WO2011161970A1 - Anti-pdgf receptor antibody - Google Patents

Abstract

Disclosed are antibodies against PDGFRα and the use of same. Specifically disclosed are an anti-PDGFRα antibody that recognizes an amino acid sequence in an amino acid sequence from the 106^th to the 311^th position in a PDGFRα as an epitope, an anti-PDGRFα antibody that has 50% or greater antibody dependent cellular cytotoxicity (ADCC) activity in T cells, and an anti-PDGFRα antibody that has 15% or greater complement dependent cytotoxicity (CDC) activity. Also disclosed are the DNA that code the aforementioned antibodies and a pharmaceutical composition that includes the aforementioned antibodies as an effective component.

Description

Anti-PDGF receptor antibody

The present invention relates to an antibody that recognizes platelet-derived growth factor receptor α (hereinafter referred to as “PDGFRα”) as an antigen and use thereof. Part of the present invention relates to humanized anti-PDGFRα antibodies and uses thereof. The present invention includes a gene that encodes an anti-PDGFRα antibody and a cell that produces the anti-PDGFRα antibody. The use of the antibody is particularly used for treatment and diagnosis of a disease associated with PDGFRα activity, expression or overexpression.

Platelet-derived growth factor (hereinafter referred to as “PDGF”) is a protein that controls cell growth, differentiation, migration, and the like. There are four types of PDGF, A, B, C, and D. It is known that PDGF-A and PDGF-B have a homo- or hetero-dimer structure by disulfide bonds.

PDGFRα (also referred to as PDGFR2, CD140a), which is one of the subtypes of PDGF receptor, is a tyrosine kinase-related receptor. PDGFRα can form a homodimer or a heterodimer with PDGFRβ, a structurally similar PDGF receptor subtype. PDGF-AA is known to have affinity only for PDGFRαα, and PDGF-AB, PDGF-BB, and PDGF-CC are known to have affinity for both PDGFRαα and PDGFαβ. PDGFRα has been reported to be associated with diseases associated with cell proliferation such as tumors, stromal cells, and blood vessels (see Non-Patent Document 1). For example, Gleevec has been reported to exert effects on gastrointestinal stromal tumors with mutations in the PDGFRα gene. In addition, antibodies targeting PDGFRα and cancer therapeutic agents using the antibodies have been reported (see Patent Documents 1 to 5 and Non-Patent Documents 2 and 3).

International Publication No. 1992/013867 Pamphlet International Publication No. 1995/000659 Pamphlet International Publication No. 2004/046356 Pamphlet International Publication No. 2006/138729 Pamphlet International Publication No. 2008/112003 Pamphlet

However, there has been no specific use of these antibodies as pharmaceuticals, showing higher therapeutic effects on diseases associated with cell proliferation such as tumors, stromal cells, blood vessels, etc., and having side effects such as toxicity. A low anti-PDGFRα antibody has been sought.
Although WO 1992/013867 and WO 2004/046356 refer to the association of anti-PDGFRα antibodies with diseases associated with cell proliferation, antibodies that can specifically treat such diseases However, there is a need for antibodies useful for actual treatment. WO 1995/000659 pamphlet, WO 2006/138729 pamphlet, and WO 2008/112003 pamphlet are prepared by preparing fully human antibodies or antibodies with high affinity as therapeutic antibodies. Is disclosed. Thus, so far, anti-PDGFRα antibodies having a high therapeutic effect have been attempted mainly by focusing on affinity in anticipation of the drug efficacy due to the neutralizing activity of anti-PDGFRα antibodies. However, in actual treatment, a clinically sufficient therapeutic effect is not obtained only by high affinity. The present inventors, together with appropriate affinity, have excellent antibody-dependent cytotoxic activity (hereinafter referred to as “ADCC activity”) and / or complement-dependent cytotoxic activity (hereinafter referred to as “CDC activity”). ) Was considered to be able to exert a high therapeutic effect in clinical practice, and such an antibody was searched. As a result, an antibody having excellent ADCC and / or CDC activity in addition to affinity was found, and it was confirmed that the antibody exhibited a more excellent therapeutic effect.

Therefore, in one aspect, the present invention relates to an anti-PDGFRα antibody having excellent ADCC activity, for example, an anti-PDGFRα antibody having an ADCC activity (%) of 25% or more. The ADCC activity (%) exhibited by the anti-PDGFRα antibody of the present invention can be 30% or more, 35% or more, 40% or more, or 45% or more, and more preferably 50% or more. Here, “ADCC activity (%)” means ADCC activity compared to the control antibody of the test antibody, and 100 − {(measured value that is an index of ADCC activity obtained for the test antibody) / (control antibody) Measured value obtained as an index of ADCC activity)} × 100 (%). The “control antibody” is not limited as long as it is an antibody known not to exhibit ADCC activity, and may be, for example, an anti-GST antibody. In the present specification, “ADCC activity (%)” may be simply referred to as “ADCC activity”.

Further, the anti-PDGFRα antibody excellent in ADCC activity may be an antibody having ADCC activity equivalent to or higher than that of the 24A1 antibody. In the present specification, “an antibody having ADCC activity equivalent to that of the 24A1 antibody” means that the ADCC activity compared to the control antibody of the test antibody (hereinafter referred to as “ADCC activity (%) of the test antibody”) is that of the 24A1 antibody. It is an antibody included in a range of ± 20% of ADCC activity (hereinafter referred to as “ADCC activity (%) of 24A1 antibody”) compared with a control antibody. In the present specification, “an antibody having ADCC activity equal to or higher than that of the 24A1 antibody” means an antibody in which the ADCC activity (%) of the test antibody is larger than the ADCC activity (%) of the 24A1 antibody. Here, the “control antibody” is not limited as long as it is an antibody known not to exhibit ADCC activity, and may be, for example, an anti-GST antibody. The ADCC activity (%) of the 24A1 antibody is 100-{(measured value as an index of ADCC activity obtained for the 24A1 antibody) / (measured value as an index of ADCC activity obtained for the control antibody)} × 100 ( %). The ADCC activity (%) of the test antibody is 100-{(measured value as an index of ADCC activity obtained for the test antibody) / (measured value as an index of ADCC activity obtained for the control antibody)} × It can be calculated by 100 (%). “The ADCC activity (%) of the test antibody falls within the range of ± 20% of the ADCC activity (%) of the 24A1 antibody” means that the ADCC activity (%) of the test antibody is the ADCC activity (%) of the 24A1 antibody. It is included between 0.8 and 1.2 times. Preferably, an antibody having ADCC activity equivalent to that of the 24A1 antibody is such that the ADCC activity (%) of the test antibody is within ± 15%, ± 10%, or ± 8% of the ADCC activity (%) of the 24A1 antibody. More preferably, it is an antibody contained within a range of ± 5%. In the present specification, being contained within the range of ± X% means -X% or more and + X% or less. Therefore, an antibody having ADCC activity equal to or higher than that of the 24A1 antibody has, for example, an ADCC activity (%) of the test antibody of −20% or more, −15% or more of the ADCC activity (%) of the 24A1 antibody, −10 % Or more, −8% or more, or −5% or more of the antibody. Further, the anti-PDGFRα antibody having excellent ADCC activity may be an antibody having ADCC activity equivalent to or higher than that of the humanized 24A1 antibody.

Furthermore, in another aspect, the present invention relates to an anti-PDGFRα antibody excellent in CDC activity, for example, an anti-PDGFRα antibody having a CDC activity (%) of 10% or more. The CDC activity (%) exhibited by the anti-PDGFRα antibody of the present invention can be 15% or more, or 20% or more, and more preferably 30% or more. Here, the CDC activity (%) means the CDC activity compared to the control antibody of the test antibody, and 100-{(measured value that is an index of CDC activity obtained for the test antibody) / (obtained for the control antibody). Measured value that is an index of CDC activity)} × 100 (%). The “control antibody” is not limited as long as it is an antibody known not to exhibit CDC activity, and may be, for example, an anti-GST antibody. In the present specification, “CDC activity (%)” may be simply referred to as “CDC activity”.

Further, the anti-PDGFRα antibody excellent in CDC activity may be an antibody having CDC activity equivalent to or higher than that of 21A1 antibody. In this specification, “an antibody having a CDC activity equivalent to that of the 21A1 antibody” means that the CDC activity compared to the control antibody of the test antibody (hereinafter referred to as “CDC activity (%) of the test antibody”) is that of the 21A1 antibody. It is an antibody that is included within a range of ± 20% of the CDC activity compared to the control antibody (hereinafter referred to as “CDC activity (%) of 21A1 antibody”). In the present specification, the “CDC activity equal to or higher than that of the 21A1 antibody” means an antibody in which the CDC activity (%) of the test antibody is larger than the CDC activity (%) of the 21A1 antibody. The control antibody is not limited as long as it is an antibody known not to exhibit CDC activity, and may be, for example, an anti-GST antibody. The CDC activity (%) of 21A1 antibody is {(measured value as an index of CDC activity obtained for 21A1 antibody) / (measured value as an index of CDC activity obtained for antibody non-administered group)} × 100 ( %). The CDC activity (%) of the test antibody is {(measured value as an index of CDC activity obtained for the test antibody) / (measured value as an index of CDC activity obtained for the antibody non-administered group)} × It can be calculated by 100 (%). “The CDC activity (%) of the test antibody falls within the range of ± 20% of the CDC activity (%) of the 21A1 antibody” means that the CDC activity (%) of the test antibody is the CDC activity (%) of the 21A1 antibody. It is included within the range of 0.8 to 1.2 times. Preferably, an antibody having CDC activity equivalent to 21A1 antibody is such that the CDC activity (%) of the test antibody is within ± 15%, ± 10%, or ± 8% of the CDC activity (%) of 21A1 antibody. More preferably, it is an antibody contained within a range of ± 5%. Therefore, an antibody having a CDC activity equal to or higher than that of the 21A1 antibody has, for example, a CDC activity (%) of the test antibody of −20% or more, −15% or more of the CDC activity (%) of the 21A1 antibody, −10 % Or more, −8% or more, or −5% or more of the antibody.

Preferably, the anti-PDGFRα antibody excellent in CDC activity exhibits appropriate ADCC activity in addition to CDC activity. As such an antibody, for example, an anti-PDGFRα antibody having a CDC activity (%) of 10% or more and an ADCC activity (%) of 25% or more can be mentioned. The CDC activity (%) in an antibody excellent in CDC activity and ADCC activity can be 10% or more, 15% or more, or 20% or more, and more preferably 30% or more. In addition, the ADCC activity (%) in an antibody excellent in CDC activity and ADCC activity can be 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more, more preferably 50%. % Or more. The antibody excellent in CDC activity and ADCC activity may be an antibody having CDC activity and ADCC activity equivalent to or higher than 21A1 antibody. Here, whether the subject antibody has CDC activity equal to or higher than 21A1 antibody and ADCC activity equal to or higher than 21A1 antibody can be determined according to the above-described method.

In addition, as a result of examining the relationship between the ADCC activity and antitumor activity and the epitope, the present inventors were superior in the anti-PDGFRα antibody that recognizes the amino acid sequence from the 213rd to 311th amino acids (SEQ ID NO: 3) of PDGFRα as the epitope It was found to produce ADCC activity.
Until now, as the epitope of PDGFRα, amino acids 959-973 and 1074-1089 have been disclosed in Science 243: 800-804 (1989). In addition, in International Publication No. 1995/000659, it is disclosed that a region common to humans, monkeys, pigs, dogs, cats and rabbits, a region not common to hamsters and rats, and an extracellular domain are useful as epitopes. ing. However, there is no report disclosed regarding the epitope recognized by the antibody of the present invention. The present invention provides a novel anti-PDGFRα antibody epitope that exhibits a higher therapeutic effect and is expected to have lower side effects such as toxicity.

Accordingly, in one aspect, the present invention relates to an antibody that recognizes a specific epitope of PDGFRα, specifically, an anti-PDGFRα antibody that recognizes the amino acid sequence from position 106 to 311 of PDGFRα (SEQ ID NO: 1) as an epitope, Relates to an anti-PDGFRα antibody that recognizes the 106th to 213rd amino acid sequence (SEQ ID NO: 2) of PDGFRα or the 213th to 311th amino acid sequence (SEQ ID NO: 3) of PDGFRα as an epitope. In particular, an anti-PDGFRα antibody that recognizes the amino acid sequence from 213 to 311 of PDGFRα (SEQ ID NO: 3) as an epitope is particularly excellent in ADCC activity, and thus is useful for the treatment of diseases caused by cell proliferation (for example, tumors). is there. For example, an anti-PDGFRα antibody that recognizes the amino acid sequence from 213 to 311 of PDGFRα (SEQ ID NO: 3) as an epitope further has ADCC activity of 25% or more, 30% or more, 35% or more, 40% or more, 45% Or 50% or more. The anti-PDGFRα antibody that recognizes the amino acid sequence from 213 to 311 of PDGFRα (SEQ ID NO: 3) as an epitope may have ADCC activity equal to or higher than that of the 24A1 antibody. Throughout the present specification, the amino acid recognized by an antibody that recognizes a specific epitope of PDGFRα may be a part of the amino acid sequence that constitutes the epitope. For example, the amino acid recognized by the antibody may be 3 amino acids or more selected from amino acids constituting the epitope.

More specifically, the present inventors have found an antibody having a sequence excellent in antitumor activity. Therefore, in another aspect, the present invention relates to an antibody specified by the amino acid sequence of a complementarity determining region (hereinafter referred to as “CDR”). In the present invention, “CDR” refers to Kabat et al. (“Sequences of Proteins of Immunological Interest”, Kabat, E. et al., U.S. Department of Health and Human Services, Cho & L et al., 1983). Mol. Biol., 196, 901-917, 1987). Specifically, in the present invention, CDR1, CDR2, and CDR3 of the heavy chain are respectively SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: The present invention relates to an antibody having the amino acid sequence of No. 36. Further, in the present invention, the light chain CDR1, CDR2, and CDR3 are respectively represented by SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, or SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38. It relates to an antibody having the amino acid sequence described. Preferably, the present invention provides that the heavy chain CDR1, CDR2, and CDR3 have the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, and the light chain CDR1. , CDR2 and CDR3 have the amino acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, or heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 33, The amino acids having the amino acid sequences described in SEQ ID NO: 34 and SEQ ID NO: 35, and the light chain CDR1, CDR2, and CDR3 are described in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38 An antibody having a sequence.

In one embodiment, the antibody of the present invention is an antibody in which the amino acid sequences of heavy and light chains are specified, and includes the following antibodies:
(I) an antibody whose heavy chain variable region (hereinafter referred to as “VH”) has an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10;
(Ii) an antibody having an amino acid sequence encoded by a nucleic acid sequence in which a light chain variable region (hereinafter referred to as “VL”) hybridizes with a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 11 under stringent conditions;
(Iii) an antibody having both a VH of (i) and a VL of (ii);
(Iv) an antibody in which VH has an amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO: 10;
(V) an antibody wherein the VL has an amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO: 11; and
(Vi) An antibody having both a VH of (iv) and a VL of (v).
The antibodies (i) to (vi) are antibodies that bind to PDGFRα, and preferably have cell growth inhibitory activity. Of these antibodies, most preferred is an antibody in which the heavy chain has the amino acid sequence of SEQ ID NO: 10 and the light chain has the amino acid sequence of SEQ ID NO: 11. The antibodies (i) to (vi) may have CDC activity of 10% or more, 15% or more, or 20% or more, 30% or more, and / or ADCC activity of 25% or more, It may be 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more.

In the present specification, “24A1 antibody” refers to an antibody in which the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 30 and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 32 unless otherwise specified. is there. “Humanized 24A1 antibody” has the sequences of SEQ ID NOs: 4, 5, and 6 as heavy chain CDRs 1, 2, and 3, respectively, and the sequences of SEQ ID NOs: 7, 8, and 9, respectively. It refers to an antibody having as light chain CDRs 1, 2 and 3. Preferably, the “humanized 24A1 antibody” is an antibody whose heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 24 and whose light chain consists of the amino acid sequence set forth in SEQ ID NO: 26. Further, “21A1 antibody” means the 21A1 antibody obtained in Example 1 of the present specification, unless specifically stated otherwise. Specifically, the heavy chain is derived from the amino acid sequence set forth in SEQ ID NO: 40. The light chain is an antibody consisting of the amino acid sequence set forth in SEQ ID NO: 42. The “humanized 21A1 antibody” has the sequences of SEQ ID NOs: 33, 34, and 35 as the heavy chain CDRs 1, 2, and 3, respectively, and the sequences of SEQ ID NOs: 36, 37, and 38, respectively. It refers to an antibody having as light chain CDRs 1, 2 and 3. . The ADCC activity (%) and CDC activity (%) of the 24A1 antibody and the 21A1 antibody can be measured by the measurement method described in the present specification after preparing the antibody. ) Can be determined by referring to the description.

The antibodies of the present invention further include the following antibodies:
An anti-PDGFRα antibody satisfying at least one of the following (A) to (C):
(A) an anti-PDGFRα antibody that recognizes the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3 as an epitope;
(B) The heavy chain CDR1, CDR2, and CDR3 have the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, and / or the light chain CDR1, CDR2 And CDR3 are the antibodies having the amino acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, or heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 33. , SEQ ID NO: 34, and SEQ ID NO: 35, and / or light chain CDR1, CDR2, and CDR3 are SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: An antibody having the amino acid sequence described in 38; or (C) the antibody according to any one of (i) to (vi),
And an antibody exhibiting one or more properties selected from the following (a) to (e):
(A) ADCC activity (%) is 25% or more, 30% or more, 35% or more, 40% or more, 45%, or 50% or more;
(B) has ADCC activity equal to or greater than 24A1 antibody or humanized 24A1 antibody;
(C) CDC activity (%) is 10% or more, 15% or more, 20% or more, or 30% or more;
(D) has ADCC and CDC activity equal to or higher than 21A1 antibody; or
(E) It binds to PDGFRα with a dissociation constant (Kd) of 7 × 10 ⁻⁹ or less, 6.5 × 10 ⁻⁹ or less, or 6.2 × 10 ⁻⁹ or less.

In the present specification, to hybridize under stringent conditions means to hybridize under hybridization conditions usually used by those skilled in the art. For example, whether or not to hybridize can be determined by a method described in Molecular Cloning, Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). For example, hybridization conditions are 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0, ₂ M NaH ₂ PO ₄ , 20 mM EDTA · 2Na, pH 7.4). The condition may be such that the medium is hybridized at 42 ° C. and then washed at 42 ° C. with 0.5 × SSC. Moreover, the homology of an amino acid sequence can be determined using well-known programs, such as BLAST and FASTA, for example.

For example, the present invention relates to the following (1) to (17).
(1) An anti-PDGFRα antibody that recognizes the amino acid sequence contained in the 106th to 311th amino acid sequences of PDGFRα as an epitope.
(2) An anti-PDGFRα antibody that recognizes the amino acid sequence contained in the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 as an epitope.
(3) An anti-PDGFRα antibody whose ADCC activity (%) by T cells is 25% or more.
(4) An anti-PDGFRα antibody having ADCC activity equal to or higher than that of the 24A1 antibody.
(5) An anti-PDGFRα antibody having a CDC activity (%) of 10% or more.
(6) The anti-PDGFRα antibody according to (5), wherein the ADCC activity (%) by T cells is 25% or more.
(7) The anti-PDGFRα antibody according to any one of (3) to (5), which recognizes an amino acid sequence contained in the 106th to 311th amino acid sequences of PDGFRα as an epitope.
(8) The anti-PDGFRα antibody according to any one of (3) to (5), which recognizes the amino acid sequence contained in the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 as an epitope.
(9) The heavy chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 4, the heavy chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 5, and the heavy chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 6 And / or the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 7, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 8, and / or A light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 9, or a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 33 and a heavy chain CDR2 set forth in SEQ ID NO: 34. And the heavy chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 35 and / or the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 36, CDR2 of the chain is arranged An antibody having the amino acid sequence set forth in SEQ ID NO: 37 and the light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 38, particularly a humanized antibody having such a CDR sequence.
(10) (I) the variable region of the heavy chain has one amino acid sequence selected from:
The amino acid sequence encoded by the nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10;
An amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO: 10; or
The amino acid sequence of SEQ ID NO: 10, and / or
(II) The light chain variable region has one amino acid sequence selected from:
A nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 11;
An amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO: 11; or
The amino acid sequence of SEQ ID NO: 11,
An antibody capable of binding to PDGFα.
(11) The anti-PDGFRα antibody according to any one of claims 1 to 7, which binds to PDGFRα with a dissociation constant (Kd) of 7 × 10 ⁻⁹ or less.
(12) A pharmaceutical composition comprising the antibody according to any one of (1) to (11) or (14) to (16).
(13) The pharmaceutical composition according to (12), which is a therapeutic agent for a disease based on cell proliferation.
(14) The amino acid sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 3 is recognized as an epitope, and ADCC activity (%) is 25% or more, 30% or more, 35% or more, 40% An anti-PDGFRα antibody that is 45% or more, or 50% or more.
(15) The amino acid sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 3 is recognized as an epitope, and the CDC activity (%) is 10% or more, 15% or more, 20%, or 30% Anti-PDGFRα antibody as described above.
(16) The amino acid sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 3 is recognized as an epitope, and ADCC activity (%) is 25% or more, 30% or more, 35% or more, 40% Or more, 45% or more, or 50% or more, and an antibody whose CDC activity (%) is 10% or more, 15% or more, 20%, or 30% or more.
(17) A DNA encoding the antibody according to any one of (1) to (11) or (14) to (16).

In the present specification, the “antibody” includes non-human animal antibodies, antibodies having the amino acid sequences of non-human animal antibodies and human antibodies, and human antibodies. An antibody having an amino acid sequence of a non-human animal antibody and an amino acid sequence of a human antibody is a human chimeric antibody obtained by replacing the antigen-binding domain Fv of a human antibody with the antigen-binding domain Fv of a non-human animal monoclonal antibody. It includes humanized antibodies in which CDRs are incorporated into the frame region of human antibodies (hereinafter referred to as “FR”). The immunoglobulin class of the antibody of the present invention is not particularly limited, and may be any immunoglobulin class of IgG, IgM, IgA, IgE, IgD, and IgY. In addition, the antibody of the present invention encompasses antibodies of any isotype. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. In the present invention, the term “antibody” refers to a full-length antibody, an antibody fragment (F (ab ′) ₂ , Fab ′, Fab, scFv, dsFv or a polymer thereof, Diabody, etc., and CDR). Including peptides).

In the present specification, the “disease caused by cell proliferation” is a disease that substantially develops or worsens due to cell proliferation, and includes, for example, tumors (cancer, sarcoma). .

The present invention also includes DNA having a nucleic acid sequence encoding the above antibody (including antibody fragments), a vector having the DNA, a host cell having the vector, and the like. For example, the present invention includes DNA having the nucleic acid sequence described in SEQ ID NO: 23 and / or 25, or SEQ ID NO: 29 and / or 31, a vector having the DNA, a host cell having the vector, and the like. The present invention also relates to a DNA having a nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence of 23 and / or 25 or SEQ ID NO: 29 and / or 31, a vector having the DNA, and It includes a host cell having a vector. In addition, the present invention has 80%, 85%, 90%, 95%, 98%, or 99% homology with the nucleic acid sequence shown in 23 and / or 25 or SEQ ID NO: 29 and / or 31. It includes a DNA having a nucleic acid sequence, a vector having the DNA, a host cell having the vector, and the like. Further, according to the present invention, the heavy chain CDR1, CDR2, and CDR3 have the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, and / or the light chain. CDR1, CDR2, and CDR3 are antibodies having the amino acid sequences described in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, or heavy chain CDR1, CDR2, and CDR3 are respectively Having the amino acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, and / or the light chain CDR1, CDR2, and CDR3 are SEQ ID NO: 36, SEQ ID NO: 37, and DNA encoding an antibody having the amino acid sequence set forth in SEQ ID NO: 38, a vector having the DNA, a host cell having the vector, and the like .
DNA having the nucleic acid sequence hybridizing under stringent conditions with the above-described DNA (SEQ ID NO: 29 and / or 31), or 23 and / or 25, or SEQ ID NO: 29 and / or 31 Any of the nucleic acid sequences described herein, including DNA having a nucleic acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% homology, is preferably encoded by the sequence. The antibody binds to PDGFRα, and more preferably, the antibody encoded by the sequence exhibits one or more properties selected from the following (a) to (e):
(A) ADCC activity is 25% or more, 30% or more, 35% or more, 40% or more, 45%, or 50% or more;
(B) has ADCC activity equal to or greater than 24A1 antibody or humanized 24A1 antibody;
(C) CDC activity is 10% or more, 15% or more, 20% or more, or 30% or more;
(D) has ADCC and CDC activity equal to or higher than 21A1 antibody; or
(E) It binds to PDGFRα with a dissociation constant (Kd) of 7 × 10 ⁻⁹ or less, 6.5 × 10 ⁻⁹ or less, or 6.2 × 10 ⁻⁹ or less.

The antibody of the present invention can exhibit an excellent PDGFRα activity inhibitory action. In addition, since the antibody of the present invention is excellent in ADCC activity and / or CDC activity, it can exhibit a superior cell killing effect on PDGFRα-expressing cells. Therefore, the antibody of the present invention can be used as a therapeutic or prophylactic agent for a disease associated with the onset or exacerbation of PDGFRα mutation or expression, particularly a disease (tumor etc.) caused by cell proliferation.

In order to measure the neutralizing activity of the antibody of the present invention, it is a photograph in which phosphorylation of PDGFRα by PDGF-AA was confirmed by Western blotting. As detection antibodies, human PDGFRα polyclonal antibody C-20 (left photo) and phosphorylated tyrosine antibody 4G10 (right photo) were used. “IP with Anti-PDGFRa 10A1” at the top of each photo refers to a sample immunoprecipitated with anti-PDGFRα monoclonal antibody 10A1, and “TNE Lysate” refers to a cell extract obtained by lysing cells with TNE buffer. From the left, each lane in each photograph shows normal mouse IgG (negative control), monoclonal antibodies 1A1, 5A1, 8A1, 9A1, 10A1, 18A1, 21A1, 22A1, 24A1, 40A1, and PDGF-AA administered cells, and PDGF -AA untreated cells, PDGF-AA treated cells. The number on the left of each photograph indicates the molecular weight (kDa). It is a graph which shows the result of having measured the ADCC activity of the antibody of this invention. The vertical axis represents ADCC activity (%), and the horizontal axis represents the clone number of each monoclonal antibody. It is a graph which shows the result of having measured the CDC activity of the antibody of this invention. The vertical axis represents CDC activity (%), and the horizontal axis represents the clone number of each monoclonal antibody. Each monoclonal antibody was administered at 5 μg / mL (black) or 1 μg / mL (white). It is a graph which shows the result of having measured the antitumor effect of the antibody of this invention using the nude mouse. The vertical axis represents tumor size (mm ³ ), and the horizontal axis represents elapsed time (weeks). The upward arrow below the horizontal axis indicates the time when the antibody was administered. Each symbol indicates the following specimen: black diamond is monoclonal antibody 5A1, black square is monoclonal antibody 8A1, black triangle is monoclonal antibody 9A1, cross mark is monoclonal antibody 10A1, asterisk is monoclonal antibody 18A1, black circle is monoclonal antibody 21A1, plus The mark is the monoclonal antibody 22A1, the small square is the monoclonal antibody 24A1, the horizontal square is the monoclonal antibody 40A1, and the open diamond is the monoclonal antibody 14C1. It is a graph which shows the result of having confirmed the volume dependence of the anti-tumor effect of the monoclonal antibody 24A1 of this invention using the nude mouse. The vertical axis represents tumor size (mm ³ ), and the horizontal axis represents elapsed time (weeks). The upward arrow below the horizontal axis indicates the time when the antibody was administered. Each symbol indicates the following specimen: black diamond is 1 mg administration group; black square is 0.4 mg administration group; black triangle is 0.1 mg administration group; cross is control antibody (40B3) (0.4 mg) administration group. It is a schematic diagram of a fusion protein of each deletion mutant of PDGFRα and Fc used for epitope mapping by dot blot. It is a photograph which shows the result of the epitope mapping by a dot blot. The upper part of the photograph shows the antibody used, and the left part of the photograph shows the Fc fusion protein or control (mIgG2aFc) of the deletion mutant of PDGFRα used. It is the photograph which confirmed the expression of the heavy chain (Hc) and light chain (Lc) of the cloned monoclonal antibodies 21A1 and 24A1 by Western blotting. “Cell Pellet” at the top of the photograph refers to a cell pellet sample, and “Sup (× 10)” refers to a sample obtained by concentrating the cell culture supernatant 10 times. Each lane of Cell Pellet and Sup (× 10) is from the left, pcDNA vector alone (negative control), Hc of monoclonal antibody 21A1, Lc of monoclonal antibody 21A1, Hc + Lc of monoclonal antibody 21A1, Hc of monoclonal antibody 24A1, monoclonal antibody Lc of 24A1 and Hc + Lc of monoclonal antibody 24A1 are shown. The number on the left of the photo indicates the molecular weight (kDa). The arrow head on the right side of the photograph shows the position corresponding to the molecular weight of Hc and Lc proteins. It is the figure which compared the amino acid sequence of Hc of monoclonal antibody 24A1, humanized 24A1 antibody, and IGHV4-30-4. FIG. 4 is a diagram comparing the amino acid sequences of Lc of monoclonal antibody 24A1, humanized 24A1 antibody, and IGKV 1D-12. It is the photograph which confirmed the expression of the produced heavy chain 24A1 antibody and the heavy chain (Hc) and light chain (Lc) of humanized 24A1 antibody by Western blotting. “Cell Pellet” at the top of the photograph refers to a cell pellet sample, and “Sup (× 10)” refers to a sample obtained by concentrating the cell culture supernatant 10 times. Each lane of Cell Pellet and Sup (× 10) is, from the left, pcDNA vector alone (negative control), Hc of chimeric 24A1 antibody, Lc of chimeric 24A1 antibody, Hc + Lc of chimeric 24A1 antibody, Hc of humanized 24A1 antibody, human The Lc of the 24A1 antibody and Hc + Lc of the humanized 24A1 antibody are shown. The number on the left of the photo indicates the molecular weight (kDa). The arrow head on the right side of the photograph shows the position corresponding to the molecular weight of Hc and Lc proteins. It is the figure which showed the nucleic acid sequence (hPDGFR (alpha) (21A1) MoAbLc) of the heavy chain (hPDGFR (alpha) (21A1) MoAb Hc) and the light chain of the monoclonal antibody 21A1. It is the figure which showed the amino acid sequence of the heavy chain (hPDGFR (21A1) and the light chain (hPDGFR [alpha] (21A1) MoAb Lc) of the monoclonal antibody 21A1.

The antibody of the present invention can be obtained by the following method. First, an expression vector for mammalian cells incorporating a polynucleotide encoding a polypeptide having all or part of PDGFRα is prepared and expressed to obtain an antigen. When producing a monoclonal antibody, an animal such as a mouse, rat or rabbit is immunized using this antigen according to a known method for producing a monoclonal antibody. Subsequently, immune cells obtained from the immunized animal and myeloma cells are fused to obtain a hybridoma, and a PDGFRα monoclonal antibody can be obtained from the hybridoma culture. When preparing a polyclonal antibody, the same antigen as described above is used, the animal is immunized according to a known method of polyclonal antibody preparation, blood is collected from the immunized animal, and PDGFRα polyclonal antibody is collected from the blood from the serum. Obtainable.

The PDGFRα used as the immunogen is not particularly limited as long as it is a mammalian PDGFRα, but is preferably human PDGFRα. For example, human PDGFRα has been reported for its entire amino acid sequence (SEQ ID NO: 13) and cDNA sequence (SEQ ID NO: 12). DNA encoding a polypeptide that matches all or part of PDGFRα can also be obtained by cloning from a mammalian library such as a human tumor cell line using a mammal-derived PDGFRα DNA as a probe. The immunogen used for producing the antibody of the present invention is an expression vector containing DNA encoding PDGFRα or a part thereof (for example, pGEX (for E. coli), pcDNA3.1 (for animal cell expression), etc.) It can be obtained by transforming into insect cells, animal cells, etc., and culturing and expressing the transformed host microorganism / cultured cells such as Escherichia coli in an appropriate medium (for example, LB medium).

When using a peptide having a part of PDGFRα as an immunogen, a peptide having a part of PDGFRα can be used as it is, or a peptide having a part of one or more kinds of PDGFRα, It can also be used by binding via a linker. A peptide having PDGFRα or a part thereof can be prepared using a known method such as the Fmoc method or the Boc method, or an automatic peptide synthesizer. When a peptide having a part of PDGFRα is used as an immunogen, the immunogen includes an extracellular region of PDGFRα, preferably the amino acid described in any one of SEQ ID NO: 1 to SEQ ID NO: 3. Contains an array. For example, as an immunogen, for example, PDGFRα extracellular region (1-514 amino acids) (SEQ ID NO: 14) and mouse IgG2a constant region fusion protein (PDGFRα-Fc) can be used as antigens.

The animal is immunized with the antigen by dissolving the antigen in a sodium phosphate buffer (PBS) and, if necessary, an immunostimulant (eg, mineral oil or aluminum precipitate and heat-killed bacteria or lipopolysaccharide, Freund's). Immunization of non-human mammals or birds with a complete adjuvant or Freund's incomplete adjuvant. Administration of the immunogen to the animal can be performed, for example, by subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or footpad injection. The amount of the immunogen used is not particularly limited as long as it is an amount capable of producing an antibody, but is preferably 0.1 to 1000 μg, more preferably 1 to 500 μg, and still more preferably 10 to 100 μg. Immunization can be performed once or several times at appropriate intervals. Preferably, immunization once per 1 to 5 weeks can be performed a plurality of times (preferably 2 to 5 times in total). An antibody can be obtained by purifying from the serum of an animal exhibiting a sufficient antibody titer.

The monoclonal antibody of the present invention can be obtained by culturing a hybridoma obtained by fusing an antibody-producing cell obtained from the spleen or the like of an immunized animal immunized by the above method with a myeloma cell (myeloma cell). Can do. Examples of the fusion method include the method of Milstein et al. (Galfre, G. & Milstein, C., Methods Enzymol. 73: 3-46, 1981). The antibody can be obtained by culturing the hybridoma in vitro and purifying the culture solution. The antibody can also be obtained by transplanting a hybridoma to a syngeneic animal or an immunodeficient animal to which pristane has been administered intraperitoneally in advance, then ascites, and purifying the collected ascites. Purification of the antibody can be obtained by collecting the IgG fraction using a protein A column, protein G column or the like after centrifugation. When the antibody class is IgY or IgM, the antibody can be purified by a column using mercaptopyridine as a ligand. Moreover, it can be purified using a PDGFRα solid phase column, ion exchange chromatography, hydrophobic interaction chromatography or the like regardless of the class of antibody.

(Production of human chimeric antibody)
The human chimeric antibody of the present invention is prepared by preparing DNAs encoding VH and VL of non-human animal monoclonal antibodies that bind to PDGFRα, which are combined with human immunoglobulin constant region cDNA and incorporated into an expression vector. It can be obtained by introducing the vector into a host cell and expressing it (Morrison, SL, et al., Proc. Natl. Acad. Sci. USA, 81, 6851-6855, 1984).

(Production of humanized antibody)
The humanized antibody of the present invention constructs a DNA encoding a V region in which amino acid sequences encoding CDRs of VH and VL of a non-human animal monoclonal antibody that binds to PDGFRα are grafted on VH and VL of human antibodies, The constructed DNA can be obtained by binding to a constant region cDNA of human-derived immunoglobulin and incorporating it into an expression vector, and introducing the vector into an appropriate host cell for expression (L. Rieohmann et al., Nature, 332). 323, 1988: Kettleborough, CA et al., Protein Eng., 4, 773-783, 1991; Clark M., Immunol. Today., 21, 397-402, 2000).

The CDR of the non-human animal monoclonal antibody includes the amino acid sequence predicted from the DNA sequence encoding the VH and VL of the non-human animal monoclonal antibody obtained by the above-described method, the entire amino acid sequence of VH and VL of the known antibody, Can be obtained by comparing. The amino acid sequence of a known antibody can be obtained from the amino acid sequence of an antibody registered in a database such as a protein data bank. Preferably, CDR1, CDR2 and CDR3 of the heavy chain are respectively described in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively. And / or light chain CDR1, CDR2 and CDR3 are the amino acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, or SEQ ID NO: 36, respectively. It has the amino acid sequence set forth in SEQ ID NO: 37 and SEQ ID NO: 38. More preferably, the heavy chain CDR1, CDR2, and CDR3 are each composed of the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and the light chain CDR1, CDR2, and CDR3 are respectively It consists of the amino acid sequences described in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, or the heavy chain CDR1, CDR2 and CDR3 are the amino acids described in SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively. The light chain CDR1, CDR2, and CDR3 are amino acid sequences described in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively.

Further, the FR of a human antibody is not particularly limited as long as the antibody after transplantation exhibits the effects of the present invention. Preferably, the variable region of a humanized antibody (hereinafter referred to as “V region”) is a CDR. Human antibody FR having a three-dimensional structure similar to that of the V region of the non-human animal monoclonal antibody derived from or human antibody FR having high homology with the FR amino acid sequence of the non-human animal monoclonal antibody used. Whether or not the V region of the humanized antibody having the FR of the selected human antibody has a three-dimensional structure similar to the V region of the non-human animal monoclonal antibody from which the CDR is derived can be determined by, for example, determining the FR and non- The three-dimensional structure can be predicted by computer modeling based on the DNA sequence information of the V region containing the CDR of the human animal monoclonal antibody and compared with the three-dimensional structure of the V region of the non-human animal monoclonal antibody from which the CDR is derived. it can. The FR amino acid sequence of the non-human animal monoclonal antibody can be obtained from the VH and VL amino acid sequences and the CDR amino acid sequence information obtained by the above-described method. The V region of a humanized antibody can also be designed by appropriately mutating the FR amino acid sequence of a human antibody. The antibody of the present invention preferably has the amino acid sequences described in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 as heavy chains FR1, FR2, FR3 and FR4, respectively. The antibody of the present invention preferably has the amino acid sequences described in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 as the light chains FR1, FR2, FR3 and FR4.

The DNA sequence encoding the V region of the humanized antibody to be used is designed as a DNA sequence corresponding to the amino acid sequence obtained by combining the CDR amino acid sequence of the non-human animal monoclonal antibody and the FR amino acid sequence of the human antibody. DNA encoding the V region of a humanized antibody can be prepared by a method well known to those skilled in the art based on the designed DNA sequence. For example, it can be obtained by chemically synthesizing a DNA fragment having a length of about 100 bp as a synthetic DNA based on the designed DNA and amplifying the DNA fragment by PCR. Further, the DNA fragment of about 100 bp is bound using an enzyme such as ligase, PCR is performed using primers encoding the sequences at both ends of the designed DNA sequence encoding the V region of the humanized antibody, It can also be obtained by extracting a DNA fragment having a length of. Furthermore, DNA encoding the V region of a humanized antibody used for PCR can also be obtained by a method known as CDR grafting. The DNA encoding the V region of the humanized antibody to be used can also be obtained by incorporating a DNA encoding CDR into the DNA of the V region of the human antibody by site-directed mutagenesis. Site-specific mutations include, for example, Gene Taylor Site-Directed Mutagenesis System (Gene Taylor Site-Directed Mutagenesis System (Invitrogen)), Transformer Site-Directed Mutagenesis Kit (Clontech), Site Directed A mutagenesis system (Site-Directed Mutagenesis System) (Takara Bio Inc.) or the like can be used by following the description of the kit. Preferably, VH comprises the amino acid sequence of SEQ ID NO: 10 and / or VL comprises the amino acid sequence of SEQ ID NO: 11.

As human antibodies CH and CL used in the preparation of humanized antibodies, any human antibody CH and CL can be used. Examples include human γ1, γ2 CH and human κ CL. As a gene encoding CH and CL of a human antibody, chromosomal DNA or cDNA can be used. The DNA encoding the V region of the humanized antibody obtained by the above-described method is bound to, for example, DNA encoding human antibody CH and CL, respectively, and incorporated into an expression vector for animal cells. A vector that expresses the conjugated antibody can be prepared.

Examples of enhancers and promoters used for expression of humanized antibodies include enhancers and promoters of immunoglobulin genes themselves or non-immunoglobulin enhancers and promoters. As an expression vector for animal cells, for example, pSV2-gpt (RC Mulligan and P. Berg, Science, 209, 1422, 1980) can be used. The genes encoding the H chain and L chain of the humanized antibody of the present invention produced by the above method may be incorporated into the same vector or into different vectors.

(Human antibody)
Human antibodies can be obtained, for example, by using a human antibody phage library or a human antibody-producing transgenic mouse (Tomitsuka et al., Nature Genet., 15, 146-156 (1997)).

The human antibody phage library allows human antibody Fab or scFv to be displayed on the surface as a fusion protein by introducing the VH gene and VL gene from the antibody gene pool having various sequences derived from human B cells into the phage gene. A phage library. As such a human antibody phage library, naive / non-immune obtained by amplifying the VH gene and VL gene of a normal human antibody from peripheral blood lymphocytes by RT-PCR and making it into a library. Library (Cambridge Antibody Technology, Inc .; Medical Research Counsel, Diax, etc.), select functional specific antibody genes in human B cells, and select appropriate antigen binding region such as CDR3 region of V gene fragment Synthetic library (Bioinvent; Crucell; Morphosis) substituted with an oligonucleotide encoding a random amino acid sequence in length, and a patient who has been vaccinated with a cancer patient or target antigen Library created from lymphocytes Iburari can be mentioned.

When a human antibody phage library is used, for example, PDGFRα or a peptide having an epitope sequence recognized by the antibody of the present invention is immobilized on a solid phase, the phage antibody library is reacted, and unbound phage is washed away. Thereafter, the desired phage can be obtained (panning) by recovering the bound phage. Moreover, the accuracy of the obtained clone can be raised by amplifying the obtained phage and repeatedly performing panning for the amplified library. By analyzing the VH gene and VL gene of the obtained clone, a complete human antibody having these gene sequences can be prepared.

A human antibody-producing transgenic mouse is a mouse in which an Ig gene of a human antibody is introduced into a mouse in which an endogenous immunoglobulin (Ig) gene is knocked out. A human antibody-producing transgenic mouse can be obtained, for example, by the following method. A human-mouse hybrid cell is treated with colcemid (spindle formation inhibitor) for 48 hours to form a microcell, which is a structure in which one to several chromosomes are enveloped in the nuclear membrane. Microcells isolated in the presence of cytochalasin B are fused with chromosome recipient cells (mouse ES cells) with polyethylene glycol to produce microcell hybrid ES cells, which are injected into mouse embryos. By immunizing a human antibody-producing transgenic mouse with an antigen (preferably a peptide having an epitope sequence recognized by the antibody of the present invention) according to the above-described method for producing an anti-PDGFRα antibody, an anti-PDGFRα human antibody is obtained. Obtainable.

(Preparation of antibody fragments)
An F (ab ′) ₂ fragment (an antibody fragment having an antigen binding activity with a molecular weight of about 100,000) is obtained by cleaving at the 234th amino acid residue of the H chain by treating the IgG antibody of the present invention with pepsin. be able to. The Fab ′ fragment can be obtained by treating F (ab ′) ₂ obtained by the above-described method with dithiothreitol. The Fab ′ fragment of the present invention can be obtained from DNA encoding Fab ′ of the antibody of the present invention. Fab fragments (antibody fragments having an antigen binding activity of about 50,000 molecular weight in which about half of the region on the N-terminal side of the H chain and all regions of the L chain are bound by disulfide bonds) are treated with papain. By doing so, it can be obtained by cleaving at the 224th amino acid residue of the H chain. The Fab fragment of the present invention can be obtained from DNA encoding the Fab of the antibody of the present invention. The scFv can be obtained by inserting a DNA encoding a linker sequence between cDNAs encoding the VH and VL of the antibody of the present invention to construct a DNA encoding scFv. The length of the linker is not particularly limited as long as VH and VL can associate with each other, but is preferably 10 to 20 residues, and more preferably 15 residues. The linker sequence is not particularly limited as long as it does not inhibit folding of the polypeptide chains of the two domains of VH and VL, but is preferably a linker composed of glycine and / or serine, more preferably , GGGGS (G: glycine, S: serine) or a repetitive sequence thereof. dsFv can be obtained by substituting one cysteine residue in each of VH and VL with a cysteine residue and connecting the cysteine residues with a disulfide bond. Diabody can be obtained by constructing the above-mentioned scFv-encoding DNA such that the amino acid sequence of the linker is 8 residues or less (preferably 5 residues). Bispecific diabody can be obtained by preparing scFv by combining VH and VL DNAs of two different types of scFv. The peptide containing CDR of the present invention can be obtained by designing as a peptide having the amino acid sequence of CDR of VH or VL of the antibody of the present invention.

(ADCC activity measurement)
The “ADCC activity” in the present specification can be measured by any method as long as it is a known method for measuring ADCC activity, but preferably means the activity measured by the following procedure. . Target cell line (eg, human osteosarcoma cell line) is placed in a 96-well plate in a suitable medium (eg, a medium containing 10% fetal bovine serum containing equal amounts of Ham F12 medium and Dulbecco MEM medium). The cells are seeded so as to have × 10 ³ to 1 × 10 ⁵ cells / well and cultured at 37 ° C. for 12 to 48 hours. A test antibody or a control antibody (an antibody known not to show ADCC activity. For example, an anti-GST antibody) is added so as to be 0.1 to 1.0 μg / well. Thereafter, T cells prepared in advance are added to each well and allowed to react at 37 ° C. for 8 to 24 hours. After the reaction, each well is washed with a culture medium, and a new medium and an MTS reagent are added. After culturing at 37 ° C. for 1 to 12 hours, the absorbance at 490 nm is measured. The ADCC activity (%) is calculated by 100 − ((absorbance of test antibody) / (absorbance of control antibody)) × 100 (%) based on the obtained absorbance value. In the present specification, the “measured value serving as an index of ADCC activity” means a measured value measured by the above method.

(CDC activity measurement)
In this specification, “CDC activity” can be measured by any method as long as it is a known method for measuring CDC activity, but preferably means the activity measured by the following procedure. To do. Target cell line prepared to be 10 × 10 ³ to 10 × 10 ⁶ cells / mL in an appropriate medium (for example, Ham F12 medium containing 10% fetal bovine serum and Dulbecco MEM medium mixed in equal amounts) (For example, human small cell lung cancer strain SY) was seeded, and an appropriate medium (for example, Ham F12 medium and Dulbecco MEM medium) in which a test antibody or a control antibody (for example, anti-GST antibody) was dissolved was mixed in an equal amount. Medium) (antibody solution) is added at 0.005 to 0.1 μg / well. As a control, only the medium is added to the antibody non-administered group in the same amount as the antibody solution. Fresh antiserum collected from nude mice is diluted 50-fold with an appropriate medium (for example, a medium in which Ham F12 medium and Dulbecco MEM medium are mixed in equal amounts) and added to each well. After reaction at 37 ° C. for 1 to 24 hours, the culture supernatant is collected and the degree of cell damage is measured using a lactate dehydrogenase (LDH) measurement reagent. The CDC activity (%) is calculated by (each antibody administration group / antibody non-administration group) × 100 (%). In the present specification, the “measured value serving as an index of CDC activity” means a measured value measured by the above method.

(Measurement of dissociation constant)
In the present specification, the dissociation constant can be measured by any method as long as it is a known method for measuring the dissociation constant, but preferably means a numerical value measured by the following method. In accordance with the manual of BIACORE (GE Healthcare Biosciences, BIACORE-X), the PDGFRα extracellular region (1-514 amino acids) and mouse IgG constant region fusion protein (PDGFRα-Fc) were immobilized on the CM5 chip, Flow, Kon, Koff are measured, and the dissociation constant (KD value) is determined.

(Pharmaceutical composition)
The antibody of the present invention can be used as a medicine based on knowledge usually used by those skilled in the art of antibody medicine. Therefore, in one aspect, the present invention relates to a pharmaceutical composition containing the antibody of the present invention as an active ingredient. Specifically, the pharmaceutical composition of the present invention may be a therapeutic or prophylactic agent for diseases caused by cell proliferation, for example, a therapeutic or prophylactic agent for tumors (cancer and / or sarcoma). Can do. More specifically, the pharmaceutical composition of the present invention comprises melanoma, small cell lung cancer, non-small cell lung cancer, glioma, liver cancer, prostate cancer, endometrial cancer, kidney cancer, head and neck cancer, mesothelioma, sarcoma, Bile duct cancer, small intestine adenocarcinoma, childhood malignant tumor, squamous cell carcinoma, gastrointestinal stromal tumor, esophageal cancer, thyroid cancer, bladder cancer, colon cancer, gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, breast cancer, neuroblast It may be used as a therapeutic or preventive for cell tumor (neuroblastoma), glioblastoma (glioblastoma), uterine cancer, ovarian cancer, Wilms tumor, chronic myelogenous leukemia, Philadelphia chromosome positive chronic myelogenous leukemia it can. When the antibody of the present invention is used as a pharmaceutical, examples of the administration site include oral administration, buccal administration, intratracheal administration, subcutaneous administration, intramuscular administration, and intravascular (intravenous) administration. The pharmaceutical composition can be, for example, an injection, capsule, tablet, syrup, or granule preparation. The antibody of the present invention may be administered alone or together with a pharmacologically acceptable single substance.

Hereinafter, examples will be shown in order to explain the present invention in more detail, but the present invention is not limited thereto. It should be noted that all documents cited throughout this application are incorporated herein by reference in their entirety.

Example 1 Production of Monoclonal Antibody Against Human PDGFRα As an immunizing antigen, PDGFRα extracellular region (1-514 amino acids) and mouse IgG2a constant region fusion protein (PDGFRα-Fc) were used. This was used to immunize BALB / c mice. After four immunizations of mice, the spleen monocytes of the immunized mice and a fusion partner, X63-Ag8-653, were subjected to polyethylene glycol-mediated cell fusion, and the literature (J. Immunol. 146: 3721-3728). ) Hybridomas were selected by the method described. Selection was performed by selecting cells that respond to immobilized PDGFRα-Fc.

(Example 2) Measurement of Western blotting, immunoprecipitation, FACS, and dissociation constant For the prepared monoclonal antibody, the dissociation constant was measured by Western blotting, immunoprecipitation, FACS, and BIACORE according to the following procedure.
(1) Western blotting was performed according to a conventional method of Western blotting (for example, “Molecular Biology Basic Experimental Method”, Nankodo). For Western blotting, the human osteosarcoma cell line MG-63 (ATCC, CRL-1427) whose expression of PDGFRα was confirmed was used. Cultured in an equal volume of Ham F12 medium and Dulbecco MEM medium containing 10% fetal bovine serum, 2 × Sample buffer (125 mM Tris / HCl [pH 6.8], 4% SDS, 20% Glycerol, 10 % 2-Mercaptoethanol, 0.02% Bromophenol blue), and subjected to SDS polyacrylamide electrophoresis. After the gel was transferred to a PVDF membrane (Millipore, IPVH00010), each antibody obtained in Example 1 was reacted and detected with an anti-mouse IgG HRP-labeled antibody (Immuno-Biological Laboratories, Inc., 17504).
The results are shown in Table 1. In the table, x indicates no reactivity, and Δ indicates weakly positive.

(2) Immunoprecipitation was performed according to a conventional immunoprecipitation method (for example, “Molecular Biology Basic Experiment Method”, Nankodo). For the immunoprecipitation method, a human small cell lung cancer strain SY in which PDGFRα expression was confirmed (provided by Aichi Cancer Center, see Br. J. Cancer (1991), 63, Suppl. XIV, 24-28). Using. After culturing in a medium in which Ham F12 medium containing 10% fetal bovine serum and Dulbecco MEM medium were mixed in equal amounts, the cells were collected and TNE buffer (20 mM Tris / HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0 A cell extract was prepared at 5% [v / v] NP-40). The cell extract was subjected to an absorption operation of non-specific reaction with Protein G Sepharose (GE Healthcare Biosciences, 17-0618-01), and then 5 μg of each antibody obtained in Example 1 was reacted. Thereafter, Protein G Sepharose (GE Healthcare Bioscience Co., Ltd., 17-0618-01) was added to recover the antibody and antibody reaction product. The recovered antibody was subjected to SDS polyacrylamide electrophoresis. After the gel was transferred to a PVDF membrane (Millipore, IPVH00010), human PDGFRα polyclonal antibody C-20 (Santa Cruz Biotechnology, sc-338) was reacted, and an anti-rabbit IgG HRP-labeled antibody (Immuno-Biological Laboratories, 17502). ). The results are shown in Table 1. In the table, ◯ indicates strong positive.

(3) FACS (Fluorescence Activated Cell Sorting)
Flow cytometry was performed according to a conventional method (for example, “Molecular Biology Basic Experiment”, Nanedo). For flow cytometry, human small cell lung cancer strain SY in which PDGFRα expression was confirmed (provided by Aichi Cancer Center, see Br. J. Cancer (1991), 63, Suppl. XIV, 24-28). Was used. It was cultured in a medium in which Ham F12 medium containing 10% fetal bovine serum and Dulbecco MEM medium were mixed in equal amounts. The cells were adjusted to 2 × 10 ⁶ cells / mL with staining buffer (PBS, 0.1% NaN ₃ , 5% fetal bovine serum, 1 mM EDTA) (hereinafter referred to as “SB”), and then 5 μg / mL in advance. 50 μL was added to a 96-well plate (FALCON, 353077) into which 50 μL of each antibody obtained in Example 1 prepared in 1 was dispensed. After reacting at 4 ° C. for 30 minutes, the plate was washed with SB, and anti-mouse IgG FITC-labeled antibody (Immuno-Biological Laboratories, Inc., 17621) was added. After reacting at 4 ° C. for 30 minutes, the plate was washed with SB, resuspended in 100 μL of SB, and detected with a FACS apparatus (BECTON DICKINSON, FACSort). The results are shown in Table 1. In the table, ◯ indicates strong positive.

(4) Measurement of dissociation constant by BIACORE Among the prepared monoclonal antibodies, for clone numbers 5A1, 8A1, 9A1, 18A1, 24A1 and 40A1, the dissociation constant (KD) between the antibody and the antigen is BIACORE (GE Healthcare Bioscience) Measurement was conducted using BIACORE-X). Example 1 After immobilizing PDGFRα extracellular region (1-514 amino acids) and mouse IgG constant region fusion protein (PDGFRα-Fc) on a CM5 chip according to the manual of BIACORE (GE Healthcare Biosciences, BIACORE-X) Each PDGFRα antibody prepared in (1) was flowed, Kon and Koff were measured, and the KD value was determined. The results are shown in Table 1.

(Example 3) Measurement of neutralizing activity It was confirmed that the human osteosarcoma cell line MG-63 (ATCC, CRL-1427) expresses PDGFRα (results not shown). The neutralizing activity of the antibody obtained in Example 1 was measured using human osteosarcoma cell line MG-63. The human osteosarcoma cell line MG-63 was added to a 6-well plate (Corning Inc, 3506) in 2 × 10 ⁵ cells in a medium containing 10% fetal calf serum mixed with equal amounts of Ham F12 medium and Dulbecco MEM medium. / Well, and cultured at 5% CO ₂ and 37 ° C. for 24 hours. Thereafter, the medium was removed, each antibody obtained in Example 1 adjusted to 10 μg / mL with an equal volume of Ham F12 medium and Dulbecco MEM medium, or normal mouse serum (Normal Mouse IgG as a negative control). ) (Immuno-Biological Laboratories, Inc., 17314) was added and reacted at 5% CO ₂ and 37 ° C. for 30 minutes. After the reaction, the medium containing the antibody was removed, and 1 mL of PDGF-AA (PEPROTECH, 100-13A), a PDGFRα ligand prepared to 100 ng / mL in a medium in which Ham F12 medium and Dulbecco MEM medium were mixed in equal amounts. The mixture was added and reacted at 37 ° C. for 10 minutes with 5% CO ₂ . Thereafter, the cells were collected, and a cell extract was prepared with TNE buffer (20 mM Tris / HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.5% [v / v] NP-40). Using the PDGFRα monoclonal antibody 10A1 prepared in Step 1, immunoprecipitation was performed according to the description in Test Example 1-1 (2), and SDS polyacrylamide gel electrophoresis was performed. After the gel was transferred to a PVDF membrane (Millipore, IPVH00010), PDGFRα was detected with a human PDGFRα polyclonal antibody C-20 (Santa Cruz Biotechnology, sc-338) and a phosphotyrosine antibody 4G10 (Millipore, 05-321). did.

The results are shown in FIG. Each antibody obtained in Example 1 suppressed the phosphorylation of PDGFRα by PDGF-AA as compared with the case where normal mouse serum (Normal Mouse IgG) was administered. In particular, the antibodies of clone numbers 5A1, 22A1 and 24A1 showed remarkable phosphorylation neutralizing activity. Each antibody obtained in Example 1 was shown to have neutralizing activity against PDGFRα and suppress signal transduction downstream of PDGFRα.

(Example 4) ADCC activity measurement The ADCC activity of the antibody obtained in Example 1 was measured using human osteosarcoma cell line MG-63 (ATCC, CRL-1427).
(A) Preparation of T cells T cells used in ADCC activity measurement were prepared by the following procedure. Spleen cells collected from nude mice (Nippon Charles River, Inc., 9238301 CAnN.Cg-Foxn1nu / CrlCrlj (BALB / c nude mice)) were separated using a column (Polysciences Inc., 21759) filled with nylon wool. did. The obtained non-adsorbed fraction (T cell fraction) was added with concanavalin A (Vector Laboratories Inc., L-1000) at a final concentration of 5 μg / mL and stimulated at 37 ° C. for 3 hours, and then with Ham F12 medium. It was prepared to be 1 × 10 ⁶ cells / mL in a medium mixed with an equal volume of Dulbecco MEM medium. The prepared T cells were used as T cells in the following ADCC activity measurement test.

(B) ADCC activity measurement Human osteosarcoma cell line MG-63 (ATCC, CRL-1427) was added to a 96-well plate in a medium in which Ham F12 medium and Dulbecco MEM medium were mixed in an equal amount containing 10% fetal bovine serum. (Corning Inc., 3598) was seeded at 1 × 10 ⁴ cells / well and cultured at 5% CO ₂ and 37 ° C. for 24 hours. Thereafter, the medium was removed, and each antibody obtained in Example 1 adjusted to 0.4 μg / mL with a medium in which Ham F12 medium and Dulbecco MEM medium were mixed in equal amounts was added to 50 μL / well. As a negative control, an anti-GST antibody 14C1 prepared according to a conventional method and adjusted to 0.4 μg / mL was added to 50 μL / well. Thereafter, T cells prepared in advance were added to each well at 50 μL / well and reacted at 37 ° C. for 12 hours. After the reaction, each well was washed with a culture medium, added with 100 μL of the same medium and 20 μL of MTS reagent (Promega, G3580), cultured at 37 ° C. for 2 hours, and then measured for absorbance at 490 nm (reference wavelength: 650 nm). The ADCC activity (%) was calculated by 100− (each antibody administration group / negative control) × 100 (%).

The results are shown in FIG. Compared with the negative control 14C1, administration of each PDGFRα antibody showed antibody-dependent ADCC activity. In particular, the antibodies of clone numbers 1A1, 5A1, 8A1, 22A1, 24A1 and 45A1 showed excellent ADCC activity.

(Example 5) Measurement of CDC activity Human small cell lung cancer strain SY in which PDGFRα expression was confirmed (provided by Aichi Cancer Center, see Br. J. Cancer (1991), 63, Suppl. XIV, 24-28). Then, the CDC activity of the antibody obtained in Example 1 was measured.
Human small cell lung cancer strain SY (provided by Aichi Prefectural Cancer Center) prepared to be 4 × 10 ⁵ cells / mL in a medium in which Ham F12 medium containing 10% fetal bovine serum and Dulbecco MEM medium are mixed in equal amounts , Br. J. Cancer (1991), 63, Suppl. XIV, 24-28) is seeded in a 96-well plate (Corning Inc., 3598) at 50 μL / well, Ham F12 medium, Dulbecco MEM medium, etc. Each antibody obtained in Example 1 adjusted to 4 μg / mL or 20 μg / mL with a medium mixed in an amount was added at 50 μL / well so that the final concentration was 1 μg / mL or 5 μg / mL. As a negative control, anti-GST antibody 40B3 prepared according to a conventional method and adjusted to 4 μg / mL or 20 μg / mL was added to a concentration of 50 μL / well. As a control, 50 μL / well of a medium in which Ham F12 medium and Dulbecco MEM medium were mixed in equal amounts was added to the antibody non-administered group. Fresh antiserum collected from nude mice (Charles River Japan, 9238301 CAnN.Cg-Foxn1nu / CrlCrlj (BALB / c nude mice)) was mixed with an equal amount of Ham F12 medium and Dulbecco MEM medium in a medium 50 Diluted twice and added to each well at 100 μL / well. After reacting at 5% CO ₂ and 37 ° C. for 5 hours, the culture supernatant was collected, and the degree of cell damage was measured using a lactate dehydrogenase (LDH) measurement reagent (Promega, G1780). The CDC activity (%) was calculated by (each antibody administration group / antibody non-administration group) × 100 (%).

The results are shown in FIG. Among the antibodies prepared in Example 1, complement-dependent cytotoxic activity in the antiserum was observed by adding antibodies of clone numbers 1A1, 5A1, 8A1, 9A1, 10A1, 18A1, 21A1 and 22A1. It was. In particular, it was shown that the antibodies of clone numbers 5A1, 8A1, 21A1 and 22A1 are excellent in CDC activity.

(Example 6) Measurement of anti-tumor effect The anti-tumor effect of the antibody prepared in Example 1 was confirmed in vivo using a nude mouse transplanted human tumor model (Xenograft).
1 × 10 ⁷ SY cells (provided by Aichi Cancer Center, Br.J) Cancer (1991), 63, Suppl. XIV, 24-28). Two weeks later, it was confirmed that tumors were formed, and 400 μg each of the antibody prepared in Example 1 was administered by intravenous tail injection. As a negative control, 400 μg of GST antibody 14C1 was used. Each antibody was administered once a week to 5 nude mice. The antitumor effect was measured by measuring the tumor size once a week.

The results are shown in FIG. One week after antibody administration, suppression of tumor growth was observed in all of the antibody administration groups prepared in Example 1 except 22A1. Two weeks after the antibody administration, tumor growth was slow in the antibody administration groups of clone numbers 5A1, 10A1, 18A1, and 40A1, and a marked antitumor effect was observed in the antibody administration groups of clone numbers 8A1, 21A1, and 24A1.

For the antibodies of clone numbers 21A1 and 24A1 having a high antitumor effect, the same antitumor effect measurement experiment was performed again to confirm the reproducibility of the antitumor effect. As a result, it was confirmed that the antitumor effect was exhibited with good reproducibility as in Example 5 (results not shown).

(Example 7) Measurement of dose-dependent antibody volume of antitumor effect 1 × 10 ⁷ subcutaneously in the back of nude mice (Charles River Japan, 9238301 CAnN.Cg-Foxn1nu / CrlCrlj (BALB / c nude mice)) SY cells (provided by Aichi Cancer Center, Br. J. Cancer (1991), 63, Suppl. XIV, 24-28) were transplanted. Two weeks later, it was confirmed that tumors were formed, and 1 mg, 400 μg, and 100 μg of the clone No. 24A1 antibody prepared in Example 1 (hereinafter referred to as “24A1 antibody”) were administered by tail vein injection. As a negative control, 400 μg of GST antibody 40B3 was used. Each antibody was administered once a week to 5 nude mice. The antitumor effect was measured by measuring the tumor size once a week.

The results are shown in FIG. In all the groups administered with 24A1 antibody, tumor growth was suppressed. Therefore, it was shown that the 24A1 antibody has a tumor suppressing effect even when administered at 0.1 mg / animal. In addition, as the dose of 24A1 antibody increased, the tumor growth inhibitory effect was high. Therefore, it was confirmed that the 24A1 antibody suppresses tumor growth in a dose-dependent manner.

(Example 8) Epitope mapping In Examples 5 and 6, the epitopes of the 24A1 antibody and the 21A1 antibody confirmed to have antitumor effects were determined by epitope mapping using a dot blot.
A gene encoding the extracellular region (1-514 amino acids) (SEQ ID NO: 14) of human PDGFRα and genes encoding various deletion mutants deleted from the C-terminus of the region were prepared. The generated deletion mutant genes were human PDGFRα 1-106 amino acids (PDGFRα106-Fc), 1-213 amino acids (PDGFRα213-Fc), 1-311 amino acids (PDGFRα311-Fc), 1-409 amino acids (PDGFRα409-Fc). ), Or 1-514 (PDGFRα-Fc) (see FIG. 6). COS cells (ATCC, CRL-1650) are transfected with expression vectors into which these genes have been introduced, and cultured for 2 days in serum-free Dulbecco MEM medium (Immuno-Biological Laboratories, Inc., 33252), and the culture supernatant is recovered. did. The collected culture supernatant was ultra-concentrated to a 10-fold concentration and dot-blotted onto a PVDF membrane (Millipore, IPVH00010). After reacting with 21A1 antibody or 24A1 antibody, detection was performed with an anti-mouse Igκ chain HRP-labeled antibody (Southern Biotech, 1050-05).

Results are shown in FIG. It was shown that the 21A1 antibody has an epitope in the 106-213 amino acid (SEQ ID NO: 2) region of PDGFRα, and the 24A1 antibody has an epitope in the 213-311 amino acid (SEQ ID NO: 3) region.

Example 9 Antibody Sequencing In order to determine the gene sequence of the 24A1 antibody, the heavy chain (hereinafter referred to as “Hc”) and the light chain (hereinafter referred to as “Lc”) were cloned. MRNA was extracted from the hybridoma producing the 24A1 antibody to prepare cDNA. According to a conventional method, 5′-Rapid amplification cDNA end (5′-RACE) was performed to determine the base sequences of the 5 ′ ends of the Hc and Lc genes. The full length cDNAs were cloned using primers specific for Hc and Lc. The determined gene sequences of Hc and Lc were as shown in SEQ ID NO: 29 and SEQ ID NO: 31, and the amino acid sequences were as shown in SEQ ID NO: 30 and SEQ ID NO: 32, respectively.

An expression vector was prepared by incorporating the cloned Hc and Lc genes of 24A1 antibody into pcDNA3.1 (+) (Invitrogen, V790-20), and expression was confirmed by a transient expression test. COS-1 cells (ATCC, CRL-1650) were transfected with Hc expression vector alone, Lc expression vector alone, or both Hc expression vector and Lc expression vector. After culturing in serum-free Dulbecco MEM medium (Immuno-Biological Laboratories, Inc., 33252) for 2 days, the culture supernatant and cells were recovered. The culture supernatant was ultra-concentrated to a 10-fold concentration and subjected to SDS-PAGE polyacrylamide gel electrophoresis. The gel was transferred to a PVDF membrane (Millipore, IPVH00010), and then detected with an anti-mouse IgG HRP-labeled antibody (Immuno-Biological Laboratories, Inc., 17504).

The results are shown in FIG. Expression of Hc, Lc and Hc + Lc was confirmed in the cell pellet. It was also confirmed that Hc + Lc of the 24A1 antibody was efficiently secreted into the culture supernatant.

(Example 10) Antigen specificity test To confirm the binding activity and antigen specificity of 21A1 antibody and 24A1 antibody gene products transiently expressed in COS-1 (ATCC, CRL-1650), EIA method was performed. It was. Human PDGFRα-Fc fusion protein (PDGFRα extracellular region [1-514 amino acids] and mouse IgG2a constant region fusion protein) and human c-kit-Fc fusion protein (c-Kit extracellular region [1-504 amino acids] and mouse IgG2a The constant region fusion protein) was immobilized on a 96-well plate (Nalge Nunc International KK, 430341). COS-1 cell culture supernatant in which Hc and Lc of 24A1 antibody were co-expressed was serially diluted, and 50 μL was added to each well. After reacting at 37 ° C. for 2 hours, an orthophenylenediamine · 2HCl (OPD) (SIGMA, P8787) solution washed with PBS added with tween 20 to a final concentration of 0.05% and adjusted to 400 μg / mL 100 μL was added to cause coloration.

The results are shown in Table 2. The 21A1 antibody and 24A1 antibody in the culture supernatant reacted only with the PDGFRα-Fc fusion protein, and no reaction with the c-kit-Fc fusion protein was confirmed. Therefore, it was shown that the 21A1 antibody and the 24A1 antibody expressed by the expression vector bind to PDGFRα and that the binding is antigen-specific.

(Example 11) Humanization of 24A1 antibody The gene was modified for the purpose of humanizing the 24A1 antibody cloned in Example 9. Human Hc and Lc genes having homology with the gene sequences of the Hc and Lc variable regions of 24A1 antibody were searched by IgBLAST of National Center for Biotechnology Information (NCBI). Hc had the highest homology with IGHV4-30-4 (SEQ ID NO: 27) and Lc with IGKV1D-12 (SEQ ID NO: 28). Based on this sequence, the Hc and Lc genes of the 24A1 antibody were modified and humanized at the amino acid level. FIG. 9 shows a comparison of the amino acid sequences of Hc, and FIG. 10 shows a comparison of the amino acid sequences of Lc. The CDRs were determined according to the rules of KABAT et al. (“Sequences of Proteins of Immunological Interest”, Kabat, E. et al., US Department of Health and Human services, 1983). 9 and 10, each CDR is indicated by an underline. Each CDR is as follows.

Heavy chain CDR1: SDYAWN (SEQ ID NO: 4)
Heavy chain CDR2: YISYSGSTSYNPLSKS (SEQ ID NO: 5)
Heavy chain CDR3: RDYRYGYWYFDV (SEQ ID NO: 6)
Light chain CDR1: HASQNIHVWLT (SEQ ID NO: 7)
Light chain CDR2: KASNLHT (SEQ ID NO: 8)
Light chain CDR3: QQGQSYPWT (SEQ ID NO: 9)

The CDR sequence is a sequence that determines antigen specificity, and the surrounding amino acid sequence plays an important role in taking the three-dimensional structure presenting the CDR to the antigen. Therefore, the 5 amino acids near the CDR remain mouse type. And the other framework regions (hereinafter referred to as “FR”) were replaced with human amino acid sequences. Each FR is as follows.

Heavy chain FR1: DVQLQESGPGLVKPSQTLSLTCTVSGYSIT (SEQ ID NO: 15)
Heavy chain FR2: WIRQFPGNNKLEVG (SEQ ID NO: 16)
Heavy chain FR3: RISITRDTSKNQFFFLQLNSVTTEDTATYYCAR (SEQ ID NO: 17)
Heavy chain FR4: WGAGTTVTVSSAK (SEQ ID NO: 18)
Light chain FR1: DIQMNQSPSSLSASLGDTITTC (SEQ ID NO: 19)
Light chain FR2: WYQQKPGNIIPKLLIY (SEQ ID NO: 20)
Light chain FR3: GVPSRFSGSGSGGTGFLTTISSLQPEDIATYYC (SEQ ID NO: 21)
Light chain FR4: FGGGTKLEIK (SEQ ID NO: 22)

9 and 10, the amino acid sequence after substitution is indicated by “Hu PDGFRa (24A1)”. The amino acid sequence of the heavy chain after substitution is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain is shown in SEQ ID NO: 26. Based on the constructed sequences, gene sequences corresponding to the amino acid sequences of the humanized Hc variable region and the humanized Lc variable region were prepared. The produced artificial genes Hu PDGFRa (24A1) Hc and Hu PDGFRa (24A1) Lc were used as human antibody constant region expression vectors SRH (human IgG1 constant region) and SRL-neo (human Igκ chain constant region) (The Journal of Immunology, Vol. 157, Issue 7: 3148-3152) to prepare a humanized anti-PDGFRa antibody (24A1) expression vector.

In order to confirm the expression of Hc and Lc of the prepared humanized anti-PDGFRa antibody (24A1), a transient expression test was performed. For comparison, an expression vector (chimeric anti-PDGFRa antibody (24A1)) in which the Hc and Lc variable regions of a mouse anti-PDGFRα antibody (24A1 antibody) were respectively incorporated into SRH and SRL-neo was prepared and expressed. COS-1 cells (ATCC, CRL-1650) were transfected with Hc, Lc alone or a mixture of Hc and Lc. After culturing in serum-free Dulbecco MEM medium (Immuno-Biological Laboratories, Inc., 33252) for 2 days, the culture supernatant (Sup) and cells (Cell Pellet) were collected. The culture supernatant was ultra-concentrated to a 10-fold concentration, and each was subjected to SDS-PAGE polyacrylamide gel electrophoresis. After the gel was transferred to a PVDF membrane (Millipore, IPVH00010), it was detected with an anti-human IgG constant region HRP-labeled antibody (Immuno-Biological Laboratories, Inc., 17507) and an anti-human Igκ chain HRP-labeled antibody (BIOSOURCE, AHI11804). .

As shown in FIG. 11, the expression of Hc, Lc, and both Hc and Lc was efficiently confirmed in Cell Pellet. Secretion was confirmed in the culture supernatant although the efficiency was low.

(Example 12) Activity measurement of humanized anti-PDGFRα antibody (24A1) EIA was performed to confirm the activity of humanized anti-PDGFRα antibody (24A1). Human PDGFRα-Fc fusion protein (PDGFRα extracellular region [1-514 amino acids] and mouse IgG2a constant region fusion protein) and human FLT4-Fc fusion protein (FLT4 extracellular region [1-766 amino acids] and mouse IgG2a constant region fusion protein) ) Was immobilized on a 96-well plate (Nalge Nunc International KK, 430341). According to the method described in Example 10, the COS-1 cell culture supernatant in which Hc and Lc genes were co-expressed was serially diluted and added to each well. After reacting at 37 ° C. for 2 hours, it was washed and reacted with an anti-human IgG constant region HRP labeled antibody (Immuno-Biological Laboratories, Inc., 17507). After reacting at 37 ° C. for 30 minutes, it was washed and colored with o-phenylenediamine (OPD) (SIGMA, P8787).

The results are shown in Table 3. The humanized PDGFRa antibody (24A1) and human chimeric PDGFRa antibody (24A1) in the culture supernatant reacted only with the PDGFRα-Fc fusion protein, and no reaction with the FLT4-Fc fusion protein was confirmed. This confirmed the antigen specificity of the humanized PDGFRα antibody (24A1). Further, it was shown that the humanized PDGFRα antibody (24A1) has the same activity as the human chimeric PDGFRα antibody (24A1) having a mouse-type variable region.

(Example 13) Determination of antibody sequence To determine the gene sequence of the 21A1 antibody, the heavy chain (hereinafter referred to as “Hc”) and the light chain (hereinafter referred to as “Lc”) were cloned. MRNA was extracted from the hybridoma producing the 21A1 antibody to prepare cDNA. According to a conventional method, 5′-Rapid amplification cDNA end (5′-RACE) was performed to determine the base sequences of the 5 ′ ends of the Hc and Lc genes. The full length cDNAs were cloned using primers specific for Hc and Lc. The determined gene sequences of Hc and Lc were as shown in SEQ ID NO: 39 and SEQ ID NO: 41, and the amino acid sequences were as shown in SEQ ID NO: 40 and SEQ ID NO: 42, respectively (FIGS. 12 and 13).

From the obtained sequence information, according to the rule of KABAT et al. (“Sequences of Proteins of Immunological Interest”, Kabat, E. et al., US Department of Health and Human services, 1983). In FIG. 12 and FIG. 13, each CDR is indicated by an underline. Each CDR was as follows.
Heavy chain CDR1: IFWMS (SEQ ID NO: 33)
Heavy chain CDR2: EINPDSTINYTPSLKD (SEQ ID NO: 34)
Heavy chain CDR3: QRFGGPY (SEQ ID NO: 35)
Light chain CDR1: KASQDVSTAVA (SEQ ID NO: 36)
Light chain CDR2: SASYRYT (SEQ ID NO: 37)
Light chain CDR3: QQYSTPYT (SEQ ID NO: 38)

Claims (16)

1. An anti-PDGFRα antibody that recognizes the amino acid sequence contained in the 106th to 311th amino acid sequences of PDGFRα as an epitope.
2. An anti-PDGFRα antibody that recognizes the amino acid sequence contained in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 as an epitope.
3. An anti-PDGFRα antibody having an antibody-dependent cytotoxic activity (ADCC activity) by T cells of 25% or more.
4. An anti-PDGFRα antibody having ADCC activity equal to or higher than 24A1 antibody.
5. An anti-PDGFRα antibody having a complement-dependent cytotoxicity (CDC activity) of 10% or more.
6. Furthermore, the anti-PDGFRα antibody according to claim 5, wherein the antibody-dependent cytotoxic activity (ADCC activity) by T cells is 25% or more.
7. 6. The anti-PDGFRα antibody according to any one of claims 3 to 5, which recognizes an amino acid sequence contained in the 106th to 311th amino acid sequences of PDGFRα as an epitope.
8. 6. The anti-PDGFRα antibody according to any one of claims 3 to 5, which recognizes an amino acid sequence contained in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 as an epitope.
9. The heavy chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 4, the heavy chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 5, and the heavy chain CDR3 is set forth in SEQ ID NO: 6 And / or the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 7, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 8, and the light chain An antibody having the CDR3 of SEQ ID NO: 9 as the amino acid sequence.
10. (I) The variable region of the heavy chain has one amino acid sequence selected from the following (a) to (c):
    (A) an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10;
    (B) an amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO: 10, and
    (C) the amino acid sequence of SEQ ID NO: 10; and / or
    (Ii) The variable region of the light chain has one amino acid sequence selected from the following (d) to (f):
    (D) a nucleic acid sequence that hybridizes with a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 11 under stringent conditions;
    (E) an amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO: 11, and
    (F) the amino acid sequence of SEQ ID NO: 11,
    An antibody capable of binding to PDGFα.
11. The anti-PDGFRα antibody according to any one of claims 1 to 7, which binds to PDGFRα at a dissociation constant (Kd) of 7 × 10 ^-9 or less.
12. A DNA encoding the antibody according to any one of claims 1 to 11.
13. A vector having the DNA according to claim 12.
14. A host cell having the vector according to claim 13.
15. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 11.
16. The pharmaceutical composition according to claim 15, which is a therapeutic agent for a disease based on cell proliferation.

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