Patent Application
20230365679
The present patent application claims the priority benefit of Chinese patent application No. CN202011048723.0 filed on Sep. 29, 2020, which is hereby incorporated by reference in its entirety.
The present disclosure relates to the field of biomedicine, in particular to a novel humanized anti-CD22 antibody and application thereof.
CD22 (Siglec-2) is a sialic acid-binding immunoglobulin-like lectin (Siglec) receptor, that binds specifically to sialic acid (Sia)-containing glycans, facilitating cell adhesion and/or cell signaling. CD22 expression is restricted to B cells and plays a critical role in establishing a baseline level of B-cell inhibition, and thus is a critical determinant of homeostasis in humoral immunity. CD22 is expressed in neoplastic cells in various B-cell malignancies, including B-lymphocytic leukemias/lymphomas and mature B-cell leukemias/lymphomas. The expression of CD22 can be particularly strong in hairy cell leukemia (HCL) and prolymphocytic leukemia.
Studies have shown that anti-CD22 antibodies can be used in the treatment of autoimmune diseases and B cell tumors. In addition, anti-CD22 antibodies are often developed for conjugation to other drugs, such as cytotoxic agents, enabling the targeted delivery of the drugs to target cells by means of their targeting properties.
Accordingly, there is a need in the art for a novel anti-CD22 antibody or fragment thereof that has a high affinity for CD22 and can be used in the treatment of CD22-related diseases, either alone or when conjugated to other drugs or molecules.
In order to solve the above technical problem, the present disclosure provides an isolated monoclonal antibody, which is a humanized anti-CD22 antibody that binds to CD22 and exhibits strong recognition ability and high affinity for recombinant CD22 protein and CD22-positive cells, strong internalization ability into CD22-positive cells, and potent growth inhibition and apoptosis induction effects on CD22-positive cells after fused with a bacterial exotoxin. Based on the isolated monoclonal antibody, the present disclosure provides various antibody formats comprising key domains of the antibody and corresponding uses thereof.
Technical solutions of the invention are as follows.
In one aspect, the present disclosure provides an isolated anti-CD22 antibody or antigen binding fragment thereof. As used herein, the “antigen binding fragment” of the antibody encompasses various functional fragments of the antibody that retain the binding ability of the antibody to CD22 and corresponding biological activities of the antibody.
With respect to sequence structure, the present disclosure provides an isolated anti-CD22 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) comprise respectively:
The anti-CD22 antibody or antigen-binding fragment thereof provided by the present disclosure is capable of binding to antigen CD22, particularly human CD22, with high affinity.
Preferably, in the anti-CD22 antibody or antigen binding fragment thereof provided by the present disclosure, the heavy chain variable region may comprise an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 5, or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 5; and/or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 8, or an amino acid sequence having at least 75% of identity to the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 8.
The “at least 75% identity” as used in the context of the present disclosure with respect to sequence is any percent identity no less than 75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity.
Further preferably, in the antibody or antigen binding fragment thereof provided by the present disclosure, the heavy chain variable region and the light chain variable region comprise respectively:
In particular, the antibody or antigen binding fragment thereof provided by the present disclosure comprises at least a heavy chain variable region and a light chain variable region, both comprising the CDRs as described above and framework regions in an arrangement as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further alternatively, the up to 25% difference in amino acid sequence due to the “at least 75% identity” may be present in any framework region of the heavy chain variable region or the light chain variable region, or in any domain or sequence in the antibody or antigen binding fragment thereof provided by the present disclosure outside of the heavy chain variable region and the light chain variable region. The difference may result from amino acid deletion, addition or substitution at any position.
The antibody provided by the present disclosure is a murine antibody, a chimeric antibody or a fully or partially humanized antibody; alternatively, the antibody is in the form of a scFv, dsFv, (dsFv)2, Fab, Fab′, F(ab′)2 or Fv antibody. Preferably, the antibody is a monoclonal antibody or a single chain antibody.
In addition to the variable regions, the antibody or antigen-binding fragment thereof comprises a human or murine constant region, preferably a human or murine heavy chain constant region (CH) and/or light chain constant region (CL). Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; more preferably, the antibody or antigen binding fragment thereof comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD and IgE and/or a kappa or lambda type light chain constant region. According to one particular embodiment of the invention, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody. According to one particular embodiment of the invention, the monoclonal antibody is an IgG1 antibody.
Further preferably, the antibody or antigen binding fragment thereof provided by the present disclosure comprises a heavy chain constant region comprising an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 9; and/or a light chain constant region comprising an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 10.
According to one particular embodiment of the invention, the present disclosure provides a monoclonal antibody having a heavy chain and a light chain comprising respectively:
In another aspect, the present disclosure also provides a nucleic acid molecule comprising a nucleotide sequence encoding the anti-CD22 antibody or antigen-binding fragment thereof provided by the present disclosure, or encoding a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain or a light chain comprised in the anti-CD22 antibody or antigen-binding fragment thereof. In a further aspect, the present disclosure provides a vector comprising a nucleic acid molecule as described above. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector and the like.
In yet a further aspect, the present disclosure provides a host cell comprising a nucleic acid molecule or vector as described above, or a host cell transformed or transfected with a nucleic acid molecule or vector as described above. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant or animal cell.
The antibody or antigen binding fragment thereof provided by the present disclosure can be obtained using any method known in the art. For example, the heavy chain variable region and/or the light chain variable region of the antibody, or the heavy chain and/or the light chain of the antibody, may be obtained from the nucleic acid molecule provided by the present disclosure, and then they may be assembled with the optional other domains of the antibody into an antibody; alternatively, the host cell provided by the present disclosure is cultured under conditions that allow the host cell to express the heavy chain variable region and/or the light chain variable region of the antibody or the heavy chain and/or the light chain of the antibody, and to assemble them into an antibody. Optionally, the method further comprises a step of recovering the produced antibody.
In another aspect, the present disclosure also provides a composition comprising an antibody or antigen binding fragment thereof, a nucleic acid molecule, a vector and/or a host cell provided by the present disclosure. Preferably, the composition is a pharmaceutical composition, which further optionally comprises a pharmaceutically acceptable carrier, adjuvant, and excipient.
In yet another aspect, the present disclosure also provides an immunoconjugate comprising the anti-CD22 antibody or antigen binding fragment thereof provided herein and a partner molecule which is a therapeutic agent. The therapeutic agent can be a cytotoxin (e.g., Pseudomonas aeruginosa exotoxin (e.g., PE38)), a radioisotope, an antibiotic, a small molecule toxin, other drug, etc.
In addition, the present disclosure also provides a kit comprising an antibody or antigen binding fragment thereof, a nucleic acid molecule, a vector, the host cell, a composition, and/or an immunoconjugate provided by the present disclosure.
In a further aspect, the invention also provides use of an antibody or antigen binding fragment thereof, a nucleic acid molecule, a vector, the host cell, a composition, and/or a immunoconjugate provided herein for the manufacture of a medicament for the treatment of a disease which includes a tumor or cancer expressing CD22. For example, the disease is a CD22-related B cell malignant tumor, or a B cell malignant tumor characterized by high expression of CD22, such as CD22-high expressing lymphoma or leukemia. Preferably, the lymphoma is non-hodgkin's lymphoma, small lymphocytic lymphoma or mantle cell lymphoma; and the leukemia is chronic lymphocytic leukemia, hairy cell leukemia or acute lymphocytic leukemia.
Accordingly, the present disclosure provides a method for treating a disease, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a composition, and/or an immunoconjugate provided herein. The subject is a mammal, including a human or non-human primate, and a domestic, farm or laboratory mammal such as dog, cat, cow, pig, sheep, horse, mouse, rabbit, etc. Preferably, the subject is a human. The disease includes a tumor or cancer expressing CD22. For example, the disease is a CD22-related B cell malignant tumor, or a B cell malignant tumor characterized by high expression of CD22, such as CD22 high expressing lymphoma or leukemia. Preferably, the lymphoma is non-hodgkin's lymphoma, small lymphocytic lymphoma or mantle cell lymphoma; and the leukemia is chronic lymphocytic leukemia, hairy cell leukemia or acute lymphocytic leukemia.
Embodiments of the present invention are described in detail below with reference to the attached figures, in which:
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the invention and do not limit the scope of the invention in any way.
Experimental procedures in the following examples are all conventional ones, unless otherwise specified. Raw materials and reagents used in the following examples are all commercially available products, unless otherwise specified.
In this Example, an IgG1-type monoclonal antibody was reconstructed by obtaining VH and VL region sequences and fusing them to constant regions.
A framework shuffling strategy was used to alter amino acids in the framework regions of heavy chain variable region (VH) and light chain variable region (VL) comprised in the recombinant immunotoxin HA22 from the original murine to human ones. All the CDRs, which were determined using both the Kabat and Chothia numbering systems and the online tool (http://www.bioinf.org.uk/abs/), were left unchanged. Each of the framework and J regions in both the VH and VL of HA22 was aligned to the human antibody germline sequences in the international ImMunoGeneTics information System® (IMGT: http://www.imgt.org) for the closest human antibody matches. Murine residues that were deemed as Vernier residues or important for VH/VL interactions were not changed to preserve the specificity and affinity towards CD22.
A panel of humanization designs were prepared and evaluated to select the final candidate that possessed maximum humanization while maintaining optimal affinity and efficacy profile.
Amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) contained in HA22 and humanized versions of the antibody are shown in Table 1. Elbow regions of the heavy and light chains were determined through defining antibody variable regions according to Kabat nomenclature, for subsequent mAb reconstruction; and CDR1, CDR2, and CDR3 were identified and underlined.
The antibody was determined to have a kappa-type light chain upon an analysis of the variable regions, so kappa-type light chain constant region and IgG1 heavy chain constant region were selected for the reconstruction of IgG antibodies. The constant region sequences are shown in Table 2.
Complete heavy and light chains shown in Table 3 were reconstructed through fusing the V region sequences provided in Table 1 to the constant region sequences provided in Table 2.
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK (SEQ ID NO: 11)
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC (SEQ ID NO: 12)
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK (SEQ ID NO: 13)
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC (SEQ ID NO: 12)
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK (SEQ ID NO: 13)
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC (SEQ ID NO: 14)
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK (SEQ ID NO: 15)
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC (SEQ ID NO: 14)
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK (SEQ ID NO: 15)
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC (SEQ ID NO: 16)
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK (SEQ ID NO: 17)
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC (SEQ ID NO: 16)
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK (SEQ ID NO: 18)
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC (SEQ ID NO: 16)
pCDNA 3.4 vector was used as the expression vector for the mAbs. After codon optimization, full genes were synthesized and inserted into the pCDNA 3.4 vector and expression vectors for transfection were prepared using endotoxin-free plasmid preparation kit. ExpicCHO cell strain was used as a host for transient expression host. After transient transfection, cell culture supernatants were harvested, and antibodies were obtained through affinity purification, and then identified and stored at −80° C.
In this Example, ELISA plates were coated with CD22 antigen to capture antibodies to be evaluated, and the color developed through an HRP-labeled secondary antibody capable of specifically recognizing Fd region, and the binding (EC50) of each mAb to the CD22 antigen was detected.
ELISA plates were coated with 200 ng/mL CD22 antigen (Biotinylated Human Siglec-2/CD22 Protein, Fc, Avitag™, ACROBiosystems (ARCO)) at 100 μl per well, at 4° C. overnight. Next day, the plates were washed with PBST, then blocked by the added blocking buffer (PBST+3% milk) at 37° C. for 1-2 hours, and then washed again with PBST.
Each antibody to be tested was diluted with PBST+1% BSA in gradient, to obtain antibody dilutions at concentrations of 0.01 ng/mL, 0.05 ng/mL, 0.25 ng/mL, 1.28 ng/mL, 6.40 ng/mL, 32 ng/mL, 160 ng/mL, 800 ng/mL, 4000 ng/mL, 20000 ng/mL, respectively; and 100 μl of each of the dilutions was added to each well of the ELISA plates which were then incubated at 37° C. for 1 hour.
After incubation, the plates were washed with PBST. 100 μl of 1:10000 diluted secondary antibody (Mouse anti-human IgG Fd secondary antibody, HRP; REF: SA5-10190, Invitrogen, USA) was added to each well of the ELISA plates which were then incubated at 37° C. for 1 hour. Thereafter, the plates were washed with PBST.
100 μl of TMB solution (TMB Single-Component Substrate solution, Cat #PR1200, Beijing Solarbio Science & Technology Co., Ltd.) was added to each well of the plates which were then incubated at room temperature for 15 minutes. Then 50 μl of stop buffer (ELISA stop buffer (10×), Cat #C1058, Beijing Solarbio Science & Technology Co., Ltd.) was added thereto, and the plates were shaken. Absorbance values at 450 nm were detected. Results are shown in
As shown in
EC50 value of the positive antibody HA22 mAb was 20.28 ng/mL, and EC50 values of mAb-1 to mAb-6 were all lower than that of HA22 mAb, and the EC50 values can be ranked as follows: mAb-2<mAb-6<mAb-3<mAb-4<mAb-1<mAb-5<HA22 mAb.
In this Example, the binding kinetics of the antibodies to the antigen was detected by SPR.
Following reagents and materials were mainly used:
Experimental steps were as follows:
The results are shown in
Raji cells (having a relatively high expression of CD22), CA46 cells (having a relatively high expression of CD22), and REH cells (having a relatively medium expression of CD22) were selected, and the cell binding ability of the antibodies was evaluated by curves of mean fluorescence intensity versus concentration obtained through FACS.
Reagents:
Experimental steps were as follows:
Results are shown in
As shown in panel 3A and Table 4, all the fitting parameters R2 reached above 0.95; the EC50 value of the reference antibody HA22 mAb was 152 ng/mL, and the maximum fluorescence signal of the antibody was between 7506 and 8131 and had entered a plateau phase. The maximum fluorescence signal of mAb-1 was comparable to that of the reference antibody HA22 mAb, while the maximum fluorescence signals of the remaining mAbs were all higher than that of the reference antibody HA22 mAb, and the maximum fluorescence signals can be ranked as: mAb-6>mAb-5>mAb-3>mAb-4>mAb-2>mAb-1>HA22 mAb. Higher fluorescence intensity indicates that the humanized mAbs have higher binding potential on Raji cells than HA22 mAb. At a concentration of 10 ng/mL, a fluorescence signal already could be detected, indicating that binding of each antibody to Raji cells was achieved at 10 ng/mL. Antibodies mAb-3, mAb-5, and mAb-6 still did not reach a fluorescence signal plateau at 50 μg/mL, thereby resulting in inaccurate EC50 values, but from the overall linearity evaluation, it can be determined that mAb-1 through mAb-6 had comparable or even stronger binding capacity to Raji cells.
As shown in panel 3B and Table 6-2, on REH cells with moderate expression of CD22, mAb-1 through mAb-6 had comparable or even stronger binding capacity to REH cells compared to HA22 mAb.
As shown in panel 3C and Table 6-3, on CA46 cells with high expression of CD22, mAb-1 through mAb-6 retained similar binding properties to that of HA22 mAb, all of which achieved high CA46 binding. According to the results of detection on all the three CD22 positive cell lines, humanized mAb-1 to mAb-6 can retain cell binding capacity comparable to or even superior to that of HA22 mAb.
CD22 is an efficient target for internalization, so the internalization efficiency of an antibody against CD22 is often the most important index of evaluation for the development of immunotoxins and ADCs. To evaluate the internalization efficiency of the humanized antibodies i.e. mAb-1 to mAb-6, three CD22 positive cell lines (Raji, CA46 and REH cells) were selected for the evaluation and the internalization efficiency of the antibodies was compared with that of the reference antibody HA22 mAb.
Reagents:
Experimental steps were as follows:
Results are shown in
At 0 h, no endocytosis occurred, the antibodies were 100% concentrated on the cell surface; and the antibodies were endocytosed into the cells over time and the amounts of the antibodies on the membrane surface which could be detected decreased. The lower antibody signal is detected, the more endocytosis occurs. As shown in
To evaluate whether the candidate antibodies have antibody-dependent cell-mediated cytotoxicity, three tumor cell lines, i.e., Raji, CA46 and REH cell lines having different CD22 expression levels were selected as target cells and PBMC as effector cells for ADCC assay.
Reagent:
Experimental steps were as follows:
Results are shown in
As shown in
The cytotoxicities observed at the dose of 50 μg/mL were analyzed, and as shown in
The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications to the present invention without departing from the spirit of the present invention, which should fall within the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011048723.0 | Sep 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/121826 | 9/29/2021 | WO |
