
Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.
The antibody of the invention may be coupled to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The nonselective coupling agent refers to a compound such as glutaraldehyde or the like that forms a covalent bond between the effector molecule and the antibody. The coupling agent using carboxyl can be any one or more of cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and acyl hydrazone coupling agent (the coupling site is acyl hydrazone).
Certain residues on antibodies (e.g., cys or Lys, etc.) are useful in connection with a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be directly attached to the antibody, or indirectly attached through a linker.
Antibodies can be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, an ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers typically degrade readily in the intracellular environment, e.g., the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may comprise, for example, a dipeptide, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide bonds). The non-degradable linker typically releases the drug under conditions where the antibody is hydrolyzed by the protease.
Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, the attachment being accomplished through the reactive groups. Thiol-specific reactive groups are preferred and include: such as maleimides, halogenated amides (e.g., iodine, bromine, or chlorine); halogenated esters (e.g., iodine, bromine, or chlorinated); halomethyl ketone (e.g., iodine, bromine, or chlorine), benzyl halide (e.g., iodine, bromine, or chlorine); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-di- (mercuromethyl) dioxane, while the counterion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.
The drug may be any cytotoxic, cytostatic or immunosuppressive drug. In embodiments, the linker connects the antibody and the drug, and the drug has a functional group that can bond to the linker. For example, the drug may have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that may be bonded to the linker. In the case of a drug directly attached to a linker, the drug has reactive groups prior to attachment to the antibody.
Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, the bifunctional linker compounds may be used to form ADCs in two or more step processes. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.
Typically, the functional groups on the linker are selected to facilitate specific reaction with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety may be used to specifically react with a reactive alkynyl group on a drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkyne group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as described in bioconjugate techniques, second edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for selective reaction of a drug moiety with a linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for both the linker and the drug.
Antibody formulations
Antibodies have different stability in different preparation buffers, and are characterized by changes in charge heterogeneity, degradation and polymerization of antibody molecules, and the like, and the changes in quality properties are related to physicochemical properties of the antibodies, so that in the process of developing antibody drugs, preparation buffers suitable for the antibodies need to be screened according to the physicochemical properties of different antibodies. The conventional antibody preparation buffer system comprises phosphate buffer, citric acid buffer, histidine buffer and the like, and meanwhile, salt ions with different concentrations, excipient such as sorbitol, trehalose, sucrose and the like, and surfactant such as tween 20 or tween 80 and the like with proper amount are added according to the properties of the antibody so as to maintain the stability of the antibody.
The antibody pharmaceutical composition preparation can effectively inhibit side reactions such as aggregation, precipitation, hydrolysis, oxidation, deamidation and the like of the humanized antibody, and can effectively improve the stability of the product under the conditions of pressurization (high temperature, strong light irradiation, freeze thawing and the like), acceleration and long-term refrigeration.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding CAR-T cell as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The antibodies of the invention may also be used for cellular therapy where the nucleotide sequence is expressed intracellularly, e.g., for chimeric antigen receptor T cell immunotherapy (CAR-T), etc.
The pharmaceutical composition of the present invention can be directly used for binding to claudin18.2 protein molecules, and thus can be used for preventing and treating claudin18.2 related diseases. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the monoclonal antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
Where a pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Detection application and kit
The antibodies of the invention may be used in detection applications, for example for detecting samples, thereby providing diagnostic information.
In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of an organ.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
The main advantages of the invention include
(1) The antibody of the invention specifically binds Claudin18.2, has higher internalization rate and can mediate higher ADCC action compared with the positive control antibody Zolbetuximab.
The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
EXAMPLE 1 production of mouse anti-hClaudin18.2 antibody
To generate antibodies against human Claudin18.2, 293 cell lines were transfected with Claudin18.2-PCDNA3.1 plasmid as antigen to immunize 6-8 week old Balb/c female mice, mice were immunized by standard cellular immunization procedures, spleens were taken after 5 times and hybridomas were generated by complete fusion with SP2/0 cells using the BTX electrofusion system. Cross-reactivity of hybridoma supernatants with human claudin18.2 expressing CHOK1 cells with blank CHOK1 was determined using Cell-based ELISA format. Binding of hybridoma supernatants to human claudin18.2 expressing CHOK1 cells and human claudin18.1 expressing CHOK1 was then determined using flow cytometry. Positive clones reacted only with CHOK1 cells expressing human Claudin18.2, but not with CHOK1 and blank CHOK1 expressing human Claudin18.1. The selected candidate clone is used for modulating the related antibody sequence through a specific gene amplification technology.
Example 2C 102X02 construction of expression
C102X02 VH and VL were constructed on eukaryotic expression vectors containing hIgG1 and kappa constant regions, fresh 293F cells were transfected, cultured for 3-7 days, and supernatants were collected and purified by protein A columns to give C102X02 antibody proteins.
C102X02 VH amino acid sequence (SEQ ID NO: 1)
QIQLVQSGPELKKPGETVKISCKASGYTFTNHGMNWVKQAPGKGLKWMGWINTNTGEPTYAEELKGRFAFSLETSASTAFLQIINLKNEDTATYFCARYYYGNSFAYWGQGTLVTVSA
C102X02 VL amino acid sequence (SEQ ID NO: 5)
DIVMTQSPSSLTVIAGEKVTLSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSFPLTFGAGTKLELKR
hIgG1 constant region amino acid sequence (SEQ ID NO: 8)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
kappa chain constant region amino acid sequence (SEQ ID NO: 9)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Wherein the underlined regions are CDRs (IMGT definition, single column as follows):
TABLE 1 murine anti-Claudin 18.2 antibody CDR sequences
Example 3C 102X02 test for binding to Claudin18.2 Positive cells
This example shows primarily the binding capacity of Claudin18.2 antibodies to Claudin18.2 positive cells 293-hClaudin18.2 cells. In particular, the method comprises the steps of,
taking 3×10 5 Individual cell 293-Claudin 18.2 cell line (Novoprot)ein, cat. No.: XCC 08), adding gradient diluted Claudin18.2 antibody protein, incubating for 1h, washing 1 time with PBS, adding Anti-hFc-APC (from Jackson immunology), washing 1 time with PBS, and detecting on-stream. The results are plotted as S-curves in FIG. 1. The affinity EC50 is shown in table 2.
Table 2: cell affinity results of murine Claudin18.2 antibody with 293-Claudin 18.2 cell line
EXAMPLE 4 non-specific binding detection of C102X02 to Claudin 18.1 Positive cells
Taking 3×10 5 Individual cells 293-Claudin 18.1 cell line (Novoprotein, cat. No.: XCC 07), gradient diluted Claudin18.2 antibody protein was added, incubated for 1h, washed 1 time with PBS, anti-hFc-APC (from Jackson immunology) was added, washed 1 time with PBS and then run on the machine for detection. The results are plotted as S-curves in FIG. 2.
The results show that the antibodies of the invention bind non-specifically to Claudin 18.1 positive cells.
EXAMPLE 5C 102X02 mediated cell internalization
This example shows mainly the internalization effect of C102X02 on Claudin18.2 positive cells. Specifically, 0.1. Mu.g/test Claudin18.2 antibody was added to a final concentration of secondary antibody Allophycocyanin (APC) AffiniPure F (ab') 2 Fragment Goat Anti-Human IgG (1:2000), mixed in equal volume 1:1, 4 degrees combined for 1.5h; mixing the mixture of the secondary antibody and the antibody to be detected with XCC08 cells (0.15 x 10 6 Personal/test) 4 degrees binding for 1h, pre-chilling 1% BSA wash once; then adding 37 degrees of preheated 1% BSA,100 mu L/test, placing into a 37 degrees of incubator, incubating for 0h, 1h, 2h, 3h and 4h respectively, immediately taking out after the internalization time is over, washing once with precooled 1% BSA, and adding 200 mu L of precooled PBS for vortex mixing flow type on-machine detection. The calculated result is shown in FIG. 3, with the relative fluorescence intensity (MFI) at 0h being 1.
The results show that the antibodies of the invention have a higher internalization rate than the positive control antibody Zolbetuximab.
EXAMPLE 6C 102X02 mediated ADCC
At 1.6x10 6 cell/mL Density 25. Mu.L of jurkat-NFAT-CD16A cells (40000/well) were added to a 96-well plate at 0.4X10 6 cell/mL Density 25. Mu.L XCC08 cells (293-Claudin 18.2 cell lines, 10000 cells/well) were added to 96 well plates, incubated overnight at 37℃and 50. Mu.L gradient diluted Claudin18.2 antibody was added to 96 well plates, 20. Mu.g/mL started, 5-fold 7 gradients; cells were lysed after overnight incubation and luciferase activity was detected by addition of luciferase substrate (Promega, cat. No. E1501). The results are shown in FIG. 4.
The results indicate that the antibodies of the invention are able to mediate higher ADCC compared to the positive control antibody Zolbetuximab.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (13)
1. An antibody against claudin18.2, wherein said antibody comprises a heavy chain and a light chain, and wherein the heavy chain variable region of said heavy chain comprises the following three heavy chain CDRs:
HCDR1 shown in SEQ ID NO.2,
HCDR2 shown in SEQ ID NO.3, and
HCDR3 shown in SEQ ID No. 4; and
the light chain variable region of the light chain comprises the following three light chain CDRs:
LCDR1 shown in SEQ ID NO.6,
LCDR2 with sequence WAS, and
LCDR3 as shown in SEQ ID NO. 7.
2. A recombinant protein, said recombinant protein comprising:
(i) An antibody against Claudin18.2 formed by a light chain and a heavy chain,
the heavy chain variable region of the heavy chain comprises the following three heavy chain CDRs:
HCDR1 shown in SEQ ID NO.2,
HCDR2 shown in SEQ ID NO.3, and
HCDR3 shown in SEQ ID No. 4; and
the light chain variable region of the light chain comprises the following three light chain CDRs:
LCDR1 shown in SEQ ID NO.6,
LCDR2 with sequence WAS, and
LCDR3 as shown in SEQ ID No. 7;
and (ii) tag sequences that facilitate expression and/or purification.
3. A CAR construct, wherein the scFv segment of the antigen binding region of the CAR construct is a binding region that specifically binds to claudin18.2 and the scFv segment has a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the heavy chain comprises the following three heavy chain CDRs:
HCDR1 shown in SEQ ID NO.2,
HCDR2 shown in SEQ ID NO.3, and
HCDR3 shown in SEQ ID No. 4; and
the light chain variable region of the light chain comprises the following three light chain CDRs:
LCDR1 shown in SEQ ID NO.6,
LCDR2 with sequence WAS, and
LCDR3 as shown in SEQ ID NO. 7.
4. A recombinant immune cell expressing the CAR construct of claim 3 exogenously; the immune cells are selected from the group consisting of: NK cells, T cells.
5. An antibody conjugate, comprising:
(a) An antibody moiety selected from the group consisting of: the antibody of claim 1, or the recombinant protein of claim 2, or a combination thereof; and
(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, toxin, cytokine, radionuclide, enzyme, or a combination thereof.
6. Use of an active ingredient, characterized in that the active ingredient is selected from the group consisting of: the antibody of claim 1, or the recombinant protein of claim 2, the CAR construct of claim 3, the immune cell of claim 4, the antibody conjugate of claim 5, or a combination thereof, the active ingredient being for:
(a) Preparing a detection reagent or a kit;
(b) Preparing a medicament or preparation for preventing and/or treating Claudin18.2 related diseases; claudin18.2 related disease is gastric cancer over-expressed by Claudin18.2.
7. A pharmaceutical composition comprising:
(i) An active ingredient selected from the group consisting of: the antibody of claim 1, or the recombinant protein of claim 2, the CAR construct of claim 3, the immune cell of claim 4, the antibody conjugate of claim 5, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
8. An antibody preparation, wherein the antibody preparation comprises:
(a) The antibody of claim 1; and
(b) A carrier, said carrier comprising: buffering agent, sterile water, surfactant.
9. A kit comprising the antibody preparation of claim 8 and a container for containing the antibody preparation.
10. A polynucleotide encoding a polypeptide selected from the group consisting of:
(1) The antibody of claim 1; or (b)
(2) The recombinant protein of claim 2;
(3) The CAR construct of claim 3.
11. A vector comprising the polynucleotide of claim 10.
12. A genetically engineered host cell comprising the vector of claim 11 or the polynucleotide of claim 10 integrated into the genome.
13. A method for the in vitro non-diagnostic detection of claudin18.2 protein in a sample, said method comprising the steps of:
(1) Contacting the sample with the antibody of claim 1 in vitro;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of Claudin18.2 protein in the sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109790222A (en) * | 2016-07-08 | 2019-05-21 | 科济生物医药（上海）有限公司 | The antibody of anti-claudin 18A2 and its application |
| CN110857322A (en) * | 2018-08-22 | 2020-03-03 | 瑞阳(苏州)生物科技有限公司 | Anti-human claudin18.2 monoclonal antibody and application thereof |
| CN112888458A (en) * | 2018-09-30 | 2021-06-01 | 佧珐药业有限公司 | Combination therapy of antibodies to CLDN18 and chemotherapeutic agents |
| CN116789822A (en) * | 2022-03-18 | 2023-09-22 | 广东东阳光药业股份有限公司 | Claudin18.2 humanized antibody and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109790222A (en) * | 2016-07-08 | 2019-05-21 | 科济生物医药（上海）有限公司 | The antibody of anti-claudin 18A2 and its application |
| CN110857322A (en) * | 2018-08-22 | 2020-03-03 | 瑞阳(苏州)生物科技有限公司 | Anti-human claudin18.2 monoclonal antibody and application thereof |
| CN112888458A (en) * | 2018-09-30 | 2021-06-01 | 佧珐药业有限公司 | Combination therapy of antibodies to CLDN18 and chemotherapeutic agents |
| CN116789822A (en) * | 2022-03-18 | 2023-09-22 | 广东东阳光药业股份有限公司 | Claudin18.2 humanized antibody and application thereof |
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