Antibodies that bind to fibrin and pharmaceutical compositions containing such antibodies

Abstract

The present invention provides a fibrin-binding antibody and a pharmaceutical composition containing the antibody. In particular, the present invention provides a fibrin-binding humanized antibody and a pharmaceutical composition containing the antibody.

Description

Technical Field

[0001] The present invention relates to an antibody that binds to fibrin and a pharmaceutical composition containing the antibody.

Background Art

[0002] Antibodies that bind to fibrin with a stronger affinity than to fibrinogen have been developed (Patent Documents 1 and 2). These patent documents disclose the use of the antibody for the treatment of cancer. An antibody that strongly binds to fibrin can also be used for in vivo imaging (Patent Documents 1 and 2).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

[0004] The present invention provides an antibody that binds to fibrin and a pharmaceutical composition containing the antibody. In particular, the present invention provides a humanized antibody that binds to fibrin and a pharmaceutical composition containing the antibody.

[0005] According to the present invention, the following inventions are provided. [1] An antibody or an antigen-binding fragment thereof that binds to insoluble fibrin, (1) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6; or (2) A heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 11, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 14; An antibody or an antigen-binding fragment thereof that binds to insoluble fibrin. [2] An insoluble fibrin antibody or an antigen-binding fragment thereof, (3) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8; or (4) An antibody or an antigen-binding fragment thereof that binds to insoluble fibrin, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 16. [3] Binds to insoluble fibrin with a stronger affinity than to fibrinogen, or Binds to insoluble fibrin with a stronger affinity than to fibrinogen, and (5) Comprising a heavy chain variable region having an amino acid sequence having 90% or more identity with the heavy chain variable region of the antibody according to any one of (1) to (4) above, and a light chain variable region of the antibody. (6) Comprising a heavy chain variable region of the antibody according to any one of (1) to (4) above, and a light chain variable region having an amino acid sequence having 90% or more identity with the light chain variable region of the antibody. (7) Comprising a heavy chain variable region having an amino acid sequence having 90% or more identity with the heavy chain variable region of the antibody according to any one of (1) to (4) above, and a light chain variable region having an amino acid sequence having 90% or more identity with the light chain variable region of the antibody. (8) Comprising a heavy chain variable region having an amino acid sequence having 95% or more identity with the heavy chain variable region of the antibody according to any one of (1) to (4) above, and a light chain variable region of the antibody. (9) An antibody or antigen-binding fragment thereof according to any one of (1) to (4) above, comprising a heavy-chain variable region of the antibody and a light-chain variable region having an amino acid sequence having 95% or more identity with the light-chain variable region of the antibody, or (10) An antibody or antigen-binding fragment thereof according to any one of (1) to (4) above, comprising a heavy-chain variable region having an amino acid sequence having 95% or more identity with the heavy-chain variable region of the antibody and a light-chain variable region having an amino acid sequence having 95% or more identity with the light-chain variable region of the antibody. An antibody or antigen-binding fragment thereof according to any one of [1] or [2] above, which binds to insoluble fibrin. [4] An antibody or antigen-binding fragment thereof according to any one of [1] to [3] above, which is a humanized antibody or antigen-binding fragment thereof. [5] An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof according to any one of [1] to [4] above. [6] A pharmaceutical composition comprising the antibody-drug conjugate according to [5] above. [7] The pharmaceutical composition according to [6] above, for use in treating cancer. [8] A pharmaceutical composition for use in treating cancer, comprising an anticancer agent comprising an antibody or antigen-binding fragment thereof according to any one of [1] to [4] above. [9] An in vivo diagnostic agent comprising an antibody or antigen-binding fragment thereof according to any one of [1] to [4] above.

Brief Description of the Drawings

[0006]

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[0007] As used herein, "subject" is a mammal and can be, for example, a dog, a cat, a cow, a horse, a pig, a primate (e.g., a monkey, a gorilla, an orangutan, a bonobo, a chimpanzee, and a human), and can be, for example, a human.

[0008] As used herein, "treatment" is used in the sense including treatment (therapeutic treatment) and prevention (preventive treatment). As used herein, "treatment" means the treatment, cure, prevention, or improvement of remission of a disease or disorder, or the reduction of the progression rate of a disease or disorder. As used herein, "prevention" means reducing the likelihood of onset of a disease or condition or delaying the onset of a disease or condition.

[0009] As used herein, "disease" means a condition for which treatment is beneficial. As used herein, "cancer" means a malignant tumor.

[0010] As used herein, "therapeutically effective amount" means an amount of a drug that is effective for treating (preventing or treating) a disease or condition. A therapeutically effective amount of a drug can reduce the rate of worsening of symptoms of a disease or condition, stop the worsening of said symptoms, improve said symptoms, cure said symptoms, or suppress the onset or development of said symptoms.

[0011] As used herein, "fibrin" is an insoluble coagulum formed by cleavage of the C-terminus of the three types of polypeptide chains (Aα chain, Bβ chain, and γ chain) that make up fibrinogen. In this specification, fibrin may sometimes be referred to as insoluble fibrin. More specifically, cleavage of the C-terminus of fibrinogen results in a state called a fibrin monomer, and polymerization of the fibrin monomer by the action of calcium forms a poorly soluble fibrin polymer. The fibrin polymer is cross-linked between polymers by the action of factor XIII to form stabilized fibrin (insoluble fibrin or fibrin gel as defined in this specification). Insoluble fibrin is degraded by plasmin. Plasmin is contained in plasma as its precursor, plasminogen. When the peptide between Arg and Val of plasminogen is degraded by a plasminogen activator (e.g., urokinase, tissue plasminogen activator, and streptokinase), plasmin is produced. Plasmin can be inhibited by a protein called a plasmin inhibitor and its action can be restricted.

[0012] The Aα chain of fibrinogen can be the Aα chain of human fibrinogen. Examples of the Aα chain of human fibrinogen include the Aα chain of human fibrinogen having the amino acid sequence registered under GenBank accession number: AAI01936.1, and the Aα chain of human fibrinogen having an amino acid sequence corresponding to said amino acid sequence.

[0013] The Bβ chain of fibrinogen can be the Bβ chain of human fibrinogen. Examples of the Bβ chain of human fibrinogen include the Bβ chain of human fibrinogen having the amino acid sequence registered under NCBI reference number: NP_005132.2, and the Bβ chain of human fibrinogen having an amino acid sequence corresponding to the said amino acid sequence.

[0014] The γ chain of fibrinogen can be the γ chain of human fibrinogen. Examples of the γ chain of human fibrinogen include the γ chain of human fibrinogen having the amino acid sequence registered under GenBank accession number: AAH07044.1, and the γ chain of human fibrinogen having an amino acid sequence corresponding to the said amino acid sequence.

[0015] As used herein, "antibody" means immunoglobulin. The antibody can be various isotypes of antibodies, for example, it can be IgG. Preferably, the antibody can be a monoclonal antibody. The antibody can be a human chimeric antibody, a humanized antibody, or a human antibody. A human chimeric antibody can be produced by replacing the constant region of a non-human antibody with the constant region of a human antibody. A humanized antibody can be produced by replacing the six CDRs of a human antibody with the six corresponding CDRs of a non-human antibody. A human antibody can be produced using an animal (e.g., mouse) in which at least the heavy chain variable region of immunoglobulin is replaced with the corresponding region of a human locus. When the constant region is non-human, a human antibody can be obtained by replacing the constant region with the amino acid sequence of a human antibody. As used herein, the antibody can preferably be a humanized antibody. As used herein, the antibody can preferably be a human antibody. When the antibody is produced intracellularly, it has a signal peptide, but when secreted extracellularly, the signal peptide is excised. Therefore, when administered as a medicine, the antibody does not require a signal peptide.

[0016] In this specification, "CDR" refers to the complementarity-determining regions present in the heavy-chain variable region and the light-chain variable region of an antibody. There are three each in the heavy-chain and light-chain variable regions, which are called CDR1, CDR2, and CDR3 from the N-terminus. The CDRs can be determined, for example, based on the numbering by Kabat et al. (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, Bethesda: US Dept. of Health and Human Services, PHS, NIH.).

[0017] As used herein, the "insoluble fibrin-specific antibody" refers to an antibody that binds to insoluble fibrin and has a higher affinity for insoluble fibrin than for fibrinogen. Such an insoluble fibrin-specific antibody can be easily obtained by screening based on the affinity for insoluble fibrin and the affinity for fibrinogen. Fibrin undergoes a conformational change in insoluble fibrin and has an epitope site that is exposed only after becoming insoluble. Therefore, the "insoluble fibrin-specific antibody" can be obtained by immunizing with this exposed domain, i.e., the D domain (hereinafter also referred to as the "D-domain"), as an immunogen. It is also possible to obtain it using a linear peptide. For example, when immunizing with a partial peptide of the fibrin Bβ chain corresponding to positions 231 to 246 of the amino acid sequence of the fibrin Bβ chain (for example, the human fibrin Bβ chain may have the amino acid sequence represented by SEQ ID NO: 17), an "insoluble fibrin-specific antibody" can be obtained. In addition, an "insoluble fibrin-specific antibody" can also be obtained by immunizing with a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 18 as an immunogen. Such an insoluble fibrin-specific antibody can be an antibody that has a higher affinity for insoluble fibrin than for any of fibrinogen, fibrin monomer, and fibrin polymer. An antibody having a ratio of the affinity for insoluble fibrin to the affinity for fibrinogen of, for example, more than 1, 1.5 or more, 2 or more, 3 or more, 4 or more, or 5 or more can be obtained as an insoluble fibrin-specific antibody. Affinity means binding affinity (KD) and can be determined by well-known methods such as ELISA and the binding equilibrium exclusion method. As used herein, strong affinity means a low KD. As used herein, weak affinity means a high KD.

[0018] As used herein, "competing" means competing with other binding antibodies for binding to an antigen. Competition can occur when the binding sites of two antibodies overlap for a particular antigen. Such antibodies can be obtained by immunization using an epitope as described above and / or by confirming whether the binding of one antibody to an antigen is reduced by the other antibody in a competition assay. Examples of competing antibodies include antibodies that compete with each other.

[0019] As used herein, "antibody-drug conjugate" (hereinafter also referred to as "ADC") means a substance in which an antibody and a cytotoxic agent are linked. In an ADC, the antibody and the cytotoxic agent can be linked via an appropriate linker. As the cytotoxic agent, a chemotherapeutic agent, a radioisotope, and a toxin can be used. An ADC also includes a conjugate of an antigen-binding fragment of an antibody and a drug.

[0020] As used herein, "antigen-binding fragment of an antibody" means a fragment of an antibody that maintains the ability to bind to an antigen. Examples of antigen-binding fragments include Fab, Fab’, F(ab’)2, Fv, scFv (single-chain Fv), diabody, and sc(Fv)2 (single-chain (Fv)2). For example, when an antibody is digested with papain, Fab can be obtained. Alternatively, when an antibody is digested with pepsin, F(ab’)2 can be obtained, and further reduction of F(ab’)2 can yield Fab’. Other antigen-binding fragments of an antibody can be prepared by methods well known to those skilled in the art. Such antigen-binding fragments of an antibody can be used in the present invention.

[0021] In the present invention, an antibody that binds to insoluble fibrin binds to insoluble fibrin with a stronger affinity than to fibrinogen (i.e., a lower dissociation constant K for insoluble fibrin than for fibrinogen). DAn antibody that binds to insoluble fibrin with a stronger affinity than to fibrinogen can be, for example, an antibody that binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17 or 18 (see WO2014 / 133093). The amino acid sequence set forth in SEQ ID NO: 17 corresponds to the amino acid sequence set forth in SEQ ID NO: 1 of WO2014 / 133093, and the amino acid sequence set forth in SEQ ID NO: 18 corresponds to the amino acid sequence set forth in SEQ ID NO: 2 of WO2014 / 133093.

[0022] Examples of an antibody or an antigen-binding fragment thereof that binds to insoluble fibrin with a stronger affinity than to fibrinogen include an antibody or an antigen-binding fragment thereof having, as corresponding CDRs, the heavy-chain CDR1-3 and the light-chain CDR1-3 of an antibody selected from the group consisting of the 10-102 antibody, the 34-105 antibody, and the Fib-0355 antibody disclosed in WO2014 / 133093.

[0023] Examples of an antibody or an antigen-binding fragment thereof that binds to insoluble fibrin with a stronger affinity than to fibrinogen also include an antibody or an antigen-binding fragment thereof having, as corresponding CDRs, the heavy-chain CDR1-3 and the light-chain CDR1-3 of an antibody selected from the group consisting of the 99 antibody, the 1101 antibody, and the 0211 antibody disclosed in WO2018 / 203517.

[0024] Furthermore, examples of an antibody or an antigen-binding fragment thereof that binds to insoluble fibrin with a stronger affinity than to fibrinogen include a heavy-chain variable region comprising a heavy-chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a heavy-chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy-chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3, a light-chain variable region comprising a light-chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a light-chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and a light-chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 and Examples include an antibody or an antigen-binding fragment thereof. The antibody can be a human chimeric antibody or a humanized antibody. According to the present invention, a humanized antibody having the above heavy-chain variable region and light-chain variable region is also provided.

[0025] Examples of an antibody or an antigen-binding fragment thereof that binds to insoluble fibrin with a stronger affinity than to fibrinogen include, for example, a heavy-chain variable region having the amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 7, and a light-chain variable region having the amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 8 and include an antibody or an antigen-binding fragment thereof. The antibody may further include a heavy-chain constant region or a part thereof within the Fab fragment in addition to the heavy-chain variable region. The antibody can be a human chimeric antibody or a humanized antibody. According to the present invention, a humanized antibody having the above heavy-chain variable region and light-chain variable region is also provided.

[0026] Examples of an antibody or an antigen-binding fragment thereof that binds to insoluble fibrin with a stronger affinity than to fibrinogen further include a heavy-chain variable region including a heavy-chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 9, a heavy-chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 10, and a heavy-chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 11, and a light-chain variable region including a light-chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 12, a light-chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 13, and a light-chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 14 and include an antibody or an antigen-binding fragment thereof. The antibody can be a human chimeric antibody or a humanized antibody. According to the present invention, a humanized antibody having the above heavy-chain variable region and light-chain variable region is also provided.

[0027] Examples of an antibody or an antigen-binding fragment thereof that binds to insoluble fibrin with a stronger affinity than to fibrinogen include, for example, A heavy chain variable region having the amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region having the amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 16, and comprising antibodies or antigen-binding fragments thereof. The antibody may further comprise a heavy chain constant region or a part thereof within the Fab fragment in addition to the heavy chain variable region. The antibody can be a human chimeric antibody or a humanized antibody. According to the present invention, a humanized antibody having the above heavy chain variable region and light chain variable region is also provided.

[0028] According to the present invention, antibodies that compete with the antibody described in (3) above for binding to insoluble fibrin, preferably antibodies that compete with each other, can be provided. Here, the antibody is preferably a humanized antibody.

[0029] According to the present invention, antibodies that compete with the antibody described in (4) above for binding to insoluble fibrin, preferably antibodies that compete with each other, can be provided. Here, the antibody is preferably a humanized antibody.

[0030] According to the present invention, the antibody described in (1) above can be a humanized antibody and an antibody specific for insoluble fibrin. According to the present invention, the antibody described in (2) above can be a humanized antibody and an antibody specific for insoluble fibrin. According to the present invention, the antibody described in (3) above can be a humanized antibody and an antibody specific for insoluble fibrin. According to the present invention, the antibody described in (4) above can be a humanized antibody and an antibody specific for insoluble fibrin. According to the present invention, antibodies that compete with the antibody described in (3) above for binding to insoluble fibrin, preferably antibodies that compete with each other, can be humanized antibodies and antibodies specific for insoluble fibrin. According to the present invention, antibodies that compete with the antibody described in (4) above for binding to insoluble fibrin, preferably antibodies that compete with each other, can be humanized antibodies and antibodies specific for insoluble fibrin.

[0031] The antibody of the present invention can bind to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17. Here, the antibody of the present invention can be a humanized antibody. The antibody of the present invention is a humanized antibody that can bind to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, and can be selected from any of the above antibodies. The antibody of the present invention is a humanized antibody that can bind to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, and can have 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more amino acid identity in the heavy or light chain variable region with the above antibody. The antibody of the present invention is a humanized antibody that can bind to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, and can be an antibody having one amino acid insertion, deletion, substitution or addition in the heavy or light chain variable region with the above antibody. The antibody of the present invention is a humanized antibody that can bind to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, and can be an antibody having two amino acid insertions, deletions, substitutions or additions in the heavy or light chain variable region with the above antibody.

[0032] Any of the antibodies of the present invention can be used as the antibody moiety in an antibody-drug conjugate.

[0033] In the present invention, in an antibody-drug conjugate, an antibody and a cytotoxic agent are linked via a linker. Examples of cytotoxic agents include chemotherapeutic agents (such as anticancer agents like commercially available anticancer drugs, such as auristatins (auristatin E, auristatin F phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin F and their derivatives), maytansinoids DM1 and DM4 and their derivatives), camptothecins (SN-38, irinotecan, rutotecan, DB67, BMP1350, ST1481, CKD602, topotecan and exatecan, and their derivatives), DNA minor groove binders (enediynes, lexitropsins, duocarmycins and their derivatives), taxanes (paclitaxel and docetaxel and their derivatives), polyketides (discodermolide and its derivatives), anthraquinones (mitoxantrone and its derivatives), benzodiazepines (pyrrolobenzodiazepines, indolinobenzodiazepines, and oxazolidinobenzodiazepines and their derivatives), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine and their derivatives), doxorubicins (doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin and their derivatives), cardiac glycosides (digitoxin and its derivatives), calicheamicin, epothilone, cryptophycin, semadotin, semadotin, lysocine, netropsin, combretastatin, erythrobins, etoposide, T67 (tularemycin), and nocodazole), radioisotopes (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.) can be mentioned and can be used as cytotoxic agents in the ADC of the present invention. As the cytotoxic agent in the ADC of the present invention, preferably, for example, camptothecin, particularly SN-38, or exatecan can be used. Any of the cytotoxic agents that can be used for the treatment of cancer can be used. As the cytotoxic agent, a pharmaceutically acceptable salt, solvate (e.g., hydrate), ester, or prodrug of the above cytotoxic agent may be used.

[0034] In the present invention, in the ADC, the antibody and the cytotoxic agent may be linked via a linker. In the present invention, the linker may be a non-cleavable linker or a cleavable linker. Various linker structures used in general ADCs are known and these can be used as appropriate.

[0035] In one aspect of the present invention, the linker of the ADC contains a plasmin cleavage sequence and is cleavable in the presence of plasmin. In the present invention, for the linker of the ADC, the parts other than the plasmin cleavage sequence consist of stable chemical bonds during the process until it is delivered to insoluble fibrin after administration. With such a configuration, the ADC of the present invention is stable until it is delivered to insoluble fibrin after administration, and after binding to insoluble fibrin, it is cleaved by plasmin and releases the cytotoxic agent only in the vicinity of insoluble fibrin. The plasmin cleavage sequence is an amino acid sequence, specifically, a peptide chain containing an amino acid sequence such as a plasmin cleavage sequence selected from the group consisting of valine-leucine-lysine, glycine-proline-lysine, glutamic acid-lysine-lysine, lysine-phenylalanine-lysine, norvaline-chlorohexylalanyl-lysine, and norleucine-hexahydrotyrosine-lysine. Such a linker can be appropriately selected and synthesized by those skilled in the art in the preparation of the ADC. In one aspect, for example, a first spacer may be introduced between the antibody and the plasmin cleavage sequence in the linker. For example, polyethylene glycol (PEG), for example, PEG with about 5 to 40 repeating units per molecule can be used as the first spacer. A second spacer may be introduced between the plasmin cleavage sequence and the cytotoxic agent. For example, p-aminobenzyloxycarbonyl (PABC) can be used as the second spacer. In one aspect, the linker contains a first spacer and a plasmin cleavage sequence. In one aspect, the linker contains a first spacer, a plasmin cleavage sequence, and a second spacer. In a particular subject, the linker contains PEG, a plasmin cleavage sequence, and PABC. In one aspect, the linker does not contain a cleavable moiety other than the plasmin cleavage sequence. The binding between the antibody and the linker can be linked, for example, via a maleimide group to the sulfhydryl group of the antibody. In one aspect, the antibody is linked to the anti-cancer agent via its sulfhydryl group with a linker having a maleimide-PEG-plasmin cleavage sequence. In one aspect, the antibody is linked to the anti-cancer agent via its sulfhydryl group with a linker having a maleimide-PEG-plasmin cleavage sequence-PABC. In any case, in the ADC of the present invention, the anti-cancer agent is linked to the anti-cancer agent with a linker having a plasmin cleavage site cleavable by plasmin. When the ADC reaches a location where insoluble fibrin accumulates, the linker is cleaved at the plasmin cleavage site by plasmin present therearound, and the anti-cancer agent is released to the periphery of the insoluble fibrin. It is considered that there are many locations where insoluble fibrin accumulates based on bleeding due to cancer invasion around the cancer tissue (see FIG. 8 of WO2018 / 203517). The antibody of the present invention (i.e., an insoluble fibrin-specific antibody) is useful for targeting cancer in a drug delivery system, and the ADC of the present invention is considered useful as a therapeutic agent for cancer.

[0036] The cancer to be treated by the ADC or pharmaceutical composition of the present invention is not particularly limited, and examples include cancer, such as lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, uterine cancer, ovarian cancer, skin cancer, thyroid cancer, thymic cancer, kidney cancer, testicular cancer, penile cancer, liver cancer, biliary tract cancer, brain tumor, osteosarcoma, retroperitoneal tumor, angio-lymphangiosarcoma, and metastatic cancers thereof.

[0037] The subject of the present invention can be a subject who does not suffer from a thrombotic disorder or a disease associated with a thrombotic disorder, or a subject who has not been diagnosed as having a thrombotic disorder or a disease associated with a thrombotic disorder. Thereby, it can be expected to reduce side effects in tissues other than cancer. Therefore, it may be determined whether a subject having cancer suffers from a thrombotic disorder or a disease associated with a thrombotic disorder, and then the ADC of the present invention may be administered to a subject who does not suffer from a thrombotic disorder or a disease associated with a thrombotic disorder. Whether a subject suffers from a thrombotic disorder or a disease associated with a thrombotic disorder can be appropriately determined by a physician.

[0038] In one aspect of the present invention, the pharmaceutical composition comprises the ADC of the present invention and an excipient. The pharmaceutical composition of the present invention can be administered by administration methods such as intravenous administration, subcutaneous administration, intratumoral administration, intraperitoneal administration, intracerebroventricular administration, intramuscular administration, etc. The dosage can be appropriately determined by a doctor in consideration of the patient's age, sex, weight, severity of the disease, etc.

[0039] The ADC of the present invention targets insoluble fibrin that accumulates in the stroma of these cancers and accumulates a cytotoxic agent at the targeted site. Moreover, it has a linker that can be cleaved by plasmin, which is activated at the location where insoluble fibrin is present, and releases the cytotoxic agent at the targeted site. Thereby, the cancer around the release site can be damaged site-specifically.

[0040] According to the present invention, there is provided the use of an insoluble fibrin-specific antibody in the manufacture of a medicament for treating cancer. According to the present invention, there is provided the use of an ADC of an insoluble fibrin-specific antibody and a cytotoxic agent in the manufacture of a medicament for treating cancer, wherein the antibody and the cytotoxic agent have a plasmin cleavage site cleavable by plasmin.

[0041] According to the present invention, there is provided a method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the ADC of the present invention to the subject. According to the present invention, there is provided a method for treating cancer in a subject in need thereof, comprising determining whether a subject having cancer has a thrombotic disorder or a disease associated with a thrombotic disorder, and then administering a therapeutically effective amount of the ADC of the present invention to a subject not suffering from a thrombotic disorder or a disease associated with a thrombotic disorder.

[0042] According to the present invention, there is provided the use of the ADC of the present invention for use in a method for treating cancer.

[0043] According to the present invention, the antibody of the present invention can be prepared by methods well known to those skilled in the art. For example, the antibody of the present invention can be expressed in cells (insect cells, avian cells, Escherichia coli, yeast, and mammalian cells), preferably mammalian cells (for example, mammalian cells suitable for protein expression, such as Chinese hamster ovary cells (CHO cells) and 293 cells and their derived cells, and human cells such as those suitable for protein expression). Expression can be carried out, for example, using an expression vector containing a nucleic acid encoding the fusion protein of the present invention operably linked to a promoter drivable in the expression cells. When a light chain is required for the fusion protein, the light chain can be co-expressed in the expression cells. According to the present invention, the fusion protein of the present invention can be purified by purification methods well known to those skilled in the art. Purification can be carried out using an affinity column containing an antigen, an affinity column for a tag attached to the fusion protein, and the like.

[0044] According to the present invention, there are provided a composition and a pharmaceutical composition comprising the antibody of the present invention or an antigen-binding fragment thereof. The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable excipient in addition to the antibody of the present invention or an antigen-binding fragment thereof. Examples of pharmaceutically acceptable excipients include salts, tonicity agents, pH adjusters, and water. In one aspect, the pharmaceutical composition of the present invention can be a kit (i.e., a ready-to-prepare kit) containing a lyophilized pharmaceutical composition and water for injection.

[0045] The pharmaceutical composition comprising the antibody of the present invention or an antigen-binding fragment thereof can be administered by parenteral administration (for example, intravenous administration). Also, when the pharmaceutical composition of the present invention is for intravenous administration, it can be a formulation suitable for intravenous administration. Such a pharmaceutical composition can be appropriately prepared by those skilled in the art. For example, it can be prepared in the same manner as protein formulations and antibody formulations. The dosage, administration timing, administration route, and the like can be appropriately determined by a physician.

[0046] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of an antibody or an antigen-binding fragment thereof.

[0047] In certain embodiments of the invention, the antibody can be linked to a detectable label for in vivo imaging. Detectable labels include, for example, 177 Lu, 111 indium, 67 Cu, 18 F, 99m Tc, 124 I, 125 I and 131 I and include isotopes selected from the group consisting of. The detectable label may be a positron emission tomography (PET) agent, which includes 124 I, 89 Zr, etc., but are not limited thereto. In certain embodiments, the first and / or second antibody or antigen-binding portion or derivative thereof is radiolabeled. The radiolabel is 177 Lu, 111 indium, 67 Cu, 18 F, 99m Tc, 124 I, 125 I, 131 I, and 99m Tc may be at least one of. The detectable label can be any label suitable for in vitro and / or in vivo imaging. Imaging can include any one or more of the following: planar radionuclide imaging, positron emission tomography (PET), echo planar imaging (EPI), single photon emission computed tomography (SPECT), ultrasound imaging (e.g., non-radioactive, contrast-specific, high-frequency, two-dimensional), magnetic resonance imaging (MRI; also called magnetic resonance tomography or MRT), X-ray, computed tomography (CT) scan, fluorescence imaging, near-infrared imaging, and other imaging methods useful or applicable in medicine. Thus, these contrast agents and imaging agents suitable therefor can be linked to the antibody.

[0048] According to the present invention, there are provided an antibody of the present invention conjugated with a detectable label for in vivo imaging, and a diagnostic agent or diagnostic kit containing the antibody.

[0049] According to the present invention, there is provided a method for observing fibrin in a subject in vivo, the method comprising administering an effective amount of an antibody of the present invention conjugated with a detectable label for in vivo imaging to the subject.

Examples

[0050] Example 1: Preparation of Humanized 1101 Antibody and Humanized 99 Antibody The antibodies produced by clone 1101 (1101 antibody) and the monoclonal antibody produced by clone 99 (99 antibody) described in WO2018 / 203517 were humanized to obtain a humanized 1101 antibody and a humanized 99 antibody, respectively. In the humanization process, the sequence of the CDR was changed to improve the binding affinity to insoluble fibrin.

[0051] Using the obtained humanized 1101 antibody and humanized 99 antibody, the binding to insoluble fibrin was confirmed by an ELISA assay. ELISAs coated with bovine serum albumin (BSA), fibrinogen (FNG), soluble fibrin (SF), fibrin degradation product (FDP), and insoluble fibrin (IF) were prepared. The surface of the ELISA plate was blocked with TBS-T containing 1% BSA and 0.05% Tween 20, and the HRP-labeled humanized 1101 antibody and the HRP-labeled humanized 99 antibody were added and reacted. The TMB reagent, which is a substrate for HRP, was added to cause color development, and the color development was detected using a microplate reader (SpectraMax Paradigm, molecular Device). The results of the color development intensity were as shown in FIG. 1. As shown in FIG. 1, both the humanized 99 antibody and the humanized 1101 antibody specifically bound to insoluble fibrin.

[0052] Next, the binding of the humanized 1101 antibody and the humanized 99 antibody to denatured fibrinogen was confirmed by Western blotting. The fibrinogen solution was diluted with a loading buffer containing 2-mercaptoethanol and SDS, and heated at 95 °C to obtain denatured fibrinogen. The obtained denatured fibrinogen was subjected to SDS-PAGE, transferred to a PVDF membrane, and then blocked with StartingBlock TM (TBS) Blocking Buffer (Thermo Fisher). Thereafter, the humanized 1101 antibody and the humanized 99 antibody were reacted with the membrane. Denatured fibrinogen exposes the sites specifically exposed to insoluble fibrin to which the humanized 1101 antibody and the humanized 99 antibody bind, similar to insoluble fibrin, so that the humanized 1101 antibody and the humanized 99 antibody can bind. After washing and blocking the membrane, Western blotting was performed using an HRP-labeled anti-human IgG antibody (Bethyl) as the secondary antibody and ECL™ Prime Western Blotting Detection Reagent (Thermo Fisher) as the luminescent reagent. Photography and image analysis were performed using a ChemiDoc imaging system (Bio-rad). As shown in Fig. 2, the results showed that both the humanized 1101 antibody and the humanized 99 antibody were able to detect denatured fibrin at a concentration of 0.04 μg / mL.

[0053] Furthermore, immunohistochemical staining (IHC staining) was attempted using humanized 1101 antibody and humanized 99 antibody. Pancreatic tissue sections of pancreatic cancer model mice (HE-PKC mice) were fixed with paraformaldehyde by a conventional method to prepare sections. The sections were blocked with PBS containing 1% BSA and reacted with the obtained tissue sections with humanized 1101 antibody and humanized 99 antibody (10 mg or 1 mg). The sections were stained using Alexa 647-labeled anti-human IgG (Jackson Immunotechnology) as a secondary antibody and DAPI as a nuclear staining reagent. The sections were photographed using a virtual slide (Olympus Life Science), and image analysis was performed using OlyVIA. The results were as shown in Fig. 3. As shown in Fig. 3, it was found that the obtained humanized 1101 antibody and humanized 99 antibody were useful for IHC staining.

[0054] In addition, a location where a thrombus was present was found in the blood vessels of the normal tissue part of a human lung cancer resection specimen. Immunohistochemical staining was performed on this thrombus using humanized 1101 antibody and humanized 99 antibody in the same manner as above. The results were as shown in Fig. 4. As shown in Fig. 4, the humanized 1101 antibody and humanized 99 antibody were each able to visualize the thrombus site. That is, it was also shown that the humanized 1101 antibody and humanized 99 antibody bind to human thrombi.

[0055] Next, recombinant CHO cells expressing humanized 99 antibody and humanized 1101 antibody were prepared. The particle sizes (nm) of the produced humanized 1101 antibody and humanized 99 antibody were measured using Delsa (trademark) Nano HC (Beckman coulter). The results were as shown in Fig. 5. As shown in Fig. 5, the humanized 1101 antibody and humanized 99 antibody showed a monodisperse particle size distribution, and it was revealed that the humanized 1101 antibody had a more uniform particle size.

[0056] Sequence Listing SEQ ID NO: 1: Amino acid sequence of heavy chain CDR1 of humanized anti-fibrin antibody 1101 clone Sequence number 2: Amino acid sequence of the heavy-chain CDR2 of the humanized anti-fibrin antibody clone 1101 Sequence number 3: Amino acid sequence of the heavy-chain CDR3 of the humanized anti-fibrin antibody clone 1101 Sequence number 4: Amino acid sequence of the light-chain CDR1 of the humanized anti-fibrin antibody clone 1101 Sequence number 5: Amino acid sequence of the light-chain CDR2 of the humanized anti-fibrin antibody clone 1101 Sequence number 6: Amino acid sequence of the light-chain CDR3 of the humanized anti-fibrin antibody clone 1101 Sequence number 7: Amino acid sequence of the heavy-chain variable region of the anti-fibrin antibody clone 1101 Sequence number 8: Amino acid sequence of the light-chain variable region of the anti-fibrin antibody clone 1101 Sequence number 9: Amino acid sequence of the heavy-chain CDR1 of the humanized anti-fibrin antibody clone 99 Sequence number 10: Amino acid sequence of the heavy-chain CDR2 of the humanized anti-fibrin antibody clone 99 Sequence number 11: Amino acid sequence of the heavy-chain CDR3 of the humanized anti-fibrin antibody clone 99 Sequence number 12: Amino acid sequence of the light-chain CDR1 of the humanized anti-fibrin antibody clone 99 Sequence number 13: Amino acid sequence of the light-chain CDR2 of the humanized anti-fibrin antibody clone 99 Sequence number 14: Amino acid sequence of the light-chain CDR3 of the humanized anti-fibrin antibody clone 99 Sequence number 15: Amino acid sequence of the heavy-chain variable region of the humanized anti-fibrin antibody clone 99 Sequence number 16: Amino acid sequence of the light-chain variable region of the humanized anti-fibrin antibody clone 99 Sequence number 17: An example of an amino acid sequence exposed in insoluble fibrin (β chain) Sequence number 18: An example of an amino acid sequence exposed in insoluble fibrin (γ chain)

Claims

**Claim 1**: An antibody or an antigen-binding fragment thereof that binds to humanized insoluble fibrin, comprising: (1) A heavy-chain variable region comprising a heavy-chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a heavy-chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy-chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and A light-chain variable region comprising a light-chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a light-chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and a light-chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 6; or (2) A heavy-chain variable region comprising a heavy-chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 9, a heavy-chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 10, and a heavy-chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 11; and A light-chain variable region comprising a light-chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 12, a light-chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 13, and a light-chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 14; An antibody or an antigen-binding fragment thereof that binds to insoluble fibrin, comprising the same. **Claim 2**: A humanized insoluble fibrin antibody or an antigen-binding fragment thereof, comprising: (3) A heavy-chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light-chain variable region having the amino acid sequence set forth in SEQ ID NO: 8; or (4) A heavy-chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light-chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, which is an antibody or an antigen-binding fragment thereof that binds to the insoluble fibrin according to Claim 1. **Claim 3**: An antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof according to Claim 1 or 2. **Claim 4**: A pharmaceutical composition comprising the antibody-drug conjugate according to Claim 3. **Claim 5** The pharmaceutical composition according to Claim 4, which is used for treating cancer. **Claim 6** A pharmaceutical composition for treating cancer, comprising an anti-cancer agent comprising the antibody or an antigen-binding fragment thereof according to Claim 1 or 2. **Claim 7**: An in vivo diagnostic agent comprising the antibody or an antigen-binding fragment thereof according to Claim 1 or 2.

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