HK1193833B - Antibody specifically recognising transferrin receptor - Google Patents
Antibody specifically recognising transferrin receptor Download PDFInfo
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- HK1193833B HK1193833B HK14107189.0A HK14107189A HK1193833B HK 1193833 B HK1193833 B HK 1193833B HK 14107189 A HK14107189 A HK 14107189A HK 1193833 B HK1193833 B HK 1193833B
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Description
Technical Field
The present invention relates to anti-TfR antibodies that specifically react with human TfR antigen. The invention also relates to pharmaceutical compositions comprising anti-TfR antibodies, in particular pharmaceutical compositions related to the treatment of malignancies.
Background
Cancer is the first cause of death in japan, and the number of patients increases with increasing age, and development of a drug and a treatment method with high efficacy and safety is strongly desired. Current chemotherapy, radiotherapy, and the like kill cancer cells and damage normal cells, causing strong side effects. In order to solve the above problems, studies on molecular target therapy have been actively conducted in which a molecule specifically expressed in cancer cells is used as a target drug for designing therapy. Such a molecular target cancer therapeutic agent has received much attention because of its advantages such as long half-life and few side effects of antibody drugs. Examples of successful development include a chimeric antibody rituximab (non-patent document 1) targeting CD20, a humanized antibody trastuzumab (non-patent document 2) targeting Her2/neu, and a humanized antibody bevacizumab targeting Vascular Endothelial Growth Factor (VEGF). These antibodies were used as patients with cancer, and their therapeutic effects were confirmed.
The use of antibodies as therapeutic agents is divided into non-labeled and labeled antibodies. The mechanism of action of the unlabeled antibody is believed to be: (1) antibody-dependent cell-damage-associated Activities (ADCC) (non-patent document 3) and complement-dependent cell-damage-associated activities (CDC) (non-patent document 4) of cells and molecules of the immune system, inhibition of signals associated with intracellular survival and proliferation by target molecules, (3) induction of apoptosis, and (4) regulation of cytokine secretion. Through the combination of various mechanisms, tumor cells are killed and proliferation is stopped, thereby exerting a therapeutic effect. The labeled antibody is a substance that binds to a cell-damaging substance such as a radioactive substance, a toxin, an enzyme, or a drug, and delivers the substance to a cancer tissue by utilizing the specificity of the antibody, thereby improving the therapeutic effect and reducing side effects.
Transferrin receptor (TfR) is originally found on reticulocytes as an intracellular membrane structure for entry of iron bound to transferrin (Tf) (non-patent document 5). It has been known that the expression is caused in trophoblast cells of placenta (non-patent documents 10 to 12), activated lymphocytes (non-patent document 12), various tumor cells, and the like. For example, it has been reported that transferrin receptors are highly expressed in breast cancer (non-patent document 6), prostate cancer (non-patent document 7), lung cancer (non-patent document 8), pancreatic cancer (non-patent document 9), colorectal cancer (non-patent documents 30 and 31), gastric cancer (non-patent document 31), bladder cancer (non-patent documents 32 and 33), liver cancer (non-patent document 34), cervical cancer (non-patent document 35), brain tumor (non-patent document 36), chronic lymphocytic leukemia (non-patent documents 37 and 38), non-hodgkin lymphoma (non-patent documents 38 and 39), and adult T-cell leukemia (non-patent document 40). Further, since TfR is highly expressed on the surface of various cancer cells and is low in normal cells, it has long been known as a molecular target for cancer therapy (non-patent documents 13 to 16, patent documents 1 and 2). However, anti-human TfR antibodies developed to date are all animal-derived antibodies. In general, it is known that when an antibody against an animal other than a human, for example, a mouse antibody is administered to a human, a human antibody against the mouse antibody is induced in the human (human antibody: hereinafter, referred to as HAMA). It is known that HAMA reacts with an administered mouse antibody to cause side effects (non-patent documents 17 to 20), accelerates the disappearance of the administered mouse antibody from the body (non-patent documents 18, 21, and 22), and thereby reduces the therapeutic effect of the mouse antibody (non-patent documents 23 and 24). In a clinical first-stage test using a mouse anti-human TfR antibody actually present, HAMA production was observed, and no significant therapeutic effect was found (non-patent document 25).
In order to avoid such a problem, chimeric antibodies have been developed (patent documents 3 and 4). The chimeric antibody includes a part of an antibody derived from 2 or more species (a variable region of a mouse antibody, a constant region of a human antibody, and the like). Such chimeric antibodies have the advantage of retaining the characteristics of mouse antibodies and being able to stimulate human complement or cell damaging activity due to the presence of human Fc. However, such a chimeric antibody still causes HACA (human anti-chimeric antibody) response, which is a "human anti-chimeric antibody" (non-patent document 26). Further, recombinant antibodies in which only a part of the substituted antibody is a complementarity determining region (i.e., "CDR") have been developed (patent documents 5 and 6). Using a CDR grafting technique, an antibody composed of a mouse CDR, a human variable region framework, and a constant region, i.e., "humanized antibody" was produced (non-patent document 27). However, such humanized antibodies are also immunogenic to humans and cause HAHA (human-human antibody) reaction (non-patent documents 28 and 29). Therefore, a safer and more effective antibody therapeutic agent without immunogenicity is expected in clinical applications.
However, in the development of new antibody drugs, although it is essential to obtain an antibody that recognizes a "intact" target cancer antigen present on the surface of a cell membrane, since the target cancer antigen is a membrane protein, it is difficult to obtain even an antibody against a known cancer antigen. In order to solve these problems, the present inventors have so far created a huge human antibody library composed of 1000 hundred million independent clones, and established a comprehensive acquisition method of antibodies against proteins (cell surface antigens) present on the cell membrane surface of cancer cells and tissues using this database (patent documents 7 to 9).
Documents of the prior art
Patent document
Patent document 1: U.S. Pat. No. 5667781
Patent document 2: U.S. Pat. No. 7976841
Patent document 3: european patent No. 120694
Patent document 4: european patent 125023
Patent document 5: british patent GB21886638A
Patent document 6: U.S. Pat. No. 5585089
Patent document 7: international publication No. 01/062907 pamphlet
Patent document 8: international publication No. 2001/096401 pamphlet
Patent document 9: japanese patent laid-open publication No. 2005-185281
Non-patent document
Non-patent document 1: MassR, et al, ProcAmSeciClinOncol 19,75a,2000
Non-patent document 2: BernsteinNL, et al, annals of Oncology1998,9: 1995-.
Non-patent document 3: bruggemannm, et, j.exp.med.,166, 1351-.
Non-patent document 4: LoosM. (1982), prog. allergy,30,135-192.MolImmunol.1982May;19(5):651-7.
Non-patent document 5: JClininvest1963;42,314-
Non-patent document 6: IntJcancer1981, 27:329-,334,
non-patent document 7: jurol1990;143:381-385,
non-patent document 8: cancer GeneTher2000, 7:59-65;
non-patent document 9: EurJcancer2004, 40 (9) 1418-
Non-patent document 10: JClinInvest1980;65: 1182-.
Non-patent document 11: planta 1986, 7:391-403
Non-patent document 12: JClininvest (1980)66,1135-1143.10
Non-patent document 13: natl Acad SciUSA1982, 79:1175-,
non-patent document 14: cancer Res1986, 46:1759-
Non-patent document 15: cancer Res1990, 50:6295-
Non-patent document 16: blood2004, 103:1838-
Non-patent document 17: J.Clin.Oncol.,2,881 (1984)
Non-patent document 18: blood,65,1349 (1985)
Non-patent document 19: natl cancer Inst, 80,932 (1988)
Non-patent document 20: proc.Natl.Acad.Scl., U.S.A.,82,1242 (1985)
Non-patent document 21: J.Nucl.Med.,26,1011 (1985)
Non-patent document 22: natl cancer Inst.,80,937 (1988)
Non-patent document 23: j. Immunol.,135,1530 (1985)
Non-patent document 24: cancer Res, 46,6489 (1986)
Non-patent document 25: clini cancer Res 1995;1:1259-
Non-patent document 26: med.,170,2153-2157,1989
Non-patent document 27: nature,332,323-327,1988
Non-patent document 28: cancer Res.2001;61:6851-
Non-patent document 29: JPharmBiomedAnal.2006;41:1347-
Non-patent document 30: IntJOncol.199813 (4): 871-5
Non-patent document 31: TohokuJ. exp. Med.1987;153:239-
Non-patent document 32: urol. Res.1987, 15:341-344
Non-patent document 33: Br.J.Urol.1990;65:339-
Non-patent document 34: histopathology1988, 12:53-63
Non-patent document 35: J.Clin.Pathol.1984, 37:131-
Non-patent document 36: APathol, Ant, Histopathiol, 1990, 416:491-
Non-patent document 37: leukemia1993, 7:2019-2025
Non-patent document 38: hematol. Pathol.1990;4:37-41
Non-patent document 39: lancet1983, 1:498-501
Non-patent document 40: blood2004, 103:1838-
Disclosure of Invention
Problems to be solved by the invention
The object of the present invention is to provide a fully human anti-human TfR antibody that specifically recognizes human TfR, inhibits the survival and proliferation of cancer cells highly expressing TfR, and is not immunogenic to humans. It is also intended to provide a method for producing these antibodies and a therapeutic agent for diseases such as cancer using these antibodies.
Means for solving the problems
As described above, although an antibody targeting TfR has been developed as an antitumor agent, development has been unsuccessful due to production of HAMA and insufficient drug efficacy, etc., since the antibody is derived from an animal. Accordingly, the present inventors have made an intensive study on a method for producing an antibody alone, and found that: phage antibodies reactive with TfR on cancer cell membranes (scFv antibodies) were obtained by phage display of human antibody libraries. By analyzing the gene sequences of these antibodies, the CDRs of the antibodies were found to have novel amino acid sequences. Further, these scFv antibodies were IgG-converted to complete human IgG antibodies. The anti-tumor effect was investigated in vitro (invitro) and in vivo (invivo) using these IgG antibodies. As a result, it was found that the antibody had a strong antitumor effect. Thus, the present invention has been completed by finding utility in the treatment of various cancers in which TfR is highly expressed using these antibodies.
That is, according to the present invention, there is provided an antibody specifically reacting with human TfR, wherein the first complementarity determining region (VHCDR1), the second complementarity determining region (VHCDR2) and the third complementarity determining region (VHCDR3) of the heavy chain each comprise an amino acid sequence of any one of SEQ ID Nos. 1 to 3, 7 to 9, 13 to 15, 19 to 21, 25 to 27, 31 to 33, 37 to 39, 43 to 45, 49 to 51, 55 to 57, 61 to 63, 67 to 69, 73 to 75, 79 to 81, 85 to 87, 91 to 93, 97 to 99, 103 to 105, 109 to 111, and 115 to 117.
According to the present invention, there is provided an antibody specifically reacting with human TfR, wherein the first complementarity determining region (VHCDR1), the second complementarity determining region (VHCDR2) and the third complementarity determining region (VHCDR3) of the heavy chain each comprise an amino acid sequence selected from the group consisting of SEQ ID Nos. 1 to 3, 7 to 9, 13 to 15, 19 to 21, 25 to 27, 31 to 33, 37 to 39, 43 to 45, 49 to 51, 55 to 57, 61 to 63, 67 to 69, 73 to 75, 79 to 81, 85 to 87, 91 to 93, 97 to 99, 103 to 105, 109 to 111, and 115 to 117, and the first complementarity determining region (VLCDR1) of the light chain, the second complementarity determining region (VLCDR2) of the light chain, the third complementarity determining region (VLCDR3) of the light chain each comprise SEQ ID Nos. 4 to 6,10 to 12, 16 to 18, 22 to 24, 28 to 30, 34 to 36, 40 to 42, 46 to 48, 54 to 70 to 76, 60 to 64, 70 to 78, and 70 to 78, 82 to 84, 88 to 90, 94 to 96, 100 to 102, 106 to 108, 112 to 114, 118 to 120.
According to the present invention, there is provided an antibody specifically reactive with human TfR, which is selected from the antibodies of the following items (1) to (20).
(1) An antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 1, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 2, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 3, or substantially the same CDR as same, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 4, a light chain second complementarity determining region (VLCDR2) of sequence No. 5, a light chain third complementarity determining region (VLCDR3) of sequence No. 6, or substantially the same CDR as same;
(2) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 7, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 8, a heavy chain third complementarity determining region (VHCDR3) of sequence No.9, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 10, a light chain second complementarity determining region (VLCDR2) of sequence No. 11, a light chain third complementarity determining region (VLCDR3) of sequence No. 12, or substantially the same CDR as thereof;
(3) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 13, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 14, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 15, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 16, a light chain second complementarity determining region (VLCDR2) of sequence No. 17, a light chain third complementarity determining region (VLCDR3) of sequence No. 18, or substantially the same CDR as thereof;
(4) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 19, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 20, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 21, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 22, a light chain second complementarity determining region (VLCDR2) of sequence No. 23, a light chain third complementarity determining region (VLCDR3) of sequence No. 24, or substantially the same CDR as thereof;
(5) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 25, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 26, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 27, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 28, a light chain second complementarity determining region (VLCDR2) of sequence No. 29, a light chain third complementarity determining region (VLCDR3) of sequence No. 30, or substantially the same CDR as thereof;
(6) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 31, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 32, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 33, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 34, a light chain second complementarity determining region (VLCDR2) of sequence No. 35, a light chain third complementarity determining region (VLCDR3) of sequence No. 36, or substantially the same CDR as thereof;
(7) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 37, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 38, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 39, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 40, a light chain second complementarity determining region (VLCDR2) of sequence No. 41, a light chain third complementarity determining region (VLCDR3) of sequence No. 42, or substantially the same CDR as thereof;
(8) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 43, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 44, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 45, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 46, a light chain second complementarity determining region (VLCDR2) of sequence No. 47, a light chain third complementarity determining region (VLCDR3) of sequence No. 48, or substantially the same CDR as thereof;
(9) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 49, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 50, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 51, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 52, a light chain second complementarity determining region (VLCDR2) of sequence No. 53, a light chain third complementarity determining region (VLCDR3) of sequence No. 54, or substantially the same CDR as thereof;
(10) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 55, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 56, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 57, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 58, a light chain second complementarity determining region (VLCDR2) of sequence No. 59, a light chain third complementarity determining region (VLCDR3) of sequence No. 60, or substantially the same CDR as thereof;
(11) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 61, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 62, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 63, or substantially the same CDR as same, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 64, a light chain second complementarity determining region (VLCDR2) of sequence No. 65, a light chain third complementarity determining region (VLCDR3) of sequence No. 66, or substantially the same CDR as same;
(12) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 67, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 68, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 69, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 70, a light chain second complementarity determining region (VLCDR2) of sequence No. 71, a light chain third complementarity determining region (VLCDR3) of sequence No. 72, or substantially the same CDR as thereof;
(13) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 73, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 74, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 75, or substantially the same CDR as same, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 76, a light chain second complementarity determining region (VLCDR2) of sequence No. 77, a light chain third complementarity determining region (VLCDR3) of sequence No. 78, or substantially the same CDR as same;
(14) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence number 79, a heavy chain second complementarity determining region (VHCDR2) of sequence number 80, a heavy chain third complementarity determining region (VHCDR3) of sequence number 81, or substantially the same CDR as same, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence number 82, a light chain second complementarity determining region (VLCDR2) of sequence number 83, a light chain third complementarity determining region (VLCDR3) of sequence number 84, or substantially the same CDR as same;
(15) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 85, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 86, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 87, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 88, a light chain second complementarity determining region (VLCDR2) of sequence No. 89, a light chain third complementarity determining region (VLCDR3) of sequence No. 90, or substantially the same CDR as thereof;
(16) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No.91, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 92, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 93, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 94, a light chain second complementarity determining region (VLCDR2) of sequence No. 95, a light chain third complementarity determining region (VLCDR3) of sequence No. 96, or substantially the same CDR as thereof;
(17) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 97, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 98, a heavy chain third complementarity determining region (VHCDR3) of sequence No. 99, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 100, a light chain second complementarity determining region (VLCDR2) of sequence No. 101, a light chain third complementarity determining region (VLCDR3) of sequence No. 102, or substantially the same CDR as thereof;
(18) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence number 103, a heavy chain second complementarity determining region (VHCDR2) of sequence number 104, a heavy chain third complementarity determining region (VHCDR3) of sequence number 105, or substantially the same CDR as same, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence number 106, a light chain second complementarity determining region (VLCDR2) of sequence number 107, a light chain third complementarity determining region (VLCDR3) of sequence number 108, or substantially the same CDR as same;
(19) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence number 109, a heavy chain second complementarity determining region (VHCDR2) of sequence number 110, a heavy chain third complementarity determining region (VHCDR3) of sequence number 111, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence number 112, a light chain second complementarity determining region (VLCDR2) of sequence number 113, a light chain third complementarity determining region (VLCDR3) of sequence number 114, or substantially the same CDR as thereof;
(20) an antibody having a heavy chain variable region having a CDR comprising a heavy chain first complementarity determining region (VHCDR1) of sequence number 115, a heavy chain second complementarity determining region (VHCDR2) of sequence number 116, a heavy chain third complementarity determining region (VHCDR3) of sequence number 117, or substantially the same CDR as thereof, and a light chain variable region having a CDR comprising a light chain first complementarity determining region (VLCDR1) of sequence number 118, a light chain second complementarity determining region (VLCDR2) of sequence number 119, a light chain third complementarity determining region (VLCDR3) of sequence number 120, or substantially the same CDR as thereof.
The invention also provides an antibody specifically reacting with human TfR, wherein 1 or more amino acids are deleted, added, substituted and/or inserted in any amino acid sequence of sequence numbers 1-3, 7-9, 13-15, 19-21, 25-27, 31-33, 37-39, 43-45, 49-51, 55-57, 61-63, 67-69, 73-75, 79-81, 85-87, 91-93, 97-99, 103-105, 109-111, 115-117, 4-6, 10-12, 16-18, 22-24, 28-30, 34-36, 40-42, 46-48, 52-54, 58-60, 64-66, 70-72, 76-78, 82-84, 88-90, 94-96, 100-102, 106-108, 112-114, 118-120, and the activity of the antibody is equivalent to that of any one of claims 1-3.
Preferably, the antibody of the present invention is a human antibody or a humanized antibody.
Preferably, the antibody of the invention is selected from Fab, Fab ', F (ab')2Single chain antibody (scFv), dimerizationV regions (diabodies), disulfide stabilized V regions (dsfvs), and antibody fragments comprising peptides of CDRs.
According to the present invention, there is provided a DNA encoding the antibody of the present invention as described above.
According to the present invention, there is provided a recombinant vector containing the DNA of the present invention.
According to the present invention, there is provided a transformant obtained by introducing the recombinant vector of the present invention into a host cell.
According to the present invention, there is provided a method for producing an antibody of the present invention, comprising the steps of culturing the transformant of the present invention in a medium, producing and accumulating the antibody of the present invention in the culture, and collecting the antibody from the culture.
The present invention provides a pharmaceutical composition comprising the antibody of the present invention.
According to the present invention, there is provided the above-mentioned pharmaceutical composition in which a cell-damaging substance is bound to an antibody.
Preferably, the cell-damaging agent is a drug, toxin, or radioactive substance.
Preferably, the pharmaceutical composition of the present invention is used as an anticancer agent.
Preferably, the cancer is a solid cancer or a hematological cancer.
Preferably, the solid cancer is lung cancer, carcinoma of large intestine, gastric cancer, bladder cancer, pancreatic cancer, prostatic cancer, hepatocarcinoma, cervical cancer, metrocarcinoma, ovarian cancer, breast cancer, head and neck cancer, and skin cancer.
Preferably, the hematological cancer is leukemia, lymphoma, myeloma.
More preferably, the hematologic cancer is adult T cell leukemia (ATL).
According to the present invention, there is also provided a method for inhibiting or treating cancer, comprising the step of administering the antibody of the present invention described above to a subject.
The present invention also provides use of the antibody of the present invention for producing a pharmaceutical composition or an anticancer agent.
ADVANTAGEOUS EFFECTS OF INVENTION
The antibodies of the invention are fully human antibodies that specifically recognize human TfR, preventing the survival or proliferation of cancer cells expressing TfR. When a human antibody is administered to a human, the antibody avoids its antigenicity and does not produce HAHA, and therefore, the antibody can exert a high antitumor effect with less side effects. That is, the anti-human TfR antibody of the present invention is useful as an anticancer agent.
Drawings
Fig. 1 is a result of confirming TfR binding of an anti-TfR phage antibody using immunoprecipitation and western blotting.
FIG. 2 shows human antibody genes. The J chain (junction) gene and the C chain (constant) gene of the light chain lambda of the human antibody gene are 5J, C which are sequentially arranged in parallel, and J4-CL4 and J5-CL5 are pseudogenes and have no functions.
Fig. 3 shows the flow cytometric analysis results of TfRIgG antibody and cancer cell lines.
Figure 4 shows the anti-tumor effect of TfRIgG antibodies in various cancer models.
Figure 5 shows the anti-tumor effect of TfR006IgG antibody in the ATL model.
Figure 6 shows the reactivity of lung cancer tissue samples from the clinic with TfR006 phage antibody.
Detailed Description
Next, the present invention will be described in more detail.
Definitions and general techniques
In the present specification, unless defined otherwise, scientific and technical terms used in relation to the present invention include meanings commonly understood by those skilled in the art. In general, nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described in the present specification are those commonly used in the art.
The methods and techniques of the present invention are generally, and not exclusively, practiced in the absence of any other indication that is known in the art by employing the teachings of the various general and more specific references that are cited throughout the specification.
TfR
Human transferrin receptor (TfR) is a one-pass transmembrane protein (seq id No. 125) composed of 760 amino acids encoded by human chromosome three. This protein is known as the CD71 antigen, and is involved in the acquisition of iron by cells and in the proliferation of cells. The TfR of the present invention is not particularly limited in structure, and includes monomers, multimers, intatforms expressed on cell membranes, solubilizing forms composed of extracellular regions, trunctform, mutationforms due to mutations, deletions, etc., forms subjected to post-translational modification due to phosphorylation, etc., and all represent human tfrs.
Reaction and reactivity
In the present specification, "the reaction" and "reactivity" are not particularly limited, and mean the same. That is, the antibody recognizes the antigen. The antigen may be intactTfR expressed on the cell membrane, or may be truncatedform or soluble form. Further, TfR or modified TfR may be used in order to maintain the steric structure. Examples of the method for investigating reactivity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), western-blot, Fluorescence Microassay (FMAT) surface plasmon resonance (BIAcore), immunostaining, and immunoprecipitation.
The antibody used in the flow cytometry analysis may be an antibody labeled with a fluorescent substance such as FITC, biotin or the like, or may be an unlabeled antibody. Depending on the presence or absence of a label and the type of antibody used, avidin, an anti-human immunoglobulin antibody, or the like, which is fluorescently labeled, is used. The reactivity was evaluated by the following method: a sufficient amount of an anti-TfR antibody (usually at a final concentration of 0.01 to 10. mu.g/mL) was added to the sample to compare the reactivity of the negative control antibody and the positive control antibody.
Antibodies
In the present specification, the following notations (in parentheses) are used as required according to the convention.
A heavy chain (H chain), a light chain (L chain), a heavy chain variable region (VH), a light chain variable region (VL), Complementarity Determining Regions (CDRs), a first complementarity determining region (CDR 1), a second complementarity determining region (CDR 2), a third complementarity determining region (CDR 3), a first complementarity determining region of the heavy chain (VHCDR1), a second complementarity determining region of the heavy chain (VHCDR2), a third complementarity determining region of the heavy chain (VHCDR3), a first complementarity determining region of the light chain (VLCDR1), a second complementarity determining region of the light chain (VLCDR2), and a third complementarity determining region of the light chain (VLCDR 3).
In the present specification, "antibody" is synonymous with immunoglobulin and can be understood as generally known in the art. Specifically, the term antibody is not limited to any particular method of producing an antibody. For example, the antibody may be a recombinant antibody, a monoclonal antibody, and a polyclonal antibody.
In the present specification, the term "human antibody" refers to any antibody in which the sequences of the variable region and the constant region are human sequences. This term encompasses antibodies that have sequences derived from human genes, but which undergo changes that may cause removal of undesirably folded cysteine, etc., in view of, for example, reduction in immunogenicity and increase in affinity. The term also includes antibodies that can be produced by non-human cell-specific glycosylation and recombination in non-human cells. These antibodies can be prepared in various ways.
In the present specification, the term "humanized antibody" refers to a non-human antibody in which a characteristic amino acid residue in the sequence of the antibody of a non-human species is substituted for a residue identified at a position corresponding to a human antibody. In this "humanization" step, the resulting antibody reduces the immunogenicity of the human. It is understood that antibodies from non-human sources can be humanized using techniques well known in the art. For example, reference may be made to Winter et al, Immunol. Today14:43-46 (1993). The subject antibodies can be engineered by recombinant DNA technology that replaces human sequences corresponding to CH1, CH2, CH3, hinge regions, and/or framework regions. See, for example, WO92/02190, and U.S. Pat. Nos. 5530101, 5585089, 5693761, 5693792, 5714350, and 5777085. In the present specification, "humanized antibody" is within the meaning thereof, and includes chimeric antibodies and CDR-grafted antibodies.
In the antibody variable region of the present invention, the sequence of the Framework Region (FR) is not particularly limited as long as it does not substantially affect the specific binding property to the corresponding antigen. The FR region of a human antibody is preferably used, but it is also possible to use the FR region of an animal species other than human (e.g., mouse or rat).
In the present specification, the term "phage antibody" refers to an scFv antibody produced by a phage. I.e., antibody fragments comprising VH and VL amino acid sequences. The fragment includes an amino acid sequence as a tag in addition to the amino acid as Linker.
One embodiment of an antibody of the invention comprises a variable region and a constant region (e.g., an IgG-type antibody). The sequence of the constant region is not particularly limited. For example, the constant region of a known human antibody can be used. The heavy chain constant region (CH) of a human antibody may be of the hIgG type as long as it is a human immunoglobulin (hereinafter, referred to as hlg), and any of the subtypes hIgG1, hIgG2, GhIgG3, and hIgG4, which are hIgG types, can be used. Further, as the light chain constant region (CL), any one belonging to hIg may be used, and a kappa cluster or a lambda cluster may be used. In addition, constant regions from animal species other than human (e.g., mouse or rat) can also be used.
The amino acid sequence of the FR or constant region used in the antibody of the present invention may be the original amino acid sequence of the original FR or constant region as it is, or may be a different amino acid sequence in which 1 or several (for example, 1 to 8, preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 or 2) amino acids are deleted, added, substituted and/or inserted.
In the present invention, "activity equivalent to an antibody of the claim" means that the binding activity to human TfR and/or antitumor activity is equivalent. The binding activity means recognition of an antigen. The antigen may be intactTfR expressed in cell membrane, or trunctedform or solubilized form. Further, the TfR may be a TfR that retains a steric structure, or a modified TfR. Examples of the method for examining the binding activity include flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), western-blot, and surface plasmon resonance (BLAcore) using Fluorescence Microassay (FMAT). The antitumor activity means an activity of inhibiting the proliferation or survival of tumor cells. The proliferation or survival of the tumor cell is invitro or invivo. For example, the invitro includes an activity of reducing the number of tumor cells, an activity of inhibiting the increase of the number of tumor cells, an activity of inducing cell death of tumor cells, an Antibody-dependent cell-damaging Activity (ADCC), and a Complement-dependent cell-damaging activity (CDC). Examples of the activity of Invivo include an activity of reducing the weight or volume of a tumor, an activity of inhibiting an increase in the weight or volume of a tumor, an activity of promoting a reduction in the weight or volume of a tumor by another drug, and an activity of inhibiting death of an individual by tumor cells.
Examples of the animal model of Invivo include a xenograft model in which a cultured cell line derived from human cancer tissue is transplanted into an immunodeficient mouse such as a nude mouse, and a homograft model in which a cultured mouse cancer cell line is transplanted into a wild-type mouse having a normal immune system.
The xenograft model can be prepared by transplanting a human cancer cell line to various sites such as subcutaneous, intradermal, intraperitoneal, and intravenous sites of an immunodeficient mouse such as a nude mouse.
In the present invention, "equivalent" means not necessarily the same degree of activity, and the activity may be enhanced, or the activity may be decreased as long as it has the activity. Examples of the antibody having a decreased activity include an antibody having an activity of 30% or more, preferably 50% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more, as compared with the original antibody.
The above antibody may have 1 or several amino acids substituted, deleted, added and/or inserted in the amino acid sequence of the variable region (CDR sequence and/or FR sequence) as long as it has an equivalent binding activity to TfR or an equivalent antitumor activity. Methods well known to those skilled in the art for preparing an amino acid sequence of an antibody having a binding activity to TfR and/or an antitumor activity by deleting, adding, substituting and/or inserting 1 or several (for example, 1 to 8, preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 or 2) amino acids in the amino acid sequence are known as methods for introducing mutations into a protein. For example, if the skilled person is involved, an antibody having a site-specific mutation-inducing activity (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, anDNAkagawa, M. (1995) against a tumor-binding activity of an antibody having a tumor-binding activity against a nucleotide sequence of Escherichia coli, DNA fragment, V, Jansen, HW, Pfcymer, B, P fragment, M, F fragment, Hpprox (Hpprox) and DNA fragment, DNA fragment, DNA fragment, DNA fragment, DNA fragment, DNA fragment, DNA, fragment, DNA fragment, DNA, fragment, DNA, fragment, DNA, fragment, DNA, fragment, DNA, fragment.
Thus, antibodies having binding activity for TfR and/or anti-tumor activity, which have a mutation in the variable region of 1 or more amino acids, are also included in the antibodies of the present invention.
The phrase "CDR substantially identical thereto" in the present specification means a CDR constituting an antibody having a binding activity to TfR and/or an antitumor activity, in which 1 or more amino acids are deleted, added, substituted and/or inserted as described above.
The antibody of the present invention is not limited by its origin, and may be an antibody derived from any animal, such as a human antibody, a mouse antibody, or a rat antibody. Alternatively, a chimeric antibody, a humanized antibody, or the like may be used. Preferred embodiments of the antibodies of the invention are human antibodies.
The antibody of the present invention may vary in amino acid sequence, molecular weight, isoelectric point, presence or absence of a sugar chain, form, etc., depending on the cell or host producing the antibody described later or the purification method. The antibody obtained is included in the present invention as long as it has an activity equivalent to that of the antibody of the present invention. For example, the amino acid sequence described in the present invention is also included in the present invention when it is subjected to post-translational modification. In addition, post-translational modifications to a portion other than the known post-translational modifications are also included in the present invention as long as they have activity equivalent to that of the antibody of the present invention. When the antibody of the present invention is expressed in a prokaryotic cell such as E.coli, a methionine residue is added to the N-terminus of the amino acid sequence of the original antibody. The antibody of the present invention also encompasses such an antibody. The present invention also encompasses the use of a posttranslational modification other than a known posttranslational modification, as long as the modification has an activity equivalent to that of the antibody of the present invention.
Production of antibodies
(1) Generation of scFv reactive with antigen by phage display library
The antibodies of the invention can be obtained by any method known in the art. For example, using phage display technology, libraries can be provided that contain reservoirs of antibodies with varying affinities for TfR. These libraries are then screened to enable the identification and isolation of antibodies against TfR. Preferably, the phage library is a scFv phage display library generated using human VL and VHcDNA prepared from mRNA isolated from human B cells. Methods for preparing such libraries and screening are known in the art. Human TfR was used as an antigen and genetic material was recovered from phage clones showing screening reactivity. Analysis of the genes of the selected phage enables determination of the DNA sequences encoding VH and VL of the variable regions of human antibodies that bind to the antigen. Using this scFv sequence, a human antibody can be obtained by IgG-converting scFv.
(2) IgG conversion of scFv (production of human antibody)
Human antibodies are obtained by preparing expression vectors for H chain and L chain, expressing the vectors in host cells, collecting the secreted supernatant, and purifying the supernatant. Human antibodies can also be obtained by expressing VH and VL (concatemeric) on the same vector. These methods are well known and reference can be made to WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388, WO97/10354 and the like.
Specifically, a completely long heavy chain gene can be obtained by linking a DNA encoding VH to another DNA molecule encoding a heavy chain constant region (CH 1, CH2, and CH 3). The sequences of human heavy chain constant region genes are known in the art (e.g., Kabat, e.a. (1991) sequencesof proteins of immunologicalcalemtest, fifth edition, u.s.department of health and humanservices, nihpublications No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, and most preferably a constant region of IgG1 or IgG 2. The constant region sequence of IgG1 may be any of various alleles or allotypes known to occur among individuals, such as Gm (1), Gm (2), Gm (3), and Gm (17). These isoforms correspond to amino acid substitutions naturally occurring in the constant region of IgG 1.
The full-length light chain gene (and Fab light chain gene) can be obtained by linking the DNA encoding VL to another DNA molecule encoding a light chain constant region. The sequence of the human light chain constant region gene is known in the art (e.g., Kabat, e.a. (1991) sequencesof proteins of immunologicalcalemtest, fifth edition, u.s.department of health and humanservices, nihpublications No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. The kappa constant region may be any of various alleles known to occur between individuals, such as Inv (1), Inv (2), or Inv (3). The lambda constant region can be any from the 3 lambda genes.
The human antibody can be obtained by inserting the DNA encoding the H chain or L chain obtained as described above into an expression vector, preparing an expression vector, expressing the vector in a host cell, and recovering and purifying the secretion supernatant. Examples of the expression vector include plasmids, retrovirus, adenovirus, adeno-associated virus (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YAC, and EBV-derived episomes. The expression vector and the expression regulatory sequence are selected in a manner suitable for the host cell for expression to be used. The antibody light chain gene and the antibody heavy chain gene can be inserted into a vector, respectively, or both genes can be inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of a complementary restriction enzyme site on the antibody gene fragment and the vector, or blunt-ended ligation in the absence of a restriction enzyme site).
Suitable vectors are those which have a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction enzyme sites and which can be engineered to allow for the easy insertion and expression of any VH or VL sequences, as described above. Such vectors, in general, initiate splicing between the splice donor site in the inserted J region and the preceding splice acceptor site in the human C region, or in a splice region present within the human CH exon. Polyadenylation and termination of transcription occur at natural chromosomal sites downstream of the coding region. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned in a vector in such a way that a signal peptide is linked in frame to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide, or may also be a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
The expression vector of the antibody of the present invention may have, in addition to the antibody gene and the control sequence, a sequence that controls replication of the vector in a host cell (for example, an origin of replication), a selection marker gene, and the like. The selectable marker gene facilitates selection of the host cell into which the vector is introduced. For example, generally, a selectable marker gene confers resistance to drugs such as G418, hygromycin, methotrexate, and the like, on a host cell into which the vector is introduced. Preferred selectable marker genes include, for example, dihydrofolate reductase (DHFR) gene (used simultaneously with methotrexate selection/amplification in DHFR host cells), neomycin phosphotransferase gene (for G418 selection), and glutamate synthase gene.
Host cells were transformed with the antibody gene expression vector prepared in the above manner. The host cell may be any cell such as bacteria, yeast, animal cells, insect cells, plant cells, etc., as long as it can produce the antibody of the present invention, and animal cells are preferred. Examples of the animal cells include Chinese hamster ovary (CHO/dhfr) (-) cells, CHO/DG44 cells, monkey-derived COS cells (A.Wright & S.L.Morrison, J.Immunol.160,3393-3402 (1998)), SP2/O (mouse myeloma) (K.Motmansetotal., Eur.J.Cancer Prev.5,512-5199 (1996)), R.P.Junghanset., Cancer Res.50,1495-1502 (1990)), and the like. In addition, liposomes (R.W.Maloneet al, Proc.Natl.Acad.Sci, USA86,6007 (1989)), P.L.Felgneret al, Proc.Natl.Acad.Sci, USA84,7413 (1987)), electroporation method, calcium phosphate method (F.L.Graham & A.J.vanderEb, virology52,456-467 (1973)), DEAE-Dextran method, etc. can be suitably used for transformation.
After culturing the transformant, the human antibody is isolated from the cells or culture medium of the transformant. The separation and purification of the antibody can be carried out by a suitable combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.
Antibody fragments
Antibody fragments can be produced based on the antibody of the present invention or based on sequence information of the gene encoding the antibody of the present invention. Examples of the antibody fragment include Fab, Fab ', and F (ab')2scFv, dsFv antibodies.
The Fab is a fragment having a molecular weight of about 5 ten thousand, which is composed of an L chain and an H chain fragment obtained by digesting IgG with papain in the presence of cysteine, wherein the H chain fragment is composed of an H chain variable region, a CH1 region, and a part of a hinge portion. In the present invention, the antibody can be obtained by digesting the above antibody with papain. The Fab can be produced by recombining DNA encoding the L chain and a part of the H chain of the antibody into an appropriate vector, transforming the recombinant with the vector to obtain a transformant, and using the transformant.
Fab 'is F (ab')2The disulfide bond between H chains of (1) to give a fragment having a molecular weight of about 5 ten thousand. In the present invention, the antibody can be obtained by digesting the above antibody with pepsin and cleaving the disulfide bond with a reducing agent. In addition, the Fab-encoding DNA can be used to prepare the Fab-encoding DNA by genetic engineering.
F(ab’)2A fragment (Fab') consisting of L chain and H chain fragments obtained by pepsin digestion of IgG and having a molecular weight of about 10 ten thousand bound via disulfide bondsA segment wherein the H chain fragment consists of the H chain variable region, the CH1 region, and a portion of a hinge. In the present invention, the antibody can be obtained by digesting the above antibody with pepsin. In addition, similarly to Fab, F (ab')2The DNA of (4) is prepared by a genetic engineering method.
The scFv is a single-chain antibody fragment in which Fv composed of an H chain variable region and an L chain variable region is connected to the C-terminus of one chain and the N-terminus of the other chain via a peptide linker. As the peptide linker, for example, a peptide linker having high flexibility (GGGGS)3And the like. For example, a DNA encoding an scFv antibody is constructed using a DNA encoding the H chain variable region and the L chain variable region of the above antibody and a DNA encoding a peptide linker, and the DNA is recombined into an appropriate vector, and a transformant is transformed using the vector, and from the transformant, an scFv can be produced.
The dsFv is an Fv fragment stabilized by introducing a Cys residue into the H chain variable region and the L chain variable region at an appropriate position, and binding the H chain variable region and the L chain variable region via a disulfide bond. The position of Cys residue introduction in each chain is determined by the steric structure predicted by molecular simulation. In the present invention, for example, the three-dimensional structures composed of the amino acid sequences of the H chain variable region and the L chain variable region of the above-mentioned antibody are predicted, DNAs encoding the H chain variable region and the L chain variable region, respectively, into which mutations have been introduced based on the prediction, are constructed, and these DNAs are recombined into an appropriate vector, and a transformant is obtained by transformation using the vector, and dsFv can be produced from the transformant.
Furthermore, antibody fragments can be quantified in large amounts by linking them with an scFv antibody, dcFv antibody, or the like, or by fusing streptavidin using an appropriate linker.
Pharmaceutical composition
According to the present invention, there is provided a pharmaceutical composition comprising the antibody of the present invention. One embodiment of the present invention is the treatment of cancer, but is not limited thereto. Diseases other than cancer caused by high expression of TfR are also included in the scope of the present invention. In a more preferred embodiment, the cancer is a solid cancer (e.g., lung cancer, colorectal cancer, stomach cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, cervical cancer, uterine cancer, ovarian cancer, breast cancer, head and neck cancer, skin cancer, etc.), or a hematological cancer (e.g., leukemia, lymphoma, myeloma, etc.). In another preferred embodiment of the present invention, the cancer is adult T-cell leukemia (ATL).
In one embodiment of the pharmaceutical composition of the present invention, the antibody of the present invention is used as an active ingredient. This exerts an antitumor effect by utilizing the cell growth inhibitory activity, cell death inducing activity, ADCC activity, CDC activity and the like of the antibody. The antibody may have only one of the above activities, or may have a plurality of activities at the same time. That is, the naked antibody is an active ingredient of the pharmaceutical composition.
In another embodiment, the antibody of the present invention can be used as a cancer therapeutic agent for a cancer cell targeted destruction therapy (missiletherapy) that specifically targets cancer tissues. That is, a therapeutic method in which an antibody bound to a substance that damages cancer cells is administered to specifically move the antibody to a cancer site, thereby achieving a therapeutic effect and reducing side effects.
Examples of the substance that damages cancer cells include cell-damaging substances such as drugs, toxins, and radioactive substances. The binding of the cell-damaging agent to the antibody can be carried out by a method known to those skilled in the art (ClinCancerRes.2004Jul 1;10 (13): 4538-49).
As the agent that binds to the antibody, a known substance that damages cancer cells can be used. Examples thereof include Duocarmycin, analogues and inducers of Duocarmycin, CC-1065, Duocarmycin mimetics comprising CBI as the main component, Duocarmycin analogues comprising MCBI as the main component, Duocarmycin analogues comprising CCBI as the main component, doxorubicin conjugates, morpholino doxorubicin, cyanomorpholino doxorubicin, dolastatin-10, combretastatin, calicheamicin (calicheamicin), maytansine analogues, DM1, DM2, DM3, DM4, DMI, auristatin E (auristatin E), auristatin EB (AEEFP), and auristatin E EFP (AEFP)) Monomethylauristatin (MMAE), monomethyl auristatin F (MMAF), AE ester of 5-benzoylvaleric Acid (AEVB), tubulysin, bissalazole (Disorazole), epothilone, paclitaxel, docetaxel, SN-38, topotecan, rhizomycin, echinomycin, colchicine, vinblastine, vindesine, estramustine, cimadrol, leuziroside, methotrexate, methyl folic acid, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytarabine, milfoil, lomonon, lomustine, actinomycin, daunomycin conjugates, mitomycin C, mitomycin A, carminomycin, aminopterin, talithromycin, podophyllotoxin derivatives, etoposide phosphate, vincristine, paclitaxel, tretinomycin, tyrosol, tyrosinic acid, N is a radical of8-acetylspermidine, camptothecin and the like, but are not limited thereto.
The antibody and the agent may be directly bound via a linker or the like, or may be indirectly bound via a linker or other substance.
Examples of the linking group to which the agent is directly bonded include a disulfide bond using an SH group and a maleimide bond. For example, the intramolecular disulfide bond of the Fc region of the reducing antibody binds to the disulfide bond of the agent via a disulfide bond. Further, there is a method by maleimide. In addition, another method is a method of introducing cysteine into an antibody by genetic engineering.
The antibody and the agent may be indirectly bound via another substance (linker). The linker preferably has 1 or more than 2 functional groups that react with the antibody or the agent or both. Examples of the functional group include an amino group, a carboxyl group, a mercapto group, a maleimide group, and a pyridyl group.
Examples of linkers include N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate) (LC-SMCC), N-succinimidyl kappa-maleimidoundecanoate (KMUA), N-succinimidyl gamma-maleimidobutyrate (GMBS), N-hydroxysuccinimidyl maleimidohexanoate (EMCS), N-hydroxysuccinimidyl m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), N- (. alpha. -maleimidoacetoxy) -succinimidyl ester (AMAS), succinimidyl-6- (. beta. -maleimidopropionamido) hexanoate (SMPH), N-succinimidyl-4- (p-maleimidophenyl) -butyl ester (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI), 6-Maleimidocaproyl (MC), Maleimidopropanoyl (MP), p-aminophenoxycarboxyl (PAB), N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), and the like, but are not limited thereto. Further, the linker may be, for example, a peptide linker such as valine-citrulline (Val-Cit) or alanine-phenylalanine (ala-phe), and the above-mentioned linkers may be used in combination.
As for the binding method of the agent and the antibody, for example, it can be based on cancer research, 68(22)9280(2008), Nature Biotechnology, 26(8)925(2008), BioConjudgeTehemistry, 19, 1673 (2008), cancer research;68 (15) 6300 (2008), or Japanese patent application laid-open No. 2008-516896.
Examples of the toxin include so-called immunotoxins that are chemically or genetically bound to an antibody. Examples of the toxin include diphtheria toxin a chain, pseudomonas toxin, ricin chain, ricin a chain without sugar chain, gelonin saporin, and the like.
The radioactive substance to be used can be any known substance to those skilled in the art. For example, yttrium 90: (90Y), rhenium 186 (186Re), rhenium 188 (188Re), copper 67(67Cu), iron 5959Fe), strontium 89 (89Sr), gold 198198Au), mercury 203: (203Hg), lead 212: (212Pb) and dysprosium 165 (165Dy), ruthenium 103（103Ru), bismuth 212 (212Bi), bismuth 213 (213Bi), holmium 166 (166Ho), samarium 153 (153Sm), lutetium 177 (177Lu), and the like. Preference is given to90Y、153Sm、177Lu。
The binding of the antibody to the radioactive substance can be carried out by a method known to those skilled in the art (BioconjugChem.1994Mar-Apr; 5 (2): 101-4.)
Cancer treatment using antibodies conjugated to compounds containing radioisotopes can be performed by methods well known to those skilled in the art (bioconjugchem.1998 Nov-Dec;9(6): 773-82). Specifically, an antibody to which a compound containing a radioisotope is bound is initially administered to a patient in a small amount, and whole body scintigraphy is performed. It was confirmed that the binding of the antibody to the cells of the normal tissue was small and the binding of the antibody to the cancer cells was large, and further, the antibody to which the compound containing a radioisotope was bound was administered in a large amount.
Formulations comprising pharmaceutical compositions of anti-human TfR antibodies of the invention are also within the scope of the invention. Such a preparation preferably contains a physiologically acceptable diluent or carrier in addition to the pharmaceutical composition containing the antibody, and may be a mixture with another agent such as another antibody or an anticancer agent. Suitable carriers may include, but are not limited to, saline, phosphate buffered saline dextrose solution, and buffered saline. Alternatively, the antibody may be lyophilized (freezedry) and, if necessary, reconstituted by adding an aqueous buffer solution as described above. Examples of administration forms include oral administration such as tablets, capsules, granules, powders, syrups, and the like, or parenteral administration such as injections (subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, and the like), transdermal administration, transmucosal administration, nasal administration, pulmonary administration, suppositories, and the like. The pharmaceutical composition of the present invention may be formulated for administration alone or in combination with other agents.
The dosage of the pharmaceutical composition of the present invention varies depending on the symptoms, age, body weight, etc., but in general, when administered orally, the amount of the antibody is about 0.01mg to 1000mg per 1 day of an adult, and is administered 1 time or several times. When the non-oral administration is carried out, about 0.01mg to 1000mg is administered by subcutaneous injection, intramuscular injection or intravenous injection 1 time.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
Examples
Example 1: phage antibody screening using cancer cell lines
(1) Screening of phage antibody binding to cancer cells (hepatoma cell line HepG 2)
HepG2 cells were cultured in a 15cm dish, detached from the dish by 2mg/ml of collagenasel/cellsissociationBuffer (GibcoBRL), recovered, washed with chilled PBS, and mixed with 1 × 1013The reaction solution (1% BSA, 0.1% NaN) was added to a human antibody phage library of cfu (see Japanese patent laid-open Nos. 2005-185281, WO2008/007648, and WO 2006/090750)3MEM) was added so that the final volume was 1.6mL, and the reaction was carried out by slowly rotating the flask at 4 ℃ for 4 hours. After completion of the reaction, the reaction mixture was divided into two portions, 0.6mL of an organic solvent (dibutyl phthalate cycloheximide9: 1) was added to each portion, and the mixture was centrifuged by a microcentrifuge for 2 minutes (3000 rpm). The supernatant was removed, and the cells precipitated on the bottom of the tube were suspended in 0.7mL of 1% BSA/MEM, and 0.7mL of an organic solvent was added thereto. The supernatant was removed by centrifugation as well, and the cells were suspended in 0.3mL of PBS and frozen with liquid nitrogen.
The frozen cells were thawed at 37 ℃ and 20mL of E.coli DH12S (OD 0.5) were infected with 1 hour, phage-infected E.coli was placed in 600mL of 2 × YTGA medium (2 × XYT, 200. mu.g/mLampicisulte, 1% Glucose), and cultured at 30 ℃ overnight, after which 10mL were removed and 200mL of 2 × YT was placedA Medium (2 × YT, 200. mu.g/mLambicisulfate), incubated at 37 ℃ for 1.5 hours, 1 × 1011The helper phage KO7 was cultured at 37 ℃ for 1 hour, 800mL of 2 × YTGAK medium (2 × YT, 200. mu.g/mLamicissute, 0.05% Glucose, 50. mu.g/mLkanamycin) was added, and the culture was carried out at 30 ℃ overnight, 10 minutes centrifugation (8000 rpm) was performed to recover the supernatant, 200mL of PEG solution (20% polyethyleneglycol6000, 2.5 MNaCl) was added to the recovered supernatant, followed by sufficient stirring and 10 minutes centrifugation (8000 rpm) was performed to precipitate the phage, which was suspended in 10mL of PBS to prepare the first round of selected phage.
Next, a second round of sifting, blend 2 × 10 was performed7Cultured cells and 1 × 1010The phage selected in the first round were added with the reaction solution (1% BSA, 0.1% NaN)3MEM) to a final volume of 0.8 mL. Thereafter, the second round of screening was performed in the same manner as in the first round of screening to obtain phages.
Third round of screening used 1 × 109The second round of selected phages was performed in the same manner as described above.
(2) Analysis of phage antibodies
The phage obtained in the third round of screening was collected, and the DNA sequence was analyzed by a known method to discharge an incomplete antibody or an antibody having a repeated sequence, which retains a defective region, and obtain phage antibodies having independent antibody sequences (see patent No. 4870348).
In the same manner, phage antibodies reactive with cancer antigens were screened using 21 types of cancer cells shown in table 1 below. As a result, 1863 phage antibodies having independent sequences were obtained as shown in table 1.
[ Table 1]
| Cancer cell | Number of phage obtained |
| CO-2 | 102 |
| MKN45 | 90 |
| OCTH-16 | 82 |
| HepG2 | 410 |
| NCI-H441 | 80 |
| K562 | 33 |
| U937 | 107 |
| HL-60 | 107 |
| MV4-11 | 46 |
| KF28 | 62 |
| NCI-N87 | 50 |
| RERF-LC-AI | 73 |
| SW480 | 46 |
| MCF7 | 73 |
| LNCap.FGC | 60 |
| MDA-MB-231 | 78 |
| U-87MG | 62 |
| T98G | 71 |
| DU-145 | 96 |
| MMAc | 76 |
| G-361 | 59 |
Example 2: screening of phages reactive with soluble human TfR
(1) Production of soluble TfR antigen-producing cells
TfR cDNA was prepared by PCR using cancer cell lines MIAPaCa2 and SKOV-3. A soluble TfR antigen expression vector was prepared by inserting pCMV-Script (Clonetech) into a cDNA prepared in the extracellular region of TfR by a conventional method. This expression vector was introduced into cell line 293T to prepare expression cells producing a soluble TfR antigen.
(2) Positive phage selection by ELISA
The supernatant of the soluble TfR-producing cells was recovered and purified to obtain soluble TfR antigen. Using this soluble TfR antigen, the reactivity of the antigen antibody was studied by ELISA. That is, the soluble TfR antigen was adjusted to 10. mu.g/mL with PBS, 50. mu.L/well was added to ImmunoModule/StripPlates (NUNK), and the mixture was allowed to stand at 37 ℃ for 2 hours. Thereafter, the soluble TfR antigen was removed and blocking solution (5% skim milk/0.05% NaN) was added at 200. mu.L/well3PBS), blocking at 37 ℃ for 2 hours. The blocking solution was removed, washed with PBS, and 100. mu.L/well of the culture supernatant of the above phage (Table 1) was added and reacted at 37 ℃ for 1 hour. After washing 5 times with PBS, 100. mu.L/well of 1. mu.g/mLRabbitanti-cp 3 diluted with PBS/0.05% Tween20 was added and reacted at 37 ℃ for 1 hour. After washing 5 times with PBS, 2000-fold dilution of anti-RabbitIgG (H + L) -HRP with PBS/0.05% Tween20 was added in an amount of 100. mu.L/well, and the reaction was carried out at 37 ℃ for 1 hour. After washing 5 times with PBS, 100. mu.L/well of OPDin0.1M citrate phosphate buffer (pH 5.1) +0.01% H was added2O2And reacted at room temperature for 5 minutes. Add 100. mu.L/well of 2NH2SO2Thereafter, absorbance at 492nm was measured by SPECTRAma × 340PC (molecular devices). As a result, 20 of the 1863 phage strains showed a significant positive reaction with the soluble TfR antigen, and the DNA sequences of the 20 phage strains were analyzed to confirm that the respective CDR sequences were novel.
(1) Tfr001 antibodies
VHCDR 1: sequence number 1, VHCDR 2: sequence number 2, VHCDR 3: sequence No. 3
VLCDR 1: sequence number 4, VLCDR 2: sequence number 5, VLCDR 3: sequence number 6
(2) TfR002 antibody
VHCDR 1: sequence number 7, VHCDR 2: sequence number 8, VHCDR 3: serial number 9
VLCDR 1: sequence number 10, VLCDR 2: sequence number 11, VLCDR 3: serial number 12
(3) Tfr003 antibodies
VHCDR 1: sequence number 13, VHCDR 2: sequence number 14, VHCDR 3: serial number 15
VLCDR 1: sequence number 16, VLCDR 2: sequence number 17, VLCDR 3: serial number 18
(4) Tfr004 antibodies
VHCDR 1: sequence number 19, VHCDR 2: sequence number 20, VHCDR 3: serial number 21
VLCDR 1: sequence number 22, VLCDR 2: sequence number 23, VLCDR 3: serial number 24
(5) Tfr005 antibodies
VHCDR 1: sequence number 25, VHCDR 2: sequence number 26, VHCDR 3: serial number 27
VLCDR 1: sequence number 28, VLCDR 2: sequence number 29, VLCDR 3: serial number 30
(6) Tfr006 antibodies
VHCDR 1: sequence number 31, VHCDR 2: sequence number 32, VHCDR 3: sequence number 33
VLCDR 1: sequence number 34, VLCDR 2: sequence number 35, VLCDR 3: serial number 36
(7) Tfr007 antibodies
VHCDR 1: sequence number 37, VHCDR 2: sequence number 38, VHCDR 3: serial number 39
VLCDR 1: sequence number 40, VLCDR 2: sequence number 41, VLCDR 3: serial number 42
(8) Tfr008 antibodies
VHCDR 1: sequence number 43, VHCDR 2: sequence number 44, VHCDR 3: serial number 45
VLCDR 1: sequence number 46, VLCDR 2: sequence number 47, VLCDR 3: serial number 48
(9) TfR009 antibody
VHCDR 1: sequence number 49, VHCDR 2: sequence number 50, VHCDR 3: serial number 51
VLCDR 1: sequence number 52, VLCDR 2: sequence number 53, VLCDR 3: serial number 54
(10) Tfr010 antibodies
VHCDR 1: sequence number 55, VHCDR 2: sequence number 56, VHCDR 3: serial number 57
VLCDR 1: sequence number 58, VLCDR 2: sequence number 59, VLCDR 3: serial number 60
(11) TfR011 antibodies
VHCDR 1: sequence number 61, VHCDR 2: sequence number 62, VHCDR 3: serial number 63
VLCDR 1: sequence number 64, VLCDR 2: sequence number 65, VLCDR 3: serial number 66
(12) Tfr012 antibodies
VHCDR 1: sequence number 67, VHCDR 2: sequence number 68, VHCDR 3: serial number 69
VLCDR 1: sequence number 70, VLCDR 2: sequence number 71, VLCDR 3: serial number 72
(13) TfR013 antibody
VHCDR 1: sequence number 73, VHCDR 2: sequence number 74, VHCDR 3: serial number 75
VLCDR 1: sequence number 76, VLCDR 2: sequence number 77, VLCDR 3: serial number 78
(14) Tfr014 antibody
VHCDR 1: sequence number 79, VHCDR 2: sequence number 80, VHCDR 3: serial number 81
VLCDR 1: sequence number 82, VLCDR 2: sequence number 83, VLCDR 3: serial number 84
(15) TfR015 antibodies
VHCDR 1: sequence number 85, VHCDR 2: sequence number 86, VHCDR 3: sequence No. 87
VLCDR 1: sequence number 88, VLCDR 2: sequence number 89, VLCDR 3: serial number 90
(16) TfR016 antibodies
VHCDR 1: sequence number 91, VHCDR 2: sequence number 92, VHCDR 3: serial number 93
VLCDR 1: sequence number 94, VLCDR 2: sequence number 95, VLCDR 3: serial number 96
(17) TfR017 antibodies
VHCDR 1: sequence number 97, VHCDR 2: sequence number 98, VHCDR 3: serial number 99
VLCDR 1: sequence number 100, VLCDR 2: sequence number 101, VLCDR 3: serial number 102
(18) TfR018 antibodies
VHCDR 1: sequence number 103, VHCDR 2: sequence number 104, VHCDR 3: serial number 105
VLCDR 1: sequence number 106, VLCDR 2: sequence number 107, VLCDR 3: serial number 108
(19) TfR019 antibodies
VHCDR 1: sequence number 109, VHCDR 2: sequence number 110, VHCDR 3: serial number 111
VLCDR 1: sequence number 112, VLCDR 2: sequence number 113, VLCDR 3: serial number 114
(20) Tfr020 antibodies
VHCDR 1: sequence number 115, VHCDR 2: sequence number 116, VHCDR 3: serial number 117
VLCDR 1: sequence number 118, VLCDR 2: sequence number 119, VLCDR 3: serial number 120
Sequence number 1: TSGVG
Sequence number 2: LIYWDDDKHYSPSLKS
Sequence number 3: NGDYGIEFDY
Sequence number 4: GGNNIGSKSVH
Sequence number 5: YDSDRPS
Sequence number 6: QVWDSSSDHVV
Sequence number 7: SYSMN
Sequence number 8: SISSSSSYIYYADSVKG
Sequence number 9: ARESVDAFDI
Sequence number 10: QGDSLRSYDAS
Sequence number 11: GLSDRPS
Sequence number 12: ISRDSGGNPH
Sequence number 13: SYAMS
Sequence number 14: AISGSGGSTYYADSVKG
Sequence number 15: GYYGSNYYYGMDV
Sequence number 16: SGSSSNIGSNYVY
Sequence number 17: RNNQRPS
Sequence number 18: AAWDDSLSGPV
Sequence number 19: DFVFS
Sequence number 20: WISAHDGNTNYAQKLQD
Sequence number 21: DTFTNLLGDYSYDAMDV
Sequence number 22: GSSTGAVTSGHYPY
Sequence number 23: DTTEKHS
Sequence number 24: LLSSGDGRAV
Sequence number 25: NYPSM
Sequence number 26: WISAYNGNTNYGEKLQG
Sequence number 27: DDYYGSGVDAFDI
Sequence number 28: GGNKIGSKSVH
Sequence number 29: YDRDRPS
Sequence number 30: QVWDSSSDVV
Sequence number 31: SYGMH
Sequence number 32: VISFDGSSKYYADSVKG
Sequence number 33: DSNFWSGYYSPVDV
Sequence number 34: TRSSGSIASNSVQ
Sequence number 35: YEDTQRPS
Sequence number 36: QSYDSAYHWV
Sequence number 37: SYWLS
Sequence number 38: KIDPSDSYTQYSPSFEG
Sequence number 39: HGYDAFHV
Sequence number 40: SGSSSNIGNNAVN
Sequence No. 41: YDDLLPS
Sequence number 42: AAWDDSLNGWV
Sequence number 43: DYAMH
Sequence number 44: GISWNSGSIGYADSVKG
Sequence number 45: DQHREFYYYGMDV
Sequence number 46: SGSSSNIGSNYVY
Sequence number 47: RNNQRPS
Sequence number 48: AAWDDSLSGPV
Sequence number 49: SYWIG
Sequence number 50: IIYPGDSDTRYSPSFQG
Sequence number 51: QGTNWGVGDAFDI
Sequence number 52: GGNNIGSKSVH
Sequence number 53: DDSDRPS
Sequence number 54: QVWDISSDHVV
Sequence number 55: SYAMS
Sequence number 56: AISGSGGSTYYADSVKG
Sequence number 57: DRYYYGSGSYYDAFDI
Sequence number 58: QGDSLRSYYAS
Sequence number 59: GKNNRPS
Sequence number 60: NSRDSSGNHVV
Sequence number 61: SYSMN
Sequence number 62: VISYDGSNKYYADSVKG
Sequence number 63: VDPGDRGWYFDL
Sequence number 64: SGSSSNIGSNTVN
Sequence number 65: SNNQRPS
Sequence number 66: AAWDDSLNGWV
Sequence number 67: SSPYYWG
Sequence number 68: SVYYSGNTYYNPSLTR
Sequence number 69: HSWGINDAFDV
Sequence number 70: SGSSSNIGNNYVS
Sequence number 71: DNNKRPS
Sequence number 72: GTWDSSLSVWV
Sequence number 73: DYAMH
Sequence number 74: GISWNSGSIDYADSVKG
Sequence number 75: ENLAVAGLDY
Sequence number 76: QGDSLRGYYAS
Sequence number 77: DKnPRPS
Sequence number 78: QSRDNSGEMVV
Sequence number 79: ELSMH
Sequence number 80: GFDPEDGETIYAQKFQG
Sequence number 81: DAYYGSGSPRDAFDI
Sequence number 82: GGDNVGGKSLH
Sequence number 83: DDRDRPS
Sequence number 84: QVWDDISRLVI
Sequence number 85: SYYIH
Sequence number 86: IINPRGGGTDFAQKFQG
Sequence number 87: GDCTNGVCYSGGLDV
Sequence number 88: SGSSSNIGNNYVS
Sequence number 89: DNDKRPS
Sequence number 90: GTWDNSLSGV
Sequence number 91: DYAMH
Serial number 92: GISWNSGSIGYADSVKG
Sequence number 93: DVDLWFGEYYFDY
Sequence number 94: SGSSSNIGNNYVS
Sequence number 95: DNNKRPS
Sequence number 96: GTWDSSLSAPYV
Sequence number 97: DYAMY
Sequence number 98: GINWNSAIIGYADSVKG
Sequence number 99: EALYYSAFFDS
Sequence number 100: SGSSSNIGNNYVS
Sequence number 101: DNNKRPS
Sequence number 102: GTWDSSLSAWV
Sequence number 103: DYAMH
Sequence number 104: GINWNGGSTDYADSVEG
Sequence number 105: DYADLGSGSDY
Sequence number 106: SGSRSNIGSNYVH
Sequence number 107: RNDQRPS
Sequence number 108: ASWDDKMSGRL
Sequence number 109: SYEMN
Sequence number 110: YISSSGSTIYYADSVKG
Sequence number 111: HSNYDILTGYSTDAFDI
Serial number 112: TGTSSDIGFYDSVS
Sequence number 113: DVSNRPS
Sequence number 114: TSNTKTNTLYV
Sequence number 115: RGNYWWT
Sequence number 116: SVHYSGSTNYNPSLKS
Serial number 117: DSDYGDYYFDY
Sequence number 118: QGDSLRSYYAS
Sequence number 119: GKNNRPS
Sequence number 120: NSRDSSGNHVV
Example 3: tfr reactivity confirmation of anti-Tfr phage antibodies
(1) Immunoprecipitation
To confirm that the above 20 phage antibodies recognize human TfR, immunoprecipitation and western blotting were performed. Coli was infected with 20 types of phages, and the culture supernatant was recovered and purified to obtain a purified scFv antibody. In the glass filter, relativeAntibody beads were prepared by immobilizing 5mg of the antibody on CNBr-activatedsepharose4B1 mL. Then, the mixture was recovered at 10cm3Cell lysates were prepared at 600. mu.L from SKOV-3 cells cultured in petri dishes. mu.L of biotin was placed in 600. mu.L of cell lysate and the antigen was biotinylated. The prepared antibody bead solution (150. mu.L) and biotinylated cell lysate were put into a 2mL tube and stirred at 4 ℃ for 6 hours. The tubes were centrifuged (5500 g, 1 min, 4 ℃), the supernatant removed, 800. mu.L of washing buffer (0.5 mM thiotin, 0.1% Tween 20/PBS) was added, and the beads were washed by centrifugation. After washing of the beads was repeated 3 times, 30. mu.L of citric acid solution for elution (50 mM citric acid pH 2.5) was added, and after stirring, centrifugation (5500 g, 1 minute, 4 ℃) was performed to collect the supernatant, whereby the immunocomplexes were eluted. The elution was repeated 3 times to recover the supernatant. Neutralization was performed by adding 3M Tris, and electrophoresis was performed by SDS-PAGE. The bands were confirmed by SDS-PAGE and silver staining. The sample was subjected to Western blotting using Streptavidin-HRP (Anti-Streptavidin, IgGFracton, Conjugatedtoperoxidases Corbichem) at the same time. As a result, as shown in fig. 1, it was confirmed that each of the antibodies (TfR 001, TfR003, TfR 005) bound to a protein having a molecular weight of about 90KD (TfR having a molecular weight of about 90 KD).
(2) Mass analysis
Subsequently, the antigen protein obtained by the immunoprecipitation method was subjected to mass analysis.
The fraction corresponding to the membrane protein detected was subjected to trypsin digestion in the gel to recover the peptide. The bands obtained by Coomassie blue staining were excised by SDS polyacrylamide gel electrophoresis according to a conventional method. This was immersed in a 200mM ammonium bicarbonate-50% acrylonitrile solution, shaken at 37 ℃ for 45 minutes, and then the solution was discarded, and the same operation was repeated twice to remove Coomassie Brilliant blue. Drying the gel under reduced pressure per unit area (mm)2) The gel sheet was added with 4. mu.L of trypsin (20. mu.g/mL) dissolved in 40mM ammonium bicarbonate (pH 8.1) -10% acrylonitrile and left at room temperature for 1 hour to be sufficiently soaked. Adding into the previous step2.5 times of trypsin was added easily, and the mixture was left standing at 37 ℃ for 18 hours. This was filtered through a tube having a filter with a pore size of 0.22 μm, and the peptide produced by cleaving the antigen with trypsin was recovered.
The sample obtained by digestion with trypsin in the gel was placed in an HPLC connected to an electrospray ionization ion trap quadrupole mass spectrometer. The mass and internal amino acid sequence of each peptide was determined by ionizing each peptide sequentially eluted with a difference in hydrophobicity by an electrospray method from a reverse phase column of HPLC by a linear concentration gradient of 0% to 80% acrylonitrile containing 0.1% TFA. The resulting alignment of the internal amino acid sequence was searched using the published TfR amino acid sequence database. As a result, it was confirmed that the phage antibody binds to TfR.
Example 4: IgG conversion of phage antibodies (scFv)
(1) Preparation of plasmid expressing TfR006IgG antibody
IgG conversion of phage antibody was described below, taking IgG conversion of TfR006 as an example. Other antibodies were IgG-converted by the same method.
The genes of the phage antibody (scFv) of TfR006 are arranged in the order VH-VL to form the structure of scFv in which VH and VL are linked by a linker (SEQ ID NO: 121).
VH consists of V, D, J genes, VL consists of V, J genes. In the case of human light chain lambda, 5 sets of J lambda (lambda Junction) gene and C lambda (lambda constant) gene were arranged in parallel, and J4-CL4 and J5-CL5 were pseudogenes (FIG. 2).
The results of searching for the human reproductive series genes presumed to be used in VH and VL of TfR006 using IMGT (x) are shown in table 2.
（*）IMGT：http://www.imgt.org
[ Table 2]
IgG conversion of phage antibody was performed with reference to the search results of IMGT. Genes in which VH of TfR006 was linked to the constant region of human G1 (SEQ ID NO: 122), and VL of TfR006 was linked to IGLC3 (SEQ ID NO: 123) juxtaposed to the IGLJ3 gene were synthesized by Genscript. In the case of full-length artificial synthesis, optimization of codon usage frequency was carried out (according to kim et al, method of Codonotimization for high-level expression of humanerthiological spinostimulation peptides, Cene, VoL199, 1997p 293-301), DNA sequence for efficient translation (Kozak, Atleatsixnucletotitid expression of Aunititiationgenetic modification of proteins) was added to the 5' side of heavy and light chain genes as a signal for secretion (SEQ ID NO: 124), and a consensus sequence of signal peptides of human antibody heavy chain three lines (substoup 3). In addition, for recombination into an expression vector, NheI was added to the 5 '-side and EcoRI was added to the 3' -side of both ends of the synthesized heavy and light chain genes.
As an expression vector for an antibody Gene, pCAGGS (Niwa et al, effective selection for high-expression transfection and transformation vector, Gene, Vol108, p 193-200) was used, and a mouse DHFR Gene expression region for Gene amplification was inserted into the HindIII site of the vector.
(2) Transient expression of TfR006IgG antibody
Transient expression of TfR006IgG antibody was passaged the day before using Freestyle (Life technologies), 293-F as suspension cell for gene introduction (Life technologies), day of transfection, adjusted to 1 × 106cell/mL cell concentration 400mL cell suspension. A solution (I) of 100. mu.g of TfR006 heavy chain expression vector and 100. mu.g of TfR006 light chain expression vector totaling 200. mu.g of plasmid suspended in OptiProSFM was prepared. Next, 200. mu.L of MAXreagen was added to 8mL of OptiPROSFM (solution II). Mixing the solution (I) and the solution (II) at room temperatureAnd standing for 10 to 20 minutes. The total of 16mL of the reaction solution was added to 400mL of 293 expression medium in which 293-F cells were suspended, and the reaction solution was incubated at 37 ℃ and 8% CO for 6 to 7 days2Culturing in a cell culture shaker TAITECBioshaker BR-43 FL. After 6 to 7 days, the culture supernatant containing the recombinant antibody of TFR006 was recovered and purified into a material.
(3) Establishment of Stable production Strain of TfR006IgG antibody
CHOdhfr (-) cells (G.Urlaub et al, isolationof Chinesebamse cells and recombinant plasmid DNA, Proc.Natl.Acad.Sci.USA77, p4216-4220, 1980) were used to simultaneously transform a pCAGGS-IGL-CMV-dhfr-A vector for expressing TFR006L chain and a pCAGGS-IGH-CMV-dhfr-A vector for expressing TFR006H chain, by cleaving the plasmids at Pvul in ampicillin resistance gene and converting them from circular plasmids to linear plasmids, and electroporation was carried out by adding DNA (0.002 mg/sample of each of L chain and H chain) to 3 × 10 using Amaxa. manufactured by Lonza30.1mL of Amaxa electroporation CHO buffer was added to the cells, and electric pulses were applied.
The electroporation-treated cells were added to Iscove's Modified Dulbecco's Medium (IMDM) containing 10% dialyzed FBS, not containing HT (H: hypoxanthine, T: thymine). After 3 days from the gene transfer, neo + transformed cells were selected using 1mg/mL of G418 by replacing the medium with IMDM without 10% dialyzed FBS, 2 mML-glutamine, and HT, to obtain colonies of TfR006IgG antibody-producing positive cell lines. Subsequently, the colonies selected with G418 were used for gene amplification. After two rounds of amplification (2 round) of Methotrexate (MTX) at 0.25mM, 1mM, cell lines producing approximately 50mg of TfR006IgG antibody per 1 liter were established.
(4) Purification of TfR006IgG antibody
The TfR6IgG antibody protein contained in the culture supernatant of the transiently expressed or stably expressed strain was purified by using Ab-CapcherExTra (protenova) affinity column of AKTAprime. The peak fraction obtained was gel-filtered using a Sephadex S-300 column equilibrated with Dulbecco' S PBS as a solvent, and then purified. The absorbance coefficient of the purified TfR006IgG antibody was calculated using the total amino acid sequence of TfR006 at ProtParam (http:// web. EXPASY. org/ProtParam /) of EXPASY, and was = 1.607.
(5) Quantification of TfR006IgG antibody by enzyme immunoassay (ELISA)
The concentration of the antibody contained in the culture supernatant of the TfR006IgG antibody-producing cells, the purified antibody was quantified by absorbance, and also quantified by enzyme immunoassay (ELISA). As solid phase antibody, goat anti-human IgG (H + L) (complete absorption of mouse, rabbit, bovine, mouse IgG) (Cosmobio: American Qualex International, Inc.: AQI, Cat. No. A-110 UD) 100. mu.L/well (concentration of 5. mu.g/mL) was added to the plate and allowed to stand at 4 ℃ overnight. Subsequently, a blocking agent was added at 200. mu.L/well, blocking was performed at room temperature for 1 hour, and then the antibody of the sample was diluted in stages, added to each well, and incubated for 1 hour to allow reaction. After washing 5 times with PBST (0.05% Tween20, PBS), a detection antibody solution obtained by diluting goat anti-human IgG (H + L) (absorption of mouse, rabbit, cow, mouse IgG was completed) -HRP (Cosmobio: AQI, Cat. A-110 PD) with PBST in 10000 times was added at a ratio of 100. mu.L/well. After 1 hour incubation, 5 washes with PBST were performed, and substrate buffer TMB was added at 100. mu.L/well. After 15 minutes of incubation at room temperature in a dark room, the reaction was stopped by adding 100. mu.L/well of the reaction-stopping solution, and absorbance at 450nm was measured. A standard curve was prepared using purified human IgG as a standard, and the concentration of human antibody was calculated using the standard curve.
Example 5: reactivity of IgG-modified TfR antibodies
Reactivity of IgG-raised anti-TfR antibodies was studied using 2 TfR-expressing cells, K562 (ATCCCL-243: CML), MIAPaCa (ATCCCRL-1420: pancreatic cancer). Recovery of K by centrifugation562 cells. MIAPaCa-2 was peeled off with 2mM EDTA/PBS and recovered by centrifugation. The recovered cells were washed with PBS 1 time, and then FACSBuffer (1% BSA, 2mM EDTA, 0.1% NaN was added)3PBS) to 1 × 106And (mL). mu.L of this cell suspension was dispensed into a 96-well V bottom petri dish (Costar 3897), and then 1 to 0.01. mu.g/mL of TfR001IgG antibody, TfR005IgG antibody and TfR006IgG antibody was prepared by FACSBuffer, and 100. mu.L of each antibody solution was added to the cells, followed by incubation at 4 ℃ for 1 hour. The cells were washed 2 times with FACSBuffer, and then 100. mu.L of Alexa-anti-humanIgG (Invitrogen) solution diluted to 750 times with FACSBuffer was added, followed by stirring and incubation at 4 ℃ for 1 hour. The wells were washed 2 times by FACSBuffer centrifugation, loaded into the HTS of FACSCalibur (BD), and the FL1 fluorescence intensity was measured for each well. As shown in FIG. 3, each antibody (a: 500 ng/mL; b: 50 ng/mL; c: 5 ng/mL) showed strong reactivity with K562 and MIAPaCa-2. Anti-humanIgG (1. mu.g/mL) and Anti-humanTfR (1. mu.g/mLMBLD 259-3) were used as a negative control (negative control) and a positive control (positive control), respectively.
Example 6: INVTRO proliferation inhibitory Effect of IgG-modified TfR antibody
TfR-expressing cell 13 strains, namely Ramos (ATCCRL-1596), K-562 (ATCCCL-243), NCI-H358 (ATCCRL-5807), A549 (ATCCCL-185), MIAPaCa-2 (ATCCRL-1420), PK-45P (KG 0493, university of northeast aged medical research institute), KLM-1 (RCB), A431 (ATCCRL-1555), DU145 (ATCCHTB-81), HT-29 (ATCCHTB-38), BFTC905 (DSMMZACC 361), MKN45 (JCRBJCRB 0254) MT-2 were adjusted to densities of 2500 to 10000/mL in culture medium and injected into a 96-well flat bottom plate (NUNC 008) at a density of 100. mu.L/well. At 37 deg.C, 5% CO2After 24-hour incubation in 95% air, a 20. mu.g/mL-1.52 ng/mL dilution series of TfR006 antibody was prepared, and 100. mu.L of the dilution series was added to the plate under culture, followed by incubation at 37 ℃ with 5% CO2And further cultured in 95% air for 96 hours. After the completion of the culture, the plate was centrifuged at 1200rpm for 3 minutes, and after taking out the supernatant by standing, 1 was added00. mu.LPBS, centrifugation again, PBS was dispensed into a 96-well V-plate (Costar 3897), 0.25% Trypsin EDTA 50. mu.L was added, cells were detached, the total amount of cells was transferred into a V-plate containing PBS after stirring with a pipette, the wells were washed with 50. mu.L of culture solution, and the total amount was transferred into a V-plate, the V-plate was placed into an HTS of FACSCalibur (BD), after stirring, 40. mu.L of cells were aspirated by each well, and the number of cells was measured, the number of cells × 5 obtained was the number of cells per 1 well, the proliferation rate obtained by addition of the antibody at each concentration was calculated by the following calculation formula, the antibody concentration (IC 50) indicating 50% of the proliferation rate was obtained by using Masterex Pluronins software (Hitachi solutions), and as a result, each antibody showed a strong cell proliferation inhibitory effect, and each IC50 is shown in Table 3.
The proliferation rate was 100% of the number of cells (antibody addition)/number of cells (no antibody) × 100%
[ Table 3]
Example 7: antitumor effect in transplanted tumor model
The antitumor effect of the human anti-TfR antibody was confirmed by the following transplantation tumor model into which each TfR positive-expressing cancer cell line was transplanted.
[ Table 4]
| Cell line | Cancer species | Culture solution |
| PK-45P | Pancreatic cancer | RPMI1640+10%FBS |
| HT-29 | Large intestine cancer | McCoy’s 5A+10%FBS |
| K562 | Leukemia (leukemia) | RPMI1640+10%FBS |
| MIAPaCa-2 | Pancreatic cancer | DMEM+10%FBS |
| DU145 | Prostate cancer | MEM+10%FBS |
| RAMOS | Lymphoma (lymphoma) | RPMI1640+10%FBS25 --> |
| BFTC905 | Cancer of the bladder | DMEM+10%FBS |
The cells were cultured in each of the culture media shown in Table 4, and at the time of transplantation, each cell was suspended in RPMI1640, and the suspension of each cancer cell was transplanted subcutaneously on the right flank of a SCID mouse (female, 7-week-old, Japanese air) at a rate of 100. mu.L per mouse, to give 5 × 106One/mouse. After transplantation, tumor diameter was measured with a vernier caliper, and volume was calculated by the following formula.The average tumor volume reaches 150mm3In the above, one cancer cell tumor-bearing mouse was classified into two groups (n = 5) by grouping software (exsasversion 7.6cac). Antibody administration group TfR006 antibody diluted with PBS was administered in tail vein so that each mouse was 15 mg/kg. The negative control group was administered PBS through the tail vein at 0.2mL/20g mouse. Dosing was performed twice a week (every 3 or 4 days) for a total of 6 times. Tumor diameters were measured twice a week after the administration by a vernier caliper to determine the tumor volume of each group. The antitumor effect was determined by tumor volume measurement.
Tumor volume was calculated by the following formula:
tumor volume (short diameter)2× Long diameter × 0.5.5
The change with time of the mean value of the tumor volume of each group is shown in fig. 4. In any cancer cell line-transplanted tumor model, inhibition of tumor growth was confirmed in the antibody-administered group. From this result, it can be seen that the TfR006 antibody has a strong proliferation inhibitory effect on various cancer cells.
Example 8: anti-tumor effect of anti-TfR antibodies in ATL model
The ATL cell line MT-2 cells were cultured in RPMI1640 medium supplemented with 10% FBS, and at the time of transplantation, the cells were recovered by centrifugation, and the cells were suspended in RPMI1640 to give 1 × 108and/mL. The same amount of matrigel (Becton Dickinson) as the cell suspension was mixed and transplanted subcutaneously in the right ventral side of NOG/Jic mice (female, 7-week-old, Central institute of animal experiments). After transplantation, the tumor diameter was measured twice a week by means of a vernier caliper, and the mean tumor volume reached 150mm3Time points before and after, were divided into groups by randomly dividing the tumor volume, 5 mice each, into 4 groups. The TfR006 antibody was administered to 3 groups at 15mg/kg, 5mg/kg and 1.5mg/kg via the tail vein, and the remaining group was used as a negative control group, and PBS was administered to 0.2mL/20g of mice via the tail vein. The administration was performed 2 times per week (every 3 or 4 days) for a total of 6 times. To giveAfter the administration, the tumor diameter was measured with a vernier caliper 2 times per week in the same manner as before the grouping, and the tumor volume of each group was determined. Determination of antitumor effect the determination was made by means of Dunnet multiple comparison measurement of the reference variable of the PBS group as the control group, by measuring the tumor volume of the final day.
Tumor volume was calculated by the following formula
Tumor volume (short diameter)2× Long diameter × 0.5.5
In addition, random grouping and multiple comparison measurements were performed by the bioassay data statistical analysis software EXSUS (CAC, Inc.).
The change with time of the mean value of the tumor volume for each group is shown in fig. 5. As shown in fig. 5, tumor proliferation was dose-dependently controlled by TfR006 antibody.
Example 9: immunostaining using clinical specimens
(1) Slicing production
The excised lung cancer tissue was cut into a size of 5mm × 5mm × 10mm, placed in a 4% PFA/0.01% glutaraldehyde/0.1M arsenodicarbonic acid buffer (PFA is Wako pure chemical industries, glutaraldehyde is Kanto chemical industries, and sodium arsenicum is SIGMA) at 4 ℃, fixed with a microgrid using an electric furnace (SHARP), and fixed again at 4 ℃ for 1 hour using the same fixing solution. The cells were transferred to 10% sucrose/PBS, immersed at 4 ℃ for 4 hours, then replaced with 15% sucrose/PBS, immersed at 4 ℃ for 4 hours, and then replaced with 20% sucrose/PBS, immersed at 4 ℃ for 1 night, embedded by OTCcomp, and snap-frozen in dry ice-hexane. It was cut to a thickness of 4 μm in a cryostat (Reichert-Jung 2800 FRIGCUTE), attached to a silane-coated slide glass, and dried by a cold air dryer for 30 minutes.
(2) Dyeing process
The slide sheet to which the section was attached was immersed 3 times in PBS every 5 minutes to be hydrophilized. Next, 50. mu.L of 0.3% H was added dropwise2O2/0.1%NaN3The reaction was carried out at room temperature for 10 minutes to block endogenous peroxidase. Thereafter, the cells were washed 3 times with PBS every 5 minutes, and 2% BSA2/PBS was added to block the nonspecific reaction at room temperature for 10 minutes. The remaining liquid was added dropwise, and the phage antibody TfR006 (50. mu.L) was added dropwise, followed by reaction at room temperature for 1 hour. After washing 3 times with PBS, 50. mu.L of anti-cp3 rabbit antibody (5. mu.g/mL) was added dropwise thereto, and a secondary antibody reaction was carried out at room temperature for 45 minutes. After washing 3 times with PBS, 50. mu.L of peroxidase-labeled dextran conjugated anti-rabbit immunoglobulin-goat polyclonal antibody (DAKO) was dropped, and three antibody reactions were performed at room temperature for 30 minutes. After washing 3 times with PBS, 50. mu.L of DAB. H solution was added dropwise2O2After the color development liquid develops brown, the liquid is moved to a short tube filled with distilled water to stop the reaction. After washing with water for 10 minutes, the specimen was stained with hematoxylin, dehydrated, transparent, mounted on Marinol, and observed under a microscope. As shown in FIG. 6, the antibody reacted with cancer cells of lung cancer and did not react with non-cancer cells.
Claims (14)
1. An antibody that specifically reacts with human TfR, wherein:
the antibody has a heavy chain variable region having CDRs comprising a heavy chain first complementarity determining region (VHCDR1) of sequence No. 31, a heavy chain second complementarity determining region (VHCDR2) of sequence No. 32, and a heavy chain third complementarity determining region (VHCDR3) of sequence No. 33, and a light chain variable region having CDRs comprising a light chain first complementarity determining region (VLCDR1) of sequence No. 34, a light chain second complementarity determining region (VLCDR2) of sequence No. 35, and a light chain third complementarity determining region (VLCDR3) of sequence No. 36.
2. The antibody of claim 1, wherein:
the antibody is a human antibody or a humanized antibody.
3. The antibody of claim 1 or 2, wherein:
the antibody is selected from Fab, Fab ', F (ab')2Single chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsfvs), and antibody fragments of peptides comprising CDRs.
4. A DNA encoding the antibody according to any one of claims 1 to 3.
5. A recombinant vector comprising the DNA according to claim 4.
6. A transformant obtained by introducing the recombinant vector according to claim 5 into a host cell.
7. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 3.
8. The pharmaceutical composition of claim 7, wherein:
the antibody has a cell-damaging substance bound thereto.
9. The pharmaceutical composition of claim 8, wherein:
the cell-damaging agent is a drug, toxin, or radioactive substance.
10. The pharmaceutical composition according to any one of claims 7 to 9, wherein:
the pharmaceutical composition is used as an anticancer agent.
11. The pharmaceutical composition of claim 10, wherein:
the cancer is solid cancer or hematological cancer.
12. The pharmaceutical composition of claim 10, wherein:
the solid cancer is lung cancer, carcinoma of large intestine, gastric cancer, bladder cancer, pancreatic cancer, prostatic cancer, hepatocarcinoma, cervical cancer, metrocarcinoma, ovarian cancer, breast cancer, head and neck cancer, and skin cancer.
13. The pharmaceutical composition of claim 10, wherein:
the blood cancer is leukemia, lymphoma, or myeloma.
14. The pharmaceutical composition of claim 10, wherein:
the hematologic cancer is adult T cell leukemia (ATL).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011104007 | 2011-05-09 | ||
| JP2011-104007 | 2011-05-09 | ||
| PCT/JP2012/061676 WO2012153707A1 (en) | 2011-05-09 | 2012-05-07 | Antibody specifically recognising transferrin receptor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1193833A1 HK1193833A1 (en) | 2014-10-03 |
| HK1193833B true HK1193833B (en) | 2017-07-21 |
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