Anti-CD98 antibody and application thereof

Abstract

The present disclosure provides an anti-CD98 antibody and an antigen-binding fragment thereof. Further provided are a nucleotide encoding the antibody or the antigen-binding fragment thereof, a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, and a method for using the antibody or the antigen-binding fragment thereof in the prevention and / or treatment of diseases such as cancer. The antibody or antigen-binding fragment thereof of the present disclosure has increased antibody stability.

Description

Anti-CD98 antibodies and their applications Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-CD98 antibody and its applications, as well as their applications. Background Technology

[0002] The type II transmembrane protein CD98 (also known as CD98 heavy chain (CD98hc) or 4F2hc, encoded by the SLC3A2 gene) can interact with various light chains to form different heterodimeric amino acid transporters (HATs). Light chains play a role in amino acid transport, and CD98 participates in transport activity by stabilizing the structure of light chains and helping them to be localized to the cell membrane.

[0003] Existing research has found that CD98 participates in tumorigenesis, tumor development, and metastasis by promoting amino acid transport activity, thereby promoting cell survival, enhancing integrin signaling, and increasing cell diffusion, migration, survival, and growth. CD98 protein is widely expressed in normal tissues, including the brain, spleen, kidney, small intestine, testes, and hematopoietic system. Furthermore, CD98 expression is upregulated in various types of solid tumors and hematologic malignancies and is associated with adverse clinical outcomes, thus making it an attractive target for developing cancer treatments.

[0004] Currently, the demand for anti-CD98 antibodies as an anti-tumor therapy has not been met, and there is also a need to further develop anti-CD98 antibodies with higher stability. Summary of the Invention

[0005] This disclosure provides a new series of anti-CD98 antibodies that further improve antibody stability.

[0006] According to one aspect of this disclosure, an antibody or antigen-binding fragment thereof targeting and binding to CD98 is provided, said antibody or antigen-binding fragment comprising:

[0007] (1) The following three heavy chain variable region complementarity-determining regions (HCDRs):

[0008] HCDR1, having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in any of SEQ ID NO:3-7, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region.

[0009] HCDR2, having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in any of SEQ ID NO:3-7, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region.

[0010] HCDR3, having the amino acid sequence of HCDR3 contained in the heavy chain variable region as shown in any of SEQ ID NO:3-7, or having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region; and / or

[0011] (2) The following three light chain variable region complementarity-determining regions (LCDRs):

[0012] LCDR1, having the amino acid sequence of LCDR1 contained in the light chain variable region as shown in SEQ ID NO:2, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR1 contained in the light chain variable region.

[0013] LCDR2, having the amino acid sequence of LCDR2 contained in the light chain variable region as shown in SEQ ID NO:2, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR2 contained in the light chain variable region.

[0014] LCDR3 having the amino acid sequence of LCDR3 contained in the light chain variable region as shown in SEQ ID NO:2, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR3 contained in the light chain variable region.

[0015] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in any one of SEQ ID NO:3-7, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:2.

[0016] In some implementations, the HCDR1-3 and / or the LCDR1-3 are defined by the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, the Contact numbering system, or a combination thereof.

[0017] In some embodiments, the antibody or its antigen-binding fragment comprises the following heavy chain variable regions and / or light chain variable regions, wherein CDRs are defined according to the Kabat numbering system:

[0018] (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:8, HCDR2 with sequence SEQ ID NO:29, and HCDR3 with sequence SEQ ID NO:10; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:11, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or

[0019] (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:8, HCDR2 with sequence SEQ ID NO:9, and HCDR3 with sequence SEQ ID NO:33; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:11, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or

[0020] (c) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:8, HCDR2 with sequence SEQ ID NO:9, and HCDR3 with sequence SEQ ID NO:36; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:11, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or

[0021] (d) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:8, HCDR2 with sequence SEQ ID NO:29, and HCDR3 with sequence SEQ ID NO:33; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:11, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or

[0022] (e) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:8, HCDR2 with sequence SEQ ID NO:29, and HCDR3 with sequence SEQ ID NO:36; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:11, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13.

[0023] In some embodiments, the antibody or its antigen-binding fragment comprises the following heavy chain variable regions and / or light chain variable regions, wherein CDRs are defined according to the IMGT numbering system:

[0024] (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:14, HCDR2 of SEQ ID NO:30, and HCDR3 of SEQ ID NO:16; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:17, LCDR2 of SEQ ID NO:18, and LCDR3 of SEQ ID NO:19; or

[0025] (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:14, HCDR2 of SEQ ID NO:15, and HCDR3 of SEQ ID NO:34; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:17, LCDR2 of SEQ ID NO:18, and LCDR3 of SEQ ID NO:19; or

[0026] (c) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:14, HCDR2 of SEQ ID NO:15, and HCDR3 of SEQ ID NO:37; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:17, LCDR2 of SEQ ID NO:18, and LCDR3 of SEQ ID NO:19; or

[0027] (d) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:14, HCDR2 of SEQ ID NO:30, and HCDR3 of SEQ ID NO:34; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:17, LCDR2 of SEQ ID NO:18, and LCDR3 of SEQ ID NO:19; or

[0028] (e) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:14, HCDR2 with sequence SEQ ID NO:30, and HCDR3 with sequence SEQ ID NO:37; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:17, LCDR2 with sequence SEQ ID NO:18, and LCDR3 with sequence SEQ ID NO:19.

[0029] In some embodiments, the antibody or its antigen-binding fragment comprises the following heavy chain variable regions and / or light chain variable regions, wherein CDRs are defined according to the Chothia numbering system:

[0030] (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:20, HCDR2 of SEQ ID NO:31, and HCDR3 of SEQ ID NO:10; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:12, and LCDR3 of SEQ ID NO:13; or

[0031] (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:20, HCDR2 of SEQ ID NO:21, and HCDR3 of SEQ ID NO:33; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:12, and LCDR3 of SEQ ID NO:13; or

[0032] (c) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:20, HCDR2 of SEQ ID NO:21, and HCDR3 of SEQ ID NO:36; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:12, and LCDR3 of SEQ ID NO:13; or

[0033] (d) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:20, HCDR2 with sequence SEQ ID NO:31, and HCDR3 with sequence SEQ ID NO:33; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:22, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or

[0034] (e) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:20, HCDR2 with sequence SEQ ID NO:31, and HCDR3 with sequence SEQ ID NO:36; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:22, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13.

[0035] In some embodiments, the antibody or its antigen-binding fragment comprises the following heavy chain variable regions and / or light chain variable regions, wherein CDRs are defined according to the Contact numbering system:

[0036] (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 of sequence SEQ ID NO:23, HCDR2 of sequence SEQ ID NO:32, and HCDR3 of sequence SEQ ID NO:25; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of sequence SEQ ID NO:26, LCDR2 of sequence SEQ ID NO:27, and LCDR3 of sequence SEQ ID NO:28; or

[0037] (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 of sequence SEQ ID NO:23, HCDR2 of sequence SEQ ID NO:24, and HCDR3 of sequence SEQ ID NO:35; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of sequence SEQ ID NO:26, LCDR2 of sequence SEQ ID NO:27, and LCDR3 of sequence SEQ ID NO:28; or

[0038] (c) A heavy chain variable region comprising the following three HCDRs: HCDR1 of sequence SEQ ID NO:23, HCDR2 of sequence SEQ ID NO:24, and HCDR3 of sequence SEQ ID NO:38; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of sequence SEQ ID NO:26, LCDR2 of sequence SEQ ID NO:27, and LCDR3 of sequence SEQ ID NO:28; or

[0039] (d) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:23, HCDR2 with sequence SEQ ID NO:32, and HCDR3 with sequence SEQ ID NO:35; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:26, LCDR2 with sequence SEQ ID NO:27, and LCDR3 with sequence SEQ ID NO:28; or

[0040] (e) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:23, HCDR2 with sequence SEQ ID NO:32, and HCDR3 with sequence SEQ ID NO:38; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:26, LCDR2 with sequence SEQ ID NO:27, and LCDR3 with sequence SEQ ID NO:28.

[0041] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO:3-7, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the heavy chain, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the heavy chain variable region; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:2, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the light chain variable region, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the light chain variable region.

[0042] In some embodiments, the antibody is of the IgA, IgD, IgE, IgG, or IgM type.

[0043] In some embodiments, the antibody or its antigen-binding fragment is scFv, Fab, Fab', (Fab')2, Fv fragment, dsFv, biantibody, bispecific antibody, and multispecific antibody.

[0044] In some embodiments, the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody.

[0045] In some embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0046] In some embodiments, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4.

[0047] In some embodiments, the heavy chain constant region is selected from the heavy chain constant regions of hIgG1, hIgG2, hIgG3, or hIgG4.

[0048] In some embodiments, the antibody or its antigen-binding fragment binds to hCD98 in a pH-dependent manner, wherein the binding activity of the antibody or its fragment is higher at acidic pH than at neutral pH. For example, the binding activity at neutral pH is at least 2, 3, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than the binding activity at acidic pH. The binding activity can be determined by any method known in the art, such as ELISA, FACS, or surface plasmon resonance (SPR).

[0049] In some embodiments, the antibody or a fragment thereof binds to hCD98 and is subjected to EC at neutral pH. 50 EC at acidic pH 50 At least 2 times higher, preferably 5 times higher, more preferably 10 times higher (e.g., at least 2 times, 3 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times higher), wherein EC 50 Measured by ELISA.

[0050] In some embodiments, the acidic pH is 5.8-6.8, preferably 6.0-6.8, and more preferably 6.0-6.5. In some embodiments, the acidic pH is 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, or 6.8, particularly 6.5.

[0051] In some embodiments, the neutral pH is 7.0-7.6, preferably 7.2-7.6, and more preferably 7.2-7.5. In some embodiments, the neutral pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6, particularly 7.4.

[0052] In some embodiments, the antibody or its antigen-binding fragment has higher charge property stability and / or binding activity stability than a reference antibody, wherein the reference antibody has a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:2.

[0053] In some embodiments, the antibody or its antigen-binding fragment is labeled. Preferably, the antibody or its antigen-binding fragment is labeled with a detectable marker, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0054] According to another aspect of this disclosure, a chimeric antigen receptor is provided, comprising an extracellular antigen-binding fragment, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding fragment comprises an antibody or an antigen-binding fragment thereof as described in this disclosure.

[0055] According to another aspect of this disclosure, a modified immune cell is provided that comprises the chimeric antigen receptor described in this disclosure.

[0056] According to another aspect of this disclosure, an isolated nucleic acid is provided that encodes the antibody or antigen-binding fragment thereof described in this disclosure.

[0057] According to another aspect of this disclosure, an expression vector is provided that comprises the isolated nucleic acid described in this disclosure.

[0058] In some embodiments, the expression vector may be a eukaryotic cell expression vector and / or a prokaryotic cell expression vector, such as a retroviral vector, lentiviral vector, bacteriophage vector, adenovirus vector, adeno-associated vector, or herpes simplex vector.

[0059] According to another aspect of this disclosure, a host cell is provided that contains the isolated nucleic acid or the expression vector described in this disclosure.

[0060] In some embodiments, the host cell is a conventional host cell in the art, as long as the expression vector can stably express the nucleic acid molecule it carries as the antibody or its antigen-binding fragment as described in this disclosure.

[0061] In some embodiments, the host cells are prokaryotic and / or eukaryotic cells. The prokaryotic cells are preferably E. coli cells such as TG1 or BL21 (expressing single-chain antibodies or Fab antibodies), and the eukaryotic cells are preferably HEK293 cells or CHO cells (expressing full-length IgG antibodies). Transforming the expression vector into the host cells yields the host cells of this disclosure. The transformation method is a conventional method in the art, preferably a chemical transformation, heat shock, or electroporation.

[0062] According to another aspect of this disclosure, a conjugate is provided comprising: an antibody or an antigen-binding fragment thereof as described in this disclosure; and a conjugation portion.

[0063] In some embodiments, the coupling portion is selected from detectable markers, radioisotopes, fluorescent substances, luminescent substances, colored substances, enzymes, polyethylene glycol (PEG), radionuclides, nucleic acids, small molecule toxins, polypeptides with binding activity, proteins, receptors, ligands, and other active substances that inhibit tumor cell growth or promote tumor cell apoptosis or necrosis.

[0064] According to another aspect of this disclosure, a multispecific antibody is provided, said antibody being formed by coupling a first antibody or a fragment thereof with other antibodies or fragments thereof or antibody analogs, each antibody or fragment thereof or antibody analog retaining its original binding specificity, wherein the first antibody or a fragment thereof is the antibody or antigen-binding fragment thereof described in this disclosure.

[0065] In some embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0066] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising: the antibody or antigen-binding fragment thereof described in this disclosure, the chimeric antigen receptor, the expression vector, the host cell, the conjugate or the multispecific antibody; and a pharmaceutically acceptable carrier and / or excipient.

[0067] In some embodiments, the pharmaceutical composition can be prepared in a form suitable for administration, such as a solid, semi-solid, or liquid form, and can be in the form of an aqueous solution, non-aqueous solution, or suspension, powder, tablet, capsule, granule, injection, or infusion. It can be administered intravascularly, subcutaneously, intraperitoneally, intramuscularly, by inhalation, intranasal, airway instillation, or intrapleural instillation. The pharmaceutical composition can also be administered in the form of an aerosol or spray, for example, by nasal administration; or by intrathecal, intramedullary, or intraventricular administration, and can also be administered transdermally, percutaneously, locally, intraentrantly, sublingually, or rectally. The pharmaceutical composition can be formulated into various dosage forms as needed, and the physician can determine the beneficial dosage for the patient based on factors such as patient type, age, weight, general disease condition, and route of administration.

[0068] In some embodiments, the pharmaceutical composition further includes other active ingredients, such as a second antitumor agent.

[0069] In some embodiments, the second antitumor agent may be a reagent that modulates immune checkpoint proteins, including but not limited to PD-1, PD-L1, CTLA-4, 4-1BB, 4-1BBL, CD28, CD40, CD40L, CD47, OX40, OX40L, TIM-3, TIGIT, NKG2A, B7-H3, B7-H4, VISTA, LAG3, and 2B4. In some embodiments, the reagent that modulates immune checkpoint proteins is an antibody that specifically binds to an immune checkpoint protein.

[0070] In some embodiments, the second antitumor agent may be an agent that enhances the phagocytic function of macrophages or an agent that enhances the activity of CD8+ T cells. In some embodiments, the second antitumor agent enhances the phagocytic function of macrophages by targeting phagocytic inhibitory factors (such as CD47). In some embodiments, the agent that enhances the phagocytic function of macrophages is an anti-CD47 antibody. In some embodiments, the second antitumor agent enhances the activity of CD8+ T cells, for example, by blocking or reversing the negative regulation of cell-mediated immune responses, such as by targeting inhibitory receptors. In some embodiments, the second antitumor agent is an antibody that specifically binds to PD-1, CTLA-4, PD-L1, or 4-1BB.

[0071] In some embodiments, the antibody or its antigen-binding fragment in the pharmaceutical composition may be administered simultaneously or sequentially with other active ingredients.

[0072] According to another aspect of this disclosure, the use of the antibody or antigen-binding fragment thereof described herein, the chimeric antigen receptor, the expression vector, the host cell, the conjugate, the multispecific antibody, or the pharmaceutical composition thereof in the preparation of a medicament for reducing tumors, inhibiting tumor cell growth, preventing and / or treating cancer or autoimmune diseases, or preventing cancer recurrence is provided.

[0073] According to another aspect of this disclosure, a method is provided for reducing tumors, inhibiting tumor cell growth, treating cancer, or preventing cancer recurrence in a subject in need, comprising administering to the subject a therapeutically effective amount of the antibody or a fragment thereof described in this disclosure, or the chimeric antigen receptor described therein, or the conjugate described therein, or the multispecific antibody described therein, or the pharmaceutical composition described therein.

[0074] In some embodiments, the tumor or cancer is a tumor or cancer expressing CD98, particularly a tumor or cancer expressing hCD98.

[0075] In some embodiments, the antibody or its antigen-binding fragment exhibits the desired binding activity in the tumor microenvironment and has no or very low binding activity at normal physiological pH. In some embodiments, anti-CD98 antibodies or their antigen fragments show preferential binding to CD98 on or within tumor cells, relative to CD98 not present on or within tumor cells. Specifically, this binding preference is achieved through pH-dependent binding of the antibody or its fragment to hCD98. For example, at the pH of the tumor microenvironment of a subject, such as a human, the binding activity of the anti-CD98 antibody or its fragment to hCD98 is significantly greater than the binding activity at normal physiological pH in the subject, such as a human.

[0076] In some implementations, the pH of the tumor or cancer microenvironment is lower than the subject's normal physiological pH.

[0077] In some embodiments, the tumor or cancer has an acidic pH microenvironment, more preferably, the acidic pH is 5.8-6.8, more preferably 6.0-6.8, and even more preferably 6.0-6.5. In some embodiments, the acidic pH is 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, or 6.8, particularly 6.5.

[0078] In some embodiments, the normal physiological pH can be the pH of normal blood or serum, such as neutral pH. In some embodiments, the neutral pH is 7.0-7.6, preferably 7.2-7.6, more preferably 7.2-7.5. In some embodiments, the neutral pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6, particularly 7.4.

[0079] In some embodiments, the tumor or cancer includes, but is not limited to: lymphoma, acute promyelocytic leukemia, hepatocellular carcinoma, pancreatic cancer, pancreatic epithelioid carcinoma, breast cancer, colorectal cancer, epidermoid carcinoma of the skin, melanoma, fibrosarcoma, non-small cell lung cancer, gastric cancer, acute myeloid leukemia, glioma, head and neck cancer, bile duct cancer, osteomalacia, osteosarcoma, renal cancer, or neuroblastoma.

[0080] In some embodiments, the autoimmune disease is caused by the abnormal proliferation of immune cells such as T cells or B cells.

[0081] In one embodiment, the autoimmune disease includes, but is not limited to, multiple sclerosis, type 1 diabetes, or rheumatoid arthritis.

[0082] According to another aspect of this disclosure, a diagnostic or therapeutic kit is provided, the kit comprising the antibody or antigen-binding fragment thereof described in this disclosure, the chimeric antigen receptor, the expression vector, the host cell, the conjugate, the multispecific antibody, or the pharmaceutical composition described herein, and optionally, instructions for use. Attached Figure Description

[0083] Figure 1 shows the flow cytometry analysis results of antibodies binding to CD98 cell lines. MFI is the median fluorescence intensity. A represents the analysis results of Raji cells, and B represents the analysis results of A549 cells.

[0084] Figure 2 shows the results of CEX-HPLC detection of the charge characteristics of H15L54.

[0085] Figure 3 shows the results of CEX-HPLC detection of the charge characteristics of H15L54-019.

[0086] Figure 4 shows the results of CEX-HPLC detection of the charge characteristics of H15L54-020.

[0087] Figure 5 shows the binding activity of H15L54 and H15L54 variant to human CD98 by ELISA analysis. T0 represents the sample collected after purification, and T1 represents the sample collected after treatment at 40°C for one week. Detailed Implementation

[0088] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in numerous publications.

[0089] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0090] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0091] The term "antibody" in this disclosure encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single-chain molecules, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0092] The terms “antibody or antigen-binding fragment thereof” and “antibody” are used interchangeably herein to refer to antibodies that are substantially similar in structure to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant domains) from the N-terminus to the C-terminus. Each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain constant domain) from the N-terminus to the C-terminus. The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: κ light chain and λ light chain.

[0093] Within the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0094] The term "variable region" or "variable domain" in this disclosure refers to a domain of the heavy or light chain of an antibody involved in the binding of an antigen-binding molecule to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0095] The term "variable" in this disclosure refers to the fact that certain segments of the variable domain are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the variable domains of the light and heavy chains. The more highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the native heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are held together tightly by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, MD (1991)). Constant domains do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0096] The term "hypervariant region" or "HVR" in this disclosure refers to a region in the variable domain region of an antibody that is highly variable in sequence and / or forms a structurally defined loop ("hypervariant loop"). Typically, a natural tetrachain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariant loop and / or from the "complementarity-determining region (CDR)," the amino acid residues from the CDR having the highest sequence variability and / or being involved in antigen recognition.

[0097] As used herein, the term “complementarity-determining region (CDR)” refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, the Martin numbering system, the Contact numbering system, the Honegger numbering system, the Gelfand numbering system, or combinations thereof. For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. The correspondence between different numbering systems is well known to those skilled in the art, and some commonly used software can be used to define HCDR1-3 and LCDR1-3. For example, the Kabat numbering system is an immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The Chothia numbering system, proposed by Chothia et al., is a classic rule for identifying CDR boundaries based on the location of structural loop regions (see, for example, Chothia & Lesk, (1987), J.Mol.Biol.196:901-917; Chothia et al., (1989), Nature 342:878-883; Al-Lazikani et al., (1997), JMB 273:927-948). The IMGT numbering system is based on the international ImMunoGeneTics information (IMGT) system initiated by Lefranc et al. (see, for example, Lefranc et al., (2003), Dev.Comparat.Immunol.27:55-77). The Martin numbering system is a new numbering scheme recommended by Martin et al., focusing on the structural alignment of different frame regions of unconventional lengths; it can be completed using the Chothia numbering system corrected by ABnum software. The Contact numbering system defines the CDR region based on existing antibody complex crystal structure data. The Honegger numbering system (also known as the AHo numbering system) is based on structural alignment of 3D structures covering observed length variations of immunoglobulin variable regions, allowing the definition of structurally conserved Cα positions, thus deriving appropriate FR regions and CDR lengths.The Gelfand numbering system is a relatively complex numbering scheme described by Gelfand et al., in which a variable chain sequence is divided into 21 segments called “words”, each of which matches a secondary structure element (chain or loop). Chains are defined by alphabetical order of letters (e.g., A, B, C), and loops are defined by two letters corresponding to adjacent chains (e.g., AB, BC…). This numbering system does not include gaps or missing points, but allows for precise comparison of the secondary structures (loops and chains) between aligned sequences.

[0098] The term "framework" or "FR" in this disclosure refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences typically appear in the VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0099] The term "humanized antibody" in this disclosure includes amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In some embodiments, a humanized antibody includes at least one, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all FRs correspond to the FRs of a human antibody. A humanized antibody may optionally include at least a portion of the antibody constant region derived from a human antibody. An antibody in a "humanized form," such as a non-human antibody, refers to an antibody that has undergone humanization.

[0100] This disclosure also relates to amino acid sequence variants that can be prepared by introducing appropriate modifications into the nucleotide sequence of a coding molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions, and / or substitutions of residues in the amino acid sequence of an antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain a final construct with desired properties, such as antigen-binding activity. Sites used for substitution typically include HVRs and frames (FRs). Possible amino acid substitutions include, but are not limited to: conserved substitutions of Ala (A) including Val, Leu, and Ile, preferably Val; conserved substitutions of Arg (R) including Lys, Gln, and Asn, preferably Lys; conserved substitutions of Asn (N) including Gln, His, Asp, Lys, and Arg, preferably Gln; conserved substitutions of Asp (D) including Glu and Asn, preferably Glu; and conserved substitutions of Cys (C) including S... er, Ala, preferably conservatively substituted with Ser; conservative substitutions of Gln(Q) include Asn, Glu, preferably conservatively substituted with Asn; conservative substitutions of Glu(E) include Asp, Gln, preferably conservatively substituted with Asp; conservative substitutions of Gly(G) include Ala; conservative substitutions of His(H) include Asn, Gln, Lys, Arg, preferably conservatively substituted with Arg; conservative substitutions of Ile(I) include Leu, Val, Met, Ala, Phe, Nle, preferably Conservative substitution for Leu (L) includes Nle, Ile, Val, Met, Ala, and Phe, with Ile being the preferred conservative substitution; conservative substitution for Lys (K) includes Arg, Gln, and Asn, with Arg being the preferred conservative substitution; conservative substitution for Met (M) includes Leu, Phe, and Ile, with Leu being the preferred conservative substitution; conservative substitution for Phe (F) includes Trp, Leu, Val, Ile, Ala, and Tyr; Tyr; conservative substitution for Pro (P) includes... Conservative substitutions include Ala; conservative substitutions of Ser(S) include Thr; conservative substitutions of Thr(T) include Val and Ser, with Ser being the preferred conservative substitution; conservative substitutions of Trp(W) include Tyr and Phe, with Tyr being the preferred conservative substitution; conservative substitutions of Tyr(Y) include Trp, Phe, Thr, and Ser, with Phe being the preferred conservative substitution; conservative substitutions of Val(V) include Ile, Leu, Met, Phe, Ala, and Nle, with Leu being the preferred conservative substitution.

[0101] The terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” in this disclosure refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0102] The term "antibody fragment" or "antigen-binding fragment" in this disclosure includes a portion of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0103] The terms "antigen-binding fragment" or "antigen-binding site" used in this disclosure refer to a portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding fragment" refers to a portion of an antibody containing a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen molecule is large, the antigen-binding molecule may bind only a specific portion of the antigen, referred to as an epitope. The antigen-binding fragment may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding fragment comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one aspect, the antigen-binding fragment is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0104] The term "specific binding" in this disclosure refers to binding selectivity for an antigen, which can be distinguished from unwanted or nonspecific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen.

[0105] The term "affinity" or "binding affinity" in this disclosure refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its bound ligand (e.g., an antigen). Binding affinity is typically expressed as a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constants (Koff and Kon, respectively). Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured using conventional methods known in the art, such as surface plasmon resonance (SPR).

[0106] The term "isolated" nucleic acid molecule or polynucleotide in this disclosure refers to a nucleic acid molecule, DNA, or RNA, that has been separated from its native environment. In this disclosure, the recombinant polynucleotide encoding a polypeptide contained in a vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their native chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this disclosure, in positive and negative strand form, and in double strand form. Isolated polynucleotides or nucleic acids of this disclosure further include synthetically generated molecules of this type. Additionally, polynucleotides or nucleic acids may be or may include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0107] The terms "vector" or "expression vector" and "expression construct" used herein are used interchangeably to describe a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector comprises a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vectors of this disclosure contain an expression cassette. The expression vector can be transcribed into a large amount of stable mRNA. Once the expression vector is within the target cell, a ribonucleic acid molecule or protein encoded by the gene is generated by cellular transcription and / or translation mechanisms. The term "expression cassette" of this disclosure refers to a recombinant or synthetically produced polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in the target cell. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter.

[0108] The terms “host cell,” “host cell line,” and “host cell culture” used herein are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, and also include the progeny of such cells. Host cells include “transformants” and “transformed cells,” including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells are any type of cell that can be used to generate the antibodies or antigen-binding fragments disclosed herein. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, and also include cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0109] The term "antibody-drug conjugate" or "ADC" in this disclosure refers to a binding protein (such as an antibody or its antibody- or antigen-binding fragment) chemically linked to one or more chemical drugs. In a preferred embodiment, an ADC includes a binding protein, a drug, and a connector linking the binding protein to the drug.

[0110] The term "chimeric antigen receptor" or "CAR" in this disclosure refers to a receptor having desired antigen specificity and signal transduction domains to propagate intracellular signals upon antigen binding. For example, T lymphocytes recognize specific antigens via the interaction of T cell receptors (TCRs) with short peptides presented by class I or II major histocompatibility complex (MHC) molecules. For initial activation and clonal expansion, naïve T cells depend on antigen-presenting cells (APCs) that provide additional co-stimulatory signals. In some embodiments, monocytes and macrophages may be engineered to express, for example, chimeric antigen receptors (CARs). Modified cells can be recruited to the tumor microenvironment, where they act as potent immune effectors by infiltrating the tumor and killing target cancer cells. CARs may include antigen-binding fragments, transmembrane domains, and intracellular domains. The antigen-binding fragment binds to the antigen on the target cell. Examples of cell surface markers that can be used as antigens for binding to the antigen-binding fragment of a CAR include those associated with viruses, bacteria, parasitic infections, autoimmune diseases, and cancer cells (e.g., tumor antigens).

[0111] The term "effective amount" of the drug disclosed herein refers to the amount necessary to induce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes an amount sufficient to improve or prevent symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0112] The "therapeutic effective amount" of a drug (such as a pharmaceutical composition) refers to the amount necessary to effectively achieve the desired therapeutic or preventive effect in terms of dosage, dosing intervals, and time. For example, a therapeutically effective amount of a drug eliminates, mitigates / reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0113] The terms “individual” or “subject” in this disclosure refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, an individual or subject is a human being.

[0114] The term "pharmaceutical composition" in this disclosure refers to a mixture containing one or more antibodies of this disclosure or antibodies or antigen-binding fragments thereof, along with other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0115] The term "pharmaceuticalally acceptable carrier" in this disclosure refers to a component in a pharmaceutical composition that, other than the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0116] Example

[0117] The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0118] Materials and methods

[0119] Cell culture

[0120] A549 cells (human non-small cell lung cancer cells) were cultured in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Raji cells (human Burkitt's lymphoma cells) were cultured in RPMI 1640 medium supplemented with 10% FBS. These cells were cultured in a humidified incubator at 37°C with a 5% CO2 atmosphere. FreeStyle 293F cells were obtained from Life Technologies and cultured according to the manufacturer's instructions.

[0121] Protein expression and purification

[0122] A His6-Avi-tagged fusion protein, CD98 ECD (SEQ ID NO:39), was generated by transient transfection of FreeStyle 293F cells and purified by affinity chromatography and molecular sieve chromatography. For the full-length IgG antibody, the VH and VL coding sequences were subcloned into human IgG1 H-chain (HC) and L-chain (LC) expression vectors, respectively. The two IgG expression plasmids (HC plasmid + LC plasmid) were co-transfected into 293F cells at a 1:1 ratio. Three to six days after transfection, the cell culture supernatant was collected, and IgG1 was purified by protein A affinity chromatography and molecular sieve chromatography.

[0123] The sequence of SEQ ID NO:39 is:

[0124] Example 1: Preparation of Antibody

[0125] H15L54 is a pH-dependent anti-CD98 monoclonal antibody (mAb) that selectively targets the acidic tumor microenvironment (WO2022252167A1, which is incorporated herein by reference in its entirety). In this embodiment, based on H15L54, amino acid site mutations were performed on its heavy chain variable region to obtain a series of new anti-CD98 antibodies. From these H15L54 antibody-derived variants, H15L54 variants with higher stability than H15L54 were screened.

[0126] The heavy chain variable region sequences of each H15L54 variant antibody are shown in Table 1 below. The light chain variable region is the same as that of H15L54, and its corresponding CDR sequence is shown in Table 2.

[0127] Table 1

[0128] Table 2

[0129] Example 2: Flow cytometry detection of antibody binding activity to human CD98-expressing cells

[0130] Binding assays based on flow cytometry were performed, in which Raji cells and A549 cells were serially diluted with different concentrations of the antibody prepared in Example 1 in 1% BSA / PBS (pH 6.5 or pH 7.4, respectively) and incubated at 4°C for 1 hour. The cells were then washed three times with PBS containing 1% BSA. Antibody binding to the cells was detected by adding PE-labeled goat anti-human IgG antibody (Invitrogen, CAT#12-4998-82).

[0131] The results in Figure 1A and Figure 1B show that the binding activity of antibodies H15L54-004, H15L54-008, and H15L54-009 is similar to that of H15L54.

[0132] Example 3

[0133] Based on the results of the H15L54-004 (N52S), H15L54-008 (N104T), and H15L54-009 (N104Q) variants in Example 2, variants H15L54-019 and H15L54-020 were further prepared, wherein H15L54-019 has amino acid substitutions of N52S and N104T in its heavy chain, and H15L54-020 has amino acid substitutions of N52S and N104Q in its heavy chain. The thermal stability of these two variants was evaluated by cation exchange chromatography (CEX-HPLC) and enzyme-linked immunosorbent assay (ELISA).

[0134] In short, the antibody concentration was adjusted to approximately 20 mg / mL, and the sample was incubated at 40°C for one week (T1). Samples were collected before and after incubation, and their stability was analyzed. Its charge properties were determined using CEX-HPLC, and its binding activity to human CD98 was determined using ELISA.

[0135] (1) Stability determination by CEX-HPLC

[0136] The CEX-HPLC method was used. Samples were injected into an Agilent 1260 Infinity II high-performance liquid chromatography system, employing a Thermo Scientific ProPac WCX-10 column (4 x 250 mm) and a 280 nm UV detector. Mobile phase A consisted of 50 mM HAc / NaAc buffer at pH 5.5, and mobile phase B consisted of 50 mM HAc / NaAc buffer containing 500 mM NaCl at pH 5.5. Gradient elution was performed after injection (the gradient elution program is shown in Table 3 below), with the flow rate maintained at 0.8 mL / min.

[0137] Table 3. Gradient elution program

[0138] The results of determining the charge properties by CEX-HPLC are shown in Table 4 and Figures 2-4.

[0139] Table 4. Determination of charge properties by CEX-HPLC method

[0140] Where T0 represents the sample collected after purification; T1 represents the sample collected after treatment at 40℃ for one week.

[0141] The results of CEX-HPLC analysis of charge characteristics showed that at T0, the main peak purity of the H15L54 variants (H15L54-019 and H15L54-020) was significantly higher than that of H15L54, while the proportion of acidic peaks was significantly lower, indicating that the H15L54 variants had better charge homogeneity than H15L54. At T1, i.e., after one week of treatment at 40℃, the charge heterogeneity of H15L54 increased significantly, manifested as a significant decrease in main peak purity and a significant increase in the proportion of acidic peaks. At this time, the H15L54 variants (H15L54-019 and H15L54-020) had better charge stability than H15L54.

[0142] (2) ELISA combination test

[0143] For ELISA-based binding assays, the biotinylated protein antigen (CD98 ECD) was captured using a streptavidin (Sigma)-coated 96-well plate (Nunc, MaxiSorp™). Serial dilutions of H15L54, H15L54-019, and H15L54-020 were then added, and binding signals were detected using HRP-labeled goat polyclonal anti-human IgG Fc (Thermo Fisher Scientific).

[0144] The results of ELISA assays for binding activity are shown in Table 5 and Figure 5.

[0145] Table 5. ELISA assay for binding activity

[0146] Where T0 represents the sample collected after purification; T1 represents the sample collected after treatment at 40℃ for one week; NA represents unusable.

[0147] ELISA assays of binding activity showed that, at T0, H15L54 and its variants exhibited good pH dependence (EC50). 50The ratio was 6.3 to 9.1; at T1, although H15L54 and its variants still remained pH-dependent (EC). 50 The ratio was 12.0 to 15.8, but at pH 6.5, the binding activity of H15L54 at T1 was significantly lower than that at T0 (EC). 50 The binding activity of H15L54 variants (H15L54-019 and H15L54-020) increased from 14.1 ng / mL at T0 to 29.0 ng / mL at T1, while the binding activity of H15L54-019 and H15L54-020 showed no significant change between T0 and T1. These results indicate that the H15L54 variants (H15L54-019 and H15L54-020) have better binding activity stability than H15L54.

[0148] As can be seen from this embodiment, the anti-CD98 antibody disclosed herein has superior antibody stability.

[0149] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that targets and binds to CD98, said antibody or antigen-binding fragment comprising: (1) The following three heavy chain variable region complementarity-determining regions (HCDRs): HCDR1, having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in any of SEQ ID NO:3-7, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region. HCDR2, having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in any of SEQ ID NO:3-7, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region. HCDR3, having the amino acid sequence of HCDR3 contained in the heavy chain variable region as shown in any of SEQ ID NO:3-7, or having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region; and / or (2) The following three light chain variable region complementarity-determining regions (LCDRs): LCDR1, having the amino acid sequence of LCDR1 contained in the light chain variable region as shown in SEQ ID NO:2, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR1 contained in the light chain variable region. LCDR2, having the amino acid sequence of LCDR2 contained in the light chain variable region as shown in SEQ ID NO:2, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR2 contained in the light chain variable region. LCDR3 having the amino acid sequence of LCDR3 contained in the light chain variable region as shown in SEQ ID NO:2, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR3 contained in the light chain variable region.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or its antigen-binding fragment comprises: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in any of SEQ ID NO:3-7, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:2; Preferably, the HCDR1-3 and / or the LCDR1-3 are defined by the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, the Martin numbering system, the Contact numbering system, the Honegger numbering system, the Gelfand numbering system, or a combination thereof.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody or its antigen-binding fragment comprises: (1) The following are the variable regions of heavy chains and / or light chains, where the CDR is defined according to the Kabat numbering system: (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:8, HCDR2 with sequence SEQ ID NO:29, and HCDR3 with sequence SEQ ID NO:10; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:11, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:8, HCDR2 with sequence SEQ ID NO:9, and HCDR3 with sequence SEQ ID NO:33; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:11, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or (c) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:8, HCDR2 with sequence SEQ ID NO:9, and HCDR3 with sequence SEQ ID NO:36; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:11, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or (d) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:8, HCDR2 with sequence SEQ ID NO:29, and HCDR3 with sequence SEQ ID NO:33; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:11, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or (e) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:8, HCDR2 with sequence SEQ ID NO:29, and HCDR3 with sequence SEQ ID NO:36; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:11, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or (2) The following heavy chain variable regions and / or light chain variable regions, wherein the CDR is defined according to the IMGT numbering system: (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:14, HCDR2 of SEQ ID NO:30, and HCDR3 of SEQ ID NO:16; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:17, LCDR2 of SEQ ID NO:18, and LCDR3 of SEQ ID NO:19; or (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:14, HCDR2 of SEQ ID NO:15, and HCDR3 of SEQ ID NO:34; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:17, LCDR2 of SEQ ID NO:18, and LCDR3 of SEQ ID NO:19; or (c) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:14, HCDR2 of SEQ ID NO:15, and HCDR3 of SEQ ID NO:37; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:17, LCDR2 of SEQ ID NO:18, and LCDR3 of SEQ ID NO:19; or (d) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:14, HCDR2 of SEQ ID NO:30, and HCDR3 of SEQ ID NO:34; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:17, LCDR2 of SEQ ID NO:18, and LCDR3 of SEQ ID NO:19; or (e) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:14, HCDR2 with sequence SEQ ID NO:30, and HCDR3 with sequence SEQ ID NO:37; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:17, LCDR2 with sequence SEQ ID NO:18, and LCDR3 with sequence SEQ ID NO:19; or (3) The following heavy chain variable regions and / or light chain variable regions, wherein the CDR is defined according to the Chothia numbering system: (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:20, HCDR2 of SEQ ID NO:31, and HCDR3 of SEQ ID NO:10; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:12, and LCDR3 of SEQ ID NO:13; or (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:20, HCDR2 of SEQ ID NO:21, and HCDR3 of SEQ ID NO:33; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:12, and LCDR3 of SEQ ID NO:13; or (c) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:20, HCDR2 of SEQ ID NO:21, and HCDR3 of SEQ ID NO:36; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:12, and LCDR3 of SEQ ID NO:13; or (d) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:20, HCDR2 with sequence SEQ ID NO:31, and HCDR3 with sequence SEQ ID NO:33; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:22, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or (e) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:20, HCDR2 with sequence SEQ ID NO:31, and HCDR3 with sequence SEQ ID NO:36; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:22, LCDR2 with sequence SEQ ID NO:12, and LCDR3 with sequence SEQ ID NO:13; or (4) The following heavy chain variable regions and / or light chain variable regions, wherein the CDR is defined according to the Contact numbering system: (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 of sequence SEQ ID NO:23, HCDR2 of sequence SEQ ID NO:32, and HCDR3 of sequence SEQ ID NO:25; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of sequence SEQ ID NO:26, LCDR2 of sequence SEQ ID NO:27, and LCDR3 of sequence SEQ ID NO:28; or (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 of sequence SEQ ID NO:23, HCDR2 of sequence SEQ ID NO:24, and HCDR3 of sequence SEQ ID NO:35; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of sequence SEQ ID NO:26, LCDR2 of sequence SEQ ID NO:27, and LCDR3 of sequence SEQ ID NO:28; or (c) A heavy chain variable region comprising the following three HCDRs: HCDR1 of sequence SEQ ID NO:23, HCDR2 of sequence SEQ ID NO:24, and HCDR3 of sequence SEQ ID NO:38; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of sequence SEQ ID NO:26, LCDR2 of sequence SEQ ID NO:27, and LCDR3 of sequence SEQ ID NO:28; or (d) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:23, HCDR2 with sequence SEQ ID NO:32, and HCDR3 with sequence SEQ ID NO:35; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:26, LCDR2 with sequence SEQ ID NO:27, and LCDR3 with sequence SEQ ID NO:28; or (e) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:23, HCDR2 with sequence SEQ ID NO:32, and HCDR3 with sequence SEQ ID NO:38; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:26, LCDR2 with sequence SEQ ID NO:27, and LCDR3 with sequence SEQ ID NO:

28.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein, The antibody or its antigen-binding fragment comprises: The heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO:3-7, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; and The light chain variable region has an amino acid sequence as shown in SEQ ID NO:2, an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein, The antibody is of type IgA, IgD, IgE, IgG, or IgM; Preferably, the antibody or its antigen-binding fragment is scFv, Fab, Fab', (Fab')2, Fv fragment, dsFv, biantibody, bispecific antibody, or multispecific antibody; Preferably, the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody. Preferably, the antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; More preferably, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3 or IgG4.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein, The antibody or its antigen-binding fragment binds to hCD98 in a pH-dependent manner, wherein the binding activity of the antibody or its fragment is higher at acidic pH than at neutral pH. Preferably, said antibody or fragment thereof binds to hCD98 with an EC 50 at least 2 times higher, preferably 5 times higher, more preferably 10 times higher than the EC 50 at neutral pH, wherein the EC 50 is measured by ELISA. Preferably, the acidic pH is 5.8-6.8, more preferably 6.0-6.8, and even more preferably 6.0-6.5; Preferably, the neutral pH is 7.0-7.6, more preferably 7.2-7.6, and even more preferably 7.2-7.5; Preferably, the antibody or its antigen-binding fragment has higher charge property stability and / or binding activity stability than the reference antibody, wherein the reference antibody has a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:

2.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein, The antibody or its antigen-binding fragment is labeled; preferably, the antibody or its antigen-binding fragment is labeled with a detectable marker, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, or biotin.

8. A chimeric antigen receptor comprising an extracellular antigen-binding fragment, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding fragment comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1-7.

9. An isolated nucleic acid that encodes an antibody or an antigen-binding fragment thereof as described in any one of claims 1-7.

10. An expression vector comprising the isolated nucleic acid as described in claim 9.

11. A host cell comprising the isolated nucleic acid of claim 9, or the expression vector of claim 10.

12. A conjugate comprising: an antibody or an antigen-binding fragment thereof as described in any one of claims 1-7; and a conjugation portion; Preferably, the coupling portion is selected from detectable markers, radioisotopes, fluorescent substances, luminescent substances, colored substances, enzymes, polyethylene glycol (PEG), radionuclides, nucleic acids, small molecule toxins, polypeptides with binding activity, proteins, receptors, ligands, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

13. A multispecific antibody, wherein the antibody is formed by coupling a first antibody or a fragment thereof with other antibodies or fragments thereof or antibody analogs, wherein each antibody or fragment thereof or antibody analog retains its original binding specificity, and the first antibody or fragment thereof is the antibody or antigen-binding fragment thereof as described in any one of claims 1-7; Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

14. A pharmaceutical composition comprising: an antibody or an antigen-binding fragment thereof as described in any one of claims 1-7, a chimeric antigen receptor as described in claim 8, an expression vector as described in claim 10, a host cell as described in claim 11, a conjugate as described in claim 12, or a multispecific antibody as described in claim 13, and a pharmaceutically acceptable carrier and / or excipient.

15. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-7, the chimeric antigen receptor of claim 8, the expression vector of claim 10, the host cell of claim 11, the conjugate of claim 12, the multispecific antibody of claim 13, or the pharmaceutical composition of claim 14 in the preparation of a medicament for reducing tumors, inhibiting tumor cell growth, preventing and / or treating cancer or autoimmune diseases, or preventing cancer recurrence.

16. A method for reducing tumors, inhibiting tumor cell growth, preventing and / or treating cancer or autoimmune diseases, or preventing cancer recurrence in subjects in need, comprising administering to the subject a therapeutically effective amount of an antibody or fragment thereof as described in any one of claims 1-7, a chimeric antigen receptor as described in claim 8, a conjugate as described in claim 12, a multispecific antibody as described in claim 13, or a pharmaceutical composition as described in claim 14.

17. The use or method of claim 15 or 16, wherein, The tumor or cancer mentioned is a tumor or cancer that expresses CD98; Preferably, the tumor or cancer has an acidic pH microenvironment; more preferably, the acidic pH is 5.8-6.8, more preferably 6.0-6.8, and even more preferably 6.0-6.

5. Preferably, the tumor or cancer includes lymphoma, acute promyelocytic leukemia, hepatocellular carcinoma, pancreatic cancer, pancreatic epithelioid carcinoma, breast cancer, colorectal cancer, epidermoid carcinoma of the skin, melanoma, fibrosarcoma, non-small cell lung cancer, gastric cancer, acute myeloid leukemia, glioma, head and neck cancer, bile duct cancer, osteomalacia, osteosarcoma, renal cancer, or neuroblastoma.

18. A diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7, the chimeric antigen receptor of claim 8, the expression vector of claim 10, the host cell of claim 11, the conjugate of claim 12, the multispecific antibody of claim 13, or the pharmaceutical composition of claim 14, and optionally, instructions for use.

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