An antibody specifically targeting cd228 and a drug conjugate thereof

Abstract

The application discloses an antibody specifically targeting CD228 and a drug conjugate thereof. The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The application also discloses an antibody conjugated drug capable of targeting CD228 and containing the antibody. The antibody conjugated drug has strong in-vitro killing effects on both a high-expression cell line SK-MEL-5 and a low-expression cell line A2058 of CD228, wherein the in-vitro killing effect on SK-MEL-5 is stronger than that of a positive control; and the in-vitro killing effects on breast cancer and colon cancer cell lines are stronger than those of the positive control. The CD228 antibody drug conjugate has an obvious inhibiting effect on subcutaneous xenotransplantation tumors of melanoma cells A2058, and is significantly better than a positive control antibody hl49 drug conjugate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antibody that specifically targets CD228 and its drug conjugate. Background Technology

[0002] Melanotransferrin (CD228), also known as melanin transferrin, is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein belonging to the serum transferrin superfamily. The full-length CD228 protein consists of 738 amino acids with a molecular weight of 80.2 kDa. It is mainly divided into four key regions: a 19-amino acid hydrophobic signal peptide, a 331-amino acid extracellular N-terminus, a 341-amino acid extracellular C-terminus, and a 29-amino acid hydrophobic GPI-anchored domain.

[0003] Melanoma is a cancer that develops from pigmented melanocytes. It is the most dangerous type of skin cancer. Surgery may be effective for early-stage melanoma, but may not be suitable for those with distant metastases. Melanoma typically spreads to the lymph nodes in the local area and then to other places. Surgical removal of lymph nodes is often used clinically to improve patient survival, but this is often accompanied by many complications such as lymphedema, infection, and nerve damage. Immunotherapy, chemotherapy, and radiation therapy have all been used, but are generally not curative, especially for advanced melanoma. The five-year survival rate for stage IV disease is only 15%-20%. Therefore, there is a need to improve the treatment of melanoma.

[0004] CD228, also known as human melanoma-associated antigen p97, is one of the first cell surface markers associated with melanoma. It is expressed in small amounts in normal tissues but in large quantities in tumor cells (especially malignant melanoma cells) and fetal tissues. This suggests that CD228 is a potential therapeutic target for tumors. Seagen developed an ADC targeting CD228 (SGN-CD228A), but its Phase I clinical trial has been terminated. In China, Boan Biotech, a subsidiary of Luye Pharma Group, has received approval from the CDE (Center for Drug Evaluation) for its CD228-targeting ADC (BA-1302) and is currently conducting Phase I clinical trials.

[0005] ADCs combine the specificity and favorable pharmacokinetic properties of monoclonal antibodies with the cytotoxicity of chemotherapeutic drugs using chemical linkers, and have shown good activity against various refractory hematologic malignancies and solid tumors. Summary of the Invention

[0006] This invention aims to screen a novel humanized monoclonal antibody targeting CD228 and prepare an antibody-drug conjugate capable of specifically killing melanoma cells, providing a new research approach for targeted therapy of CD228-positive tumors and showing promising application prospects.

[0007] The first aspect of this invention provides an antibody specifically targeting CD228, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3; the light chain variable region comprising LCDR1, LCDR2, and LCDR3; wherein:

[0008] The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:6.

[0009] In some implementations, the frame region of the heavy chain variable region and / or the light chain variable region is a mouse-derived frame region.

[0010] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7 or the frame region of the heavy chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the frame region of SEQ ID NO:7; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8 or the frame region of the light chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the frame region of SEQ ID NO:8.

[0011] In some implementations, the framework region of the heavy chain variable region and / or the light chain variable region is a human-derived framework region.

[0012] In some preferred embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:17 or 18, or the frame region of the heavy chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the frame region of SEQ ID NO:17 or 18; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, or the frame region of the light chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the frame region of SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

[0013] In a specific embodiment of the present invention, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:17, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:14.

[0014] In a specific embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:17, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:15.

[0015] In a specific embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:17, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:16.

[0016] In a specific embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:18, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14.

[0017] In a specific embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:18, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:15.

[0018] In a specific embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:18, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:16.

[0019] In some implementations, the antibody is a full-length antibody, Fab, Fab', F(ab')2, or Fv.

[0020] In some preferred embodiments, Fv is scFv.

[0021] In some preferred embodiments, the antibody is a full-length antibody whose heavy chain constant region and / or light chain constant region are derived from human antibodies.

[0022] In some preferred embodiments, the heavy chain constant region is derived from the human heavy chain IgG1 constant region; and / or, the light chain constant region is derived from the human light chain κ chain constant region.

[0023] In this invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region and a light chain constant region. The light chain constant region consists of one domain: CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins IgA, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y is formed by the second and third constant regions of the two heavy chains (and, for IgE and IgM, a fourth constant region) linked together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains and hinge regions for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of Y includes a variable region of a single heavy chain and a first constant region that binds to a variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0024] In this invention, the "Fab fragment" consists of a light chain and a heavy chain, comprising a CH1 region and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. The "Fab' fragment" contains a portion of a light chain and a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments to form the F(ab')2 molecule. The "F(ab')2 fragment" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0025] In this invention, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0026] In this invention, the scFv (single chain antibody fragment) can be a conventional single chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids. The VL and VH domains pair via linkers to form a monovalent molecule [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating G4S amino acid sequences or variants thereof. For example, linkers with the amino acid sequences (G4S)4 or (G4S)3 can be used, but variants thereof can also be used. scFv is formed by linking VH and VL together end-to-end with a linker peptide; through proper folding, VH and VL bind non-covalently to form Fv with antigen-binding ability.

[0027] The antibodies of this invention include monoclonal antibodies. The monoclonal antibody, mAb, or Ab described in this invention refers to an antibody obtained from a single clonal cell line, and the cell line is not limited to eukaryotic, prokaryotic, or bacteriophage clonal cell lines.

[0028] A second aspect of the present invention provides an isolated nucleic acid that encodes an antibody as described in the first aspect of the present invention.

[0029] A third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid described in the second aspect of the present invention.

[0030] In some preferred embodiments, the backbone of the recombinant expression vector is pCDNA3.1 and / or pCDNA3.4.

[0031] A fourth aspect of the present invention provides a transformant comprising isolated nucleic acid as described in the second aspect of the present invention or a recombinant expression vector as described in the third aspect of the present invention, wherein the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

[0032] In some preferred embodiments, the eukaryotic cells are mammalian cells.

[0033] In some embodiments, the mammalian cells are 293F cells or EXPI293 cells.

[0034] The fifth aspect of the present invention provides a method for preparing an antibody that specifically targets CD228, the method comprising culturing a transformant as described in the fourth aspect of the present invention.

[0035] A sixth aspect of the present invention provides a chimeric antigen receptor comprising an antibody as described in the first aspect of the present invention.

[0036] A seventh aspect of the present invention provides a genetically modified cell comprising a chimeric antigen receptor as described in a sixth aspect of the present invention.

[0037] In some preferred embodiments, the genetically modified cells are eukaryotic cells.

[0038] In some implementations, the eukaryotic cells are isolated human cells.

[0039] In some implementations, the eukaryotic cells are immune cells such as T cells or NK cells.

[0040] The eighth aspect of the present invention provides an antibody-drug conjugate comprising a cytotoxic agent and an antibody as described in the first aspect of the present invention.

[0041] In some preferred embodiments, the antibody is connected to the cytotoxic agent via a connector.

[0042] In some preferred embodiments, the connector is valine-citrulline.

[0043] In some embodiments, the cytotoxic agent is MMAE.

[0044] A ninth aspect of the present invention provides a pharmaceutical composition comprising an antibody as described in the first aspect of the present invention and / or an antibody-drug conjugate as described in the eighth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0045] In some preferred embodiments, the pharmaceutical composition further comprises one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0046] The tenth aspect of the present invention provides a kit comprising an antibody as described in the first aspect of the present invention, a chimeric antigen receptor as described in the sixth aspect of the present invention, genetically modified cells as described in the seventh aspect of the present invention, an antibody-drug conjugate as described in the eighth aspect of the present invention, and / or a pharmaceutical composition as described in the ninth aspect of the present invention.

[0047] In some preferred embodiments, the kit further includes (i) a means for administering an antibody, antibody-drug conjugate, or pharmaceutical composition; and / or (ii) instructions for use.

[0048] The eleventh aspect of the present invention provides a medicine box set, the medicine box set comprising medicine box A and medicine box B, wherein:

[0049] The kit A contains an antibody as described in the first aspect of the present invention, a chimeric antigen receptor as described in the sixth aspect of the present invention, a genetically modified cell as described in the seventh aspect of the present invention, an antibody-drug conjugate as described in the eighth aspect of the present invention, and / or a pharmaceutical composition as described in the ninth aspect of the present invention.

[0050] The kit B contains other anti-tumor antibodies or a pharmaceutical composition containing said other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0051] The twelfth aspect of the present invention provides a drug delivery device comprising an antibody as described in the first aspect of the present invention, a chimeric antigen receptor as described in the sixth aspect of the present invention, a genetically modified cell as described in the seventh aspect of the present invention, an antibody-drug conjugate as described in the eighth aspect of the present invention, and / or a pharmaceutical composition as described in the ninth aspect of the present invention.

[0052] In some preferred embodiments, the drug delivery device further includes components, such as a syringe or infusion device, for administering the antibody, the chimeric antigen receptor, the genetically modified cell, the antibody-drug conjugate, or the drug composition to a subject.

[0053] The thirteenth aspect of the present invention provides a method for detecting CD228 protein for non-diagnostic purposes, the method comprising using an antibody as described in the first aspect of the present invention, a chimeric antigen receptor as described in the sixth aspect of the present invention, a genetically modified cell as described in the seventh aspect of the present invention, an antibody-drug conjugate as described in the eighth aspect of the present invention, and / or a pharmaceutical composition as described in the ninth aspect of the present invention.

[0054] In some preferred embodiments, the CD228 protein is human CD228.

[0055] In some preferred embodiments, the CD228 protein is the full-length CD228 protein.

[0056] The fourteenth aspect of the present invention provides the use of an antibody as described in the first aspect of the present invention, a chimeric antigen receptor as described in the sixth aspect of the present invention, a genetically modified cell as described in the seventh aspect of the present invention, an antibody-drug conjugate as described in the eighth aspect of the present invention, and / or a pharmaceutical composition as described in the ninth aspect of the present invention in the preparation of a medicament for killing CD228-positive tumors.

[0057] In some preferred embodiments, the CD228-positive tumor is one or more of melanoma, colorectal cancer, and breast cancer.

[0058] In this invention, the amino acid sequences of the CDRs listed above are all in accordance with the Kabat definition rules (the sequences in the claims of this invention are also in accordance with the Kabat definition rules). However, it is well known to those in the art that antibody CDRs can be defined in various ways, such as Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (imgt.cines.fr / ), and Northrop Grumman's method based on affinity propagation clustering using a large number of crystal structures. CDR Definition. Those skilled in the art will understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described above in any of the known embodiments of this invention. While the scope of protection claimed in the claims of this invention is based on the sequence shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0059] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0060] The reagents and raw materials used in this invention are all commercially available.

[0061] The positive and progressive effects of this invention are as follows:

[0062] 1. A novel antibody sequence targeting CD228;

[0063] 2. It binds with high affinity to CD228;

[0064] 3. An antibody-drug conjugate targeting CD228-positive tumors, comprising the humanized monoclonal antibody described in this invention, exhibits strong in vitro killing effects against both the CD228-high expression cell line (SK-MEL-5) and the low expression cell line A2058, with the in vitro killing effect against SK-MEL-5 being stronger than the positive control; the in vitro killing effects against breast cancer and colon cancer cell lines (MDA-MB-231, HCT-116) are also stronger than the positive control. The CD228 antibody-drug conjugate shows a significant inhibitory effect on subcutaneous xenograft tumors of melanoma cells A2058, significantly superior to the positive control antibody hl49 drug conjugate. Attached Figure Description

[0065] Figure 1 It exhibits murine antibody-protein binding activity.

[0066] Figure 2 It exhibits humanized antibody protein binding activity.

[0067] Figure 3 It exhibits humanized antibody cell-binding activity.

[0068] Figure 4 To perform SDS-PAGE analysis on antibody-drug conjugates.

[0069] Figure 5 This demonstrates the binding activity of the antibody-drug conjugate to the CD228 protein.

[0070] Figure 6 The cell-binding activity of the antibody-drug conjugate with CD228.

[0071] Figure 7 The study aimed to assess the cytotoxic effects of antibody-drug conjugates on different tumor cells. A: HCT-116; B: MDA-MB-231; C: A2058; D: SK-MEL-5. Cells were co-incubated with serially diluted antibodies for 72 hours, and cell viability was assessed using a CCK8 assay.

[0072] Figure 8 Comparison of ADCC effects of antibody-drug conjugates.

[0073] Figure 9 The growth curve of a subcutaneous xenograft tumor of human melanoma cell A2058.

[0074] Figure 10 Tumor weight at the experimental endpoint. Data are presented as mean ± SEM, n = 5, ***P < 0.001 Detailed Implementation

[0075] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0076] Example 1: Screening of mouse monoclonal antibodies

[0077] Mouse monoclonal antibodies were prepared using hybridoma cell technology. For experimental protocols, please refer to the literature (Ed Harlow, David Lane. Antibody: A laboratory manual. 1988).

[0078] Construction of expression plasmids: The full-length CD228 expression plasmid (pCMV3-CD228) was purchased from Beijing Sinocare Biotechnology Co., Ltd. (HG12200-UT). The extracellular region-His fragment of CD228 (containing the HIS tag) was amplified by PCR and cloned into the pcDNA3.4 expression plasmid through homologous recombination to construct the expression plasmid pcDNA3.4-CD228-ECD-HIS. The extracellular region of CD228 (CD228-EDC) was amplified by PCR and cloned into the corresponding restriction sites of pcDNA3.4-Fc (containing the Fc tag) through homologous recombination to construct the expression plasmid pcDNA3.4-CD228-ECD-Fc. The pcDNA3.4-CD228-EDC-HIS and pcDNA3.4-CD228-EDC-Fc plasmids were transiently transfected into EXPI293 cells (Thermo Fisher Scientific Inc.) and cultured for 5 days. The supernatant was collected, and the CD228-HIS protein was purified using a Ni affinity chromatography column, while the CD228-FC protein was purified using a Protein A affinity chromatography column. The pCMV3-CD228 plasmid was then transiently transfected into EXPI293 cells to obtain CD228-overexpressing cells, which were then used in the immunization process.

[0079] Recombinant CD228-FC protein was mixed with complete Freund's adjuvant (Sigma) at a volume ratio of 1:1 and injected intraperitoneally at a concentration of 500 μg / ml (200 μl) to BALB / c mice for the first immunization. Subsequently, on days 14 and 35, mice were given booster immunizations by intraperitoneal injection of recombinant protein CD228-HIS and incomplete Freund's adjuvant (Sigma) at a volume ratio of 1:1 (200 μl) at a concentration of 500 μg / ml. On day 56, BALB / c mice were given the final immunization by intravenous injection of 100 μl of CD228-FC (diluted to 1 mg / ml with PBS). Four days later, spleens were harvested for fusion.

[0080] Mouse spleen cells were fused with SP2 / 0 cells at a ratio of 4:1 and cultured in 96-well Corning plates using HAT (GBICO) medium. Hybridoma cell selection was then performed, and the resulting cell clones were subjected to further selection. The identification and screening process consisted of two steps:

[0081] ① Recombinant CD228-HIS was immobilized on a 96-well enzyme-linked immunosorbent assay (ELISA) plate. After incubation for 1 hour with the clonal expression supernatant, the cells were washed three times with PBST. The supernatant of cell clones with CD228 binding activity was identified by goat anti-mouse IgG-HRP (Jackson Immuno). The cells were washed three times with PBST and then developed with TMB to obtain positive clones that could directly bind to CD228.

[0082] ②Then, the positive clones from step ① were transferred into 24-well (Corning) plates for culture to obtain more expression products. The cell supernatant was incubated with CD228-overexpressing cells at 4°C for 1 h, washed twice with PBS, and then incubated with goat anti-mouse IgG–HRP at 4°C for 0.5 h. After washing twice with PBS, TMB was used for color development to identify positive clones that could bind to CD228 on the cells.

[0083] Through the above screening process, candidate mouse CD228 antibodies were obtained: 2F6, 3B9, 7C2 and 11H11.

[0084] Example 2: Binding activity of murine antibody to CD228 protein

[0085] The protein binding activity of the prepared murine antibody was identified. After coating with CD228-HIS protein and blocking, serially diluted murine antibody (starting at 1 μg / ml, 3-fold serial dilution) was added and incubated for 1 h. After washing three times, secondary antibody (goat anti-mouse IgG–HRP) diluted 1:10000 was added and incubated for 1 h. TMB color development was performed, and the reaction was stopped with 1M H2SO4. OD450 readings were recorded. The EC50 of the selected murine monoclonal antibody binding to the purified antigen was measured. The results showed ( Figure 1 (As shown in Table 1), 2F6 exhibits better protein binding activity.

[0086] Table 1. Binding activity of murine antibody to CD228 protein

[0087] serial number 3B9 2F6 11H11 7C2 EC50 (ng / ml) 15.75 15.16 22.66 63.27

[0088] Example 3: Antibody gene acquisition and preparation of chimeric antibodies

[0089] Based on the above murine antibody-protein binding activity, candidate antibody 2F6 was selected for antibody gene acquisition and chimeric antibody preparation. RNA was extracted from hybridoma cells of antibody 2F6 and reverse transcribed to obtain its cDNA. Using this cDNA as a template, the light and heavy chain variable region nucleic acid sequences of the murine antibody were amplified separately using PCR, and the heavy and light chain variable region sequences were analyzed.

[0090] The heavy chain variable region encoded by 2F6 contains heavy chain CDR1 (NYWIE) as shown in SEQ ID NO:1, heavy chain CDR2 (EIRPGNGDTNYNEKFKG) as shown in SEQ ID NO:2, and heavy chain CDR3 (NDGYYGRFPN) as shown in SEQ ID NO:3; the light chain variable region contains light chain CDR1 (RAGQDISNYLN) as shown in SEQ ID NO:4, light chain CDR2 (YTSRLHS) as shown in SEQ ID NO:5, and light chain CDR3 (QQGKTLPYT) as shown in SEQ ID NO:6.

[0091] The heavy chain variable region was constructed into the pcDNA3.1-HC plasmid (containing a signal peptide and a heavy chain IgG1 constant region) to obtain the heavy chain plasmid pCDNA3.1-2F6-HC for the chimeric antibody. The light chain variable region was constructed into the pcDNA3.1-LC plasmid (containing a signal peptide and a light chain κ chain constant region) to obtain the light chain plasmid pcDNA3.1-2F6-LC for the chimeric antibody.

[0092] Expression and purification of chimeric antibodies: The heavy and light chain plasmids of the antibodies were co-transfected into 293F cells and cultured for 5 days. The supernatant was collected and purified by protein A affinity chromatography to finally obtain the recombinant anti-human CD228 chimeric antibody 2F6-CA.

[0093] The heavy chain variable region sequence of the anti-human CD228 chimeric antibody 2F6-CA is shown in SEQ ID NO:7, specifically as follows:

[0094]

[0095] The light chain variable region sequence of the aforementioned anti-human CD228 chimeric antibody 2F6-CA is shown in SEQ ID NO:8, specifically as follows:

[0096]

[0097] Example 4: Humanization Design and Antibody Expression and Purification

[0098] Homology modeling of the heavy and light chains of M-2F6 was performed using Discovery Studio 2019 software to obtain festa files for the variable region models of the mouse antibody heavy and light chains, namely 2F6-VH and 2F6-VL. Two sequences were designed for the heavy chain and three sequences for the light chain, as shown in Table 2. The variable regions in Table 2 were synthesized and directly constructed into the vector pCDNA3.4 containing the constant regions of the heavy and light chains to obtain full-length heavy and light chain expression plasmids. The plasmids were extracted, and then plasmids of different heavy and light chains were combined and paired for transfection into EXPI293 cells. After culturing for 5 days, the supernatant was collected and purified by affinity chromatography using a Protein A column. A total of 6 humanized antibodies were obtained, and the sequences of the 6 antibodies are shown in Table 3.

[0099] Table 2. Variable region sequence of 2F6 humanized antibody

[0100]

[0101]

[0102] Table 3 Full-length sequence of humanized antibodies

[0103] Antibody name Light chain sequence Heavy chain sequence 2F6A1 SEQ ID NO:14 SEQ ID NO:17 2F6A2 SEQ ID NO:15 SEQ ID NO:17 2F6A3 SEQ ID NO:16 SEQ ID NO:17 2F6B1 SEQ ID NO:14 SEQ ID NO:18 2F6B2 SEQ ID NO:15 SEQ ID NO:18 2F6B3 SEQ ID NO:16 SEQ ID NO:18

[0104] SEQ ID NO:14

[0105]

[0106] SEQ ID NO:15

[0107]

[0108] SEQ ID NO:16

[0109]

[0110]

[0111] SEQ ID NO:17

[0112]

[0113] SEQ ID NO:18

[0114]

[0115] Example 5 Humanized antibody protein binding activity

[0116] Protein binding activity of the prepared humanized antibody was identified: CD228-HIS protein was coated onto an immunoassay plate, blocked, and then serially diluted antibody (starting at 1 μg / ml, 3-fold serial dilution) was added and incubated for 1 h. After washing twice, a 1:10000 dilution of secondary antibody (goat anti-human IgG–HRP, Abbkine Scientific, A21050) was added and incubated for 1 h. Finally, TMB was used for color development, and the incubation was stopped with 1M H2SO4. OD450 readings were recorded. The results showed that ( Figure 2 (See Table 4) All six humanized antibodies exhibited strong protein-binding activity.

[0117] Table 4. Humanized antibody protein binding activity

[0118] 2F6-A1 2F6-A2 2F6-A3 2F6-B1 2F6-B2 2F6-B3 EC50 (ng / ml) 1.746 5.300 7.055 77.683 7.395 5.828

[0119] Example 6: Humanized Antibody Cell Binding Activity

[0120] CD228 overexpressing cells were obtained by transiently transfecting the full-length pCMV3-CD228 expression plasmid into 293F cells (Thermo Fisher). 1×10⁻⁶ cells were added to each well. 5 Cells were incubated with serially diluted antibody (starting at 10 μg / ml, 3-fold serial dilution) at 4°C for 1 h. After washing twice with PBS, secondary antibody (goat anti-human IgG–HRP) diluted 1:10000 was added, and incubation was continued at 4°C for 1 h. Finally, TMB was used for color development, and the incubation was stopped with 1M H2SO4. OD450 readings were performed, indicating that ( Figure 3 As shown in Table 5, 2F6-A1 and 2F6-A2 exhibited the strongest cell-binding activity, with EC50 values ​​of 83.80 ng / ml and 78.59 ng / ml, respectively, both higher than the positive control (POS, i.e., hl49) at 585.5 ng / ml. Based on the combined antibody-cell binding assay data, 2F6-A1 demonstrated the strongest affinity.

[0121] Table 5. Cell-binding activity of humanized antibodies

[0122] 2F6-A1 2F6-A2 2F6-A3 2F6-B1 2F6-B2 2F6-B3 POS EC50 (ng / ml) 83.80 78.59 88.87 113.2 110.9 103.1 585.5

[0123] hl49 heavy chain sequence (SEQ ID NO:19):

[0124]

[0125] hl49 light chain sequence (SEQ ID NO:20):

[0126]

[0127] Example 7 Preparation of Antibody-Drug Conjugates

[0128] The 2F6A1 antibody and the positive control antibody hl49 were conjugated via MMAE (MCE) using a thiol-based coupling method, with valine-citrulline (VC) as the linker. Different DAR values ​​were achieved by adjusting the amounts of reducing agent TCEP and small molecules. The MMAE-conjugated ADCs were named Ab-2F6A1-MMAE and Ab-hl49-VcMMAE, with a target DAR value of 4. The conjugation status of the ADCs was preliminarily analyzed by reducing SDS-PAGE. The SDS-PAGE results showed that ( Figure 4 The Ab-2F6A1-MMAE heavy chain band was slightly larger than the 2F6A1 heavy chain band, and several bands of different sizes were observed, indicating that the small molecule had been successfully conjugated to the antibody. HIC assay showed that the average DAR value of Ab-2F6A1-VcMMAE was 4.9, and the average DAR value of Ab-hl49-VcMMAE was 4.37.

[0129] Example 8: Antigen-binding activity of antibody-drug conjugate with CD228

[0130] The binding activity of the CD228 antibody-drug conjugate to the CD228 protein was verified by ELISA, using the same method as in Example 5. The results showed that ( Figure 5 (As shown in Table 6), the EC50 values ​​for the binding of Ab-2F6A1 and Ab-2F6A1-VcMMAE to the antigen were 8.866 ng / ml and 10.09 ng / ml, respectively. The binding activity of the CD228 antibody-drug conjugate to cells was verified by ELISA, using the same method as in Example 6. The results showed that ( Figure 6 (See Table 7) The EC50 values ​​of Ab-2F6A1 and Ab-2F6A1-VcMMAE binding to the antigen were 95.21 ng / ml and 248.9 ng / ml, respectively, indicating that after small molecule conjugation, the binding activity of ADC to antigen decreased but still remained at a high level. Figure 5 and Figure 6 The Isotype in the text refers to respiratory syncytial virus (RSV) antibody.

[0131] Table 6. Binding activity of antibody-drug conjugates to CD228 protein.

[0132] protein Ab-2F6A1 Ab-2F6A1-VcMMAE Isotype EC50 (ng / ml) 8.866 10.09 \

[0133] Table 7. Cell-binding activity of antibody-drug conjugates with CD228.

[0134] cell Ab-2F6A1 Ab-hl49 Ab-2F6A1-VcMMAE Ab-hl49-VcMMAE Isotype EC50 (ng / ml) 95.21 122.2 248.9 252.2 \

[0135] Example 9: In vitro cytotoxic activity of antibody-drug conjugates

[0136] The healthy rectal cancer cell line (HCT-116, purchased from ATCC), breast cancer cell line (MDA-MB-231, purchased from ATCC), and melanoma CD228 low-expression cell line (A2058, purchased from Enzyme Research Biotechnology Co., Ltd., CC-Y1688) were counted and diluted to 2×10⁻⁶. 5 After layering the ADC drug at a concentration of 50 μL / well (10,000 cells / well), 50 μL of diluted ADC drug was added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator. The ADC drug was diluted with culture medium, starting at 25 μg / mL, with three-fold serial dilutions for a total of eight dilutions. After 3 days, the 96-well plates were removed, and 10 μL of CCK8 was added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 1 hour. After 1 hour, the absorbance at 450 nm was measured using a microplate reader. The results showed that (…). Figure 7 (NEG was the blank control). In colorectal cancer cell line (HCT-116) and breast cancer cell line (MDA-MB-231), Ab-2F6A1-VcMMAE showed stronger killing effect than the positive control Ab-hl49-VcMMAE. However, in the melanoma CD228 low-expression cell line (A2058), its inhibitory effect was slightly inferior to the positive control. In the melanoma CD228 high-expression cell line (SK-MEL-5), the IC50 of Ab-2F6A1-VcMMAE was 1.964 ng / ml, while that of the positive control Ab-hl49-VcMMAE was 15.51 ng / ml, which was stronger than the positive control. Specific data are shown in Table 8.

[0137] Table 8. IC50 data of antibody-drug conjugates in different tumors.

[0138]

[0139] Example 10: ADCC effect of antibody-drug conjugates

[0140] SK-MEL-5 cells (ATCC) were seeded into 96-well black transparent plates, 100 μL (containing 1×10⁶ cells) 4 Cells were collected in each well (1 cell per well), with blank wells reserved. The wells were incubated at 37°C in a 5% CO2 incubator. The next day, the culture medium was aspirated from the 96-well plate, and Jurkat medium containing the test antibody was added at 60 μL / well. The plate was incubated at 37°C for 1 hour. The antibody was diluted with blank Jurkat medium, starting at a concentration of 2 μg / mL, and serially diluted 3-fold to obtain a total of 8 concentrations. Effector cells (Jurkat-ADCC cells) were counted by blowing them into the plate at a density of 7.5 × 10⁻⁶ cells / well. 4Cells were collected per well and placed in a 15 mL centrifuge tube. Centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in blank Jurkat medium. 40 μL of the resuspended cells were added to each well of a 96-well plate prepared in the previous step. The plate was incubated at 37°C with 5% CO2 for 6 h. 100 μL of Bright-One step assay reagent (Yisheng Biotechnology Co., Ltd., 11412ES60) was added to each well of the 96-well plate and incubated at room temperature for 5 minutes. The results were read using a microplate reader with the Luminescence assay program. The results showed that (…). Figure 8 (See Table 9). The mean fluorescence intensity (MFI) of Ab-2F6A1 was 221857, which mediated the strongest ADCC effect, followed by its conjugate Ab-2F6A1-VcMMAE; the positive control showed a lower level of ability to mediate the ADCC effect.

[0141] Table 9 Comparison of ADCC effects of antibody-drug conjugates

[0142] drug Ab-2F6A1 Ab-2F6A1-VcMMAE hl49-VcMMAE Isotype NEG MFI 221857 123231 66870 34990 34162

[0143] Example 11: In vivo tumor-suppressive activity of antibody-drug conjugates

[0144] A subcutaneous tumor model of A2058 cells in nude mice (purchased from Shanghai Biocare Biotechnology Co., Ltd.) was used to examine the in vivo inhibitory effect of CD228 antibody-drug conjugate on the growth of human melanoma. A2058 cells (Shanghai Enzyme Research Biotechnology Co., Ltd., CC-Y1688) were cultured to the logarithmic growth phase in DMEM medium (Wuhan Sewell Biotechnology Co., Ltd., catalog number: G4524-500ML). A2058 cells in the logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with PBS before being inoculated into nude mice at a seeding density of 1×10⁶ cells / mL. 7 / 200μl / tumor (added with 50% matrix gel, purchased from Thermo Fisher Scientific). After 7 days, the tumor will grow to an average tumor volume of 150mm. 3 Mice with tumors reaching the standard size were randomly divided into four groups: control group (PBS), isotype control group (Isotype-MMAE 5 mg / kg), experimental group (Ab-2F6A1-VcMMAE 5 mg / kg), and positive control group (Ab-hl49-VcMMAE 5 mg / kg). Administered the medication once daily on days 0, 5, and 10 via tail vein injection, with an injection volume of 100 μl. Mouse weight and tumor volume were measured every three days. Tumor size was measured using calipers, and the volume was calculated using the formula: V = (L × W) / ( ... 2) / 2, where L is the longest diameter of the tumor, and W is the shortest diameter perpendicular to the longest diameter. The results show ( Figure 9 and Figure 10 The experimental group Ab-2F6A1-VcMMAE showed a significant inhibitory effect on tumors, while the positive control group Ab-hl49-VcMMAE showed a weaker inhibitory effect. The overall curves of Isotype-MMAE and PBS groups were similar, and no significant anti-tumor activity was observed. This indicates that the CD228 antibody-drug conjugate has significant in vivo anti-melanoma activity, and is significantly superior to the positive control antibody hl49 drug conjugate.

Claims

1. An antibody that specifically targets CD228, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3; wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

6.

2. The antibody as described in claim 1, characterized in that, The frame region of the heavy chain variable region and / or the light chain variable region is a mouse-derived frame region; Preferably, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, or the frame region of the heavy chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the frame region of SEQ ID NO:7; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8, or the frame region of the light chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the frame region of SEQ ID NO:

8.

3. The antibody as described in claim 1, characterized in that, The framework region of the heavy chain variable region and / or the light chain variable region is a human-derived framework region; Preferably, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:17 or 18, or the frame region of the heavy chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the frame region of SEQ ID NO:17 or 18; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, or the frame region of the light chain variable region has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the frame region of SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16; More preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:15; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:15; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

4. The antibody according to any one of claims 1 to 3, characterized in that, The antibody is a full-length antibody, Fab, Fab', F(ab')2, or Fv; the Fv is preferably scFv. Preferably, the antibody is a full-length antibody, the heavy chain constant region and / or light chain constant region of which are derived from human antibodies; More preferably, the heavy chain constant region is derived from the human heavy chain IgG1 constant region; and / or, the light chain constant region is derived from the human light chain κ chain constant region.

5. An isolated nucleic acid, characterized in that, The nucleic acid encodes an antibody as described in any one of claims 1-4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 5; Preferably, the backbone of the recombinant expression vector is pCDNA3.1 and / or pCDNA3.

4.

7. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 5 or the recombinant expression vector as described in claim 6, and the host cell of the transformant is a eukaryotic cell or a prokaryotic cell; Preferably, the eukaryotic cells are mammalian cells, such as 293F cells or EXPI293 cells.

8. A method for preparing an antibody specifically targeting CD228, characterized in that, The method includes culturing the transformant as described in claim 7.

9. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an antibody as described in any one of claims 1-4.

10. A genetically modified cell, characterized in that, The genetically modified cells contain the chimeric antigen receptor as described in claim 9; Preferably, the genetically modified cells are eukaryotic cells, more preferably isolated human cells; more preferably immune cells such as T cells or NK cells.

11. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate comprises a cytotoxic agent and an antibody as described in any one of claims 1-4; Preferably, the antibody is connected to the cytotoxic agent via a connector; More preferably, the connector is valine-citrulline.

12. The antibody-drug conjugate as described in claim 11, characterized in that, The cytotoxic agent is MMAE.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody as described in any one of claims 1-4 and / or the antibody-drug conjugate as described in claim 11, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

14. A reagent kit, characterized in that, The kit comprises the antibody as described in any one of claims 1-4, the chimeric antigen receptor as described in claim 9, the genetically modified cell as described in claim 10, the antibody-drug conjugate as described in claim 11 or 12, and / or the pharmaceutical composition as described in claim 13; Preferably, the kit further includes (i) a device for using an antibody, antibody-drug conjugate, or pharmaceutical composition; and / or (ii) instructions for use.

15. A medicine box set, characterized in that, The pillbox set includes pillbox A and pillbox B, wherein: The kit A contains the antibody as described in any one of claims 1-4, the chimeric antigen receptor as described in claim 9, the genetically modified cell as described in claim 10, the antibody-drug conjugate as described in claim 11 or 12, and / or the pharmaceutical composition as described in claim 13; The kit B contains other anti-tumor antibodies or a pharmaceutical composition containing said other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

16. A drug delivery device, characterized in that, The drug delivery device comprises an antibody as described in any one of claims 1-4, a chimeric antigen receptor as described in claim 9, a genetically modified cell as described in claim 10, an antibody-drug conjugate as described in claim 11 or 12, and / or a pharmaceutical composition as described in claim 13; Preferably, the drug delivery device further includes a component for administering the antibody, the chimeric antigen receptor, the genetically modified cell, the antibody-drug conjugate, or the drug composition to the subject, such as a syringe or infusion device.

17. A method for detecting CD228 protein for non-diagnostic purposes, characterized in that, The method includes using the antibody as described in any one of claims 1-4, the chimeric antigen receptor as described in claim 9, the genetically modified cell as described in claim 10, the antibody-drug conjugate as described in claim 11 or 12, and / or the pharmaceutical composition as described in claim 13; Preferably, the CD228 protein is a human CD228; and / or, the CD228 protein is a full-length CD228 protein.

18. The use of the antibody as described in any one of claims 1-4, the chimeric antigen receptor as described in claim 9, the genetically modified cell as described in claim 10, the antibody-drug conjugate as described in claim 11 or 12, and / or the pharmaceutical composition as described in claim 13 in the preparation of a medicament for killing CD228-positive tumors; preferably, the CD228-positive tumor is one or more of melanoma, colorectal cancer, and breast cancer.

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