Antibody and fragment thereof that bind to CD320, and antibody-drug conjugate including said antibody

Abstract

The present disclosure provides an antibody and an antibody fragment thereof that bind to CD320. The present disclosure also provides an antibody-drug conjugate including said antibody and a cytotoxic agent (in particular, an anticancer agent).

Description

Antibodies and fragments thereof that bind to CD320, and antibody-drug conjugates containing the antibodies

[0001] The present disclosure relates to antibodies and fragments thereof that bind to CD320, as well as antibody-drug conjugates comprising the antibodies and fragments, pharmaceuticals comprising the antibodies and fragments, pharmaceuticals comprising the conjugates, and the like.

[0002] CD320 is a receptor for transcobalamin, a carrier protein that transports vitamin B12. Because vitamin B12 is essential for nucleic acid and protein synthesis and mitochondrial metabolism (Reference 1), it is known that CD320 is highly expressed in rapidly proliferating cells, especially cancer cells. Furthermore, overexpression of vitamin B12-transcobalamin-CD320 is associated with chemotherapy resistance, and it has been reported that this complex may be an important indicator of cancer diagnosis and prognosis, as well as a potential therapeutic target (References 2-3).

[0003] Nutrients. 2023. 15(12):2734.Anticancer Res. 2013 Oct;33(10):4203-12.Nat Commun. 2016 Jul 14;7:12100.Nutrients. 2022. 14(10):2058.

[0004] The present disclosure provides antibodies and fragments thereof that bind to CD320.

[0005] According to the present disclosure, for example, the following inventions are provided: [1] An antibody or antigen-binding fragment of an antibody that binds to CD320. [2] An antibody or antigen-binding fragment of an antibody that binds to CD320, comprising: (1) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 6, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 8; (2) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 10, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 14, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 16; (3) A heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 20, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 22, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 23, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 24; (4) An antibody or antigen-binding fragment of the antibody comprising a heavy chain variable region and a light chain variable region having 1 to 5 amino acid mutations from the antibody of any of (1) to (3) above; or (5) An antibody or antigen-binding fragment of the antibody capable of competing for binding to CD320 with the antibody of any of (1) to (3) above. [3] The antibody or antigen-binding fragment of the antibody according to [1] or [2] above, which is a human chimeric antibody or an antigen-binding fragment thereof. [4] The antibody or antigen-binding fragment of the antibody according to [1] or [2] above, which is a humanized antibody or an antigen-binding fragment thereof.[5] An antibody-drug conjugate comprising an antibody or its antigen-binding fragment and a drug, wherein the antibody or fragment is linked to the drug via a linker, and the antibody or fragment comprises the antibody or its antigen-binding fragment described in any of [1] to [4] above. [6] A pharmaceutical composition comprising the antibody or its antigen-binding fragment described in any of [1] to [4] above. [7] The pharmaceutical composition described in [6] above, wherein the antibody or its antigen-binding fragment has antibody-dependent cellular cytotoxicity and / or complement-dependent cytotoxicity. [8] A pharmaceutical composition comprising the antibody-drug conjugate described in [5] above. [9] The pharmaceutical composition described in any of [6] to [8] above, for use in treating cancer in a subject in need thereof.

[0006] Shows the reactivity of the obtained monoclonal anti-CD320 antibody to CD320. Shows the results of immunostaining showing the binding of the monoclonal anti-CD320 antibody to CD320-positive cells. Shows the expression of CD320 in various cancer cell lines. Shows the uptake of fluorescently labeled clone 8-1-2 antibody into cells. Shows the binding of clone 8-1-2 antibody to pancreatic cancer cell lines. Shows the accumulation of clone 8-1-2 antibody in tumors in vivo. Shows the active targeting of clone 8-1-2 antibody to tumors in vivo. Shows the antitumor effect of an antibody-drug conjugate (ADC) of clone 8-1-2 antibody and SN-38 in a pancreatic cancer cell xenograft model.

[0007] Definition of Terms As used herein, "subject" means a mammal, which may in particular be a human.

[0008] As used herein, the term "treatment" includes both therapy (therapeutic treatment) and prevention (prophylactic treatment). As used herein, "treatment" refers to the treatment, cure, prevention, or improvement in remission of a disease or disorder, or the reduction in the rate of progression of a disease or disorder. As used herein, "prevention" refers to reducing the likelihood of developing a disease or condition, or delaying the onset of a disease or condition.

[0009] As used herein, "disease" refers to a condition for which treatment is beneficial. Diseases include, for example, cancer, inflammatory diseases, and immune disorders.

[0010] As used herein, a "therapeutically effective amount" refers to an amount of a drug effective to treat (prevent or cure) a disease or condition. A therapeutically effective amount of a drug is capable of slowing the rate of worsening of symptoms of a disease or condition, halting the worsening of the symptoms, ameliorating the symptoms, curing the symptoms, or inhibiting the onset or development of the symptoms. A pharmaceutical composition includes a therapeutically effective amount of a therapeutically active ingredient. The therapeutically active ingredient may be a pharmaceutically acceptable salt. The therapeutically active ingredient may be a pharmaceutically acceptable solvate (e.g., hydrate). The drug moiety of an ADC may be in the free form or a pharmaceutically acceptable salt. The drug moiety of an ADC may be a pharmaceutically acceptable solvate (e.g., hydrate).

[0011] As used herein, the term "antibody" refers to an immunoglobulin, including polyclonal and monoclonal antibodies, with monoclonal antibodies being preferred.

[0012] The origin of the antibody is not particularly limited, but examples include antibodies from non-human animals (e.g., antibodies from non-human mammals) and human antibodies. The antibody may also be a chimeric antibody or a humanized antibody. The antibody may also be a bispecific antibody. The origin of the antibody is preferably a humanized antibody or a human antibody.

[0013] Antibodies are classified into subclasses IgG, IgA, IgE, IgM, and IgD. Human IgG is classified into subclasses IgG1, IgG2, IgG3, and IgG4. Human IgG1, IgG2, and IgG4 are preferably used in pharmaceuticals, and antibodies having sequences of these subclasses can be used as human chimeric antibodies and humanized antibodies.

[0014] The antibody is preferably a chimeric monoclonal antibody, a humanized monoclonal antibody, or a human monoclonal antibody. When used as a pharmaceutical, a chimeric monoclonal antibody is preferably used, more preferably a humanized monoclonal antibody or a human monoclonal antibody, and each is preferably isolated. Note that chimeric monoclonal antibodies and humanized monoclonal antibodies can be prepared from antibodies of non-human animals by methods known per se.

[0015] A humanized antibody has heavy and light chain variable regions containing CDRs from a non-human animal antibody and framework regions from a human antibody, a heavy chain constant region and Fc region from a human antibody, and a light chain constant region. Humanized antibodies are typically produced by replacing the CDRs of a human antibody with the corresponding CDRs from a non-human animal antibody. Humanized antibodies may be produced by introducing amino acid modifications selected from the group consisting of addition, insertion, deletion, and substitution into one or more of the CDRs or framework regions. Amino acid modifications are typically made to enhance or restore the affinity of the humanized antibody for the antigen.

[0016] A human chimeric antibody has the heavy and light chain variable regions of a non-human animal antibody and the heavy and Fc regions and light chain constant regions of a human antibody, and can be produced by replacing the heavy and Fc regions and light chain constant regions of a human antibody with the corresponding regions of a non-human animal.

[0017] An antibody has a structure in which two heavy chains and two light chains are associated. The heavy chain consists of a heavy chain variable region (VH), a heavy chain constant region (CH1, CH2, and CH3), and a hinge region located between the heavy chain variable region and the heavy chain constant region. The light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The heavy chain variable region and the light chain variable region each have three complementarity-determining regions (CDRs), which characterize the antigen specificity of the antibody. The CDRs are called heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and light chain CDR1, light chain CDR2, and light chain CDR3, respectively, from the N-terminus of the heavy chain and light chain. Antibodies include full-length antibodies.

[0018] As used herein, a "bispecific binding protein" refers to a protein that has two or more domains that bind to two different antigens or epitopes and is capable of binding to the two different antigens. The domains are typically derived from an antibody, preferably comprising the heavy and light chain variable regions of an antibody. A bispecific binding protein has a first binding domain that binds to a first antigen and a second binding domain that binds to a second antigen, where the first and second antigens bind to different epitopes on the same molecule or different epitopes on the same molecule. The first and second binding domains are derived from antibodies, respectively, and comprise, for example, the heavy and light chain variable regions of antibodies that bind to the first and second antigens, respectively. Examples of bispecific binding proteins include antibodies or antigen-binding fragments thereof that have, in addition to the heavy and light chain variable regions of antibodies that bind to the first and second antigens, heavy chain constant regions and Fc regions, and light chain constant regions, preferably of subclasses selected from the group consisting of human IgG1, IgG2, and IgG4. Bispecific binding proteins also include fusion proteins of an scFv that binds to a first antigen and an scFv that binds to a second antigen.

[0019] As used herein, the term "antigen-binding fragment" refers to a portion of an antibody fragment that retains its ability to bind to an antigen. The antigen-binding fragment may include the heavy chain variable region, the light chain variable region, or both of the antibodies of the present disclosure. The antigen-binding fragment may be chimerized or humanized. Examples of antigen-binding fragments include Fab, Fab', F(ab'), Fv, scFv (single-chain Fv), diabody, sc(Fv) (single-chain (Fv)), and half-molecule Ig. Antigen-binding fragments of these antibodies may be used in the present disclosure. Such antibody fragments can be obtained, for example, by treating the antibody with an enzyme, but are not limited thereto. For example, Fab can be obtained by digesting the antibody with papain. Alternatively, F(ab') can be obtained by digesting the antibody with pepsin, and Fab' can be obtained by further reduction. These fragments can be expressed and purified as recombinant proteins in prokaryotic or eukaryotic expression systems.

[0020] As used herein, "antibody-drug conjugate" (ADC) refers to a substance in which a monoclonal antibody or its antigen-binding fragment (hereinafter sometimes simply referred to as "antibody, etc.") is directly or indirectly linked to a drug. In an ADC, the monoclonal antibody, etc. and the drug can be linked via an appropriate linker. The ADC binds to a membrane component (e.g., a transmembrane protein such as a receptor) on the cell membrane, is taken up into the cell by endocytosis or internalization, and can be released intracellularly after detaching from the antibody, etc. By introducing a cleavable linker between the antibody, etc. and the drug intracellularly, the linker can be cleaved intracellularly, for example, in an endosome, allowing the drug to be separated from the antibody, etc. and released into the cytoplasm. Even if the ADC does not internalize intracellularly, the bystander effect can still produce the desired efficacy of the drug (e.g., a small molecule compound such as a cytotoxic agent) on the target cell. Using a cytotoxic agent as the drug can kill the cell to which the drug is delivered. Chemotherapeutic agents, radioisotopes, and toxins can be used as cytotoxic agents. Furthermore, when a signal transduction inhibitor is used as the drug, the signal transduction can be inhibited in the cells to which the drug is delivered.

[0021] As used herein, "CD320" refers to a transcopalamine receptor expressed on the cell surface. CD320 mediates the cellular uptake of bound copalamine (i.e., vitamin B12) and is involved in B cell differentiation and proliferation, etc. Examples of CD320 include human CD320, and an example of human CD320 is one having the amino acid sequence registered in UniProtKB under registration number Q9NPF0.

[0022] <Antibody or antibody fragment of the present disclosure> The present disclosure provides an isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD320 (preferably human CD320). The antibody or antibody fragment of the present disclosure has a means of binding to CD320 (preferably human CD320). The antibody or antibody fragment or means for binding to CD320 (preferably human CD320) of the present disclosure comprises: (1) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:2, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:3, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:4, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:6, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:8; (2) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:10, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:11, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:12, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:14, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:15, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:16; or (3) A heavy chain variable region including a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 20; and a light chain variable region including a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 22, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 23, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 24.

[0023] In a preferred embodiment, the antibody and its antigen-binding fragment may be a human chimeric antibody or an antigen-binding fragment thereof. In a preferred embodiment, the antibody and its antigen-binding fragment may be a humanized antibody or an antigen-binding fragment thereof. As an active ingredient of a pharmaceutical composition, a human chimeric antibody and its antigen-binding fragment may be preferably used, and a humanized antibody and its antigen-binding fragment may be more preferably used.

[0024] Antibodies from non-human mammals can be humanized using techniques well known in the art (e.g., the techniques described in Winter et al., Immunol. Today, 14:43-46 (1993)). Humanized monoclonal antibodies can be engineered, for example, by using recombinant DNA techniques to replace the CH1, CH2, CH3, hinge, and / or framework domains of an antibody from a non-human mammal with corresponding human sequences (e.g., the techniques described in WO 92 / 02190 and U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085).

[0025] In a preferred embodiment, the human chimeric antibody has a heavy chain constant region and Fc region derived from human IgG1 and a human light chain constant region. In a preferred embodiment, the human chimeric antibody has a heavy chain constant region and Fc region derived from human IgG2 and a human light chain constant region. In a preferred embodiment, the human chimeric antibody has a heavy chain constant region and Fc region derived from human IgG4 and a human light chain constant region.

[0026] In a preferred embodiment, the humanized antibody has a heavy chain constant region, Fc region, and light chain constant region of a subclass selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4. In a preferred embodiment, the humanized antibody has a heavy chain constant region, Fc region, and light chain constant region of a subclass selected from the group consisting of human IgG1, IgG2, and IgG4. In a preferred embodiment, the humanized antibody has a heavy chain constant region, Fc region, and light chain constant region of human IgG1. In a preferred embodiment, the humanized antibody has a heavy chain constant region, Fc region, and light chain constant region of human IgG2. In a preferred embodiment, the humanized antibody has a heavy chain constant region, Fc region, and light chain constant region of human IgG4.

[0027] In some embodiments, the humanized or chimeric antibody may have antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To this end, the humanized or chimeric antibody may have an Fc region derived from human IgG1. ADCC and CDC activities can be measured by conventional methods.

[0028] The heavy chain variable region of an antibody typically comprises heavy chain framework region 1 (heavy chain FR1), heavy chain CDR1, heavy chain FR2, heavy chain CDR2, heavy chain FR3, heavy chain CDR3, and heavy chain FR4, as in the heavy chain variable region of a humanized antibody.The light chain variable region of an antibody typically comprises light chain framework region 1 (light chain FR1), light chain CDR1, light chain FR2, light chain CDR2, light chain FR3, light chain CDR3, and light chain FR4, as in the light chain variable region of a humanized antibody.

[0029] In a preferred embodiment, the heavy chain framework region 1 (heavy chain FR1), heavy chain CDR1, heavy chain FR2, heavy chain CDR2, heavy chain FR3, heavy chain CDR3, and heavy chain FR4, and the light chain framework region 1 (light chain FR1), light chain CDR1, light chain FR2, light chain CDR2, light chain FR3, light chain CDR3, and light chain FR4 are selected from the group consisting of avituzumab, abrilumab, adalimumab, alemtuzumab, alirocumab, anifrolumab, atezolizumab, bapineuzumab, belimumab, benralizumab, bevacizumab, bimagrumab, brosozumab, bococizumab, briakinumab, Brodalumab, canakinumab, carlumab, certolizumab, cixutumumab, clazakizumab, codrituzumab, crenezumab, dacetuzumab, daclizumab, dalotuzumab, daratuzumab, denosumab, drozizumab, durigotuzumab, dupilumab, eculizumab, efalizumab, eldelumab, elotuzumab, emibetuzumab, enokizumab, epratuzumab, etrolizumab, evolocumab, farletuzumab, fezakinumab, fezakinumab, ficlatuzumab, figituzumab, fretikumab, foralumab, fresolimumab, fluticamic acid, Ranumab, ganitumab, gantenerumab, gemtuzumab, gevokizumab, gelenbatumumab, golimumab, guselkumab, ivalizumab, imgatuzumab, inotuzumab, ipilimumab, ixekizumab, lampalizumab, lebrikizumab, lenzilumab, rindilumab, lirilumab, matuzumab, mavrilimumab, mepolizumab, mogamulizumab, motavizumab, muromonab, natalizumab, necituzumab, nimotuzumab, nivolumab, obinutuzumab, ocrelizumab, octatumumab, olaratumab, olokizumab, omalizumab, onartuzumab , otelixizumab, otraltuzumab, ozanezumab, palivizumab, panitumumab, panobacumab, palsatuzumab, patrituzumab, pembrolizumab, pertuzumab, pinatuzumab, polatuzumab, ponezumab, radletumab, ramucirumab, ranibizumab, reslizumab, rilotumumab, lobatumumab, romosozumab, sarilumab, secukinumab, seribantumab, sifalimumab, simtuzumab, simtuzumab, tabalumab, tanezumab, teplizumab, tigatuzumab, tildrakizumab, tocilizumab, tobetumab, tralokinumab,The heavy chain framework region 1 (heavy chain FR1), heavy chain CDR1, heavy chain FR2, heavy chain CDR2, heavy chain FR3, heavy chain CDR3, and heavy chain FR4 of one or more antibodies selected from the group consisting of trastuzumab, tremelimumab, urelumab, ustekinumab, vedolizumab, veltuzumab, visilizumab, zalutuzumab, and zanolimumab (antibody group A), and the light chain framework region 1 (light chain FR1), light chain CDR1, light chain FR2, light chain CDR2, light chain FR3, light chain CDR3, and light chain FR4 of one or more antibodies selected from the above group. Heavy chain framework regions 1 to 4 may each be derived from different antibodies or from the same antibody. Light chain framework regions 1 to 4 may each be derived from different antibodies or from the same antibody. Heavy chain framework regions 1-4 and light chain framework regions 1-4 may each be derived from different antibodies or from the same antibody.

[0030] In a preferred embodiment, heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4 are means for maintaining heavy chain CDR1-CDR3 on the antibody framework and are derived from a heavy chain constant region of a subclass selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4. In a preferred embodiment, heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4 are derived from a heavy chain constant region of a subclass selected from the group consisting of human IgG1, IgG2, and IgG4. In a preferred embodiment, light chain FR1, light chain FR2, light chain FR3, and light chain FR4 are derived from a kappa chain or a lambda chain.

[0031] In a preferred embodiment, heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4 have the amino acid sequences set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively. In a preferred embodiment, light chain FR1, light chain FR2, light chain FR3, and light chain FR4 have the amino acid sequences set forth in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively.

[0032] In a preferred embodiment, heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4 have the amino acid sequences set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively. In a preferred embodiment, light chain FR1, light chain FR2, light chain FR3, and light chain FR4 have the amino acid sequences set forth in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively.

[0033] In a preferred embodiment, heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4 have the amino acid sequences set forth in SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively. In a preferred embodiment, light chain FR1, light chain FR2, light chain FR3, and light chain FR4 have the amino acid sequences set forth in SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively.

[0034] The heavy chain FR1-FR4 and light chain FR1-FR4 may have amino acid sequences that are 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the corresponding amino acid sequences of the heavy chain FR1-FR4 and light chain FR1-FR4, respectively. Alternatively, the heavy chain FR1-FR4 and light chain FR1-FR4 may have one to several (e.g., 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acid mutations relative to the corresponding amino acid sequences of the heavy chain FR1-FR4 and light chain FR1-FR4, respectively. The amino acid mutations may be one or more selected from the group consisting of amino acid addition, insertion, substitution, and deletion.

[0035] In some embodiments, the antibody or antibody fragment may be a recycling antibody.

[0036] In certain aspects, antigen-binding fragments of any of the above-described humanized antibodies are provided. In certain preferred aspects, the antigen-binding fragment may comprise, for example, Fab, Fab', or scFv. In certain preferred aspects, the antigen-binding fragment may comprise scFv. In certain preferred aspects, the antigen-binding fragment may be, for example, Fab, Fab', or scFv, e.g., scFv. These fragments may be fused to other proteins or other fragments. For example, by fusing them to other fragments that bind to other epitopes or antigens, multispecific binding proteins (e.g., bispecific binding proteins) can be obtained. Furthermore, these fragments (typically scFv) or multispecific binding proteins (e.g., bispecific binding proteins) containing such fragments may be fused to other proteins and incorporated into chimeric antigen receptors (first, second, and third generation).

[0037] As used herein, a "chimeric antigen receptor" (CAR) is a chimeric molecule comprising an antigen-binding fragment of an antibody (particularly, an scFv) and an immune cell activation domain. A CAR is generally a molecule comprising an scFv, an extracellular hinge domain, a transmembrane domain (e.g., CD8α or CD28), and an activation signaling domain (e.g., CD3ζ) linked together. A CAR can be introduced into a cell and expressed on the cell surface. A cell expressing a CAR can target a specific antigen. A CAR is introduced into immune cells, such as T cells or NK cells, to target the immune cells, such as T cells or NK cells, to target cells, such as cancer cells. While first-generation CARs comprise an scFv, an extracellular hinge domain, a transmembrane domain (e.g., CD8α or CD28), and an activation signaling domain (e.g., CD3ζ), second-generation CARs further comprise a costimulatory molecule signaling domain to activate the immune cells into which the CAR is introduced. Costimulatory factors such as CD28, 4-1BB, OX40, CD27, and ICOS are used as costimulatory molecule signaling domains. Third-generation CARs incorporate multiple costimulatory factors (e.g., a combination of the above costimulatory factors). Thus, improvements have been made to CARs to enable sustained proliferation of CAR-introduced immune cells in vivo. It is preferable that all domains other than the scFv portion are derived from human proteins.

[0038] Antibody-Drug Conjugates (ADCs) of the Present Disclosure In certain aspects, an antibody or its antigen-binding fragment may be in the form of a conjugate with a drug. According to the present disclosure, a conjugate (ADC) comprising an antibody or its antigen-binding fragment and a drug is provided, where the antibody may be an antibody or its antigen-binding fragment of the present disclosure. The drug may be a molecule to be delivered to CD320-positive cells by the antibody. Examples of drugs include, but are not limited to, physiologically active substances, preferably small molecular weight compounds, cytokines, and nucleic acids. Contrast agents or labeling agents (e.g., radioisotopes) may be used as or in place of drugs. Examples of small molecular weight compounds include cytotoxic agents. Examples of cytotoxic agents include, but are not limited to, toxins, anticancer drugs, and radioisotopes.

[0039] Examples of toxins include Pseudomonas aeruginosa exotoxin (PE) or cytotoxic fragments thereof (eg, PE38), diphtheria toxin, ricin A, and the like.

[0040] Examples of anticancer agents include chemotherapeutic agents (e.g., anticancer agents such as commercially available anticancer agents, e.g., auristatins (auristatin E, auristatin F phenylenediamine (AFP), monomethylauristatin E, monomethylauristatin F, and derivatives thereof), maytansinoids DM1 and DM4, and derivatives thereof), camptothecins (SN-38, topotecan, and exotecan, and derivatives thereof (e.g., deruxtecan)), DNA minor groove binders (enediynes, lexitropsin, duocarmycin, and derivatives thereof), taxanes (paclitaxel and docetaxel, and derivatives thereof), polyketides (discodermolide and derivatives thereof), anthraquinones (mitoxantrone and derivatives thereof), and benzodiazepines. benzodiazepines (pyrrolobenzodiazepines, indolinobenzodiazepines, and oxazolidinobenzodiazepines and their derivatives), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine and their derivatives), doxorubicins (doxorubicin, morpholino-doxorubicin, PNU-159682 (CAS Registry Number: 202350-68-3), and cyanomorpholino-doxorubicin and their derivatives), cardiac glycosides (digitoxin and its derivatives), calechiamycin, epothilones, cryptophycins, cemadotin, rhizoxin, netropsin, combretastatin, ereutherin, etoposide, T67 (Tularik), and nocodazole), radioactive isotopes (e.g., ３２ P. ６０ C. ９０ Y. １１１ In, １３１ I, １２５ I, １５３ Sm, １８６ Re, １８８ Re, and ２１２ Bi), and toxins (e.g., diphtheria toxin A, Pseudomonas endotoxin, ricin, saporin, etc.), which can be used as cytotoxic agents in the ADCs of the present disclosure. Any cytotoxic agent used in the treatment of cancer can be used.

[0041] Examples of radioisotopes include, but are not limited to, therapeutic nuclides. ３２ P. １４ C. ６０ C. １１１ I, 1 ２５ I, ３ H. １３１ I, ２１１ At, ９０ Y. １８６ Re, １８８ Re, and ２１２ Bi and the like.

[0042] Nucleic acids include natural nucleic acids such as natural DNA and natural RNA, antisense oligonucleotides (ASOs), siRNA, shRNA, microRNA, gapmers, mixmers, modified nucleic acids such as modified DNA and modified RNA, artificial nucleic acids, and combinations thereof. Examples of modified nucleic acids include fluorescent dye-modified nucleic acids, biotinylated nucleic acids, and nucleic acids into which cholesteryl groups have been introduced. To enhance the stability of RNA, bases may be modified with 2'-O-methyl, 2'-fluoro, or 2'-methoxyethyl (MOE), and the phosphodiester bond in the nucleic acid backbone may be replaced with a phosphorothioate bond. Examples of artificial nucleic acids include nucleic acids in which the oxygen atom at the 2' position is bridged to the carbon atom at the 4' position. Examples of such artificial nucleic acids include locked nucleic acids, which are cross-linked DNA in which the oxygen atom at the 2' position is bridged to the carbon atom at the 4' position via a methylene bridge. bridged nucleic acids (BNA) such as LNA, ENA in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via ethylene, BNACOC in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CHOCH-, BNANC in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -NR-CH- (where R is a methyl or hydrogen atom), and cMO in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH(OCH)-. Examples of RNA include E, cEt in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH2(CH3)-, AmNA in which the carbon atoms at the 2' position and the 4' position are bridged via an amide, scpBNA in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via a methylene, forming a cyclopropane at the 6' position, and peptide nucleic acids (PNAs) in which the backbone of a polymer is N-(2-aminoethyl)glycine amide-linked instead of deoxyribose or ribose. Examples of RNA include artificial RNAs for gene silencing, such as siRNA and shRNA, non-coding RNAs, such as microRNA (miRNA) and aptamers, and natural RNAs, such as mRNA. These RNAs can be modified to be stabilized in vivo.

[0043] The targets of the conjugate drug of the present disclosure are not particularly limited, and examples thereof include the following: Clusterin gene, Nucleolin gene, AKT1 protein kinase gene, BIRC5 gene, MAGEC1 gene, MAGEC2 gene, CTAG1 gene, TPBG gene, Hsp27 gene, β-Catenin gene, CXCL12 (SDF-1) gene, STAT-3 gene, PKN3 gene, PLK1 gene, mutant KRAS (G12D) gene, Grb-2 gene, androgen receptor gene, TGFβ gene (TGFβ-1 gene, TGFβ-2 gene, TGFβ-3 gene), STAT-3 gene, VEGF gene, KSP (E g5) gene, CEBPA gene, Nek2 gene, p53 gene, MUC1 gene, TPBG gene, HIF-1α gene, RPN2 gene, EphA2 gene, RRM1 gene, CDC45 gene, six-1 gene, IGF-1 receptor gene, HoxA1 ​​gene, IGFBP-2 gene, IGFBP-5 gene, EGF receptor gene, Raf-1 gene, mTOR gene, Bcl-2 gene, Casein kinase-2 gene, KRAS gene, c-Myc gene, COX-2 gene, β-3tubin gene, ITCH gene, VEGF gene, VEGF receptor 2 gene, SIP1 gene, AGT gene, ERV-9 LTR gene, EVI1 gene, TNF-α gene, PAX-2 gene, Srpx2 gene, IRS-1 gene, Survivin gene, DUSP6 gene, HPV E6 / E7 gene, HSPA9 gene, mitochondrial RNA (non-coding mitochondrial RNA), EWS FLI1 gene, SRC-3 gene, MDR1 gene, NTRK1 gene, NTRK2 gene, MDX3 gene, NR2F6 gene, MYD88 gene, NOTCH1 gene, β-3 integrin gene, c-FLIP gene, MADD gene, HER2 gene, CCAT2 gene, CTCFL gene, HIF-2α gene, BMI-1 gene, NETO-2 gene, CTFR gene, PD-1 gene, PD-L1 gene, PD-L2 (B7-DC (CD273)) gene, CLTA4 gene, HLA gene (HLA-A,HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, HLA-E, HLA-G), MCH gene, gene, 4-1BB gene, 4-1 BBL gene, CD3 gene (CD3α, CD3β, CD3γ, CD3δ, CD3ε, CD3ζ gene), IL-6 gene, IL-17 gene, IL-23 gene, ICOS gene, CD70 gene, CD27 gene, OX40 gene, OX40L gene, TL1A gene, DR3 gene, GITRL gene, GITR gene, CD30L gene, CD30 gene, TIM1 gene, TIM1L gene, TIM4 gene, SLAM gene, CD48 gene, CD58 gene, CD2 gene, CD155 gene, CD112 gene, CD226 gene, CD80 (B7-1) gene, CD86 (B7-2) gene, B7-H2 gene, LIGHT gene, HVEM gene, C D40 gene, CD40L gene, Galectin9 gene, CD113, Collagen gene, CD160 gene, LAG3 gene, PSA gene, PSMA gene, PSCA gene, STEAP gene, BIRC5 gene, MAGEC1 gene, MAGEC2 gene, CTAG1 gene, TPBG gene, or molecules such as proteins encoded by these genes, or mRNA encoding proteins encoded by these genes, proteins known as the HDGF family (prototype protein, HDGF; HRP-1, HRP-2, HRP-3, HRP-4 (HRP = HDGF-related protein (HDGF Related Protein)) Protein); and LEDGF (particularly HRP-3), TrkB receptor, or genes or mRNAs encoding these receptors, miR-34, miR-34a, miR-16, miR-155, miR-17, miR-17-92, miR-215, let-7, miR-34, miR-10b, miR-3157, miR-34, miR-7, miR-21, miR-574-5p, miR-221, miR-484, miR-205, miR-210, miR-3189-3p, miR-3151, miR-199, miR-101, miR-96, miR-182, and other miRNAs.

[0044] In the ADCs of the present disclosure, the drug and antibody are linked by a linker. The linker may be a cleavable linker or a non-cleavable linker. Examples of cleavable linkers include linkers having an -S-S- bond in their structure that is cleaved in the reducing environment within a cell (e.g., an SS linker or a DMSS linker), linkers having a hydrazone bond in their structure that is cleaved by the low pH within an endosome, linkers having an orthoester bond in their structure, and linkers having a peptide bond in their structure that is cleaved by cathepsin B (e.g., a linker having a valine-citrulline bond in the molecule (Val-Cit linker)), which can be preferably used in the present disclosure. In addition to the above, other cleavable linkers include linkers having a sugar chain in their structure that is cleaved by a glycolytic enzyme such as glucuronidase, which can be preferably used in the present disclosure.

[0045] Examples of non-cleavable linkers include linkers (sometimes referred to herein as maleimide linkers) that do not have a binding site that is cleavable in an intracellular environment (for example, the low pH environment in an endosome and the reducing environment in a cell) in their structure, and can be preferably used in the present disclosure.

[0046] An example of a cleavable linker is a linker having a succinimidyl group obtained by reacting the S atom of a cysteine ​​residue of an antibody with a maleimide group, a first spacer, valine-citrulline, and a second spacer. The first spacer can be polyalkylene glycol (preferably polyethylene glycol). The second spacer can be p-aminobenzyl carbamate (PABC). An example of a cleavable linker is a linker containing a succinimidyl group obtained by reacting the S atom of a cysteine ​​residue of an antibody with a maleimide group, polyalkylene glycol (preferably polyethylene glycol), valine-citrulline, and p-aminobenzyl carbamate (PABC). In some embodiments, the drug-antibody ratio (DAR) can be a natural number from 1 to 8. The DAR indicates the number of drugs linked to one antibody.

[0047] In some embodiments, the ADC can have the structure: {In the formula, "mAb" represents a monoclonal antibody, "PEG12" represents polyethylene glycol with a degree of polymerization of 12, and "t" represents the drug-antibody ratio. Here, t is a natural number from 1 to 8 when the mAb is IgG1. PEG may be replaced with an alkyl having 1 to 10 carbon atoms (e.g., a lower alkyl having 1 to 6 carbon atoms).}

[0048] Those skilled in the art will understand that an antigen-binding fragment of an antibody can be used instead of an antibody in the ADC. That is, the ADC comprises an antigen-binding fragment of an antibody and a drug, and the fragment and the drug can be linked via the linker. The drug may be directly linked to the linker, or the drug may be encapsulated in a drug delivery carrier (e.g., lipid nanoparticles), and the lipid nanoparticles may be directly linked to the linker. The linker may be directly linked to the thiol group of a cysteine ​​residue or the amino group (preferably the thiol group) of a lysine residue. In a preferred embodiment, the linker is linked to the thiol group of a cysteine ​​residue of the antibody via a succinimidyl group.

[0049] <Bispecific Binding Proteins of the Present Disclosure> According to the present disclosure, an antibody or antigen-binding fragment thereof of the present disclosure may be a bispecific protein. That is, an antibody or antigen-binding fragment thereof of the present disclosure has a first binding domain that binds to a first antigen or epitope and a second binding domain that binds to a second antigen or epitope. In some embodiments, the first and second antigens are different. In some embodiments, the first and second antigens are identical, but the epitope of the first binding domain is different from the epitope of the second binding domain. In some preferred embodiments where the first and second antigens are identical, the first and second binding domains can bind to the antigen simultaneously (do not compete for binding to the antigen).

[0050] In a preferred embodiment, the first antigen binds to CD320 and the second antigen binds to a T cell antigen (e.g., the T cell receptor complex, preferably CD3, e.g., the CD3 epsilon chain (CD3ε)). In this embodiment, the bispecific antibody links tumor cells or immune cells to T cells, thereby capable of killing the tumor cells or immune cells.

[0051] In a preferred embodiment, the first binding domain and the second binding domain can simultaneously bind to CD320. In this embodiment, the bispecific binding protein may have ADCC activity. Also in this embodiment, the bispecific binding protein may be in the form of a conjugate with a drug (ADC), which may or may not have ADCC activity. For details about ADCs, see the section on antibody drug conjugates (ADCs) of this disclosure above.

[0052] In a preferred embodiment, the bispecific binding protein has an Fc region. In a preferred embodiment, the Fc region contains a mutation that reduces binding to at least one Fcγ receptor compared to the wild type. Various such mutations are known, and examples include those disclosed in WO2012 / 073985A.

[0053] The first and second binding domains may comprise antibody heavy and light chain variable regions, respectively. The first and second binding domains may comprise antibody Fab or scFv fragments, respectively.

[0054] In a preferred embodiment, the bispecific binding protein may be a whole IgG-type bispecific antibody. In a whole IgG-type bispecific antibody, the first binding domain and the second binding domain each comprise a Fab fragment. In a preferred embodiment, the bispecific binding protein comprises a first binding domain and a second binding domain each comprise an scFv fragment, and preferably, the first binding domain and the second binding domain are linked via a peptide linker (e.g., a GS linker or a Whitlow linker (see, e.g., US 2019 / 0092818A)).

[0055] <Pharmaceutical Compositions of the Present Disclosure> The present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the present disclosure. The present disclosure also provides a pharmaceutical composition comprising an ADC of the present disclosure. The present disclosure further provides a pharmaceutical composition comprising a bispecific binding protein of the present disclosure. The pharmaceutical composition may further comprise a pharmaceutically acceptable additive.

[0056] Pharmaceutically acceptable additives include, but are not limited to, salts, solvents (e.g., water and ethanol), buffers, sugars and sugar alcohols, surfactants, isotonicity agents, preservatives, antioxidants, chelating agents, and excipients.The pharmaceutical composition of the present disclosure may be formulated according to conventional methods as a medicament for intravenous, subcutaneous, intraperitoneal, or intratumoral administration.According to the present disclosure, the conjugate may be prepared as a medicament using a vehicle suitable for administration (e.g., sterilized pyrogen-free water) before use.

[0057] The pharmaceutical compositions of the present disclosure can be used, for example, to treat tumors.

[0058] To identify a target population with high efficacy, in certain aspects of the present disclosure, the cancer may be a CD320-positive cancer or a cancer confirmed to be CD320-positive. CD320-positive cancers include pancreatic cancer, gastric cancer, colorectal cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, renal cancer, urinary tract cancer, Ewing's sarcoma, glioblastoma, leukemia / lymphoma (Anticancer Res. 2013 Oct;33(10):4203-12), and multiple myeloma (Cell Rep Med. 2023 4(7):101110). Whether a cancer is CD320-positive or -negative can be easily determined using techniques such as immunostaining or FACS using an anti-CD320 antibody.

[0059] In one aspect of the present disclosure, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof of the present disclosure and a drug. The conjugate can be administered in a therapeutically effective amount.

[0060] Certain aspects of the present disclosure provide use of an antibody or antigen-binding fragment thereof of the present disclosure in the manufacture of a medicament for use in treating cancer, the medicament comprising an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of the present disclosure and a drug. Certain aspects of the present disclosure provide use of an antibody or antigen-binding fragment thereof of the present disclosure and a cytotoxic agent in the manufacture of a medicament for use in treating cancer, the medicament comprising an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of the present disclosure and a drug. Certain aspects of the present disclosure provide use of an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of the present disclosure and a drug in the manufacture of a medicament for use in treating cancer.

[0061] In one aspect of the present disclosure, there is provided the use of an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof of the present disclosure and a drug in a method for treating cancer.

[0062] According to the present disclosure, a bispecific binding protein that binds to a CD320-expressing cell and a T cell receptor (TCR) complex (preferably CD3, e.g., CD3 epsilon chain (CD3ε)) may be used instead of an ADC. For details of such bispecific binding proteins, see the section on bispecific binding proteins of the present disclosure. According to the present disclosure, a pharmaceutical composition comprising the bispecific binding protein of the present disclosure is provided. Additives to be added to the pharmaceutical composition and uses of the pharmaceutical composition are as described above. Note that the bispecific binding protein of the present specification may be replaced with a multispecific binding protein.

[0063] According to the present disclosure, T cells (CAR-T cells) or NK cells (CAR-NK cells) expressing a chimeric antigen receptor that binds to CD320 may be used instead of ADCs. CARs can activate CAR-expressing cells when they bind to cells expressing a target antigen. According to the present disclosure, there is provided a pharmaceutical composition comprising the CAR-expressing cells of the present disclosure (e.g., T cells (e.g., cytotoxic T cells) or NK cells). Additives to be added to the pharmaceutical composition and uses of the pharmaceutical composition are as described above.

[0064] Immunization of BALB / c mice: BALBc mice (Jackson Laboratory) were immunogenized with recombinant human CD320 Fc Chimera (R&D Systems) containing an Fc tag attached to the C-terminus, produced in HEK293 cells. For the first immunization, the antigen was diluted with sterile phosphate-buffered saline (PBS) to prepare a 50 μg / ml antigen solution, which was placed in a 1 ml syringe. An equal volume of Freund's Complete Adjuvant (Difco) was placed in another 1 ml syringe, and the two syringes were connected with an adapter to form an emulsion. 100 μl of the immunogen was administered intraperitoneally. For subsequent immunizations, the immunogen was adjusted to 250 μg / ml with sterilized PBS, mixed with an equal volume of GERBU ADJUVANT 100 (Nacalai Tesque) in a 1.5 ml tube, and administered using a 1 ml syringe. 200 μl of the immunogen was administered intraperitoneally.

[0065] Blood was collected from the tail vein of immunized mice. After centrifugation at 13,000 × g for 10 minutes at 4°C, the supernatant was collected and used as a sample. Antibody titers were measured by indirect ELISA using samples serially diluted in two-fold increments from 1:500 to 1:64,000. Recombinant Human CD320 Fc Chimera (R&D Systems) was diluted with 0.1 M phosphate buffer to a final concentration of 0.1 μg / ml and added at 50 μl per well to a 96-well immunoplate (MaxiSorp™, Thermo Fisher Scientific). The plate was then allowed to stand at room temperature for 1 hour for immobilization. After washing three times with 200 μL / well of TBS-T, 200 μL / well of PBS / 0.1% Tween 20 / 0.25% skim milk (BD) blocking solution was added and the plate was incubated at room temperature for 30 minutes. After discarding the solution and washing three times with TBS-T, 50 μL / well of serially diluted samples in blocking solution was added and the plate was incubated at room temperature for 1 hour. After discarding the samples and washing three times, 50 μL / well of secondary antibody diluted 5000-fold in blocking solution was added and the plate was incubated at room temperature for 30 minutes. The secondary antibody used was Peroxidase-conjugated AffiniPure Goat anti-mouse IgG(H+L) (Jackson ImmunoResearch). The solution was discarded and the plate was washed three times with TBS-T. 100 μl / ml of colorimetric substrate solution (0.2 M sodium citrate / TMB / 30% H2O2) was added and incubated at room temperature for 15 minutes. 30 μl / well of 2N H2SO4 was added to stop the reaction. Absorbance at 450 nm was then measured using a Spectra Max paradigm (Molecular Devices).

[0066] Cell fusion and generation of anti-CD320 antibody-producing hybridomas. The mouse myeloma cell line P3X63-Ag8.653 (hereafter referred to as x63) was purchased from the JCRB Cell Bank. It was cultured in 440 ml of RPMI 1640 (Wako) supplemented with 50 ml of inactivated fetal bovine serum (FBS, Gibco) and 10 ml of 100 units / ml penicillin, 100 μg / ml streptomycin, and 250 ng / ml amphotericin B (Wako) at 37°C in a 5% CO2 incubator (Sanyo). When the cells reached semiconfluence, filling approximately 80% of a 150 mm dish (Corning), they were passaged in a clean bench. The cells were detached by pipetting and placed in a 15 ml centrifuge tube (Corning). They were centrifuged at 270 × g for 3 min at 4°C using a centrifuge (Universal Centrifuge 5800, Kubota). After removing the supernatant, the cells were suspended in 5 ml of RPMI 1640. A new 150 mm dish was prepared, and an appropriate amount of the cell suspension was added. The dish was then gently shaken. The dish was then cultured in a CO2 incubator.

[0067] Mice showing elevated antibody titers in CD320 ELISA tests were given a booster immunization by diluting 25 μg of recombinant human CD320 Fc Chimera (R&D Systems) in 300 μl of sterile phosphate-buffered saline (PBS). 30 μl of the immunization was administered intraperitoneally to each mouse, followed by intravenous administration of the remaining 270 μl. Four days later, the mice were sacrificed by cardiac exsanguination using a 1 mL syringe (Terumo) under inhalation anesthesia. The spleens were then surgically removed and placed in RPMI 1640 medium supplemented with 200 units / ml penicillin, 200 μg / ml streptomycin, and 500 ng / ml amphotericin B. To prevent infection, the spleen was immersed in 70% ethanol for a few seconds, washed with RPMI 1640, and then transferred to a 100 mm dish containing RPMI 1640. RPMI 1640 was injected into the spleen using a 10 mL syringe (Terumo) and a 22G needle (Terumo) to extract splenocytes. The cells were then passed through an Easy strainer with a 70 μm mesh (Greiner). The collected cell suspension was centrifuged at 270 × g for 5 min at room temperature, and the supernatant was removed. The cells were then suspended in 10 mL of RPMI 1640. This washing with RPMI 1640 was repeated twice and the cells were suspended in 5 mL of RPMI 1640. Next, P3X63-Ag8.653 was prepared for cell fusion with splenocytes. The cells were detached by pipetting and centrifuged at 270 × g for 3 minutes at room temperature. The supernatant was removed and the cells were suspended in 10 ml of RPMI 1640. This washing with RPMI 1640 was repeated twice and the cells were suspended in 5 ml of RPMI 1640. The cells were mixed so that the ratio of lymph node cells to P3X63-Ag8.653 cells was 5:1 to 7:1, and the cells were centrifuged at 270 × g for 3 minutes at room temperature. The supernatant was removed.1.8 g of PEG 4000 was dissolved in 2 ml of RPMI 1640 and filtered through a 0.22 μm MILLEX-GV Syringe Driven Filter Unit. One ml of this PEG 4000 solution was added to the cells, which had been thoroughly loosened by tapping, at a rate of 1 ml / min while vortexing. Next, 2 ml of RPMI 1640 was added at a rate of 1 ml / min and 8 ml at a rate of 4 ml / min while vortexing. The mixture was centrifuged at 170 × g for 5 min at room temperature, and the supernatant was removed. Fusion medium was prepared with the composition listed in Table 2-2 (Table 1). Cells were suspended in Fusion medium and seeded onto Costar 96-well cell culture plates (Corning).

[0068]

[0069] One week after cell fusion, the culture supernatants were screened by ELISA and flow cytometry. Limiting dilution was performed on wells with strong CD320 binding, and 416 clones were isolated from 1633 wells. Flow cytometry was performed again, and four clones, 8-1-2, 8-4-23, 44A, and 1322E, were selected for their CD320 binding activity (Figure 1). On the other hand, only clones 44A and 1322E bound strongly to the CD320-positive gastric cancer cell line HCG27 fixed with 4% paraformaldehyde, resulting in strong immunostaining. Clones 8-1-2 and 8-4-23 either did not bind or only weakly bound, resulting in no cell staining (Figure 2). Clones 8-1-2 and 8-4-23 likely recognize the three-dimensional structure of CD320. In addition, various cancer cell lines were CD320 positive (Figure 3). Furthermore, clone 8-1-2 was fluorescently labeled with Alexa647 and subjected to antibody uptake experiments into the pancreatic cancer cell line PSN1. Although the antibody was internalized within 30 minutes after administration, some of the antibody redistributed to the cell membrane, demonstrating recycling properties (Figure 4). When CD320 expression in pancreatic cancer cells was confirmed by immunochemical staining, CD320 expression was confirmed in almost all pancreatic cancer cell lines (15 strongly positive, 31 moderately positive, 40 weakly positive, and 3 negative). CD320 expression was also confirmed in gastric scirrhous carcinoma.

[0070] Cloning of antibody genes: Total RNA was extracted from hybridoma cell clones using the RNeasy mini Kit (QIAGEN). Gene fragments encoding the antibody heavy and light chain variable regions were isolated by 5'-RACE using the SMARTer RACE cDNA Amplification Kit (Clontech). Polymerase amplification was performed using KOD One (TOYOBO) in the first step and Platinum Taq (Thermo Fisher) in the second step.

[0071] Primer sequences used: Forward primer (SEQ ID NO: 49): CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT Reverse primer 1 (SEQ ID NO: 50): ACATTGATGTCTTTGGGGTAGAAG Reverse primer 2 (SEQ ID NO: 51): GGGATCCAGAGTTCCAGGTC (See PLoS One. 2019 14(6):e0218717.)

[0072] The gene fragment was cloned into the pTA2 vector (TOYOBO) and the sequence was determined using a DNA sequencer.

[0073] The antibody gene fragments of the human chimeric antibody clones 8-1-2, 8-4-23, 44A, and 1322E were amplified by PCR and cloned into the pcDNA3.3 vector (Thermo Fisher) incorporating the human antibody constant region to create expression vectors. The sequences were confirmed by DNA sequencing. The constructed expression vectors were transfected into CHO cells using the Expi CHO Expression System (Thermo Fisher) for transient antibody expression. For clone 8-1-2, an antibody format of the FC region of human IgG1 was prepared and antibody production in CHO cells was performed. The resulting antibodies were collected from the culture supernatant, affinity purified using Protein A, and purified by gel filtration column chromatography before use in experiments.

[0074] The binding of the anti-human CD320 antibody clone 8-1-2 in IgG1 format to the CD320-positive pancreatic cancer cell line PSN1 was measured by flow cytometry (Figure 5). Furthermore, the antibody was fluorescently labeled with Alexa647 and injected intravenously into a xenograft model transplanted with the pancreatic cancer cell line PSN1, and in vivo imaging was performed (IVIS Lumina S5, PerkinElmer). An anti-CD20 antibody (rituximab, Zenyaku Kogyo) that does not bind to PSN1 was used as a control antibody. The tumor accumulation of the 8-1-2 antibody was stronger than that of the control antibody (passive targeting: tumor-selective accumulation dependent on the antibody's size as a macromolecule) (Figure 6). This indicates active targeting over passive targeting: an additional effect of CD320-specific binding on tumor accumulation. Furthermore, in a similar experiment, tumor tissue was excised 24 hours after administration of a fluorescently labeled anti-CD320 antibody (BZ-X710, Keyence) and the distribution of the anti-CD320 antibody was confirmed. It was found to bind to the cancer cell membrane, confirming active targeting (Figure 7). These results demonstrate that the chimeric anti-CD320 antibody (clone 8-1-2) can also bind strongly to CD320 in vivo. Next, we constructed an antibody-drug conjugate (ADC) with SN-38 as its payload and confirmed its in vitro cytotoxicity against PSN-1. It demonstrated a potent cytotoxic effect similar to that of free SN-38 (Table 2). Furthermore, a strong antitumor effect was observed in an animal model of PSN-1 (Figure 8).

[0075]

[0076] Sequence Listing 8-1-2 / 8-4-23 heavy chain variable region (SEQ ID NO: 1): EVHLVESGGGLGKPGGSLKLSCAVSGFAFSSYDMSWVRQTPERRLEWVAYISSGGGITYHPDTVKGRFTISRDIAKNTLYLQMSSLKSEDTAMYYCSRHNREYDDAMDYWGQGTSVTVSSA 8-1-2 / 8-4-23 heavy chain CDR1 (SEQ ID NO: 2): SYDMS 8-1-2 / 8-4-23 heavy chain CDR2 (SEQ ID NO: 3): YISSGGGITYHPDTVKG 8-1-2 / 8-4-23 heavy chain CDR3 (SEQ ID NO: 4): HNREYDDAMDY 8-1-2 / 8-4-23 light chain variable region (SEQ ID NO: 5): DIVMTQAAFSNAVTLGTPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSCGGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKRADAAPTV 8-1-2 / 8-4-23 light chain CDR1 (SEQ ID NO: 6): RSSKSLLHSNGITYLY 8-1-2 / 8-4-23 light chain CDR2 (SEQ ID NO: 7): QMSNLAS 8-1-2 / 8-4-23 light chain CDR3 (SEQ ID NO: 8): AQNLELPLT

[0077] 1322E heavy chain variable region (SEQ ID NO: 9): QVQLQQSGPELVKPGALVKISCKASGYTFTSYDINWVKQRPGQGLEWIGWNYPGDGSTKYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARRGHYYAYFDYWGQGTTLTVSSAK 1322E heavy chain CDR1 (SEQ ID NO: 10): SYDIN 1322E heavy chain CDR2 (SEQ ID NO: 11): WNYPGDGSTKYNEKFKG 1322E heavy chain CDR3 (SEQ ID NO: 12): RGHYYAYFDY 1322E light chain variable region (SEQ ID NO: 13): ENVLTQSPAIMSASPGEKVTMTCRASSSVSSSYLHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISNVEAEDAATYFCQHFSNYPLTFGAGTKLELQRADAAPTVSI 1322E light chain CDR1 (SEQ ID NO: 14): RASSSVSSSYLH 1322E light chain CDR2 (SEQ ID NO: 15): STSNLAS 1322E light chain CDR3 (SEQ ID NO: 16): QHFSNYPLT

[0078] 44A heavy chain variable region (SEQ ID NO: 17): QVQLQQSGPELVKPGALVKISCKASGYTFTSYDINWVKQRPGQGLEWIGWSYPGDGSTKYNEKFEGKATLTADRASSTAYMQLSSLTSEDSAVYFCARRGHYYAYFDFWGQGTTLTVSSAK 44A heavy chain CDR1 (SEQ ID NO: 18): SYDIN 44A heavy chain CDR2 (SEQ ID NO: 19): WSYPGDGSTKYNEKFEG 44A heavy chain CDR3 (SEQ ID NO: 20): RGHYYAYFDF 44A light chain variable region (SEQ ID NO: 21): ENVLTQSPAIMSASPGEKVTMTCRASSSVSSSYLHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISNVEAEDAATYFCQHFSNYPLTFGAGTKLELQRADAAPTVSI 44A light chain CDR1 (SEQ ID NO: 22): RASSSVSSSYLH 44A light chain CDR2 (SEQ ID NO: 23): STSNLAS 44A light chain CDR3 (SEQ ID NO: 24): QHFSNYPLT

Claims

1. An antibody or antigen-binding fragment of an antibody that binds to CD320.

2. An antibody or antigen-binding fragment of an antibody that binds to CD320, comprising: (1) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:2, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:3, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:4, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:6, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:8; (2) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:10, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:11, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:12, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:14, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:15, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:16; (3) A heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 20, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 22, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 23, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 24; (4) An antibody or antigen-binding fragment of the antibody comprising a heavy chain variable region and a light chain variable region having 1 to 5 amino acid mutations from any of the antibodies set forth in (1) to (3) above; or (5) An antibody or antigen-binding fragment of the antibody that can compete for binding to CD320 with any of the antibodies set forth in (1) to (3) above.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a human chimeric antibody or an antigen-binding fragment thereof.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a humanized antibody or antigen-binding fragment thereof.

5. An antibody-drug conjugate comprising an antibody or its antigen-binding fragment and a drug, wherein the antibody or fragment is linked to the drug via a linker, and the antibody or fragment comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

7. The pharmaceutical composition according to claim 6, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity and / or complement-dependent cytotoxicity.

8. A pharmaceutical composition comprising the antibody-drug conjugate of claim 5.

9. A pharmaceutical composition according to any one of claims 6 to 8 for use in treating cancer in a subject in need thereof.

Images