Anti-podoprotein antibody
阅读说明：本技术 抗平足蛋白抗体 (Anti-podoprotein antibody ) 是由 刑部伸彦 藤田直也 柿野卫 川岛爱 藤原伸哉 合田直树 于 2019-06-14 设计创作，主要内容包括：本发明的课题在于,提供作为人源化抗体或小鼠-人嵌合抗体的抗平足蛋白抗体、或包含其抗原结合区域的抗体片段,通过一种抗平足蛋白抗体、或包含其抗原结合区域的抗体片段来解决该课题,所述抗平足蛋白抗体、或包含其抗原结合区域的抗体片段为包含规定的氨基酸序列的、经分离的人源化抗体或小鼠-人嵌合抗体。(The present invention addresses the problem of providing an anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof, which is a humanized antibody or a mouse-human chimeric antibody, and solves the problem by providing an anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof, which is an isolated humanized antibody or a mouse-human chimeric antibody containing a predetermined amino acid sequence.)
1. An anti-podophyllin antibody, or an antibody fragment containing an antigen-binding region thereof, which is an isolated humanized antibody or a mouse-human chimeric antibody comprising an amino acid sequence,
wherein the amino acid sequences of CDR 1-CDR 3 of the heavy chain and CDR 1-CDR 3 of the light chain are respectively selected from amino acid sequences having 90% identity with the amino acid sequences shown in the sequence numbers 1-6,
the amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence 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 CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6.
2. The anti-podoprotein antibody of claim 1, or an antibody fragment comprising an antigen binding region thereof, that is a humanized antibody and the variable region comprises the amino acid sequence of (I) or (II),
wherein in the following (I), the amino acid sequences of FR 1-FR 4 of the heavy chain and FR 1-FR 4 of the light chain are respectively selected from amino acid sequences having 90% or more identity with the amino acid sequences represented by the following sequence Nos. 7-14,
in the following (II), the amino acid sequences of FR 1-FR 4 of the heavy chain and FR 1-FR 4 of the light chain are each optionally an amino acid sequence having 90% or more identity to the amino acid sequences represented by the following SEQ ID Nos. 7, 15, 16, 10-14,
(I)：
the amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence No. 4,
the amino acid sequence of CDR2 of the light chain is the amino acid sequence of SEQ ID NO. 5,
the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6,
the amino acid sequence of FR1 of heavy chain is the amino acid sequence of SEQ ID NO. 7,
the amino acid sequence of FR2 of heavy chain is the amino acid sequence of SEQ ID NO. 8,
the amino acid sequence of FR3 of heavy chain is the amino acid sequence of SEQ ID NO. 9,
the amino acid sequence of FR4 of heavy chain is the amino acid sequence of SEQ ID NO. 10,
the amino acid sequence of FR1 of light chain is the amino acid sequence of SEQ ID NO. 11,
the amino acid sequence of FR2 of light chain is the amino acid sequence of SEQ ID NO. 12,
the amino acid sequence of FR3 of light chain is the amino acid sequence of SEQ ID NO. 13, and
the amino acid sequence of FR4 of the light chain is the amino acid sequence of seq id No. 14;
(II)：
the amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence No. 4,
the amino acid sequence of CDR2 of the light chain is the amino acid sequence of SEQ ID NO. 5,
the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6,
the amino acid sequence of FR1 of heavy chain is the amino acid sequence of SEQ ID NO. 7,
the amino acid sequence of FR2 of heavy chain is the amino acid sequence of SEQ ID NO. 15,
the amino acid sequence of FR3 of heavy chain is the amino acid sequence of SEQ ID NO. 16,
the amino acid sequence of FR4 of heavy chain is the amino acid sequence of SEQ ID NO. 10,
the amino acid sequence of FR1 of light chain is the amino acid sequence of SEQ ID NO. 11,
the amino acid sequence of FR2 of light chain is the amino acid sequence of SEQ ID NO. 12,
the amino acid sequence of FR3 of light chain is the amino acid sequence of SEQ ID NO. 13, and
the amino acid sequence of FR4 of light chain is the amino acid sequence of SEQ ID NO. 14.
3. The anti-podophyllin antibody according to claim 1, or an antibody fragment comprising an antigen-binding region thereof, which is a mouse-human chimeric antibody and the variable region comprises an amino acid sequence,
wherein the amino acid sequences of FR 1-FR 4 of the heavy chain and FR 1-FR 4 of the light chain are respectively selected from amino acid sequences having 90% or more identity with the amino acid sequences represented by the following SEQ ID NO. 17-24,
the amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence No. 4,
the amino acid sequence of CDR2 of the light chain is the amino acid sequence of SEQ ID NO. 5,
the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6,
the amino acid sequence of FR1 of heavy chain is the amino acid sequence of SEQ ID NO. 17,
the amino acid sequence of FR2 of heavy chain is the amino acid sequence of SEQ ID NO. 18,
the amino acid sequence of FR3 of heavy chain is the amino acid sequence of SEQ ID NO. 19,
the amino acid sequence of FR4 of heavy chain is the amino acid sequence of SEQ ID NO. 20,
the amino acid sequence of FR1 of light chain is the amino acid sequence of SEQ ID NO. 21,
the amino acid sequence of FR2 of light chain is the amino acid sequence of SEQ ID NO. 22,
the amino acid sequence of FR3 of light chain is the amino acid sequence of SEQ ID NO. 23, and
the amino acid sequence of FR4 of light chain is the amino acid sequence of SEQ ID NO. 24.
4. The anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof according to any one of claims 1 to 3, wherein the anti-podophyllin antibody comprises a constant region of a human IgG antibody.
5. An inhibitor of binding of podoprotein to CLEC-2 comprising: the anti-podophyllin antibody according to any one of claims 1 to 4, or an antibody fragment containing an antigen-binding region thereof.
6. A pharmaceutical composition comprising: the anti-podophyllin antibody according to any one of claims 1 to 4, or an antibody fragment containing an antigen-binding region thereof.
7. The pharmaceutical composition according to claim 6, for use in inhibiting proliferation or metastasis of cancer cells.
8. The pharmaceutical composition according to claim 6, for use in inhibiting platelet aggregation, anti-thrombosis, or anti-inflammation.
9. The pharmaceutical composition of claim 7, wherein the cancer cell is a podophyllin positive tumor cell.
10. A DNA encoding a protein portion of the anti-podophyllin antibody according to any one of claims 1 to 4, or an antibody fragment comprising an antigen-binding region thereof.
11. A recombinant vector comprising the DNA of claim 10.
12. A host cell comprising the DNA of claim 10 or the recombinant vector of claim 11.
Technical Field
The present invention relates to anti-podophyllin antibodies. More specifically, the present invention relates to an anti-podophyllin antibody which is a humanized antibody or a mouse-human chimeric antibody, or an antibody fragment containing an antigen binding region thereof.
Background
It has been reported that platelets aggregate and cover the cell membrane of cancer cells, thereby contributing to resistance to shear stress of blood flow, evading attack from immune cells, promoting survival of metastatic cancer cells, promoting formation of tumor mass and embolization, and further accelerating formation of metastatic foci by promoting mechanisms such as tumor proliferation, epithelial-mesenchymal transition (EMT), angiogenesis, and the like (non-patent documents 1 and 2).
On the other hand, it is reported that: identified on cancer cells is a podoprotein that is a protein that induces platelet aggregation, and binds CLEC-2, a receptor on platelets, via its PLAG domain, preferably the PLAG4 domain (non-patent documents 1 to 4).
There are also reports that indicate: the podophyllotoxin is highly expressed in cancers such as mesothelioma, brain tumor, testicular tumor, kaposi's sarcoma, lymphangioma, cavernous hemangioma, angiosarcoma, lung squamous carcinoma, ovarian carcinoma dysgerminoma, glioma, germ cell tumor, head and neck cancer, lung cancer, bladder cancer, bone and soft tissue tumor (primary bone tumor (osteosarcoma, chondrosarcoma), soft tissue sarcoma), esophageal cancer, etc., and enhances infiltration ability and metastatic ability of cancer cells (non-patent documents 5 to 17).
Therefore, it is suggested that the inhibition of the binding of the podoprotein on cancer cells to CLEC-2 on platelets can inhibit the formation of tumor mass and embolism, and can inhibit tumor proliferation and epithelial-mesenchymal transition (EMT), and inhibit the formation of metastasis by inhibiting angiogenesis, thereby preventing cancer-related thrombosis (non-patent documents 1 and 4). It is known that, in venous thrombosis, the expression of podophyllin is promoted in the vascular wall, and it is also reported that inflammation is caused by the presence of thrombus, and therefore prevention of inflammation is also expected by prevention of thrombus (non-patent documents 4 and 18). Furthermore, it is expected that the infiltration and metastasis of the cancer can be suppressed.
Under such circumstances, development of a novel therapeutic agent for inhibiting binding of podophyllin on cancer cells to CLEC-2 on platelets has been carried out. As one of them, PG4D2 antibody (IgG2a subclass) as an anti-human podophyllin antibody targeting the PLAG4 domain was established (non-patent document 1, patent document 1). Equilibrium dissociation constant (K) of the antibodyD) An antibody having a binding activity of 0.3nM or less was found to be very strong, and the activity of inhibiting the incidence of platelet aggregation and metastasis dependent on podophyllin-positive tumor cells was also confirmed for this antibody.
However, this PG4D2 antibody is a mouse PG4D2 antibody obtained by immunizing a mouse, and a humanized antibody and a mouse-human chimeric antibody are not established. Mouse antibodies have a short half-life in humans. In addition, mouse antibodies are highly immunogenic to humans, which limits their therapeutic value in humans. There is a fear that if a mouse antibody is administered for a long period or a plurality of times, it will cause an immune response which brings an undesirable risk of allergy to a patient. Thus, there is a need in the art for humanized or mouse-human chimeric antibodies.
Documents of the prior art
Patent document
Patent document 1: international publication No. 2017/010463
Non-patent document
Non-patent document 1: medicine 'Tao' あゆみ, 265(1),60-65(2018)
Non-patent document 2: zhi of Japan society for thrombosis and hemostasis, 27(1),3-10(2016)
Non-patent document 3: zhi of Japan society for thrombosis and hemostasis, 27(1),11-17(2016)
Non-patent document 4: zhi of Japan society for thrombosis and hemostasis, 28(4),518-
Non-patent document 5: 30 osteosarcoma gene に, , which is a する high-sensitivity anti- ポドプラニン antibody (establishment of an antibody against high-sensitivity antipodactylin against osteosarcoma), [ 31 years, 1 month, 21 days, search ], internet < URL: https:// www.jstage.jst.go.jp/article/jsmtmr/30/0/30_70/_ pdf >)
Non-patent document 6: histopathology, Apr; 46(4):396-402(2005)
Non-patent document 7: hum. pathol, Apr; 36(4):372-80(2005)
Non-patent document 8: tumour biol., Jul-Aug; 26(4):195-200(2005)
Non-patent document 9: am.j.pathol., Mar; 166(3):913-21(2005)
Non-patent document 10: acta neuropathohol, May; 111(5):483-8(2006)
Non-patent document 11: cancer Cell, Apr; 9(4):261-72(2006)
Non-patent document 12: acta neuropathol, Jan; 113(1):87-94(2007)
Non-patent document 13: int.j.cancer, Jun 1; 134(11):2605-14(2014)
Non-patent document 14: int, Mar; 60(3):193-202(2010)
Non-patent document 15: rep., Mar; 25(3):599-607(2011)
Non-patent document 16: am.j.pathol., Aug; 179(2):1041-9(2011)
Non-patent document 17: monoclon.Antib.Immunodiagn.Immunotherer, Jun; 34(3):154-61(2015)
Non-patent document 18: blood, Apr 6; 129(14):1896-1898(2017)
Disclosure of Invention
Problems to be solved by the invention
The present invention addresses the problem of providing an anti-podoprotein antibody that is a humanized antibody or a mouse-human chimeric antibody, or an antibody fragment that contains the antigen binding region thereof.
Means for solving the problems
The present inventors have conducted intensive studies and, as a result, have found that the above problems can be solved by humanizing a mouse PG4D2 antibody or by mouse-human chimerizing it using a predetermined sequence, and have completed the present invention.
The present invention is as follows.
The present invention is an anti-podophyllin antibody, or an antibody fragment containing an antigen-binding region thereof, which is an isolated humanized antibody or a mouse-human chimeric antibody containing the following amino acid sequence.
Wherein the amino acid sequences of CDR 1-CDR 3 of the heavy chain and CDR 1-CDR 3 of the light chain are respectively selected from amino acid sequences having 90% or more identity with the amino acid sequences shown in the following sequence numbers 1-6.
The amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence 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 CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6.
Preferred embodiments of the anti-podophyllin antibody or the antibody fragment including the antigen binding region thereof are as follows: it is a humanized antibody and the variable region comprises the amino acid sequence of the following (I) or (II).
Wherein in the following (I), the amino acid sequences of FR 1-FR 4 of the heavy chain and FR 1-FR 4 of the light chain are respectively selected from amino acid sequences having 90% or more identity with the amino acid sequences represented by the following sequence Nos. 7-14,
in the following (II), the amino acid sequences of FR 1-FR 4 of the heavy chain and FR 1-FR 4 of the light chain are optionally amino acid sequences having 90% or more identity to the amino acid sequences represented by the following SEQ ID Nos. 7, 15, 16, 10-14, respectively.
(I)：
The amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence No. 4,
the amino acid sequence of CDR2 of the light chain is the amino acid sequence of SEQ ID NO. 5,
the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6,
the amino acid sequence of FR1 of heavy chain is the amino acid sequence of SEQ ID NO. 7,
the amino acid sequence of FR2 of heavy chain is the amino acid sequence of SEQ ID NO. 8,
the amino acid sequence of FR3 of heavy chain is the amino acid sequence of SEQ ID NO. 9,
the amino acid sequence of FR4 of heavy chain is the amino acid sequence of SEQ ID NO. 10,
the amino acid sequence of FR1 of light chain is the amino acid sequence of SEQ ID NO. 11,
the amino acid sequence of FR2 of light chain is the amino acid sequence of SEQ ID NO. 12,
the amino acid sequence of FR3 of light chain is the amino acid sequence of SEQ ID NO. 13, and
the amino acid sequence of FR4 of the light chain is the amino acid sequence of seq id No. 14;
(II)：
the amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence No. 4,
the amino acid sequence of CDR2 of the light chain is the amino acid sequence of SEQ ID NO. 5,
the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6,
the amino acid sequence of FR1 of heavy chain is the amino acid sequence of SEQ ID NO. 7,
the amino acid sequence of FR2 of heavy chain is the amino acid sequence of SEQ ID NO. 15,
the amino acid sequence of FR3 of heavy chain is the amino acid sequence of SEQ ID NO. 16,
the amino acid sequence of FR4 of heavy chain is the amino acid sequence of SEQ ID NO. 10,
the amino acid sequence of FR1 of light chain is the amino acid sequence of SEQ ID NO. 11,
the amino acid sequence of FR2 of light chain is the amino acid sequence of SEQ ID NO. 12,
the amino acid sequence of FR3 of light chain is the amino acid sequence of SEQ ID NO. 13, and
the amino acid sequence of FR4 of light chain is the amino acid sequence of SEQ ID NO. 14.
Preferred embodiments of the anti-podophyllin antibody or the antibody fragment including the antigen binding region thereof are as follows: it is a mouse-human chimeric antibody and the variable region comprises the following amino acid sequence.
Wherein the amino acid sequences of FR 1-FR 4 of the heavy chain and FR 1-FR 4 of the light chain are each optionally an amino acid sequence having 90% or more identity to the amino acid sequences represented by the following SEQ ID Nos. 17-24.
The amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence No. 4,
the amino acid sequence of CDR2 of the light chain is the amino acid sequence of SEQ ID NO. 5,
the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6,
the amino acid sequence of FR1 of heavy chain is the amino acid sequence of SEQ ID NO. 17,
the amino acid sequence of FR2 of heavy chain is the amino acid sequence of SEQ ID NO. 18,
the amino acid sequence of FR3 of heavy chain is the amino acid sequence of SEQ ID NO. 19,
the amino acid sequence of FR4 of heavy chain is the amino acid sequence of SEQ ID NO. 20,
the amino acid sequence of FR1 of light chain is the amino acid sequence of SEQ ID NO. 21,
the amino acid sequence of FR2 of light chain is the amino acid sequence of SEQ ID NO. 22,
the amino acid sequence of FR3 of light chain is the amino acid sequence of SEQ ID NO. 23, and
the amino acid sequence of FR4 of light chain is the amino acid sequence of SEQ ID NO. 24.
Preferred embodiments of the anti-podophyllin antibody or the antibody fragment including the antigen binding region thereof are as follows: the anti-podophyllin antibody described above contains the constant region of a human IgG antibody.
In addition, the present invention provides an inhibitor of binding of pellitorin to CLEC-2, comprising: the anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof.
In addition, the present invention provides a pharmaceutical composition comprising: the anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof.
The preferable modes of the pharmaceutical composition are as follows: it is used for inhibiting proliferation or metastasis of cancer cells.
In addition, the preferable mode of the pharmaceutical composition is as follows: can be used for inhibiting platelet aggregation, resisting thrombosis, or resisting inflammation.
In addition, the preferable mode of the pharmaceutical composition is as follows: the cancer cell is a tumor cell positive for pellegumin.
The present invention also provides a DNA encoding a protein portion of the above-described anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof.
In addition, the present invention provides a recombinant vector comprising the above DNA.
In addition, the present invention provides a host cell comprising the above DNA or the above recombinant vector.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, an anti-podophyllin antibody which is a humanized antibody or a mouse-human chimeric antibody, or an antibody fragment containing an antigen-binding region thereof can be provided. The anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof has an effect of inhibiting binding between podophyllin on cancer cells and CLEC-2 on platelets, and is therefore useful, for example, as a pharmaceutical composition for inhibiting proliferation or metastasis of cancer cells, inhibiting platelet aggregation, and inhibiting thrombosis or inflammation.
Drawings
Fig. 1 is a graph showing the effect of inhibiting platelet aggregation in vitro using the mouse PG4D2 antibody in an example of the present invention.
Fig. 2 is a graph showing the effect of inhibiting platelet aggregation in vitro using humanized antibody K4 in the examples of the present invention.
Fig. 3 is a graph showing the effect of in vitro inhibition of platelet aggregation using humanized antibody K5 in an example of the present invention.
Fig. 4 is a graph showing the effect of inhibiting a haematopoietic lung metastasis to the lung using humanized antibodies K4 or K5 in an example of the present invention.
Fig. 5 is a graph showing the antitumor effect using the humanized antibody K4 or K5 in the example of the present invention.
Detailed Description
The present invention will be described in detail below.
The amino acid sequence of the antibody in the present specification is defined based on Kabat.
The present invention is an anti-podophyllin antibody, or an antibody fragment containing an antigen-binding region thereof, which is an isolated humanized antibody (including fully humanized antibody) or a mouse-human chimeric antibody comprising the following amino acid sequence.
The amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence 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 CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6.
The amino acid sequences of the CDRs 1-3 of the heavy chain and the CDRs 1-3 of the light chain are respectively selected from amino acid sequences having 90% or more, preferably 95% or more identity with the amino acid sequences represented by the sequence numbers 1-6.
That is, the amino acid sequences of the CDRs 1 to 3 of the heavy chain and the CDRs 1 to 3 of the light chain may be amino acid sequences having 90% or more, preferably 95% or more identity to the amino acid sequences represented by the sequence numbers 1 to 6, respectively, as long as the antigen binding activity of the anti-podophyllin antibody or the antibody fragment containing the antigen binding region thereof, which is constituted by these amino acid sequences, is substantially the same as that of the anti-podophyllin antibody or the antibody fragment containing the antigen binding region thereof.
The amino acid sequences of the CDRs 1 to 3 of the heavy chain and the CDRs 1 to 3 of the light chain may be amino acid sequences having 90% or more, preferably 95% or more identity to the amino acid sequences represented by the sequence nos. 1 to 6, respectively, as long as the anti-podophyllin antibody or an antibody fragment containing an antigen binding region thereof has an activity of inhibiting the binding between the podophyllin and CLEC-2 present on platelets.
An amino acid sequence having a defined identity to a defined amino acid sequence is: an amino acid sequence formed by substitution, deletion, insertion or addition to the predetermined amino acid sequence.
The substitution is preferably a conservative substitution. "conservative substitutions" refer to: the amino acid residues are substituted with other chemically similar amino acid residues in a manner that does not substantially alter the activity of the peptide. Examples thereof include: the case where a certain hydrophobic residue is substituted with another hydrophobic residue; the case where a certain polar residue is substituted with another polar residue having the same charge; and the like. 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 the polar (neutral) amino acid include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of the (basic) amino acid having a positive charge include arginine, histidine, and lysine. Examples of the (acidic) amino acid having a negative charge include aspartic acid and glutamic acid.
The term "isolated" in the context of the present invention means: not in the organism. For example, antibodies produced by an individual and present in that individual are not included.
The "humanized antibody" of the present invention is an antibody in which the variable region is composed 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 is composed of a constant region derived from a human antibody. The subtype of the human antibody is not particularly limited as long as the effect of the present invention can be exerted, and is preferably IgG, and more preferably IgG 4. The amino acid sequence of the constant region is, for example, the amino acid sequence of the constant region of the IgG4 antibody, the amino acid sequence of the heavy chain thereof is the amino acid sequence of sequence No. 25, and the amino acid sequence of the light chain thereof is the amino acid sequence of sequence No. 26. The "humanized antibody" of the present invention may be a "fully humanized antibody".
In the "mouse-human chimeric antibody" of the present invention, the variable region is a variable region derived from a known mouse PG4D2 antibody, and the constant region is a constant region derived from a human antibody. The subtype of the human antibody is not particularly limited as long as the effect of the present invention can be exerted, and is preferably IgG, and more preferably IgG 4. The amino acid sequence of the constant region is, for example, the amino acid sequence of the constant region of the IgG4 antibody, and as described above, the amino acid sequence of the heavy chain includes the amino acid sequence of sequence No. 25, and as the amino acid sequence of the light chain includes the amino acid sequence of sequence No. 26.
The "antibody fragment comprising an antigen-binding region" in the present invention means: a protein comprising a portion of an antibody and capable of binding to an antigen. Examples of the antibody fragment include F (ab')2Fab', Fab, Fv (variable fragment of antibody),Disulfide bond antibody (disulfide-bond Fv)Single-chain antibodies (scFv), and polymers thereof. The antibody fragment may be chemically or genetically coupled with functional molecules such as non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors, albumin, enzymes, and other antibodies.
The anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof is preferably a humanized antibody as long as the effect of the present invention can be exerted, and the variable region preferably contains the amino acid sequence of the following (I) or (II).
(I)：
The amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence No. 4,
the amino acid sequence of CDR2 of the light chain is the amino acid sequence of SEQ ID NO. 5,
the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6,
the amino acid sequence of FR1 of heavy chain is the amino acid sequence of SEQ ID NO. 7,
the amino acid sequence of FR2 of heavy chain is the amino acid sequence of SEQ ID NO. 8,
the amino acid sequence of FR3 of heavy chain is the amino acid sequence of SEQ ID NO. 9,
the amino acid sequence of FR4 of heavy chain is the amino acid sequence of SEQ ID NO. 10,
the amino acid sequence of FR1 of light chain is the amino acid sequence of SEQ ID NO. 11,
the amino acid sequence of FR2 of light chain is the amino acid sequence of SEQ ID NO. 12,
the amino acid sequence of FR3 of light chain is the amino acid sequence of SEQ ID NO. 13, and
the amino acid sequence of FR4 of the light chain is the amino acid sequence of seq id No. 14;
(II)：
the amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence No. 4,
the amino acid sequence of CDR2 of the light chain is the amino acid sequence of SEQ ID NO. 5,
the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6,
the amino acid sequence of FR1 of heavy chain is the amino acid sequence of SEQ ID NO. 7,
the amino acid sequence of FR2 of heavy chain is the amino acid sequence of SEQ ID NO. 15,
the amino acid sequence of FR3 of heavy chain is the amino acid sequence of SEQ ID NO. 16,
the amino acid sequence of FR4 of heavy chain is the amino acid sequence of SEQ ID NO. 10,
the amino acid sequence of FR1 of light chain is the amino acid sequence of SEQ ID NO. 11,
the amino acid sequence of FR2 of light chain is the amino acid sequence of SEQ ID NO. 12,
the amino acid sequence of FR3 of light chain is the amino acid sequence of SEQ ID NO. 13, and
the amino acid sequence of FR4 of light chain is the amino acid sequence of SEQ ID NO. 14.
In the above (I), the amino acid sequences of FR 1-FR 4 of the heavy chain and FR 1-FR 4 of the light chain are each optionally an amino acid sequence having 90% or more, preferably 95% or more identity to the amino acid sequences represented by the above sequence numbers 7-14, and in the above (II), the amino acid sequences of FR 1-FR 4 of the heavy chain and FR 1-FR 4 of the light chain are each optionally an amino acid sequence having 90% or more, preferably 95% or more identity to the amino acid sequences represented by the above sequence numbers 7, 15, 16, 10-14. The details are the same as described above.
The anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof is preferably a mouse-human chimeric antibody as described above as long as the effect of the present invention is exerted, and the variable region preferably contains the following amino acid sequence.
The amino acid sequence of CDR1 of the heavy chain is the amino acid sequence of sequence No. 1,
the amino acid sequence of CDR2 of the heavy chain is the amino acid sequence of sequence 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 sequence No. 4,
the amino acid sequence of CDR2 of the light chain is the amino acid sequence of SEQ ID NO. 5,
the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO. 6,
the amino acid sequence of FR1 of heavy chain is the amino acid sequence of SEQ ID NO. 17,
the amino acid sequence of FR2 of heavy chain is the amino acid sequence of SEQ ID NO. 18,
the amino acid sequence of FR3 of heavy chain is the amino acid sequence of SEQ ID NO. 19,
the amino acid sequence of FR4 of heavy chain is the amino acid sequence of SEQ ID NO. 20,
the amino acid sequence of FR1 of light chain is the amino acid sequence of SEQ ID NO. 21,
the amino acid sequence of FR2 of light chain is the amino acid sequence of SEQ ID NO. 22,
the amino acid sequence of FR3 of light chain is the amino acid sequence of SEQ ID NO. 23, and
the amino acid sequence of FR4 of light chain is the amino acid sequence of SEQ ID NO. 24.
The amino acid sequences of FR 1-FR 4 of the heavy chain and FR 1-FR 4 of the light chain are each optionally an amino acid sequence having 90% or more, preferably 95% or more identity to the amino acid sequences represented by SEQ ID Nos. 17 to 24. The details are the same as described above.
The anti-podophyllin antibody of the present invention or an antibody fragment containing an antigen-binding region thereof can be produced by a known genetic engineering method based on an amino acid sequence constituting the structure.
The production may further comprise a step of recovering the obtained anti-podophyllin antibody or an antibody fragment containing an antigen binding region thereof. The recovery step may be a purification step, a concentration step, or the like. In this purification step, the anti-podophyllin antibody of the present invention or an antibody fragment including an antigen-binding region thereof can be purified until the antibody or the antibody fragment is homogeneous by using a known purification technique.
The anti-podophyllin antibody provided by the invention comprises a multispecific antibody, a functional modified antibody and a conjugated antibody. The production method may be a known method.
Another embodiment of the present invention is an inhibitor of binding of pellitorin to CLEC-2 (hereinafter, may be referred to as "the binding inhibitor of the present embodiment" or the like), which comprises the above-described anti-pellitorin antibody or an antibody fragment comprising an antigen-binding region thereof.
The binding inhibitor of the present embodiment may be a composition for inhibiting binding. The binding inhibitor and the binding-inhibiting composition of the present embodiment may be a mixture, and the components thereof may be uniform or non-uniform.
The binding inhibitor of the present embodiment has an effect of inhibiting the binding of pelargonium protein to CLEC-2, and therefore has an effect of inhibiting the binding of pelargonium protein to CLEC-2.
The binding inhibitor of the present embodiment may contain one of the above-described anti-podophyllin antibodies or antibody fragments containing an antigen binding region thereof, or may contain a plurality of kinds.
The content of the anti-podophyllin antibody or the antibody fragment including the antigen-binding region thereof relative to the total amount of the binding inhibitor of the present embodiment is not particularly limited as long as binding of the podophyllin to CLEC-2 can be inhibited, and is usually 0.5mg/mL or more, preferably 1mg/mL or more, more preferably 10mg/mL or more, and usually 500mg/mL or less, preferably 200mg/mL or less, more preferably 100mg/mL or less, based on the total amount of the anti-podophyllin antibody or the antibody fragment including the antigen-binding region thereof.
As for other details of the binding inhibitor of the present embodiment, the following description of "a pharmaceutical composition comprising the above-mentioned anti-podophyllin antibody or an antibody fragment comprising an antigen-binding region thereof" is cited.
Another embodiment of the present invention is a pharmaceutical composition (hereinafter, sometimes referred to as "pharmaceutical composition of the present embodiment", etc.) comprising: the anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof.
The pharmaceutical composition of the present embodiment has an effect of inhibiting the binding of pellitorin to CLEC-2, and therefore has an effect obtained by inhibiting the binding of pellitorin to CLEC-2. Examples thereof include an inhibitory effect on the proliferation or metastasis of cancer cells, an inhibitory effect on platelet aggregation, an antithrombotic effect, and an anti-inflammatory effect. Therefore, the pharmaceutical composition of the present embodiment can be used as a pharmaceutical composition for inhibiting the proliferation or metastasis of cancer cells, inhibiting platelet aggregation, resisting thrombus, resisting inflammation, or the like.
The cancer cell is preferably a tumor cell positive for podophyllin. Examples of tumor cells positive for pelotendin include mesothelioma, brain tumor, testicular tumor, kaposi's sarcoma, lymphangioma, cavernous hemangioma, angiosarcoma, lung squamous carcinoma, ovarian carcinoma dysgerminoma, glioma, germ cell tumor, head and neck cancer, lung cancer, bladder cancer, bone and soft tissue tumor (primary bone tumor (osteosarcoma, chondrosarcoma), soft tissue sarcoma)), cells of esophageal cancer, and the like.
Therefore, the pharmaceutical composition of the present embodiment can be used for the prevention or treatment of diseases that can be prevented or treated by inhibiting the proliferation or metastasis of cancer cells, inhibiting platelet aggregation, resisting thrombosis, resisting inflammation, and the like. Included in "treatment" is amelioration. Examples of the disease include mesothelioma, brain tumor, testicular tumor, kaposi's sarcoma, lymphangioma, cavernous hemangioma, angiosarcoma, lung squamous carcinoma, ovarian carcinoma dysgerminoma, glioma, germ cell tumor, head and neck cancer, lung cancer, bladder cancer, bone and soft tissue tumor (primary bone tumor (osteosarcoma, chondrosarcoma), soft tissue sarcoma)), and esophageal cancer.
The pharmaceutical composition of the present embodiment contains: the anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof. That is, the pharmaceutical composition of the present embodiment may contain one of the above-described anti-podophyllin antibodies or antibody fragments containing an antigen binding region thereof, or may contain a plurality of kinds.
The mode of administration of the pharmaceutical composition of this embodiment is not particularly limited, and administration can be performed by oral administration or parenteral administration. Examples of parenteral administration include intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, intracerebral administration, intraspinal administration, and other local administrations.
Dosage forms for oral administration and parenteral administration and methods for producing the same are well known to those skilled in the art, and a pharmaceutical composition can be produced by blending the above-mentioned anti-podophyllin antibody or antibody fragment containing an antigen-binding region thereof with a pharmaceutically acceptable carrier or the like.
Dosage forms for parenteral administration include injectable preparations, external preparations, and suppositories、Inhalant, eye drop, eye ointment, nose drop, ear drop, liposome, etc.
Examples of the preparations for injection include drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracerebral preparations, and intraspinal preparations.
Examples of the external preparation include ointment, cataplasm, and lotion.
In particular, where it is desired to act directly on the central nervous tissue, an osmotic pressure pump may be usedAnd the likeThe micro-pump for medical use may be used for continuous injection, or may be mixed with fibrin paste or the like to prepare a sustained release preparation, which is then left in the affected tissue.
Among them, for example, an injection preparation is usually prepared by dissolving an antibody in distilled water for injection, and a dissolution aid, a buffer, a pH adjuster, an isotonic agent, a soothing agent, a preservative, a stabilizer, and the like may be added as necessary. In addition, the composition can be made into lyophilized preparation for preparation before use.
The dosage form for oral administration includes solid or liquid dosage forms, and specifically includes tablets, coated tablets, pills, fine granules, powders, capsules, syrups, emulsions, suspensions, injections, troches, and the like.
The pharmaceutical composition of this embodiment may further contain other therapeutically effective agents, and may further contain components such as bactericides, anti-inflammatory agents, vitamins, amino acids, and the like as necessary.
Examples of the pharmacologically acceptable carriers include excipients, lubricants, binders, and disintegrants in solid preparations, and solvents, dissolution aids, suspending agents, isotonic agents, buffers, and soothing agents in liquid preparations. If necessary, additives such as usual preservatives, antioxidants, coloring agents, sweeteners, adsorbents, and wetting agents may be used in appropriate amounts.
The dose of the pharmaceutical composition of the present embodiment can be determined by various factors such as the physician according to the route of administration, the type of disease, the degree of symptoms, the age, sex, body weight, severity of the disease, pharmacological manifestations such as pharmacokinetic and toxicological profiles, whether or not a drug delivery system is used, and whether or not the drug is administered as part of a combination with other drugs.
The pharmaceutical composition of the present embodiment can be administered orally or parenterally. In the case of parenteral administration, administration can be carried out by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, and the like, but is not limited thereto. The dose is not particularly limited, and is usually about 0.01. mu.g/kg to about 1000mg/kg per day, preferably about 0.01mg/kg to about 100mg/kg per day, and more preferably about 0.1mg/kg to about 20mg/kg per day. The pharmaceutical composition may be administered at intervals of daily, 1 time per week, 1 to 4 times per month, or 1 to 7 times per year, but is not limited thereto.
The pharmaceutical composition of this embodiment can be administered alone or in combination with other pharmaceutical compositions or drugs, and for example, can be used in combination with a pharmaceutical composition or drug for preventing or treating skin tumor, brain tumor, testicular tumor, kaposi's sarcoma, lymphangioma, cavernous hemangioma, angiosarcoma, lung squamous carcinoma, ovarian carcinoma dysgerminoma, glioma, germ cell tumor, head and neck cancer, lung cancer, bladder cancer, bone and soft tissue tumor (primary bone tumor (osteosarcoma, chondrosarcoma), soft tissue sarcoma)), esophageal cancer, and the like.
The present invention includes the following aspects.
Use of the above-mentioned anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof for producing an inhibitor of binding of podophyllin to CLEC-2 or a composition for inhibiting binding of podophyllin to CLEC-2.
[ 2] the anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof is used for the prevention or treatment of a disease which can be prevented or treated by inhibiting the binding of the podophyllin to CLEC-2.
[ 3 ] A method for preventing or treating a disease which can be prevented or treated by inhibiting the binding of pellitorin to CLEC-2, which comprises: administering a prophylactically or therapeutically effective amount of the above-described anti-podophyllin antibody, or an antibody fragment comprising an antigen-binding region thereof, to a human or patient in need of prophylaxis or treatment.
Use of the anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof for inhibiting binding of the podophyllin to CLEC-2.
The anti-podophyllin antibody or an antibody fragment containing an antigen-binding region thereof is used for inhibiting binding of the podophyllin to CLEC-2.
[ 6 ] A method for inhibiting the binding of pellagdin to CLEC-2, which comprises administering to a human (i) the above-mentioned anti-pellagdin antibody or an antibody fragment comprising an antigen-binding region thereof, (ii) an inhibitor of the binding of pellagdin to CLEC-2, or (iii) a composition for inhibiting the binding of pellagdin to CLEC-2.
Another embodiment of the present invention is a DNA encoding a protein portion of the above-described anti-podophyllin antibody or an antibody fragment including an antigen binding region thereof. The base sequence of the above-mentioned DNA encoding the protein portion of the anti-podophyllin antibody can be easily designed by those skilled in the art based on the amino acid sequence of the protein. Specific examples thereof include the nucleotide sequences (SEQ ID NOS: 27 to 52) described in examples.
In addition, a sequence encoding a signal peptide or a Kozak sequence may be added to the DNA of the present embodiment.
The DNA of this embodiment can be produced by a known genetic engineering method.
Another embodiment of the present invention is a recombinant vector comprising the above DNA.
The recombinant vector of the present embodiment may be a known vector, and examples thereof include a plasmid vector and a viral vector. The vector may be one capable of expression in prokaryotic cells such as E.coli, preferably one capable of expression in eukaryotic cells, and more preferably one capable of expression in mammalian-derived cells.
Another embodiment of the present invention is a host cell (transformant) comprising the above DNA or the above recombinant vector.
The host cell of the present embodiment is a cell transformed by introducing the above DNA or the above recombinant vector. The host cell of the present embodiment may be a known cell, and may be a prokaryotic cell such as Escherichia coli or Bacillus subtilis, but is preferably a eukaryotic cell, and more preferably a cell derived from a mammal.
Examples of the host cell include CHO cells (such as CHO-S cells as a strain thereof), and examples of a vector for expression in the cell include Freedom (registered trademark) pCHO1.0(Thermo Fisher Scientific).
Examples
The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
[ example 1]
(determination of amino acid sequence of humanized antibody as an anti-podophyllin antibody)
In order to prepare a humanized antibody by engineering the mouse PG4D2 antibody, each CDR region and each FR region of the heavy chain and light chain of the mouse PG4D2 antibody were determined using antibody databases IMGT and Abysis database using Kabat encoding. Then, the FR regions of human Germline (GL) having high identity with the amino acid sequences were selected based on the sequences of the FR regions of the heavy and light chains, respectively, using the IMGT database. FR sequences with the highest amino acid identity are selected for FR 1-4 respectively by using an FR Shuffling (Shuffling) method, and the combination of FR 1-4 is determined. For the heavy chain and the light chain, the complementarity determining regions of the mouse PG4D2 antibody were grafted into selected FR regions of the human germline, respectively, to design candidate sequences for a humanized antibody. Further, the amino acid sequences of 2 humanized antibodies K4, K5 were determined based on the following studies: whether all of the specified amino acid residues are completely conserved; whether the isoelectric point (pI) of the FR region is not significantly high; whether the FR sequence of the selected GL is not used significantly less frequently; presence or absence of chemical modification sites; whether or not there are 2 or more residues having different properties from 3 viewpoints of hydrophobicity, volume, and physicochemical properties of the amino acid side chain for non-conserved amino acid residues as compared with the mouse PG4D2 antibody; whether the identity of the amino acid sequences is large; and the like. That is, the humanized antibodies K4 and K5 were not obtained by simply replacing the amino acid sequences of the FR regions of the heavy chain and the light chain of the mouse PG4D2 antibody.
Production example 1 production of humanized antibody as anti-podophyllin antibody
The amino acid sequences of the humanized antibodies K4 and K5 thus produced are shown below.
The variable region of the amino acid sequence of humanized antibody K4 was: CDR1 of heavy chain: amino acid sequence of sequence No. 1, CDR2 of heavy chain: amino acid sequence of sequence No. 2, CDR3 of heavy chain: amino acid sequence of seq id No. 3, CDR1 of light chain: amino acid sequence of seq id No. 4, CDR2 of light chain: amino acid sequence of seq id No. 5, CDR3 of light chain: amino acid sequence of seq id No. 6, FR1 of heavy chain: amino acid sequence of seq id No. 7, FR2 of heavy chain: amino acid sequence of seq id No. 8, FR3 of heavy chain: amino acid sequence of seq id No. 9, FR4 of heavy chain: amino acid sequence of seq id No. 10, FR1 of light chain: amino acid sequence of seq id No. 11, FR2 of light chain: amino acid sequence of seq id No. 12, FR3 of light chain: amino acid sequence of seq id No. 13, FR4 of light chain: the amino acid sequence of sequence number 14, the constant region is the amino acid sequence of human IgG4 antibody, the amino acid sequence of its heavy chain is the amino acid sequence of sequence number 25, and the amino acid sequence of its light chain is the amino acid sequence of sequence number 26.
The variable region of the amino acid sequence of humanized antibody K5 was: CDR1 of heavy chain: amino acid sequence of sequence No. 1, CDR2 of heavy chain: amino acid sequence of sequence No. 2, CDR3 of heavy chain: amino acid sequence of seq id No. 3, CDR1 of light chain: amino acid sequence of seq id No. 4, CDR2 of light chain: amino acid sequence of seq id No. 5, CDR3 of light chain: amino acid sequence of seq id No. 6, FR1 of heavy chain: amino acid sequence of seq id No. 7, FR2 of heavy chain: amino acid sequence of seq id No. 15, FR3 of heavy chain: amino acid sequence of seq id No. 16, FR4 of heavy chain: amino acid sequence of seq id No. 10, FR1 of light chain: amino acid sequence of seq id No. 11, FR2 of light chain: amino acid sequence of seq id No. 12, FR3 of light chain: amino acid sequence of seq id No. 13, FR4 of light chain: the amino acid sequence of SEQ ID NO. 14, wherein the constant region is identical to the amino acid sequence of the constant region of humanized antibody K4 described above.
Further, as the nucleotide sequence encoding each of the above amino acid sequences, the nucleotide sequences of the following sequences were used.
As the nucleotide sequences encoding the amino acid sequences of SEQ ID Nos. 1 to 16, the nucleotide sequences of SEQ ID Nos. 27 to 42 were used, respectively.
As the nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 25 (constant region of heavy chain), the nucleotide sequence of SEQ ID NO. 43 was used, and
as the nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 26 (constant region of light chain), the nucleotide sequence of SEQ ID NO. 44 was used.
For expression in CHO cells (Thermo Fisher Scientific), free (registered trademark) pCHO1.0(Thermo Fisher Scientific) was used as an expression vector.
CHO cells expressing humanized antibodies K4 and K5 were thawed, and then scale-up culture was performed, followed by 10L-scale fed-batch culture in a stirred cell culture tank. The culture conditions were set at a stirring speed of 100rpm, a culture temperature of 37 ℃ and a pH of 7.0, and the culture was carried out for 6 to 8 days, after which the culture broth was collected.
The collected culture solution was purified to obtain humanized antibodies K4 and K5. The collected supernatant of the culture solution was used in the measurement in examples 2 and 3 described later.
Production example 2 production of mouse-human chimeric antibody as anti-podophyllin antibody
The amino acid sequences of the mouse-human chimeric antibody chPG4D2 are shown below.
The variable region of the amino acid sequence of the mouse-human chimeric antibody chPG4D2 is: CDR1 of heavy chain: amino acid sequence of sequence No. 1, CDR2 of heavy chain: amino acid sequence of sequence No. 2, CDR3 of heavy chain: amino acid sequence of seq id No. 3, CDR1 of light chain: amino acid sequence of seq id No. 4, CDR2 of light chain: amino acid sequence of seq id No. 5, CDR3 of light chain: amino acid sequence of seq id No. 6, FR1 of heavy chain: amino acid sequence of seq id No. 17, FR2 of heavy chain: amino acid sequence of seq id No. 18, FR3 of heavy chain: amino acid sequence of seq id No. 19, FR4 of heavy chain: amino acid sequence of seq id No. 20, FR1 of light chain: amino acid sequence of seq id No. 21, FR2 of light chain: amino acid sequence of seq id No. 22, FR3 of light chain: amino acid sequence of seq id No. 23, FR4 of light chain: the amino acid sequence of SEQ ID NO. 24, wherein the constant region is identical to the amino acid sequence of the constant region of humanized antibody K4 described above.
It can be easily understood by those skilled in the art that when the respective nucleotides encoding the respective amino acid sequences of the aforementioned sequence Nos. 1 to 6 and 17 to 24 and the respective amino acid sequences of the constant region of the aforementioned humanized antibody K1 are designed, the mouse-human chimeric antibody chPG4D2 can be produced in the same manner as in production example 1.
In this case, as the nucleotide sequence encoding each of the above amino acid sequences, for example, the nucleotide sequences described below can be used.
As the nucleotide sequences encoding the amino acid sequences of SEQ ID Nos. 1 to 6 and 17 to 24, the nucleotide sequences of SEQ ID Nos. 27 to 32 and 45 to 52 can be used, respectively.
The nucleotide sequence encoding the amino acid sequence of the constant region may be the same as the amino acid sequence encoding the constant region described in preparation example 1.
For expression in CHO cells (Thermo Fisher Scientific), free (registered trademark) pCHO1.0(Thermo Fisher Scientific) was used as an expression vector.
CHO cells expressing the mouse-human chimeric antibody chPG4D2 were thawed, and then scale-up culture was performed, followed by batch culture on a 5L scale in a stirred cell culture tank. The culture conditions were: the stirring speed was 100rpm, the culture temperature was 37 ℃ and pH was 7.0, and the culture broth was recovered after 7 days of culture.
The recovered culture solution was purified to obtain a mouse-human chimeric antibody chPG4D 2. The collected culture supernatant was used for the measurement in examples 2 and 3 described later.
Example 2 the podophyllin-binding Activity of the antibodies
The binding activity to the domain of podophyllin PLAG4 was measured by the ELISA method described in patent document 1 using the supernatants of the culture solutions recovered in production examples 1 and 2. As the antigenic peptide, Human platypodium planopodin PLAG4 domain peptide (WT-hPLGA 4(Genscript,788349-1)) was immobilized, and pre-absorbed Goat Anti-Human IgG Fc (Biotin) preadsorbed (Abcam, ab98618) and Streptavidin β -galactosidase complex (Streptavidin β -Gal conjugate) (Roche Diagnostics, 11112481001) were used in the detection.
The results are shown in Table 1. The binding activity (antibody titer) was shown by assuming that the case of the mouse-human chimeric antibody chPG4D2 was 100%.
[ Table 1]
TABLE 1
Antibodies
Binding activity (antibody titer) (%)
Humanized antibody K4
84.8-171.8
Humanized antibody K5
90.0-111.5
Mouse-human chimeric antibody chPG4D2
100
Example 3 Activity of Each antibody for inhibiting binding of CLEC-2 to podophyllin
The activity of inhibiting binding of CLEC-2 to podophyllin was measured by the ELISA method described in patent document 1 using each of the culture supernatants obtained in production example 1 and production example 2. As the antigen peptide, Recombinant Human CLEC-2(R & D, 1718-CL-050) was immobilized, and as a protein to be bound thereto, Recombinant Human podophyllin Fc Chimera (Recombinant Human Podoplanin Fc Chimera) (R & D, 3670-PL) was used, and at the time of detection, pre-absorbed Goat Anti-Human IgG Fc (Biotin)) (coat Anti-Human IgG Fc (Biotin) preadsorbed) (Abcam, ab98618) and Streptavidin β -galactosidase complex (Streptavidin- β -galactosidase conjugate) (SIGMA, S3887, Roche, 11112481001) were used.
As a positive control, mouse PG4D2 antibody (PG4D2) was used. The mouse PG4D2 antibody was a monoclonal antibody obtained from a hybridoma of NITE P-02071(PG4D 2).
The results are shown in Table 2.
[ Table 2]
TABLE 2
Example 4 Effect of Each antibody on inhibition of platelet aggregation in vivo
It is known that platelet aggregation is caused when a human podophyllin gene is introduced into CHO cells, a cell line which has not caused platelet aggregation (J.biol.chem., Dec 19; 278 (51): 51599-605 (2003)).
Human panopodin-positive osteoma cell strain SJSA-1(ATCC CRL-2098) was added to human whole blood, and whether each antibody inhibited the induced platelet aggregation was analyzed using a whole blood platelet aggregation assay device WBA carpa (TAIYO). The osteosarcoma cell line SJSA-1(ATCC CRL-2098) can be obtained from American Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America).
As a negative control in the system using humanized antibodies K4 and K5, human IgG4 antibody (CrownBio, C0004) was used.
In addition, the in vitro assay system calculates the aggregation rate as follows: the whole blood containing the cell-induced aggregate was aspirated through the micro-mesh filter, and the aggregation rate was calculated by measuring the aspiration pressure that was increased until the aggregate was clogged.
As a positive control, mouse PG4D2 antibody (PG4D2) was used. As a negative control of the system using the mouse PG4D2 antibody, a mouse IgG2a antibody (Sigma, M9144) was used.
The results are shown in FIGS. 1 to 3. The humanized antibodies K4 and K5 inhibited platelet aggregation in a concentration-dependent manner in the same manner as the positive control.
Example 5 Effect of inhibiting Each antibody against pulmonary hematogenous metastasis
The inhibitory effect of hematogenous metastasis of each antibody was analyzed using a hematogenous metastasis model by severe combined immunodeficiency mouse SCID-Beige (CB17. Cg-Prkdc)scidLystbg-JA model in which a human panfoot protein-positive osteoma cell line SJSA-1(ATCC CRL-2098) was injected into the tail vein of/CrlCrlj) (Charles River, female, 6 weeks old, Japan) and the number of metastatic nodules formed on the lung surface was measured after 20 days.
The day before the SJSA-1 cell injection, 6 mice of each group were administered with a control antibody (human IgG4 antibody (CrownBio)), a humanized antibody K4 or K5 from the tail vein route, and the effect on lung metastasis in the podophyllin-dependent experiment was studied.
The results are shown in FIG. 4. Lung metastasis of SJSA-1 cell line was significantly inhibited by prior administration of humanized antibodies K4 or K5.
Example 6 antitumor Effect of Each antibody
Will be 5X 106Transplanting the human podophyllum positive osteoma cell strain SJSA-1(ATCC CRL-2098) toSevere complex immunodeficiency mouse SCID-Beige (CB17. Cg-Prkdc)scidLystbg-JA xenograft mouse was prepared subcutaneously from/CrlCrlj) (Charles River, female, 5-week-old, Japan), and the in vivo antitumor effects of the humanized antibodies K4 and K5 were evaluated.
The humanized antibodies K4 and K5 were administered at 5mg/kg 2 times per week (day 1, day 4, day 8, day 11, and day 15) from the first day of transplantation in the K4 administration group and the K5 administration group, respectively. Tumor size was measured 2 times per week (1/2 Long diameter X (short diameter)2) And body weight (n ═ 6). At the end (after 20 days) photographs were taken and the weight of the extirpated tumor was measured.
As a negative control, human IgG4 antibody (Sino Biological, HG4K) was used.
The results (days post-transplantation versus mean tumor size) are shown in figure 5 (p <0.05, p <0.01 using Kruskal-Wallis test). The effect of inhibiting tumor growth was confirmed in the K4-administered group and the K5-administered group. Note that, at this time of the experiment, no significant reduction in the body weight of the mice was observed.
Sequence listing
<110> Research society of public welfare group legal For Cancer (Japanese Foundation For Cancer Research)
Asia Bi corporation (API Co., Ltd.)
<120> anti-podophyllin antibody
<130> C4701-19231
<150> JP 2019-048861
<151> 2019-03-15
<160> 52
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<213> Intelligent (Homo sapiens)
<400> 16
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 17
<211> 25
<212> PRT
<213> mouse (Mus musculus)
<400> 17
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Arg Lys Leu Ser Cys Ala Ala Ser
20 25
<210> 18
<211> 14
<212> PRT
<213> mouse (Mus musculus)
<400> 18
Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val Ala
1 5 10
<210> 19
<211> 32
<212> PRT
<213> mouse (Mus musculus)
<400> 19
Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe Leu Gln
1 5 10 15
Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg
20 25 30
<210> 20
<211> 11
<212> PRT
<213> mouse (Mus musculus)
<400> 20
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
1 5 10
<210> 21
<211> 23
<212> PRT
<213> mouse (Mus musculus)
<400> 21
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys
20
<210> 22
<211> 15
<212> PRT
<213> mouse (Mus musculus)
<400> 22
Trp Tyr Leu Gln Gln Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
1 5 10 15
<210> 23
<211> 32
<212> PRT
<213> mouse (Mus musculus)
<400> 23
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys
20 25 30
<210> 24
<211> 10
<212> PRT
<213> mouse (Mus musculus)
<400> 24
Phe Gly Gly Gly Thr Lys Leu Glu Met Lys
1 5 10
<210> 25
<211> 327
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 25
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 26
<211> 107
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 26
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 27
<211> 30
<212> DNA
<213> mouse (Mus musculus)
<400> 27
ggattcactt tcagtggctt tggaatgctc 30
<210> 28
<211> 51
<212> DNA
<213> mouse (Mus musculus)
<400> 28
tacattagta gtgtcagtag tagaatctac tatgcagaca cagtgaaggg t 51
<210> 29
<211> 30
<212> DNA
<213> mouse (Mus musculus)
<400> 29
gaatcctacg gccccgcctg gtttgcttac 30
<210> 30
<211> 48
<212> DNA
<213> mouse (Mus musculus)
<400> 30
agatctagtc agcacattgt acatattgat ggaaacacct atttagaa 48
<210> 31
<211> 21
<212> DNA
<213> mouse (Mus musculus)
<400> 31
aaagtttcca accgattttc t 21
<210> 32
<211> 27
<212> DNA
<213> mouse (Mus musculus)
<400> 32
tttcaaggtt cacatgttcc gtacacg 27
<210> 33
<211> 75
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 33
gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtgcag cctct 75
<210> 34
<211> 42
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 34
tgggtccgcc aggctccagg gaaggggctg gagtgggtgg cc 42
<210> 35
<211> 96
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 35
cgattcacca tctccagaga caacgccaag aactcactgt atctgcaaat gaacagcctg 60
agagccgagg acacggctgt gtattactgt gcgaga 96
<210> 36
<211> 33
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 36
tggggccagg gcaccctggt caccgtctcc tca 33
<210> 37
<211> 69
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 37
gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60
atctcctgc 69
<210> 38
<211> 45
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 38
tggtacctgc agaagccagg gcagtctcca cagctcctga tctat 45
<210> 39
<211> 96
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 39
ggagtcccag acaggttcag tggcagtggg tcaggcactg atttcacact gaaaatcagc 60
agggtggagg ctgaggatgt tggagtttat tactgc 96
<210> 40
<211> 30
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 40
ttcggcggag ggaccaaggt ggagatcaaa 30
<210> 41
<211> 42
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 41
tgggtccgcc aggctccagg gaaggggctg gagtgggtct ca 42
<210> 42
<211> 96
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 42
agattcacca tctccagaga caattccaag aacacgctgt atcttcaaat gaacagcctg 60
agagccgagg acacggctgt gtattactgt gcgaga 96
<210> 43
<211> 981
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 43
gcttccacca agggaccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 60
agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctcc 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240
tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300
aaatatggtc ccccatgccc accatgccca gcacctgagt tcctgggggg accatcagtc 360
ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420
tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600
tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660
gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720
aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840
gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 900
aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960
ctctccctgt ctctgggtaa a 981
<210> 44
<211> 321
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 44
cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60
ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120
tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180
agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240
aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300
agcttcaaca ggggagagtg t 321
<210> 45
<211> 75
<212> DNA
<213> mouse (Mus musculus)
<400> 45
gatgtgcaac tggtggagtc tgggggaggc ttggtgcagc ctggagggtc ccggaaactc 60
tcctgtgcag cctct 75
<210> 46
<211> 42
<212> DNA
<213> mouse (Mus musculus)
<400> 46
tgggttcgtc aggctccaga gaaggggctg gagtgggtcg ca 42
<210> 47
<211> 96
<212> DNA
<213> mouse (Mus musculus)
<400> 47
cgattcacca tctccagaga caatcccaag aacaccctgt tcctgcaaat gaccagtctg 60
aggtctgagg acacggccat gtattactgt gcaaga 96
<210> 48
<211> 33
<212> DNA
<213> mouse (Mus musculus)
<400> 48
tggggccaag ggactctggt cactgtctct gca 33
<210> 49
<211> 69
<212> DNA
<213> mouse (Mus musculus)
<400> 49
gatgtcttga tgacccaaac tccactctcc ctgccggtca gtcttggaga tcaagcctcc 60
atctcttgc 69
<210> 50
<211> 45
<212> DNA
<213> mouse (Mus musculus)
<400> 50
tggtacctgc agcaaccagg ccagtctcca aagctcctga tctac 45
<210> 51
<211> 96
<212> DNA
<213> mouse (Mus musculus)
<400> 51
ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caggatcagc 60
agagtggagg ctgaggatct gggagtttat tactgc 96
<210> 52
<211> 30
<212> DNA
<213> mouse (Mus musculus)
<400> 52
ttcggagggg ggaccaagct ggaaatgaaa 30
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