CN117897403A - Claudin 18.2-resistant antibody and application thereof - Google Patents
Claudin 18.2-resistant antibody and application thereof Download PDFInfo
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- CN117897403A CN117897403A CN202280052157.1A CN202280052157A CN117897403A CN 117897403 A CN117897403 A CN 117897403A CN 202280052157 A CN202280052157 A CN 202280052157A CN 117897403 A CN117897403 A CN 117897403A
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Abstract
An anti-Claudin18.2 antibody or antigen binding fragment thereof and uses thereof are provided. Bispecific antibodies, multispecific antibodies, multifunctional fusion proteins, and compositions thereof comprising the antibodies, and uses thereof in the manufacture of a medicament for treating cancer are also provided.
Description
The invention belongs to the field of biological medicine, and particularly relates to an anti-Claudin 18.2 antibody and application thereof.
Cell-cell tight junctions are a transmembrane protein complex, the stability of tight junctions requires coordinated activity of several different proteins to be maintained, while Claudin protein is the main protein that ensures tight junction permeability with specificity. To date, 24 members of the Claudin family have been found in mammals, each of which is specifically expressed on a tissue. The extracellular loop structure of the Claudin protein is linked to this structure of the neighboring cells, serving to bridge the cell layer and regulate the parallel transport between the chamber and basal layer.
Claudin18 protein structure includes four transmembrane regions, two extracellular loops with N-and C-termini in the cytosol, and has a molecular weight of about 26KD, which can be selectively sheared to change Claudin protein into Claudin subtypes with different properties: claudin18.1 and claudin18.2. Although there are only eight amino acid differences between the first extracellular domains of claudin18.1 and claudin18.2, the expression profile is different, claudin18.1 is selectively expressed in normal lung and stomach epithelium, claudin18.2 is expressed only on transiently differentiated stomach epithelial cells, and is not detected at all in any other normal human organ, but claudin18.2 is significantly upregulated in a variety of malignancies, including 80% gastrointestinal adenomas, 60% pancreatic tumors, 30% esophageal cancer, and 25% non-small cell lung cancer. In tumors, the tight junctions between cells are disrupted and claudin18.2 fails to perform its normal function. Claudin18.2 is therefore a suitable tumor therapeutic target.
Ganymed Pharmaceuticals AG an IgG1 chimeric antibody Zolbetuximab (also known as IMAB 362) against Claudin18.2 was developed, and IMAB362 recognizes the first extracellular domain (ECD 1) of Claudin18.2 with high affinity and specificity. After target binding, IMAB362 mediates cell killing by ADCC, CDC, induction of apoptosis induced by target cross-linking on tumor cell surfaces, and proliferation. Thus, IMAB362 effectively lyses claudin18.2 positive cells, including human gastric cancer cell lines in vitro and in vivo. Mice with a claudin18.2 positive cancer cell line have survival benefits, and up to 40% of mice exhibit regression of their tumors when treated with IMAB 362.
Stomach cancer and gastroesophageal junction (GEJ) adenocarcinoma are one of the most medical malignancies that are currently not met, and stomach cancer is also the third leading cause of cancer death worldwide. Therefore, the development of the Claudin18.2 antibody and other medicines such as multifunctional and bifunctional antibodies thereof has great significance for the treatment of partial solid tumors, especially stomach-related tumors. However, no corresponding antibody development is currently marketed.
Disclosure of Invention
In response to the deficiencies of the prior art, in this application, the inventors developed an anti-claudin 18.2 antibody with good properties that does not bind to claudin18.1, but is capable of binding claudin18.2 with high affinity and specificity and mediating killing of target cells (e.g., tumor cells) expressing claudin18.2 by effector cells.
The present invention provides an anti-Claudin 18.2 antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein,
(a) HCDR1 of the heavy chain variable region selected from SEQ ID NO: 1. 9, 15, 19, or an amino acid sequence identical to any one of SEQ ID NOs: 1. 9, 15, 19, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO: 1. 9, 15, 19, an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences;
(b) HCDR2 of the heavy chain variable region selected from SEQ ID NO: 2. 10, 16, 20, 21, 22, 23, 24, 25, or an amino acid sequence identical to SEQ ID NO: 2. 10, 16, 20, 21, 22, 23, 24, 25, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NOs: 2. 10, 16, 20, 21, 22, 23, 24, 25, has one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences;
(c) HCDR3 of the heavy chain variable region selected from SEQ ID NO: 3. 11, 17, 26, 27, 63, or an amino acid sequence identical to SEQ ID NO: 3. 11, 17, 26, 27, 63, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NOs: 3. 11, 17, 26, 27, 63, an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences;
(d) LCDR1 of the light chain variable region selected from SEQ ID NO: 4. 28, or an amino acid sequence that hybridizes to any one of SEQ ID NOs: 4. 28, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO: 4. 28, having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence;
(e) LCDR2 of the light chain variable region selected from SEQ ID NO:5, or an amino acid sequence identical to SEQ ID NO:5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO:5, having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences; and/or
(f) LCDR3 of the light chain variable region selected from SEQ ID NO: 6. 12, or an amino acid sequence that hybridizes to any one of SEQ ID NOs: 6. 12, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO: 6. 12, having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence.
In some embodiments, HCDR1, HCDR2, HCDR3 of the heavy chain variable region, and LCDR1, LCDR2, LCDR3 of the light chain variable region are selected from any one of the amino acid sequences (1) - (11) below:
(1) SEQ ID NO:1, HCDR1 shown in SEQ ID NO:2, HCDR2 shown in SEQ ID NO:3, HCDR3, SEQ ID NO:4, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;
(2) SEQ ID NO:9, HCDR1, SEQ ID NO:10, HCDR2 shown in SEQ ID NO:11, HCDR3 shown in SEQ ID NO:4, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 12;
(3) SEQ ID NO:15, HCDR1, SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17, HCDR3, SEQ ID NO:4, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 12;
(4) SEQ ID NO:19, HCDR1, SEQ ID NO:20, HCDR2 shown in SEQ ID NO:63, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;
(5) SEQ ID NO:19, HCDR1, SEQ ID NO:21, HCDR2 shown in SEQ ID NO:63, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;
(6) SEQ ID NO:19, HCDR1, SEQ ID NO:22, HCDR2 shown in SEQ ID NO:26, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;
(7) SEQ ID NO:19, HCDR1, SEQ ID NO:23, HCDR2 shown in SEQ ID NO:63, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;
(8) SEQ ID NO:19, HCDR1, SEQ ID NO:24, HCDR2 shown in SEQ ID NO:63, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;
(9) SEQ ID NO:19, HCDR1, SEQ ID NO:25, HCDR2 shown in SEQ ID NO:63, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;
(10) SEQ ID NO:19, HCDR1, SEQ ID NO:23, HCDR2 shown in SEQ ID NO:27, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;
(11) SEQ ID NO:19, HCDR1, SEQ ID NO:24, HCDR2 shown in SEQ ID NO:27, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in fig. 6.
The invention also provides an anti-claudin 18.2 antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, characterized in that wherein:
(a) The heavy chain variable region has the amino acid sequence of SEQ ID NO: 7. 13, 18, 29, 30, 31, 33, 34, 35, 36, 37,
or with SEQ ID NO: 7. 13, 18, 29, 30, 31, 33, 34, 35, 36, 37, a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,
or with SEQ ID NO: 7. 13, 18, 29, 30, 31, 33, 34, 35, 36, 37, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences;
(b) The light chain variable region has the amino acid sequence of SEQ ID NO: 8. 14, 32, 38;
or with SEQ ID NO: 8. 14, 32, 38 has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of the amino acid sequences set forth herein,
or with SEQ ID NO: 8. 14, 32, 38, and an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).
In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of the amino acid sequences of (1) - (11) below:
(1) SEQ ID NO:7 and SEQ ID NO:8, 8;
(2) SEQ ID NO:13 and SEQ ID NO:14;
(3) SEQ ID NO:18 and SEQ ID NO:14;
(4) SEQ ID NO:29 and SEQ ID NO:32;
(5) SEQ ID NO:30 and SEQ ID NO:32;
(6) SEQ ID NO:31 and SEQ ID NO:32;
(7) SEQ ID NO:33 and SEQ ID NO:38, a step of carrying out the process;
(8) SEQ ID NO:34 and SEQ ID NO:38, a step of carrying out the process;
(9) SEQ ID NO:35 and SEQ ID NO:38, a step of carrying out the process;
(10) SEQ ID NO:36 and SEQ ID NO:38, a step of carrying out the process;
(11) SEQ ID NO:37 and SEQ ID NO:38.
In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
In some embodiments, the antibody is a monoclonal antibody.
In some embodiments, the anti-claudin 18.2 antibody or antigen binding fragment thereof further comprises an Fc region selected from the group consisting of mouse IgG1, igG2a, igG2b, and/or IgG3, or from the group consisting of rat IgG1, igG2a, igG2b, and/or IgG2c.
In some embodiments, the anti-claudin 18.2 antibody or antigen binding fragment thereof further comprises an Fc region selected from human IgG1, igG2, igG3 and/or IgG4.
The invention also provides a nucleic acid molecule encoding an anti-claudin 18.2 antibody or antigen binding fragment thereof according to any of the preceding claims.
The invention also provides a multifunctional fusion protein comprising an anti-claudin 18.2 antibody or antigen binding fragment thereof according to any of the preceding claims.
In some embodiments, the anti-claudin 18.2 antibody or antigen binding fragment thereof further comprises one or more secondary antibodies or antigen binding portions thereof that specifically bind to other antigens.
In some embodiments, the antigen that binds to the second antibody or antigen binding portion thereof is selected from a tumor-associated antigen (TAA) or an immune checkpoint.
In some embodiments, the immune checkpoint is PD-L1, CTLA4, PD-L2, PD-1, 4-1BB, CD47, TIGIT, GITR, TIM3, ILT4, TNFR2, TREM2, LAG3, CD27, B7H3, or B7H4.
In some embodiments, the composition further comprises a cytokine.
In some embodiments of the present invention, in some embodiments, the cytokine is selected from the group consisting of IL-1, IL-2Rα, IL-2Rβ, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-4Rα, IL-5Rα, IL-4Rα, and IL-5Rα IL-7, IL-7Rα, IL-8, IL-9Rα, IL-10R1, IL-10R2, IL-11Rα, IL-12Rα, IL-11R 1, IL-11R 2, IL-11R 1, IL-12R 1, IL-10R1, IL-11R 1, IL-12Rβ2, IL-12Rβ1, IL-13, IL-13Rα, IL-13Rα2, IL-14, IL-15, IL-15Rα sushi, IL-16, IL-17, IL-18, IL-19, IL-20R1, IL-20R2, IL-21, IL-21Rα, IL-22, IL-23R, IL-27R, IL-31R, TGF, VEGF, IFN γ, IFNα, or GM-CSF.
The invention also provides the use of an anti-claudin 18.2 antibody or antigen binding fragment thereof according to any one of the above claims or a multifunctional fusion protein according to any one of the above claims in the preparation of a medicament for the treatment of cancer.
In some embodiments, the cancer is lung cancer, liver cancer, melanoma, glioblastoma, head and neck cancer, esophageal cancer, gastric cancer, prostate cancer, ovarian cancer, bladder cancer, pancreatic cancer, gastric cancer, colon cancer, cervical cancer, or a related tumor.
In some embodiments, the use is achieved by one or more of tumor immunotherapy, cell therapy, and gene therapy.
The invention also provides a pharmaceutical composition comprising an anti-claudin 18.2 antibody or antigen-binding fragment thereof as described in any of the preceding claims and a pharmaceutically acceptable carrier, diluent or excipient.
The invention also provides a pharmaceutical composition comprising the multifunctional fusion protein of any one of the above, and a pharmaceutically acceptable carrier, diluent or excipient.
Abbreviations and term definitions
The following abbreviations are used herein:
mAb: monoclonal antibodies
VH: antibody heavy chain variable region
VL: antibody light chain variable regions
CDR: complementarity determining regions in immunoglobulin variable regions
FR: antibody framework regions, i.e. amino acid residues other than CDR residues in the variable regions of antibodies
IgG: immunoglobulin G
The term "antibody" refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis. Antibodies can be isolated by reconstitution from natural sources such as plasma or serum in which the antibodies are naturally present, or culture supernatants of antibody-producing hybridoma cells, animal immune serum, or phage library screening. Alternatively, it may be partially or completely synthesized by using a technique of gene recombination or the like. Preferred antibodies include, for example, antibodies to the isotype of immunoglobulins or subclasses of these isotypes. Human immunoglobulins are known to include the 9 classes (isotypes) IgGl, igG2, igG3, igG4, igAl, igA2, igD, igE, igM. Among these isotypes, the antibodies of the invention may include IgGl, igG2, igG3 and/or IgG4.
The term "monoclonal antibody" refers to a homogeneous antibody directed against only one specific epitope. In contrast to common polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope on the antigen. The modifier "monoclonal" refers to a homogeneous characteristic of the antibody and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies of the invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere herein.
The term "murine antibody" is herein a monoclonal antibody prepared according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with an antigen, and then isolating hybridomas expressing antibodies having the desired sequence or functional properties.
The term "chimeric antibody" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce the immune response induced by the murine antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting the mouse-derived specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to requirements, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into a human vector, and finally expressing chimeric antibody molecules in a eukaryotic industrial system or a prokaryotic industrial system.
The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting the CDR sequences of a mouse into the framework of the variable region of a human antibody, i.e., the framework sequences of a different type of human germline antibody. The strong heterologous reaction induced by chimeric antibodies due to the large number of mouse protein components can be overcome.
As used herein, a portion of an antibody is an immunoglobulin molecule that consists of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL), or only a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively.
The term "antigen-binding fragment" of an antibody refers to a polypeptide fragment of an antibody, e.g., a polypeptide fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. Antigen binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') 2, fd, fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabodies), linear antibodies (linear antibodies), nanobodies (e.g., technology from Ablynx), domain antibodies (e.g., technology from domanis), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide.
The term "polypeptide" refers to an amino acid chain of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. Polypeptides may be "exogenous", meaning that they are "heterologous", i.e. foreign to the host cell utilized, e.g. human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the amino-to carboxy-terminal direction. Amino acid residue sequences are named according to standard nomenclature with three-letter or one-letter codes as follows: alanine (Ala, a), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
The term "host cell" refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a selected gene of interest. The term includes progeny of a parent cell, whether or not the progeny is identical in morphology or genetic composition to the original parent cell, as long as the progeny has the selected gene of interest present. Common host cells include bacteria, yeast, mammalian cells, and the like.
The term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked, referred to herein as "expression vectors".
The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline solutions, water and emulsions, as well as various types of wetting agents.
The term "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example using publicly available computer software, such as the BLAST software or FASTA packages.
There are a variety of methods/systems in the art to define and describe CDRs, and these systems and/or definitions have been developed and refined for many years, including Kabat, chothia, IMGT, abM and contacts. Kabat is the most commonly used, defining CDRs based on sequence variability; chothia defines CDRs based on sequence variability based on the position of structural loop regions; the IMGT system defines CDRs based on sequence variability and position within the variable domain structure; abM is defined based on AbM antibody modeling software from oxford molecular corporation, a compromise between Kabat and Chothia; contacts define CDRs based on analysis of complex crystal structures, similar in many respects to Chothia.
The CDRs of the anti-claudin 18.2 antibody of the invention refer to the hypervariable regions of the heavy and light chains of immunoglobulins, wherein the amino acid sequences of SEQ ID NO: 1. 2, 3, 4, 5, 6, 9, 10, 11, 12, 15, 16, 17 according to Kabat definition, SEQ ID NO: 19. 20, 21, 22, 23, 24, 25, 26, 27, 63 are defined in terms of AbM.
The term "specific" means that one of the molecules involved in specific binding does not show any significant binding to a molecule other than one or several of the binding partner molecules. In addition, the term is also used when the domain comprising the antibody variable region is specific for a particular epitope of a plurality of epitopes in an antigen. When an epitope to which a domain comprising an antibody variable region binds is contained in several different antigens, an antigen binding molecule comprising a domain comprising an antibody variable region can bind to various antigens having the epitope.
The term "epitope" refers to an antigenic determinant in an antigen, and refers to an antigenic site to which the domains of an antigen binding molecule comprising antibody variable regions disclosed in the present specification bind. Thus, epitopes can be defined according to their structure. Alternatively, the epitope may be defined in terms of antigen binding activity in an antigen binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope may be specified by the amino acid residues that form the epitope; when the epitope is a sugar chain, the epitope can be determined by its specific sugar chain structure.
The term "positive control antibody" refers to a natural or engineered cell capable of binding or expressing a target protein.
The term "isotype control" refers to the use of the same species source, same subtype, same dose, same immunoglobulin and subtype of immunoglobulin, same label, etc. as the experimental sample in the same experiment to eliminate the experimental background effect of the non-specific binding sample on the experimental value in the experiment as a negative control for more explaining the experimental effect.
The murine, chimeric or humanized monoclonal antibodies that bind to Claudin18.2 provided by the invention have higher Claudin18.2 binding activity, ADCC activity and CDC activity than prior art antibodies. Meanwhile, the anti-Claudin 18.2 antibody is subjected to humanized transformation, and the transformed antibody has Claudin18.2 binding activity equivalent to that of the original antibody, has relatively stable affinity, can ensure the specificity of the antibody, and has CDC activity basically consistent with that of the original antibody.
FIG. 1 shows the binding activity of murine antibodies 1, 2, 3 to human Claudin18.2-PANC 1.
FIG. 2 shows the binding activity of chimeric antibodies 4, 5 to human Claudin18.2-CHOK 1.
FIG. 3 shows the binding activity of chimeric antibody 6 to human Claudin18.2-CHOK 1.
FIG. 4 shows the binding activity of chimeric antibodies 4, 5 to human Claudin18.2-PANC 1.
FIG. 5 shows the binding activity of humanized antibodies 7, 8, 9 to human Claudin18.2-CHOK 1.
FIG. 6 is a graph showing the binding activity of humanized antibody 10 to human Claudin18.2-CHOK 1.
FIG. 7 shows the binding activity of humanized antibodies 11, 12 to human Claudin18.2-CHOK 1.
FIG. 8 shows CDC activity of murine antibodies 1, 2, 3 against human Claudin18.2-CHOK 1.
FIG. 9 shows CDC activity of chimeric antibodies 4, 5 on human Claudin18.2-CHOK 1.
FIG. 10 shows CDC activity of chimeric antibody 6 on human Claudin18.2-CHOK 1.
FIG. 11 shows CDC activity of humanized antibody 10 on human Claudin18.2-CHOK 1.
FIG. 12 shows CDC activity of humanized antibodies 11, 12 on human Claudin18.2-CHOK 1.
FIG. 13 shows ADCC activity of chimeric antibodies 4, 5 against human Claudin18.2-PANC-1.
FIG. 14 shows ADCC activity of chimeric antibody 6 against human Claudin 18.2-PANC-1.
FIG. 15 shows the binding activity of chimeric antibody A, B, C, D, E, F, G to human Claudin18.2-CHOK 1.
FIG. 16 shows the binding activity of humanized antibody H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1 using Z1 as the control antibody.
FIG. 17 shows the binding activity of humanized antibody H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1 using IMAB362 as a control antibody.
FIG. 18 shows CDC activity of humanized antibody H, J, L on human Claudin18.2-CHOK 1.
The present invention will be further described with reference to the accompanying drawings and specific examples, to which the invention is not limited. It is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. Variations and advantages that will occur to those skilled in the art are included within the following claims and any equivalents thereof without departing from the spirit and scope of the inventive concept. In the description and claims of the present invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge to those skilled in the art, except where specifically mentioned below, and the present invention is not particularly limited.
Example 1 preparation of an anti-Claudin 18.2 antibody
293 cells overexpressing human Claudin18.2 were used as immunogens, balb/c mice were immunized 3-4 times, and serum titers were detected 1 week after each immunization using CHOK1 and a cell-ELISA coated with CHOK1 overexpressing human Claudin 18.2. After the titers reached the fusion requirement, the cells were used to boost after 2 weeks from the last immunization. Three days later, spleen was removed, spleen cells and mouse myeloma cells SP2/0 were fused by electrofusion, and after culturing in a HAT-containing medium for 7-10 days, the culture supernatant was removed, and screening with CHOK1 cells expressing human Claudin18.2 and counter screening with CHOK1 and CHOK1 cells expressing human Claudin18.1, candidate antibody 1, candidate antibody 2 and candidate antibody 3 specifically binding to human Claudin18.2 were obtained. Chimeric antibodies 4, 5, and 6 were constructed on the basis of candidate antibodies (murine antibodies) 1, 2, and 3, humanized antibodies 7, 8, and 9 were constructed on the basis of chimeric antibody 4, and humanized antibodies 10, 11, and 12 were constructed on the basis of chimeric antibody 5, respectively.
TABLE 1 antibody sequence listing
| Heavy chain amino acid sequence | Light chain amino acid sequence | |
| Antibody 1 | SEQ ID NO：7 | SEQ ID NO：8 |
| Antibody 2 | SEQ ID NO：13 | SEQ ID NO：14 |
| Antibody 3 | SEQ ID NO：18 | SEQ ID NO：14 |
| Antibody 4 | SEQ ID NO：39 | SEQ ID NO：40 |
| Antibody 5 | SEQ ID NO：41 | SEQ ID NO：42 |
| Antibody 6 | SEQ ID NO：43 | SEQ ID NO：42 |
| Antibody 7 | SEQ ID NO：44 | SEQ ID NO：45 |
| Antibody 8 | SEQ ID NO：44 | SEQ ID NO：46 |
| Antibody 9 | SEQ ID NO：47 | SEQ ID NO：46 |
| Antibody 10 | SEQ ID NO：48 | SEQ ID NO：49 |
| Antibody 11 | SEQ ID NO：50 | SEQ ID NO：51 |
| Antibody 12 | SEQ ID NO：50 | SEQ ID NO：49 |
Example 2 detection of antibody binding Capacity to cells
The antibody to be tested was taken and diluted 6-8 gradients with 40ug/ml as initial final concentration. Cells overexpressing human Claudin18.2 (human Claudin18.2-PANC1, human Claudin18.2-CHOK 1) in the incubator were removed, the cell suspension was transferred to a 15ml centrifuge tube, centrifuged, and counted in PBS for resuspension. A Blank control group (Blank), a negative control group (NC), an experimental group, an irrelevant antibody group, and the control antibody of the antibodies 1-6 were FUT8 gene-silenced IMAB362 obtained by the knockout process described in patent application 202110752316.6, and the control antibody of the humanized antibodies 7-12 was IMAB362. According to about 2X 10 5 Cell/well, cell suspensions were plated in 96-well plates. Centrifugation (1000 rpm,5 min), followed by washing with PBS, and further centrifugation were repeated twice to remove the medium residue. The supernatant was discarded, 100. Mu.l of the primary antibody solution and the independent antibody solution were added to each of the experimental group and the independent antibody solution, and the cells were resuspended and incubated at room temperature for 1 hour. Blank, NC groups were incubated with equal amounts of PBS. After 1h, the mixture was centrifuged, and washed twice with PBS. After discarding the supernatant, 100. Mu.l of fluorescent secondary antibody diluent (goat anti-mouse IgGH) was added to each of the remaining samples except for the Blank group to which 100. Mu.l of PBS was added &L-FITC was from Abcam ab6785, goat anti-human IgG Fc gamma-FITC was from bioleged 398006), incubated at room temperature in the dark for 0.5h, and after centrifugation, washed twice with PBS. After discarding the supernatant, 120. Mu.l of PBS was added for resuspension, and flow cytometry was performed in order to measure the average fluorescence intensity. The results are shown in FIGS. 1-7.
The results showed that the binding curves of each murine, chimeric and humanized antibody were concentration gradient dependent. The binding activity of the murine antibodies 1, 2 and 3 to human Claudin18.2-PANC1, the chimeric antibodies to human Claudin18.2-PANC1 and human Claudin18.2-CHOK1, and the binding activity of the humanized antibodies to human Claudin18.2-CHOK1 are obviously superior to those of the control antibodies.
Example 3 CDC Activity assay of antibodies
Taking out the 96-well plate, and starting the antibody to be detected, the control antibody (the control antibody of the antibody 1-6 is FUT8 gene-silenced IMAB362 obtained by adopting the knockout technology described in patent application 202110752316.6, and the control antibody of the humanized antibody 7-12 is IMAB 362) and the isotype control antibody according to 10ug/ml, diluting 3 times, carrying out 7 gradient dilutions, and setting 2 parallel wells and 50 ul/well. Taking out CHOK1A2HIS cell count, and setting cell number to 2×10 4 Each 100 ul/well was incubated with antibody at 37℃for 15min. Complement was added to retain human serum at a concentration of 20%/50 ul/well, i.e., a final concentration of 5%/well. Incubate at 37℃for 1h. CCK-8, 20 ul/well, was added and incubated at 37℃for 4h, and the microplate reader was read at a wavelength of 450 nm. The results are shown in FIGS. 8-12.
The results showed that CDC activity was higher for each of the murine, chimeric and humanized antibodies than for the control antibody.
Example 4 ADCC Activity assay of antibodies
The 96-well plate was removed, and control antibody (FUT 8 gene silenced IMAB362 using the knock-out procedure described in patent application 202110752316.6), isotype control antibody and chimeric antibodies 4, 5, 6 were initially diluted 5-fold at 10ug/ml, 7 dilutions were made in a gradient, and 2 parallel wells were set up at 100 ul/well. The PANC-1.2 cell count was taken out and the cell count was set to 1X 10 4 And/50 ul/well.
Taking out target cell NK cell count, setting cell number 5×10 4 And/50 ul/well. Centrifuge at 1000rpm for 4min. Incubate at 37℃for 3.25h. ST wells (containing only target cells), SE wells (containing only effector cells), BV wells (200 ul of diluent), BM wells (200 ul of diluent) and M wells (50 ul of effector cells+150 ul of diluent) were set. BM wells were incubated with BV Kong Jiaru lysate 20ul for 45min at 37 ℃. And (3) centrifuging at 1000rpm for 4min, taking 50ul of supernatant, adding 50ul of substrate, placing in an ELISA plate, standing at room temperature for 30min, adding stop solution 50 ul/hole, and detecting at 490nm of the ELISA. The results are shown in FIGS. 13 and 14.
The ADCC activity results showed that chimeric antibodies 4, 5, and 6 had equivalent in vitro ADCC activity inhibitory effect as the control antibodies. Wherein the control antibody is FUT8 gene silenced IMAB362 according to the knockout process described in patent application 202110752316.6, and wherein the ADCC effect of FUT8 gene silenced IMAB362 is increased 5-fold compared to IMAB362 without knockout of FUT8 gene. The control antibodies showed 5-fold improvement in the in vitro ADCC activity inhibitory effect, and the chimeric antibodies 4, 5, and 6 showed a remarkable in vitro ADCC activity inhibitory effect, as compared with the control antibodies.
CDR engineering of the sequence of example 5
CDR-engineering the murine sequence of murine antibody 1 to obtain chimeric antibodies A, B, C, D, E, F, G and Z1 (reference); CDR-engineering of the humanized sequence of humanized antibody 7 resulted in humanized antibody H, I, J, K, L, M, N, O, P, Q, R. The specific sequences of the antibodies are shown in Table 2.
TABLE 2 antibody sequence listing
| Antibody name | Heavy chain SEQ ID NO. | Light chain SEQ ID NO. |
| Antibody A | 52 | 61 |
| Antibody B | 53 | 40 |
| Antibody C | 53 | 61 |
| Antibody D | 54 | 40 |
| Antibody E | 54 | 61 |
| Antibody F | 55 | 40 |
| Antibody G | 55 | 61 |
| Antibody H | 44 | 62 |
| Antibody I | 56 | 45 |
| Antibody J | 56 | 62 |
| Antibody K | 57 | 45 |
| Antibody L | 57 | 62 |
| Antibody M | 58 | 45 |
| Antibody N | 58 | 62 |
| Antibody O | 59 | 45 |
| Antibody P | 59 | 62 |
| Antibody Q | 60 | 45 |
| Antibody R | 60 | 62 |
| Z1 (reference) | 52 | 40 |
Example 6 chimeric antibody A, B, C, D, E, F, G binding Capacity to cells assay
The antibody to be tested is taken, diluted by 2 times and then diluted by 4 times according to the initial final concentration of 40ug/mL, and diluted by 8 gradients. Cells overexpressing human Claudin18.2 (human Claudin18.2-CHOK 1) in the incubator were removed, the cell suspension was transferred to a 15ml centrifuge tube, centrifuged, and counted in PBS for resuspension. A Blank control (Blank), a Negative Control (NC), an experimental group, an irrelevant antibody group, and Z1 as a control antibody were set aside. According to about 2X 10 5 Cell/well, cell suspensions were plated in 96-well plates. Centrifugation (1000 rpm,5 min), followed by washing with PBS, and further centrifugation were repeated twice to remove the medium residue. The supernatant was discarded, 100. Mu.L of the primary antibody solution and the independent antibody solution were added to each of the experimental group and the independent antibody solution, and the cells were resuspended and incubated at room temperature for 1 hour. Blank, NC groups were incubated with equal amounts of PBS. After 1h, the mixture was centrifuged, and washed twice with PBS. After discarding the supernatant Except for the blank group, 100. Mu.L of PBS was added, 100. Mu.L of a fluorescent secondary antibody diluent (humanized antibody secondary antibody is derived from Biolegend 398006) was added to each of the rest samples, and after incubation for 0.5h at room temperature in the absence of light, the samples were centrifuged and washed twice with PBS. After discarding the supernatant, 120. Mu.L of PBS was added for resuspension and flow cytometry was performed sequentially to determine the average fluorescence intensity. The results are shown in FIG. 15.
The results showed that the binding curves of each chimeric antibody were concentration gradient dependent. The binding activity of chimeric antibody A, B, C, D, E to human Claudin18.2-CHOK1 was substantially identical to the binding activity of the control antibody to human Claudin18.2-CHOK1, and the binding activity of chimeric antibody F and chimeric antibody G to human Claudin18.2-CHOK1 was lower than the binding activity of the control antibody to human Claudin18.2-CHOK 1.
EXAMPLE 7 detection of the binding Capacity of humanized antibody H, I, J, K, L, M, N, O, P, Q, R to cells
The antibody to be tested was taken and diluted 6 times, 6 gradients, with 20ug/mL as initial final concentration. Cells overexpressing human Claudin18.2 (human Claudin18.2-CHOK 1) in the incubator were removed, the cell suspension was transferred to a 15mL centrifuge tube, centrifuged, and counted in PBS for resuspension. A Blank control (Blank), a Negative Control (NC), an experimental group, an irrelevant antibody group, and Z1 and IMAB362 were used as control antibodies. According to about 2X 10 5 Cell/well, cell suspensions were plated in 96-well plates. Centrifugation (1000 rpm,5 min), followed by washing with PBS, and further centrifugation were repeated twice to remove the medium residue. The supernatant was discarded, 100. Mu.L of the primary antibody solution and the independent antibody solution were added to each of the experimental group and the independent antibody solution, and the cells were resuspended and incubated at room temperature for 1 hour. Blank, control were incubated with equal amounts of PBS. After 1h, the mixture was centrifuged, and washed twice with PBS. After discarding the supernatant, 100. Mu.L of a fluorescent secondary antibody diluent (murine secondary antibody is derived from Abcam ab6785, chimeric and humanized antibody secondary antibodies are derived from Biolegend 398006) was added to each of the remaining samples except for the blank, and after incubation for 0.5h at room temperature in the absence of light, the samples were washed twice by centrifugation with PBS. After discarding the supernatant, 120. Mu.L of PBS was added for resuspension and flow cytometry was performed sequentially to determine the average fluorescence intensity. The results are shown in FIGS. 16 and 17.
The results showed that the binding curves of each humanized antibody were concentration gradient dependent. The binding activity of humanized antibody H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1 is substantially identical to the binding activity of control antibody Z1 to human Claudin18.2-CHOK 1; the binding activity of humanized antibody H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1 was substantially identical to the binding activity of control antibody IMAB362 to human Claudin18.2-CHOK 1.
Example 8 CDC Activity assay of humanized antibody H, J, L
The 96-well plate was removed, and the test antibody H, antibody J, antibody L, positive control antibody (IMAB 362) and isotype control antibody were diluted 4-fold starting at 10ug/ml, 7-fold gradient dilutions were performed, and 2 parallel wells, 50 ul/well, were set. Taking out CHOK1A2HIS cell count, and setting cell number to 2×10 4 Each 100 ul/well was incubated with antibody at 37℃for 15min. Complement was added to retain human serum at a concentration of 20%/50 uL/well, i.e. a final concentration of 5%/well. Incubate at 37℃for 1h. CCK-8, 20 ul/well, was added and incubated at 37℃for 4h, and the microplate reader was read at a wavelength of 450 nm. The results are shown in FIG. 18.
The results showed that the CDC activity of the humanized antibody H, J, L was substantially identical to that of the control antibody.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.
Claims (20)
- An anti-Claudin 18.2 antibody or antigen binding fragment thereof, comprising a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein,(a) HCDR1 of the heavy chain variable region selected from SEQ ID NO: 1. 9, 15, 19, or an amino acid sequence identical to any one of SEQ ID NOs: 1. 9, 15, 19, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO: 1. 9, 15, 19, an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences;(b) HCDR2 of the heavy chain variable region selected from SEQ ID NO: 2. 10, 16, 20, 21, 22, 23, 24, 25, or an amino acid sequence identical to SEQ ID NO: 2. 10, 16, 20, 21, 22, 23, 24, 25, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NOs: 2. 10, 16, 20, 21, 22, 23, 24, 25, has one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences;(c) HCDR3 of the heavy chain variable region selected from SEQ ID NO: 3. 11, 17, 26, 27, 63, or an amino acid sequence identical to SEQ ID NO: 3. 11, 17, 26, 27, 63, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NOs: 3. 11, 17, 26, 27, 63, an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences;(d) LCDR1 of the light chain variable region selected from SEQ ID NO: 4. 28, or an amino acid sequence that hybridizes to any one of SEQ ID NOs: 4. 28, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO: 4. 28, having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence;(e) LCDR2 of the light chain variable region selected from SEQ ID NO:5, or an amino acid sequence identical to SEQ ID NO:5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO:5, having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences; and/or(f) LCDR3 of the light chain variable region selected from SEQ ID NO: 6. 12, or an amino acid sequence that hybridizes to any one of SEQ ID NOs: 6. 12, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO: 6. 12, having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence.
- The anti-claudin 18.2 antibody or antigen-binding fragment thereof according to claim 1, wherein the HCDR1, HCDR2, HCDR3 of the heavy chain variable region, and the LCDR1, LCDR2, LCDR3 of the light chain variable region are selected from any one of the amino acid sequences (1) - (11) as follows:(1) SEQ ID NO:1, HCDR1 shown in SEQ ID NO:2, HCDR2 shown in SEQ ID NO:3, HCDR3, SEQ ID NO:4, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;(2) SEQ ID NO:9, HCDR1, SEQ ID NO:10, HCDR2 shown in SEQ ID NO:11, HCDR3 shown in SEQ ID NO:4, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 12;(3) SEQ ID NO:15, HCDR1, SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17, HCDR3, SEQ ID NO:4, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 12;(4) SEQ ID NO:19, HCDR1, SEQ ID NO:20, HCDR2 shown in SEQ ID NO:63, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;(5) SEQ ID NO:19, HCDR1, SEQ ID NO:21, HCDR2 shown in SEQ ID NO:63, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;(6) SEQ ID NO:19, HCDR1, SEQ ID NO:22, HCDR2 shown in SEQ ID NO:26, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;(7) SEQ ID NO:19, HCDR1, SEQ ID NO:23, HCDR2 shown in SEQ ID NO:63, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;(8) SEQ ID NO:19, HCDR1, SEQ ID NO:24, HCDR2 shown in SEQ ID NO:63, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;(9) SEQ ID NO:19, HCDR1, SEQ ID NO:25, HCDR2 shown in SEQ ID NO:63, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;(10) SEQ ID NO:19, HCDR1, SEQ ID NO:23, HCDR2 shown in SEQ ID NO:27, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6;(11) SEQ ID NO:19, HCDR1, SEQ ID NO:24, HCDR2 shown in SEQ ID NO:27, HCDR3 shown in SEQ ID NO:28, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in fig. 6.
- An anti-claudin 18.2 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:(a) The heavy chain variable region has the amino acid sequence of SEQ ID NO: 7. 13, 18, 29, 30, 31, 33, 34, 35, 36, 37,or with SEQ ID NO: 7. 13, 18, 29, 30, 31, 33, 34, 35, 36, 37, a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,or with SEQ ID NO: 7. 13, 18, 29, 30, 31, 33, 34, 35, 36, 37, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences;(b) The light chain variable region has the amino acid sequence of SEQ ID NO: 8. 14, 32, 38; or with SEQ ID NO: 8. 14, 32, 38 has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of the amino acid sequences set forth herein,or with SEQ ID NO: 8. 14, 32, 38, and an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).
- An anti-claudin 18.2 antibody or antigen binding fragment thereof according to claim 3, wherein the heavy chain variable region and the light chain variable region are selected from any one of the amino acid sequences of (1) to (11):(1) SEQ ID NO:7 and SEQ ID NO:8, 8;(2) SEQ ID NO:13 and SEQ ID NO:14;(3) SEQ ID NO:18 and SEQ ID NO:14;(4) SEQ ID NO:29 and SEQ ID NO:32;(5) SEQ ID NO:30 and SEQ ID NO:32;(6) SEQ ID NO:31 and SEQ ID NO:32;(7) SEQ ID NO:33 and SEQ ID NO:38, a step of carrying out the process;(8) SEQ ID NO:34 and SEQ ID NO:38, a step of carrying out the process;(9) SEQ ID NO:35 and SEQ ID NO:38, a step of carrying out the process;(10) SEQ ID NO:36 and SEQ ID NO:38, a step of carrying out the process;(11) SEQ ID NO:37 and SEQ ID NO:38.
- the anti-claudin 18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody is a murine, chimeric, humanized or fully human antibody.
- The anti-claudin 18.2 antibody or antigen binding fragment thereof according to any one of claims 1-5, wherein the antibody is a monoclonal antibody.
- The anti-claudin 18.2 antibody or antigen binding fragment thereof according to any one of claims 1-6, further comprising an Fc region selected from the group consisting of mouse IgG1, igG2a, igG2b and/or IgG3, or from the group consisting of rat IgG1, igG2a, igG2b and/or IgG2c.
- The anti-claudin 18.2 antibody or antigen binding fragment thereof according to any one of claims 1-6, further comprising an Fc region selected from the group consisting of human IgG1, igG2, igG3 and/or IgG4.
- A nucleic acid molecule encoding the anti-claudin 18.2 antibody or antigen binding fragment thereof of any one of claims 1-8.
- A multifunctional fusion protein comprising the anti-claudin 18.2 antibody or antigen-binding fragment thereof of any one of claims 1-8.
- The multifunctional fusion protein of claim 10 further comprising one or more secondary antibodies or antigen-binding portions thereof that specifically bind to other antigens.
- The multifunctional fusion protein of claim 11 wherein the antigen that binds to the second antibody or antigen binding portion thereof is selected from a Tumor Associated Antigen (TAA) or an immune checkpoint.
- The multifunctional fusion protein of claim 12 wherein the immune checkpoint is PD-L1, CTLA4, PD-L2, PD-1, 4-1BB, CD47, TIGIT, GITR, TIM3, ILT4, TNFR2, TREM2, LAG3, CD27, B7H3 or B7H4.
- The multifunctional fusion protein of any one of claims 10-13 further comprising a cytokine.
- The multifunctional fusion protein according to claim 14, the cytokine is selected from the group consisting of IL-1, IL-2R alpha, IL-2R beta, IL-3R alpha, IL-4R alpha, IL-5R alpha, IL-6 IL-6R α, IL-7R α, IL-8, IL-9R α, IL-10R1, IL-10R2, IL-11R α, IL-12R α, IL-10R2, IL-11, R α, IL-12, IL-9R α, IL-10R1, IL-12, IL-3, and IL-3 IL-12R beta 2, IL-12R beta 1, IL-13R alpha, IL-13R alpha 2, IL-14, IL-15R alpha sushi, IL-16, IL-17, IL-18, IL-19, IL-20R1, IL-20R2, IL-21R alpha, IL-22, IL-23R, IL-27R, IL-31R, TGF, VEGF, IFN gamma, IFN alpha or GM-CSF.
- Use of an anti-claudin 18.2 antibody or antigen binding fragment thereof according to any one of claims 1-8 or a multifunctional fusion protein according to any one of claims 10-15 for the manufacture of a medicament for the treatment of cancer.
- The use according to claim 16, wherein the cancer is lung cancer, liver cancer, melanoma, glioblastoma, head and neck cancer, esophageal cancer, gastric cancer, prostate cancer, ovarian cancer, bladder cancer, pancreatic cancer, gastric cancer, colon cancer, cervical cancer or related tumors.
- The use according to claim 16 or 17, wherein the use is achieved by one or more of tumor immunotherapy, cell therapy and gene therapy.
- A pharmaceutical composition comprising the anti-claudin 18.2 antibody or antigen-binding fragment thereof of any one of claims 1-8 and a pharmaceutically acceptable carrier, diluent or excipient.
- A pharmaceutical composition comprising the multifunctional fusion protein of any one of claims 10-15 and a pharmaceutically acceptable carrier, diluent or excipient.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2021120034 | 2021-09-24 | ||
| CNPCT/CN2021/120034 | 2021-09-24 | ||
| PCT/CN2022/120428 WO2023045997A1 (en) | 2021-09-24 | 2022-09-22 | Anti-claudin18.2 antibody and use thereof |
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| CN117897403A true CN117897403A (en) | 2024-04-16 |
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| CN202280052157.1A Pending CN117897403A (en) | 2021-09-24 | 2022-09-22 | Claudin 18.2-resistant antibody and application thereof |
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| CN (1) | CN117897403A (en) |
| WO (1) | WO2023045997A1 (en) |
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| WO2013167153A1 (en) * | 2012-05-09 | 2013-11-14 | Ganymed Pharmaceuticals Ag | Antibodies useful in cancer diagnosis |
| 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 |
| CN112912396B (en) * | 2018-10-22 | 2023-03-14 | 上海吉倍生物技术有限公司 | anti-CLDN 18.2 antibodies and uses thereof |
| CN109762067B (en) * | 2019-01-17 | 2020-02-28 | 北京天广实生物技术股份有限公司 | Antibodies that bind human Claudin18.2 and uses thereof |
| CN112574307B (en) * | 2019-09-29 | 2023-11-28 | 迈威(上海)生物科技股份有限公司 | Anti-human Claudin18.2 antibody and application thereof |
| WO2021130291A1 (en) * | 2019-12-23 | 2021-07-01 | SOTIO a.s. | Tumor-specific claudin 18.2 antibodies |
| CN115461373B (en) * | 2020-02-10 | 2024-03-29 | 上海诗健生物科技有限公司 | Antibodies to claudin18.2 and uses thereof |
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| WO2023045997A1 (en) | 2023-03-30 |
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