Anti-podoplanin antibody

Abstract

To provide anti-podoplanin antibodies being humanized antibodies or mouse-human chimeric antibodies or antigen binding fragments thereof.SOLUTION: Disclosed is an isolated, humanized or mouse-human chimeric anti-podoplanin antibody comprising certain amino acid sequences or an antibody fragment including the antigen binding region thereof.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an anti-podoplanin antibody. Specifically, it relates to an anti-podoplanin antibody that is a humanized antibody or a mouse-human chimeric antibody, or an antibody fragment containing its antigen-binding region.

Background Art

[0002] Platelets aggregate to cover the cancer cell membrane, promoting resistance to shear stress in the bloodstream and avoidance of attack from immune cells, promoting the survival of metastatic cancer cells, promoting the formation of tumor masses and embolization, and further promoting the formation of metastatic foci through mechanisms such as promoting tumor growth, epithelial-mesenchymal transition (EMT), and angiogenesis (Non-Patent Documents 1, 2). On the other hand, podoplanin, a protein that induces platelet aggregation, has been identified on cancer cells, and it has been reported that podoplanin binds to CLEC-2, a receptor on platelets, via its PLAG domain, preferably the PLAG4 domain (Non-Patent Documents 1 to 4). Podoplanin is highly expressed in cancers such as mesothelioma, brain tumor, testicular tumor, Kaposi sarcoma, lymphangioma, cavernous hemangioma, angiosarcoma, lung squamous cell carcinoma, ovarian cancer undifferentiated embryonal cell tumor, glioma, embryonal cell tumor, head and neck cancer, lung cancer, bladder cancer, bone and soft tissue tumors (primary bone tumors (osteosarcoma, chondrosarcoma), soft tissue sarcoma)), esophageal cancer, etc., and it has also been reported that it enhances the invasion ability and metastasis ability of cancer cells (Non-Patent Documents 5 to ). Therefore, it is suggested that by inhibiting the binding between podoplanin on cancer cells and CLEC-2 on platelets, the formation of tumor masses and embolization can be suppressed, and further, the formation of metastatic foci can be suppressed by inhibiting tumor growth, epithelial-mesenchymal transition (EMT), and angiogenesis, and cancer-related thrombosis can be prevented (Non-Patent Documents 1, 4). In venous thrombosis, it is known that the expression of podoplanin is promoted in the blood vessel wall, and it has also been reported that the presence of thrombus causes inflammation, so prevention of thrombus is expected to prevent inflammation (Non-Patent Documents 4, 18). Furthermore, it is expected that the invasion and metastasis of the above cancers can be suppressed.

[0003] Under these circumstances, the development of new therapeutic agents that inhibit the binding of podoplanin on cancer cells to CLEC-2 on platelets is underway. One such agent is the PG4D2 antibody (IgG2a subclass), an anti-human podoplanin antibody that targets the PLAG4 domain (Non-Patent Literature 1, Patent Literature 1). This antibody has an equilibrium dissociation constant (K D This antibody exhibits extremely strong binding activity, with a binding ratio of 0.3 nM or less. Furthermore, it has been shown to suppress the incidence of podoplanin-positive tumor cell-dependent platelet aggregation and metastasis. However, the PG4D2 antibody in question is a mouse PG4D2 antibody obtained by immunizing mice, and humanized antibodies and mouse-human chimeric antibodies have not yet been established. Mouse antibodies have a relatively short half-life in vivo in humans. Furthermore, mouse antibodies are highly immunogenic to humans, which limits their therapeutic value in humans. There are concerns that long-term or repeated administration of mouse antibodies may trigger an immune response that carries the risk of undesirable allergic reactions in patients. Therefore, there is a need for humanized antibodies or mouse-human chimeric antibodies in this field. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2017 / 010463 [Non-patent literature]

[0005] [Non-Patent Document 1] Progress in Medicine, 265(1), 60-65 (2018) [Non-Patent Document 2] Journal of the Japanese Society on Thrombosis and Hemostasis, 27(1), 3-10 (2016) [Non-Patent Document 3] Journal of the Japanese Society on Thrombosis and Hemostasis, 27(1), 11-17 (2016) [Non-Patent Document 4] Journal of the Japanese Society of Thrombosis and Hemostasis, 28(4), 518-526 (2017) [Non-licensed Document 5] "Establishment of 30 osteosarcoma high-sensitivity anti-Hydritis antibodies", [January 21, 2011], インターネット<URL:https: / / www.jstage.jst.go.jp / article / jsmtmr / 30 / 0 / 30_70 / _pdf> [Non-licensed Document 6] Histopathology, Apr;46(4):396-402 (2005) [Non-licensed Document 7] Hum. Pathol., Apr;36(4):372-80 (2005) [Non-licensed Document 8] Tumour Biol., Jul-Aug;26(4):195-200 (2005) [Non-licensed Document 9] Am. J. Pathol., Mar;166(3):913-21 (2005) [Non-licensed Document 10] Acta Neuropathol., May;111(5):483-8 (2006) [Non-licensed Document 11] Cancer Cell, Apr;9(4):261-72 (2006) [Non-licensed Document 12] Acta Neuropathol., Jan;113(1):87-94 (2007) [Non-licensed Document 13] Int. J. Cancer, Jun 1;134(11):2605-14 (2014) [Non-licensed Document 14] Pathol. Int., Mar;60(3):193-202 (2010) [Non-licensed Document 15] Oncol. Rep., Mar;25(3):599-607 (2011) [Non-licensed Document 16] Am. J. Pathol., Aug;179(2):1041-9 (2011)

Non-Patent Document 17

Non-Patent Document 18

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a humanized antibody or a mouse-human chimeric antibody, which is an anti-podoplanin antibody or an antibody fragment containing an antigen-binding region thereof.

Means for Solving the Problems

[0007] As a result of intensive studies, the present inventors have found that the above problems can be solved by humanizing or mouse-human chimerizing a mouse PG4D2 antibody using a predetermined sequence, and have completed the present invention. The present invention is as follows.

[0008] The present invention is an isolated humanized antibody or mouse-human chimeric antibody, which is an anti-podoplanin antibody containing the following amino acid sequence or an antibody fragment containing an antigen-binding region thereof. However, the amino acid sequences of CDR1 to CDR3 of the heavy chain and CDR1 to CDR3 of the light chain may each be an amino acid sequence having 90% or more identity with the amino acid sequences represented by SEQ ID NOs: 1 to 6 below. The amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 1, The amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 2, The amino acid sequence of CDR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 3, The amino acid sequence of CDR1 of the light chain is the amino acid sequence of SEQ ID NO: 4, The amino acid sequence of CDR2 of the light chain is the amino acid sequence of SEQ ID NO: 5, and The amino acid sequence of the CDR3 of the light chain is the amino acid sequence of SEQ ID NO: 6.

[0009] The anti-podoplanin antibody or an antibody fragment containing its antigen-binding region is a humanized antibody, and a preferred embodiment is that the variable region contains the amino acid sequence of the following (I) or (II). However, in the following (I), the amino acid sequences of the FR1 to FR4 of the following heavy chain and the FR1 to FR4 of the light chain may each be an amino acid sequence having 90% or more identity with the amino acid sequences represented by the following SEQ ID NOs: 7 to 14. In the following (II), the amino acid sequences of the FR1 to FR4 of the following heavy chain and the FR1 to FR4 of the light chain may each be an amino acid sequence having 90% or more identity with the amino acid sequences represented by the following SEQ ID NOs: 7, 15, 16, 10 to 14. (I): The amino acid sequence of the CDR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 1, The amino acid sequence of the CDR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 2, The amino acid sequence of the CDR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 3, The amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO: 4, [[ID=②2]]The amino acid sequence of the CDR2 of the light chain is the amino acid sequence of SEQ ID NO: 5, The amino acid sequence of the CDR3 of the light chain is the amino acid sequence of SEQ ID NO: 6, The amino acid sequence of the FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 7, The amino acid sequence of the FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 8, The amino acid sequence of the FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 9, The amino acid sequence of the FR4 of the heavy chain is the amino acid sequence of SEQ ID NO: 10, The amino acid sequence of the FR1 of the light chain is the amino acid sequence of SEQ ID NO: 11, The amino acid sequence of the FR2 of the light chain is the amino acid sequence of SEQ ID NO: 12, The amino acid sequence of the FR3 of the light chain is the amino acid sequence of SEQ ID NO: 13, and The amino acid sequence of FR4 in the light chain is the same as the amino acid sequence of SEQ ID NO: 14; (II): The amino acid sequence of the heavy chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of the heavy chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of the heavy chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of the light chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 4. The amino acid sequence of the light chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 5. The amino acid sequence of the light chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of FR1 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 7. The amino acid sequence of FR2 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 15. The amino acid sequence of FR3 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 16. The amino acid sequence of FR4 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 10. The amino acid sequence of FR1 in the light chain is the same as the amino acid sequence of SEQ ID NO: 11. The amino acid sequence of FR2 in the light chain is the same as the amino acid sequence of SEQ ID NO: 12. The amino acid sequence of FR3 in the light chain is the amino acid sequence of SEQ ID NO: 13, and The amino acid sequence of FR4 in the light chain is the same as the amino acid sequence of SEQ ID NO: 14.

[0010] The anti-podoplanin antibody or the antibody fragment containing its antigen-binding region is a mouse-human chimeric antibody, and in a preferred embodiment, the variable region contains the following amino acid sequence. However, the amino acid sequences of FR1-FR4 of the heavy chain and FR1-FR4 of the light chain described below may each be amino acid sequences that have 90% or more identity with the amino acid sequences represented by SEQ ID NOs. 17-24 described below. The amino acid sequence of the heavy chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of the heavy chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of the heavy chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of the light chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 4. The amino acid sequence of the light chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 5. The amino acid sequence of the light chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of FR1 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 17. The amino acid sequence of FR2 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 18. The amino acid sequence of FR3 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 19. The amino acid sequence of FR4 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 20. The amino acid sequence of FR1 in the light chain is the same as the amino acid sequence of SEQ ID NO: 21. The amino acid sequence of FR2 in the light chain is the same as the amino acid sequence of SEQ ID NO: 22. The amino acid sequence of FR3 in the light chain is the amino acid sequence of SEQ ID NO: 23, and The amino acid sequence of FR4 in the light chain is the same as the amino acid sequence of SEQ ID NO: 24.

[0011] In a preferred embodiment, the anti-podoplanin antibody or the antibody fragment containing its antigen-binding region preferably includes a constant region of a human IgG antibody in the anti-podoplanin antibody.

[0012] Furthermore, the present invention provides a podoplanin-CLEC-2 binding inhibitor comprising the anti-podoplanin antibody or an antibody fragment containing the antigen-binding region thereof.

[0013] Furthermore, the present invention provides a pharmaceutical composition comprising the anti-podoplanin antibody or an antibody fragment containing its antigen-binding region. In a preferred embodiment, the pharmaceutical composition is intended to suppress the proliferation or metastasis of cancer cells. Furthermore, it is preferable that the pharmaceutical composition is for the purpose of inhibiting platelet aggregation, antithrombotic effects, or anti-inflammatory effects. Furthermore, the pharmaceutical composition preferably includes the fact that the cancer cells are podoplanin-positive tumor cells.

[0014] Furthermore, the present invention provides DNA encoding the protein portion of the anti-podoplanin antibody or an antibody fragment containing its antigen-binding region. Furthermore, the present invention provides a recombinant vector containing the aforementioned DNA. Furthermore, the present invention provides a host cell containing the DNA or the recombinant vector. [Effects of the Invention]

[0015] According to the present invention, it is possible to provide an anti-podoplanin antibody, which is a humanized antibody or a mouse-human chimeric antibody, or an antibody fragment containing the antigen-binding region thereof. Since the anti-podoplanin antibody or the antibody fragment containing the antigen-binding region thereof has the effect of inhibiting the binding of podoplanin on cancer cells to CLEC-2 on platelets, it is useful as a pharmaceutical composition for, for example, inhibiting the proliferation or metastasis of cancer cells, inhibiting platelet aggregation, antithrombotic, or anti-inflammatory purposes. [Brief explanation of the drawing]

[0016] [Figure 1] A graph showing the in vitro platelet aggregation inhibitory effect of mouse PG4D2 antibody in an example of the present invention. [Figure 2] A graph showing the in vitro platelet aggregation inhibitory effect of humanized antibody K4 in an example of the present invention. [Figure 3] A graph showing the in vitro platelet aggregation inhibitory effect of humanized antibody K5 in an example of the present invention. [Figure 4] A graph showing the inhibitory effect of humanized antibody K4 or K5 on hematogenous metastasis to the lungs in an example of the present invention. [Figure 5] A graph showing the antitumor effect of humanized antibody K4 or K5 in an example of the present invention. [Modes for carrying out the invention]

[0017] The present invention will be described in detail below. Furthermore, the amino acid sequences of antibodies in this specification are based on the Kabat definition.

[0018] The present invention relates to an isolated anti-podoplanin antibody, which is a humanized antibody (including a fully human antibody) or a mouse-human chimeric antibody, or an antibody fragment containing the antigen-binding region thereof, comprising the following amino acid sequence. The amino acid sequence of the heavy chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of the heavy chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of the heavy chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of the light chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 4. The amino acid sequence of CDR2 in the light chain is the amino acid sequence of SEQ ID NO: 5, and The amino acid sequence of the light chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 6.

[0019] The amino acid sequences of CDR1 to CDR3 of the heavy chain and CDR1 to CDR3 of the light chain may each have an amino acid sequence that is 90% or more, preferably 95% or more, identical to the amino acid sequences represented by Sequence ID Nos. 1 to 6. In other words, the amino acid sequences of CDR1 to CDR3 of the heavy chain and CDR1 to CDR3 of the light chain may be amino acid sequences that have 90% or more, preferably 95% or more, identity with the amino acid sequences represented by SEQ ID NOs: 1 to 6, respectively, as long as the antigen-binding activity of the anti-podoplanin antibody or antibody fragment containing the antigen-binding region thereof that they constitute is substantially the same as that of the anti-podoplanin antibody or antibody fragment containing the antigen-binding region thereof. Furthermore, the amino acid sequences of CDR1 to CDR3 of the heavy chain and CDR1 to CDR3 of the light chain may be 90% or more, preferably 95% or more, identical to the amino acid sequences represented by SEQ ID NOs: 1 to 6, respectively, as long as the anti-podoplanin antibody or antibody fragment containing its antigen-binding region has the activity to inhibit the binding of podoplanin to CLEC-2 present on platelets. An amino acid sequence having a predetermined identity with a predetermined amino acid sequence is one resulting from substitution, deletion, insertion, or addition in the predetermined amino acid sequence.

[0020] Conservative substitutions are preferred. A "conservative substitution" is the replacement of an amino acid residue with another chemically similar amino acid residue in a manner that does not substantially alter the activity of the peptide. Examples include replacing one hydrophobic residue with another hydrophobic residue, or replacing one polar residue with another polar residue having the same charge. Examples of functionally similar amino acids that can be substituted in this way include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0021] In this invention, "isolated" means not present in a living organism. For example, it does not include antibodies that are produced in an individual and present within that individual.

[0022] The "humanized antibody" of the present invention is an antibody in which the variable region consists of a complementarity-determining region derived from a known mouse PG4D2 antibody and a framework region derived from a human antibody, and the constant region consists of a constant region derived from a human antibody. The subtype of the human antibody is not particularly limited as long as the effects of the present invention are exhibited, but is preferably IgG, and more preferably IgG4. The amino acid sequence of the constant region is, for example, the amino acid sequence of the constant region of the IgG4 antibody, with the amino acid sequence of SEQ ID NO: 25 as the amino acid sequence of the heavy chain and the amino acid sequence of SEQ ID NO: 26 as the amino acid sequence of the light chain. The "humanized antibody" of the present invention may also be a "fully human antibody".

[0023] The "mouse-human chimeric antibody" of the present invention has a variable region derived from a known mouse PG4D2 antibody and a constant region derived from a human antibody. The subtype of the human antibody is not particularly limited as long as the effects of the present invention are exhibited, but is preferably IgG, and more preferably IgG4. As for the amino acid sequence of the constant region, for example, the IgG4 antibody These are amino acid sequences of the constant region, and as mentioned above, the amino acid sequence of SEQ ID NO: 25 is the amino acid sequence of the heavy chain, and the amino acid sequence of SEQ ID NO: 26 is the amino acid sequence of the light chain.

[0024] In the present invention, "antibody fragment containing an antigen-binding region" refers to a protein containing a part of an antibody that can bind to an antigen. Examples of antibody fragments include F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-bonded Fv, single-chain antibodies (scFv), and polymers thereof. The antibody fragment may be chemically or genetically engineered to functional molecules such as non-peptide polymers like polyethylene glycol (PEG), radioactive materials, toxins, small molecule compounds, cytokines, growth factors, albumin, enzymes, and other antibodies.

[0025] The anti-podoplanin antibody or the antibody fragment containing its antigen-binding region is preferably a humanized antibody, insofar as the effects of the present invention are exhibited, and the variable region preferably contains the following amino acid sequence (I) or (II). (I): The amino acid sequence of the heavy chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of the heavy chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of the heavy chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of the light chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 4. The amino acid sequence of the light chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 5. The amino acid sequence of the light chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of FR1 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 7. The amino acid sequence of FR2 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 8. The amino acid sequence of FR3 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 9. The amino acid sequence of FR4 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 10. The amino acid sequence of FR1 in the light chain is the same as the amino acid sequence of SEQ ID NO: 11. The amino acid sequence of FR2 in the light chain is the same as the amino acid sequence of SEQ ID NO: 12. The amino acid sequence of FR3 in the light chain is the amino acid sequence of SEQ ID NO: 13, and The amino acid sequence of FR4 in the light chain is the same as the amino acid sequence of SEQ ID NO: 14; (II): The amino acid sequence of the heavy chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of the heavy chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of the heavy chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of the light chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 4. The amino acid sequence of the light chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 5. The amino acid sequence of the light chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of FR1 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 7. The amino acid sequence of FR2 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 15. The amino acid sequence of FR3 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 16. The amino acid sequence of FR4 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 10. The amino acid sequence of FR1 in the light chain is the same as the amino acid sequence of SEQ ID NO: 11. The amino acid sequence of FR2 in the light chain is the same as the amino acid sequence of SEQ ID NO: 12. The amino acid sequence of FR3 in the light chain is the amino acid sequence of SEQ ID NO: 13, and The amino acid sequence of FR4 in the light chain is the same as the amino acid sequence of SEQ ID NO: 14.

[0026] In (I) above, the amino acid sequences of FR1 to FR4 of the heavy chain and FR1 to FR4 of the light chain may each be amino acid sequences that have 90% or more, preferably 95% or more, identity with the amino acid sequences represented by SEQ ID NOs: 7 to 14, and in (II) above, the amino acid sequences of FR1 to FR4 of the heavy chain and FR1 to FR4 of the light chain may each be 90% or more, preferably, identity with the amino acid sequences represented by SEQ ID NOs: 7, 15, 16, and 10 to 14. The amino acid sequences may have more than 95% identity. The details are the same as described above.

[0027] Furthermore, the anti-podoplanin antibody or the antibody fragment containing its antigen-binding region is preferably a mouse-human chimeric antibody, as long as the effects of the present invention are exhibited, as described above, and the variable region is preferably the following amino acid sequence. The amino acid sequence of the heavy chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of the heavy chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of the heavy chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of the light chain CDR1 is the same as the amino acid sequence of SEQ ID NO: 4. The amino acid sequence of the light chain CDR2 is the same as the amino acid sequence of SEQ ID NO: 5. The amino acid sequence of the light chain CDR3 is the same as the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of FR1 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 17. The amino acid sequence of FR2 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 18. The amino acid sequence of FR3 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 19. The amino acid sequence of FR4 in the heavy chain is the same as the amino acid sequence of SEQ ID NO: 20. The amino acid sequence of FR1 in the light chain is the same as the amino acid sequence of SEQ ID NO: 21. The amino acid sequence of FR2 in the light chain is the same as the amino acid sequence of SEQ ID NO: 22. The amino acid sequence of FR3 in the light chain is the amino acid sequence of SEQ ID NO: 23, and The amino acid sequence of FR4 in the light chain is the same as the amino acid sequence of SEQ ID NO: 24.

[0028] The amino acid sequences of FR1-FR4 of the heavy chain and FR1-FR4 of the light chain may each have an amino acid sequence that is 90% or more, preferably 95% or more, identical to the amino acid sequences represented by Sequence ID Nos. 17-24. The details are the same as described above.

[0029] The anti-podoplanin antibody of the present invention or an antibody fragment containing its antigen-binding region can be produced using known genetic engineering techniques based on the amino acid sequence constituting the structure. Furthermore, the manufacturing process may include a step of recovering the obtained anti-podoplanin antibody or antibody fragment containing its antigen-binding region. This recovery step may be a purification step, a concentration step, etc. In the purification step, known purification techniques can be used, and the antibody fragment containing the anti-podoplanin antibody or its antigen-binding region may be purified until it is homogeneous.

[0030] The anti-podoplanin antibody of the present invention includes a multispecific antibody, a functionally modified antibody, and a conjugate antibody. Known methods can be used to produce it.

[0031] Another aspect of the present invention is a podoplanin-CLEC-2 binding inhibitor (hereinafter sometimes referred to as "the binding inhibitor of this aspect") comprising the anti-podoplanin antibody or an antibody fragment including the antigen-binding region thereof. Furthermore, the binding inhibitor in this embodiment may be a binding-inhibiting composition. Also, the binding inhibitor and binding-inhibiting composition in this embodiment may be a mixture, and their components may be homogeneous or heterogeneous.

[0032] The binding inhibitor of this embodiment has the effect of inhibiting the binding of podoplanin to CLEC-2, and therefore has the effect obtained by inhibiting the binding of podoplanin to CLEC-2.

[0033] The binding inhibitor of this embodiment may contain one or more of the anti-podoplanin antibody or antibody fragments containing its antigen-binding region. The amount of the anti-podoplanin antibody or antibody fragment containing its antigen-binding region relative to the total amount of the binding inhibitor in this embodiment is not particularly limited as long as the binding of podoplanin to CLEC-2 is inhibited, but the total amount of the anti-podoplanin antibody or antibody fragment containing its antigen-binding region is, The concentration is typically 0.5 mg / mL or more, preferably 1 mg / mL or more, more preferably 10 mg / mL or more, and also typically 500 mg / mL or less, preferably 200 mg / mL or less, more preferably 100 mg / mL or less. Further details of the binding inhibitor in this embodiment should be referenced to the description of "a pharmaceutical composition comprising the anti-podoplanin antibody or an antibody fragment containing its antigen-binding region" described later.

[0034] Another aspect of the present invention is a pharmaceutical composition comprising the anti-podoplanin antibody or an antibody fragment containing its antigen-binding region (hereinafter sometimes referred to as "the pharmaceutical composition of this aspect"). The pharmaceutical composition of this embodiment has the effect of inhibiting the binding of podoplanin to CLEC-2, and therefore has the effects obtained by inhibiting the binding of podoplanin to CLEC-2. For example, these effects include suppression of cancer cell proliferation or metastasis, inhibition of platelet aggregation, antithrombotic effect, and anti-inflammatory effect. Therefore, the pharmaceutical composition of this embodiment can be used as a pharmaceutical composition for suppressing cancer cell proliferation or metastasis, for inhibiting platelet aggregation, for antithrombotic effects, or for anti-inflammatory effects.

[0035] The cancer cells are preferably podoplanin-positive tumor cells. Examples of podoplanin-positive tumor cells include cells from mesothelioma, brain tumors, testicular tumors, Kaposi's sarcoma, lymphangioma, cavernous hemangioma, angiosarcoma, pulmonary squamous cell carcinoma, ovarian cancer undifferentiated germ cell tumor, glioma, germ cell tumor, head and neck cancer, lung cancer, bladder cancer, bone and soft tissue tumors (primary bone tumors (osteosarcoma, chondrosarcoma), soft tissue sarcoma)), and esophageal cancer.

[0036] Therefore, the pharmaceutical composition of this embodiment can be used to prevent or treat diseases that can be prevented or treated by inhibiting the proliferation or metastasis of cancer cells, inhibiting platelet aggregation, antithrombotic, anti-inflammatory, etc. "Treatment" includes improvement. Examples of such diseases include mesothelioma, brain tumors, testicular tumors, Kaposi's sarcoma, lymphangioma, cavernous hemangioma, angiosarcoma, pulmonary squamous cell carcinoma, ovarian cancer undifferentiated germ cell tumor, glioma, germ cell tumor, head and neck cancer, lung cancer, bladder cancer, bone and soft tissue tumors (primary bone tumors (osteosarcoma, chondrosarcoma), soft tissue sarcoma)), esophageal cancer, etc.

[0037] The pharmaceutical composition of this embodiment contains the anti-podoplanin antibody or an antibody fragment containing its antigen-binding region. That is, the pharmaceutical composition of this embodiment may contain one or more of the anti-podoplanin antibody or antibody fragments containing its antigen-binding region.

[0038] The administration method of the pharmaceutical composition according to this embodiment is not particularly limited and can be administered orally or parenterally. Examples of parenteral administration include intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, intracerebral administration, intraspinal administration, and other local administration.

[0039] Dosage forms for oral and parenteral administration and methods for preparing them are well known to those skilled in the art, and pharmaceutical compositions can be manufactured by combining the anti-podoplanin antibody or an antibody fragment containing its antigen-binding region with a pharmaceutically acceptable carrier or the like.

[0040] Dosage forms for parenteral administration include injectable preparations, topical preparations, suppositories, inhaled preparations, eye preparations, eye ointments, nasal drops, ear drops, and liposomal preparations. Examples of injectable formulations include intravenous infusion formulations, intravenous injection formulations, intramuscular injection formulations, subcutaneous injection formulations, intradermal injection formulations, intracerebral administration formulations, and intraspinal administration formulations. Examples of topical preparations include ointments, poultices, and lotions. In particular, if you want to directly act on the central nervous system tissue, you can continuously infuse it using a medical micropump osmotic pump, or you can mix it with fibrin glue or other materials to create a sustained-release formulation and leave it in the affected tissue. Of these, for example, injectable formulations are usually prepared by dissolving antibodies in distilled water for injection, but may also contain solubilizers, buffers, pH adjusters, isotonic agents, analgesics, preservatives, and stabilizers as needed. These can be added. It can also be prepared as a freeze-dried formulation for immediate use.

[0041] Dosage forms for oral administration include solid or liquid forms, specifically tablets, coated tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions, injections, lozenges, and the like.

[0042] The pharmaceutical composition of this embodiment may further contain other therapeutically effective agents, and may also contain, if necessary, components such as bactericides, anti-inflammatory agents, vitamins, and amino acids.

[0043] Examples of pharmacologically acceptable carriers include excipients, lubricants, binders, and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics in liquid formulations. Furthermore, if necessary, conventional additives such as preservatives, antioxidants, colorants, sweeteners, adsorbents, and wetting agents may be used in appropriate amounts.

[0044] The dosage of the pharmaceutical composition according to this embodiment is determined by a physician based on various factors, such as the route of administration, the type of disease, the severity of symptoms, the patient's age, sex, weight, the severity of the disease, pharmacological information such as pharmacokinetic and toxicological characteristics, whether or not a drug delivery system is used, and whether or not it is administered as part of a combination of other drugs.

[0045] The pharmaceutical composition of this embodiment can be administered orally or parenterally. When administered parenterally, it can be administered by intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, endothelial infusion, local infusion, intranasal infusion, intrapulmonary infusion, and intrarectal infusion, but is not limited to these. The dosage is not particularly limited, but is usually about 0.01 μg / kg to about 1000 mg / kg per day, preferably about 0.01 mg / kg to about 100 mg / kg per day, and more preferably about 0.1 mg / kg to about 20 mg / kg per day. The pharmaceutical composition can be administered daily, once a week, once to four times a month, or once to seven times a year, but is not limited to these.

[0046] The pharmaceutical composition of this embodiment may be administered alone, or may be used in combination with other pharmaceutical compositions or pharmaceuticals, such as pharmaceutical compositions or pharmaceuticals for the prevention or treatment of skin tumors, brain tumors, testicular tumors, Kaposi's sarcoma, lymphangioma, cavernous hemangioma, angiosarcoma, pulmonary squamous cell carcinoma, ovarian cancer undifferentiated germ cell tumor, glioma, germ cell tumor, head and neck cancer, lung cancer, bladder cancer, bone and soft tissue tumors (primary bone tumors (osteosarcoma, chondrosarcoma), soft tissue sarcoma)), and esophageal cancer.

[0047] Furthermore, the present invention includes the following embodiments. [1] Use of the anti-podoplanin antibody or an antibody fragment containing the antigen-binding region thereof for producing a composition for inhibiting the binding of podoplanin to CLEC-2 or for inhibiting the binding of podoplanin to CLEC-2. [2] An anti-podoplanin antibody or an antibody fragment comprising its antigen-binding region for use in the prevention or treatment of a disease that can be prevented or treated by inhibiting the binding of podoplanin to CLEC-2. [3] A method for preventing or treating a disease that can be prevented or treated by inhibiting the binding of podoplanin to CLEC-2, comprising administering a prophylactic or therapeutically effective amount of the anti-podoplanin antibody or an antibody fragment containing its antigen-binding region to a person or patient in need of prevention or treatment. [4] Use of the anti-podoplanin antibody or an antibody fragment containing the antigen-binding region thereof for inhibiting the binding of podoplanin to CLEC-2. [5] An anti-podoplanin antibody or an antibody fragment containing its antigen-binding region, used to inhibit the binding of podoplanin to CLEC-2. [6] (i) the anti-podoplanin antibody or an antibody fragment containing its antigen-binding region, (ii A method for inhibiting the binding of podoplanin to CLEC-2, comprising the step of administering to a human being an inhibitor of the binding of podoplanin to CLEC-2, or (iii) a composition for inhibiting the binding of podoplanin to CLEC-2.

[0048] Another aspect of the present invention is DNA encoding the protein portion of the anti-podoplanin antibody or an antibody fragment containing its antigen-binding region. The base sequence of the DNA encoding the protein portion of the anti-podoplanin antibody can be easily designed by those skilled in the art based on the amino acid sequence of the protein. Specific examples include the nucleotide sequences described in the examples (SEQ ID NOs: 27-52). Furthermore, the DNA in this embodiment may have sequences encoding signal peptides or Kozak sequences added to it. The DNA of this embodiment can be produced using known genetic engineering techniques.

[0049] Another aspect of the present invention is a recombinant vector comprising the DNA. The recombinant vector in this embodiment may be any known vector, including plasmid vectors and viral vectors. A vector expressible in prokaryotic cells such as E. coli may be used, but a vector expressible in eukaryotic cells is preferred, and a vector expressible in mammalian cells is more preferred.

[0050] Another aspect of the present invention is a host cell (transformed organism) containing the DNA or the recombinant vector. The host cells in this embodiment are transformed by the introduction of the DNA or the recombinant vector. The host cells in this embodiment may be any known cell, such as prokaryotic cells like Escherichia coli or Bacillus subtilis, but eukaryotic cells are preferred, and mammalian cells are more preferred.

[0051] Examples of the host cells include CHO cells (such as CHO-S cells), and examples of vectors expressed in these cells include Freedom® pCHO 1.0 (Thermo Fisher Scientific). [Examples]

[0052] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0053] [Example 1] (Determination of the amino acid sequence of humanized antibodies that are anti-podoplanin antibodies) To create humanized antibodies by modifying mouse PG4D2 antibodies, the CDR and FR regions of the heavy and light chains of the mouse PG4D2 antibody were determined using Kabat numbering with the antibody databases IMGT and Abysis database. Next, using the IMGT database, the FR regions of human germline (GL) with high amino acid identity were selected from the sequences of the FR regions of the heavy and light chains. Using the FR shuffling method, the FR sequence with the highest amino acid identity for each of FR1-4 was selected, and the combinations of FR1-4 were determined. For each of the heavy and light chains, the complementarity-determining region of the mouse PG4D2 antibody was transplanted into the selected human germline FR region to design candidate sequences for humanized antibodies. Furthermore, the amino acid sequences of the two humanized antibodies K4 and K5 were determined based on the following considerations: whether all predetermined amino acid residues are completely conserved; whether the isoelectric point (pI) of the FR region is not excessively high; whether the frequency of use of the selected GL FR sequence is not excessively low; whether it has no chemical modification sites; whether, for amino acid residues that are not conserved compared to the mouse PG4D2 antibody, there are two or more residues with different properties from three perspectives: hydrophobicity, volume, and physicochemical properties of the amino acid side chain; and whether there is a high degree of amino acid sequence identity. In other words, the amino acid sequences of the two humanized antibodies K4 and K5 are determined based on the FR regions of the heavy and light chains of the mouse PG4D2 antibody. It was not obtained by simply substituting the amino acid sequence.

[0054] [Manufacturing Example 1] Production of humanized antibodies that are anti-podoplanin antibodies The amino acid sequences of the manufactured humanized antibodies K4 and K5 are shown below. The amino acid sequence of the humanized antibody K4 is as follows: The variable region consists of the amino acid sequence of the heavy chain CDR1: the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of the heavy chain CDR2: the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of the heavy chain CDR3: the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of the light chain CDR1: the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of the light chain CDR2: the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of the light chain CDR3: the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of the heavy chain FR1: the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of the heavy chain FR2: the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of the heavy chain FR3: the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of the heavy chain FR4: the amino acid sequence of SEQ ID NO: 10, the amino acid sequence of the light chain FR1: the amino acid sequence of SEQ ID NO: 11, the amino acid sequence of the light chain FR2: the amino acid sequence of SEQ ID NO: 12, the amino acid sequence of the light chain FR3: the amino acid sequence of SEQ ID NO: 13, and the amino acid sequence of the light chain FR4: the amino acid sequence of SEQ ID NO: 14.

[0055] The amino acid sequence of humanized antibody K5 is as follows: the variable region consists of the amino acid sequence of the heavy chain CDR1: SEQ ID NO: 1, CDR2: SEQ ID NO: 2, CDR3: SEQ ID NO: 3, CDR1: SEQ ID NO: 4, CDR2: SEQ ID NO: 5, CDR3: SEQ ID NO: 6, FR1: SEQ ID NO: 7, FR2: SEQ ID NO: 15, FR3: SEQ ID NO: 16, FR4: SEQ ID NO: 10, FR1: SEQ ID NO: 11, FR2: SEQ ID NO: 12, FR3: SEQ ID NO: 13, and FR4: SEQ ID NO: 14. The constant region is the same as the constant region amino acid sequence of humanized antibody K4.

[0056] Furthermore, the following nucleotide sequences were used as the nucleotide sequences encoding each of the above amino acid sequences. The nucleotide sequences of SEQ ID NOs. 27 to 42 were used as the nucleotide sequences encoding the amino acid sequences of SEQ ID NOs. 1 to 16, respectively. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25 (the constant region of the heavy chain) is the nucleotide sequence of SEQ ID NO: 43, and The nucleotide sequence of SEQ ID NO: 44 was used as the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 26 (the constant region of the light chain).

[0057] For expression in CHO cells (Thermo Fisher Scientific), Freedom® pCHO 1.0 (Thermo Fisher Scientific) was used as the expression vector.

[0058] After thawing, CHO cells expressing humanized antibodies K4 and K5 were cultured in large batches in a 10 L-scale fed-batch culture vessel. The culture conditions were a stirring speed of 100 rpm, a culture temperature of 37 °C, and a pH of 7.0. After 6-8 days of culture, the culture medium was collected. The recovered culture medium was purified to obtain humanized antibodies K4 and K5. The supernatant of the recovered culture medium was used for the measurements in Examples 2 and 3, which will be described later.

[0059] [Manufacturing Example 2] Production of mouse-human chimeric antibody, which is an anti-podoplanin antibody. The amino acid sequences of the mouse-human chimeric antibody chPG4D2 are shown below. The amino acid sequence of the mouse-human chimeric antibody chPG4D2 is as follows: The variable region consists of: heavy chain CDR1: amino acid sequence of SEQ ID NO: 1, heavy chain CDR2: amino acid sequence of SEQ ID NO: 2, heavy chain CDR3: amino acid sequence of SEQ ID NO: 3, light chain CDR1: amino acid sequence of SEQ ID NO: 4, light chain CDR2: amino acid sequence of SEQ ID NO: 5, light chain CDR3: amino acid sequence of SEQ ID NO: 6, heavy chain FR1: amino acid sequence of SEQ ID NO: 17, heavy chain FR2: amino acid sequence of SEQ ID NO: 18, heavy The amino acid sequences are as follows: FR3 of the main chain: amino acid sequence of SEQ ID NO: 19; FR4 of the heavy chain: amino acid sequence of SEQ ID NO: 20; FR1 of the light chain: amino acid sequence of SEQ ID NO: 21; FR2 of the light chain: amino acid sequence of SEQ ID NO: 22; FR3 of the light chain: amino acid sequence of SEQ ID NO: 23; and FR4 of the light chain: amino acid sequence of SEQ ID NO: 24. The constant region is identical to the amino acid sequence of the constant region of the humanized antibody K4 described above.

[0060] Those skilled in the art will readily understand that the mouse-human chimeric antibody chPG4D2 can be manufactured in the same manner as in Production Example 1 by designing each nucleotide that codes for each amino acid sequence of Sequence IDs 1-6 and 17-24 and each amino acid sequence of the constant region of the humanized antibody K1. In this case, the following nucleotide sequences can be used as the nucleotide sequences that encode each of the above amino acid sequences. The nucleotide sequences of SEQ ID NOs. 27-32 and 45-52 can be used as the nucleotide sequences encoding the amino acid sequences of SEQ ID NOs. 1-6 and 17-24, respectively. Furthermore, the nucleotide sequence encoding the amino acid sequence of the constant region can be the same as that described in Production Example 1.

[0061] For expression in CHO cells (Thermo Fisher Scientific), Freedom® pCHO 1.0 (Thermo Fisher Scientific) was used as the expression vector.

[0062] CHO cells expressing the mouse-human chimeric antibody chPG4D2 were thawed, expanded, and then batch cultured in a 5 L-scale cell culture vessel. The culture conditions were: stirring speed 100 rpm, culture temperature 37°C. The culture medium was collected after being incubated at °C and pH 7.0 for 7 days. The collected culture medium was purified to obtain the mouse-human chimeric antibody chPG4D2. The supernatant of the collected culture medium was used for the measurements in Examples 2 and 3, which will be described later.

[0063] [Example 2] Podoplanin-binding activity of each antibody The binding activity to the podoplanin PLAG4 domain was measured using the supernatant of the culture medium recovered in Production Example 1 and Production Example 2, by a method similar to the ELISA method described in Patent Document 1. Human podoplanin PLAG4 domain peptide (WT-hPLAG4 (Genscript, 788349-1)) was immobilized as the antigen peptide, and Goat Anti-Human IgG Fc (Biotin) preadsorbed (Abcam, ab98618) and Streptavidin β-Gal conjugate (Roche Diagnostics, 11112481001) were used for detection. The results are shown in Table 1. Note that binding activity (antibody titer) is expressed with the mouse-human chimeric antibody chPG4D2 as 100%.

[0064] [Table 1]

[0065] [Example 3] Inhibitory activity of each antibody in binding CLEC-2 to podoplanin The inhibitory activity of CLEC-2 binding to podoplanin was measured using the culture supernatants obtained in Production Example 1 and Production Example 2, according to the ELISA method described in Patent Document 1. Recombinant Human CLEC-2 (R&D, 1718-CL-050) was immobilized as the antigen peptide, and the protein that binds to it was measured. Recombinant Human Podoplanin Fc Chimera (R&D, 3670-PL) was used as the reagent, and Goat Anti-Human IgG Fc (Biotin) preadsorbed (Abcam, ab98618) and Streptavidin-β-galactosidase conjugate (SIGMA, S3887, Roche, 11112481001) were used for detection. Furthermore, mouse PG4D2 antibody (PG4D2) was used as a positive control. Mouse PG4D2 antibody is a monoclonal antibody obtained from the hybridoma of NITE P-02071 (PG4D2). The results are shown in Table 2.

[0066] [Table 2]

[0067] [Example 4] In vitro platelet aggregation inhibitory effect of each antibody It is known that introducing the human podoplanin gene into CHO cells, a cell line that does not induce platelet aggregation, can induce platelet aggregation (J. Biol. Chem., Dec 19;278(51):51599-605 (2003)). Human podoplanin-positive osteosarcoma cell line SJSA-1 (ATCC CRL-2098) was added to human whole blood, and the suppression of induced platelet aggregation by each antibody was analyzed using a whole blood platelet aggregation analyzer, WBA CARNA (Taiyo). The osteosarcoma cell line SJSA-1 (ATCC CRL-2098) is from the American Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852). It can be obtained from the United States of America. Furthermore, human IgG4 antibody (CrownBio, C0004) was used as a negative control in the systems using humanized antibodies K4 and K5. Furthermore, this in vitro analysis system calculates the agglutination rate by aspirating whole blood containing cell-induced agglutinations through a micromesh filter and measuring the increased suction pressure due to clogging by the agglutins. Mouse PG4D2 antibody (PG4D2) was used as a positive control. Mouse IgG2a antibody (Sigma, M9144) was used as a negative control in the system using mouse PG4D2 antibody. The results are shown in Figures 1 to 3. Humanized antibodies K4 and K5 inhibited platelet aggregation in a concentration-dependent manner, similar to the positive control.

[0068] [Example 5] Inhibitory effect of each antibody on hematogenous transfer to the lungs Human podoplanin-positive osteosarcoma cell line SJSA-1 (ATCC CRL-2098) was used in severe combined immunodeficiency mouse SCID-Beige (CB17.Cg-Prkdc scid Lyst bg-J The hematogenous metastasis inhibitory effect of each antibody was analyzed using a hematogenous metastasis model in which the antibody was injected into the tail vein of a 6-week-old female Charles River hamster (CrlCrlj) and the number of metastatic nodules formed on the lung surface 20 days later was measured. On the day before SJSA-1 cell injection, a control antibody (human IgG4 antibody (CrownBio)), humanized antibody K4, or K5 was administered to 6 mice in each group via the tail vein to examine its effect on podoplanin-dependent experimental lung metastasis. The results are shown in Figure 4. Prior administration of humanized antibodies K4 or K5 significantly suppressed lung metastasis of the SJSA-1 cell line.

[0069] [Example 6] Antitumor effect of each antibody 5×10 chromosomes positive for human podoplanin 6 Individual osteosarcoma cell lines SJSA-1 (ATCC CRL-2098) were used in severe combined immunodeficiency mice SCID-Beige (CB17.Cg-Prkdc scid Lyst bg-J Xenograft mice were created by subcutaneous transplantation of humanized antibodies K4 and K5 (Charles River, Japan, female, 5 weeks old) and their in vivo antitumor effects were evaluated. The K4-administered group and the K5-administered group were each given humanized antibodies K4 and K5, respectively, at a dose of 5 mg / kg twice a week (days 1, 4, 8, 11, and 15) starting from day 1 of transplantation. Tumor size (1 / 2 long diameter × (short diameter)) was measured twice a week. 2 Weight and body weight were measured (n=6). At the endpoint (20 days later), photographs were taken and the weight of the removed tumor was measured. Furthermore, a human IgG4 antibody (Sino Biological, HG4K) was used as a negative control. The results (relationship between the number of days after transplantation and the mean tumor size) are shown in Figure 5 (Kruskal-Wallis test: * p < 0.05, ** p < 0.01). Tumor growth inhibition was observed in both the K4 and K5 administration groups. No significant weight loss was observed in mice during this study.

Claims

1. A humanized antibody, an isolated anti-podoplanin antibody or an antibody fragment containing its antigen-binding region, wherein the variable region contains the amino acid sequence (I) or (II) below. However, in (I) below, the amino acid sequences of FR1 to FR4 of the heavy chain and FR1 to FR4 of the light chain may each be amino acid sequences that have 90% or more identity with the amino acid sequences represented by SEQ ID NOs: 7 to 14 below. In (II) below, the amino acid sequences of FR1 to FR4 of the heavy chain and FR1 to FR4 of the light chain may be amino acid sequences that have 90% or more identity with the amino acid sequences represented by SEQ ID NOs: 7, 15, 16, and 10 to 14 below. (I): The amino acid sequence of the heavy chain CDR1 is the same as the amino acid sequence of SEQ ID NO:

1. The amino acid sequence of the heavy chain CDR2 is the same as the amino acid sequence of SEQ ID NO:

2. The amino acid sequence of the heavy chain CDR3 is the same as the amino acid sequence of SEQ ID NO:

3. The amino acid sequence of CDR1 in the light chain is the same as the amino acid sequence of SEQ ID NO:

4. The amino acid sequence of CDR2 in the light chain is the same as the amino acid sequence of SEQ ID NO:

5. The amino acid sequence of CDR3 in the light chain is the same as the amino acid sequence of SEQ ID NO:

6. The amino acid sequence of FR1 in the heavy chain is the same as the amino acid sequence of SEQ ID NO:

7. The amino acid sequence of FR2 in the heavy chain is the amino acid sequence of SEQ ID NO:

8. The amino acid sequence of FR3 in the heavy chain is the same as the amino acid sequence of SEQ ID NO:

9. The amino acid sequence of FR4 in the heavy chain is the same as the amino acid sequence of SEQ ID NO:

10. The amino acid sequence of FR1 in the light chain is the same as the amino acid sequence of SEQ ID NO:

11. The amino acid sequence of FR2 in the light chain is the same as the amino acid sequence of SEQ ID NO:

12. The amino acid sequence of FR3 in the light chain is the amino acid sequence of SEQ ID NO: 13, and The amino acid sequence of FR4 in the light chain is the same as the amino acid sequence of SEQ ID NO: 14; (II): The amino acid sequence of the heavy chain CDR1 is the same as the amino acid sequence of SEQ ID NO:

1. The amino acid sequence of the heavy chain CDR2 is the same as the amino acid sequence of SEQ ID NO:

2. The amino acid sequence of the heavy chain CDR3 is the same as the amino acid sequence of SEQ ID NO:

3. The amino acid sequence of CDR1 in the light chain is the same as the amino acid sequence of SEQ ID NO:

4. The amino acid sequence of CDR2 in the light chain is the same as the amino acid sequence of SEQ ID NO:

5. The amino acid sequence of CDR3 in the light chain is the same as the amino acid sequence of SEQ ID NO:

6. The amino acid sequence of FR1 in the heavy chain is the same as the amino acid sequence of SEQ ID NO:

7. The amino acid sequence of FR2 in the heavy chain is the same as the amino acid sequence of SEQ ID NO:

15. The amino acid sequence of FR3 in the heavy chain is the same as the amino acid sequence of SEQ ID NO:

16. The amino acid sequence of FR4 in the heavy chain is the same as the amino acid sequence of SEQ ID NO:

10. The amino acid sequence of FR1 in the light chain is the same as the amino acid sequence of SEQ ID NO:

11. The amino acid sequence of FR2 in the light chain is the same as the amino acid sequence of SEQ ID NO:

12. The amino acid sequence of FR3 in the light chain is the amino acid sequence of SEQ ID NO: 13, and The amino acid sequence of FR4 in the light chain is the same as the amino acid sequence of SEQ ID NO:

14.

2. The anti-podoplanin antibody according to claim 1, or an antibody fragment comprising the antigen-binding region thereof, wherein the anti-podoplanin antibody comprises a constant region of a human IgG antibody.

3. A podoplanin-CLEC-2 binding inhibitor comprising an anti-podoplanin antibody according to claim 1 or 2, or an antibody fragment comprising the antigen-binding region thereof.

4. A pharmaceutical composition comprising an anti-podoplanin antibody according to claim 1 or 2, or an antibody fragment comprising the antigen-binding region thereof.

5. The pharmaceutical composition according to claim 4 for suppressing the proliferation or metastasis of cancer cells.

6. The pharmaceutical composition according to claim 4 for inhibiting platelet aggregation, antithrombotic effects, or anti-inflammatory effects.

7. The pharmaceutical composition according to claim 5, wherein the cancer cells are podoplanin-positive tumor cells.

8. DNA encoding the protein portion of an anti-podoplanin antibody or an antibody fragment containing the antigen-binding region thereof, as described in claim 1 or 2.

9. A recombinant vector comprising the DNA described in claim 8.

10. A host cell comprising the DNA described in claim 8 or the recombinant vector described in claim 9.

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