Patent Application
20240050472
The present application belongs to the field of biomedicines and in particular, relates to an anti-Claudin 18.2 antigen-binding fragment and antibody, and use thereof.
Tight junction is one of the forms of cell adhesion and is normally present in the junctional complexes in epithelial cells and endothelial cells. Tight junction molecules are composed of three types of whole membrane proteins including Occludin, Claudin proteins and junctional adhesion molecules and peripheral cytoplasmic proteins such as zonula occludens proteins. At present, it has been proven that Claudin proteins are important components of tight junctions of cells and establish the paracellular barrier that controls the flow of molecules between the cells. They have four transmembrane domains, with the NH2-terminus and the COOH-terminus both located intracellularly. The abnormal expression of Claudin proteins may lead to structural destruction and functional damage of epithelial cells and endothelial cells, and thus Claudin proteins play an important role in the pathogenesis of various diseases.
Claudin-18 (CLDN18) belongs to the claudin family, and its encoding gene can form two subtypes of proteins: Claudin 18.1 (CLD18A1, GENBANK®: NM 016369) and Claudin 18.2 (CLD18A2, GENBANK®: NM 001002026) by alternative splicing. Claudin 18.1 is mainly expressed in normal lung tissues; Claudin 18.2, as a highly specific cell surface molecule, is only expressed on differentiated gastric epithelial cells (short-lived gastric epithelial cells) in normal tissues, but not on gastric stem cells. Claudin 18.2 is expressed in most of primary gastric cancers and their metastatic cancers, and the expression of such a target is present in 50%-80% of patients with gastric cancer. In addition, the activation and expression of Claudin 18.2 are often observed in pancreatic cancer, esophageal cancer, ovarian cancer and lung cancer. These characteristics indicate that Claudin 18.2 is an ideal drug target for tumor therapy.
Therefore, providing an antibody specifically binding to Claudin 18.2 has important therapeutic significance for cancer caused by Claudin 18.2-positive tumor cells.
The present application provides an anti-Claudin 18.2 antigen-binding fragment and antibody, and use thereof. The antigen-binding fragment and antibody can specifically bind to Claudin 18.2 proteins, prepare the Claudin 18.2 proteins into chimeric antigen receptors and CAR-T cells, and have significant cytotoxicity on target cells expressing the Claudin 18.2 proteins.
In a first aspect, the present application provides an anti-Claudin 18.2 antigen-binding fragment including a heavy chain variable region and a light chain variable region;
wherein the heavy chain variable region of the antigen-binding fragment includes CDR3, and CDR3 includes an amino acid sequence shown in SEQ ID NO. 3; and
the light chain variable region of the antigen-binding fragment includes CDR3, and CDR3 includes an amino acid sequence shown in SEQ ID NO. 6.
In the present application, the antigen-binding fragment can specifically bind to Claudin 18.2, the antibody containing the antigen-binding fragment can specifically bind to a variety of sources of Claudin 18.2 proteins including human, murine, monkey and the like, and the antigen-binding fragment does not bind to Claudin 18.1. The antigen-binding fragment has a high specificity and is important for the research of drugs or vaccines targeting Claudin 18.2.
In some specific embodiments, the heavy chain variable region of the antigen-binding fragment further includes CDR1, and CDR1 includes an amino acid sequence shown in SEQ ID NO. 1.
In some specific embodiments, the light chain variable region of the antigen-binding fragment further includes CDR1, and CDR1 includes an amino acid sequence shown in SEQ ID NO. 4.
In some specific embodiments, the heavy chain variable region of the antigen-binding fragment further includes CDR2, and CDR2 includes an amino acid sequence shown in SEQ ID NO. 2.
In some specific embodiments, the light chain variable region of the antigen-binding fragment further includes CDR2, and CDR2 includes an amino acid sequence shown in SEQ ID NO. 5.
Corresponding sequences are shown in Table 1 below.
In some specific embodiments, CDR1 of the heavy chain variable region of the antigen-binding fragment is an amino acid sequence shown in SEQ ID NO. 1, CDR2 is an amino acid sequence shown in SEQ ID NO. 2, and CDR3 is an amino acid sequence shown in SEQ ID NO. 3; CDR1 of the light chain variable region of the antigen-binding fragment is an amino acid sequence shown in SEQ ID NO. 4, CDR2 is an amino acid sequence shown in SEQ ID NO. 5, and CDR3 is an amino acid sequence shown in SEQ ID NO. 6.
In a second aspect, the present application provides an anti-Claudin 18.2 antibody including the antigen-binding fragment described in the first aspect.
In some specific embodiments, an amino acid sequence of a heavy chain variable region of the anti-Claudin 18.2 antibody is shown in SEQ ID NO. 7, and an amino acid sequence of a light chain variable region is shown in SEQ ID NO. 8.
The amino acid sequence of the heavy chain variable region (VH) of the antibody is as follows (SEQ ID NO. 7):
INPYNDGTK
YNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRG
YYGPYFDY
WGQGTTLTVSS.
The amino acid sequence of the light chain variable region (VL) of the antibody is as follows (SEQ ID NO. 8):
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNGGNQKNYLTWYQQKPGQPPKLLIYWA STRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYYYPYTFGGGTKLEIK. The underlines in the preceding sequences are complementarity-determining regions.
Preferably, the anti-Claudin 18.2 antibody further includes a constant region.
Preferably, the anti-Claudin 18.2 antibody is modified with a glycosylation group.
The anti-Claudin 18.2 antibody may be present in the form of monomers or polymers. If the anti-Claudin 18.2 antibody is present in the form of polymers, one of its heavy chains and one of its light chains form an interchain disulfide bond, another heavy chain and another light chain form an interchain disulfide bond, and the former heavy chain and the later heavy chain form two interchain disulfide bonds.
Meanwhile, the present application further provides a method for preparing the anti-Claudin 18.2 antibody described in the second aspect, and the method specifically includes the following steps:
In a third aspect, the present application provides a nucleic acid molecule including a DNA fragment for encoding the antigen-binding fragment described in the first aspect or the anti-Claudin 18.2 antibody described in the second aspect.
In a fourth aspect, the present application provides an expression vector including the nucleic acid molecule described in the third aspect.
In a fifth aspect, the present application provides a chimeric antigen receptor (CAR) including the anti-Claudin 18.2 antibody described in the second aspect.
In the present application, CAR-T cells containing the chimeric antigen receptor can highly express the anti-Claudin 18.2 antibody and has significant cytotoxicity on Claudin 18.2-positive cells.
Preferably, the chimeric antigen receptor further includes a signal peptide (Leader), a hinge region (Hinge), a transmembrane domain (TM), a co-stimulatory domain (ICD) and a signal transduction domain.
Preferably, the signal peptide includes a CD8α signal peptide and/or an IgGκ light chain signal peptide, and preferably is an IgGκ light chain signal peptide.
Preferably, the hinge region includes any one of a CD8α, CD28, human IgG1, IgG2, IgG4 or IgA hinge region, and preferably is a CD8α hinge region.
Preferably, the transmembrane domain includes a CD8α transmembrane region and/or a CD28 transmembrane region, and preferably is a CD8α transmembrane region.
Preferably, the signal conduction domain includes a CD3ζ signal conduction domain.
Preferably, the signal transduction domain further includes a co-stimulatory domain, for example, any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10 or OX40.
In the present application, the chimeric antigen receptor targeting Claudin 18.2 includes an IgGκ light chain signal peptide, an anti-Claudin 18.2 antibody (scFv), a CD8α hinge region, a CD8α transmembrane region, 4-1BB and CD3ζ.
In the present application, the chimeric antigen receptor includes an IgGκ light chain signal peptide sequence, an antibody sequence (8D2-scFv) specifically binding to a Claudin 18.2 antigen, a CD8a hinge region sequence, a CD8α transmembrane region sequence, a 4-1BB co-stimulatory domain sequence and a CD3ζ signal transduction domain sequence.
The amino acid sequence of the IgGκ light chain signal peptide (SEQ ID NO. 9) is as follows:
The amino acid sequence of the CD8α hinge region (hinge) (SEQ ID NO. 11) is as follows:
The amino acid sequence of the CD8α transmembrane region (TM) (SEQ ID NO. 13) is as follows:
The amino acid sequence of the 4-1BB intracellular co-stimulatory domain (ICD) (SEQ ID NO. 15) is as follows:
The amino acid sequence of the CD3ζ signal transduction domain (SEQ ID NO. 17) is as follows:
In a sixth aspect, the present application provides a host cell including the nucleic acid molecule described in the third aspect, the expression vector described in the third aspect or the chimeric antigen receptor described in the fifth aspect.
In a seventh aspect, the present application provides a pharmaceutical composition including the anti-Claudin 18.2 antibody described in the second aspect.
Preferably, the pharmaceutical composition further includes an anti-tumor drug.
In the present application, the pharmaceutical compositions may also be used in combination with other anti-tumor drugs in a manner of simultaneous administration, separate administration or sequential administration.
Preferably, the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.
In an eighth aspect, the present application provides use of the antigen-binding fragment described in the first aspect, the anti-Claudin 18.2 antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the host cell described in the sixth aspect or the pharmaceutical composition described in the seventh aspect in the preparation of a cancer detection reagent and/or a cancer treatment drug.
Preferably, the cancer includes a Claudin 18.2-positive cancer.
Preferably, the cancer includes any one of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer or gallbladder cancer.
Any numerical range described in the present application includes not only the above-listed point values but also any point values within the numerical range which are not listed. Due to the limitation of space and the consideration of simplicity, specific point values included in the range are not exhaustively listed in the present application.
Compared with the prior art, the present application has at least the beneficial effects described below.
(1) The antigen-binding fragment and anti-Claudin 18.2 antibody provided in the present application can specifically bind to a variety of species sources (including human, murine and cynomolgus monkey) of Claudin 18.2 proteins and have more choices in the selection of subsequent animal models; the EC50 value of the antibody binding to 293T-Hu18.2 is 2.303, the EC50 value of the antibody binding to 293T-Mu18.2 is 7.331, the EC50 value of the antibody binding to 293T-RM18.2 is 9.159, and the EC50 value of the antibody binding to MFC-Hu18.2 is 2.727 E-12; moreover, the antibody does not bind to 293T and murine MFC cells which stably express human Claudin 18.1, and through the membrane proteome array experiment, it can be seen that 8D2-scFv-hFc can specifically bind to Claudin 18.2 and does not bind to other non-target proteins, indicating that the anti-Claudin 18.2 antibody has significant specificity.
(2) The present application provides a chimeric antigen receptor 8D2 CAR. After the chimeric antigen receptor is transferred into T cells through lentivirus vectors, CAR-T cells expressing 8D2 CAR are obtained. The CAR-T cells have significant cytotoxicity on cells stably expressing Claudin 18.2 proteins and thus have a significant therapeutic value for Claudin 18.2-positive cancers such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer and gallbladder cancer.
wherein
Technical solutions of the present application are further described below through embodiments in conjunction with drawings. However, the following examples are simple examples of the present application and do not represent or limit the protection scope of the present application. The protection scope of the present application is subject to the claims.
In the following examples, unless otherwise specified, all reagents and consumables used are purchased from conventional reagent manufacturers in the art; and unless otherwise specified, the experimental methods and technical means used are conventional experimental methods and to technical means known in the art.
In this example, BALB/c mice were immunized with DNA to prepare hybridoma monoclonal cells, hybridoma cells were cultured by using a selective culture medium R1640-HAT and then were cultured in an HT culture medium solution, and then hybridoma supernatant samples were detected by ELISA to obtain 99 positive clones;
subsequently, the flow screening experiment was carried out to subclone 7 parent clones (which bound to Claudin 18.2 and did not bind to Claudin 18.1) to obtain 6 subclones.
The correct sequence clone 8D2 was obtained by sequencing, that is, the anti-Claudin 18.2 antibody. After sequencing and identification, it is found that the sequence of the anti-Claudin 18.2 antibody 8D2 was as follows:
INPYNDGTK
YNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRG
YYGPYFDY
WGQGTTLTVSS;
PYT
FGGGTKLEIK.
In this example, the construction of cell lines stably expressing Claudin 18.2 proteins was detected by flow cytometry, and the steps were as follows.
(1) Construction of expression vectors of Claudin 18.1 and Claudin 18.2 and preparation of lentivirus
The complete coding sequence of human Claudin 18.1 (GENBANK®: NM 016369, referred to as “Hu18.1” hereinafter), the complete coding sequence of human Claudin 18.2 (GENBANK®: NM_001002026, referred to as “Hu18.2” hereinafter), the complete coding sequence of murine Claudin 18.2 (GENBANK®: NM_001194921.1, referred to as “Mu18.2” hereinafter) and the complete coding sequence of monkey Claudin 18.2 (GENBANK®: XM 001114708.4, referred to as “RM18.2” hereinafter) were synthesized by the complete series of PCR-based gene synthesis technology;
after enzyme digestion, ligation and transformation, the clones were selected for PCR identification and sequencing to confirm that the correct lentivirus vector plasmids pCDH-Claudin 18.1 and pCDH-Claudin 18.2 were obtained;
the above plasmids were co-transfected into 293T cells with gag/pol, Rev and VSV-G vectors which were required for a four-plasmid system to package lentivirus vectors, respectively; after 72 h of transfection, Claudin 18.1 and Claudin 18.2 virus solutions were collected, concentrated, subpackaged and stored at −80° C.
(2) Construction of Claudin 18.1 and Claudin 18.2 exogenous stable expression lines and flow assay
The collected Claudin 18.1 and Claudin 18.2 virus solutions were added to 293T cells and mouse gastric cancer cells MFC (purchased from NANJING CO-BIOER, CBP60882) spread in T75 cell culture flasks, respectively;
in addition, human gastric cancer cells NCI-N87 (purchased from NANJING CO-BIOER, CBP60491) and human gastric cancer cells MKN45 (purchased from NANJING CO-BIOER, CBP60488) were infected with the Claudin 18.2 virus solution for constructing 293T-Hu18.1, MFC-Hu18.1, 293T-Hu18.2, MFC-Hu18.2, NCI-N87-Hu18.2, MKN45-Hu18.2, 293T-Mu18.2 and 293T-RM18.2 cell lines, respectively. Specific information is shown in Table 2 below.
After continuous screening and culture with puromycin, the construction of the above cell lines was detected by flow antibodies, and the results are as shown in
In this example, the binding ability of the antibody 8D2 to each cell line was analyzed by a flow cytometer (BECKMAN COULTER, CytoFLEX S Flow Cytometer).
The specific method is as follows:
Flow cytometry analysis showed that the antibody 8D2 could specifically recognize 293T, MKN45, NCI-N87 and murine MFC cells stably expressing human Claudin 18.2 and did not bind to 293T and murine MFC cells stably expressing human Claudin 18.1; therefore, the 8D2 antibody could specifically recognize human Claudin 18.2 proteins; meanwhile, the antibody 8D2 could also bind to cells stably expressing murine and monkey Claudin 18.2, indicating that the antibody 8D2 could also specifically recognize murine and monkey Claudin 18.2.
Some flow cytometry analysis results are shown in
As shown in
As shown in
In this example, an anti-Claudin 18.2 chimeric antigen receptor (8D2 CAR) and its expression vector were constructed.
(1) Sequence Design
The chimeric antigen receptor included an IgGκ light chain signal peptide sequence (Leader), an antibody sequence (8D2-scFv) specifically binding to a Claudin 18.2 antigen, a CD8α hinge region sequence (Hinge), a CD8α transmembrane region sequence, a 4-1BB co-stimulatory domain sequence and a CD3ζ signal transduction domain sequence.
The specific structure is shown in
(2) Construction of an Anti-Claudin 18.2 Chimeric Antigen Receptor Expression Vector
First, the 8D2 CAR sequence was synthesized by gene synthesis, and the 8D2 CAR synthesized by gene synthesis and empty vectors were double digested with EcoRI and BamHI. After 30 min of digestion in a water bath at 37° C., DNA electrophoresis was carried out with 1.5% agarose gel, and then the 8D2 CAR and the empty vectors were purified and recovered with an agarose gel kit from TIANGEN;
then, the pCDH-EF1 vectors were ligated to the 8D2 CAR gene fragments, and the specific connection system is shown in Table 4 below:
In this example, the lentivirus expression vector was packaged with lentivirus, CAR-T cells were constructed, and the infection efficiency of lentivirus to T cells was detected.
(1) Lentivirus Packaging with a Four-Plasmid System
The four-plasmid system expressed gag/pol, Rev and VSV-G which were required for lentivirus vector packaging as well as an artificial chimeric antigen receptor composed of engineered stable single-chain antibodies, respectively. The four plasmids were transfected into 293T cells instantaneously, with a total mass of 10 μg;
the plasmids were added into serum-free DMEM, mixed and placed for 15 min, and then the mixture was added to a T75 culture flask spread with 293T cells, gently mixed and cultured in a 5% CO2 cell incubator at 37° C. for 6 h;
after 6 h, a fresh culture medium was replaced for continued culture, and 10 mM of sodium butyrate solution was added; after 72 h, the culture supernatant of lentivirus was collected for purification and detection.
(2) Amplification of CAR-T Cells
30 mL of whole blood was collected, peripheral blood was diluted with normal saline at 1:1, Ficoll was added to a centrifuge tube, the diluted peripheral blood was slowly added to the centrifuge tube and centrifuged at 1500 rpm for 30 min, and the PBMC layer was gently pipetted into another centrifuge tube;
the PBMC was washed with normal saline several times and then transferred into a CAR-T cell medium (containing 50 ng/mL OKT3 and 300 IU/mL IL-2) for culture;
after the PBMC was isolated, the PBMC needed to be activated with the CAR-T cell medium containing 50 ng/mL OKT3 and 300 IU/mL IL-2;
after 2 days, the medium was replaced with a CAR-T cell medium containing 300 IU/mL for expanded culture;
then, the cells were counted every two days, the CAR-T cell culture medium containing 300 IU/mL was also replaced every two days, the cell concentration was maintained at 0.5×106-1×106/mL, and the cells were observed continuously for 10 days.
(3) Infection of T Cells with Lentivirus
The infection efficiency of lentivirus to T cells was improved using RetroNectin, and 30 μg of RetroNectin was coated in a 6-well plate and placed in a cell incubator at 37° C. for 2 h;
RetroNectin was absorbed, and the coated 6-well plate was sealed with the Hank's solution containing 2.5% BSA and placed in a cell incubator at 37° C. for 0.5 h;
the sealant was absorbed, the 6-well plate was washed with the Hank's solution containing 2% Hepes, an X-VIVO medium was added, an appropriate amount of lentivirus solution was added, and the mixture was centrifuged at 2000 g for 2 h;
the supernatant was discarded, 1×106 T cells were added, centrifuged at 1000 g for 10 min and cultured in a 5% CO2 cell incubator at 37° C., and the above procedure was repeated on the second day; the expression of 8D2 CAR was determined 5 days after infection, 8D2 CAR bound to FITC-Protein L, and the expression of 8D2 CAR was detected by flow cytometry.
The results are shown in
In this example, a cytotoxicity assay was carried out using 8D2 CAR, with the blank control NC (untransfected T cells) as a control.
The cytotoxicity of CAR-T cells to 293T-Hu18.1 cells (293T cells stably expressing human Claudin 18.1), 293T-Hu18.2 cells (293T cells stably expressing human Claudin 18.2) and 293T-Mu18.2 cells (293T cells stably expressing murine Claudin 18.2) cells was measured by LDH.
The detection method adopted in this example was carried out with reference to the detection method described in CN104877032A and included the following steps.
The cells were centrifuged, washed with a serum-free phenol red-free DMEM medium and then counted.
1×106 293T-Hu18.1, 293T-Hu18.2 and 293T-Mu18.2 cells were taken in 50 μL each and spread in a 96-well plate as target cells.
Untransfected T cells and each CAR-T cells were added according to the ratio of target cell:effector cell=1:1/4/8.
The cells were cultured at 37° C., 5% CO2 and a certain humidity for 12 h. The lysate was added as a positive control, and then the mixture was centrifuged at 250 g for 5 min. 100 μL of culture supernatant was taken from each well and added to a new 96-well plate, 20 μL of reaction solution was added, and the mixture reacted in a darkroom for 2030 min and detected with a microplate reader at 590 nm.
The dissolution percentage was calculated according to the following formula:
cytotoxicity (%)=[(experimental wells-medium background wells)−(effector cell spontaneous LDH release wells-medium background wells)−(target cell spontaneous LDH release wells-medium background wells)]/[(target cell maximum LDH release wells-volume correction wells)−(target cell spontaneous LDH release wells-medium background wells)]×100%.
The results are shown in
In this example, the non-target binding interaction of the antibody was verified using a Membrane Proteome Array.
First, the antibody fusion protein 8D2-scFv-hFc was expressed, and 8D2-scFv-hFc was the Fc segment of a single-chain antibody sequence of 8D2 fused with human IgG1; the membrane proteome array (MPA) was a platform for analyzing the targeting of specific antibodies and other ligands to human membrane proteins and could be used to determine the specificity of antibody targets.
Plasmids containing approximately 6000 membrane protein clones (94% or more of the human membrane protein group) were transfected into HEK-293T cells (ATCC, CRL-3216), respectively or to QT6 cells (ATCC, CRL-1708) in a 384-well cell culture plate (Corning, 3764), 18,000 cells/well.
After 36 h of incubation, the test antibody was added to the membrane proteome array matrix plate at a predetermined concentration, and the binding of the antibody 8D2-scFv-hFc to about 6000 types of membrane protein expression cells was directly detected by flow cytometry. Therefore, all target proteins had a native state and an appropriate post-translational modification.
The membrane proteome array results are shown in
In conclusion, the anti-Claudin 18.2 antibody provided by the present application can specifically bind to a variety of sources of Claudin 18.2 proteins, including human, murine and monkey ones, basically has no binding ability to other proteins and has a high specificity; meanwhile, the CAR provided herein and the T cell containing the CAR have significant cytotoxicity to cells expressing the Claudin 18.2 protein; therefore, the anti-Claudin 18.2 antibody provided herein has a specific therapeutic effect on diseases targeting the Claudin 18.2 protein.
The applicant states that the above are only the embodiments of the present application and are not intended to limit the protection scope of the present application. Those skilled in the art should understand that any changes or substitutions easily conceivable by those skilled in the art within the technical scope disclosed in the present application fall within the protection scope and the disclosed scope of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011486494.0 | Dec 2020 | CN | national |
This is a National Stage Application filed under 35 U.S.C. 371 based on International Patent Application No. PCT/CN2020/138240, filed on Dec. 22, 2020, which claims priority to Chinese Patent Application No. 202011486494.0, filed with the China National Intellectual Property Administration (CNIPA) on Dec. 16, 2020, the disclosures of which are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/138240 | 12/22/2020 | WO |
