Antibodies and antibody-drug conjugates targeting CD228

Abstract

The invention relates to the technical field of biology, in particular to a CD228 targeting antibody and an antibody-drug conjugate. The antibody has good FACS binding capacity, internalization activity and affinity, has good tumor cell killing capacity when being used for preparing an antibody drug conjugate, and can have a good detection effect when being used for preparing a detection reagent for CD228.

Description

[0001] This application claims priority to Chinese Patent Application No. 202411165325.5, filed on August 23, 2024, entitled "Antibody and Antibody-Drug Conjugate Targeting CD228", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biotechnology, and more particularly to antibodies and antibody-drug conjugates targeting CD228. Background Technology

[0003] CD228, also known as melanotransferrin, MELTF, p97, and MFI2, is a cell surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein belonging to the transferrin superfamily. It was initially identified as a protein highly expressed on malignant melanoma cells. CD228 is overexpressed in most clinical melanoma isolates and is also expressed in many other solid tumors, such as mesothelioma, non-small cell lung cancer, breast cancer, colorectal cancer, and pancreatic cancer, while it is not expressed or is expressed at low levels in normal tissues. This provides strong evidence for CD228 to be a target for antibody drugs, especially antibody-drug conjugates (ADCs).

[0004] According to incomplete statistics, there are three drugs under development targeting CD228. Among them, Seagen (which has been acquired by Pfizer) has an antibody-drug conjugate (ADC) and a bispecific antibody. Boan Bio's BA1302 is the first domestically produced CD228 ADC.

[0005] Seagen's SGN-CD228A antibody, hL49, is a humanized antibody targeting CD228. It is conjugated to a monomethyl guanylate (MMAE) molecule via a PEG-modified β-glucuronidase, with a mean drug-to-antibody ratio (DAR) of 8. MMAE is a microtubule inhibitor and a commonly used chemical payload in investigational and clinical ADCs. This clinical trial, initiated in August 2019, enrolled patients with relapsed or refractory metastatic cutaneous melanoma, malignant pleural mesothelioma, Her2-negative metastatic breast cancer, advanced non-small cell lung cancer, metastatic colon cancer, and advanced pancreatic cancer. The planned enrollment was 240 patients, aiming to evaluate the safety, tolerability, pharmacokinetic (PK), and antitumor activity of SGN-CD228A in advanced solid tumors. The study was intended to include dose escalation and dose expansion, with multiple disease-specific expansion cohorts, but was terminated on March 23, 2023.

[0006] Seagen's other antibody, SGN-BB228, is a bispecific antibody (CD228 / 4-1BB) designed to provide an effective co-stimulatory bridge between tumor-specific T cells and CD228-expressing tumor cells. A Phase I clinical trial is underway to evaluate its tolerability and safety in patients with melanoma and other solid tumors.

[0007] Boan Biotech's BA1302 is also an ADC drug, and its antibody component is screened from Boan Biotech's proprietary fully human antibody transgenic mice. This antibody exhibits superior binding specificity, binding only to the membrane-bound CD228 and not its secreted form. The chemical component utilizes the linker-loader (BNLD11) selected by Boan Biotechnology, demonstrating excellent in vitro and in vivo stability. Preclinical studies have shown that BA1302 exhibits excellent tumor-suppressive effects in tumor models such as lung cancer, gastric cancer, and melanoma, and demonstrates good safety and tolerability in cynomolgus monkey toxicology pre-tests, with an MTD dose exceeding 10 mg / kg, showcasing excellent clinical therapeutic potential.

[0008] Although several antibody drugs targeting CD228 have been disclosed in existing technologies, there is still an urgent need to develop high-quality anti-CD228 antibodies, as they are a specific marker highly expressed in various solid tumor cells. This would enrich the methods and approaches for treating tumors, and also serve as a tumor diagnostic tool by detecting the expression of CD228 in tumors. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide antibodies and antibody-drug conjugates targeting CD228.

[0010] In a first aspect, the present invention provides an antibody or antigen-binding molecule targeting CD228.

[0011] At least one of the CDR1-3 regions of its heavy chain has an amino acid sequence as shown in any one of SEQ ID NO:1-9, or a sequence based on the amino acid sequence shown in SEQ ID NO:1-9 by substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) homology with the amino acid sequence shown in any one of SEQ ID NO:1-9;

[0012] At least one of its light chain CDR1-3 regions has an amino acid sequence as shown in any one of SEQ ID NO:10-18, or a sequence based on the amino acid sequence shown in SEQ ID NO:10-18 by substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence that is more than 80% homologous (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to the amino acid sequence shown in any one of SEQ ID NO:10-18.

[0013] In this invention, relevant indications are determined by quantifying surface proteins of natural tumor cells and using microarray staining. Cells required for antibody development are selected, and anti-human CD228 monoclonal antibodies are screened using mouse hybridomas. Antibodies with good binding activity and cross-linking with monkeys are selected. After sequencing alignment, human-mouse chimeric antibodies are constructed. Cell-level FACS binding, internalization, and protein-level affinity assays are performed on the chimeric antibodies. Antibodies with good binding activity, affinity, and internalization activity are screened for antibody-ADC conjugation. The internalization activity and killing activity of the antibody-ADC against the natural tumor cells are compared, and antibodies with good activity are selected.

[0014] In this invention, the antibody or antigen-binding molecule targeting CD228,

[0015] Its heavy chain CDR1 has an amino acid sequence as shown in any one of SEQ ID NO:1 to 3;

[0016] Its heavy chain CDR2 has an amino acid sequence as shown in any one of SEQ ID NO:4 to 6;

[0017] Its heavy chain CDR3 has an amino acid sequence as shown in any one of SEQ ID NO:7 to 9;

[0018] Its light chain CDR1 has an amino acid sequence as shown in any one of SEQ ID NO10-12;

[0019] Its light chain CDR2 has an amino acid sequence as shown in any one of SEQ ID NO:13-15;

[0020] Its light chain CDR3 has an amino acid sequence as shown in any one of SEQ ID NO:16-18.

[0021] In some embodiments, the antibody or antigen-binding molecule targeting CD228,

[0022] The amino acid sequence of its heavy chain CDR1 is shown in SEQ ID NO:1, the amino acid sequence of its heavy chain CDR2 is shown in SEQ ID NO:4, and the amino acid sequence of its heavy chain CDR3 is shown in SEQ ID NO:7.

[0023] The amino acid sequence of its light chain CDR1 is shown in SEQ ID NO:10, the amino acid sequence of its light chain CDR2 is shown in SEQ ID NO:13, and the amino acid sequence of its light chain CDR3 is shown in SEQ ID NO:16.

[0024] In other embodiments, the antibody or antigen-binding molecule targeting CD228,

[0025] The amino acid sequence of its heavy chain CDR1 is shown in SEQ ID NO:2, the amino acid sequence of its heavy chain CDR2 is shown in SEQ ID NO:5, and the amino acid sequence of its heavy chain CDR3 is shown in SEQ ID NO:8.

[0026] The amino acid sequence of its light chain CDR1 is shown in SEQ ID NO:11, the amino acid sequence of its light chain CDR2 is shown in SEQ ID NO:14, and the amino acid sequence of its light chain CDR3 is shown in SEQ ID NO:17.

[0027] In other embodiments, the antibody or antigen-binding molecule targeting CD228,

[0028] The amino acid sequence of its heavy chain CDR1 is shown in SEQ ID NO:3, the amino acid sequence of its heavy chain CDR2 is shown in SEQ ID NO:6, and the amino acid sequence of its heavy chain CDR3 is shown in SEQ ID NO:9.

[0029] The amino acid sequence of its light chain CDR1 is shown in SEQ ID NO:12, the amino acid sequence of its light chain CDR2 is shown in SEQ ID NO:15, and the amino acid sequence of its light chain CDR3 is shown in SEQ ID NO:18.

[0030] In some specific embodiments, the amino acid sequences of the three CDR regions of the heavy chain are NYFIH, YINPYNGGTKSSEKVRG, and NYGYFDY, respectively, and the amino acid sequences of the three CDR regions of the light chain are shown in SEQ ID NO:11, 14, and 17, respectively.

[0031] In other specific embodiments, the amino acid sequences of the three CDR regions of the heavy chain are NYFIH, YINPYNGGTKSSEKVRG, and NYGYFDY, respectively, and the amino acid sequences of the three CDR regions of the light chain are shown in SEQ ID NO:11, 14, and 17, respectively.

[0032] In other specific embodiments, the amino acid sequences of the three CDR regions of the heavy chain are NYFIH, YINPYNGGTKSSEKVRG, and NYGYFDY, respectively, and the amino acid sequences of the three CDR regions of the light chain are shown in SEQ ID NO:11, 14, and 17, respectively.

[0033] In this invention, the antibody or antigen-binding molecule targeting CD228 is a chimeric antibody.

[0034] At least one of its heavy chains FR1 to 4 has an amino acid sequence as shown in any one of SEQ ID NO:19 to 30; or has a sequence based on the amino acid sequence shown in any one of SEQ ID NO:19 to 30 by substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence that is more than 80% homologous (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to the amino acid sequence shown in any one of SEQ ID NO:19 to 30.

[0035] At least one of its light chains FR1 to 4 has an amino acid sequence as shown in any one of SEQ ID NO:31 to 41; or has a sequence based on the amino acid sequence shown in any one of SEQ ID NO:31 to 41 by substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence that is more than 80% homologous (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to the amino acid sequence shown in any one of SEQ ID NO:31 to 41.

[0036] In this embodiment of the invention, the antibody or antigen-binding molecule targeting CD228,

[0037] Its heavy chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:19-21;

[0038] Its heavy chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:22-24;

[0039] Its heavy chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:25-27;

[0040] Its heavy chain FR4 has an amino acid sequence as shown in any one of SEQ ID NO:28-30;

[0041] Its light chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:31 to 33;

[0042] Its light chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:34-36;

[0043] Its light chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:37-39;

[0044] Its light chain FR4 has an amino acid sequence as shown in SEQ ID NO:40 or 41.

[0045] In some embodiments, the antibody or antigen-binding molecule targeting CD228,

[0046] Its heavy chain FR1-4 has the amino acid sequences shown in SEQ ID NO:19, 22, 25, 28, and its light chain FR1-4 has the amino acid sequences shown in SEQ ID NO:31, 34, 37, 40, respectively.

[0047] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:20, 23, 26, 29, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:32, 35, 38, 41, respectively;

[0048] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:21, 24, 27, 30, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:33, 36, 39, 40.

[0049] More specifically, the antibody or antigen-binding molecule targeting CD228,

[0050] Its heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:58 to 60;

[0051] Its light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:61 to 63.

[0052] In one specific embodiment, the antibody or antigen-binding molecule targeting CD228,

[0053] Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:58, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:61.

[0054] Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:59, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:62;

[0055] Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:60, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:63.

[0056] In this invention, the antibody or antigen-binding molecule targeting CD228 is a chimeric antibody or a humanized antibody.

[0057] Its heavy chain FR1 has an amino acid sequence as shown in SEQ ID NO:42 or 43;

[0058] Its heavy chain FR2 has an amino acid sequence as shown in SEQ ID NO:45 or 46;

[0059] Its heavy chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:50-53;

[0060] Its heavy chain FR4 has the amino acid sequence shown in SEQ ID NO:56;

[0061] Its light chain FR1 has the amino acid sequence shown in SEQ ID NO:44;

[0062] Its light chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:47-49;

[0063] Its light chain FR3 has an amino acid sequence as shown in SEQ ID NO:54 or 55;

[0064] Its light chain FR4 has an amino acid sequence as shown in SEQ ID NO:57.

[0065] In some embodiments, the antibody or antigen-binding molecule targeting CD228 contains:

[0066] Its heavy chain FR1-4 has the amino acid sequences shown in SEQ ID NO:42, 45, 50, 56, and its light chain FR1-4 has the amino acid sequences shown in SEQ ID NO:44, 47, 54, 57, respectively.

[0067] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:42, 45, 50, 56 in sequence, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:44, 47, 55, 57 in sequence;

[0068] Its heavy chain FR1-4 has the amino acid sequences shown in SEQ ID NO:42, 45, 51, 56, and its light chain FR1-4 has the amino acid sequences shown in SEQ ID NO:44, 47, 54, 57, respectively.

[0069] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:42, 45, 51, 56 in sequence, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:44, 47, 55, 57 in sequence;

[0070] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:43, 46, 52, 56 in sequence, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:44, 48, 54, 57 in sequence;

[0071] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:43, 46, 53, 56 in sequence, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:44, 48, 54, 57 in sequence;

[0072] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:43, 46, 52, 56 in sequence, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:44, 49, 55, 57 in sequence;

[0073] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:43, 46, 53, 56 in sequence, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:44, 49, 55, 57 in sequence;

[0074] In some specific embodiments, the antibody or antigen-binding molecule has a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 64 to 69; and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 70 to 73.

[0075] More specifically, the antigen or antigen-binding molecule:

[0076] Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:64, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:70.

[0077] Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:64, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:71;

[0078] Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:65, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:71;

[0079] Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:66, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:72;

[0080] Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:66, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:73;

[0081] Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:67, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:72;

[0082] Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:67, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:73;

[0083] Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:68, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:73;

[0084] Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:69, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:73.

[0085] In this invention, the heavy chain constant region of the antibody or antigen-binding molecule targeting CD228 is human IgG1, and the light chain constant region is human Kappa type.

[0086] The heavy chain constant region of the antibody or antigen-binding molecule targeting CD228 has an amino acid sequence as shown in SEQ ID NO:74, or a sequence based on the amino acid sequence shown in SEQ ID NO:74 with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology to the amino acid sequence shown in SEQ ID NO:74.

[0087] The light chain constant region of the antibody or antigen-binding molecule targeting CD228 has an amino acid sequence as shown in SEQ ID NO:75, or a sequence based on the amino acid sequence shown in SEQ ID NO:75 with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology to the amino acid sequence shown in SEQ ID NO:75.

[0088] Secondly, the present invention also provides nucleic acid molecules that encode antibody or antigen-binding molecules as described above.

[0089] Thirdly, the present invention also provides a plasmid vector containing the nucleic acid as described above.

[0090] Fourthly, the present invention also provides a host cell transformed or transfected with the plasmid vector, or with the nucleic acid molecule integrated into the genome.

[0091] Fifthly, the present invention also provides a method for preparing the antibody or antigen-binding molecule, comprising: culturing the host cells to obtain a culture containing the antibody or antigen-binding molecule.

[0092] Sixthly, the present invention also provides a coupling element.

[0093] It includes the antibody or antigen-binding molecule and the drug;

[0094] Or may include the antibody or antigen-binding molecule and the marker;

[0095] It may include the antibody or antigen-binding molecule and the medium.

[0096] In the conjugate of the present invention, the drug is an antitumor drug; the marker is a chemical marker or a biological marker; and the medium is a solid-phase medium or a non-solid-phase medium.

[0097] In this embodiment of the invention, the drug is at least one of MMAE, Dxd, DM1, DM4 or PBD.

[0098] In this invention, the drug and antibody are directly linked by an amide bond, or they can be linked together by a linker; this invention does not limit the specific method used.

[0099] The chemical label is a chemiluminescent indicator, a fluorescent indicator, an isotope, and / or a colloidal indicator.

[0100] The chemiluminescent indicator is selected from one or more of acridine ester, acridine sulfonamide and its derivatives, luminol, isoluminol, isoluminol isothiocyanate and its derivatives, N-(4-aminobutyl)-N-ethyl isoluminol, 4,5-diaminophthalic acid hydrazide or aminobutylethyl benzoyl hydrazide.

[0101] The fluorescent indicator is selected from one or more of the following: AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red, or PerCP dye.

[0102] Isotopes selected 125 I, 131 I, 124 I, 3 H, 14 C 111 In、 89 Zr or 32 One or more of P.

[0103] Colloidal indicators are selected from one or more of colloidal gold, colloidal carbon, or colloidal selenium.

[0104] The biomarker is biotin, avidin, or an enzyme label.

[0105] The enzyme label is selected from one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridge, alkaline phosphatase-antialkaline phosphatase bridge, and β-galactosidase-antiβ-galactosidase bridge.

[0106] The medium can be colloidal gold, an ELISA plate, magnetic beads, or latex microspheres. In this invention, the antibody can be linked to the medium via an amide bond or a streptavidin-biotin system; this invention does not limit the specific method used.

[0107] The seventh aspect is the use of the antibody or antigen-binding molecule, or a conjugate comprising the antibody or antigen-binding molecule and a drug, in the preparation of a drug for treating tumors.

[0108] Eighthly, the present invention also provides a medicine,

[0109] It includes: the antibody or antigen-binding molecule;

[0110] Or include: a conjugate comprising the antibody or antigen-binding molecule and the drug.

[0111] In this invention, the drug also includes pharmaceutically acceptable excipients.

[0112] In this invention, the dosage form of the drug includes, but is not limited to, oral preparations, injections, aerosols, or suppositories. Oral preparations include, but are not limited to, tablets, capsules, pills, oral liquids, or dispersants. Injections include, but are not limited to, injectable solutions or powders for injection.

[0113] In this invention, the medicament also includes other therapeutic agents. Multiple drugs and therapeutic agents may exist in combination or independently; this invention does not limit this. Multiple drugs may be administered simultaneously or sequentially; this invention also does not limit this. The other therapeutic agents are antitumor drugs. For example, the antitumor drugs include, but are not limited to, at least one of: cisplatin, pertuzumab, oxaliplatin, doxorubicin, nivolumab, pembrolizumab, niraparib, etoposide, tamoxifen, paclitaxel, vinorelbine, gefitinib, atezolizumab, carboplatin, erlotinib, rituximab, trastuzumab, olaparib, toremifene, fluorouracil, fulvestrant, or letrozole.

[0114] Ninthly, the present invention also provides a method of treating a disease, comprising administering the medicament described herein. The administration methods of the medicament or pharmaceutical composition include, but are not limited to: oral, vaginal, rectal, aerosol inhalation, buccal, nasal, topical, and non-enteric administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intrasternal, and intracranial injection or infusion, or administration via an external implantation device. Oral, intraperitoneal, or intravenous administration is preferred.

[0115] The disease is a tumor, including but not limited to bladder urothelial carcinoma (BLCA), cervical squamous cell carcinoma and adenocarcinoma (CESC), cholangiocarcinoma (CHOL), colorectal cancer (COAD), diffuse large B-cell lymphoma (DLBC), esophageal cancer (ESCA), head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), gastric cancer (STAD), and / or thymic carcinoma (THYM).

[0116] In a tenth aspect, the present invention also provides the use of the antibody or antigen-binding molecule, including a conjugate of the antibody or antigen-binding molecule and a label, or a conjugate including an antibody or antigen-binding molecule and a medium, in the preparation of a CD228 detection reagent.

[0117] In its eleventh aspect, the present invention also provides a CD228 detection reagent.

[0118] It includes: the antibody or antigen-binding molecule;

[0119] Or it may include conjugates of antibodies or antigen-binding molecules and markers as described above;

[0120] Or it may include conjugates of antibodies or antigen-binding molecules and mediators as described above.

[0121] The detection reagent of the present invention also includes buffer solutions to facilitate the reaction. Examples include phosphate buffer, carbonate buffer, acetate buffer, Tris-HCl buffer, and HEPES buffer. The reagent also includes agents to maintain reaction stability, such as BSA, DDT, β-mercaptoethanol, glycerol, vitamin C, or Tween.

[0122] In a twelfth aspect, the present invention also provides a CD228 detection method, which includes detecting a sample with the detection reagents as described above.

[0123] The samples include, but are not limited to, cells, blood, tissue sections, or tissue homogenates. The detection methods include, but are not limited to, ELISA, flow cytometry, and / or immunohistochemistry.

[0124] This invention provides an antibody targeting CD228 and an antibody-drug conjugate. The antibody exhibits good FACS binding ability, internalization activity, and affinity. When used to prepare antibody-drug conjugates, it demonstrates excellent tumor cell killing ability. When used as a detection reagent for CD228, it provides good detection results. Attached Figure Description

[0125] Figure 1 Central AC staining for immunohistochemical (IHC) microarray staining;

[0126] Figure 2 This shows CD228 expression data for different tumors in the TCGA and GTEx databases;

[0127] Figure 3 The results of serum titer detection in immunized mice are shown in AB.

[0128] Figure 4 The AC indicates the FACS binding result of the chimeric antibody;

[0129] Figure 5 The results of the chimeric antibody affinity test are shown.

[0130] Figure 6 The results of the AB assay for chimeric antibody internalization are shown.

[0131] Figure 7 The results of ADC internalization detection are shown in AB.

[0132] Figure 8 The results of in vitro killing assays of natural tumor cell lines using ADC are shown.

[0133] Figure 9 The results of humanized antibody FACS binding are shown in A and B, where A represents the FACS binding result of 479D3G1 humanized antibody and B represents the FACS binding result of 560D11G6 humanized antibody.

[0134] Figure 10 The results of the humanized antibody internalization test are shown in AB, where A shows the internalization of 497D3G1 humanized antibody and B shows the internalization of 560D11G6 humanized antibody. Detailed Implementation

[0135] This invention provides antibodies and antibody-drug conjugates targeting CD228. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve these conjugates. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0136] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0137] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0138] In this application, the terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0139] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0140] In this application, "specific binding" refers to the fact that antigen-binding molecules (e.g., antibodies) typically bind antigens specifically to substantially the same antigens with high affinity, but do not bind unrelated antigens with high affinity. Affinity is usually reflected by the equilibrium dissociation constant (KD), where a lower KD indicates higher affinity. For example, for antibodies, high affinity typically refers to a KD of approximately 10. -6 M or lower, approximately 10 -7 M or lower, approximately 10 -8 M or lower, approximately 1×10 -9 M or lower, approximately 1×10-10 M or lower, 1×10 -11 M or lower or 1×10 -12 M or lower KD. KD is calculated as follows: KD = Kd / Ka, where Kd represents the dissociation rate and Ka represents the binding rate. The equilibrium dissociation constant KD can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

[0141] In this application, "antigen-binding molecule" refers to a molecule that specifically binds to an antigen. For example, the antigen-binding molecule includes, but is not limited to, antibodies or antibody mimics. "Antibody mimic" refers to a biomolecule that mimics the structure and function of a natural antibody through non-natural synthetic methods. For example, antibody mimics include, but are not limited to, affibody, affitin, affilin, designed ankylosing spondylamine repeat (DARPin), nucleic acid aptamers, or Kunitz-type domain peptides.

[0142] In this application, "antibody" includes a typical "quadruple-chain antibody," which belongs to the immunoglobulin class composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. Heavy chains are linked to each other and to each other with disulfide bonds, forming a "Y"-shaped structure. Because the amino acid composition and sequence of the immunoglobulin heavy chain constant region differ, their antigenicity also differs. Based on this, the "immunoglobulins" in this article can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

[0143] In this application, "antibody" encompasses various forms and structures, including complementary-determining regions (CDRs) and antibody framework regions (FRs). CDRs are hypervariable regions of the antibody, located at the amino termini of the heavy chain (VH) and light chain (VL). These regions contain highly variable amino acid sequences, allowing for precise binding of the antibody to the antigen. The three main CDR regions are CDR-H1, CDR-H2, and CDR-H3 (in the heavy chain) and CDR-L1, CDR-L2, and CDR-L3 (in the light chain). FRs are regions in the antibody heavy and light chains other than the CDRs. While not directly involved in antigen binding, FRs significantly influence the conformation of the CDRs and the specificity of antigen binding.

[0144] In this application, "antibody" also includes antibodies that do not contain light chains or antibodies that do not contain constant regions.

[0145] In this application, the "antibody" can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0146] In this application, the "heavy chain constant region" refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in the binding of the antibody to the antigen but exhibits effector functions, such as interaction with the Fc receptor. It has a more conserved amino acid sequence compared to the variable domains of the antibody. The "heavy chain constant region" can be selected from: the CH1 domain, the hinge region, the CH2 domain, the CH3 domain, or variants or fragments thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to the natural antibody constant region, while the latter includes only a portion of the full-length heavy chain constant region. Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" can be selected from the Fc or CH3 domain.

[0147] In this application, the "light chain constant region" refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0148] In this application, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0149] In this application, "identity" can be calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. Taking into account the number of vacancies that need to be introduced for optimal alignment of the two sequences and the length of each vacancy, the percentage of identity between the two sequences varies with the common positions of the sequences.

[0150] In this application, "nucleic acid" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5′ to 3′.

[0151] In this application, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, and single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbones bonded or chemically modified residues. Nucleic acid molecule also encompasses DNA and RNA molecules suitable as carriers for the direct expression of the antibodies of the present invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, thereby allowing the mRNA to be injected into the subject to generate antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2101823 B1).

[0152] In this application, "vector" or "plasmid vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which the vector has been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them.

[0153] In this application, "host" or "host cell" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be completely identical to parental cells in their nucleic acid contents and may contain mutations. This document includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0154] In this application, "medicine" allows the biologically active ingredients contained therein to exist in an effective form and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the pharmaceutical composition.

[0155] In this application, "prevention and treatment" includes prevention and / or treatment. "Treatment" refers to surgical or therapeutic treatment aimed at preventing, mitigating (reducing) undesirable physiological changes or lesions, such as cancer and tumors, in the treated individual. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as mitigation, reduction, weakening, mitigation, and relief are used, they also include elimination, disappearance, and non-occurrence.

[0156] In this application, "subject" refers to an organism receiving treatment for a specific disease or symptom as described in this invention. Exemplarily, "subject" includes mammals receiving treatment for a disease or symptom, including bovine, equine, sheep, suidae, canine, feline, rodent, and primate animals, with preferred mammals being humans, cats, dogs, or pigs.

[0157] In this application, the terms "therapeutic effective amount" or "preventive effective amount" refer to an amount sufficient, within reasonable medical judgment, to treat or prevent a patient's disease while avoiding serious side effects with a sufficiently low risk-reward ratio. The preventive or therapeutic effective amount of a compound will vary depending on the specific compound chosen (e.g., considering the compound's potency, effectiveness, and half-life), the chosen route of administration, the disease being prevented or treated, the severity of the disease, the patient's age, size, weight, and physical condition, the patient's medical history, the duration of prevention or treatment, the nature of concurrent therapies, the desired preventive or therapeutic effect, etc., but can still be conventionally determined by those skilled in the art. When an active ingredient is administered to an individual alone, the therapeutic effective amount refers to that ingredient alone. When a combination is used, the therapeutic effective amount refers to the combined amount of active ingredients that produce the therapeutic effect, regardless of whether they are administered in combination, continuously, or simultaneously.

[0158] In this application, "cancer" refers to or describes a physiological condition in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers. The terms "tumor" or "tumor" herein refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0159] The test materials used in this invention are all commercially available products, and the amino acid sequences of the antibodies involved are as follows:

[0160] Table 1. Amino acid sequences (underlined regions are CDR regions)

[0161]

[0162]

[0163]

[0164] Table 2 CDR regions of each antibody

[0165]

[0166] Table 3. FR region of chimeric antibody

[0167]

[0168]

[0169] Table 4. FR region of humanized chimeric antibody

[0170]

[0171] The heavy and light chain variable regions of the positive control antibody hL49 are as follows (partitioning method: Kabat):

[0172] hL49-VH:

[0173] QVQLQESGPGLVKPSETLSLTCTVSGDSIT SGYWN WIRQPPGKGLEYIG YISDSGITYYNPSLKS RVTISRDTSKNQYSLKLSSVTAADTAVYYCAR RTLATYYAMDY WGQGTLVTVSS (SEQ ID NO.80)

[0174] hL49-VL:

[0175] DFVMTQSPLSLPVTLGQPASISC RASQSLVHSDGNTYLH WYQQRPGQSPRLLIY RVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQSTHVPPT FGQGTKLEIK (SEQ ID NO.81)

[0176] The present invention will be further illustrated below with reference to the embodiments:

[0177] Example 1. CD228 expression

[0178] 1.1 Expression of CD228 in native tumor cell lines

[0179] The expression abundance of CD228 on the surface of various tumor cells was measured using the human CD228 antibody hL49 (Seagen). The hL49 antibody was labeled with PE (Dojindo, Cat#LK23), and binding curves were plotted between the labeled antibody and tumor cells until saturation concentration was reached. BD Quantibrite was then used as the final concentration. TM A standard curve was created using PE beads (BD, Cat#340495) to calculate the expression level of CD228 on the cell surface. The quantitative experimental results are shown in Table 5.

[0180] Table 5. Expression levels of CD228 on the surface of tumor cells

[0181] Cellline Surface CD228 number / cell SK-MEL-28 63835 HCC70 90301 Calu-1 55529 CorL23 35923 SK-CO-1 30078 A375 23551 HCT116 12677 A2058 12692 HT1080 8210

[0182] 1.2 CD228 Immunohistochemical (IHC) Array Staining

[0183] The tissue microarray IHC staining was performed by Shanghai Yuanxi Biotechnology Co., Ltd., and the results are as follows: Figure 1 As shown in the AC diagram: melanoma (SKCM) has very high CD228 expression, as do rectal cancer (CRC) and pancreatic cancer (PAAD). These results are consistent with those from the Human Genome Atlas (TCGA) and GTEx databases. Figure 2 The consistency between these findings suggests that the aforementioned cancers could be included in subsequent indication studies. Furthermore, the TCGA and GTEx databases also indicate differences in CD228 expression between tumors and normal tissues in bladder urothelial carcinoma (BLCA), cervical squamous cell carcinoma and adenocarcinoma (CESC), cholangiocarcinoma (CHOL), colorectal cancer (COAD), diffuse large B-cell lymphoma (DLBC), esophageal cancer (ESCA), head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), gastric cancer (STAD), and thymic carcinoma (THYM), suggesting these could serve as an expansion cohort for indications.

[0184] Example 2. Anti-CD228 antibody

[0185] 2.1 Mouse-derived anti-CD228 monoclonal antibody

[0186] Animal Immunization: The immunogen hCD228-mFc (humanCD228 Uniprot ID: P08582) was produced in-house by Maiwei. SJL and BALB / c mice were immunized separately, with five mice in each group. Group 22 (G22) consisted of five SJL mice (12301-12305), and Groop 23 (G23) consisted of five BALB / c mice (12306-12310). hCD228-mFc, along with Freund's complete adjuvant (for the first immunization) and Freund's incomplete adjuvant (for the second and third immunizations), was administered intraperitoneally every two weeks. The first immunization dose was 50 μg / mouse, and the second and third doses were 25 μg / mouse. One week after the second immunization, orbital blood samples were collected, and the serum was used for antigen-specific titer testing.

[0187] Serum titers were detected using FACS and ELISA methods, respectively, using CHOK1 cells overexpressing humanCD228 (CHOK1-hCD228, self-constructed) and hCD228-his protein (self-produced).

[0188] FACS method: Take CHOK1-hCD228 cells and CHOK1-blank cells in logarithmic growth phase, centrifuge to remove supernatant, wash three times with FACS buffer (1X PBS + 1% BSA), resuspend cells in FACS buffer for the last wash, and count them at 2 x 10⁻⁶. 5 Three cell lines were seeded into 96-well U-bottom round plates, and the supernatant was removed by centrifugation. Harvested mouse serum was diluted 1:100 or 1:1000 and added to the corresponding wells. After mixing, the plates were incubated at 4°C for 1 hour. After 1 hour of incubation, the 96-well plates were removed, centrifuged, and the supernatant was discarded. The plates were washed three times with FACS Buffer, discarding the supernatant after the last wash. Diluted AF488 donkey anti-mouse IgG (INVITROGEN, Cat#A21202) secondary antibody was added to the corresponding wells, mixed, and incubated at 4°C in the dark for 30 minutes. After incubation, the 96-well plates were removed, centrifuged, and the supernatant was discarded. The plates were washed three times with FACS Buffer, and the cells were resuspended in an appropriate amount of FACS Buffer before flow cytometry analysis using FACS CantoII. The results are as follows: Figure 3 As shown in Figure A, both groups of mice showed a strong immune response, with 12304 and 12309 showing a slightly lower response than the other mice in their groups.

[0189] ELISA Method: RhesusCD228-his and mouseCD228-his proteins were diluted at 1 μg / mL in PBS. 100 μL was added to each well of a 96-well ELISA plate (Corning, catalog number 3590) and incubated overnight at 4°C. The next day, the coating solution was removed, and the plate was washed three times with washing buffer PBST (Guangdong Eyou Biotechnology, catalog number PBT-2050). Blocking was performed at room temperature for 1-2 hours with blocking buffer (5% BSA in PBS, pH 7.4). After washing the plate three times again, serially diluted mouse serum was added to the corresponding wells and incubated at room temperature for 1 hour. After washing three times, diluted secondary antibody (Peroxidase Affini Pure Goat Anti-Mouse IgG (subclasses 1+2a+2b+3), FcγFragment Specific, Cat#115-035-164) was added and incubated at room temperature for 1 hour. The plate was washed three times, and TMB peroxidase substrate (Thermo, catalog number 34029) was added for color development for 5 minutes. The reaction was then terminated with 1N hydrochloric acid, and the absorbance was measured at 450 nm. The detection results were consistent with those obtained by FACS. Figure 3 (B)

[0190] Preparation of anti-CD228 hybridomas: Based on the FACS and ELISA results of immunized mouse serum, mice with high serum antibody titers were selected. Spleen cells were mixed with mouse myeloma cells SP2 / 0 (ATCC, Cat#CRL-1581) and fused using a high-efficiency electrofusion method. The fused cells were diluted in DMEM (Corning-cellgro, Cat#10-013-CVR) medium containing HT (Corning-cellgro, Cat#25-047-Cl), and added at a ratio of 1 x 102 spleen cells. 8 The plates were spread into 96-well plates at a ratio of 40 plates per well and incubated overnight in a 5% CO2, 37°C incubator. After 24 hours, DMEM medium containing 2×HAT (purchased from Sigma, Cat#H-0262) was added.

[0191] Hybridoma screening: After 10 days of culture, the expression of anti-hCD228 antibody in the cell supernatant was detected using an Acumen Explorer microplate cytometer (TTPLabtech). The positive and negative control cells were CHOK1-hCD228 and CHOK1 cells, respectively. The specific Acumen experimental method is as follows: The night before the experiment, CHOK1-hCD228 and CHOK1 cells were seeded into 384-well plates at a concentration of 3000 cells / well. Cells were washed once with PBS solution (100 μL / well) before the experiment. 20 μL of the hybridoma / subclone cell supernatant to be tested was added to each well, and the cells were incubated at 37°C for 2 hours. Cells were washed twice with 1X PBST (PBS + 0.05% Tween 20). 20 μL of a 1:1000 diluted secondary antibody (AF488 donkey anti-mouse IgG, AF488 donkey anti-human IgG) was added to each well, and the cells were incubated at 37°C for 1 hour. 1X PBST was then used to wash the cells. Cells were washed twice with PBST (PBS + 0.05% Tween 20); cells were fixed with 20 μL of 4% PFA at room temperature for 2-8 min, and washed once with PBS; 20 μL of LPI (9 μM) + RNase A (200 μg / mL) mixture was added to each well and incubated at 37°C for 30 min; then the fluorescence value of each well was detected using an Acumen Explorer microplate cytometer.

[0192] Secondary screening: Based on the Acumen analysis results, positive clones were selected and transferred to 24-well plates for expanded culture. After 3 days of culture, the supernatant from the 24-well plates was used for FACS detection. The positive and negative control cells were CHOK1-hCD228 and CHOK1-blank cells, respectively. Another portion of the supernatant was used for ELISA screening. The screening proteins were rhesus monkey CD228-his (rhesusCD228, Unoprot ID: F6VPZ9) and mouse CD228-his (mouseCD228, Unoprot ID: Q9R0R1).

[0193] Subcloning: Based on the screening results of 24-well plates, hybridoma cells were selected and subcloned in 96-well plates through limiting dilution. After 7-10 days of subcloning culture, Acumen was used for screening, and positive single clones were selected and amplified into 24-well plates for further culture.

[0194] Three days later, the supernatant from the 24 wells was analyzed: 1) Acumen was used to detect the binding ability of the supernatant to the antigen. The cells used for testing were CHOK1-hCD228, and the negative control cells were CHOK1-blank. 2) FACS was used to detect the binding ability of the supernatant to the antigen. The cells used for testing were CHOK1-hCD228, CHOK1-rhesusCD228, and tumor cells Calu-1. According to the quantification results of cell surface proteins, the expression level of CD228 on the surface of individual cells was above 51K. The negative control cells were CHOK1-blank. 3) ELISA was used to detect the cross-binding of the supernatant with rhesus monkey CD228 and mouse CD228.

[0195] Based on the results of the 24-well plate samples, the screened positive clones were cultured in DMEM medium containing 10% (w / w) FBS at 37°C and 5% (v / v) CO2. The cells were then suspended in cryopreservation solution and cryopreserved in liquid nitrogen according to standard methods for subsequent antibody production, purification and amino acid sequencing.

[0196] According to the above protocol, this project conducted two immunizations: SJL mice and BALB / c mice. Based on the serum titer test results, mice with higher serum titers were selected for hybridoma fusion, and a total of four fusions were performed. A total of 548 clones were initially screened, and 120 positive monoclonal clones were selected after subcloning. After antibody subtype detection and sequence alignment of the positive monoclonal clones, 84 mouse antibodies with sequence specificity were selected for human-mouse chimeric antibody construction.

[0197] 2.2 Production of chimeric antibodies

[0198] 2.2.1 Extraction of candidate cloned genes

[0199] Extraction of total RNA and synthesis of cDNA from hybridoma cells

[0200] After culturing the positive monoclonal hybridoma cells obtained in Example 1.1 to the logarithmic growth phase, the cells (approximately 1 x 10⁻⁶) were collected. 6 (cells / clones), utilizing Total RNA was extracted from hybridoma cells using RNAPlus (MN, Cat#740984.250). Using 1 μg of extracted RNA, [the following was observed]: III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Cat#R323-01) was used for cDNA synthesis.

[0201] Amplifying the VH and VL sequences encoding the antibody genes and constructing a T-vector clone.

[0202] Using the synthesized cDNA as a template, a CRO company was commissioned to perform specific amplification of the cDNA using primers Primer A+S mix (containing universal primers for heavy and light chains, primers for the constant regions of human hIgG1, IgG2a, IgG2b, and IgG3, and specific primers for human kappa) and Ex Taq enzyme (TaKaRa, Cat#RR902A). The PCR reaction system and cycle are as follows.

[0203]

[0204] PCR products were extracted using a gel extraction kit after electrophoresis on a 1% agarose gel. Gel and PCRClean-up (MN, Cat#740609.250) were used to recover the target fragment.

[0205] The recovered fragment was then cloned into the pMD19-T (TaKaRa, Cat#3271) vector using Solution I (TaKaRa, Cat#6022Q). The cloned plasmid was then transformed into competent DH5α cells (Yestern, Cat#FYE607-80VL) using a heat stimulation method and evenly spread on 2YT solid plates containing ampicillin. The plates were then sent to Genewiz sequencing company for sequencing using universal primers PMAL-C2X-R.

[0206] 2.2.2 Construction, expression, and purification of chimeric antibodies

[0207] Constructing vectors for expressing chimeric antibodies

[0208] Analyze the VH and VL antibody sequencing sequences, select clone plasmids with correct antibody sequencing, and amplify the target fragment using the universal primer mix for VH / VL antibodies (containing the expression vector signal peptide) under the action of the high-fidelity enzyme PrimeSTAR (TaKaRa, Cat#R045) and primers. The PCR program is as follows:

[0209]

[0210] After electrophoresis, the VH and VL fragments were recovered using a gel extraction kit (MN, Cat#740609.250). The recovered VH and VL fragments were then inserted, respectively, into linearized vectors containing the human heavy chain constant region (pTT5_hIgG1.G1m3) and light chain constant region (pTT5_hKappa.Km3) using recombinase (Vazyme, Cat#C112-02). The recombinant vectors were transformed into competent DH5α cells and evenly spread on 2YT solid plates containing ampicillin. The plates were then sent to Genewiz sequencing company for sequencing using pTT5-F and pTT5-R primers.

[0211] 2.2.3 Expression and purification of chimeric antibodies

[0212] Sequencing sequences were analyzed, and plasmids with correct sequencing were amplified. Plasmids containing chimeric heavy and light chains were transfected into HEK293 cells at a density of 2×10e6 / ml using PEI reagent (1 μg plasmid: 3 μg PEI) at a ratio of 2:3. Transfected cells were cultured at 37°C in a 5% CO2 incubator for 5–7 days to express the chimeric antibody. The culture supernatant was centrifuged and filtered through a 0.22 μm filter, then purified using a Protein G agarose column (GE Healthcare Bio-sciences, 17-0618-05). The column was first equilibrated with 1×PBS (pH 7.4), and the filtered culture supernatant was loaded onto the column. The column was then washed with 1×PBS (pH 7.4), followed by elution with 50 nM sodium citrate (pH 2.5). The eluted sample was then neutralized with 1 M Tris-HCl (pH 9.0). The neutralized sample was replaced with 1×PBS (pH 7.4), filtered through a 0.22μm filter for sterilization, and the concentration of the purified antibody was determined using Nanodrop (Thermo Fisher Scientific Inc). The purified antibody was then aliquoted and frozen for later use.

[0213] 2.3 Characterization of chimeric antibody activity

[0214] The activity of the chimeric antibodies was characterized using FACS, Octet, and internalization assays. FACS was performed using tumor cells SK-MEL-28, Calu-1, and A375 (with CD228 expression levels of 63835, 55529, and 23551 on their cell surfaces, respectively), as well as CHOK1-humanCD228, CHOK1-rhesusCD228, and CHOK1-blank cells. The positive control antibody was Seagen's hL49, and the negative control was hIgG1. Results are as follows: Figure 4 As shown in Figure AC, chimeric antibodies 497D3G1, 560D11G6, and 45C11G12 all exhibited stronger affinity than the positive control, and all three showed cross-binding with rhesus monkey CD228. 539A3A2 and 545C2A5 showed weaker binding activity, but also cross-binding activity with rhesus monkeys. None of the antibodies showed non-specific binding to CHOK1. (Only data for some chimeric antibodies are listed here.)

[0215] The binding affinity of the chimeric antibodies was determined by Octet assay, using hCD228-his protein as the analyte, and the binding kinetics of each chimeric antibody were measured. The results are shown in Table 6. Figure 5As shown, the dissociation constants of 497D3G1, 560D11G6, 45C11G12, 545C2A5, and 539A3A2 are less than 10. -3 All of them have good affinity.

[0216] Table 6. Results of chimeric antibody affinity assay

[0217] Ab kdis(1 / s) hL49 5.09E-04 497D3G1 3.57E-04 560D11G6 4.34E-05 45C11G12 3.99E-04 545C2A5 <1.0E-07 539A3A2 9.95E-06

[0218] Internalization of the chimeric antibody was determined by FACS. Equal concentrations of antibody and internalization marker (Sartorius, Cat#90565) were incubated with cells at 37°C for 2 hours or 24 hours, and internalization activity was detected by flow cytometry. Results are as follows: Figure 6 As shown in Figure AB, 497D3G1, 560D11G6 and 45C11G12 all have good internalization activity. Among them, the internalization activity of 560D11G6 is comparable to that of the control hL49, while the internalization activities of 545C2A5 and 539A3A2 are weaker.

[0219] Example 3. Intracellular activity of antibody-drug conjugates

[0220] Antibodies with good internalization activity (497D3G1-ADC, 560D11G6-ADC, 45C11G12-ADC, and the positive control antibody hL49) were conjugated to the cytotoxic drug MMAE via PEG(12)-modified β-glucuronidase, with a DAR value of 8. The activity of antibody-drug conjugates (ADCs) was first determined by an internalization assay, and the results are as follows: Figure 7 As shown in Figure AB, 497D3G1-ADC, 560D11G6-ADC, and 45C11G12-ADC all exhibited good internalization activity. Among them, the internalization activity of 560D11G6-ADC and 497D3G1-ADC was comparable to or slightly better than that of the positive control hL49-ADC.

[0221] Example 4. Killing effect of antibody-drug conjugate on native tumor cell lines targeting CD228

[0222] The tumor-killing activity of antibody-drug conjugates (ADCs) was determined using an in vitro killing assay with native tumor cell lines expressing the CD228 target. Tumor cells were incubated with serially diluted ADCs at 37°C for 5 days. Cell viability was then assessed using the CCK8 assay. An isotype control, hIgG1-ADC, with the same conjugation method, served as a negative control. Cell viability was normalized using untreated wells. The IC50 of the killing assay and the percentage of cells surviving at the highest effective concentration were statistically analyzed. Figure 8As shown in Table 7, when the abundance of the CD228 target on the cell surface was low (A2058, A375), the IC50 of the three ADCs in vitro were all higher than that of the positive control hL49-ADC. However, in terms of the percentage of cells still surviving at the highest effective concentration, 497D3G1-ADC and 45C11G12-ADC were stronger than the positive control. For cells with high abundance of the target on the cell surface (SK-CO-1, SK-MEL-28), the IC50 of 497D3G1-ADC in vitro was lower than that of the positive control, but the percentage of cells surviving at the highest effective concentration was slightly higher than that of the positive control. In summary, the killing activity of 497D3G1-ADC is comparable to that of the positive control hL49-ADC.

[0223] Table 7. Results of tumor cell killing by ADC native tumor lines

[0224] Example 5. Production and identification of humanized anti-CD228 antibody

[0225] (I) Humanization design of candidate clones

[0226] The CDRs of candidate molecules (497D3G1 and 560D11G6) were divided using the Kabat partitioning method, and the human germline sequence and J region sequence closest to their FR region were searched in the database. The CDRs of the murine candidate antibodies were then completely transplanted into the corresponding human template sequences. Simultaneously, to ensure that the humanized antibodies retain their affinity and activity for human CCR8, key amino acid sites (amino acid sites numbered according to Kabat) affecting the 3D conformation of the antibody's variable region in the human template sequence were restored to the murine parent amino acid sequences. Finally, to avoid potential post-translational modifications (PTM) later, amino acids with similar structures or properties were substituted.

[0227] (II) Construction of humanized antibody expression vectors, expression and purification of antibodies

[0228] The variable region sequences of the aforementioned humanized antibodies were codon-optimized and finally synthesized and constructed into corresponding linearized vectors containing human heavy chain constant regions (pTT5_hIgG1.G1m3) and light chain constant regions (pTT5_hKappa.Km3) (gene synthesis and vector construction were both completed by the sequencing company Genewiz).

[0229] The plasmid synthesized by the sequencing company was amplified and prepared. The heavy and light chain plasmids were transfected into HEK293 cells at a density of 2×10⁶ / ml using PEI reagent (Polysciences, Cat#24885) at a ratio of 2:3 (1 μg plasmid: 3 μg PEI). The transfected cells were incubated at 37°C in a 5% CO₂ incubator for 5–7 days. The culture supernatant was centrifuged and filtered through a 0.22 μm filter, and purified using a purification column (Bio-Rad, 731-1553) filled with MabSelect SuRe agarose gel (GE Healthcare Bio-sciences, 17-5438-02). First, equilibrate the purification column with 1×PBS (pH 7.4). Load the filtered culture supernatant onto the purification column, wash the column with 1×PBS (pH 7.4), and then elute the sample with elution solution (0.1M sodium citrate, pH 2.5). Neutralize the eluted sample with 1M Tris-HCl, pH 9.0 solution. Replace the neutralized sample with 1×PBS (pH 7.4), filter sterilize using a 0.22µm filter, and determine the concentration of the purified antibody using Nanodrop (Thermo Fisher Scientific Inc.).

[0230] (III) Detection of humanized antibody binding and internalization activities

[0231] The candidate derived antibodies obtained above were first evaluated for their ability to specifically bind to human CD228 using a FACS binding assay, the same method as the FACS binding assay for chimeric antibodies. The results are as follows: Figure 9 As shown, both the 497D3G1 and 560D11G6 humanized antibodies maintained binding activity comparable to that of the chimeric antibodies.

[0232] The internalization activity of the fully humanized antibodies described above was evaluated using the same method as the chimeric antibody internalization assay. The results are as follows: Figure 10 As shown, the humanized antibodies hz497D3G1-H0L0, hz497D3G1-H0L1, and hz497D3G1-H1L1 all maintained good internalization activity; the humanized antibodies hz560D11G6-H0L0, hz560D11G6-H0L1, hz560D11G6-H1L0, hz560D11G6-H1L1, hz560D11G6-H2L1, and hz560D11G6-H3L1 all maintained good internalization activity.

[0233] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding molecule targeting CD228, characterized in that, The amino acid sequence of its heavy chain CDR1 is shown in SEQ ID NO:1, the amino acid sequence of its heavy chain CDR2 is shown in SEQ ID NO:4, the amino acid sequence of its heavy chain CDR3 is shown in SEQ ID NO:7, the amino acid sequence of its light chain CDR1 is shown in SEQ ID NO:10, the amino acid sequence of its light chain CDR2 is shown in SEQ ID NO:13, and the amino acid sequence of its light chain CDR3 is shown in SEQ ID NO:

16. Alternatively, the amino acid sequence of its heavy chain CDR1 is shown in SEQ ID NO:2, the amino acid sequence of its heavy chain CDR2 is shown in SEQ ID NO:5, the amino acid sequence of its heavy chain CDR3 is shown in SEQ ID NO:8, the amino acid sequence of its light chain CDR1 is shown in SEQ ID NO:11, the amino acid sequence of its light chain CDR2 is shown in SEQ ID NO:14, and the amino acid sequence of its light chain CDR3 is shown in SEQ ID NO:

17. Alternatively, the amino acid sequence of its heavy chain CDR1 is shown in SEQ ID NO:3, the amino acid sequence of its heavy chain CDR2 is shown in SEQ ID NO:6, the amino acid sequence of its heavy chain CDR3 is shown in SEQ ID NO:9, the amino acid sequence of its light chain CDR1 is shown in SEQ ID NO:12, the amino acid sequence of its light chain CDR2 is shown in SEQ ID NO:15, and the amino acid sequence of its light chain CDR3 is shown in SEQ ID NO:

18.

2. The antibody or antigen-binding molecule according to claim 1, characterized in that, It is a chimeric antibody, and its heavy chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:19-21; Its heavy chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:22-24; Its heavy chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:25-27; Its heavy chain FR4 has an amino acid sequence as shown in any one of SEQ ID NO:28-30; Its light chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:31 to 33; Its light chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:34-36; Its light chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:37-39; Its light chain FR4 has an amino acid sequence as shown in SEQ ID NO:40 or 41.

3. The antibody or antigen-binding molecule according to claim 1 or 2, characterized in that, Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:58, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:61; Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:59, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:62; Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:60, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:

63.

4. The antibody or antigen-binding molecule according to claim 1 or 2, characterized in that, Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:64, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:70; Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:64, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:

71. Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:65, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:71; Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:66, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:

72. Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:66, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:

73. Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:67, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:72; Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:67, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:

73. Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:68, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:

73. Alternatively, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:69, and its light chain variable region has an amino acid sequence as shown in SEQ ID NO:

73.

5. The antibody or antigen-binding molecule according to claim 1 or 2, characterized in that, Its heavy chain constant region has an amino acid sequence as shown in SEQ ID NO:74, or a sequence based on the amino acid sequence as shown in SEQ ID NO:74 with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology to the amino acid sequence as shown in SEQ ID NO:

74. Its light chain constant region has an amino acid sequence as shown in SEQ ID NO:75, or a sequence based on the amino acid sequence as shown in SEQ ID NO:75 with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology to the amino acid sequence as shown in SEQ ID NO:

75.

6. Biomaterials, including at least one of the following: I) A nucleic acid molecule encoding the antibody or antigen-binding molecule as described in any one of claims 1 to 5; II) A plasmid vector containing the nucleic acid described in claim I); III) Transformation or transfection with the plasmid vector of claim II), or a host cell whose genome integrates the nucleic acid molecule of claim 9; IV) It includes the antibody or antigen-binding molecule and drug as described in any one of claims 1 to 5; Or it may include the antibody or antigen-binding molecule and marker as described in any one of claims 1 to 5; Or it may include the antibody or antigen-binding molecule and mediator as described in any one of claims 1 to 5.

7. A method for preparing the antibody or antigen-binding molecule according to any one of claims 1 to 5, comprising: Culture the host cells as described in claim 6 to obtain a culture containing the antibody or antigen-binding molecule.

8. The coupling according to claim 7, characterized in that, The drug is an anti-tumor drug; The marker is a chemical marker or a biological marker; The medium is either a solid medium or a non-solid medium.

9. The coupling according to claim 8, characterized in that, The drug is at least one of MMAE, Dxd, DM1, DM4 or PBD; The chemical label is a chemiluminescent indicator, a fluorescent indicator, an isotope and / or a colloidal indicator; The biomarker is a biotin, avidin, or an enzyme marker; The medium is colloidal gold, enzyme-labeled plate, magnetic beads, or latex microspheres.

10. The use of the antibody or antigen-binding molecule according to any one of claims 1 to 5, or the conjugate of claim 6 comprising the antibody or antigen-binding molecule according to any one of claims 1 to 5 and a drug, in the preparation of a medicament for treating tumors.

11. Medicine, It includes: The antibody or antigen-binding molecule according to any one of claims 1 to 5; Or include: a conjugate of the antibody or antigen-binding molecule and the drug as described in any one of claims 1 to 5 as in claim 6.

12. The antibody or antigen-binding molecule according to any one of claims 1 to 5, the conjugate of the antibody or antigen-binding molecule according to any one of claims 1 to 5 and the labeling agent according to claim 6, or the conjugate of the antibody or antigen-binding molecule according to any one of claims 1 to 5 and the medium according to claim 6, in the preparation of CD228 detection reagent.

Images