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HK1086283B - Antibodies that bind cell-associated ca 125/0722p and methods of use thereof - Google Patents

Antibodies that bind cell-associated ca 125/0722p and methods of use thereof Download PDF

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Publication number
HK1086283B
HK1086283B HK06106277.5A HK06106277A HK1086283B HK 1086283 B HK1086283 B HK 1086283B HK 06106277 A HK06106277 A HK 06106277A HK 1086283 B HK1086283 B HK 1086283B
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Hong Kong
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antibody
hybridoma
accession number
number pta
seq
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HK06106277.5A
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Chinese (zh)
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HK1086283A1 (en
Inventor
Earl F. Albone
Daniel A. Soltis
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Purdue Pharma L.P.
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Priority claimed from PCT/US2003/032945 external-priority patent/WO2004035537A2/en
Publication of HK1086283A1 publication Critical patent/HK1086283A1/en
Publication of HK1086283B publication Critical patent/HK1086283B/en

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Description

Antibodies that bind cell-associated CA125/0772P and methods of use thereof
This application claims priority to U.S. provisional patent application 60/418,828 filed on 16/10/2002 and 60/485,986 filed on 10/7/2003 in accordance with 35u.s.c.119(e), which are all incorporated herein by reference in their entirety.
1. Field of the invention
The invention provides antibodies and antigen-binding fragments of antibodies that preferentially bind to cell-associated CA 125/0772P polypeptide over shedding CA 125/0772P polypeptide, methods of identifying such antibodies and antigen-binding fragments, and methods of making such antibodies and antigen-binding antibody fragments. The invention also provides methods of preventing, managing, treating, or ameliorating one or more symptoms associated with a CA 125/0772P-related disorder. In particular, the invention provides methods of preventing, treating, or ameliorating one or more symptoms associated with a cell proliferative disorder. For example, the invention provides methods of preventing, treating, or ameliorating one or more symptoms associated with cancer. In a preferred embodiment, the present invention provides a method of preventing, managing, treating or ameliorating one or more symptoms associated with ovarian cancer. The invention also provides compositions and products for preventing, managing, treating, or ameliorating one or more symptoms associated with a CA 125/0772P-related disorder, e.g., cancer, e.g., ovarian cancer. The invention also provides methods of diagnosing a CA 125/0772P-associated disorder or a predisposition for such a disorder.
2. Background of the invention
A high molecular weight polypeptide known as CA125 can be detected in about 80% of ovarian cancer patients (see Kabawat et al, am. J. Clin. Pathol. 79: 98-104 (1983); and Gadducci et al, Gynecol. Oncol. 44: 147-154 (1992)). CA125 is present on the surface of tumor cells, and elevated secreted, or "shed" forms of CA125 are present in about 80-90% of ovarian cancer patients.
Antibodies to CA125 have been generated and used to determine CA125 concentration and to purify CS125 from cell culture media. See, e.g., Bast et al, j.clin.invest.685: 1331-1337 (1981); krantz et al, j.cell.biochem. (suppll.) -12 EU: 139 (1988); U.S. patent nos. 4,921,790, 5,059,680, and 5,976,818; and JP 11014626.
In addition to antibodies that monitor the presence of CA125, U.S. patent nos. 5,858,361 and 6,241,985 describe anti-idiotypic anti-CA 125 antibodies as therapeutic agents.
Despite the above efforts, CA 125-related disorders such as ovarian cancer remain a major problem and, as such, methods and compositions for treating these disorders are highly desirable.
Citation or identification of any reference in this or any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
3. Summary of the invention
The present invention is based in part on the recognition that: the event that produced shed CA 125/0772P also left a portion of the extracellular region of CA 125/0772P amino acid sequence in a cell-associated form, i.e., cell-associated CA 125/0772P was also produced. The present invention is also based in part on the recognition that: antibodies and antigen-binding antibody fragments can be produced that preferentially bind cell-associated CA 125/0772P over shed CA 125/0772P, and these antibodies, or antigen-binding antibody fragments, can be used, for example, to prevent, treat, or ameliorate one or more symptoms of a CA 125/0772P-associated disorder or a CA 125/0772P-associated disorder, such as a cell proliferation disorder, e.g., cancer, e.g., ovarian cancer.
In a first aspect, the invention provides isolated antibodies, or antigen-binding antibody fragments, that preferentially bind to a cell-associated CA 125/0772P polypeptide over shed CA 125/0772P. Also provided are isolated antibodies or antigen-binding antibody fragments that bind the peptides of figure 1. These antibodies and antigen-binding antibody fragments of the invention are useful for a variety of therapeutic, prophylactic, diagnostic and purification purposes as described herein.
In another embodiment, the antibody or antigen-binding antibody fragment of the invention is a monoclonal antibody that binds to SEQ ID NO: 1 or SEQ ID NO: 2 and preferably an antibody that binds cell-associated CA125/0772P or an antibody fragment that binds an antigen. In a particular such embodiment, the antibody or antigen-binding antibody fragment of the invention binds to a heavy chain variable region of SEQ ID NO: 1 or SEQ ID NO: 2, non-repeating regions depicted in fig. 2. In another such embodiment, the antibody or antigen-binding antibody fragment of the invention binds to SEQ ID NO: 1 or SEQ ID NO: 2.
In a first embodiment, an antibody or antigen-binding antibody fragment of the invention inhibits binding of the peptide of FIG. 1 (SEQ ID NO: 1) by less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% in an ELISA competition assay in the presence of a 25-fold (weight/weight) excess of shed CA125/0772P for the peptide of FIG. 1 (SEQ ID NO: 1). In another embodiment, an antibody or antigen-binding antibody fragment of the invention exhibits an IC50 (as measured by percent positive cells) of at least about 0.05mg/ml, at least about 0.25mg/ml, at least about 0.5mg/ml, at least about 0.75mg/ml, or at least about 1.0mg/ml shed CA125/0772P in a flow cytometry competition assay. In a third embodiment, an antibody or antigen-binding antibody fragment of the invention binds to the peptide of fig. 1, but no binding to the split CA125/0772P polypeptide is detected.
An antibody or antigen-binding antibody fragment satisfying any one of these three embodiments constitutes an antibody or antigen-binding antibody fragment that "preferentially binds" a cell-associated CA 125/0772P polypeptide relative to an shed CA 125/0772P polypeptide.
Antibodies and antigen-binding antibody fragments of the invention include antibodies or antigen-binding antibody fragments that bind the peptide of FIG. 1 (SEQ ID NO: 1) with a Kd of less than about 100nM, less than about 10nM, less than about 1nM, less than about 100pM, or less than about 10pM, as measured by the BIAcore affinity assay, described in section 6.4 below.
Preferred embodiments of the antibodies or antigen-binding antibody fragments of the invention include antibodies or antigen-binding antibody fragments that mediate lysis of CA 125/0772P-positive tumor cells in an antibody-dependent cellular cytotoxicity (ADCC) assay. These antibodies or antigen-binding antibody fragments include, for example, antibodies that bind to an antigen in an ADCC assay at a 50: 1 effector: target ratio mediates at least about 10% lysis of CA 125/0772P positive tumor cells at a concentration of 5 μ g/ml antibody or antigen-binding antibody fragment; in an ADCC assay with a 50: 1 effector: target ratio mediates at least about 20% lysis of CA 125/0772P positive tumor cells at a concentration of 5 μ g/ml antibody or antigen-binding antibody fragment; in an ADCC assay with a 50: 1 effector: target ratio mediates at least about 10% lysis of CA 125/0772P positive tumor cells at a concentration of 5.0 μ g/ml antibody or antigen-binding antibody fragment; in an ADCC assay at a 25: 1 effector: target ratio mediates at least about 10% lysis of CA 125/0772P positive tumor cells at a concentration of 5 μ g/ml antibody or antigen-binding antibody fragment; in the ADCC assay at a 12.5: 1 effector: target ratio mediates at least about 10% lysis of CA 125/0772P positive tumor cells at a concentration of 5 μ g/ml antibody or antigen-binding antibody fragment; in the ADCC assay at a 12.5: 1 effector: target ratio mediates at least about 10% lysis of CA 125/0772P positive tumor cells at a concentration of 0.5 μ g/ml antibody or antigen-binding antibody fragment; in the ADCC assay at a 12.5: 1 effector: target ratio the antibody or antigen-binding antibody fragment mediating at least about 10% lysis of CA 125/0772P positive tumor cells at a concentration of 50ng/ml antibody or antigen-binding antibody fragment.
Preferred embodiments of the invention also include antibodies or antigen-binding antibody fragments that mediate lysis of CA 125/0772P-positive tumor cells in a complement-dependent cytotoxicity (CDC) assay. These antibodies or antigen-binding antibody fragments include, for example, antibodies or antigen-binding antibody fragments that mediate cleavage at a concentration ranging from about 15% of antibody or antigen-binding antibody fragment concentration to about 0.1 μ g/ml antibody or antigen-binding antibody fragment concentration of about 95%.
Preferred embodiments of the antibodies or antigen-binding antibody fragments of the invention also include antibodies that inhibit the growth of a CA 125/0772P-positive tumor and antigen-binding antibody fragments.
In a specific embodiment, the antibody of the invention is a monoclonal antibody consisting of hybridoma 4E7 (ATCC)Accession number PTA-5109), or hybridoma 7A11 (ATCC)Accession number PTA-5110), or hybridoma 7C6 (ATCC)Accession number PTA-5111), or from hybridomas7F10(ATCCAccession number PTA-5112), or hybridoma 7G10 (ATCC)Accession number PTA-5245), or hybridoma 7H1 (ATCC)Accession number PTA-5114), or hybridoma 8A1 (ATCC)Accession number PTA-5115), or hybridoma 8B5 (ATCC)Accession number PTA-5116), or hybridoma 8C3 (ATCC) Accession number PTA-5246), or hybridoma 8E3 (ATCC)Accession number PTA-5118), or hybridoma 8G9 (ATCC)Accession number PTA-5119), or hybridoma 15C9 (ATCC)Accession number PTA-5106), or hybridoma 16C7 (ATCC)Accession number PTA-5107), or hybridoma 16H9 (ATCC)Accession number PTA-5108), or hybridoma 117.1 (ATCC)Accession number PTA-4567), or hybridoma 325.1 (ATCC)Accession number PTA-5120), or hybridoma 368.1 (ATCC)Accession number PTA-4568), or by hybridoma 446.1 (ATCC)Accession number PTA-5549), or by hybridoma 501.1 (ATCC)Accession number PTA-4569), or hybridoma 621.1 (ATCC)Accession number PTA-5121), or hybridoma 633.1 (ATCC)Accession number PTA-5122), or hybridoma 654.1 (ATCC)Accession number PTA-5247), or by hybridoma 725.1 (ATCC)Accession number PTA-5124), or hybridoma 776.1 (ATCC)Record number PTA-4570). In another embodiment, the antibody or antigen-binding antibody fragment of the invention is conjugated to hybridoma 4E7 (ATCC)Accession number PTA-5109), or hybridoma 7A11 (ATCC)Accession number PTA-5110), or hybridoma 7C6 (ATCC)Accession number PTA-5111), or hybridoma 7F10 (ATCC)Accession number PTA-5112), or hybridoma 7G10 (ATCC)Accession number PTA-5245), or hybridoma 7H1 (ATCC) Accession number PTA-5114), or hybridoma 8A1 (ATCC)Accession number PTA-5115), or hybridoma 8B5 (ATCC)Accession number PTA-5116), or hybridoma 8C3 (ATCC)Accession number PTA-5246), or hybridoma 8E3 (ATCC)Accession number PTA-5118), or hybridoma 8G9 (ATCC)Accession number PTA-5119), or hybridoma 15C9 (ATCC)Accession number PTA-5106), or hybridoma 16C7 (ATCC)Accession number PTA-5107), or hybridoma 16H9 (ATCC)Accession number PTA-5108), or hybridoma 117.1 (ATCC)Accession number PTA-4567), or hybridoma 325.1 (ATCC)Accession number PTA-5120), or hybridoma 368.1 (ATCC)Accession number PTA-4568), or by hybridoma 446.1 (ATCC)Accession number PTA-5549), or by hybridoma 501.1 (ATCC)Accession number PTA-4569), or hybridoma 621.1 (ATCC)Accession number PTA-5121), or hybridoma 633.1 (ATCC)Accession number PTA-5122), or hybridoma 654.1 (ATCC)Accession number PTA-5247), or by hybridoma 725.1 (ATCC)Accession number PTA-5124), or hybridoma 776.1 (ATCC)Record No. PTA-4570) competes for binding to the cell-associated antibody to CA 125/0772P or to the antigen-binding antibody fragment. An antibody or antigen-binding antibody fragment of the invention is considered to compete for binding if they compete for binding in an ELISA cross-competition assay and/or a FACS cross-competition assay. If IC of competitor antibody or antigen binding fragment 50At a concentration no more than about 100-fold greater than the concentration of antibody or antigen-binding antibody fragment, the antibody or antigen-binding antibody fragment is considered to compete for binding in either an ELISA cross-competition assay or a FACS cross-competition assay. In preferred embodiments, IC of competitor antibody or antigen binding fragment50Is a concentration no more than about 10-fold higher than the concentration of the antibody or antigen-binding fragment. In a more preferred embodiment, the IC of the competitor antibody or antigen-binding antibody fragment50Is a concentration of the antibody or antibody fragment bound to the antigen that is no more than about equimolar.
In another embodiment, an antibody or antigen-binding fragment of the invention comprises a 117.1 light chain polypeptide variable region ("117.1L") comprising the amino acid sequence of SEQ ID No: 27 (117.1L). In yet another embodiment, an antibody or antigen-binding fragment of the invention comprises a 117.1 heavy chain polypeptide variable region ("117.1H") comprising the amino acid sequence of SEQ ID No: 28 (117.1H). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 27(117.1L) and a light chain polypeptide variable region comprising the amino acid sequence described in SEQ ID No: 28(117.1H) or a light chain polypeptide variable region of the amino acid sequence described in (e.g. seq id no).
In another specific embodiment, an antibody or antigen-binding fragment of the invention comprises 368.1 light chain polypeptide variable region ("368.1L") comprising the amino acid sequence set forth in SEQ ID No: 29 (368.1L). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises 368.1 heavy chain polypeptide variable region ("368.1H") comprising the amino acid sequence set forth in SEQ ID No: 30 (368.1H). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 29(368.1L) and a light chain polypeptide variable region comprising the amino acid sequence depicted in SEQ ID No: 30(368.1H) or a pharmaceutically acceptable salt thereof.
In another embodiment, an antibody or antigen-binding fragment of the invention comprises a 501.1 light chain polypeptide variable region ("501.1L") comprising the amino acid sequence of SEQ ID No: 31 (501.1L). In yet another embodiment, an antibody or antigen-binding fragment of the invention comprises a 501.1 heavy chain polypeptide variable region ("501.1H") comprising the amino acid sequence of SEQ ID No: 32 (501.1H). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 31(501.1L) and a light chain polypeptide variable region comprising the amino acid sequence depicted in SEQ ID No: 32(501.1H) or a light chain polypeptide variable region of the amino acid sequence described in seq id no.
In another specific embodiment, an antibody or antigen-binding fragment of the invention comprises 776.1 light chain polypeptide variable region ("776.1L") comprising the amino acid sequence set forth in SEQ ID No: 33 (776.1L). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises 776.1 heavy chain polypeptide variable region ("776.1H") comprising the amino acid sequence set forth in SEQ ID No: 34 (776.1H). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 33(776.1L) and a light chain polypeptide variable region comprising the amino acid sequence depicted in SEQ ID No: 34(776.1H) or a light chain polypeptide variable region of the amino acid sequence described in (776.1H).
In another specific embodiment, the antibody or antigen-binding fragment of the invention comprises a 725.1 light chain polypeptide variable region ("725.1L") comprising the amino acid sequence of SEQ ID No: 54, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a 725.1 heavy chain variable region ("725.1H") comprising the amino acid sequence of SEQ ID No: 53, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 54 and a light chain polypeptide variable region comprising the amino acid sequence depicted in SEQ ID No: 53 to a heavy chain polypeptide variable region of an amino acid sequence described in seq id no.
In another specific embodiment, an antibody or antigen-binding fragment of the invention comprises a 16H9 light chain polypeptide variable region ("16H 9L") comprising the amino acid sequence of SEQ ID No: 56, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a 16H9 heavy chain polypeptide variable region ("16H 9H") comprising the amino acid sequence of SEQ ID No: 55, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 56 and a light chain polypeptide variable region comprising the amino acid sequence depicted in SEQ ID No: 55, or a light chain polypeptide variable region of the amino acid sequence described in seq id no.
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 27(117.1L), and the heavy chain variable region comprises the amino acid sequence set forth in SEQ id no: 30(368.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 29(368.1L), and the heavy chain variable region comprises the amino acid sequence set forth in SEQ id no: 30(368.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 31(501.1L), and the heavy chain variable region comprises the amino acid sequence depicted in SEQ id no: 30(368.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 33(776.1L), and the heavy chain variable region comprises the amino acid sequence set forth in SEQ id no: 30(368.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 54(725.1L), and the heavy chain variable region comprises the amino acid sequence depicted in SEQ id no: 30(368.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 33(16H9L), and the heavy chain variable region comprises the amino acid sequence set forth in SEQ id no: 30(368.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
Antibodies of the invention may include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, trispecific antibodies, multispecific antibodies, diabodies, triabodies, single chain antibodies, or anti-idiotypic antibodies. In a preferred embodiment, the antibody of the invention is a monoclonal antibody that preferentially binds to a cell-associated CA125/0772P polypeptide relative to an shed CA125/0772P polypeptide.
Antigen-binding antibody fragments of the invention may include, but are not limited to, Fab fragments, F (ab')2Fragment, disulfide-linked FvS, single chain Fvs, a variable light chain polypeptide (VL) -containing fragment, a variable heavy chain polypeptide (VH) -containing fragment, or a Complementarity Determining Region (CDR) -containing fragment, and a fragment of any one of the antibodies of the present invention listed above.
Furthermore, the antibodies and antigen-binding antibody fragments of the invention may be of any immunoglobulin class. For example, the antibody of the invention may be an antibody of the IgG, IgM, IgE, IgD, IgA or IgY class. The antibodies of the invention may be of any isotype. For example, the antibody of the invention may be an IgG1、IgG2、IgG3、IgG4、IgA1Or IgA2Heavy chain isotype.
In addition, the antibodies of the invention may, for example, contain inserted into the framework region of the variable light chain region, for example, kappa or lambda light chain variable region, variable heavy chain region, or its CDR. For example, an antibody of the invention may contain a C γ 1 constant region or a C γ 4 constant region.
In another aspect, the invention provides a hybridoma cell that produces a monoclonal antibody of the invention. In one embodiment, the hybridoma of the invention is hybridoma 4E7 (ATCC)Accession number PTA-5109), hybridoma 7A11 (ATCC)Accession number PTA-5110), hybridoma 7C6 (ATCC)Record number PTA-5111) Hybrid cancer 7F10 (ATCC)Accession number PTA-5112), hybridoma 7G10 (ATCC)Accession number PTA-5245), hybridoma 7H1 (ATCC)Accession number PTA-5114), hybridoma 8A1 (ATCC)Accession number PTA-5115), hybridoma 8B5 (ATCC)Accession number PTA-5116), hybridoma 8C3 (ATCC)Accession number PTA-5246), hybridoma 8E3 (ATCC) Accession number PTA-5118), hybridoma 8G9 (ATCC)Accession number PTA-5119), hybridoma 15C9 (ATCC)Accession number PTA-5106), hybridoma 16C7 (ATCC)Accession number PTA-5107), hybridoma 16H9 (ATCC)Accession number PTA-5108), hybridoma 117.1 (ATCC)Accession number PTA-4567), hybridoma 325.1 (ATCC)Accession number PTA-5120), hybridoma 368.1 (ATCC)Accession number PTA-4568), hybridoma 446.1 (ATCC)Accession number PTA-5549), hybridoma 501.1 (ATCC)Accession number PTA-4569), hybridoma 621.1 (ATCC)Accession number PTA-5121), hybridoma 633.1 (ATCC)Accession number PTA-5122), hybridoma 654.1 (ATCC)Accession number PTA-5247), hybridoma 725.1 (ATCC)Accession number PTA-5124), or hybridoma 776.1 (ATCC)Record number PTA-4570). In another embodiment, the hybridoma of the invention is a monoclonal antibody-producing hybridoma that produces monoclonal antibodies that are conjugated to hybridoma 4E7 (ATCC)Accession number PTA-5109), hybridoma 7A11 (ATCC)Accession number PTA-5110), hybridoma 7C6 (ATCC)Accession number PTA-5111), hybridoma 7F10 (ATCC)Accession number PTA-5112), hybridoma 7G10 (ATCC)Accession number PTA-5245), hybridoma 7H1 (ATCC)Accession number PTA-5114), hybridoma 8A1 (ATCC)Accession number PTA-5115), hybridoma 8B5 (ATCC)Accession number PTA-5116), hybridoma 8C3 (ATCC) Accession number PTA-5246), hybridoma 8E3 (ATCC)Accession number PTA-5118), hybridoma 8G9 (ATCC)Accession number PTA-5119), hybridoma 15C9 (ATCC)Accession number PTA-5106), hybridoma 16C7 (ATCC)Accession number PTA-5107), hybridoma 16H9 (ATCC)Accession number PTA-5108), hybridoma 117.1 (ATCC)Accession number PTA-4567), hybridoma 325.1 (ATCC)Accession number PTA-5120), hybridoma 368.1 (ATCC)Accession number PTA-4568), hybridoma 446.1 (ATCC)Accession number PTA-5549), hybridoma 501.1 (ATCC)Accession number PTA-4569), hybridoma 621.1 (ATCC)Accession number PTA-5121), hybridoma 633.1 (ATCC)Accession number PTA-5122), hybridoma 654.1 (ATCC)Accession number PTA-5247), hybridoma 725.1 (ATCC)Accession number PTA-5124), or hybridoma 776.1 (ATCC)Record No. PTA-4570) competes for binding to cell-associated CA 125/0772P. An antibody or antigen-binding antibody fragment of the invention is considered to compete for binding if they compete for binding in a BLISA cross-competition assay and/or a FACS cross-competition assay. If IC of competitor antibody or antigen binding fragment50Is no more than about 100-fold higher than the concentration of antibody or antigen-binding antibody fragment, then the antibody or antigen-binding antibody fragment is considered to compete for binding in the ELISA cross-competition assay and/or the FACS cross-competition assay. In preferred embodiments, IC of competitor antibody or antigen binding fragment 50Is a concentration no more than about 10-fold higher than the concentration of the antibody or antigen-binding fragment. In a more preferred embodiment, the IC of the competitor antibody or antigen-binding antibody fragment50Is a concentration of the antibody or antibody fragment bound to the antigen that is no more than about equimolar.
In yet another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding antibody fragment of the invention.
In another aspect, the invention provides a fusion polypeptide comprising an antibody or antigen-binding antibody fragment of the invention operably linked to a heterologous agent, i.e., an antibody or antigen-binding antibody fragment that preferentially binds to a cell-associated CA125/0772P polypeptide relative to an shed CA125/0772P polypeptide. In one embodiment of the fusion polypeptide of the invention, the antibody, or antigen-binding antibody fragment, and the heterologous agent are operably linked by a covalent bond, such as a peptide bond or a disulfide bond. In another embodiment of the fusion polypeptide of the invention, the antibody, or antigen-binding antibody fragment, and the heterologous agent are operably linked by a non-covalent bond. In another embodiment of the fusion polypeptide of the invention, the heterologous agent comprises an amino acid sequence or a radioisotope. In various non-limiting embodiments, the heterologous agent of the fusion polypeptide of the invention comprises a cytotoxic agent or a detectable agent, e.g., an imaging agent.
Also included as part of the inventionAre analogs of the antibodies, antigen-binding antibody fragments, and fusion polypeptides of the invention that preferentially bind to cell-associated CA125/0772P relative to shed CA 125/0772P. In one embodiment, such analogs exhibit enhanced affinity for cell-associated CA125/0772P relative to the affinity of the antibody, antigen-binding antibody fragment, and fusion polypeptide prior to modification. In another embodiment, such analogs exhibit increased serum half-life as compared to the antibody, antigen-binding antibody fragment, and fusion polypeptide prior to modification. For example, analogs of the invention include hybridoma 4E7 (ATCC)Accession number PTA-5109), hybridoma 7A11 (ATCC)Accession number PTA-5110), hybridoma 7C6 (ATCC)Accession number PTA-5111), hybridoma 7F10 (ATCC)Accession number PTA-5112), hybridoma 7G10 (ATCC)Accession number PTA-5245), hybridoma 7H1 (ATCC)Accession number PTA-5114), hybridoma 8A1 (ATCC)Accession number PTA-5115), hybridoma 8B5 (ATCC)Accession number PTA-5116), hybridoma 8C3 (ATCC)Accession number PTA-5246), hybridoma 8E3 (ATCC)Accession number PTA-5118), hybridoma 8G9 (ATCC)Accession number PTA-5119), hybridoma 15C9 (ATCC)Accession number PTA-5106), hybridoma 16C7 (ATCC) Accession number PTA-5107), hybridoma 16H9 (ATCC)Accession number PTA-5108), hybridoma 117.1 (ATCC)Accession number PTA-4567), hybridoma 325.1 (ATCC)Accession number PTA-5120), hybridoma 368.1 (ATCC)Accession number PTA-4568), hybridoma 446.1 (ATCC)Accession number PTA-5549), hybridoma 501.1 (ATCC)Accession number PTA-4569), hybridoma 621.1 (ATCC)Record numberPTA-5121), hybridoma 633.1 (ATCC)Accession number PTA-5122), hybridoma 654.1 (ATCC)Accession number PTA-5247), hybridoma 725.1 (ATCC)Accession number PTA-5124), or hybridoma 776.1 (ATCC)Record No. PTA-4570).
In another aspect, the invention provides a pharmaceutical composition comprising an antibody, antigen-binding antibody fragment, fusion polypeptide, or the like of the invention, i.e., an antibody, antigen-binding antibody fragment, fusion polypeptide, or the like that preferentially binds to a cell-associated CA 125/0772P polypeptide over an shed CA 125/0772P polypeptide, and a pharmaceutically acceptable carrier. In another aspect, the invention provides a method of preparing a pharmaceutical composition comprising admixing an antibody or antigen-binding antibody fragment of the invention with a pharmaceutically acceptable carrier.
In another aspect, the invention provides a product comprising a packaging material and a pharmaceutical composition of the invention contained within the packaging material, in a form suitable for administration to a subject, preferably a human. In one embodiment, the product further contains printed instructions and/or labels for use or administration of the pharmaceutical composition. The instructions and/or labeling may, for example, address a dosage regimen for preventing or treating a disorder associated with a disorder of CA 125/0772P, such as a cell proliferation disorder, e.g., one or more symptoms of a cancer, e.g., breast, uterine, breast or lung cancer.
In another aspect, the invention provides methods of preventing, treating, managing or ameliorating the symptoms of a CA 125/0772P-related disorder, comprising: administering to a subject in need of such prevention, treatment, management, or amelioration an antibody or antigen-binding fragment of an antibody in an amount sufficient to prevent, treat, manage, or ameliorate symptoms of a cell proliferative disorder, wherein the antibody or antigen-binding antibody fragment preferentially binds cell-associated CA125/0772P over shed CA 125/0772P.
In one embodiment, the methods of the invention relate to preventing, treating, managing or ameliorating the symptoms of a cell proliferative disorder. In another embodiment, the methods of the invention relate to preventing, treating, managing or ameliorating the symptoms of cancer. In yet another embodiment, the methods of the invention relate to preventing, treating, managing or ameliorating the symptoms of cervical, uterine, breast or lung cancer. In preferred embodiments of these methods of the invention, the methods relate to preventing, treating, managing or ameliorating the symptoms of ovarian cancer.
In one embodiment of the methods of the invention, the administered antibody or antigen-binding fragment is a monoclonal antibody or a monoclonal antibody fragment that binds an antigen. In another embodiment of the method of the invention, the antibody or antigen-binding antibody fragment is administered at a dose concentration of about 5 μ g/kg to about 10mg/kg, preferably about 20 μ g/kg to about 5mg/kg, more preferably about 100 μ g/kg to about 5 mg/kg.
In yet another embodiment of the methods of the invention, the methods are performed as part of a combination cancer therapy. Such combination cancer therapies may include, for example, administration of a chemotherapeutic agent, e.g., paclitaxel or cisplatin. Such combination cancer therapy may alternatively include, but is not limited to, radiation therapy.
In another aspect, the invention provides methods that facilitate the identification of antibodies or antigen-binding antibody fragments that preferentially bind cell-associated CA 125/0772P relative to shed CA 125/0772P. In one embodiment, a method of facilitating identification of an antibody or antigen-binding antibody fragment that preferentially binds cell-associated CA 125/0772P comprises contacting the antibody or antigen-binding antibody fragment with a cell-associated CA 125/0772P-containing peptide (e.g., a cell-associated CA 125/0772P polypeptide or even a full-length CA 125/0772P polypeptide) in the presence of shed CA 125/0772P (preferably in excess (weight/weight) shed) under conditions that allow the antibody or antigen-binding antibody fragment to bind to the cell-associated CA 125/0772P-containing peptide or shed CA 125/0772P. After incubation, shed CA 125/0772P (with or without antibody or antigen-binding antibody fragment binding) and unbound antibody or antigen-binding antibody fragment were removed and the amount of antibody or antigen-binding antibody fragment bound to the cell-associated CA 125/0772P-containing peptide was measured. An antibody or antigen-binding antibody fragment of the method preferably binds to a cell-associated CA 125/0772P polypeptide relative to an shed CA 125/0772P polypeptide if the antibody or antigen-binding antibody fragment meets one of the three embodiments set forth above for "preferably binds". In a preferred embodiment, the ratio of shed CA 125/0772P to cell-associated CA 125/0772P in the reaction mixture is about 25: 1 (wt/wt). As part of this method, cell-associated CA 125/0772P may be immobilized on a solid surface. For example, the method may be performed in an ELISA format.
In yet another embodiment, the invention provides a method of facilitating identification of an antibody or antigen-binding antibody fragment that preferentially binds cell-associated CA125/0772P over shed CA125/0772P, the method comprising contacting the antibody or antigen-binding antibody fragment with a peptide containing cell-associated CA125/0772P and shed CA125/0772P (preferably shed CA125/0772P in excess (weight/weight), e.g., about 25 fold excess (wt/wt)), under conditions that allow binding of the peptide containing cell-associated CA125/0772P to the antibody or antigen-binding antibody fragment, removing unbound peptide containing cell-associated CA125/0772P, measuring the amount of cell-associated CA 125/0772P-containing peptide bound by the antibody or antigen-binding fragment, and comparing the measured amount to the amount lacking shed CA125/0772P (i.e., lesser amount) the amount of peptide containing cell-associated CA125/0772P that the antibody or antigen-binding antibody fragment can bind is compared. If the antibody or antigen-binding antibody fragment of the method satisfies one of the three embodiments set forth above for "preferentially binding," then the antibody or antigen-binding antibody fragment preferentially binds to the cell-associated CA125/0772P polypeptide relative to the shed CA125/0772P polypeptide. As part of the method, the antibody or antigen-binding antibody fragment may be immobilized on a solid surface, e.g., the method may be performed in an ELISA format.
In yet another embodiment, the invention provides a method of aiding in the identification of an antibody or antigen-binding antibody fragment that preferentially binds cell-associated CA 125/0772P, the method comprising contacting an antibody or antigen-binding antibody fragment with a cell expressing CA 125/0772P and an amount of a recombinant antibody under conditions that allow CA 125/0772P to bind to the antibody or antigen-binding antibody fragment, e.g., at least about 0.05mg/ml of shed CA 125/0772P (preferably excess shed CA 125/0772P (wt/wt)), removing unbound cells, measuring the amount of CA 125/0772P-expressing cells bound by the antibody or antigen-binding antibody fragment, and comparing the measurements to the amount of cells expressing CA 125/0772P that bound the antibody or antigen-binding antibody fragment in the absence of these amounts of shed CA 125/0772P (i.e., lesser amounts). An antibody or antigen-binding antibody fragment of the method preferably binds to a cell-associated CA 125/0772P polypeptide relative to an shed CA 125/0772P polypeptide if the antibody or antigen-binding antibody fragment meets one of the three embodiments set forth above for "preferably binds". Such methods can be carried out, for example, wherein the measurement is carried out by flow cytometry techniques, including, for example, Fluorescence Activated Cell Sorting (FACS).
In another aspect, the invention also provides methods of diagnosing a CA 125/0772P-related disorder or a predisposition for a CA 125/0772P-related disorder.
3.1. Term(s) for
As used herein, the term "analog" in the context of an antibody or antigen-binding antibody fragment or fusion polypeptide of the invention refers to an antibody, antigen-binding antibody fragment or fusion polypeptide that is modified relative to the corresponding antibody, antigen-binding antibody fragment or fusion polypeptide of the invention prior to the modification present in the analog (referred to in this context as the "pre-modified" antibody, antigen-binding antibody fragment or fusion polypeptide of the invention), but still preferentially binds cell-associated CA125/0772P relative to shed CA 125/0772P.
The "affinity" (K) of an antibody or antigen-binding antibody fragment of the invention is determined by the affinity assay described in section 6.4 belowd)。
As used herein, the term "antibody of the invention" refers to an antibody that preferentially binds to a cell-associated CA125/0772P polypeptide relative to an shed CA125/0772P polypeptide. Likewise, the term "antigen-binding antibody fragment of the invention" as used herein refers to an antigen-binding antibody fragment that preferentially binds to a cell-associated CA125/0772P polypeptide over an shed CA125/0772P polypeptide. Even if an antibody or antigen-binding antibody fragment binds to a CA125/0772P polypeptide, i.e., a pre-shedding CA125/0772P polypeptide, such an antibody or antigen-binding antibody fragment is considered to be an antibody or antigen-binding fragment of the invention, as long as such an antibody or antigen-binding antibody fragment preferentially binds to a cell-associated CA125/0772P polypeptide relative to a shedding CA125/0772P polypeptide. Due to the fact that: while the cell-associated CA125/0772P polypeptide prior to shedding of CA125/0772P is present as part of pre-shedding CA125/0772P, it is noted that antibodies that preferentially bind cell-associated CA125/0772P may also bind pre-shedding CA 125/0772P. Thus, an antibody or antigen-binding antibody fragment is considered to be an antibody or antigen-binding antibody fragment of the invention, independent of whether the antibody or antigen-binding fragment binds CA125/0772P, so long as the antibody or antigen-binding antibody fragment meets the criteria set forth herein for "binding cell-associated CA125/0772P relative to an shed CA125/0772P polypeptide". It is also proposed that the terms "antibody" and "immunoglobulin" are used interchangeably unless otherwise indicated.
The term "antibody-dependent cellular cytotoxicity assay" (ADCC assay) as used herein refers to the ADCC assay described in section 6.5 below. Likewise, an antibody or antigen-binding antibody fragment that mediates lysis of CA 125/0772P-positive tumor cells in an ADCC assay is considered positive when tested in the ADCC assay described in section 6.5 below.
The term "about" as used herein means no more than or less than 10% of the value modified by the term, unless otherwise indicated. If the nucleic acid or amino acid sequence length is a modified value, then the resulting modification value will be an integer no greater than or less than 10% of the original length. Furthermore, 10% of the length modified by this term results in a value that is certainly less than 1, then it is understood that, as used herein, the length modified is more or less than the original value by 1 nucleotide or amino acid residue.
The term "binding" in the context of antibody-antigen binding contexts, e.g., antibodies or antigen-binding antibody fragments that preferably bind cell-associated CA 125/0772P, refers to antibodies or antigen-binding antibody fragments that specifically bind a particular antigen (e.g., cell-associated CA 125/0772P) and do not specifically bind other antigens. Preferably, the antibody or antigen-binding antibody fragment is an antibody or antigen-binding antibody fragment with a specificity of at least 5 OD/microgram to bind CA 125/0772P as determined by an ELISA-specific assay, or an antibody or antigen-binding antibody fragment that is considered positive in a flow cytometry-specific assay. Peptides or polypeptides that bind antigen may bind other peptides or polypeptides with lower affinity, which may be determined by, for example, immunoassay, BIAcore, scatchard analysis, or other assays known in the art. An antibody or fragment that specifically binds to an antigen can cross-react with the relevant antigen. Preferably, the antibody or fragment that binds to the antigen does not cross-react with other antigens. For a discussion of antibody specificity, see, e.g., fundamentals immunology second edition, Paul, eds., Raven Press (1989) page 332-336. Preferably, the antibody or antigen-binding antibody fragment of the invention binds to the peptide of FIG. 1 with a Kd of less than about 100nM, more preferably K of the peptide of FIG. 1 dLess than about 5nM of antibody or antigen-binding antibody fragment, all as measured by a BIAcore affinity assayThe assay is described in section 6.4. It is also noted that antibodies that preferentially bind cell-associated CA125/0772P may also represent antibodies that specifically bind CA125/0772P (including shed CA 125/0772P) relative to other non-CA 125/0772P antigens. Finally, it is noted that the terms "specifically" and "immunospecifically" as used herein are used interchangeably unless otherwise indicated.
ELISA specific assay: as used herein, the assay refers to the ELIS a assay described in section 6.2 below. An antibody (or antigen-binding antibody fragment) is considered positive (i.e., specific for CA 125/0772P) if it exhibits an absorbance of at least 5 to greater than 30 OD/microgram of antibody.
Flow cytometry specific assays: as used herein, the assay refers to the flow cytometry assay described in section 6.2 below. If the antibody (or antigen-binding antibody fragment) exhibits flow cytometry specific assay results in the following positive cell range: less than 5% positive NIH/3T3 cells, and at least 60% positive NIH/3T3 cells produced SEQ ID NO: 2 a polypeptide; and/or less than 25% positive SK-OV3 cells and at least 80% positive OVCAR-3 cells, then the antibody (or antigen-binding antibody fragment) is considered positive (i.e., specific for CA 125/0772P).
The terms "competing for binding" and "competing with …" as used in the context of two antibody species or antigen-binding antibody fragment species (or combinations thereof) are used interchangeably. It is considered that a first antibody or antigen-binding antibody fragment is considered to compete with a second antibody or antigen-binding antibody fragment if the first antibody or antigen-binding antibody fragment competes with the second in an ELISA cross-competition assay and/or a FACS cross-competition assay.
ELISA cross-competition assay: as used herein, the assay refers to the ELISA assay described in section 7.0 below. A competitor antibody or antigen-binding fragment is considered to compete for binding in the assay if its IC50 is at a concentration that is no more than about 100-fold higher than the concentration of the antibody or antigen-binding antibody fragment.
FACS cross-competition assay: as used herein, the assay refers to the FACS assay described in section 7.0 below. If IC of competitor antibody or antigen binding fragment50Is no more than about 100-fold higher than the concentration of the antibody or antigen-binding antibody fragment, then the antibody or antigen-binding antibody fragment is considered to compete for binding in the assay.
The term "CA 125/0772P" or "CA 125/0772P polypeptide" as used herein refers to a pre-shedding CA125/0772P transmembrane polypeptide that, upon shedding, produces shed CA
125/0772P polypeptide and a cell-associated CA125/0772P polypeptide. It has recently been demonstrated that the amino acid sequence reported in the literature as the full-length sequence of the CA125/0772P polypeptide does not actually represent the full-length CA125/0772P sequence. See, for example, WO02/06317(PCT/US 01/22635) and US 2003/0124140, which disclose polypeptides referred to as "0772P". 0772P includes an extension of what was previously thought to be full-length CA 125. Since this polypeptide is referred to in the literature as CA125 or 0772P, it is referred to herein as CA 125/0772P.
The term "CA 125/0772P-related disorder" as used herein refers to a disorder that involves or is characterized by the presence of a differential level of cell-associated CA125/0772P relative to the corresponding normal state and/or an excess of shed CA125/0772P relative to the corresponding normal state. For example, for ovarian cancer, higher levels of cell-associated or shed CA125/0772P are observed relative to levels observed in normal (e.g., non-cancerous) states. Differential levels of cell associated and/or shed CA125/0772P may be a cause or indication of the disorder.
The term "cell-associated CA 125/0772P" as used herein refers to a CA125/0772P extracellular polypeptide species that is transient (e.g., CA125/0772P extracellular polypeptide species after a portion of the CA125/0772P polypeptide is released as shed CA125/0772P prior to sheddingPrior to switching) to maintain the cell associated form. For example, cell-associated CA125/0772P is a CA125/0772P extracellular polypeptide species that remains in cell-associated form in OVCAR-3 cell line cells (HTB-161; ATCC; see also, the disclosure herein) after release of a portion of the CA125/0772P polypeptide as shed CA125/0772P) On the surface. The CA125/0772P cell-associated polypeptide species is present in SEQ ID NO: 1 and amino acid residues 1 to 708 of SEQ id no: 2 from amino acid residue 1 to 711. Furthermore, CA125/0772P may be substituted at a position located in seq id NO: 2 at amino acid residues 659-665. See O' Brien et al, Tumour Biol.23(3): 154-169(2002). Likewise, the cell-associated CA125/0772P polypeptide can include SEQ ID NO: amino acid residues 659-711 of 2.
The term "complement dependent cytotoxicity assay" (CDC assay) as used herein refers to the CDC assay described in section 6.5 below. Likewise, an antibody or antigen-binding antibody fragment that mediates tumor cell lysis in a CDC assay is considered positive when tested in the CDC assay described in section 6.5 below.
As used herein, the terms "disorder" and "disease" are used interchangeably to refer to a condition in a subject.
As used herein, the term "fragment" in the phrase "antigen-binding antibody fragment" refers to a peptide or polypeptide that contains at least about 5 contiguous amino acid residues, at least about 10 contiguous amino acid residues, at least about 15 contiguous amino acid residues, at least about 20 contiguous amino acid residues, at least about 25 contiguous amino acid residues, at least about 40 contiguous amino acid residues, at least about 50 contiguous amino acid residues, at least about 60 contiguous amino acid residues, at least about 70 contiguous amino acid residues, at least about 80 contiguous amino acid residues, at least about 90 contiguous amino acid residues, at least about 100 contiguous amino acid residues, at least about 110 contiguous amino acid residues, or at least about 120 contiguous amino acid residues of the amino acid sequence of another polypeptide (e.g., an antibody that preferentially binds cell-associated CA 125/0772P).
The term "host cell" as used herein refers to a particular cell, including a mammalian or other eukaryotic cell, or prokaryotic cell, transformed or transfected with a nucleic acid molecule or infected with a virus, phagemid, or phage, for example, and includes progeny or potential progeny of such a cell. Progeny of such a cell may differ from the parent cell transfected with the nucleic acid molecule, due to mutations or environmental influences or additional recombination operations or integration of the nucleic acid molecule into the genome of the host cell which may occur in subsequent generations.
The term "hybridizes under stringent conditions" as used herein describes hybridization and washing conditions under which nucleotide sequences at least 75% identical to each other typically hybridize to the complement of each other. These stringent conditions are well known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al, eds, John Wiley & Sons (1989-2002) package 6.3.1-6.3.6. In one non-limiting example, stringent hybridization conditions are hybridization at about 45 ℃ in 6 XSSC/sodium citrate (SSC), followed by one or more washes in 0.1 XSSC, 0.2% SDS at 68 ℃. In a preferred, non-limiting example, stringent hybridization conditions are those in which hybridization occurs at about 45 ℃ in 6 XSSC followed by one or more washes in 0.2 XSSC, 0.1% SDS at 50-65 ℃ (i.e., one or more washes at 50 ℃, 55 ℃, 60 ℃ or 65 ℃). It will be understood that in some embodiments, the nucleic acids of the invention do not include nucleic acid molecules that hybridize under these conditions only to nucleotide sequences consisting of only a or T nucleotides.
As used herein, the term in the context of a peptide, polypeptide, fusion protein, antibody, or antigen-binding antibody fragment refers to a peptide, polypeptide, fusion protein, antibody, or antigen-binding antibody fragment that is substantially free of cellular material or contaminating proteins from a cell or tissue source from which it is derived or obtained; or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material or contaminating proteins" includes preparations of peptides, polypeptides, fusion proteins, antibodies or antigen-binding antibody fragments, wherein the peptides, polypeptides, fusion proteins, antibodies or antigen-binding antibody fragments are isolated from cellular components of the cells from which they were isolated or recombinantly produced. Thus, a peptide, polypeptide, fusion protein, antibody, or antigen-binding antibody fragment that is substantially free of cellular material or contaminating proteins includes preparations of the peptide, polypeptide, fusion protein, antibody, or antigen-binding antibody fragment that contain less than about 30%, about 20%, about 10%, or about 5% (by dry weight) of other proteins. When the peptide, polypeptide, fusion protein, antibody, or antigen-binding antibody fragment is recombinantly produced, it is also preferably free of culture medium, i.e., culture medium represents about 20%, about 10%, or less than about 5% of the volume of the protein preparation. When the chemical synthesis produces a peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment, it is preferably free of chemical precursors or other chemicals that are associated with the synthesis of the peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment. Preferably, these preparations of the peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment contain less than about 30%, about 20%, about 10%, about 5% (by dry weight) of chemical precursors or other chemicals other than the peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment of interest.
The term "isolated" as used herein in the context of nucleic acid molecules refers to a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural cellular source of the nucleic acid molecule. Alternatively, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
The term "treatment" as used herein refers to a beneficial effect that a subject derives from an agent, e.g., an antibody, antigen-binding antibody fragment, fusion polypeptide, or the like, of the present invention that does not result in a cure of the disease. In some embodiments, a subject is administered one or more of these agents to "treat" a disorder in order to prevent or delay the development or progression of the disorder.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a single cell clone (including any eukaryotic, prokaryotic, or phage clone) and which is independent of the method by which it is produced. Thus, a "monoclonal antibody" can refer to a composition comprising a population of antibodies, each antibody binding a single epitope, wherein the composition is free of antibodies that bind epitopes different from the single epitope bound by the population of antibodies. Of course, it is noted that in some cases, a single epitope is present at multiple positions in a polypeptide. In these cases, although a monoclonal antibody may bind to multiple sites, the antibody is still considered to bind to a single epitope.
As used herein, the terms "nucleic acid" and "nucleotide sequence" include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules, or hybrid DNA/RNA molecules, and analogs of the DNA or RNA molecules. These analogs can be generated using, for example, nucleotide analogs including, but not limited to, inosine or tritylated bases. These analogs can also include DNA or RNA molecules that contain modified backbones that can confer beneficial properties to the molecule, such as, for example, nuclease resistance or increased ability to cross cell membranes. The nucleic acid or nucleotide sequence may be single-stranded, double-stranded, may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but is preferably double-stranded DNA.
The term "operably linked" as used herein in the context of a fusion polypeptide refers to an antibody or antigen-binding antibody fragment that links the antibody or antigen-binding antibody fragment to a heterologous agent. The operable connection may be direct or indirect. For example, an amino acid sequence may be present between the antibody (or antigen-binding antibody fragment) and the heterologous agent.
As used herein, the terms "antibody, antigen-binding antibody fragment, fusion polypeptide, or the like that preferentially binds to cell-associated CA 125/0772P," "preferentially binds to cell-associated CA 125/0772P polypeptide," "preferentially binds to cell-associated CA 125/0772P relative to exfoliated CA 125/0772P," or "preferentially binds to cell-associated CA 125/0772P polypeptide relative to exfoliated CA 125/0772P" refer to an antibody or antigen-binding antibody fragment that is positive when tested in an ELISA competition assay or a flow cytometry competition assay as described herein. Preferably, the antibody or antigen-binding antibody fragment is positive in both assays such as the ELISA competition assay and the flow cytometry competition assay described herein.
ELISA competition assay: this assay as used herein refers to the ELISA assay described in section 6.3 below. An antibody (or antigen-binding antibody fragment) is considered positive if it exhibits less than about 25% inhibition when shed CA125/0772P is 25-fold (w/w) more excess than the peptide of FIG. 1 (SEQ ID NO: 1).
Flow cytometry competition assay: this assay as used herein refers to the flow cytometry assay described in section 6.3 below. An antibody (or antigen-binding antibody fragment) is considered positive (i.e., considered to preferentially bind cell-associated CA125/0772P) if the antibody (or antigen-binding antibody fragment) exhibits an IC50 (as measured by the percentage of positive cells) of at least 0.05mg/ml shed CA125/0772P, i.e., at least 0.05mg/ml shed CA125/0772P is required to reduce the percentage of positive cells in a flow cytometry competition assay by half.
As used herein, the terms "prevent" and "prevention" refer to arresting the recurrence or onset of one or more symptoms of a CA 125/0772P-related disorder or a CA 125/0772P-related disorder in a subject.
As used herein, "regimen" includes a schedule of administration and a regimen of administration. The regimens herein are methods of use and include prophylactic and therapeutic regimens.
As used herein, the term "shed CA125/0772P polypeptide" refers to a CA125/0772P extracellular polypeptide sequence that is separated from and released from a CA125/0772P polypeptide expressed on the surface of a cell expressing CA125/0772P, leaving cell-associated CA125/0772P species remaining on the cell surface, which then remains transient. The term as used herein refers to the species of shed CA125/0772P found in human serum and/or in culture supernatants of OVCAR-3 (HTB-161; ATCC) cell lines. By de los Frailes et al Tumour biol.14 (1): 18-29(1993), using human ascites or OVCAR-3 supernatant, these split CA125/0772P polypeptides can be obtained. Alternatively, these split CA125/0772P polypeptides may be obtained from commercial sources, such as FitzgeraldIndustries International (Concord, MA), Scripps Laboratories (La Jolla, Calif.), or United States Biochemical Corp (Cleveland, OH).
As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the term "subject" refers to an animal, preferably a mammal including a non-primate (e.g., cow, pig, horse, donkey, goat, camel, cat, dog, guinea pig, rat, mouse, sheep) and a primate (e.g., monkey, such as macaque, gorilla, chimpanzee, and human), preferably a human. In one embodiment, the subject is a subject having cancer, e.g., ovarian cancer.
As used herein, the term "treatment" refers to the administration of one or more antibodies, antigen-binding antibody fragments, fusion polypeptides or analogs that result in an improvement in the P-associated disorder of CA 125/0772.
The term "pharmaceutically acceptable" as used herein refers to compositions, e.g., carriers, excipients, or salts, which are approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally known pharmacopeia for use in animals, and more particularly in humans.
4. Brief description of the drawings
FIG. 1: the amino acid sequence of the CA125/0772P 3-repeat (SEQ ID NO: 1) is depicted. The italicized residues from amino acid 14 to amino acid 452 represent the repeat region. Each of the three repeats within the repeat region 14-452 is depicted by vertical lines and arrows as shown. Underlined residues represent transmembrane proximal non-repeat regions. The sequence after the underlined residues is not part of CA125/0772P and includes a carboxy-Myc-His tag.
FIG. 2: the amino acid sequence of CA125/0772P 3-repeat TM (SEQ ID NO: 2) is depicted. Italicized, underlined residues (i.e., from amino acid 14 to 452) represent the repeat region. Each of the three repeats within the repeat region 14-452 is depicted by vertical lines and arrows as shown. The underlined non-italicized residues (i.e., from amino acid 453 to amino acid 711) represent the transmembrane proximal non-repeat region. The underlined italicized residues (i.e., from amino acid 712 to amino acid 738) represent the transmembrane domain. Bold residues (i.e., from amino acid 739 to amino acid 769) represent the cytoplasmic region. The sequence after bold residues is not part of CA125/0772P and includes a carboxy-Myc-His tag.
FIG. 3: a schematic of the concentration of shed CA125/0772P versus the percentage of positive cells in FACS competition assay (in this case, 117.1 antibody and M11 antibody control (squares)) is shown. As shown, CA125/0772P (IC) was shed even at low concentrations500.003mg/ml) M11 also competed for binding to OVCAR-3 cells even at high concentrations of shed CA125/0772P (IC)50Greater than 1mg/ml), 117.1 also failed to compete for binding.
FIG. 4: a graphical representation of the percent lysis in the ADCC assay versus antibody concentration for 117.1 antibody (average of 4 individual donors) is shown. As shown in the figure, antibody 117.1 mediates specific lysis of OVCAR-3 cells in a dose-dependent manner.
FIG. 5A: the nucleotide sequence encoding the variable light chain region of monoclonal antibody 117.1 is depicted (SEQ ID NO: 35). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 5B: the nucleotide sequence encoding the variable heavy chain region of monoclonal antibody 117.1 is depicted (SEQ ID NO: 36). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 5C: the amino acid sequence encoding the variable light chain region of monoclonal antibody 117.1 is depicted (SEQ ID NO: 27). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 5D: the amino acid sequence encoding the variable heavy chain region of monoclonal antibody 117.1 is depicted (SEQ ID NO: 28). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 6A: the nucleotide sequence encoding the variable light chain region of monoclonal antibody 368.1 is depicted (SEQ ID NO: 37). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 6B: the nucleotide sequence encoding the variable heavy chain region of monoclonal antibody 368.1 is depicted (SEQ ID NO: 38). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 6C: the amino acid sequence of the variable light chain region encoding monoclonal antibody 368.1 is depicted (SEQ ID NO: 29). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 6D: the amino acid sequence encoding the variable heavy chain region of monoclonal antibody 368.1 is depicted (SEQ ID NO: 30). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 7A: the nucleotide sequence encoding the variable light chain region of monoclonal antibody 501.1 is depicted (SEQ ID NO: 39). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 7B: the nucleotide sequence encoding the variable heavy chain region of monoclonal antibody 501.1 is depicted (SEQ ID NO: 40). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 7C: the amino acid sequence encoding the variable light chain region of monoclonal antibody 501.1 is depicted (SEQ ID NO: 31). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 7D: the amino acid sequence encoding the variable heavy chain region of monoclonal antibody 501.1 is depicted (SEQ ID NO: 32). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 8A: the nucleotide sequence encoding the variable light chain region of monoclonal antibody 776.1 is depicted (SEQ ID NO: 41). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 8B: the nucleotide sequence encoding the variable heavy chain region of monoclonal antibody 776.1 is depicted (SEQ ID NO: 42). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 8C: the amino acid sequence of the variable light chain region encoding monoclonal antibody 776.1 is depicted (SEQ ID NO: 33). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 8D: the amino acid sequence encoding the variable heavy chain region of monoclonal antibody 776.1 is depicted (SEQ ID NO: 34). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 9A: the nucleotide sequence encoding the variable light chain region of monoclonal antibody 725.1 is depicted (SEQ ID NO: 52). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 9B: the nucleotide sequence encoding the variable heavy chain region of monoclonal antibody 725.1 is depicted (SEQ ID NO: 57). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 9C: the amino acid sequence encoding the variable light chain region of monoclonal antibody 725.1 is depicted (SEQ ID NO: 54). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 9D: the amino acid sequence encoding the variable heavy chain region of monoclonal antibody 725.1 is depicted (SEQ ID NO: 53). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 10A: the nucleotide sequence encoding the variable light chain region of monoclonal antibody 16H9 is depicted (SEQ ID NO: 59). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 10B: the nucleotide sequence encoding the variable heavy chain region of monoclonal antibody 16H9 is depicted (SEQ ID NO: 58). The nucleotide sequence encoding the leader sequence is double underlined, and the nucleotide sequence encoding the CDR sequence is single underlined.
FIG. 10C: the amino acid sequence encoding the variable light chain region of monoclonal antibody 16H9 is depicted (SEQ ID NO: 56). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 10D: the amino acid sequence encoding the variable heavy chain region of monoclonal antibody 16H9 is depicted (SEQ ID NO: 55). Leader sequences are double underlined and CDR sequences are single underlined.
FIG. 11: results of western blot analysis of OVCAR-3 supernatant are depicted. Antibody concentrations and assays are given in the working example of section 6.7 below. "3 RptPtn" in each blot refers to a lane containing 0772P 3-repeat recombinant polypeptide; the remaining lanes in each blot contained OVCAR-3 conditioned medium or control medium. The specific antibodies tested are indicated at the bottom of each blot (i.e., M11, OC125, 776.1, and 368.1 antibodies). Molecular weight markers are indicated on the left of the figure.
FIG. 12:131in vivo evaluation of I-labelled 776.1. Using saline, 100 mu C i 131I]776.1IgG1、300μCi[131I]776.1 IgG1, or 17. mu.g unlabeled 776.1 IgG1 (and 300. mu. Ci [131I ]]776.1 IgG1 group) treated NCR nu/nu mice with OVCAR-3 tumors. Treatment was a single dose administered intravenously on day 0. [131I]776.1 had a specific activity of 15mCi/mg and an immunoreactivity after 51% labeling. Results are shown as mean tumor volume +/-SD of a total of 10 mice per group. Mean tumor size at the start of treatment was 199mm for all groups3。
5. Detailed description of the invention
The present invention is based in part on the recognition that: the event that produced shed CA 125/0772P also left a portion of the extracellular region of CA 125/0772P amino acid sequence in a cell-associated form, i.e., cell-associated CA 125/0772P was also produced. The invention described in detail herein is also based in part on the recognition that: antibodies and antigen-binding antibody fragments, fusion polypeptides and analogs can be produced that preferentially bind cell-associated CA 125/0772P over shed CA 125/0772P, and these antibodies, antigen-binding antibody fragments, fusion polypeptides and analogs can be used, for example, to prevent, treat or ameliorate one or more symptoms of a CA 125/0772P-associated disorder or a CA 125/0772P-associated disorder, such as a cell proliferation disorder, e.g., cancer, e.g., ovarian cancer.
As discussed throughout, the antibodies and antigen-binding antibody fragments of the present invention preferably bind to cell-associated CA 125/0772P. Likewise, the fusion polypeptides and analogs of the invention also preferably bind cell-associated CA 125/0772P. It is also noted herein that since cell-associated CA125/0772P is present as part of pre-shedding CA125/0772P prior to shedding of CA125/0772P, it is noted that the antibodies, antigen-binding antibody fragments, fusion polypeptides, and analogs of the invention may also bind to pre-shedding CA 125/0772P. Thus, while not wishing to be bound by any particular mechanism or theory thereof, it is noted that the methods described in this section may be at least partially achieved by administering an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog of the present invention that binds to pre-shedding CA125/0772P in addition to CA125/0772P associated with the post-shedding cells, or that does not bind to CA125/0772P associated with the post-shedding cells but only to pre-shedding CA 125/0772P.
5.1 antibodies and antigen-binding antibody fragments of the invention
In a first aspect, the invention provides an isolated antibody, or antigen-binding antibody fragment, that preferentially binds to a cell-associated CA125/0772P polypeptide over an shed CA125/0772P polypeptide. These antibodies and antigen-binding antibody fragments of the invention are useful for a variety of therapeutic, prophylactic, diagnostic and purification purposes as described herein.
In one embodiment, the antibody or antigen-binding antibody fragment of the invention is a heavy chain variable region binding to SEQ ID NO: 1 or SEQ ID NO: 2 and preferably binds to cell-associated CA 125/0772P or an antibody fragment that binds an antigen. In a specific embodiment, the antibody or antigen-binding antibody fragment of the invention is a polypeptide that binds to SEQ ID NO: 1 or SEQ ID NO: 2, non-repeating regions as described in 2. In another embodiment, the antibody or antigen-binding antibody fragment of the invention binds to SEQ ID NO: 1 or SEQ ID NO: 2, or a repeat region as described in 2.
In a first embodiment, the binding of an antibody or antigen-binding antibody fragment of the invention to the peptide of fig. 1 is inhibited by less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% in an ELISA competition assay in the presence of a 25-fold (wt/wt) excess of shed CA 125/0772P over the peptide of fig. 1 (SEQ id no: 1). In another embodiment, an antibody or antigen-binding antibody fragment of the invention exhibits an IC50 (as measured by percent positive cells) of at least about 0.05mg/ml, at least about 0.1mg/ml, at least about 0.25mg/ml, at least about 0.5mg/ml, at least about 0.75mg/ml, or at least about 1.0mg/ml of shed CA 125/0772P in a flow cytometry competition assay. In a third embodiment, an antibody or antigen-binding antibody fragment of the invention binds to the peptide of fig. 1, but no binding to the split CA 125/0772P polypeptide is detected. An antibody or antigen-binding antibody fragment that satisfies any one of these three embodiments constitutes an antibody or antigen-binding antibody fragment that preferentially binds to a cell-associated CA 125/0772P polypeptide relative to an shed CA 125/0772P polypeptide.
The antibodies and antigen-binding antibody fragments of the invention include K binding to the peptide of FIG. 1 (SEQ ID NO: 1)dAn antibody or antigen-binding antibody fragment, K, of less than about 100nM, less than about 10nM, less than about 1nM, less than about 100pM, or less than about 10pMdFor measurement by the BIAcore affinity assay, the assay is described in section 6.4 below.
Preferred embodiments of the antibodies or antigen-binding antibody fragments of the invention include antibodies or antigen-binding antibody fragments that mediate lysis of CA 125/0772P-positive tumor cells in an ADCC assay. These antibodies or antigen-binding antibody fragments include, for example, antibodies that bind to an antigen in an ADCC assay at a 50: 1 effector: a target ratio that mediates at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% lysis of CA125/0772P positive tumor cells at a concentration of 5 μ g/ml antibody or antigen-binding antibody fragment; in an ADCC assay at a 25: 1 effector: a target ratio that mediates at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% lysis of CA125/0772P positive tumor cells at a concentration of 5 μ g/m l antibody or antigen-binding antibody fragment; in the ADCC assay at a 12.5: 1 effector: a target ratio that mediates at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% lysis of CA125/0772P positive tumor cells at a concentration of 5 μ g/ml antibody or antigen-binding antibody fragment; in the ADCC assay at a 12.5: 1 effector: a target ratio that mediates at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% lysis of CA125/0772P positive tumor cells at a concentration of 0.5 μ g/ml antibody or antigen-binding antibody fragment; or in an ADCC assay with a 12.5: 1 effector: the target ratio mediates at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50% lysis of the antibody or antigen-binding antibody fragment by CA 125/0772P-positive tumor cells at a concentration of 50ng/ml antibody or antigen-binding antibody fragment.
Preferred embodiments of the invention also include antibodies or antigen-binding antibody fragments that mediate lysis of CA 125/0772P-positive tumor cells in a complement-dependent cytotoxicity (CDC) assay. These antibodies or antigen-binding antibody fragments include, for example, those that mediate lysis in the range of about 15% lysis at 5 μ g/ml to about 95% lysis at 0.1 μ g/ml.
Preferred embodiments of the antibodies or antigen-binding antibody fragments of the invention also include antibodies that inhibit the growth of a CA 125/0772P-positive tumor and antigen-binding antibody fragments. For example, such antibodies or antigen-binding antibody fragments are described in, e.g., Treskes et al, eur.30A(2): 183-187 (1994); ahmad et al, oncol.11(6): 273-280 (1999); and kienit et al, int.j.radiat.oncol.biol.phys.38 (2): 419-428(1997) and OVCAR-3 xenograft tumor animal models described in section 6.8 below, antibodies or antigen-binding antibody fragments that inhibit the growth of CA 125/0772P-positive tumors.
In one embodiment, the antibody of the invention is a monoclonal antibody, which is produced by hybridoma 4E7 (ATCC)Accession number PTA-5109), or hybridoma 7A11 (ATCC)Accession number PTA-5110), or hybridoma 7C6 (ATCC) Accession number PTA-5111), or hybridoma 7F10 (ATCC)Accession number PTA-5112), or hybridoma 7G10 (ATCC)Accession number PTA-5245), or hybridoma 7H1 (ATCC)Accession number PTA-5114), or hybridoma 8A1 (ATCC)Accession number PTA-5115), or hybridoma 8B5 (ATCC)Accession number PTA-5116), or hybridoma 8C3 (ATCC)Accession number PTA-5246), or hybridoma 8E3 (ATCC)Accession number PTA-5118), or hybridoma 8G9 (ATCC)Accession number PTA-5119), or hybridoma 15C9 (ATCC)Accession number PTA-5106), or hybridoma 16C7 (ATCC)Record number PTA-5107),Or from hybridoma 16H9 (ATCC)Accession number PTA-5108), or hybridoma 117.1 (ATCC)Accession number PTA-4567), or hybridoma 325.1 (ATCC)Accession number PTA-5120), or hybridoma 368.1 (ATCC)Accession number PTA-4568), or by hybridoma 446.1 (ATCC)Accession number PTA-5549), or by hybridoma 501.1 (ATCC)Accession number PTA-4569), or hybridoma 621.1 (ATCC)Accession number PTA-5121), or hybridoma 633.1 (ATCC)Accession number PTA-5122), or hybridoma 654.1 (ATCC)Accession number PTA-5247), or by hybridoma 725.1 (ATCC)Accession number PTA-5124), or hybridoma 776.1 (ATCC)Record number PTA-4570) And (4) generating. In another embodiment, the antibody or antigen-binding antibody fragment of the invention is conjugated to hybridoma 4E7 (ATCC) Accession number PTA-5109), or hybridoma 7A11 (ATCC)Accession number PTA-5110), or hybridoma 7C6 (ATCC)Accession number PTA-5111), or hybridoma 7F10 (ATCC)Accession number PTA-5112), or hybridoma 7G10 (ATCC)Accession number PTA-5245), or hybridoma 7H1 (ATCC)Accession number PTA-5114), or hybridoma 8A1 (ATCC)Accession number PTA-5115), or hybridoma 8B5 (ATCC)Accession number PTA-5116), or hybridoma 8C3 (ATCC)Accession number PTA-5246), or hybridoma 8E3 (ATCC)Accession number PTA-5118), or hybridoma 8G9 (ATCC)Accession number PTA-5119), or hybridoma 15C9 (ATCC)Accession number PTA-5106), or hybridoma 16C7 (ATCC)Accession number PTA-5107), or hybridoma 16H9 (ATCC)Accession number PTA-5108), or hybridoma 117.1 (ATCC)Accession number PTA-4567), or hybridoma 325.1 (ATCC)Accession number PTA-5120), or hybridoma 368.1 (ATCC)Accession number PTA-4568), or by hybridoma 446.1 (ATCC)Accession number PTA-5549), or by hybridoma 501.1 (ATCC)Accession number PTA-4569), or hybridoma 621.1 (ATCC)Accession number PTA-5121), or hybridoma 633.1 (ATCC)Accession number PTA-5122), or hybridoma 654.1 (ATCC)Accession number PTA-5247), or by hybridoma 725.1 (ATCC)Accession number PTA-5124), or hybridoma 776.1 (ATCC) Record No. PTA-4570) competes for binding to the cell-associated antibody to CA 125/0772P or to the antigen-binding antibody fragment. An antibody or antigen-binding antibody fragment of the invention is considered to compete for binding if they compete for binding in an ELISA cross-competition assay and/or a FACS cross-competition assay. If IC of competitor antibody or antigen binding fragment50Is no more than about 100-fold higher than the concentration of antibody or antigen-binding antibody fragment, then the antibody or antigen-binding antibody fragment is considered to compete for binding in the ELISA cross-competition assay and/or the FACS cross-competition assay. In preferred embodiments, IC of competitor antibody or antigen binding fragment50Is a concentration no more than about 10-fold higher than the concentration of the antibody or antigen-binding fragment. In a more preferred embodiment, the IC of the competitor antibody or antigen-binding antibody fragment50Is a concentration of the antibody or antibody fragment bound to the antigen that is no more than about equimolar.
In another embodiment, an antibody or antigen-binding fragment of the invention comprises a 117.1 light chain polypeptide variable region ("117.1L") comprising the amino acid sequence of SEQ ID No: 27 (117.1L). In yet another embodiment, an antibody or antigen-binding fragment of the invention comprises a 117.1 heavy chain polypeptide variable region ("117.1H") comprising the amino acid sequence of SEQ ID No: 28 (117.1H). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 27(117.1L) and a light chain polypeptide variable region comprising the amino acid sequence described in SEQ ID No: 28(117.1H) or a light chain polypeptide variable region of the amino acid sequence described in (e.g. seq id no).
In another specific embodiment, an antibody or antigen-binding fragment of the invention comprises 368.1 light chain polypeptide variable region ("368.1L") comprising the amino acid sequence set forth in SEQ ID No: 29 (368.1L). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises 368.1 heavy chain polypeptide variable region ("368.1H") comprising the amino acid sequence set forth in SEQ ID No: 30 (368.1H). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 29(368.1L) and a light chain polypeptide variable region comprising the amino acid sequence depicted in SEQ ID No: 30(368.1H) or a pharmaceutically acceptable salt thereof.
In another embodiment, an antibody or antigen-binding fragment of the invention comprises a 501.1 light chain polypeptide variable region ("501.1L") comprising the amino acid sequence of SEQ ID No: 31 (501.1L). In yet another embodiment, an antibody or antigen-binding fragment of the invention comprises a 501.1 heavy chain polypeptide variable region ("501.1H") comprising the amino acid sequence of SEQ ID No: 32 (501.1H). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 31(501.1L) and a light chain polypeptide variable region comprising the amino acid sequence depicted in SEQ ID No: 32(501.1H) or a light chain polypeptide variable region of the amino acid sequence described in seq id no.
In another specific embodiment, an antibody or antigen-binding fragment of the invention comprises 776.1 light chain polypeptide variable region ("776.1L") comprising the amino acid sequence set forth in SEQ ID No: 33 (776.1L). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises 776.1 heavy chain polypeptide variable region ("776.1H") comprising the amino acid sequence set forth in SEQ ID No: 34 (776.1H). In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 33(776.1L) and a light chain polypeptide variable region comprising the amino acid sequence depicted in SEQ ID No: 34(776.1H) or a light chain polypeptide variable region of the amino acid sequence described in (776.1H).
In another specific embodiment, the antibody or antigen-binding fragment of the invention comprises a 725.1 light chain polypeptide variable region ("725.1L") comprising the amino acid sequence of SEQ ID No: 54, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a 725.1 heavy chain polypeptide variable region ("725.1H") comprising the amino acid sequence of SEQ id no: 53, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 54 and a light chain polypeptide variable region comprising the amino acid sequence depicted in SEQ ID No: 53 to a heavy chain polypeptide variable region of an amino acid sequence described in seq id no.
In another specific embodiment, an antibody or antigen-binding fragment of the invention comprises a 16H9 light chain polypeptide variable region ("16H 9L") comprising the amino acid sequence of SEQ ID No: 56, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a 16H9 heavy chain polypeptide variable region ("16H 9H") comprising the amino acid sequence of SEQ ID No: 55, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising SEQ ID No: 56 and a light chain polypeptide variable region comprising the amino acid sequence depicted in SEQ ID No: 55, or a light chain polypeptide variable region of the amino acid sequence described in seq id no.
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 27(117.1L), and the heavy chain variable region comprises the amino acid sequence set forth in SEQ id no: 30(368.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 29(368.1L), and the heavy chain variable region comprises the amino acid sequence set forth in SEQ id no: 28(117.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 31(501.1L), and the heavy chain variable region comprises the amino acid sequence depicted in SEQ id no: 28(117.1H), SEQ ID NO: 30(368.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 33(776.1L), and the heavy chain variable region comprises the amino acid sequence set forth in SEQ id no: 28(117.1H), SEQ ID NO: 30(368.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 54(725.1L), and the heavy chain variable region comprises the amino acid sequence depicted in SEQ ID NO: 30(368.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In another embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a light chain polypeptide variable region comprising the amino acid sequence of SEQ id no: 33(16H9L) and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 30(368.1H), SEQ ID NO: 32(501.1H), SEQ ID NO: 34(776.1H), SEQ ID NO: 53(725.1H) or SEQ ID NO: 55(16H 9H).
In a specific embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a variable light chain region comprising any one, two or three VL CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6. In another specific embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a variable heavy chain region comprising any one, two or three VH CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6. In another specific embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a variable light chain region comprising any one, two or three VL CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6 and a variable heavy chain region comprising any one, two or three VH CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6.
In a preferred embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a variable light chain region comprising any two or three VL CDRs depicted in table 1; or any two or three VL CDRs depicted in table 2; or any two or three VL CDRs depicted in table 3; or any two or three VL CDRs depicted in table 4; or any two or three VL CDRs depicted in table 5; or any two or three VL CDRs depicted in table 6. In another preferred embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a variable heavy chain region comprising any two or three VH CDRs depicted in table 1; or any two or three VH CDRs depicted in table 2; or any two or three VH CDRs depicted in table 3; or any two or three VH CDRs depicted in table 4; or any two or three VH CDRs depicted in table 5; or any two or three VH CDRs depicted in table 6.
In another preferred embodiment, an antibody or antigen-binding antibody fragment of the invention comprises a variable light chain region comprising any two or three VL CDRs depicted in table 1 and a variable heavy chain region comprising any two or three VH CDRs depicted in table 1; or the variable light chain region contains any two or three VL CDRs depicted in table 2 and the variable heavy chain region contains any two or three VH CDRs depicted in table 2; or the variable light chain region contains any two or three VLCDRs depicted in table 3 and the variable heavy chain region contains any two or three VHCDRs depicted in table 3; or the variable light chain region contains any two or three VL CDRs depicted in table 4 and the variable heavy chain region contains any two or three VH CDRs depicted in table 4; or the variable light chain region contains any two or three VL CDRs depicted in table 5 and the variable heavy chain region contains any two or three VH CDRs depicted in table 5; or the variable light chain region contains any two or three VL CDRs depicted in table 6 and the variable heavy chain region contains any two or three VH CDRs depicted in table 6.
For example, an antibody or antigen-binding antibody fragment of the invention may contain a VL1 domain comprising any one of the VL1 CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6; the antibodies or antigen-binding antibody fragments of the invention may comprise a VL2 domain comprising any one of the VL2 CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6; alternatively, the antibody or antigen-binding antibody fragment of the invention may comprise a VL3 domain comprising any one of the VL 3CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6; the antibodies or antigen-binding antibody fragments of the invention may comprise VL1 and VL2 domains comprising any one of the VL1 CDRs and VL2 CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6; the antibodies or antigen-binding antibody fragments of the invention may comprise VL1 and VL3 domains comprising any one of the VL1 CDRs and VL 3CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6; the antibodies or antigen-binding antibody fragments of the invention may comprise VL2 and VL3 domains comprising any one of the VL2 CDRs and VL 3CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6; the antibodies or antigen-binding antibody fragments of the invention may comprise VL1 and VL2 domains comprising any one of the VL1 CDRs and VL2 CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6; the antibody or antigen-binding antibody fragment of the invention may comprise a VL1 domain, a VL2 domain and a VL3 domain comprising any one of the VL1 CDRs and VL2 CDRs and VL 3CDRs depicted in table 1, table 2, table 3, table 4, table 5 and table 6.
TABLE 1
CDR sequence of 117.1
CDR sequences SEQ ID NO:
VH1 GFSLSTPGMGVG 3
VH2 HIWWDDFKRDNPALKS 4
VH3 VDGNFLSWYFDV 5
VL1 RSSQSLVHSNGNTYLH 6
VL2 KVSNRFS 7
VL3 SQTTHGPPT 8
TABLE 2
368.1 CDR sequence
CDR sequences SEQ ID NO:
VH1 GYSFTGFYMH 9
VH2 YVSCYTGATTYTQKFKG 10
VH3 EGDYYSMDF 11
VL1 RSSQSLERTNGNTYLH 12
VL2 KVSSRFS 13
VL3 SQTTHGPPT 14
TABLE 3
CDR sequences of 501.1
CDR sequences SEQ ID NO:
VH1 GYIFTDYGMN 15
VH2 CINTYTGETIYSDDFRG 16
VH3 GNYRDAIDY 17
VL1 KASQDIKSYLS 18
VL2 YATTLAD 19
VL3 LHHDESPFT 20
TABLE 4
776.1 CDR sequence
CDR sequences SEQ ID NO:
VH1 GYTFTDYNIH 21
VH2 YIYPYNGVSDYNQNF 22
VH3 RWDFGSGYYFDY 23
VL1 RASSSVIYMC 24
VL2 GTSTLAS 25
VL3 QQWSSNPFT 26
TABLE 5
725.1 CDR sequence
CDR sequences SEQ ID NO:
VH1 GYSFTNYGMN 60
VH2 WINAYIGEPTYADDFKG 61
VH3 GGNSLDF 62
VL1 RASSSVSSIH 63
VL2 ATSNLAS 64
VL3 QQWSSNPFT 65
TABLE 6
CDR sequence of 16H9
CDR sequences SEQ ID NO:
VH1 GFNIKDTYMH 66
VH2 RIDPANGNTKYDPKFQG 67
VH3 SDIYYGNPGGFAY 68
VL1 TASSSVSSSYLH 69
VL2 STSNLAS 70
VL3 HQYHRSPFT 71
the antibodies and antigen-binding antibody fragments of the invention are not as described in nutad et al tumorrbiol.17: 196: antibodies like OC125, M11 or OV197 antibodies as defined in 219(1996) and do not normally compete with these antibodies. In one embodiment, the antibodies and antigen-binding antibody fragments of the invention are not OC 125-derived or VK-8-derived single chain antibodies as described in WO 03/076465, and do not normally compete with these antibodies.
The antibodies of the invention may include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, trispecific antibodies, multispecific antibodies, single chain antibodies, disulfide bonds Linked Fvs, single chain Fvs or anti-idiotype antibodies. In a preferred embodiment, the antibody of the invention is a monoclonal antibody that preferentially binds to a cell-associated CA125/0772P polypeptide over an shed CA125/0772P polypeptide. The multispecific antibodies may be specific for different epitopes of cell-associated CA125/0772P or may be specific for both a cell-associated CA125/0772P epitope and a heterologous epitope, such as a heterologous polypeptide or a solid support material. See, e.g., Tutt et al, j.147(1): 60-69 (1991); kostelny et al, j.148(5): 1547 1553 (1992); and U.S. patent nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5,601,819, 5,798,229, 5,855,866, 5,869,620, 5,897,861, 5,959,084, 6,106,833, 6,248,332, 6,258,358, 6,303,755 and 6,420,140.
Antigen-binding antibody fragments of the invention may include, but are not limited to, Fab fragments, F (ab')2A fragment, a fragment comprising a variable light chain polypeptide (VL), a fragment comprising a variable heavy chain polypeptide (VH), or a fragment comprising a Complementarity Determining Region (CDR).
Furthermore, the antibodies and antigen-binding antibody fragments of the invention may be of any immunoglobulin class. For example, the antibody of the invention may be an antibody of the IgG, IgM, IgE, IgD, IgA or IgY class. The antibodies of the invention may be of any isotype. For example, the antibody of the invention may be an IgG 1、IgG2、IgG3、IgG4、IgA1Or IgA2Heavy chain isotype. The antibody of the invention is preferably an IgG1Isoforms.
Furthermore, an antibody or antigen-binding antibody fragment of the invention may, for example, contain one or more CDRs, e.g., CDR sequences as described herein, inserted into a naturally occurring or consensus framework region, preferably a human framework region. Furthermore, the antibodies or antigen-binding antibody fragments of the invention may, for example, comprise a variable light chain, e.g., a kappa or lambda light chain variable region, and/or a variable heavy chain as described herein, inserted within a naturally occurring or consensus framework region, preferably a human framework region. These framework regions are well known to those skilled in the art and may, for example, comprise a C γ 1 constant region or a C γ 4 constant region.
Preferably, the antibody that binds cell-associated CA 125/0772P or antibody fragment that binds an antigen can be from any animal source, including birds, e.g., chickens, and mammals, including non-primates (e.g., cows, pigs, horses, donkeys, goats, camels, cats, dogs, guinea pigs, rats, mice, sheep) and primates (e.g., monkeys, such as macaques, gorillas, chimpanzees, and humans). Preferably, the antibody or antigen-binding antibody fragment that binds cell-associated CA 125/0772P is a chimeric, human or humanized antibody (including monoclonal antibodies) or antigen-binding antibody fragment. As used herein, "human" antibodies or antigen-binding antibody fragments include antibodies or antigen-binding antibody fragments having the amino acid sequence of a human immunoglobulin, and include, for example, antibodies or antigen-binding antibody fragments isolated from a human immunoglobulin library or from a mouse expressing a human gene.
In another aspect, the invention provides a hybridoma cell that produces a monoclonal antibody of the invention. In one embodiment, the hybridoma of the invention is hybridoma 4E7 (ATCC)Accession number PTA-5109), hybridoma 7A11 (ATCC)Accession number PTA-5110), hybridoma 7C6 (ATCC)Accession number PTA-5111), hybridoma 7F10 (ATCC)Accession number PTA-5112), hybridoma 7G10 (ATCC)Accession number PTA-5245), hybridoma 7H1 (ATCC)Accession number PTA-5114), hybridoma 8A1 (ATCC)Accession number PTA-5115), hybridoma 8B5 (ATCC)Accession number PTA-5116), hybridoma 8C3 (ATCC)Accession number PTA-5246), hybridoma 8E3 (ATCC)Accession number PTA-5118), hybridoma 8G9 (ATCC)Accession number PTA-5119), hybridoma 15C9 (ATCC)Accession number PTA-5106), hybridoma 16C7 (ATCC)Accession number PTA-5107), hybridoma 16H9 (ATCC)Accession number PTA-5108), hybridoma 117.1 (ATCC)Accession number PTA-4567), hybridoma 325.1 (ATCC)Accession number PTA-5120), hybridoma 368.1 (ATCC)Accession number PTA-4568), hybridoma 446.1 (ATCC)Accession number PTA-5549), hybridoma 501.1 (ATCC)Accession number PTA-4569), hybridoma 621.1 (ATCC)Accession number PTA-5121), hybridoma 633.1 (ATCC)Accession number PTA-5122), hybridoma 654.1 (ATCC)Accession number PTA-5247), hybridoma 725.1 (ATCC) Accession number PTA-5124), or hybridoma 776.1 (ATCC)Record number PTA-4570). In another embodiment, the hybridoma of the invention is a monoclonal antibody-producing hybridoma that produces monoclonal antibodies that are conjugated to hybridoma 4E7 (ATCC)Accession number PTA-5109), hybridoma 7A11 (ATCC)Accession number PTA-5110), hybridoma 7C6 (ATCC)Accession number PTA-5111), hybridoma 7F10 (ATCC)Accession number PTA-5112), hybridoma 7G10 (ATCC)Accession number PTA-5245), hybridoma 7H1 (ATCC)Accession number PTA-5114), hybridoma 8A1 (ATCC)Accession number PTA-5115), hybridoma 8B5 (ATCC)Accession number PTA-5116), hybridoma 8C3 (ATCC)Accession number PTA-5246), hybridoma 8E3 (ATCC)Accession number PTA-5118), hybridoma 8G9 (ATCC)Accession number PTA-5119), hybridoma 15C9 (ATCC)Accession number PTA-5106), hybridoma 16C7 (ATCC)Accession number PTA-5107), hybridoma 16H9 (ATCC)Accession number PTA-5108), hybridoma 117.1 (ATCC)Accession number PTA-4567), hybridoma 325.1 (ATCC)Accession number PTA-5120), hybridoma 368.1 (ATCC)Accession number PTA-4568), hybridoma 446.1 (ATCC)Accession number PTA-5549), hybridoma 501.1 (ATCC)Accession number PTA-4569), hybridoma 621.1 (ATCC)Accession number PTA-5121), hybridoma 633.1 (ATCC)Accession number PTA-5122), hybridoma 654.1 (ATCC) Accession number PTA-5247), hybridoma 725.1 (ATCC)Accession number PTA-5124), or hybridoma 776.1 (ATCC)Record No. PTA-4570) competes for binding to cell-associated CA 125/0772P.
5.2 fusion Polypeptides of the invention
In one aspect, the invention provides a fusion polypeptide comprising an antibody or antigen-binding antibody fragment of the invention operably linked to a heterologous agent, i.e., an antibody or antigen-binding antibody fragment that preferentially binds to a cell-associated CA125/0772P polypeptide relative to an shed CA125/0772P polypeptide. The fusion polypeptides of the invention also preferably bind to cell associated CA 125/0772P. In one embodiment of the fusion polypeptide of the invention, the antibody, or antigen-binding antibody fragment, and the heterologous agent are operably linked by a covalent bond, such as a peptide bond or a disulfide bond. In another embodiment of the fusion polypeptide of the invention, the antibody, or antigen-binding antibody fragment, and the heterologous agent are operably linked by a non-covalent bond. The heterologous agent can be attached to the amino acid terminus, the carboxy terminus, or any point along the contiguous sequence of the antibody or antigen-binding antibody fragment. The operable linkage need not be directly between the antibody or antigen-binding antibody fragment and the heterologous agent, but may occur, for example, through a linker or spacer reagent or sequence.
In another embodiment of the fusion polypeptide of the invention, the heterologous agent comprises an amino acid sequence or a radioisotope. The heterologous agent of the fusion polypeptide of the invention comprises a cytotoxic agent or a detectable agent.
The fusion polypeptides of the invention can be used, for example, to produce antibodies or antigen-binding antibody fragments of the invention. Alternatively, the fusion polypeptide may be used as part of a prophylactic or therapeutic method described herein. In addition, the fusion polypeptides of the invention can be used as part of in vivo and in vitro immunoassay and purification methods using methods well known in the art. See, for example, PCT publication nos. WO 93/21232; U.S. patent nos. 5,314,995, 5,474,981, 5,514,558, 6,362,317 and 6,403,769; nakamura et al, immunol.39(1): 91-99 (1993); gillies et al, proc.natl.acad.sci.usa.89(4): 1428-1432 (1992); and Fell et al, j.immunol.146 (7): 2446-2452(1991), which are hereby incorporated by reference in their entirety.
Where the heterologous agent is a polypeptide, the heterologous polypeptide is typically at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids.
In one embodiment, the fusion polypeptide of the invention comprises an antibody or antigen-binding antibody fragment that preferentially binds cell-associated CA125/0772P operably linked to a heterologous agent that provides potential therapeutic benefit. For example, an antibody that preferably binds cell-associated CA125/0772P or an antibody fragment that binds an antigen may be operably linked to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, an agent, or a radioactive ion, e.g., an alpha-emitter. See, for example, U.S. patent nos. 5,624,827, 5,643,573, 5,789,554, 5,824,782, 5,994,151, 6,042,829, 6,074,644, 6,099,842, 6,132,722, 6,187,287, 6,197,299 and 6,207,805. A cytotoxic or cytotoxic agent includes any agent that is detrimental to cell growth or cell viability. Examples of cytotoxic or cytotoxic agents include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione, mitoxantrone, mithramycin, actinomycin D, dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof. Other agents with potential therapeutic benefit include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, melphalan, nitrosurea nitrogen mustard (BSNU) and nitrosurea (CCNU), cycloothoramide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisplatin (DDP), arinomycin (e.g., daunorubicin (pre-daunorubicin) and doxorubicin), antibiotics (e.g., actinomycin D (pre-actinomycin), bleomycin, mithramycin, and Anthranilamycin (AMC)), maytansinoids and antimitotics (e.g., vincristine and vinblastine) and radioactive substances, including, but not limited to, bismuth (A), (B) and (C) 213Bi), carbon (C: (14C) Chromium (C)51Cr), cobalt (57Co), fluorine (18F) Gadolinium (I) and (II)153Gd，159Gd), gallium (68Ga、67Ga), germanium (68Ge), holmium (166Ho), indium (115In、113In、112In、111In), iodine (131I、125I、123I、121I) Lanthanum (a)140La), lutetium (177Lu), manganese (54Mn), molybdenum (99Mo), palladium (103Pd), phosphorus (32P), praseodymium (142Pr), promethium (M)149Pm), rhenium (186Re、188Re), rhodium (II)105Rh), ruthenium (II)97Ru), samarium (153Sm, scandium (47Sc), selenium (75Se), strontium (85Sr), sulfur (S: (A)35S), technetium (99Tc), thallium (201Ti), tin (113Sn、117Sn), tritium (3H) Xenon (a)133Xe), ytterbium (169Yb、175Yb), yttrium (b)90Y), and zinc (65Zn)。
In addition, the antibody or antigen-binding antibody fragment may be conjugated to a therapeutic agent or drug moiety. The therapeutic agent or drug moiety is not to be understood as being limited to a typical chemotherapeutic agent. For example, the drug moiety may be a protein or polypeptide having a desired biological activity. These proteins may include, for example, toxins such as ormosia toxin, ricin a, pseudomonas exotoxin (i.e., PE-40), or diphtheria toxin, ricin, gelonin, and pokeweed antiviral protein; proteins such as tumor necrosis factor, interferons including, but not limited to, alpha-interferon (IFN-alpha), beta-interferon (IFN-beta), Nerve Growth Factor (NGF), platelet-derived growth factor (PDGF), Tissue Plasminogen Activator (TPA), apoptotic agents, e.g., TNF-alpha, TNF-beta, AIMI (disclosed in International publication No. WO 97/33899), AIM II (see, International publication No. WO 97/34911), Fas ligand (Takahashi et al, J.Immunol., 6: 1567-1574, 1994), and VEGF (PCT publication No. WO 99/23105), thrombogenic or anti-angiogenic agents (e.g., ") angiostatin or endostatin), or biological response modifiers such as, e.g., lymphokines (e.g.," -1 "(" IL-1 "), interleukin-2 (" IL-2 " Interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), macrophage colony stimulating factor ("M-CSF"), or a growth factor (e.g., growth hormone ("GH")); a protease or a nuclease.
The fusion polypeptides of the invention may alternatively be used in diagnostics, e.g., to monitor the development or progression of a cancer or tumor, as part of a clinical testing procedure, e.g., to determine the efficacy of a given therapeutic regimen, such as where an antibody is conjugated to a detectable agent. Examples of detectable reagents include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and non-radioactive paramagnetic metal ions. The detectable agent may be coupled or conjugated to the antibody, directly or indirectly, through an intermediate (such as, for example, a linker as is well known in the art) using techniques well known in the art. See, for example, U.S. Pat. nos. 4,741,900, 5,693,764, 5,776,095, 6,008,002, 6,013,531, 6,110,750, 6,124,105, 6,197,523, and 6,225,050.
Non-limiting examples of suitable enzymes that can be conjugated to the antibodies or antigen-binding antibody fragments of the invention include beta-lactamases, beta-galactosidases, phosphatases, peroxidases, reductases, esterases, hydrolases, isomerases, and proteases, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, green fluorescent protein, red fluorescent protein, dansyl chloride or phycoerythrin; a non-limiting example of a luminescent substance includes luminol. Non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include 125I、131I、111In、99mTc, or90Y。
The invention also includes an antibody or antigen-binding fragment thereof that preferably binds cell-associated CA 125/0772P, fused to a marker sequence, such as a peptide, for ease of purification. For example, the marker amino acid sequence can be a hexa-histidine peptide, such as the marker provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), many of which are commercially available. As in Gentz et al, proc.natl.acad.sci.usa.86(3): 821-824(1989), for example, histidine, e.g., hexa-histidine, provides a convenient means for purification of the fusion protein. Other peptide tags for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope from the influenza hemagglutinin protein (Wilson et al, cell.373: 767-778(1984)), and the "flag" tag (Brizzard et al, biotechniques.16 (4): 730-735 (1994)). Preferably, these tags or tag sequences are cleaved from the fusion polypeptide prior to use, e.g., as part of a therapeutic method.
The antibody or antigen-binding fragment thereof that preferably binds cell-associated CA 125/0772P may also, for example, be operably linked to a second antibody to form an antibody heteroconjugate (heteroconjugate) as described in U.S. patent No. 4,676,980, which is incorporated herein by reference in its entirety.
Techniques for operably linking moieties to antibodies are well known, see, e.g., Arnon et al, "monoclonal antibodies for immune targeting of drugs in Cancer Therapy," monoclonal antibodies And Cancer Therapy, Reisfeld et al, eds., Alan R.Liss, Inc. (1985) pp.243-; hellstrom et al, "antibodies for Drug Delivery", Controlled Drug Delivery (2nd Ed.), Robinson et al, eds, Marcel Dekker, Inc. (1987) page 623-; thorpe, "antibody carrier for cytotoxic agents in cancer treatment: review ", Monoclonal Antibodies' 84: biological And Clinical Applications, Pinchera et al, eds, EditriceKurtis (1985) page 475-; order et al, "future prospects For analysis, results And therapeutic use of radiolabeled antibodies in Cancer Therapy", monoclonal antibodies For Cancer Detection And Therapy, Baldwin et al, eds., Academic Press (1985) p 303-316; thorpe et al, immunol. rev.62: 119-158 (1982); and U.S. Pat. nos. 5,639,879, 5,744,119, 5,773,001, and 6,441,163.
Methods of fusing or conjugating polypeptides to the constant region of an antibody are well known in the art. See, for example, U.S. Pat. nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,648,218, 5,723,125, 5,783,181, 5,908,626, 5,844,095, 5,112,946, 6,030,613, 6,086,875, 6,194,177, 6,238,667, 6,262,026, and 6,277,375; EP 307,434; EP367,166; EP 394,827; PCT publications WO 91/06570; ashkenazi et al, proc.natl.acad.sci.usa. 88(23): 10535-10539 (1991); traunecker et al, Nature.331(6151): 84-86 (1988); zheng et al, j.154(10): 5590 + 5600(1995) and Vie et al, Proc. Natl. Acad. Sci. USA.89(23): 11337-11341(1992), which are incorporated by reference in their entirety.
5.3 analogs of the invention
The antibodies, antigen-binding antibody fragments, and fusion polypeptides of the invention include antibodies, antigen-binding antibody fragments, and fusion polypeptide analogs that preferentially bind cell-associated CA 125/0772P relative to shed CA 125/0772P. For example, the analog of the present invention is represented by hybridoma 4E7 (ATCC)Accession number PTA-5109), hybridoma 7A11 (ATCC)Accession number PTA-5110), hybridoma 7C6 (ATCC)Accession number PTA-5111), hybridoma 7F10 (ATCC)Accession number PTA-5112), hybridoma 7G10 (ATCC)Accession number PTA-5245), hybridoma 7H1 (ATCC)Accession number PTA-5114), hybridoma 8A1 (ATCC)Accession number PTA-5115), hybridoma 8B5 (ATCC)Accession number PTA-5116), hybridoma 8C3 (ATCC)Accession number PTA-5246), hybridoma 8E3 (ATCC)Accession number PTA-5118), hybridoma 8G9 (ATCC)Accession number PTA-5119), hybridoma 15C9 (ATCC)Accession number PTA-5106), hybridoma 16C7 (ATCC)Accession number PTA-5107), hybridoma 16H9 (ATCC)Accession number PTA-5108), hybridoma 117.1 (ATCC) Accession number PTA-4567), hybridoma 325.1 (ATCC)Accession number PTA-5120), hybridoma 368.1 (ATCC)Accession number PTA-4568), hybridoma 446.1 (ATCC)Accession number PTA-5549), hybridoma 501.1 (ATCC)Accession number PTA-4569), hybridoma 621.1 (ATCC)Accession number PTA-5121), hybridoma 633.1 (ATCC)Accession number PTA-5122), hybridoma 654.1 (ATCC)Accession number PTA-5247), hybridoma 725.1 (ATCC)Accession number PTA-5124), or hybridoma 776.1 (ATCC)Record No. PTA-4570), or an analog of an antigen-binding antibody fragment of the monoclonal antibody.
Such analogs have at least one of the following structural features: (a) an amino acid sequence that is preferably at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the pre-modification antibody, antigen-binding antibody fragment, or fusion polypeptide; (b) encoded by a nucleotide sequence that hybridizes under stringent conditions to the complement of a nucleotide sequence encoding at least 5 contiguous amino acid residues, at least about 10 contiguous amino acid residues, at least about 15 contiguous amino acid residues, at least about 20 contiguous amino acid residues, at least about 25 contiguous amino acid residues, at least about 40 contiguous amino acid residues, at least about 50 contiguous amino acid residues, at least about 60 contiguous amino acid residues, at least about 70 contiguous amino acid residues, at least about 80 contiguous amino acid residues, at least about 90 contiguous amino acid residues, at least about 100 contiguous amino acid residues, at least about 110 contiguous amino acid residues, or at least about 120 contiguous amino acid residues of an amino acid sequence of a pre-modified antibody, antigen-binding antibody fragment, or fusion polypeptide; or (c) is encoded by a nucleotide sequence that is at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to a nucleotide sequence encoding a pre-modified antibody, an antigen-binding antibody fragment, or a fusion polypeptide.
In particular embodiments, it is preferred that the antibody that binds cell-associated CA 125/0772P, the antigen-binding antibody fragment, or the analog of the fusion polypeptide is preferably at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to hybridoma 4E7 (ATCC) asAccession number PTA-5109), hybridoma 7A11 (ATCC)Accession number PTA-5110), hybridoma 7C6 (ATCC)Accession number PTA-5111), hybridoma 7F10 (ATCC)Accession number PTA-5112), hybridoma 7G10 (ATCC)Accession number PTA-5245), hybridoma 7H1 (ATCC)Accession number PTA-5114), hybridoma 8A1 (ATCC)Accession number PTA-5115), hybridoma 8B5 (ATCC)Accession number PTA-5116), hybridoma 8C3 (ATCC)Accession number PTA-5246), hybridoma 8E3 (ATCC)Accession number PTA-5118), hybridoma 8G9 (ATCC)Accession number PTA-5119), hybridoma 15C9 (ATCC)Accession number PTA-5106), hybridoma 16C7 (ATCC)Accession number PTA-5107), hybridoma 16H9 (ATCC)Accession number PTA-5108), hybridoma 117.1 (ATCC)Accession number PTA-4567), hybridoma 325.1 (ATCC)Accession number PTA-5120), hybridoma 368.1 (ATCC) Accession number PTA-4568), hybridoma 446.1 (ATCC)Accession number PTA-5549), hybridoma 501.1 (ATCC)Accession number PTA-4569), hybridoma 621.1 (ATCC)Accession number PTA-5121), hybridoma 633.1 (ATCC)Accession number PTA-5122), hybridoma 654.1 (ATCC)Accession number PTA-5247), hybridoma 725.1 (ATCC)Accession number PTA-5124), or hybridoma 776.1 (ATCC)Accession number PTA-4570) produced monoclonalThe amino acid sequence of an antibody.
Preferably, the analog comprises less than about 25, less than about 20, less than about 15, less than about 10, less than about 5, less than about 4, less than about 3, or less than about 2 amino acid substitutions, additions or deletions, or combinations thereof, relative to the starting molecule. In preferred embodiments, these analogs have conservative amino acid substitutions at one or more amino acid residues that are predicted to be non-essential (i.e., amino acid residues that are not critical for antibody specificity and preferred binding to cell-associated CA 125/0772P). A "conservative amino acid substitution" is a substitution in which an amino acid is replaced with an amino acid residue, mimetic, or analog whose side chain has a charge or polarity similar to that of the amino acid residue being replaced. Families of amino acid residues having similarly charged side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Furthermore, analogs of the invention may include insertions and/or arise at least in part from deletions relative to the original molecule. Insertions and/or deletions may be of any identity or combination, as long as the structural criteria set forth above for analogs of the invention are met.
To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the two molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the two sequences (i.e.,% identity-the number of identical overlapping positions/total number of positions x 100%). In one embodiment, the two sequences are the same length.
The percent identity between two sequences can also be determined using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm for comparison of two sequences is Karlin et al, proc. 87(6): 2264 2268(1990) by Karlin et al Proc. Natl. Acad. Sci. USA.90(12): 5873. sup. 5877 (1993). This algorithm is incorporated into Altschul et al, j.215(3): 403-410(1990) in the BLASTN and BLASTX programs. BLAST nucleotide searches can be performed using a BLASTN nucleotide program parameter set, e.g., a score of 100 and a word length of 12, to obtain nucleic acid sequences homologous to the nucleic acid molecules of the invention. A BLAST protein search can be performed using the BLASTX program parameter set, e.g., a score of 50 and a word length of 3, to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain a gapped alignment for comparison purposes, Altschul et al, Nucleic Acids Res, can be used.25(17): 3389 and 3402(1997) by gapped BLAST. Alternatively, an iterative search can be performed with PSI-BLAST, which detects distant relationships between molecules (Id.). When utilizing BLAST, gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., of BLASTX and BLASTX) can be used. Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is Myers and Miller (Myers et al, Compout. Appl. biosci).4(1): 11-17 (1988)). This algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, PAM1 may be used 20 weight residue table, gap length penalty of 12, and gap penalty of 4.
Percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are usually calculated.
An analog may also refer to an antibody, antigen-binding antibody fragment, or fusion protein of the invention that has been modified and still retains preferential binding to cell-associated CA125/0772P, wherein the modification is achieved by attaching, e.g., covalently attaching, either type of molecule to the corresponding pre-modified antibody or antigen-binding antibody fragment. For example, and not by way of limitation, an antibody, antigen-binding antibody fragment, or fusion polypeptide may be modified by glycosylation, acetylation, alkylation, esterification, lipidation, formylation, pegylation, phosphorylation, amidation, derivatization by protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, or the like. In addition, analogs may contain one or more atypical amino acids. Atypical amino acids include, but are not limited to, the D-isomers of the common amino acids, α -aminoisobutyric acid, 4-aminobutyric acid (4-Abu), 2-aminobutyric acid (2-Abu), 6-aminocaproic acid (Ahx), 2-aminoisobutyric acid (2-Aib), 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β -alanine, fluoro-amino acids, designer (designer) amino acids such as β -methyl amino acids, C α -methyl amino acids, N α -methyl amino acids, and amino acid analogs in general.
In one embodiment, the analogs of the invention exhibit greater affinity for cell-associated CA125/0772P relative to the corresponding pre-modified antibody, antigen-binding antibody fragment, or fusion polypeptide. In another particular embodiment, it is preferred that the antibody, antigen-binding antibody fragment or fusion polypeptide that binds cell-associated CA125/0772P has a longer serum half-life relative to the corresponding pre-modified antibody, antigen-binding antibody fragment or fusion polypeptide. For example, the analog may exhibit a half-life in an animal, preferably a mammal, most preferably a human, of greater than about 1 day, greater than about 2 days, greater than about 3 days, greater than about 7 days, greater than about 10 days, preferably greater than about 15 days, greater than about 25 days, greater than about 30 days, greater than about 35 days, greater than about 40 days, or greater than about 45 days.
To prolong the serum circulation of an antibody, antigen-binding antibody fragment or fusion polypeptide in vivo, for example, an inert polymer molecule such as high molecular weight polyethylene glycol (PEG) may be attached to the antibody, antigen-binding antibody fragment or fusion polypeptide, with or without a multifunctional linker, via PEG site-specific conjugation to the amino-or carboxyl-terminus of the antibody, antigen-binding antibody fragment or fusion polypeptide or via an epsilon-amino group present on a lysine residue. Linear or branched polymer derivatization resulting in minimal loss of biological activity is preferred. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure correct conjugation of the PEG molecule to the antibody. Untreated PEG can be separated from antibody-, antigen-binding antibody fragment-, and fusion polypeptide-PEG conjugates by size exclusion or ion exchange chromatography. The binding activity and in vivo potency of PEG-derivatized antibodies, antigen-binding antibody fragments and fusion polypeptides can be tested using methods well known in the art, for example, by the immunoassays described herein.
Introduction of one or more amino acid modifications (i.e., substitutions, insertions, or deletions) into the IgG constant domain, or FcRn binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) may also result in antibodies or antigen-binding antibody fragments with increased in vivo half-life. See, for example, PCT publication No. WO 98/23289 and U.S. patent No. 6,277,375, each of which is incorporated herein by reference in its entirety.
5.4. Nucleic acid molecules of the invention
In yet another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding antibody fragment, fusion polypeptide, or analog thereof of the invention.
In one embodiment, the nucleic acid molecule of the invention encodes an antibody or antigen-binding antibody fragment, fusion polypeptide, or analog thereof comprising at least 1, preferably 2 or 3, light chain CDRs listed in table 1, table 2, table 3, table 4, table 5, or table 6. For example, a nucleic acid molecule of the invention may comprise SEQ ID NO: 35. SEQ ID NO: 37. SEQ ID NO: 39. SEQ ID NO: 41. SEQ ID NO: 52. or SEQ ID NO: 59 encoding at least 1, preferably 2 or 3 of said light chain CDRs.
In another embodiment, the nucleic acid molecule of the invention encodes an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog thereof comprising at least 1, preferably 2 or 3 heavy chain CDRs listed in table 1, table 2, table 3, table 4, table 5, or table 6. For example, a nucleic acid molecule of the invention may comprise SEQ ID NO: 36. SEQ ID NO: 38. SEQ ID NO: 40. SEQ ID NO: 42. SEQ ID NO: 57. or SEQ ID NO: 58 which sequence encodes at least 1, preferably 2 or 3 of said heavy chain CDRs.
In another embodiment, the nucleic acid molecule of the invention encodes an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog thereof comprising the variable light chain polypeptide sequence set forth in fig. 5C, fig. 6C, fig. 7C, fig. 8C, fig. 9C, or fig. 10C. For example, a nucleic acid molecule of the invention may comprise SEQ ID NO: 35(117.1), SEQ ID NO: 37(368.1), SEQ id no: 39(501.1), SEQ ID NO: 41(776.1), SEQ ID NO: 52(725.1), or SEQ ID NO: 59(16H 9).
In another embodiment, the nucleic acid molecule of the invention encodes an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog thereof comprising the variable heavy chain polypeptide sequence set forth in figure 5D, figure 6D, figure 7D, figure 8D, figure 9D, or figure 10D. For example, a nucleic acid molecule of the invention may comprise SEQ ID NO: 36(117.1), SEQ ID NO: 38(368.1), SEQ id no: 40(501.1), SEQ ID NO: 42(776.1), SEQ ID NO: 57(725.1), or SEQ ID NO: 58(16H 9).
The nucleic acid molecules of the invention include nucleic acid molecules that are degenerate variants or that hybridize under stringent conditions to a complementary sequence of a nucleic acid molecule having a sequence that encodes an antibody or antigen-binding antibody fragment of the invention. For example, in one embodiment, the nucleic acid molecule of the invention is a nucleic acid molecule that hybridizes under stringent conditions to SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 52. SEQ ID NO: 57. SEQ ID NO: 58. or SEQ ID NO: 59, or a nucleic acid molecule that hybridizes to the complement of 59. Preferably, these hybrid nucleic acid molecules of the invention encode an antibody or antigen-binding antibody fragment of the invention.
5.5 pharmaceutical compositions of the invention
In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding antibody fragment, fusion polypeptide, analog, or nucleic acid molecule of the invention and a pharmaceutically acceptable carrier.
Preferably, the pharmaceutical composition of the invention comprises an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog of the invention, exhibiting K for the peptide of FIG. 1(SEQ ID NO: 1) dLess than about 100nM, less than about 10nM, less than about 1nM, less than about 100pM, or less than about 10pM, KdFor measurement by the BIAcore affinity assay, the assay is described in section 6.4 below. Alternatively, the pharmaceutical composition of the invention comprises an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog or nucleic acid molecule of the invention that mediates lysis of CA 125/0772P-positive tumor cells. More preferably, the pharmaceutical composition of the invention comprises an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog of the invention or a nucleic acid molecule encoding a polypeptide that inhibits the growth of a CA 125/0772P-positive tumor cell by itself or when conjugated to a cytotoxic agent.
In one embodiment, the antibody or antigen-binding antibody fragment, or fusion polypeptide or analog thereof of the invention is conjugated to a cytotoxic agent for the treatment of cell proliferative diseases, such as those cited in section 5.2 above. In a particular embodiment, the cytotoxic agent is a radioisotope. In another particular embodiment, the radioisotope is selected from the group consisting of125I、131I、111In、99mTc and90Y。
the term "carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete or incomplete)), excipient, stabilizer, preservative, binding agent, or carrier for administration of an antibody, antigen-binding antibody fragment, fusion polypeptide, or the like of the present invention. The pharmaceutically acceptable carrier can be a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are described in Remington: the Science & practice of Pharmacy, 20 th edition, Gennaro, eds., Lippincott (2000).
In a preferred embodiment, the pharmaceutical composition is in a sterile and suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, most preferably a human subject.
In certain embodiments, it may be desirable to administer the pharmaceutical compositions of the present invention topically to a site in need of treatment. This can be achieved, for example, and without limitation, by injection infusion or by implants that are porous, non-porous, or gelatinous, including films, such as sialastic films, or fibers. Preferably, when administering the pharmaceutical composition, care must be taken that the material used does not absorb one or more of the antibodies, antigen-binding antibody fragments, fusion polypeptides or the like of the pharmaceutical composition.
In another embodiment, the pharmaceutical composition may be in the form of a vesicle, particularly a liposome (see, e.g., Langer, Science249(4976): 1527 and 1533 (1990); treat et al, Liposomes in the Therapy of infection Disease and Cancer, Lopez-Berestein et al, eds, Liss (1989) p.353-365; Lopez-Berestein et al, supra, page 317-; lopez-berestan et al, supra, generally; and U.S. patent nos. RE35,338, 5,662,931, 5,759,519, 5,879,713, 6,027,726, 6,099,857, 6,132,764, 6,245,427, 6,284,375, 6,350,466, and 6,417,326).
In yet another embodiment, the composition may be delivered in a controlled or sustained release system. In one embodiment, controlled or sustained release can be achieved with a pump (see, e.g., Langer, Science249(4976)：1527-1533(1990)；Sefton，Crit.Rev.Biomed.Eng.14(3): 201-40 (1987); buchwald et al, surgery.884: 507-516 (1980); saudek et al, n.321(9): 574-579 (1989); and U.S. patent nos. 5,720,720 and 6,352,683). In another embodiment, Controlled or sustained Release of an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog or fragment thereof of the invention can be achieved with a polymeric material (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRCPres, Boca Raton, Florida (1974) Medical Applications of Controlled Release, Langer et al, eds., CRC Press (1974); Controlled drug bioavailability, drug product Designand Performance, Smolen et al, eds., Wiley (1984); ranger et al, j.macromol.sci.rev.macromol.chem.23：61(1983) (ii) a Levy et al, Science.228(4696): 190-192 (1985); during et al, ann. neurol.25 (4): 351-356 (1989); howard et al, j.neurosurg.711: 105-112 (1989); U.S. patent nos. 5,128,326, 5,679,377, 5,863,985, 5,912,015, 5,916,597, 5,989,463, 5,994,492, 6,011,011, 6,020,004, 6,066,325, 6,180,608, 6,190,702, 6,214,966, 6,221,958, 6,221,977, 6,267,981, 6,362,276, 6,365,173, 6,375,985, 6,394,997, and 6,399,103; and PCT publication No. WO 99/20253). Examples of polymers for sustained release of the formulation include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate), poly (methacrylic acid), Polyglycolide (PLG), polyanhydrides, poly (N-vinyl pyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), Polylactide (PLA), poly (lactide-co-glycolide) (PLGA), and polyorthoesters.
The pharmaceutical compositions of the present invention are prepared to be compatible with their intended route of administration. Examples of routes of administration include, but are not limited to, for example, parenteral (e.g., intravenous, intradermal, intramuscular, subcutaneous), oral, intranasal, inhalation, transdermal (topical), transmucosal, and rectal administration. In particular embodiments, the compositions are prepared according to conventional methods into pharmaceutical compositions suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans. In a preferred embodiment, the pharmaceutical composition is prepared according to conventional methods for subcutaneous administration to a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic to reduce pain at the injection site.
If the pharmaceutical composition of the present invention is to be administered topically, the composition may be prepared, for example, as an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well known to those skilled in the art. See, for example, Remington: the Science & Practice of Pharmacy, 20 th edition, Gennaro, eds., Lippincott (2000). For non-nebulized topical dosage forms, viscous to semi-solid to solid forms are generally used, which contain a carrier or one or more excipients that are compatible with topical application and preferably have a dynamic viscosity greater than water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which can be sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, moisturizers, buffers, or salts) to affect various properties, such as, for example, osmotic pressure, if desired. Other suitable topical dosage forms include sprayable aerosol formulations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in admixture with a pressurized volatile substance, e.g., a gaseous propellant such as freon, or in a squeeze bottle. Humectants or moisturizers may also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art.
If the composition of the present invention is to be administered intranasally, the composition may be formulated in the form of an aerosol, spray, mixture, or drops. In particular, the agents used in accordance with the present invention may conveniently be delivered in the form of an aerosol spray presentation from a pressurised pack or atomiser, with a suitable propellant being, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. For a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. For an inhaler or insufflator, for example, capsules or cartridges may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
If the pharmaceutical compositions of the present invention are to be administered orally, they may be formulated into oral forms such as tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Tablets or capsules may be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or a moisturizing agent (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or a suitable vehicle before use. These liquid preparations may be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous carriers (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoate or sorbic acid). The formulations may also contain buffer salts, flavoring agents, coloring agents and sweeteners, as appropriate. Formulations for oral administration may be suitably formulated for slow, controlled or sustained release of the prophylactic or therapeutic agent.
The pharmaceutical compositions of the present invention may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. These pharmaceutical compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
The pharmaceutical compositions of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described above, the pharmaceutical compositions of the present invention may be formulated as long acting formulations. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, these pharmaceutical compositions may be formulated with suitable polymeric or hydrophobic materials (e.g. as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g. as a sparingly soluble salt.
Typically, the components of the pharmaceutical compositions of the present invention are provided separately or mixed together in unit dosage form, e.g., as a lyophilized powder or anhydrous concentrate in a sealed container, such as an ampoule or sachet, indicating the amount of active agent. When the pharmaceutical composition is to be administered by infusion, it may be formulated with an infusion bottle containing pharmaceutical grade water or salt. When the pharmaceutical composition is to be administered by injection, an ampoule of sterile water or saline for injection may be provided so that the ingredients may be mixed prior to administration.
For the antibodies, antigen-binding antibody fragments, fusion polypeptides, and analogs of the invention, the dose administered is generally from about 5 μ g/kg to about 10mg/kg, more preferably from about 20 μ g/kg to about 5mg/kg of the subject's body weight, and most preferably from about 100 μ g/kg to about 5 mg/kg. The dose administered may be up to about 6 treatments over a period of weeks to months, as determined by the performing physician. Generally, human antibodies have a longer half-life in humans than antibodies from other species due to the immune response to the foreign polypeptide. Thus, human antibodies can generally be administered at lower doses and less frequently. In addition, the dosage and frequency of administration of the antibodies or fragments thereof of the invention can be reduced by modifications such as, for example, lipidation to enhance absorption and tissue penetration of the antibodies.
The precise dosage for use in the formulation will depend on the route of administration, the severity of the condition, and will be determined at the discretion of the practitioner and in view of published clinical studies for each patient. Effective doses can be extrapolated from dose-response curves in vitro or in animal model test systems.
In one embodiment, the pharmaceutical composition of the invention is packaged in a closed container, such as an ampoule or sachette, which indicates the amount of antibody, antigen-binding antibody fragment, fusion polypeptide, or the like. In another embodiment, the pharmaceutical compositions of the invention are provided as a dry sterile lyophilized powder or a water-free concentrate in a closed container and can be reconstituted with, for example, water or saline to a suitable concentration for administration to a subject. In yet another embodiment, the pharmaceutical composition is resuspended in a liquid form in a closed container indicating the amount and concentration of the antibody, antigen-binding antibody fragment, fusion polypeptide, or the like.
In yet another embodiment, the pharmaceutical composition of the invention is provided in a unit dose in a closed container, the unit dose being at least about 5mg, more preferably at least about 1mg, more preferably at least about 2mg, 5mg, 10mg, 15mg, 25mg, 35mg, 45mg, 50mg, 75mg, 100mg, 200mg, 300mg, 400mg, or 500 mg. When provided in liquid form, the pharmaceutical composition may be provided in such a closed container at a concentration of at least about 1 mg/ml.
The invention also provides a method of making a pharmaceutical composition of the invention, the method comprising admixing an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog of the invention with a pharmaceutically acceptable carrier.
5.6 products of the invention
In another aspect, the invention provides a product comprising a packaging material and a pharmaceutical composition of the invention contained in the packaging material, in a form suitable for administration to a subject, preferably a human, or in a form which can be diluted or reconstituted for administration to a subject. In one embodiment, the product further contains printed instructions and/or labels that direct the use or administration of the pharmaceutical composition. The instructions and/or labeling may, for example, address a dosing regimen for preventing or treating a CA 125/0772P-associated disorder, such as a cell proliferative disorder, e.g., one or more symptoms of a cancer, e.g., ovarian, uterine, breast, or lung cancer. Thus, the instructions and/or labels for use may provide informational material advising a physician, technician, or subject how to appropriately prevent, treat, or ameliorate a CA 125/0772P-related disorder or one or more symptoms of the disorder, e.g., a cell proliferative disorder, such as cancer, e.g., ovarian cancer.
For any pharmaceutical product, the packaging material and container of the product of the present invention are designed to preserve the stability of the product during storage and transport. More particularly, the present invention provides products comprising packaging materials such as boxes, bottles, tubes, vials, containers, nebulizers, insufflators, intravenous (i.v.) packets, and the like; and at least one unit dosage form of the pharmaceutical composition of the invention contained in the packaging material.
5.7 methods for identifying antibodies and antigen-binding antibody fragments that preferentially bind cell-associated CA 125/0722P
The present invention provides methods that facilitate the identification of antibodies or antigen-binding antibody fragments that preferentially bind cell-associated CA 125/0772P relative to shed CA 125/0772P. In one embodiment, a method of facilitating identification of an antibody or antigen-binding antibody fragment that preferentially binds cell-associated CA 125/0772P comprises contacting the antibody or antigen-binding antibody fragment with a peptide comprising cell-associated CA 125/0772P in the presence of exfoliated CA 125/0772P under conditions that allow the antibody or antigen-binding antibody fragment to bind to the peptide comprising cell-associated CA 125/0772P or exfoliated CA 125/0772P. After incubation, shed CA 125/0772P (with or without antibody or antigen-binding antibody fragment binding) and unbound antibody or antigen-binding antibody fragment were removed and the amount of antibody or antigen-binding antibody fragment bound to the cell-associated CA 125/0772P-containing peptide was measured. An antibody or antigen-binding antibody fragment of the method preferably binds to a cell-associated CA 125/0772P polypeptide relative to an shed CA 125/0772P polypeptide if the antibody or antigen-binding antibody fragment meets one of the three embodiments set forth above for "preferably binds". In a preferred embodiment, the ratio of shed CA 125/0772P to cell-associated CA 125/0772P in the reaction mixture is about 25: 1 (wt/wt). As part of this method, cell-associated CA 125/0772P may be immobilized on a solid surface. For example, the method may be performed in an ELISA format.
In another embodiment, the invention provides a method of facilitating identification of an antibody or antigen-binding antibody fragment that preferentially binds cell-associated CA125/0772P over shed CA125/0772P, the method comprising contacting the antibody or antigen-binding antibody fragment with a peptide comprising cell-associated CA125/0772P and shed CA125/0772P (e.g., about a 25-fold excess (weight/weight)) under conditions that allow binding of cell-associated CA125/0772P to the antibody or antigen-binding antibody fragment, removing unbound peptide comprising cell-associated CA125/0772P, measuring the amount of cell-associated CA 125/0772P-containing peptide bound by the antibody or antigen-binding fragment, and comparing the measured amount to the amount of cell-associated CA 125/0772P-containing peptide to which the antibody or antigen-binding antibody fragment can bind in the absence of such amount of shed CA125/0772P And (6) comparing. An antibody or antigen-binding antibody fragment of the method preferably binds to a cell-associated CA125/0772P polypeptide relative to an shed CA125/0772P polypeptide if the antibody or antigen-binding antibody fragment meets one of the three embodiments set forth above for "preferably binds". As part of the method, the antibody or antigen-binding antibody fragment may be immobilized on a solid surface, e.g., the method may be performed in an ELISA format. The teachings provided herein, in combination with standard techniques well known to those skilled in the art, can be used to perform these methods to identify antibodies, or antigen-binding antibody fragments, of the invention. For example, assays that can be used to identify these antibodies or antibody fragments that bind to an antigen include the ELISA competition assays described in section 6 below and in the subsections thereof.
In yet another embodiment, the invention provides a method of facilitating the identification of an antibody or antigen-binding antibody fragment that preferentially binds cell-associated CA125/0772P, the method comprising contacting the antibody or antigen-binding antibody fragment with CA 125/0772P-expressing cells and an amount, e.g., at least about 0.05mg/ml, of shed CA125/0772P under conditions that allow CA125/0772P to bind to the antibody or antigen-binding antibody fragment, removing unbound cells, measuring the amount of CA 125/0772P-expressing cells bound by the antibody or antigen-binding antibody fragment, and comparing the measurement to the amount of CA 125/0772P-expressing antibody or antigen-binding antibody fragment in the absence of these amounts of shed CA 125/0772P. If the antibody or antigen-binding antibody fragment of the method satisfies one of the three embodiments set forth above for "preferentially binding," then the antibody or antigen-binding antibody fragment preferentially binds to the cell-associated CA125/0772P polypeptide relative to the shed CA125/0772P polypeptide. Such methods can be performed, for example, as measurements performed by flow cytometry techniques, including, for example, Fluorescence Activated Cell Sorting (FACS). The teachings provided herein, in combination with standard techniques well known to those skilled in the art, can be used to perform these methods to identify antibodies, or antigen-binding antibody fragments, of the invention. For example, assays that can be used to identify these antibodies or antibody fragments that bind to an antigen include flow cytometry competition assays described in section 6 below and in the subsections thereof.
Embodiments of the invention include antibodies and antigen-binding antibody fragments that preferably bind cell-associated CA125/0772P and are specific for CA 125/0772P. Antibodies specific for CA125/0772P can be routinely identified using, for example, ELISA-specific assays and flow cytometry competition assays described in section 6 below and in the subsections thereof. Likewise, the invention also provides for the identification of antibodies and antigen-binding antibody fragments that are specific for CA125/0772P and that also preferably bind cell-associated CA 125/0772P. In one such embodiment, an antibody or antigen-binding antibody fragment specific for CA125/0772P is first identified, e.g., by an ELISA-specific assay and/or a flow cytometry competition assay. The antibody or antigen-binding antibody fragment is then tested for its ability to preferentially bind cell-associated CA125/0772P, for example, using one of the methods described herein.
Embodiments of the invention also include antibodies and antigen-binding antibody fragments that preferably bind cell-associated CA125/0772P and bind the peptide of FIG. 1 (SEQ ID NO: 1) with a Kd of less than about 100nM, less than about 10nM, less than about 1nM, less than about 100pM, or less than about 10pM, as measured by the BIAcore affinity assay, described in section 6 and its sections below. Likewise, embodiments of the invention also include antibodies and antigen-binding antibody fragments that preferably bind to cell-associated CA125/0772P and bind to cell-associated CA125/0772P with at least a minimum level of affinity. In one such embodiment, an antibody or antigen-binding antibody fragment that preferentially binds to cell-associated CA125/0772P is first identified, e.g., by one of the methods described herein. The antibody or antigen-binding antibody fragment is then tested for its ability to bind cell-associated CA125/0772P (or a peptide containing cell-associated CA 125/0772P) with a Kd of less than about 100nM, less than about 10nM, less than about 1nM, less than about 100pM, or less than about 10pM, using, for example, one of the methods described herein.
Embodiments of the invention also include antibodies and antigen-binding antibody fragments that preferably bind cell-associated CA 125/0772P and exhibit the ability to mediate lysis of CA 125/0772P-positive cells, e.g., tumor cells. These antibodies and antigen-binding antibody fragments can be routinely identified by, for example, performing ADCC and/or CDC assays described in section 6 below and sections hereof. Likewise, the invention provides methods for identifying antibodies or antigen-binding antibody fragments that preferentially bind cell-associated CA 125/0772P and exhibit the ability to mediate lysis of CA 125/0772P-positive cells. Antibodies or antigen-binding antibody fragments that preferentially bind cell-associated CA 125/0772P are identified using, for example, one of the methods described herein. The antibody or antigen-binding antibody fragment is then tested for its ability to mediate lysis of CA 125/0772P-positive cells by, for example, the methods described herein ADCC and/or CDC assays.
Embodiments of the invention also include antibodies and antigen-binding antibody fragments that preferably bind CA 125/0772P and exhibit the ability to inhibit or delay the growth of a CA 125/0772P-positive tumor. By, for example, an in vivo assay previously described, such as tresses et al, eur. 30A(2)：183-187 (1994); ahmad et al, oncol.11(6): 273-280 (1999); and kienit et al, int.j.radiat.onc.biol.phys.38(2): 419-428(1997) (which are all herein incorporated by reference in their entirety for routine identification of these antibodies the invention provides methods for identifying antibodies or antigen-binding antibody fragments that preferentially bind CA125/0772P and exhibit the ability to inhibit the growth of CA 125/0772P-positive cells.
5.8 methods of preventing, treating, managing or ameliorating symptoms of CA 125/0772P-related disorders
The present invention provides methods of preventing, treating, or managing a CA 125/0772P-related disorder, or ameliorating a symptom of a CA 125/0772P-related disorder. For example, the invention provides a method of preventing, treating, managing or ameliorating a symptom of a cell proliferative disorder, comprising administering to a subject in need thereof an amount of an antibody, antigen-binding antibody fragment, or analog effective to achieve a desired result in the subject.
As discussed throughout, the antibodies and antigen-binding antibody fragments of the invention are those that preferably bind cell-associated CA 125/0772P. Likewise, the fusion polypeptides and analogs of the invention also preferably bind cell-associated CA 125/0772P. Also mentioned herein, since CA125/0772P is present in CA125/0772P or is part of CA125/0772P before shedding cells associated with CA125/0772P, it is noted that the antibodies, antigen-binding antibody fragments, fusion polypeptides, and analogs of the invention can also bind CA 125/0772P. Thus, without wishing to be bound by any particular mechanism or theory thereof, it is noted that the methods described in this section may be at least partially achieved by administering an antibody, antigen-binding antibody fragment, fusion polypeptide or analog of the present invention that binds to pre-shedding CA125/0772P in addition to CA125/0772P associated with the post-shedding cells, or to CA125/0772P that is not associated with the post-shedding cells but only to pre-shedding CA 125/0772P.
In one embodiment, the methods of the invention relate to the prevention, treatment, management, or amelioration of symptoms of cancer. For example, the methods of the invention relate to the prevention, treatment, management, or amelioration of symptoms of cancer or cancer-related disorders, including, but not limited to, carcinomas, sarcomas, myelomas, leukemias, lymphomas, and mixed types of cancer. In particular embodiments, the methods of the invention relate to preventing, treating, managing or ameliorating ovarian, cervical, uterine, breast or lung cancer, or a symptom thereof. In preferred embodiments of these methods of the invention, the methods relate to preventing, treating, managing or ameliorating the symptoms of ovarian cancer.
In another embodiment, the invention provides a method of treating a CA 125/0772P-related disorder, or ameliorating a symptom thereof, the method comprising administering to a subject in need of such treatment or amelioration an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog of the invention in an amount sufficient to treat a cell proliferative disorder or ameliorate a symptom thereof. The CA 125/0772P-related disorder may be, for example, a cell proliferative disorder such as cancer, and may include, for example, ovarian, cervical, uterine, breast or lung cancer. This embodiment is preferably practiced where the antibodies, antigen-binding antibody fragments, fusion polypeptides or analogs of the invention are conjugated to cytotoxic agents for the treatment of cell proliferative diseases, such as those cited in section 5.2. In a particular embodiment, the cytotoxic agent is a radioisotope. In another embodiment, the radioisotope is selected from the group consisting of125I、131I、111In、99mTc and90and Y. This embodiment antigen is used as part of a combination cancer therapy by, for example, also administering to the subject a chemotherapeutic agent, such as paclitaxel or cisplatin, or radiation therapy.
In yet another embodiment, the invention provides a method of preventing a CA 125/0772P-related disorder or a symptom of a CA 125/0772P-related disorder, the method comprising administering to a subject in need of such prevention an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog of the invention in an amount sufficient to prevent a CA 125/0772P-related disorder or a symptom thereof. The CA 125/0772P-related disorder may be, for example, a cell proliferative disorder such as cancer, and may include, for example, ovarian, cervical, uterine, breast or lung cancer.
In additional embodiments, the CA 125/0772P-related disorder may be a bone cancer, e.g., ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, or other soft tissue sarcoma. In another embodiment, the CA 125/0772P-related disorder is a brain tumor, e.g., an oligodendroglioma, ependymoma, menengioma, lymphoma, schwannoma, or medulloblastoma. In another embodiment, the CA 125/0772P-related disorder is breast cancer, e.g., ductal carcinoma in situ of the breast. In another embodiment, the CA 125/0772P-related disorder is a cancer of the endocrine system, e.g., an adrenal cancer, a pancreatic cancer, a parathyroid cancer, a pituitary cancer, or a thyroid cancer. In another embodiment, the CA 125/0772P-related disorder is gastrointestinal cancer, e.g., anal cancer, rectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, liver cancer, pancreatic cancer, or small intestine cancer. In another embodiment, the CA 125/0772P-related disorder is a gynecological cancer, e.g., cervical cancer, endometrial cancer, uterine cancer, fallopian tube cancer, gestational trophoblastic disease, choriocarcinoma cancer, ovarian cancer, vaginal cancer, or vulval cancer. In another embodiment, the CA 125/0772P-related disorder is head and neck cancer, e.g., laryngeal, oropharyngeal, parathyroid or thyroid cancer. In another embodiment, the CA 125/0772P-related disorder is a leukemia cancer, e.g., acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, or a myeloproliferative disorder. In another embodiment, the CA 125/0772P-related disorder is lung cancer, e.g., mesothelioma, non-small cell lung cancer, or small cell lung cancer. In another embodiment, the CA 125/0772P-related disorder is a lymphoma, e.g., AIDS-related lymphoma, cutaneous T-cell lymphoma, hodgkin's disease, or non-hodgkin's disease. In another embodiment, the CA 125/0772P-related disorder is a metastatic cancer. In another embodiment, the CA 125/0772P-related disorder is myeloma, e.g., multiple myeloma. In another embodiment, the CA 125/0772P-related disorder is a pediatric cancer, e.g., a brain tumor, ewing's sarcoma, leukemia (e.g., acute lymphocytic leukemia or acute myeloid leukemia), liver cancer, lymphoma (e.g., hodgkin's lymphoma or non-hodgkin's lymphoma), neuroblastoma, retinoblastoma, sarcoma (e.g., osteosarcoma, rhabdomyosarcoma, or other soft tissue sarcoma), or wilms' tumor. In another embodiment, the CA 125/0772P-related disorder is penile cancer. In another embodiment, the CA 125/0772P-related disorder is prostate cancer. In another embodiment, the CA 125/0772P-related disorder is a skin cancer, e.g., cutaneous T-cell lymphoma, mycosis fungoides, kaposi's sarcoma, or melanoma. In another embodiment, the CA 125/0772P-related disorder is testicular cancer. In another embodiment, the CA 125/0772P-related disorder is thyroid cancer, e.g., papillary, alveolar, medullary, and degenerative or undifferentiated thyroid cancer. In another embodiment, the CA 125/0772P-related disorder is cancer of the urinary tract, e.g., cancer of the bladder, kidney, or urethra. In another embodiment, the CA 125/0772P-related disorder is ataxia telangiectasia, a cancer of unknown origin, Li-Fraumeni syndrome, or thymoma.
In one embodiment of the methods of the invention, an antibody or antigen-binding fragment of the invention is administered. In another embodiment, a monoclonal antibody or a monoclonal antibody fragment that binds an antigen is administered. Typically, the antibody or antigen-binding antibody fragment is administered at a dose concentration of about 5 μ g/kg to about 10mg/kg, more preferably about 20 μ g/kg to about 5mg/kg, and most preferably about 100 μ g/kg to about 5mg/kg of the subject's body weight.
In general, the methods described herein can be utilized by administering the pharmaceutical compositions of the present invention. By e.g. for determining LD in cell cultures or experimental animals50(lethal dose for 50% of the population) and ED50(therapeutically effective amount in 50% of the population) can be determined as part of the invention of the specific embodiment of the composition of toxicity and/or efficacy. The dose ratio between toxic and therapeutic effects is a therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions that exhibit large therapeutic indices are preferred. Although compositions that exhibit toxic side effects may be administered, it is preferred that the delivery system used target such compositions to affected tissues, e.g., ovarian tissue, thereby reducing side effects.
Data obtained from cell culture assays and animal studies can be used to formulate a range of doses of compositions for use in humans. The dosage of these compositions preferably lies within a circulating concentration range that includes ED with little or no toxicity50. The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. For any reagent used in the methods of the invention, the therapeutically effective amount can be estimated initially from cell culture assays. Doses in animal models can be formulated to achieve circulating plasma concentration ranges that include IC as determined in cell culture assays, e.g., proliferation assays50(i.e., the concentration of compound that achieves half-maximal inhibition of one or more symptoms). This information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured by, for example, high performance liquid chromatography.
Various delivery systems are known and can be used to administer the antibodies, antigen-binding antibody fragments, fusion polypeptides or analogs of the invention, e.g., encapsulation in Liposomes (see, e.g., Langer, Science 249 (4976): 1527-.
Methods of administering the antibodies, antigen-binding antibody fragments, fusion polypeptides or analogs of the invention, or pharmaceutical compositions containing the antibodies, antigen-binding antibody fragments, fusion polypeptides or analogs of the invention include, but are not limited to, parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous administration), epidural, or mucosal (e.g., intranasal or oral) routes of administration. See, for example, U.S. patent nos. 5,679,377, 5,702,727, 5,783,193, 5,817,624, 6,074,689, 6,156,731, 6,174,529, 6,187,803, 6,331,175, and 6,387,406. In a particular embodiment, the antibody, antigen-binding antibody fragment, fusion polypeptide or analog of the invention or a pharmaceutical composition thereof is administered intramuscularly, intravenously or subcutaneously. These compositions may be administered by any convenient route, e.g., by infusion or bolus injection, or by epithelial or mucocutaneous linings (e.g., oral, rectal, or intestinal mucosa, etc.), or they may be administered with other bioactive agents. Administration may be systemic or local. Furthermore, pulmonary administration may also be employed, for example, by using an inhaler or nebulizer and formulation with an aerosol. See, for example, U.S. patent nos. RE37,525, 5,290,540, 5,855,913, 5,874,064, 5,934,272, 5,985,309, 5,985,320, 6,019,968, 6,165,463, 6,358,530 and 6,402,733 and PCT publication No. WO99/66903, each of which is incorporated by reference in its entirety. In one embodiment, Alkermes AIR is used TMPulmonary drug delivery technology (Alkermes, inc., Cambridge, MA) can administer antibodies, fusion proteins, conjugate molecules, or pharmaceutical compositions.
In a preferred embodiment, the pharmaceutical composition is formulated in accordance with conventional methods such that it is suitable for intravenous administration to a human. Typically, pharmaceutical compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic to reduce pain at the injection site.
In another particular embodiment, it may be desirable to administer the pharmaceutical composition of the present invention topically to a site in need of treatment. This may be achieved by local infusion, injection or by an implant which is a porous, non-porous or gelatinous substance.
In yet another embodiment, the method is performed as part of a combination therapy, e.g., a combination cancer therapy. Such a combination cancer therapy may include, for example, administration of a chemotherapeutic agent, e.g., cisplatin, ifosfamide, paclitaxel, a taxane, a topoisomerase I inhibitor (CPT-11, topotecan, 9-AC, or GG-211), gemcitabine, mitomycin, emidine, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione, mitoxantrone, mithramycin, vinorelbine, oxaliplatin, 5-fluorouracil (5-fla), leucovorin, vinorelbine, temodal, or taxol. These combination cancer therapies may alternatively or additionally include, but are not limited to, radiation therapy.
The term "combination therapy" or "combination cancer therapy" limits the order of administration of agents or treatments to a subject having a CA 125/0772P-related disorder. For example, the agents of the combination therapy may be administered to the subject simultaneously, sequentially, in any order, or cyclically. In a preferred embodiment, two or more components of the combination therapy are administered to the subject simultaneously. The term "simultaneously" is not limited to two or more agents being administered at exactly the same time, but rather refers to the agents being administered to a subject in a sequence and within a time interval such that the agents may act together to provide an increased benefit over their otherwise administered.
The agents administered as part of a combination therapy method may, for example, be administered to the subject in the same pharmaceutical composition. Alternatively, the agents of the combination therapy may be administered to the subject as separate pharmaceutical compositions by the same or different routes of administration.
5.9 methods of diagnosing CA 125/0772P-related disorders
In another aspect, the invention also provides methods of diagnosing a CA 125/0772P-related disorder or a predisposition for such a disorder. The labeled antibodies, antigen-binding antibody fragments, fusion polypeptides, and analogs of the invention can be used for diagnostic purposes to detect, diagnose, or monitor CA 125/0772P-related disorders, such as cancer.
Typical immunohistological methods as described herein or methods well known to those skilled in the art are used (see, e.g., alkanen et al, j.cell.biol).101(3): 976-984 (1985); jalkane et al, j.cell.biol.105(6 pt 2): 3087-3096(1987)), the antibodies, antigen-binding antibody fragments, fusion polypeptides and analogues of the invention can be used to determine CA 125/0772P-related disorders in biological samples. Other antibody-based methods for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISA) and Radioimmunoassays (RIA). Suitable antibody assay labels are well known in the art and include, for example, enzyme labels such as alkaline phosphatase, glucose oxidase, radioisotopes such as iodine: (a)125I、131I) Carbon (C)14C) Sulfur (S), (S)35S), tritium (3H) Indium (I) and (II)111In), and technetium (99mTc); luminescent labels, such as luminol; and fluorescent labels such as fluorescein and rhodamine.
One aspect of the invention is the detection and diagnosis of a predisposition to cancer, particularly ovarian cancer, in humans. In one embodiment, the diagnosing comprises: a) administering (e.g., parenterally, subcutaneously or intraperitoneally) to the subject an amount of a labeled antibody, antigen-binding antibody fragment, fusion polypeptide, or analog that is effective for diagnosis, which preferably binds to cell-associated CA 125/0772P, and b) detecting the labeled antibody, antigen-binding antibody fragment, fusion polypeptide, or analog in the subject in order to make the diagnosis. According to this embodiment, the antibodies, antigen-binding antibody fragments, fusion polypeptides and the like are preferably labeled with an imaging moiety that can be detected using imaging systems well known to those skilled in the art. Background levels can be determined by various methods including comparing the amount of marker molecules detected to standard values previously determined for a particular system.
The presence of the marker molecule in the subject can be detected using methods well known in the art for in vivo scanning. These methods depend on the type of label used. The skilled person will be able to determine a suitable method of detecting a particular label. Methods that can be used in the diagnostic methods of the present invention include, but are not limited to, Computed Tomography (CT), whole body scans such as Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI), and sonography.
In certain embodiments, the molecule is labeled with a radioisotope and the molecule is detected in the subject using a radio-responsive surgical device (see, e.g., U.S. patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and the molecule is detected in the subject using a fluorescence response scanning device. In another embodiment, the molecule is labeled with a positron emitting metal and PET is used to detect the molecule in a subject. In another embodiment, the molecule is labeled with a paramagnetic label and the molecule is detected in the subject using MRI.
It will be understood in the art that the size and weight of the subject, as well as the type of imaging system used, will determine the type and amount of imaging portion needed to produce a useful diagnostic image. For use in human subjects 99mThe radioisotope fraction of Tc, the amount of radioactivity injected will typically be about 5 to 20 millicuries. The labeled antibody, antigen-binding antibody fragment, fusion polypeptide, or analog then preferentially accumulates at the cellular site displaying the cell-associated CA125/0772P polypeptide. In Burchiel et al, "immunopharmacology of radiolabeled antibodies and their fragments", tomor Imaging: in vivo tumor imaging is described in The Radiochemical Detection of Cancer, Burchiel et al, eds., Masson Publishing Inc. (1982), Chapter 13.
Depending on a number of variables, including the type of label used and the mode of administration, the time interval after administration to allow preferential concentration of the labeled molecules at the subject site and clearance of unbound labeled molecules from background levels may be about 6 to 48 hours or about 6 to 24 hours or about 6 to 12 hours. In another embodiment, the time interval after administration is about 5 to 20 days or about 5 to 10 days.
In one embodiment, monitoring for a CA 125/0772P-related disorder, e.g., cancer, may be performed at time points by repeated imaging methods, e.g., 1 month after initial diagnosis, 6 months after initial diagnosis, and/or 1 year after initial diagnosis, and the like.
The invention also includes a method of diagnosing or monitoring cancer comprising administering to a subject in need of such diagnosis or monitoring an amount of labeled antibody, antigen-binding antibody fragment, fusion polypeptide, or analog that preferentially binds cell-associated CA 125/0772P sufficient to detect and detect labeled antibody, antigen-binding antibody fragment, fusion polypeptide, or analog bound to an organ or tissue of the subject. In addition, the present invention provides a method of detecting cell-associated CA 125/0772P in a biological sample, the method comprising contacting the sample with a labeled antibody, antigen-binding antibody fragment, fusion polypeptide, or the like that preferentially binds to cell-associated CA 125/0772P, and detecting the antibody, antigen-binding antibody fragment, fusion polypeptide, or the like bound to the sample.
In these embodiments, the amount of labeled molecule bound to cell-associated CA 125/0772P can be compared to a standard amount or control, or an amount previously detected in the sample at an earlier time point.
5.10 methods of producing antibodies
The antibodies of the invention may be produced by any method of antibody synthesis known in the art, for example, by hybridoma technology, chemical synthesis or, preferably, by recombinant expression techniques.
Polyclonal antibodies can be produced by various methods well known in the art. For example, human CA 125/0772P containing a cell-associated CA 125/0772P polypeptide can be administered to various host animals, including, but not limited to, rabbits, mice, rats, and horses, to induce the production of serum containing polyclonal antibodies specific for human antigens. Depending on the variety of hosts, various adjuvants may be used to increase the immune response, including, but not limited to, freund's adjuvant (complete or incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacillus calmette guerin) and tyrobacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can be prepared using a variety of techniques well known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal Antibodies can be prepared using hybridoma techniques, including those well known in the art and described in Harlow et al, Antibodies: a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press (1988); or those hybridoma techniques taught by Hammering et al, Monoclonal Antibodies and T-Cell hybrids, Elsevier (1981)563-681, which are incorporated herein in their entirety by reference thereto.
Methods for producing and screening specific antibodies using hybridoma technology are routine and well known in the art. Briefly, in one embodiment, a mouse can be immunized with a CA 125/0772P polypeptide, e.g., a cell-associated CA 125/0772P polypeptide, and once an immune response, e.g., an antibody specific for an antigen, is detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The spleen cells are then fused by well-known techniques with any suitable myeloma cell, for example, a cell from cell line SP2/0-Ag14(ATCC accession number CRL-1581) available from ATCC. Hybridomas were selected and cloned by limiting dilution. The hybridomas are then assayed by methods well known in the art to obtain antibodies that secrete CA 125/0772P capable of binding to the cell associated. Ascites fluid, which usually contains high levels of antibodies, can be produced by injecting mice with positive hybridoma clones.
Accordingly, the present invention provides a method of producing monoclonal antibodies and antibodies produced by the method, the method comprising culturing hybridoma cells which secrete an antibody of the invention wherein, preferably, the hybridomas are produced by fusing spleen cells isolated from mice immunized with a CA 125/0772P polypeptide, e.g., a cell-associated CA 125/0772P polypeptide, with myeloma cells and then screening the resulting fusions for hybridoma clones which secrete antibodies capable of binding a CA 125/0772P polypeptide, e.g., a cell-associated CA 125/0772P polypeptide.
Antibody fragments that specifically recognize a particular epitope can be generated by any technique known to those skilled in the art. For example, Fab and F (ab ') 2 fragments can be produced by proteolytic cleavage of an immunoglobulin molecule with an enzyme such as papain (to produce Fab fragments) or pepsin (to produce F (ab') 2 fragments). The F (ab') 2 fragment contains the variable region, the light chain constant region and the CH1 domain of the heavy chain. In addition, the antibodies of the invention can also be produced using various phage display methods known in the art.
In the phage display method, the functional antibody domain is displayed on the surface of phage particles, the phage particles carrying encoding functional antibody domain polynucleotide sequences. Phage expressing an antigen binding domain that binds to a CA 125/0772P polypeptide antigen can be selected or identified using an antigen, e.g., using a labeled antigen or an antigen that is bound or captured to a solid surface or bead. Examples of phage display methods that can be modified so that they can be used to prepare or identify antibodies of the invention include those described in Brinkmann et al, j.182(1): 41-50 (1995); ames et al, j.184(2): 177-186 (1995); ketleborough et al, eur.j. 24(4): 952 and 958 (1994); persic et al, Gene.187(1): 9-18 (1997); burton et al, adv. immunol.57: 191-280 (1994); PCT publication Nos. WO 91/10737 and WO 95/15982, respectively; EP 853,661; and those disclosed in U.S. Pat. nos. 5,223,409, 5,403,484, 5,427,908, 5,516,637, 5,571,698, 5,580,717, 5,658,727, 5,667,988, 5,698,426, 5,712,089, 5,733,743, 5,780,225, 5,789,208, 5,821,047, 5,885,793, 5,969,108, 6,096,551, 6,140,470, 6,376,170, 6,265,150, and 6,335,163, which are incorporated herein by reference in their entirety.
As described in the above references, following phage selection, the antibody coding regions can be isolated from the phage and used to produce whole antibodies, including human antibodies, or desired antigen-binding fragments, and expressed in hosts, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described below. Using methods well known in the art such as U.S. patent nos. 5,595,898, 5,698,417, and 6,204,023; mullinax et al, Bio Techniques 12 (6): 864-869 (1992); sawai et al, am.J.reprod.Immunol.34(1): 26-34 (1995); and Better et al science.240 (4855): 1041 (1988), which reference is incorporated by reference in its entirety, Fab 'and F (ab') 2 fragments can also be produced recombinantly.
To generate a complete antibody, the VH or VL sequences in the scFv clone can be amplified with PCR primers (including the VH or VL nucleotide sequences, the restriction site, and flanking sequences protecting the restriction site). The PCR-amplified VH domain can be cloned into a vector expressing a VH constant region and the PCR-amplified VL domain can be cloned into a vector expressing a VL constant region, such as a human kappa or lambda constant region, using cloning techniques well known to those skilled in the art. Preferably, the vector expressing the VH or VL domain may contain the EF-1. alpha. promoter, secretion signal, cloning site for the variable domain, a constant domain, and a selectable marker such as neomycin. The VH and VL domains can also be cloned into vectors expressing the necessary constant regions. The heavy chain expression vector and the light chain expression vector are then co-transfected into a cell line to generate a stable or transient cell line that expresses a full length antibody, e.g., IgG, using techniques well known to those skilled in the art.
For some uses, including the use of antibodies in human and in vivo detection assays, it is preferred to use chimeric, humanized or fully human antibodies. In chimeric antibodies, different portions of the antibody are derived from different species of immunoglobulin. For example, and not by way of limitation, a chimeric antibody may have light and/or heavy chain variable regions from a murine antibody and light and/or heavy chain constant regions from a human immunoglobulin. Methods of producing recombinant chimeric antibodies are well known in the art. See, e.g., Morrison, science.229 (4719): 1202-1207 (1985); oi et al, BioTechniques.4 (3): 214-221 (1986); gilles et al, j. 125(1-2): 191-202 (1989); and U.S. Pat. nos. 4,816,397, 4,816,567 and 5,807,715. Each of these documents is incorporated by reference in its entirety.
A humanized antibody is an antibody that contains a human framework, including human constant regions, and one or more CDRs of an antibody from a non-human species, e.g., a mouse species. Such humanized antibodies can be routinely produced using a variety of techniques well known in the art, including, for example, CDR-grafting (EP 239,400; PCT publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, 5,585,089, 5,766,886, 5,859,205, 6,180,370, and 6,407,213), veneering or resurfacing (U.S. Pat. No. 5,639,641; EP519,596; Padlan, Padlan, mol.28(4/5): 489-498 (1991); studnicka et al, Protein Eng.7(6): 805-814 (1994); and Rogus ka et al, Proc.Natl.Acad.Sci.USA.91(3): 969-. In a preferred embodiment, the humanized antibody comprises a CDR having the amino acid sequence of any one of the CDRs listed in table 1, table 2, table 3, table 4, table 5, or table 6 and a human framework region. Typically, framework residues in the framework regions will be replaced with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. Identification of these framework substitutions by methods well known in the art, e.g., identification of framework residues important for antigen binding by modeling the interaction of CDRs and framework residues, identification of specific positions by sequence comparison An unusual framework residue of (a). See, for example, U.S. Pat. nos. 5,585,089, 5,770,196, and 5,869,619; and Riechmann et al, Nature.332(6162): 323-327(198g), which are hereby incorporated by reference in their entirety.
Fully or fully human antibodies are desirable for therapeutic treatment of human subjects. Human antibodies can be prepared by various methods known in the art, including phage display methods, using antibody libraries derived from human immunoglobulin sequences. See also U.S. Pat. nos. 4,444,887, 4,716,111, 5,916,771, 5,939,598, 6,075,181, 6,114,598, 6,150,584, 6,162,963, 6,235,883; PCT publication WO 98/46645; and EP 463,151; which are hereby incorporated by reference in their entirety.
Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins, but are capable of expressing human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells at random or by homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells. Introduction of human immunoglobulin loci by homologous recombination can render the mouse heavy and light chain immunoglobulin genes independently or simultaneously non-functional. Specifically, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to produce homozygous progeny that express the human antibody. Transgenic mice are immunized in a normal manner with an antigen of choice, e.g., all or a portion of CA 125/0772P, such as a cell-associated CA 125/0772P polypeptide. Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. The transgenic mice contain human immunoglobulin transgenes that rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using this technique, it is possible to produce IgG, IgA, IgM, and IgE antibodies that are useful for therapy. For an overview of this technology for the production of human antibodies, see Lonberg et al, int. Immunol.13(1): 65-93(1995). A detailed discussion of this technology and protocols for producing human antibodies and human monoclonal antibodies are found, for example, in U.S. patent nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,939,598, 6,075,181, 6,091,001, 6,114,598, 6,150,584, and 6,162,963, all of which are incorporated herein by reference in their entirety. In addition, companies such as Abgenix, inc. (Freemont, CA) and Genpharm (San Jose, CA) provide human antibodies to selected antigens using techniques similar to those described above.
Fully human antibodies recognizing selected epitopes can be generated using a technique known as "guided selection". In this method, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope. See, for example, Jespers et al Bio/Technology.12(4)：899-903(1994)。
Furthermore, using techniques well known to those skilled in the art, antibodies that specifically bind to an antigen can in turn be used to generate anti-idiotypic antibodies that "mimic" the antigen, and from which anti-idiotypic antibodies that bind to the antigen can be prepared. See, for example, Greenspan et al, FASEB J. 7(5): 437 444 (1993); and Nisonoff, j.147(8)：2429-2438(1991)。
5.11 recombinant expression of antibodies and polypeptides
Antibodies, antigen-binding antibody fragments, fusion polypeptides or the like that preferentially bind cell-associated CA 125/0772P can be recombinantly expressed using expression vectors containing antibodies, antigen-binding antibody fragments, fusion polypeptides or the like encoding the present invention. Once a polynucleotide encoding an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog of the present invention is obtained, a vector for producing the antibody, antigen-binding antibody fragment, fusion polypeptide, or analog can be generated by recombinant DNA techniques using techniques well known in the art. See, for example, U.S. Pat. nos. 4,816,567, 5,545,405, and 6,331,415, each of which is incorporated herein by reference in its entirety.
Methods well known to those skilled in the art can be used to construct expression vectors containing antibody, antigen-binding antibody fragment, fusion polypeptide or analog coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody of the invention, an antigen-binding antibody fragment of the invention, a fusion polypeptide or analog of the invention, a heavy or light chain of an antibody, a variable domain of a heavy or light chain of an antibody or a portion of such a domain, or a CDR of a heavy or light chain, operably linked to a promoter. These vectors may further include nucleic acid sequences encoding the constant regions of the antibody molecule (see, e.g., EP 216,846, EP 323,997, and U.S. Pat. No. 5,122,464), and the variable domains of the antibody may be cloned into such vectors to express the entire heavy chain, the entire light chain, or both.
The expression vector is transferred to a host cell by conventional techniques and the transformed or transfected cell is cultured by conventional techniques under conditions conducive to, or permitting, production of the antibody, antigen-binding antibody fragment, fusion polypeptide, or the like of the invention. Thus, the invention includes a host cell comprising a vector or polynucleotide encoding an antibody, antigen-binding antibody fragment, fusion polypeptide or analog of the invention or a fragment thereof, or a heavy or light chain thereof, or a portion thereof, or a single chain antibody of the invention, the polynucleotide molecule being operably linked to a heterologous promoter. In a preferred embodiment of diabody expression, vectors encoding the heavy and light chains may be co-expressed in a host cell to express the intact immunoglobulin molecule, as described in detail below.
Various host expression vector systems can be used to express the antibodies, antigen-binding antibody fragments, fusion polypeptides, or the like of the present invention (see, e.g., U.S. patent No. 5,807,715). These host expression systems represent vectors by which the coding sequences of interest can be produced and subsequently purified, and also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibodies, antigen-binding antibody fragments, fusion polypeptides or analogs of the invention in situ. These host expression systems include, but are not limited to, microorganisms such as bacteria (e.g., E.coli or Bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody, antigen-binding antibody fragment, fusion polypeptide or analog coding sequences; yeast (e.g., saccharomyces cerevisiae, pichia pastoris, or pichiaottianolic) transformed with a recombinant yeast expression vector containing coding sequences for antibodies, antigen-binding antibody fragments, fusion polypeptides, or analogs; insect cell systems transfected with recombinant viral expression vectors (e.g., baculovirus) containing antibody, antigen-binding antibody fragment, fusion polypeptide, or analog coding sequences; plant cell systems transfected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmids) containing antibody, antigen-binding antibody fragments, fusion polypeptide, or analog coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO or 3T3 cells) containing a promoter from the genome of a mammalian cell (e.g., the metallothionein promoter) or a recombinant expression construct from a mammalian virus (e.g., the adenovirus late promoter or the vaccinia virus 7.5K promoter). Preferably, bacterial cells, such as e.coli, more preferably eukaryotic cells, in particular for expressing intact recombinant antibody molecules, are used for expressing recombinant antibody molecules. For example, mammalian cells such as Chinese Hamster Ovary (CHO) cells in combination with a vector such as the major intermediate early gene promoter element from human cytomegalovirus are efficient expression systems for antibodies (Foecking et al, Gene.45 (1): 101-105(1986) and Cockett et al, Bio/technology.8 (7): 662-667 (1990)). In particular embodiments, a constitutive promoter, an inducible promoter, a cell-type or tissue-specific promoter regulates expression of a nucleotide sequence encoding an antibody, antigen-binding antibody fragment, fusion polypeptide, or analog of the present invention.
In bacterial systems, a number of expression vectors may be advantageously selected depending on the use of the antibody, antigen-binding antibody fragment, fusion polypeptide or the like to be expressed. For example, when large amounts of such proteins are to be produced to produce pharmaceutical compositions of antibody molecules, it is desirable that vectors which direct high levels of expression of the protein product be readily purified, wherein the protein product can be readily purified. These vectors include, but are not limited to, the E.coli expression vector pUR278(Ruther et al, EMBO J.2(10): 1791-1794(1983)), wherein the antibody coding sequence may be ligated separately to the vector and in frame with the Lac Z coding region to generate a fusion protein; pIN vector (Inouye et al, Nucleic Acids Res.13(9): 3101-; van Heeke et al, j.biol.chem.264(10): 5503-5509 (1989)); and so on. pGEX vectors can also be used to express foreign polypeptides as a complex with glutathione 5-transferase (GST) (Hakes et al, anal.202(2): 293-298 (1992)). Typically, these fusion proteins are soluble and can be easily purified from lysed cells by adsorbing and binding the fusion protein to a matrix glutathione agarose bead, followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene can be released from the GST moiety.
In the insertion system, Autographa californica karyotype virus (AcNPV) was used as a vector to express foreign genes. The virus was grown in Spodoptera frugiperda (Spodoptera frugiperda) cells. The antibody coding sequence can be cloned separately into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter). See, e.g., Kumar et al, biosci.19(3)：227-234(1999)。
In mammalian host cells, a number of viral-based expression systems can be utilized. When an adenovirus is used as an expression vector, the coding sequence for the antibody, antigen-binding antibody fragment, fusion polypeptide, or the like can be linked to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. The chimeric gene is then inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., regions E1 or E3) will result in a recombinant virus that is viable and capable of expressing antibody molecules in an infected host (see, e.g., Logan et al, Proc. Natl. Acad. Sci. USA.81 (12): 3655-3659 (1984)). Specific initiation signals may also be required for efficient translation of the inserted coding sequence. These signals include the ATG initiation codon and adjacent sequences. In addition, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins (both natural and synthetic). Expression efficiency can be enhanced by including suitable transcription enhancing elements, transcription terminators, and the like (see, e.g., Bittner et al, Methods enzymol.153: 516-544 (1987)).
In addition, strains of host cells which regulate the expression of the inserted sequences or modify and process the gene product in the desired manner can be used. These modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may be important for the function of the protein. Post-translational processing and modification of proteins and gene products of different host cells has characteristic and specific mechanisms. Appropriate cell lines or host systems may be selected to ensure proper modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells can be used whose cellular machinery allows correct processing of the initial transcription product, correct glycosylation and phosphorylation of the gene product. These mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (murine myeloma cell line that does not endogenously produce any immunoglobulin chain), CRL7030 and HsS78Bst cells.
For long-term, high-yield production of recombinant proteins, stable expression of the protein is preferred. For example, cell lines that stably express the antibody molecule can be engineered. Rather than transforming a host cell with a DNA under the control of appropriate expression control elements (e.g., promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.) and appropriate selectable markers, an expression vector containing a viral origin of replication is used. After introduction of the foreign DNA, the engineered cells can be grown in enrichment medium for 1-2 days and then switched to selection medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows the cells to stably integrate the plasmid into their chromosome and grow to form foci which in turn can be cloned and expanded into cell lines. The method can be advantageously used to engineer cell lines that stably express the antibody molecule.
Many selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al, Cell).11(1): 223- & ltwbr/& gt232 (1977)), hypoxanthine guanine phosphoribosyltransferase (Spring et al, biochim. biophysis. acta).2118(2): 158-.22(3): 817-823(1980)) gene can be used for tk-, hgprt-or aprt-cells, respectively. Furthermore, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, proc.natl.acad.sci.usa).77(6): 3567-3570 (1980); o' Hare et al, proc.natl.acad.sci.usa.78(3): 1527-1531 (1981)); gpt, which confers mycophenolic acid resistance (Mulligan et al, proc.natl.acad.sci.usa.78(4): 2072 and 2076 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu et al, Biotherapy.3(1)：87-95(1991)；Tolstoshev，Ann.Rev.Pharmacol.Toxicol.33：573-596(1993)；Mulligan，Science.260(5110): 926-; and Morgan et al, ann.rev.biochem.62: 191-217 (1993)); and hygro, which confers hygromycin resistance (Santerre et al, Gene)30(1-3): 147-156(1984)). Methods of recombinant DNA technology well known in the art are routinely used to select the desired recombinant clone and are described, for example, in Current Protocols in Molecular Biology, Ausubel et al, eds, John Wiley & Sons(1989-2002)；Kriegler，Gene Transfer and Expression, A Laboratory Manual, StocktonPress (1990); chapter 12 and 13 in Current Protocols in Human Genetics, Dracopoli et al, eds., John Wiley&Sons (1994); and colber-Garapin et al, j.mol.biol.150(1): 1-14(1981), which are herein incorporated by reference in their entirety.
Expression levels of antibodies, antibody fragments that bind antigens, or fusion polypeptide molecules can be increased by vector amplification (for review, see Bebbington and Hentschel, using vectors based on gene amplification to express cloned genes in mammalian cells, DNA cloning, volume 3(Academic Press 1987)). When the marker in the antibody-expressing vector system is amplifiable, an increase in the level of inhibitor present in the host culture will increase the copy number of the marker gene. Since the amplified region binds to the antibody gene, the antibody production will also increase (Crouse et al, mol.3(2)：257-266(1983))。
Host cells may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived from a polypeptide and the second vector encoding a light chain derived from a polypeptide. Both vectors may contain the same selectable marker that allows for equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes and is capable of expressing both the heavy and light chain polypeptides. In this case, the light chain should be placed before the heavy chain to avoid non-toxic heavy chain excess (Proudfoot, Nature.322 (6079): 562) -565 (1986); and Kohler, Proc. Natl. Acad. Sci. USA). 77(4): 2197-2199(1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA, or a combination thereof.
Once the antibody, antigen-binding antibody fragment, fusion polypeptide, or analog of the invention is produced by recombinant expression, the antibody, antigen-binding antibody fragment, fusion polypeptide, or analog can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific CA125/0772P antigen after initial purification with protein a, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. In addition, the antibodies or fragments thereof of the present invention may be fused to heterologous polypeptide sequences described herein or otherwise known in the art for ease of purification.
The following examples are intended to illustrate and not to limit the scope of the invention.
6. Examples of the embodiments
The results provided herein demonstrate that the extracellular portion of CA125/0772P remains in a cell-associated form following release of a portion of the CA125/0772P polypeptide as shed CA 125/0772P. Specifically, the presence of cell-associated CA125/0772P was demonstrated by the generation and characterization of several antibodies that preferentially bind to cell-associated CA125/0772P species relative to shed CA125/0772P species. In addition to the preferred binding, the results provided herein indicate that the antibodies exhibit high specificity for CA125/0772P and high affinity for the cell-associated CA125/0772P antigen. In addition, the results provided herein indicate that these antibodies may function to mediate the lysis of CA 125/0772P-positive tumor cells.
6.1 antibody production
The experiments provided herein describe the production of monoclonal antibodies against the extracellular portion of CA 125/0772P. As illustrated in the subsequent subsection, antibodies produced by these techniques include antibodies that are specific for CA125/0772P, preferentially bind cell-associated CA125/0772P, exhibit high affinity for cell-associated CA125/0772P, and may function to mediate lysis of CA 125/0772P-positive tumor cells.
Antigen and antigen-expression constructs:
an expression construct was generated to express the CA125/0772P antigen for antibody production. The first antigen to be expressed was designated as the 0772P 3-repeat (FIG. 1; SEQ ID NO: 1), which included the carboxy-terminal three tandem repeats of the extracellular domain of CA125/0772P up to, but not including, the CA125/0772P transmembrane sequence. The second antigen to be expressed was designated 0772P 3-repeat TM (FIG. 2; SEQ ID NO: 2), which includes the carboxy-terminal three tandem repeats of the extracellular domain of CA125/0772P and the transmembrane and cytoplasmic sequences depicted in FIG. 2. Specifically, the sequence encoding each antigen was subcloned into the pSecTag2B vector (Invitrogen). The vector encodes an Ig kappa signal sequence for secretion and myc and 6xhis tags for detection and purification of the expressed protein.
Antigen expression:
transient transfection of suspension CHO-KI cells with the constructs described above resulted in recombinant antigen production. The medium used for transfection was prochocmd (BioWhittaker inc. walker, MD) supplemented with GS (JRH Biosciences Lenexa, KS). To generate 1 liter of material, 2mg of transfection reagent Clonefectin was addedTM(Clontech, Palo Alto, Ca) rehydrated, diluted into 24ml transfection medium and incubated with 125. mu.g DNA in the same medium for 15 minutes. The transfection mixture was added to 450ml of a solution containing 1.25X 109CHO-KI cells were suspended in transfection medium and incubated for 4 hours at 37 ℃ on a rail-bound shaker. After incubation, the transfected cells were added to 500ml of ProCHO 4-CDM containing GS supplement, penicillin-streptomycin, and 10% ultra-low IgGFBS (Life Technologies Rockville, Md.), and the cultures were transferred to shake flasks. Samples were collected on day 3 and cultures were harvested on day 7.
Antigen purification:
the transfection supernatant was concentrated to 250ml using a 50K cut-off membrane with a Millipore Pellicon system. 2ml of TALON resin (Clontech, Palo Alto, Calif.) obtained from TALON purification kit (Cat. No. K1253-1) was transferred to a 2ml column and extracted with 20ml of 1 × Wash/extraction buffer (supplied with kit, pH 7.0). The concentrated sample was then applied to the column at a flow rate of 1 ml/min. The column was washed with 15ml extraction/wash buffer. Bound protein was then eluted with 4X 1ml of elution buffer (50mM sodium phosphate, 300mM NaCl, 150mM imidizole, pH 7.0). Half-ml fractions were collected and analyzed by SDS-PAGE and visualized using Coomassie Brilliant blue G-250. The fraction containing 0772P 3-repeat recombinant protein was further purified by ConA Sepharose chromatography. 1ml of ConASepharose (Vector Laboratories, Inc., cat # AC-1003, lot # K0425) was transferred to a 15ml conical centrifuge tube and washed with 10ml of 1 XPS buffer (PBS), pH 7.2. The wash buffer was removed by centrifugation. The fraction from TALON purification containing 0772P 3-repeat protein was diluted 1: 1 with 1 XPBS (pH 7.2) and added to washed ConA Sepharose and spun overnight at 4 ℃. The resin slurry was then transferred to a 5ml gravity flow column and washed with 10ml 1 XPBS, pH 7.2. The sample was eluted with 0.6M methyl α -D-mannopyranoside in 1 XPBS, pH 7.2, in a volume of 6ml each fraction of 0.5 ml. Fractions were analyzed by SDS-PAGE and stained with Coomassie Brilliant blue G-250. Fractions containing 0772P 3-repeat protein were pooled and dialyzed against 2L 1 XPBS, pH 7.2, and stored at 4 ℃.
Immunization:
BALB/c mice were immunized intraperitoneally (i.p.) on days 0, 21, 42, and 63. The first injection used NIH: OVCAR-3(ATCC HTB-161), subsequent injections used a 0772P 3-repeat without a transmembrane domain. Complete Freund's adjuvant was used for the first protein injection and incomplete Freund's adjuvant was used for the remaining injections. Sera were collected on days 35, 56 and 77 and analyzed by ELISA and by flow cytometry as described below. Mice with the best serum titers were selected for cell fusion. On the first and second days prior to fusion, selected mice were boosted intraperitoneally and intravenously (i.v.) with mammalian-expressed 0772P 3-repeat protein. Mice were boosted intravenously the day before fusion.
Hybridoma production:
the mouse spleen was removed and splenocytes harvested by cutting with forceps and filtered through a mesh. Cells were washed twice in IMDM medium and cell counts were performed. P3x63ag8.653 mouse myeloma cells (ATCC CRL-1580) at logarithmic growth phase were harvested, washed twice in IMDM medium and the cells counted. Spleen cells and bone marrowTumor cells were mixed together at a ratio of 5: 1 and centrifuged at 200 Xg for 5 minutes. After aspiration, the bottom of the tube was tapped to loosen the pellet. 1ml of 50% PEG (molecular weight 1450) solution was added dropwise over 30 seconds, and the precipitate was gently mixed with a pipette for 30 seconds. The resulting suspension was allowed to stand for an additional 30 seconds. 5ml of IMDM were added over 90 seconds, followed immediately by 5ml more. The resulting cell suspension was allowed to stand for 5 minutes. After centrifugation, the cells were resuspended in HAT medium (IMDM containing 10% FBS, 2mM L-glutamine, 0.6% 2-mercaptoethanol (0.04% solution), hypoxanthine, aminopterin, thymidine, and 10% ORIGENO hybridoma cloning factor (IGEN International, Gaithersburg, Md.) to a concentration of 5X 10 5At 1X 10 cells/ml5Individual cells/well were plated in 96-well plates. 96-well plates were incubated at 37 ℃ with 7% CO2Incubate in air with 100% humidity. 7 days after fusion, the medium was removed and replaced with IMDM containing 10% FBS, 2mM L-glutamine, 0.6% 2-mercaptoethanol stock solution (0.04% solution), hypoxanthine, and thymidine. 10 to 14 days after fusion, supernatants were removed from wells containing growing hybridoma colonies and tested for binding to CA125/0772P as described herein.
Antibody purification from hybridoma supernatants:
0.5ml of protein G resin (Sigma, St. Louis, MO) was loaded into a 5ml disposable column (Bio-Rad, Hercules, Calif.). The column was pre-equilibrated with 20ml binding buffer (20mM PBS, pH 7.0). The hybridoma supernatant was added to the column at a flow rate of less than 0.5 ml/min. The column was then washed with 20ml of binding buffer at a flow rate of 1 ml/min. Alternatively, a pre-packed protein G column (Amersham Pharmacia Biotech) was used. The antibody was then eluted with 3ml of elution buffer (0.1M glycine, pH 2.7). The 0.5ml fractions were collected into 1ml tubes containing 50. mu.l of 1M Tris, pH 9.0. Samples were dialyzed against PBS (0.5L) and concentrated to about 1mg/ml protein.
Antibody concentration in hybridoma supernatant:
The concentration of antibody was determined using Easy-Titer mouse I gG assay kit (Pierce Biotechnology, Rockford, IL). Briefly, mouse IgG whole molecule standards (Pierce Biotechnology, Rockford, IL) were diluted to 500ng/ml with dilution buffer (provided with kit). The standard was serially diluted 6 times 1: 2 in dilution buffer to generate a standard curve. 20 μ l of each standard was added to the corresponding well of a 96-well plate. Dilutions of hybridoma supernatant (20. mu.l) were also added to the plates. Duplicate wells were made for each standard and sample. Add 20. mu.l of polystyrene beads to each well (provided with the kit), mix the samples, seal the plates, and incubate the plates on a plate shaker for 5 minutes at room temperature. 100 μ l blocking reagent from the kit was then added to each well. The plate was again shaken at room temperature for 5 minutes. The absorbance at 405nm was then read on a Vmax plate reader (Molecular Devices corp., Sunnyvale, CA) and a 4-parameter fit was used to generate a standard curve.
6.2 CA125/0772P specificity
The results presented here indicate that the preparation of antibodies by the techniques described above results in the production of antibodies specific for CA 125/0772P.
ELISA specific assay
The method comprises the following steps:
The 96-well plate was dissolved in sodium bicarbonate buffer (0.2 MNa) with 100. mu.l (per well)2CO3/NaHCO3pH 9.6, Sigma) of 0772P 3-repeat protein (seq id NO: 1) (affinity purified) at 4 degrees C overnight incubation and coating. The following day, plates were washed three times with 200. mu.l 1 XPBST (1 XPBS buffer (PBS), 0.05% Tween 20) and blocked with 100. mu.l 1 XPBST containing 1% Bovine Serum Albumin (BSA) for 2 hours at 37 ℃. After washing the plates three times with 1 XPBST, murine anti-CA 125/0772P selected hybridoma-produced antibody (0.04 mg/. mu.l) was added to the plates (single well). After incubation for 1 hour at 37 ℃, plates were washed three times with 1 × PBST. To detect the signal, 100. mu.l of HRP (horseradish peroxidase) -conjugated sheep anti-mouse IgG (1: 2000 diluted to 1 XPBST + 1% BSA; Amarsham biosciences) was added to each well and incubated for 1h at 37 ℃. Plates were washed again 3 times with 1 × PBST. Finally, TMB (3, 3 ', 5, 5' -tetramethylbenzidine) substrate and H2O2 (1: 1 ratio, KPLKirkguard Perry) were added to each wellLaboratories) and incubated for 5 minutes, and absorbance was measured at 405nm using a plate reader (Molecular Device corp., Sunnyvale, CA). The antibodies produced by each 0772P hybridoma were assayed in triplicate and data collected and analyzed as a kinetic assay, measured over 5 minutes. The average is calculated and given. Blank and control for each reagent were also included in each experiment.
As a result:
table 7 below gives the results of ELISA-specific assays of the antibodies (117.1, 368.1, 501.1, 776.1) produced by the four selected anti-CA 125/0772P hybridomas. The table also shows the results of ELISA-specific assays for two commercially available CA125/0772P antibodies (OC125 and M11, Dako corp., Carpinteria, CA). An antibody (or antigen-binding antibody fragment) is considered positive in this assay (i.e., specific for CA 125/0772P) if it exhibits an absorbance of at least 5 to greater than 30 OD/microgram of antibody. These results indicate that each antibody tested is specific for CA 125/0772P. Note that, as set forth below, while OC125 and M11 are believed to be specific for CA125/0772P, neither preferentially binds cell-associated CA125/0772P over shed CA 125/0772P. SD is standard error.
TABLE 7
Ab name Absorbance (OD) SD Absorbance (OD)/μ gAb
OC125 0.73 0.003 18
M11 0.974 0.008 24
117.1 0.619 0.033 15
368.1 1.293 0.004 32
501.1 0.856 0.005 21
776.1 1.178 0.043 29
The results in table 8 below show the absorbance data for 20 additional antibodies generated using the above technique. As the absorbance data show, each of these antibodies is also specific for CA 125/0772P.
TABLE 8
Ab name Absorbance (OD)/μ gAb
325.1 24
446.1 27
621.1 27
633.1 18
654.1 22
725.1 25
8G9 22
7F10 19
8A1 18
8C3 23
15C9 28
8E3 18
8B5 18
7G10 20
16C7 22
7C6 23
7H1 26
16H9 22
7A11 22
4E7 19
Flow cytometry specific assay:
the method comprises the following steps:
cells (OVCAR-3(ATCC accession number HTB-161), SK-OV3(ATCC accession number HTB-77), NIH/3T3(ATCC accession number CRL-1658), and NIH/3T3 cells transfected with a sequence expressing 0772P 3-repeat protein (SEQ ID NO: 2)) were removed from the plates by digestion with trypsin (0.25%). Cells were counted and viability assessed by trypan blue (0.2%) exclusion. Cells were centrifuged (500 Xg, 5 min) and resuspended in FACS buffer (1 XDPBS containing 1% BSA and 0.1% sodium azide) to a concentration of 5-10X 107Individual cells/ml. Cells were then distributed into 96-well plates at 100. mu.l/well and centrifuged at 500 Xg for 3 min. Antibody supernatants were removed by aspiration, 50 μ l hybridoma supernatants were diluted to 1 μ g/ml to 0.5 μ g/μ l in FACS buffer and added to each well containing cells. Murine IgG1 kappa (Sigma, St. Louis, MO) (2.0, 1.0, 0.5, 0.1. mu.g/. mu.l) was included as negative controls, and OC125 and M11(DAKO Corp, Carpinteria, CA) were included as positive controls. Plates were incubated at 4 ℃ for 30 minutes with shaking. The cells were then washed twice with FACS buffer (200 μ Ι/well), centrifuged and buffer aspirated after each wash). Goat anti-mouse IgG (Fc) -biotin (Sigma, St. Louis, Mo.) was diluted 1: 1000 in FACS buffer and 50. mu.l was added to each well containing cells. Incubate the plate at 4 ℃ for 30 minutes with shaking. The cells were then washed with FACS buffer as above. streptavidin-Alexa-Four 488(Molecular Probes, Eugene, OR) was diluted 1: 1000 into FACS buffer and 50. mu.l was added to each well containing cells. Incubate the plate at 4 ℃ for 30 minutes with shaking. The cells were then washed with FACS buffer as above. The cells were then resuspended in 1ml FACS buffer and transferred to Falcon 2052 tubes and plated in Becton-Dick The inson immune cytometry system was analyzed on a FACSCalibur flow cytometer (San Jose, CA).
As a result:
table 9 below gives the results of flow cytometry specific assays obtained from the antibodies (117.1, 368.1, 501.1, 776.1) produced by the four selected anti-CA 125/0772P hybridomas. The table also shows the results of flow cytometry specific assays for OC125 and M11 that are commercially available. NIH/3T3 cells and SK-OV3 cells (an ovarian cancer cell line) were considered negative controls because they did not produce CA 125/0772P.
If the antibody (or antigen-binding antibody fragment) shows a flow cytometry specific assay result in the following positive cell range: less than about 5% positive NIH/3T3 cells, and at least about 60% positive NIH/3T3 cells produce SEQ ID NO: 2 a polypeptide; or less than about 25% positive SK-OV3 cells and at least about 80% positive OVCAR-3 cells, then these antibodies (or antigen-binding antibody fragments) are considered positive (i.e., specific for CA 125/0772P) (nd-undetermined).
These results indicate that each of the antibodies tested is specific for CA 125/0772P. Note that, as set forth below, while OC125 and M11 were thought to be specific for CA 125/0772P, neither of them preferentially bound cell-associated CA 125/0772P over shed CA 125/0772P.
TABLE 9
Antibodies % Positive-NIH/3T 30772P 3-repeats % positive-NIH/3T 3 % positive-OVCAR-3 % positive SK-OV3
OC125(1μg/ml) nd nd 98 16
OC125(0.1μg/ml) nd nd 85 10
M11(1μg/ml) 84 0.1 98 17
M11(0.1μg/ml) nd nd 91 11
117.2(2μg/ml) 83 0.1 93 6
117.2(0.5μg/ml) 63 0 nd nd
368.1(2μg/ml) 86 0.2 89 5
368.1(0.5μg/ml) 75 0.2 nd nd
501.1(2μg/ml) 89 0 95 5
501.1 85 0.2 nd nd
(0.5μg/ml)
776.1(2μg/ml) 86 0 94 9
776.1(0.5μg/ml) 84 0 nd nd
The results in Table 10 below, which give OVCAR-3/SK-Oy3 data for 20 additional antibodies generated using the above technique, indicate that these antibodies are also specific for CA 125/0772P.
Watch 10
Ab name % Positive OVCAR-3 (0.5. mu.g/ml) % positive SK-OV3 (2.0. mu.g/ml)
325.1 98 6
446.1 94 5
621.1 97 9
633.1 89 9
654.1 86 8
725.1 96 10
8G9 97 4
7F10 96 3
8A1 97 3
8C3 97 3
15C9 95 3
8E3 95 1
8B5 94 1
7G10 96 2
16C7 96 3
7C6 96 3
7H1 96 0
16H9 96 3
7A11 94 1
4E7 94 2
6.3 Competition assay demonstrates successful production of antibodies that preferentially bind CA 125/0772P
The results presented herein indicate that antibodies produced by the techniques described above can produce antibodies that preferentially bind cell-associated CA 125/0772P over shed CA 125/0772P. The fact that antibodies could be produced also suggests for the first time that there was a cell-associated CA 125/0772P polypeptide, i.e. that after release of a portion of CA 125/0772P polypeptide as shed CA 125/0772P, the extracellular portion of CA 125/0772P remained in a cell-associated form, however, this retention was transient.
ELISA competition assay:
the method comprises the following steps:
the 96-well plate was dissolved in sodium bicarbonate buffer (0.2 MNa) with 100. mu.l (per well)2CO3/NaHCO3pH 9.6, Sigma) of 1 μ g/ml 0772P 3-repeat (SEQ id no: 1) the polypeptide (affinity purified) was coated overnight at 4 ℃. The following day, plates were washed three times with 200. mu.l of 1 XPBST (1 XPBST buffer (PBS), 0.05% Tween 20) and blocked with 100. mu.l of 1 XPBST containing 1% Bovine Serum Albumin (BSA) for 2 hours at 37 ℃. After washing the plate three times with 1 XPBST, CA 125/0772P (Fitzgerald Industries International, Concord, MA; Scripps Laboratories, La Jolla, CA; and/or United State Stat) has been shed with excess (e.g., 10-50 times w/w) Biological Corp.) pre-incubated wells were added with the selected anti-CA 125/0772P hybridoma-produced antibody at the indicated concentration (e.g., 0.04 μ g/ml). After incubation for 1 hour at 37 ℃, plates were washed three times with 1 × PBST. To detect the signal, 100. mu.l of HRP-conjugated sheep anti-mouse IgG (1: 2000 diluted 1 XPBST + 1% BSA; Amarsham biosciences) was added to each well and incubated for 1h at 37 ℃. Plates were washed again 3 times with 1 × PBST. Finally, TMB substrate and H were added to each well2O2100 μ l of the mixture (1: 1 ratio, KPL) and absorbance at 405nm using a plate reader (Molecular Device Corp., Sunnyvale, Calif.). Each selected antibody was assayed in triplicate, calculated and the average given. Percent inhibition compared to no competition was calculated for the respective antibodies based on the mean values. Blank and control for each reagent were also included in each experiment.
As a result:
table 11 below gives the results of ELISA competition assays for the four selected anti-CA 125/0772P hybridoma-produced antibodies (117.1, 368.1, 501.1, 776.1). The table also shows the results of an ELISA competition assay for the commercially available CA125/0772P antibody (OC 125; Dako Corp., Carpinteria, Calif.). An antibody (or antigen-binding antibody fragment) is considered positive in this assay (i.e., preferentially binds cell-associated CA 125/0772P) if binding of the antibody (or antigen-binding antibody fragment) is less than about 25% inhibition in the presence of a 25-fold (w/w) excess of shed CA 125/0772P. These results indicate that each of antibodies 117.1, 368.1, 501.1, and 776.1 preferentially binds to cell-associated CA 125/0772P. These results also indicate that OC125 antibody is not able to preferentially bind cell-associated CA 125/0772P. (SD-Standard error)
TABLE 11
Antibodies Absorbance of the solution SD Absorbance w/Absorbance of CA125/0772P competitor (25 fold w/w excess) SD Percent inhibition of binding Absorbance w/0772P 3-repeat competitor (10 Xw/w excess) SD Percent inhibition of binding
OC125 0.73 0.003 0.074 0.001 95 0.047 0.002 99
117.1 0.619 0.033 0.554 0.007 11 0.071 0.001 94
368.1 1.293 0.004 1.333 0.009 0 0.915 0.016 30
501.1 0.856 0.005 0.735 0.008 15 0.065 0.002 96
776.1 1.178 0.043 0.977 0.01 17 0.077 0.001 96
The results presented in table 12 below give CA125/0772P competitor data for 20 additional antibodies generated by the above technique, indicating that these antibodies also preferentially bind cell-associated CA 125/0772P.
TABLE 12
Ab name % inhibition binding to w/shedding CA125/0772P competitor (25-fold excess)
325.1 2
446.1 7
621.1 2
633.1 7
654.1 9
725.1 7
8G9 7
7F10 6
8A1 8
8C3 5
15C9 5
8E3 4
8B5 6
7G10 3
16C7 3
7C6 2
7H1 4
16H9 0
7A11 5
4E7 7
Flow cytometry competition assay:
the method comprises the following steps:
mixing the components of NIH: OVCAR-3(ATCC accession number HTB-161) cells were removed from the plates by digestion with trypsin (0.25%). Cells were counted and viability assessed by trypan blue (0.2%) exclusion. Cells were centrifuged (500 Xg, 5 min) and resuspended in FACS buffer (1 XDPBS containing 1% BSA and 0.1% sodium azide) to a concentration of 5-10X 107Individual cells/ml. Cells (100. mu.l/well) were then distributed into 96-well plate round bottom plates and centrifuged at 500 Xg for 3 min. The supernatant was removed by aspiration and 50. mu.l of hybridoma supernatant was diluted to 0.2. mu.g/ml in FACS buffer. CA125 (Fizgerald industries International, Concord, Mass.) was diluted to 1000. mu.g/ml, 500. mu.g/ml, 200. mu.g/ml, 60. mu.g/ml, 20. mu.g/ml, 6. mu.g/ml, or 2. mu.g/ml in FACS buffer. Mu.l of the antibody solution was incubated with 30. mu.l of diluted CA125 or buffer only for 30 minutes at 4 ℃. To each well containing cells, 50. mu.l of the mixture was added. Murine IgG1 κ (Sigma, st. louis, MO) and M11(DAKO Corp, Carpinteria, CA) were included as negative and positive controls, respectively. The plates were incubated at 4 ℃ for 30 minutes with shaking. The cells were then washed twice with FACS buffer (200 μ Ι/well), centrifuged and the buffer aspirated after each wash. Goat anti-mouse IgG (Fc) -biotin (Sigma, St. Louis, Mo.) was diluted 1: 1000 in FACS buffer and 50. mu.l was added to each well containing cells. Incubate the plate at 4 ℃ for 30 minutes with shaking. The cells were then washed with FACS buffer as above. streptavidin-Alexa-Four 488(Molecular Probes, Eugene, OR) was diluted 1: 1000 into FACS buffer and 50. mu.l was added to each well containing cells. Incubate the plate at 4 ℃ for 30 minutes with shaking. The cells were then washed with FACS buffer as above. Cells were resuspended in 1ml FACS buffer and transferred to Falcon 2052 tubes and analyzed on a Becton-Dickinson immune cytometry system FACSCalibur flow cytometer (San Jose, CA). Percentage positive cells were plotted as a function of CA125/0772P concentration using GraphPad plotting software. By linear loops Determination of IC by regression analysis50，IC50Expressed as the concentration of shed CA125/0772P at 50% inhibition of binding.
As a result:
FIG. 3 shows a representative plot of shed CA125/0772P concentration versus percent positive cells for the 117.1 antibody and M11 antibody controls (squares).
Table 13 below gives an overview of the results of the flow cytometry competition assay. An antibody (or antigen-binding antibody fragment) is considered positive (i.e., considered to preferentially bind cell-associated CA 125/0772P) if the antibody (or antigen-binding antibody fragment) exhibits an IC50 (as measured by percentage positive cells) of at least about 0.05mg/ml shed CA 125/0772P.
The results shown in table 13 below indicate that each of the 117.1, 501.1, 776.1, 8C3, 16H9, 325.1, 633.1, and 725.1 antibodies preferentially binds cell-associated CA 125/0772P. Note that the results in table 13 also indicate that OC125 and M11 antibodies do not preferentially bind cell-associated CA 125/0772P.
Watch 13
Antibodies IC of% Positive cells(mg/ml CA125) function
OC125 0.005
M11 0.01
117.1 ＞1.0
368.1 nd
501.1 0.13
776.1 0.19
8C 3 ＞0.5
16H9 ＞0.5
325.1 0.36
621.1 ＞0.5
633.1 0.18
725.1 0.42
446.1 nd
654.1 nd
8G9 nd
7F10 nd
8A1 nd
15C9 nd
8E3 nd
8B5 nd
7G10 nd
16C7 nd
7C6 nd
7H1 nd
7A11 nd
6.4 affinity assay
The results presented herein indicate that antibodies generated that preferentially bind CA125/0772P include antibodies that exhibit high affinity for cell-associated CA 125/0772P.
BIAcore affinity assay: the method comprises the following steps:
The GM5 BIAcore biosensor chip was docked into the BIAcore X instrument and activated with 55. mu.l of 1: 1 NHS/EDC at room temperature. 10. mu.g/ml 0772P3 repeat region protein and BSA dissolved in 0.05M acetate buffer (pH 4.5) were immobilized on the Flow Cell (FC)1 and FC2 of the activated chip, respectively, at a flow rate of 5. mu.l/min to achieve a resonance reaction of 1000-2000 RU. The chip was blocked by injecting 55. mu.l of ethanolamine-HCl (pH 8.5), and then washed 5 times with 50mM NaOH-1M NaCl. To measure the binding of anti-0722P mAbs to the 0772P3 repeat region immobilized to the chip, 30. mu.l of anti-0772P in various concentrations of BIAcore running buffer (HBS-EP, Cat. #1001-080, BIAcore, Piscataway, NJ) was injected onto the sensor surface at a flow rate of 5. mu.l/min. After completion of the injection period, dissociation was monitored in BIAcore running buffer at the same flow rate for 360 seconds. The surface was regenerated between two injections with 30. mu.l of 50mM NaOH-1M NaCl. The respective sensorgrams were analyzed using BIAevaluation (sensorgram).
BIAcore affinity assay: as a result:
table 14 below gives a summary of BIAcore affinity assays for the 117.1, 368.1, 501.1 and 776.1 antibodies, as well as the M11 and OC125 antibodies. As shown in the table, each of antibodies 117.1, 368.1, 501.1, 776.1, 4E7, 7C6, 7F10, 7G10, 7H1, 8a1, 8B5, 8C3, 8E3, 15C9, 16C7, 16H9, 325.1, 621.1, 633.1, and 725.1 has high affinity for binding to the CA 125/0772P polypeptide.
TABLE 14
Antibodies K(nM)
M11 1.6
OC125 4
117.1 12
368.1 0.7
501.1 70
776.1 0.4
4E7 30
7A11 nd
7C6 73
7F10 3.7
7G10 47
7H1 69
8A1 2.8
8B5 32
8C3 5.0
8E3 33
8G9 14
15C9 14
16C7 44
16H9 3.9
325.1 15
446.1 nd
621.1 40
633.1 26
654.1 190
725.1 2.6
6.5 functional assay:
the results presented herein indicate that the antibodies generated that preferentially bind cell-associated CA 125/0772P include antibodies that function to mediate lysis of CA 125/0772P-positive tumor cells.
ADCC assay:
the method comprises the following steps:
human leukocytes were isolated from peripheral blood of normal donors by Histopaque-1077 gradient centrifugation method (Sigma co., st. louis, MO) and used as effector cells. OVCAR-3 target cells (5X 10)3Counts/well) were mixed with Histopaque-purified human leukocytes in a ratio of effector cells to target (E/T) of 12.5: 1 to 50: 1 in the U-shaped bottom of a 96-well plate, with or without various concentrations of monoclonal antibody in the wells, dissolved in RPMI1640 supplemented with 10% FBS, in a total volume of 120. mu.l. Wetting 96-well plates at 37 ℃ with 5% CO2Incubation in air. Target cells and effector cells without the tested antibody were used as negative controls. After 16-18 hours of incubation, 50 μ l aliquots of the culture supernatants were collected and lactate dehydrogenase activity was measured in flat bottom 96 well plates using the Cytotox 96 nonradioactive cytotoxicity assay kit (Promega co., Madison, WI) according to the manufacturer's instructions. Tumors were counted as follows Percentage lysis of cells: % cytotoxicity ═ 100 (experimental release-effector cell spontaneous release-target cell spontaneous release)/(target cell maximal release-target cell spontaneous release). Results are expressed as mean percent lysis ± s.d. of duplicate samples.
As a result: figure 4 shows a representative plot of the antibody concentration (average of 4 different donors) of the percent lysis versus 117.1 antibody. As shown in the figure, the 117.1 antibody mediated lysis of OVCAR-3 ovarian cancer cells in a dose-dependent manner.
And (3) CDC measurement:
OVCAR-3 target cells (2X 10)4One/well) was mixed with diluted (15: 1, 20: 1, 25: 1) human or guinea pig complement in a U-shaped bottom of a 96-well plate with or without various concentrations of antibody in the wells, dissolved in RPMI 1640 supplemented with 10% FBS, in a total volume of 120. mu.l. Wetting 96-well plates at 37 ℃ with 5% CO2Incubation in air. Target cells without added antibody were used as negative controls. After 4 hours of incubation, 50 μ l aliquots of the culture supernatants were collected and lactate dehydrogenase activity was measured in flat bottom 96 well plates using the Cytotox96 nonradioactive cytotoxicity assay kit (Promega co., Madison, WI) according to the manufacturer's instructions. The percentage lysis of tumor cells was calculated as follows: % cytotoxicity ═ 100 (experimental release-effector cell spontaneous release-target cell spontaneous release)/(target cell maximal release-target cell spontaneous release). Results are expressed as mean percent lysis ± s.d. of duplicate samples.
Sequences of antibodies that preferably bind cell-associated CA 125/0772P
The results presented herein provide the amino acid and nucleotide sequences of the variable regions of the 6 monoclonal antibodies described herein: 117.1, 368.1, 501.1, 776.1, 725.1 and 16H9, including CDR sequences.
The method comprises the following steps:
the hybridomas were harvested and centrifuged at 1800rpm for 10 minutes at 4 ℃. To every 1071ml of TRIzol (Invitrogen) was added to each cell and the total RNA was processed. To the direction of200 μ l chloroform was added to 1ml TRIzol reagent, shaken vigorously by hand for 15 seconds and centrifuged at 12,000 Xg for 15 minutes at 4 ℃. The aqueous phase containing the RNA was transferred to a new tube and precipitation was performed by adding 500. mu.l isopropanol to each 1ml of T RIzol reagent used for the initial homogenization. The RNA pellet was washed 1 time with 70% ethanol and briefly air dried before being resuspended in DEPC water. Mu.g of total RNA was treated with 10 units of Calf Intestinal Phosphatase (CIP) at 50 ℃ for 1 hour to remove 5' phosphate. This step removes truncated mRNA and non-mRNA from subsequent steps. Dephosphorylated RNA was treated with 0.5 units of Tobacco Acid Pyrophosphatase (TAP) for 1 hour at 37 ℃ to remove the 5' cap structure from the intact full-length mRNA. GeneRacer RNA Oligo (5'-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3'; SEQ ID NO: 43) was ligated to the 5 ' end of the mRNA using 5 units of T4RNA ligase at 37 ℃ for 1 hour. Using AMV-reverse transcriptase and GeneRacer Oligo dT primer (5' -GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T)) 18-3'; SEQ ID NO: 44) the ligated mRNA was reverse transcribed at 42 ℃ for 1 hour to generate cDNA with known priming sites at the 5 'and 3' ends. The 5 'end was amplified with gene-specific 3' primers (heavy chain 5 '-AYCTCCACACACAGGRRCCAGTGGATAGAC (SEQ ID NO: 45), light chain 5'-GGATACAGTTGGTGCAGCATC-3'(SEQ ID NO: 46)) located in the constant region of the desired gene and GeneRacer 5' primers (5'-CGACTGGAGCACGAGGACACTGA-3'; SEQ ID NO: 47) homologous to GeneRacer RNA Oligo. The PCR reaction was performed on a GeneAmp 9700 PCR system using 2. mu.l of cDNA, following the procedure: 5 min at 94 ℃ followed by 30 sec denaturation of the template at 94 ℃, 30 sec annealing at 55 ℃, 1 min extension at 72 ℃ for 30 cycles, and 7 min at 72 ℃ for the last cycle. The gel target band was purified using Qiagen gel purification kit and cloned using TOPO-4 cloning kit (Invitrogen). The resulting isolated colonies were screened for inserts of the correct size by PCR in a GeneAmp 9700 instrument. PCR was performed by the following steps: the bacteria were lysed at 94 ℃ for 8 minutes, then denatured at 94 ℃ for 30 seconds, annealed at 55 ℃ for 30 seconds, extended at 72 ℃ for 1-4 minutes for 25 cycles, and extended at 72 ℃ for 7 minutes for the last cycle. Primers used for the screening were: sense, 5'-ATTAACCCTCACTAAAGGGA-3' (SEQ ID NO: 48) or 5 ' -TAATACGACTCACTATA GGG-3' (SEQ ID NO: 49), antisense heavy or light chain constant region primers (see above). Positive clones were grown overnight in 4ml medium to expand the clones and subjected to SNAP Miniprep (Invitrogen). Clones were then sequenced in the GeneAmp 9700 PCR system using BigDye (Perkin Elmer) chemistry by denaturing the DNA at 94 ℃ for 10 seconds, annealing the primers at 50 ℃ for 5 seconds (5'-ATTAACCCTCACTAAAGGGA-3' (SEQ ID NO: 50) or 5'-TAATACGACTCA CTATAGGG-3' (SEQ ID NO: 51)), and extending the primers at 72 ℃ for 4 minutes for 25 cycles. The reactions were passed through DyeE × columns (Qiagen) and sequenced on an Applied Biosystems 310 automated DNA sequencer.
As a result:
the nucleic acid sequences of the variable regions of the 6 monoclonal antibodies that preferentially bind cell-associated CA 125/0772P were obtained and these sequences are depicted in fig. 5-10. Specifically, fig. 5A, 6A, 7A, 8A, 9A, and 10A depict the nucleotide sequences encoding the variable light chain regions of monoclonal antibodies 117.1, 368.1, 501.1, 776.1, 725.1, and 16H9, respectively, while fig. 5B, 6B, 7B, 8B, 9B, and 10B depict the nucleotide sequences encoding the variable heavy chain regions of monoclonal antibodies 117.1, 368.1, 501.1, 776.1, 725.1, and 16H9, respectively. The nucleotide sequence encoding the leader sequence is underlined and the nucleotide sequence encoding the CDR sequence is underlined.
The amino acid sequences encoding the variable regions of the 6 monoclonal antibodies that preferentially bind cell-associated CA 125/0772P were obtained and these sequences are depicted in fig. 5-10. Specifically, fig. 5C, 6C, 7C, 8C, 9C, and 10C depict the amino acid sequences of the variable light chain regions of monoclonal antibodies 117.1, 368.1, 501.1, 776.1, 725.1, and 16H9, respectively, while fig. 5D, 6D, 7D, 8D, 9D, and 10D depict the amino acid sequences of the variable heavy chain regions of monoclonal antibodies 117.1, 368.1, 501.1, 776.1, 725.1, and 16H9, respectively. The leader sequence is double underlined and the CDR sequences are underlined. Note that the leader sequence does not become part of the mature antibody and is not considered part of the variable region of the antibody as such.
6.7 Western blot analysis of OVCAR-3 supernatant
The working examples presented herein provide a western blot analysis designed to directly test the ability of antibodies 368.1 or 776.1 to bind to split CA 125/0772P. The data presented here directly demonstrate that neither 368.1 nor 776.1 antibodies recognize the high molecular weight species corresponding to split CA 125/0772P. In contrast, antibodies OC125 and M11 recognized this high molecular weight species, although all antibodies tested strongly bound to the control, recombinant 0772P polypeptide containing the 3-repeat. Thus, these data additionally demonstrate that the 368.1 and 776.1 antibodies preferentially bind to the cell-associated CA 125/0772P polypeptide.
The method comprises the following steps:
media (RPMI supplemented with 10% fetal bovine serum) was removed from the cultured OVCAR-3 cells and replaced with freshly supplemented media. Aliquots of conditioned media were removed at 1 hour, 6 hours, 24 hours, 48 hours, and 72 hours. The supplemented medium was used as time 0 point.
To 4-12% Bis-Tris (Invitrogen) gel 10. mu.l of conditioned media from each time point was added and separated by electrophoresis at 200 volts for 45 minutes. 100ng, 10ng and 1.0ng of purified 0772P 3-repeat polypeptide were added as positive controls.
Proteins were transferred to nitrocellulose membranes at 30 volts for 1 hour and then blocked in non-fat milk overnight at 4 ℃. The primary antibody was adjusted to 400. mu.g/ml with PBS and diluted 1: 1000 with non-fat milk (OC125Dako # M3519, M11 Dako # M3520). The blot was washed three times for 10 minutes each in PBS/Tween. The secondary antibody (anti-mouse IgG Fc-specific, Sigma # B-7410) was diluted 1: 1000 in non-fat milk and incubated for 1 hour at room temperature. Washing was carried out as above. NeutrAvidin-HRP (Molecular Probes # A-2664) was diluted 1: 1000 in PBS/Tween and incubated for 15 min at room temperature. The blot was washed in bulk PBS-Tween and developed by chemiluminescence (30 seconds exposure).
As a result:
to determine whether the 368.1 or 776.1 antibodies bound to the shedding CA 125/0772P polypeptide, western blot analysis was performed on the supernatants of cultured OVCAR-3 cells, which are known to shed CA 125/0772P from their surfaces. This assay tests the ability of the antibody to bind directly to shed CA 125/0772P.
As shown in fig. 11, the results of western blot analysis showed that neither 368.1 nor 776.1 antibodies recognized the high molecular weight species corresponding to exfoliated CA 125/0772P. In contrast, antibodies M11 and OC125 (i.e., antibodies that do not preferentially bind cell-associated CA 125/0772P) recognize this high molecular weight species. All 4 antibodies tested strongly bound to a control-recombinant 0772P polypeptide containing a 3-repeat, i.e. 0772P 3-repeat, which contains the extracellular domain sequence immediately adjacent to the CA 125/0772P transmembrane domain. Thus, these data additionally demonstrate that the 368.1 and 776.1 antibodies preferentially bind to the cell-associated CA 125/0772P polypeptide.
6.8 radiolabeled 776.1 antibody retards tumor growth
The results presented herein indicate that radiolabeled 776.1 antibody successfully delayed tumor growth in an animal model of human ovarian cancer.
The method comprises the following steps:
animal(s) production
Female NCr nu/nu ("nude") mice (Taconic Farms, Germantown, n.y.) 6-7 weeks old were used for all studies. All animals were fed food and water ad libitum.
Tumor cell transplantation
For efficacy studies, the OVCAR-3 human ovarian cancer cell line was used as a model for human ovarian cancer. The OVCAR-3 cell line (Hamilton, et al, Cancer Res.43: 5379-5389(1983)) was derived from human-derived ovarian adenomas and purchased from ATCC (catalog number HTB-161). OVCAR-3 cells were maintained in RPMI-1640 supplemented with 10% FBS at 37 ℃ in 5% CO 2. OVCAR-3 expresses tumor-associated CA 125/0772P on the cell surface. OVCAR-3 xenografts were implanted subcutaneously and grown as ectopic tumors in immunodeficient NCr nude mice. Subcutaneous OVCAR-3 tumorsThe main criteria for growth were 150-250mm at 2 weeks post-transplantation3Tumors, at which point experimental treatment will begin.
To facilitate subcutaneous tumor formation, OVCAR-3 was serially propagated in vivo in the peritoneal cavity of NCr nu/nu mice (Burbridge et al, int.j.15: 1155-1162 (1999); guichard et al, Clin.7: 3222-3228(2001)). Before subcutaneous transplantation, 10X 10 in 0.9% saline6In vitro cultured OVCAR-3 cells (passage 32) were injected intraperitoneally into NCr nu/nu mice (passage one). After 7 weeks, tumor cells were harvested by peritoneal lavage and 5X 10 cells were plated6Individual cells are injected into a new set of receptors (second generation). After 4 weeks, cells were harvested by peritoneal lavage and 5X 10 cells were removed 6Individual cells were injected into a new set of receptors (third generation). Third generation cells were harvested after 3 weeks and analyzed for CA 125/0772P expression and viability.
For radioimmunotherapy studies, third generation cells were implanted for subcutaneous tumor growth. Third generation cells were typically > 95% viable and maintained high levels of CA 125/0772P expression as confirmed by flow cytometry. For ectopic, solid tumor growth, cells were resuspended in a mixture of Matrigel (Matrigel, BD Biosciences: Lot #005002, 14.6mg/mL) and 0.9% saline to a final concentration of 15X 10 cells6Individual cells/mL, final Matrigel concentration 7.3 mg/mL. Mice were injected with 0.2ml volumes of cell suspension at a final dose of 3X 106And (4) cells. The cell suspension was injected subcutaneously ventrally in the abdominal region using a 23 gauge needle. The injection site was sterilized by swabbing with sterile gauze in 70% ethanol. Approximately 10 days after implantation, palpable tumors were measured across two dimensions with an electronic caliper (Fowler Instruments). Mice were grouped according to tumor volume, 10 per group. There were no significant differences between the mean tumor volumes for all groups in one study. Tumor measurements and observations were recorded twice weekly. Using standard formula (length x width) 2) X 0.5 tumor volume was calculated.
776.1 and131coupling of iodine
By means of an improved IODO-GEN method(Visser et al, J.Nucl. Med.42: 509-519(2001)) in Perkin-Elmer iodinated murine IgG1776.1 by high dose131An effective method of minimizing chemical and radiation-induced damage by conjugation to monoclonal antibodies. 10 microliters of 1.41mg/ml ascorbic acid solution (pH 5.0) was added to 10mCi131I and the mixture was incubated for 1 minute. Then 100. mu.l of 0.5M phosphate (pH 7.4) was added. 0.5mg 776.1 mAb (calculated as antibody concentration to give the required volume) was then added. Then 35. mu.l of a 1mg/ml solution of IODO-GEN in acetonitrile was added. After 3 minutes of incubation, 100. mu.l of ascorbic acid solution (25mg/ml, pH 5.0) was added. After a further 5 minutes, 100. mu.l of 0.1% Murine Serum Albumin (MSA) in 50mM PBS was added. After 4 min incubation, analysis by Instant Thin Layer Chromatography (ITLC) in physiological saline131And (I) doping. Unincorporated iodine was removed by Sephadex G-25 chromatography using a pre-packed NAP-10 column (Amersham-Pharmacia) with PBS containing 0.1% MSA as a buffer. All steps were performed at room temperature. The purified mAb was again analyzed for free iodine content by ITLC and was deemed appropriate if free iodine < 5% of the total iodine present.
Measurement of immunoreactivity of radiolabeled 776.1 by ELISA
Immunoreactivity of radiolabeled 776.1 was determined by ELISA assay. Immunlon 4(Dynatech) 96-well plates were coated with 1. mu.g/ml of 0772P 3-replicate (affinity purification) labeled with Hemagglutinin (HA) dissolved in DPBS at 100. mu.l/well. The following day, plates were blocked with 200. mu.l/well blocking buffer (1 XPBS, containing 1% BSA) for 1 hour at room temperature. Unlabeled and radiolabeled 776.1 was diluted to 3. mu.g/ml in blocking buffer and added to the first row of blocked plates in triplicate at 150. mu.l/well, and 100. mu.l blocking buffer was added to the remaining wells. The antibody was then serially diluted three-fold for a total of 7 dilutions. The plate was incubated at room temperature for 1 hour, followed by three washes with DPBS containing 0.05% Tween-20 (PBST; 200. mu.l/well). To detect the signal, 100. mu.l of HRP-conjugated goat anti-mouse IgG (Amarsham biosciences) diluted 1: 2000 was added to each well and incubated for 1h at room temperature. Plates were washed 3 times with PBS and washed by adding TMB (KPL) substrate and H2O2The HRP conjugate was detected in a mixture (1: 1 ratio, 100. mu.l/well). The plate was incubated for 10 min and absorbance was measured at 650nm with a plate reader (Molecular Device). Immunoreactivity was determined by comparing the concentration of radiolabeled and unlabeled antibody at 50% saturation.
Use 2131I]776.1 carrying out single dose Radioimmunotherapy (RIT)
For tumors carrying mature OVCAR-3 (ideal volume of 150 mm)3To 250mm3) Is administered to a mouse by a single intravenous injection of 0.2ml of 0.9% sodium chloride131I]776.1 (mouse IgG 1). For all studies, 10 mice in one group received 100 or 300 μ Ci of sodium chloride in 0.9% sodium chloride131I]776.1. Mice in the control group were injected with only 0.9% sodium chloride or unlabeled 776.1 at a dose equivalent to the protein dose given in the high dose radiolabeled 776.1 group. Tumors were measured twice weekly. When the tumor volume was greater than 10% of their body weight, mice were sacrificed.
As a result:
the [ 2 ] administered as a single intravenous dose in an OVCAR-3 xenograft tumor model of human ovarian cancer131I]The 776.1IgG1 antibody was effective in delaying tumor growth compared to the 300 μ Ci dose of the IgG1 control from three studies and the 100 μ Ci dose of the IgG1 control in two of the three studies. See fig. 12 for an overview of one of these studies. Compared to the saline control, [ 2 ]131I]776.1IgG1 was effective in delaying tumor growth in two of three studies at doses of 100. mu. Ci and 300. mu. Ci. Two of the three studies, at a dose of 300 μ Ci131I]776.1IgG1 indicated tumor regression, which was defined as obtaining an average tumor volume that was less than the initial tumor volume at the start of the study. In one study, the [ alpha ], [ beta 131I]776.1 IgG1 treated group did not observe any statistical delay in tumor growth, and was resistant to 300. mu. Ci [ solution ]131I]No regression was observed for the 776.1 IgG1 dose group. However, the mean initial tumor volume at the beginning of this particular study was greater than that in the remaining two studiesThe initial mean tumor volume was significantly larger.
Use 290Y]776.1 radiolabeled antibody gave similar results. In particular, a significant reduction in tumor growth was observed (p < 0.05). In three of the four studies, a significant reduction in growth was observed at doses of 50 μ Ci and 150 μ Ci of antibody. In these same three studies, at the highest dose of [ 2 ]90Y]776.1 regression of tumor growth was observed and the effect on tumor growth was equal to or better than 3 doses of 6mg/kg cisplatin.
7.0 antibody/antigen-binding antibody fragment competition assay
ELISA Cross-Competition assay
The antibody to be tested was biotinylated using the EZ-Link Sulfo-NHS-LC-biotinylation kit (Pierce Biotechnology, Rockford, Ill.) according to the manufacturer's instructions and then dialyzed against 1L phosphate buffer at 4 ℃ for 48 hours to remove unreacted biotinylation reagent, with two buffer changes. Dissolved in bicarbonate buffer (0.2M Na) 2CO3/NaHCO3pH 9.6, Sigma) 3 rpt-0772P 100. mu.l (per well) at 4 ℃ overnight in 96-well plates.
The following day, plates were washed 3 times with 200 μ l 1 XPBST (1 XPBST buffer (PBS), 0.05% Tween20) and blocked with 100 μ l 1 XPBST containing 1% Bovine Serum Albumin (BSA) for 1 hour at room temperature. After 3 washes with 1 XPBST, a titration curve was obtained from a 0 to 1000 fold excess of competitor antibody (relative to the labeled antibody added in the later step) in 95. mu.l of 1 XPBST + 1% BSA, which competitor antibody was added to separate wells and incubated for 1 hour at 37 ℃. Biotinylated antibody was then added to 5. mu.l of 1 XPBST + 1% BSA and incubated for an additional 1 hour at room temperature. The concentration of biotinylated antibody added was such that 70% of the maximum binding of 0772P3-rpt protein was achieved without competitors using the detection conditions described below. The amount of antibody added depends on the binding characteristics and is usually determined empirically in studies of experimental properties.
Plates were then washed three times with 1 × PBST. To detect the signal, 100. mu.l of streptavidin-HRP (diluted 1: 4000-1: 8000 to 1 XPBST + 1% BSA, Southern Biotechnology Associates, Inc. (Birmingham, Alabama)) was added to each well and incubated for 1 hour at room temperature. Plates were washed 3 times with 1 × PBST. Finally, TMB substrate and H were added to each well 2O2100 μ l of the mixture (1: 1 ratio, KPL) and absorbance at 405nm was measured using a plate reader (Molecular Devices Corp., Sunnyvale, Calif.). The assay was performed in triplicate for each antibody and the average was calculated.
Non-specific competition was determined by replacing specific competitors with normal mouse IgG 1. Blanks, controls for the respective reagents, and self-competition were also included in each experiment. Percent inhibition (minus non-specific competition) was plotted as a function of competitor competition and IC was determined50Or the concentration of competitor at which 50 competitions were observed.
FACS cross-competition assay
The antibody to be tested was biotinylated using the EZ-Link Sulfo-NHS-LC-biotinylation kit (Pierce Biotechnology, Rockford, Ill.) according to the manufacturer's instructions and then dialyzed against 1L phosphate buffer at 4 ℃ for 48 hours to remove unreacted biotinylation reagent, with two buffer changes. Cultured OVCAR-3 cells were harvested and plated in FACs buffer (1 × Dulbecco's Phosphate Buffer (DPBS), 0.05% NaN32% BSA). The competition assay was prepared in a 96-well plate in a total volume of 50. mu.l.
Mu.l of 0-to 1000-fold excess unlabelled competitor antibody (relative to the biotinylated antibody added in the following step) was titrated to 25. mu.l of OVCAR-3 cells (4X 10) suspended in FACS buffer 5) (in separate wells), mixed well, dispensed into 96-well plates, and incubated for 30 minutes at room temperature. To each well containing cells, 5 μ l of biotinylated antibody in FACS buffer was then added, mixed well and incubated at room temperature for 30 minutes. The amount of antibody used was the minimum concentration that achieved the maximum binding of OVCAR-3 cells (expressed as percentage positive cells). Added withThe amount of antibody introduced depends on the binding characteristics and is usually determined empirically in studies of the nature of the assay.
The cells were collected and washed twice with 200. mu.l FACS buffer. To detect the signal, cells were incubated with 50. mu.l of 1. mu.g/m l FITC conjugated streptavidin (prepared with FACS buffer, Molecular Probes, Eugene, OR) for 30 min at room temperature. After washing 2 times with 200 μ l FACS buffer, cells from each well were resuspended in 400 μ l FACS buffer and FACS analyzed. FACS analysis was performed using FACScan instruments and CellQuest software (Becton Dickinson) according to the manufacturer's recommendations. The data obtained under each experimental condition represent 10,000 events. Blanks, controls for the respective reagents, and self-competition were also included in each experiment. Percent inhibition (minus non-specific competition), or percent-positive staining reduction of OVCAR-3 cells as a function of competitor competition, and determination of IC 50Or the concentration of competitors at which 50% competition is observed.
If IC of competitor50At a concentration no more than about 100-fold higher than that of the labeled antibody, the antibodies are considered to compete. More preferably, if the IC of the competitor50At a concentration no more than about 10-fold higher than that of the labeled antibody, the antibodies are considered to compete. More preferably, if the IC of the competitor50At no more than about equimolar concentrations of the labeled antibody, the antibody is considered to compete.
8.0 hybridoma Collection
Hybridoma 117.1, which secretes monoclonal antibody 117.1, was deposited at the American Type Culture Collection (ATCC) on 8.2.2002 under the International recognized Budapest treaty on the deposit of microorganisms for the purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 368.1 secretes a monoclonalAntibody 368.1, deposited at the American Type Culture Collection (ATCC) on 8/2.2002 according to the International recognized Budapest treaty on the deposit of microorganisms for the purposes of patent procedure) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 501.1, which secretes monoclonal antibody 501.1, was deposited at the American Type Culture Collection (ATCC) on 8.2.2002 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedure) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 776.1 secretes monoclonal antibody 776.1, which was deposited at the American Type Culture Collection (ATCC) on day 2/8 2002 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 15C9, which secretes monoclonal antibody 15C9, was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 16C7 secretes monoclonal antibody 16C7 according to the protocol for patentingThe hybridoma is deposited with the American Type Culture Collection (ATCC) at 3/4/2003 under the International Budapest treaty on the deposit of microorganisms for the purposes of sequencing ) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 16H9 secretes monoclonal antibody 16H9, which was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 4E7, which secretes monoclonal antibody 4E7, was deposited at the American Type Culture Collection (ATCC) on 3/4.2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 7A11 secretes monoclonal antibody 7A11, which was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 7C6 secretes monoclonal antibody 7C6 according to the internationally recognized cloth of microbial deposits for purposes of patent procedure The hybridoma was deposited in the American Type Culture Collection (ATCC) at 3/4/2003 under the dapest treaty) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 7F10 secretes monoclonal antibody 7F10, which was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 7G10 secretes monoclonal antibody 7G10, which was deposited at the American Type Culture Collection (ATCC) on day 4/6 of 2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 7H1 secretes monoclonal antibody 7H1, which was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 8A1, which secretes monoclonal antibody 8A1, is deposited on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedureDeposited at American Type Culture Collection (ATCC)) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 8B5 secretes monoclonal antibody 8B5, which was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 8C3 secretes monoclonal antibody 8C3, which was deposited at the American Type Culture Collection (ATCC) on day 4/6 of 2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 8E3 secretes monoclonal antibody 8E3, which was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures ) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 8G9 secretes monoclonal antibody 8G9, which was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 325.1, which secretes monoclonal antibody 325.1, was deposited at the American Type Culture Collection (ATCC) on 3/4.2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-. Hybridoma 325.1, which secretes monoclonal antibody 325.1, was deposited at the American Type Culture Collection (ATCC) on 3/4.2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 621.1 secretes monoclonal antibody 621.1, which was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures ) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 633.1 secretes monoclonal antibody 633.1, which was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 654.1 secretes monoclonal antibody 654.1, which was deposited at the American Type Culture Collection (ATCC) on day 4/6 of 2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 725.1 secretes monoclonal antibody 725.1, which was deposited at the American Type Culture Collection (ATCC) on 3/4/2003 under the International recognized Budapest treaty on the deposit of microorganisms for the purposes of patent procedures) (10801 Universal boulevard, Manassass, Virginia 20110-.
Hybridoma 446.1 secretes monoclonal antibody 446.1, which was deposited with the American Type Culture Collection (ATCC) on 25/9/2003 under the International recognized Budapest treaty on the deposit of microorganisms for purposes of patent procedures ) (10801 Universal boulevard, Manassass, Virginia 20110-.
The scope of the invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. Citation or discussion of a reference herein shall not be construed as an admission that such reference is prior art to the present application.
Sequence listing
<110>Euro-Celtique S.A.
<120> antibodies that bind cell-associated CA 125/0772P and methods of use thereof
<130>6750-214-228
<140> to be distributed
<141>2003-10-15
<150>60/485,986
<151>2003-07-10
<150>60/418,828
<151>2003-10-12
<160>71
<170>FastSEQ for Windows Version 4.0
<210>1
<211>748
<212>PRT
<213> Artificial sequence
<220>
<223> CA 125/0772P 3-repeat
<400>1
Ala Ala Gln Pro Ala Arg Arg Ala Arg Arg Thr Lys Leu Phe Thr His
1 5 10 15
Arg Ser Ser Val Ser Thr Thr Ser Thr Pro Gly Thr Pro Thr Val Tyr
20 25 30
Leu Gly Ala Ser Lys Thr Pro Ala Ser Ile Phe Gly Pro Ser Ala Ala
35 40 45
Ser His Leu Leu Ile Leu Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu
50 55 60
Arg Tyr Glu Glu Asn Met Trp Pro Gly Ser Arg Lys Phe Asn Thr Thr
65 70 75 80
Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Leu Phe Lys Asn Thr Ser
85 90 95
Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu
100 105 110
Lys Asp Gly Glu Ala Thr Gly Val Asp Ala Ile Cys Thr His Arg Pro
115 120 125
Asp Pro Thr Gly Pro Gly Leu Asp Arg Glu Gln Leu Tyr Leu Glu Leu
130 135 140
Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp
145 150 155 160
Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser Val Pro
165 170 175
Thr Thr Ser Thr Gly Val Val Ser Glu Glu Pro Phe Thr Leu Asn Phe
180 185 190
Thr Ile Asn Asn Leu Arg Tyr Met Ala Asp Met Gly Gln Pro Gly Ser
195 200 205
Leu Lys Phe Asn Ile Thr Asp Asn Val Met Lys His Leu Leu Ser Pro
210 215 220
Leu Phe Gln Arg Ser Ser Leu Gly Ala Arg Tyr Thr Gly Cys Arg Val
225 230 235 240
Ile Ala Leu Arg Ser Val Lys Asn Gly Ala Glu Thr Arg Val Asp Leu
245 250 255
Leu Cys Thr Tyr Leu Gln Pro Leu Ser Gly Pro Gly Leu Pro Ile Lys
260 265 270
Gln Val Phe His Glu Leu Ser Gln Gln Thr His Gly Ile Thr Arg Leu
275 280 285
Gly Pro Tyr Ser Leu Asp Lys Asp Ser Leu Tyr Leu Asn Gly Tyr Asn
290 295 300
Glu Pro Gly Pro Asp Glu Pro Pro Thr Thr Pro Lys Pro Ala Thr Thr
305 310 315 320
Phe Leu Pro Pro Leu Ser Glu Ala Thr Thr Ala Met Gly Tyr His Leu
325 330 335
Lys Thr Leu Thr Leu Asn Phe Thr Ile Ser Asn Leu Gln Tyr Ser Pro
340 345 350
Asp Met Gly Lys Gly Ser Ala Thr Phe Asn Ser Thr Glu Gly Val Leu
355 360 365
Glu His Leu Leu Arg Pro Leu Phe Gln Lys Ser Ser Met Gly Pro Phe
370 375 380
Tyr Leu Gly Cys Gln Leu Ile Ser Leu Arg Pro Glu Lys Asp Gly Ala
385 390 395 400
Ala Thr Gly Val Asp Thr Thr Cys Thr Tyr His Pro Asp Pro Val Gly
405 410 415
Pro Gly Leu Asp Ile Gln Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr
420 425 430
His Gly Val Thr Gln Leu Gly Phe Tyr Val Leu Asp Arg Asp Ser Leu
435 440 445
Phe Ile Asu Gly Tyr Ala Pro Gln Asn Leu Ser Ile Arg Gly Glu Tyr
450 455 460
Gln Ile Asn Phe His Ile Val Asn Trp Asn Leu Ser Asn Pro Asp Pro
465 470 475 480
Thr Ser Ser Glu Tyr Ile Thr Leu Leu Arg Asp Ile Gln Asp Lys Val
485 490 495
Thr Thr Leu Tyr Lys Gly Ser Gln Leu His Asp Thr Phe Arg Phe Cys
500 505 510
Leu Val Thr Asn Leu Thr Met Asp Ser Val Leu Val Thr Val Lys Ala
515 520 525
Leu Phe Ser Ser Asn Leu Asp Pro Ser Leu Val Glu Gln Val Phe Leu
530 535 540
Asp Lys Thr Leu Asn Ala Ser Phe His Trp Leu Gly Ser Thr Tyr Gln
545 550 555 560
Leu Val Asp Ile His Val Thr Glu Met Glu Ser Ser Val Tyr Gln Pro
565 570 575
Thr Ser Ser Ser Ser Thr Gln His Phe Tyr Leu Asn Phe Thr Ile Thr
580 585 590
Asn Leu Pro Tyr Ser Gln Asp Lys Ala Gln Pro Gly Thr Thr Asn Tyr
595 600 605
Gln Arg Asn Lys Arg Asn Ile Glu Asp Ala Leu Asn Gln Leu Phe Arg
610 615 620
Asn Ser Ser Ile Lys Ser Tyr Phe Ser Asp Cys Gln Val Ser Thr Phe
625 630 635 640
Arg Ser Val Pro Asn Arg His His Thr Gly Val Asp Ser Leu Cys Asn
645 650 655
Phe Ser Pro Leu Ala Arg Arg Val Asp Arg Val Ala Ile Tyr Glu Glu
660 665 670
Phe Leu Arg Met Thr Arg Asn Gly Thr Gln Leu Gln Asn Phe Thr Leu
675 680 685
Asp Arg Ser Ser Val Leu Val Asp Gly Tyr Ser Pro Asn Arg Asn Glu
690 695 700
Pro Leu Thr Gly Asn Ser Ala Asp Ile Gln His Ser Gly Gly Arg Ser
705 710 715 720
Ser Leu Glu Gly Pro Arg Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp
725 730 735
Leu Asn Met His Thr Gly His His His His His His
740 745
<210>2
<211>809
<212>PRT
<213> Artificial sequence
<220>
<223> CA 125/0772P 3-repeat TM
<400>2
Ala Ala Gln Pro Ala Arg Arg Ala Arg Arg Thr Lys Leu Phe Thr His
1 5 10 15
Arg Ser Ser Val Ser Thr Thr Ser Thr Pro Gly Thr Pro Thr Val Tyr
20 25 30
Leu Gly Ala Ser Lys Thr Pro Ala Ser Ile Phe Gly Pro Ser Ala Ala
35 40 45
Ser His Leu Leu Ile Leu Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu
50 55 60
Arg Tyr Glu Glu Asn Met Trp Pro Gly Ser Arg Lys Phe Asn Thr Thr
65 70 75 80
Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Leu Phe Lys Asn Thr Ser
85 90 95
Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu
100 105 110
Lys Asp Gly Glu Ala Thr Gly Val Asp Ala Ile Cys Thr His Arg Pro
115 120 125
Asp Pro Thr Gly Pro Gly Leu Asp Arg Glu Gln Leu Tyr Leu Glu Leu
130 135 140
Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp
145 150 155 160
Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser Val Pro
165 170 175
Thr Thr Ser Thr Gly Val Val Ser Glu Glu Pro Phe Thr Leu Asn Phe
180 185 190
Thr Ile Asn Asn Leu Arg Tyr Met Ala Asp Met Gly Gln Pro Gly Ser
195 200 205
Leu Lys Phe Asn Ile Thr Asp Asn Val Met Lys His Leu Leu Ser Pro
210 215 220
Leu Phe Gln Arg Ser Ser Leu Gly Ala Arg Tyr Thr Gly Cys Arg Val
225 230 235 240
Ile Ala Leu Arg Ser Val Lys Asn Gly Ala Glu Thr Arg Val Asp Leu
245 250 255
Leu Cys Thr Tyr Leu Gln Pro Leu Ser Gly Pro Gly Leu Pro Ile Lys
260 265 270
Gln Val Phe His Glu Leu Ser Gln Gln Thr His Gly Ile Thr Arg Leu
275 280 285
Gly Pro Tyr Ser Leu Asp Lys Asp Ser Leu Tyr Leu Asn Gly Tyr Asn
290 295 300
Glu Pro Gly Pro Asp Glu Pro Pro Thr Thr Pro Lys Pro Ala Thr Thr
305 310 315 320
Phe Len Pro Pro Leu Ser Glu Ala Thr Thr Ala Met Gly Tyr His Leu
325 330 335
Lys Thr Leu Thr Leu Asn Phe Thr Ile Ser Asn Leu Gln Tyr Ser Pro
340 345 350
Asp Met Gly Lys Gly Ser Ala Thr Phe Asn Ser Thr Glu Gly Val Leu
355 360 365
Gln His Leu Leu Arg Pro Leu Phe Gln Lys Ser Ser Met Gly Pro Phe
370 375 380
Tyr Leu Gly Cys Gln Leu Ile Ser Leu Arg Pro Glu Lys Asp Gly Ala
385 390 395 400
Ala Thr Gly Val Asp Thr Thr Cys Thr Tyr His Pro Asp Pro Val Gly
405 410 415
Pro Gly Leu Asp Ile Gln Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr
420 425 430
His Gly Val Thr Gln Leu Gly Phe Tyr Val Leu Asp Arg Asp Ser Leu
435 440 445
Phe Ile Asn Gly Tyr Ala Pro Gln Asn Leu Ser Ile Arg Gly Glu Tyr
450 455 460
Gln Ile Asn Phe His Ile Val Asn Trp Asn Leu Ser Asn Pro Asp Pro
465 470 475 480
Thr Ser Ser Glu Tyr Ile Thr Leu Leu Arg Asp Ile Gln Asp Lys Val
485 490 495
Thr Thr Leu Tyr Lys Gly Ser Gln Leu His Asp Thr Phe Arg Phe Cys
500 505 510
Leu Val Thr Asn Leu Thr Met Asp Ser Val Leu Val Thr Val Lys Ala
515 520 525
Leu Phe Ser Ser Asn Leu Asp Pro Ser Leu Val Glu Gln Val Phe Leu
530 535 540
Asp Lys Thr Leu Asn Ala Ser Phe His Trp Leu Gly Ser Thr Tyr Gln
545 550 555 560
Leu Val Asp Ile His Val Thr Glu Met Glu Ser Ser Val Tyr Gln Pro
565 570 575
Thr Ser Ser Ser Ser Thr Gln His Phe Tyr Leu Asn Phe Thr Ile Thr
580 585 590
Asn Leu Pro Tyr Ser Gln Asp Lys Ala Gln Pro Gly Thr Thr Asn Tyr
595 600 605
Gln Arg Asn Lys Arg Asu Ile Glu Asp Ala Leu Asn Gln Leu Phe Arg
610 615 620
Asn Ser Ser Ile Lys Ser Tyr Phe Ser Asp Cys Gln Val Ser Thr Phe
625 630 635 640
Arg Ser Val Pro Asn Arg His His Thr Gly Val Asp Ser Leu Cys Asn
645 650 655
Phe Ser Pro Leu Ala Arg Arg Val Asp Arg Val Ala Ile Tyr Glu Glu
660 665 670
Phe Leu Arg Met Thr Arg Asn Gly Thr Gln Leu Gln Asn Phe Thr Leu
675 680 685
Asp Arg Ser Ser Val Leu Val Asp Gly Tyr Ser Pro Asn Arg Asn Glu
690 695 700
Pro Leu Thr Gly Asn Ser Asp Leu Pro Phe Trp Ala Val Ile Leu Ile
705 710 715 720
Gly Leu Ala Gly Leu Leu Gly Leu Ile Thr Cys Leu Ile Cys Gly Val
725 730 735
Leu Val Thr Thr Arg Arg Arg Lys Lys Glu Gly Glu Tyr Asn Val Gln
740 745 750
Gln Gln Cys Pro Gly Tyr Tyr Gln Ser His Leu Asp Leu Glu Asp Leu
755 760 765
Gln Asn Ser Ala Asp Ile Gln His Ser Gly Gly Arg Ser Ser Leu Glu
770 775 780
Gly Pro Arg Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Met
785 790 795 800
His Thr Gly His His His His His His
805
<210>3
<211>12
<212>PRT
<213> Artificial sequence
<220>
<223>117.1 VH1 CDR
<400>3
Gly Phe Ser Leu Ser Thr Pro Gly Met Gly Val Gly
1 5 10
<210>4
<211>16
<212>PRT
<213> Artificial sequence
<220>
<223>117.1 VH2 CDR
<400>4
His Ile Trp Trp Asp Asp Phe Lys Arg Asp Asn Pro Ala Leu Lys Ser
1 5 10 15
<210>5
<211>12
<212>PRT
<213> Artificial sequence
<220>
<223>117.1 VH3 CDR
<400>5
Val Asp Gly Asn Phe Leu Ser Trp Tyr Phe Asp Val
1 5 10
<210>6
<211>16
<212>PRT
<213> Artificial sequence
<220>
<223>117.1 VL1 CDR
<400>6
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
1 5 10 15
<210>7
<211>7
<212>PRT
<213> Artificial sequence
<220>
<223>117.1 VL2 CDR
<400>7
Lys Val Ser Asn Arg Phe Ser
1 5
<210>8
<211>9
<212>PRT
<213> Artificial sequence
<220>
<223>117.1 VL3 CDR
<400>8
Ser Gln Ser Arg Tyr Val Pro Glu Thr
1 5
<210>9
<211>10
<212>PRT
<213> Artificial sequence
<220>
<223>368.1 VH1 CDR
<400>9
Gly Tyr Ser Phe Thr Gly Phe Tyr Met His
1 5 10
<210>10
<211>17
<212>PRT
<213> Artificial sequence
<220>
<223>368.1 VH2 CDR
<400>10
Tyr Val Ser Cys Tyr Thr Gly Ala Thr Thr Tyr Thr Gln Lys Phe Lys
1 5 10 15
Gly
<210>11
<211>9
<212>PRT
<213> Artificial sequence
<220>
<223>368.1 VH3 CDR
<400>11
Glu Gly Asp Tyr Tyr Ser Met Asp Phe
1 5
<210>12
<211>16
<212>PRT
<213> Artificial sequence
<220>
<223>368.1 VL1 CDR
<400>12
Arg Ser Ser Gln Ser Leu Glu Arg Thr Asn Gly Asn Thr Tyr Leu His
1 5 10 15
<210>13
<211>7
<212>PRT
<213> Artificial sequence
<220>
<223>368.1 VL2 CDR
<400>13
Lys Val Ser Ser Arg Phe Ser
1 5
<210>14
<211>9
<212>PRT
<213> Artificial sequence
<220>
<223>368.1 VL3 CDR
<400>14
Ser Gln Thr Thr His Gly Pro Pro Thr
1 5
<210>15
<211>10
<212>PRT
<213> Artificial sequence
<220>
<223>501.1 VH1 CDR
<400>15
Gly Tyr Ile Phe Thr Asp Tyr Gly Met Asn
1 5 10
<210>16
<211>17
<212>PRT
<213> Artificial sequence
<220>
<223>501.1 VH2 CDR
<400>16
Cys Ile Asn Thr Tyr Thr Gly Glu Thr Ile Tyr Ser Asp Asp Phe Arg
1 5 10 15
Gly
<210>17
<211>9
<212>PRT
<213> Artificial sequence
<220>
<223>501.1 VH3 CDR
<400>17
Gly Asn Tyr Arg Asp Ala Ile Asp Tyr
1 5
<210>18
<211>11
<212>PRT
<213> Artificial sequence
<220>
<223>501.1 VL1 CDR
<400>18
Lys Ala Ser Gln Asp Ile Lys Ser Tyr Leu Ser
1 5 10
<210>19
<211>7
<212>PRT
<213> Artificial sequence
<220>
<223>501.1 VL2 CDR
<400>19
Tyr Ala Thr Thr Leu Ala Asp
1 5
<210>20
<211>9
<212>PRT
<213> Artificial sequence
<220>
<223>501.1 VL3 CDR
<400>20
Leu His His Asp Glu Ser Pro Phe Thr
1 5
<210>21
<211>10
<212>PRT
<213> Artificial sequence
<220>
<223>77G.1 VH1 CDR
<400>21
Gly Tyr Thr Phe Thr Asp Tyr Asn Ile His
1 5 10
<210>22
<211>15
<212>PRT
<213> Artificial sequence
<220>
<223>776.1 VH2 CDR
<400>22
Tyr Ile Tyr Pro Tyr Asn Gly Val Ser Asp Tyr Asn Gln Asn Phe
1 5 10 15
<210>23
<211>12
<212>PRT
<213> Artificial sequence
<220>
<223>776.1 VH3 CDR
<400>23
Arg Trp Asp Phe Gly Ser Gly Tyr Tyr Phe Asp Tyr
1 5 10
<210>24
<211>10
<212>PRT
<213> Artificial sequence
<220>
<223>776.1 VL1 CDR
<400>24
Arg Ala Ser Ser Ser Val Ile Tyr Met Cys
1 5 10
<210>25
<211>7
<212>PRT
<213> Artificial sequence
<220>
<223>776.1 VL2 CDR
<400>25
Gly Thr Ser Thr Leu Ala Ser
1 5
<210>26
<211>9
<212>PRT
<213> Artificial sequence
<220>
<223>776.1 VL3 CDR
<400>26
Gln Gln Trp Ser Ser Asn Pro Phe Thr
1 5
<210>27
<211>131
<212>PRT
<213> Artificial sequence
<220>
<223>117.1 light chain polypeptide variable region (117.1L)
<400>27
Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Gly
1 5 10 15
Ser Ser Ser Asp Ala Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45
Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
50 55 60
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
100 105 110
Ser Gln Ser Arg Tyr Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys
130
<210>28
<211>141
<212>PRT
<213> Artificial sequence
<220>
<223>117.1 heavy chain polypeptide variable region (117.1H)
<400>28
Met Gly Arg Leu Thr Ser Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr
1 5 10 15
Val Leu Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln
20 25 30
Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu
35 40 45
Ser Thr Pro Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys
50 55 60
Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Phe Lys Arg Asp
65 70 75 80
Asn Pro Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser
85 90 95
Ser Gln Val Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala
100 105 110
Thr Tyr Tyr Cys Val Arg Val Asp Gly Asn Phe Leu Ser Trp Tyr Phe
115 120 125
Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
130 135 140
<210>29
<211>131
<212>PRT
<213> Artificial sequence
<220>
<223>368.1 light chain polypeptide variable region (368.1L)
<400>29
Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala
1 5 10 15
Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45
Glu Arg Thr Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
50 55 60
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Ser Arg Phe Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys
100 105 110
Ser Gln Thr Thr His Gly Pro Pro Thr Cys Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys
130
<210>30
<211>137
<212>PRT
<213> Artificial sequence
<220>
<223>368.1 heavy chain polypeptide variable region (368.1H)
<400>30
Met Gly Trp Ile Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg
20 25 30
Thr Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Phe Tyr Met His Trp Val Lys Gln Ser Leu Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Tyr Val Ser Cys Tyr Thr Gly Ala Thr Thr Tyr Thr
65 70 75 80
Gln Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser
85 90 95
Thr Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Glu Gly Asp Tyr Tyr Ser Met Asp Phe Trp Gly
115 120 125
Gln Gly Thr Ser Val Thr Val Ser Ser
130 135
<210>31
<211>128
<212>PRT
<213> Artificial sequence
<220>
<223>501.1 light chain polypeptide variable region (501.1L)
<400>31
Met Asp Met Arg Ala Pro Ala Gln Phe Phe Gly Ile Leu Leu Leu Trp
1 5 10 15
Phe Pro Gly Ile Arg Cys Asp Ile Lys Met Thr Gln Ser Pro Ser Ser
20 25 30
Ile Tyr Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser
35 40 45
Gln Asp Ile Lys Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Trp Lys
50 55 60
Sar Pro Lys Thr Leu Ile Tyr Tyr Ala Thr Thr Leu Ala Asp Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Ile
85 90 95
Ile Asn Ser Leu Glu Ser Asp Asp Ile Ala Thr Tyr Phe Cys Leu His
100 105 110
His Asp Glu Ser Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
115 120 125
<210>32
<211>137
<212>PRT
<213> Artificial sequence
<220>
<223>501.1 heavy chain polypeptide variable region (501.1H)
<400>32
Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser
1 5 10 15
Ala Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys
20 25 30
Pro Gly Glu Thr Val Gln Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe
35 40 45
Thr Asp Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu
50 55 60
Lys Trp Met Gly Cys Ile Asn Thr Tyr Thr Gly Glu Thr Ile Tyr Ser
65 70 75 80
Asp Asp Phe Arg Gly Arg Phe Ala Ile Ser Leu Glu Thr Ser Ala Ser
85 90 95
Thr Ala Phe Ile Gln Ile Asn Asn Leu Lys Asn Glu Asp Ala Ala Thr
100 105 110
Tyr Phe Cys Ala Arg Gly Asn Tyr Arg Asp Ala Ile Asp Tyr Trp Gly
115 120 125
Gln Gly Thr Ser Val Thr Val Ser Ser
130 135
<210>33
<211>127
<212>PRT
<213> Artificial sequence
<220>
<223>776.1 light chain polypeptide variable region (776.1L)
<400>33
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Phe Ala Ser Pro Gly Glu Thr Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ile Tyr Met Cys Trp Asn Gln Gln Lys Pro Gly Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro
65 70 75 80
Thr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
115 120 125
<210>34
<211>139
<212>PRT
<213> Artificial sequence
<220>
<223>776.1 heavy chain polypeptide variable region (776.1H)
<400>34
Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Asp Tyr Asn Ile His Trp Val Lys Gln Ser His Gly Lys Ile Leu
50 55 60
Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Val Ser Asp Tyr Asn
65 70 75 80
Gln Asn Phe Lys Ser Lys Ala Thr Leu Ile Val Asp Asn Ser Ser Asn
85 90 95
Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Trp Asp Phe Gly Ser Gly Tyr Tyr Phe Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
130 135
<210>35
<211>393
<212>DNA
<213> Artificial sequence
<220>
<223>117.1 light chain polypeptide variable region (117.1L)
<400>35
atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctggttc cagcagtgat 60
gctgtgatga cccaaactcc actctccctg cctgtcagtc ttggagatca ggcctccatc 120
tcttgcagat ctagtcagag ccttgtacac agtaatggaa acacctattt acattggtac 180
ctgcagaagc caggccagtc tccaaaactc ctgatctaca aagtttccaa ccgattttct 240
ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caggatcagc 300
agagtggagg ctgaggatct gggagtttat ttctgctctc aaagtagata tgttccgtgg 360
acgttcggtg gaggcaccaa gctggaaatc aaa 393
<210>36
<211>423
<212>DNA
<213> Artificial sequence
<220>
<223>117.1 heavy chain polypeptide variable region (117.1H)
<400>36
atgggcaggc ttacttcttc attcctgcta ctgattgtcc ctgcatatgt cctgtcccag 60
gttactctga aagagtctgg ccctgggata ttgcagccct cccagaccct cagtctgact 120
tgttctttct ctgggttttc actgagcact cctggtatgg gtgtaggctg gattcgtcag 180
ccatcaggga agggtctgga gtggctggca cacatttggt gggatgattt caagcgcgat 240
aatccagccc ttaagagccg actgactatc tctaaggata cctccagcag ccaggttttc 300
ctcaaaatcg ccagtgtgga cactgcagat actgccacat attactgtgt tcgagtggat 360
ggtaacttcc tctcctggta tttcgatgtc tggggcgctg ggaccacggt caccgtctcc 420
tca 423
<210>37
<211>393
<212>DNA
<213> Artificial sequence
<220>
<223>368.1 light chain polypeptide variable region (368.1L)
<400>37
atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60
gttgtgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120
tcttgcagat ctagtcagag ccttgaacgc actaatggaa acacctattt acattggtac 180
ctgcagaagc caggccagtc tccaaaactc ctgatctaca aagtttccag ccgattttct 240
ggggtcccag ataggttcag tggcagtgga tcagggacag atttcacact caagatcagt 300
agagtggagg ctgaggatct gggaatttat ttctgttctc aaactacaca tggtcctccg 360
acgtgcggtg gaggcaccaa gctggaaatc aaa 393
<210>38
<211>411
<212>DNA
<213> Artificial sequence
<220>
<223>368.1 heavy chain polypeptide variable region (368.1H)
<400>38
atgggatgga tctggatctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactctgag 60
gtccagctgc agcagtctgg acctgagtta gtgaggactg gggcttcagt gaagatatcc 120
tgcaaggctt ctggttactc attcactggt ttctacatgc actgggtcaa gcagagcctt 180
ggaaagagcc ttgagtggat tggatatgtt agttgttaca ctggtgctac tacctacacc 240
cagaagttca agggcaaggc cacatttact gttgacacat cctccagcac agcctacatg 300
caactcaaca gcctgacatc tgaagactct gcggtctatt actgtgcaag agaaggggat 360
tactattcta tggacttctg gggtcaagga acctcagtca ccgtctcctc a 411
<210>39
<211>386
<212>DNA
<213> Artificial sequence
<220>
<223>501.1 light chain polypeptide variable region (501.1L)
<400>39
atggacatga gggcccctgc tcagtttttt gggatcttgt tgctctggtt tccaggtatc 60
agatgtgaca tcaagatgac ccagtctcca tcgtccattt atgcatcgct gggagagagg 120
gtcactataa cttgcaaggc gagtcaggac attaaaagct atttaagctg gtaccaacag 180
aaaccctgga aatctcctaa gaccctgatc tattatgcaa caaccttggc agatggggtc 240
ccatcaagat tcagtggcag tggatctggg caagattatt ctctaatcat caacagcctg 300
gagtctgacg atatagctac ttatttctgt ctacaccatg atgagagccc attcacgttc 360
ggctcgggga caaaattgga aataaa 386
<210>40
<211>411
<212>DNA
<213> Artificial sequence
<220>
<223>501.1 heavy chain polypeptide variable region (501.1H)
<400>40
atggcttggg tgtggacctt gctgttcctg atggcagctg cccaaagtgc ccaagcacag 60
atccagttgg tgcagtctgg acctgagctg aagaagcctg gagagacagt ccagatctcc 120
tgcaaggctt ctggctatat cttcacagac tatggaatga actgggtgaa acaggctcca 180
ggaaagggtt taaaatggat gggctgtata aacacctaca ctggagagac aatatatagt 240
gatgacttca ggggacggtt tgccatctct ttggaaacct ctgccagcac tgcctttatt 300
cagatcaaca acctcaaaaa tgaggacgcg gcaacatatt tctgtgcaag gggaaattac 360
agggatgcta ttgactattg gggtcaagga acctcagtca ccgtctcctc a 411
<210>41
<211>383
<212>DNA
<213> Artificial sequence
<220>
<223>776.1 light chain polypeptide variable region (776.1L)
<400>41
atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcttcagt cataatgtcc 60
agaggacaaa ttgttctctc ccagtctcca gcaatcctgt ttgcatctcc aggggagacg 120
gtcacaatga cttgcagggc cagttcaagt gtaatttaca tgtgttggaa tcagcagaag 180
ccaggatcct cccccaaacc ctggatttat ggcacatcca ccctggcttc tggagtccct 240
actcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagagtagag 300
gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccatt cacgttcggc 360
tcggggacaa agttggaaat aaa 383
<210>42
<211>417
<211>DNA
<213> Artificial sequence
<220>
<223>776.1 heavy chain polypeptide variable region (776.1H)
<400>42
atgggatgga gctggatctt tctcttcctc ctgtcaggaa ctgcaggcgt ccactctgag 60
gtccagcttc agcagtcagg acctgagctg gtgaaacctg gggcctcagt gaagatatcc 120
tgcaaggctt ctggatacac attcactgac tacaacattc actgggtgaa acagagccat 180
ggaaagatcc ttgagtggat tggatatatt tatccttata atggtgtttc tgactacaac 240
cagaatttca agagcaaggc cacattgatt gtagacaatt cctccaacac agcctacatg 300
gaactccgca gcctgacatc tgaggactct gcagtctatt attgtgcaag atgggacttc 360
ggtagtggct actactttga ctactggggc caaggcacca ctctcacagt ctcctca 417
<210>43
<211>45
<212>RNA
<213> Artificial sequence
<220>
<223> primer (see section 6.6)
<400>43
rcgacuggag cacgaggaca cugacaugga cugaaggagu agaaa 45
<210>44
<211>54
<212>DNA
<213> Artificial sequence
<220>
<223> primer (see section 6.6)
<400>44
gctgtcaacg atacgctacg taacggcatg acagtgtttt tttttttttt tttt 54
<210>45
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> primer (see section 6.6)
<400>45
ayctccacac acaggrrcca gtggatagac 30
<210>46
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> primer (see section 6.6)
<400>46
ggatacagtt ggtgcagcat c 21
<210>47
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> primer (see section 6.6)
<400>47
cgactggagc acgaggacac tga 23
<210>48
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> primer (see section 6.6)
<400>48
attaaccctc actaaaggga 20
<210>49
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> primer (see section 6.6)
<400>49
taatacgact cactataggg 20
<210>50
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> primer (see section 6.6)
<400>50
attaaccctc actaaaggga 20
<210>51
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> primer (see section 6.6)
<400>51
taatacgact cactataggg 20
<210>52
<211>383
<212>DNA
<213> Artificial sequence
<220>
<223>725.1 light chain polypeptide variable region (725.1L)
<400>52
atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcttcagt cataatgtcc 60
agaggacaaa ttattctctc ccagtctcca gcaatcctgt ctgcatctcc aggggagaag 120
gtcacaatga cttgcagggc cagttcaagt gtaagttcca ttcactggta ccagcagaag 180
ccagaatcct cccccaaacc ctggatttac gccacatcca acctggcttc tggagtccct 240
gttcgcttca gtggcagtgg gtctgggacc tcttatactc tcacaatcag cagaatggag 300
gctgcagatg ctgccactta ttactgccag cagtggagta ttgatccagc cacgttcgga 360
ggggggacca agctggaaat aaa 383
<210>53
<211>135
<212>PRT
<213> Artificial sequence
<220>
<223>725.1 heavy chain polypeptide variable region (725.1H)
<400>53
Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser
1 5 10 15
Ala Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys
20 25 30
Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu
50 55 60
Lys Trp Met Gly Trp Ile Asn Ala Tyr Ile Gly Glu Pro Thr Tyr Ala
65 70 75 80
Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Ala Ser Thr His
85 90 95
Thr Ala Tyr Leu Gln Ile Asn Ser Leu Lys Ser Glu Asp Thr Ala Thr
100 105 110
Tyr Phe Cys Ala Ser Gly Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly
115 120 125
Thr Thr Leu Thr Val Ser Ser
130 135
<210>54
<211>127
<212>PRT
<213> Artificial sequence
<220>
<223>725.1 light chain polypeptide variable region (725.1L)
<400>54
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Ile Leu Ser Gln Ser Pro Ala Ile
20 25 30
Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
35 40 45
Ser Ser Val Ser Ser Ile His Trp Tyr Gln Gln Lys Pro Glu Ser Ser
50 55 60
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
65 70 75 80
Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile
85 90 95
Ser Arg Met Glu Ala Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ile Asp Pro Ala Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
<210>55
<211>141
<212>PRT
<213> Artificial sequence
<220>
<223>16H9 heavy chain polypeptide variable region (16H9H)
<400>55
Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly
1 5 10 15
Val Asn Ser Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile
35 40 45
Lys Asp Thr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu
50 55 60
Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp
65 70 75 80
Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn
85 90 95
Thr Ala Tyr Val Gln Leu Ser Sar Leu Thr Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ser Ser Asp Ile Tyr Tyr Gly Asn Pro Gly Gly Phe
115 120 125
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
130 135 140
<210>56
<211>129
<212>PRT
<213> Artificial sequence
<220>
<223>16H9 light chain polypeptide variable region (16H9L)
<400>56
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
20 25 30
Met Ser Ala Ser Leu Gly Glu Arg Val Thr Met Thr Cys Thr Ala Ser
35 40 45
Ser Ser Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly
50 55 60
Ser Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly
65 70 75 80
Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
85 90 95
Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His
100 105 110
Gln Tyr His Arg Ser Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu
115 120 125
Ile
<210>57
<211>406
<212>DNA
<213> Artificial sequence
<220>
<223>725.1 heavy chain polypeptide variable region (725.1H)
<400>57
atggcttggg tgtggacctt gctattcctg atggcagctg cccaaagtgc ccaagcacag 60
atccagttgg tgcagtctgg acctgaactg aagaagcctg gagagacagt caagatctcc 120
tgcaaggctt ctggatattc cttcacaaac tatggaatga actgggtgaa gcaggctcca 180
gggaagggtt taaagtggat gggctggata aacgcctaca ttggagagcc aacatatgct 240
gatgacttca agggacgatt tgccttctct ctggaagcct ctacccacac tgcctatttg 300
cagatcaaca gcctcaaaag tgaggacacg gctacatatt tctgtgcaag tgggggtaac 360
tcccttgact tttggggcca aggcaccact ctcacagtct cctcag 406
<210>58
<211>423
<212>DNA
<213> Artificial sequence
<220>
<223>16H9 heavy chain polypeptide variable region (16H9H)
<400>58
atgaaatgca gctgggttat cttcttcctg atggcagtgg ttacaggggt caattcagag 60
gttcagctgc agcagtctgg ggcagagctt gtgaagccag gggcctcagt caagttgtcc 120
tgcacagctt ctggcttcaa cattaaagac acctatatgc actgggtgaa gcagaggcct 180
gaacagggcc tggagtggat tggaaggatt gatcctgcga atggtaatac taaatatgac 240
ccgaagttcc agggcaaggc cactataaca gcagacacat cctccaacac agcctacgtg 300
cagctcagca gcctgacatc tgaggacact gccgtctatt actgtgctag tagtgacatc 360
tactatggta accccggggg gtttgcttac tggggccaag ggactctggt cactgtctct 420
gca 423
<210>59
<211>389
<212>DNA
<213> Artificial sequence
<220>
<223>16H9 light chain polypeptide variable region (16H9L)
<400>59
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctct aggggaacgg 120
gtcaccatga cctgcactgc cagctcaagt gtaagttcca gttacttgca ctggtaccag 180
cagaagccag gatcctcccc caaactctgg atttatagca catccaacct ggcttctgga 240
gtcccagctc gcttcagtgg cagtgggtct gggacctctt actctctcac aatcagcagc 300
atggaggctg aagatgctgc cacttattac tgccaccagt atcatcgttc cccattcacg 360
ttcggctcgg ggacaaagtt ggaaataaa 389
<210>60
<211>10
<212>PRT
<213> Artificial sequence
<220>
<223>725.1 VH1 CDR
<400>60
Gly Tyr Ser Phe Thr Asn Tyr Gly Met Asn
1 5 10
<210>61
<211>17
<212>PRT
<213> Artificial sequence
<220>
<223>725.1 VH2 CDR
<400>61
Trp Ile Asn Ala Tyr Ile Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys
1 5 10 15
Gly
<210>62
<211>7
<212>PRT
<213> Artificial sequence
<220>
<223>725.1 VH3 CDR
<400>62
Gly Gly Asn Ser Leu Asp Phe
1 5
<210>63
<211>10
<212>PRT
<213> Artificial sequence
<220>
<223>725.1 VL1 CDR
<400>63
Arg Ala Ser Ser Ser Val Ser Ser Ile His
1 5 10
<210>64
<211>7
<212>PRT
<213> Artificial sequence
<220>
<223>725.1 VL2 CDR
<400>64
Ala Thr Ser Asn Leu Ala Ser
1 5
<210>65
<211>9
<212>PRT
<213> Artificial sequence
<220>
<223>725.1 VL3 CDR
<400>65
Gln Gln Trp Ser Ile Asp Pro Ala Thr
1 5
<210>66
<211>10
<212>PRT
<213> Artificial sequence
<220>
<223>16H9 VH1 CDR
<400>66
Gly Phe Asn Ile Lys Asp Thr Tyr Met His
1 5 10
<210>67
<211>17
<212>PRT
<213> Artificial sequence
<220>
<223>16H9 VH2 CDR
<400>67
Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln
1 5 10 15
Gly
<210>68
<211>13
<212>PRT
<213> Artificial sequence
<220>
<223>16H9 VH3 CDR
<400>68
Ser Asp Ile Tyr Tyr Gly Asn Pro Gly Gly Phe Ala Tyr
1 5 10
<210>69
<211>12
<212>PRT
<213> Artificial sequence
<220>
<223>16H9 VL1 CDR
<400>69
Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His
1 5 10
<210>70
<211>7
<212>PRT
<213> Artificial sequence
<220>
<223>16H9 VL2 CDR
<400>70
Ser Thr Ser Asn Leu Ala Ser
1 5
<210>71
<211>9
<212>PRT
<213> Artificial sequence
<220>
<223>16H9 VL3 CDR
<400>71
His Gln Tyr His Arg Ser Pro Phe Thr
1 5

Claims (113)

1. An isolated antibody that preferentially binds to a cell-associated CA125/O772P polypeptide over an shed CA125/O772P polypeptide, wherein the antibody binds to SEQ ID NO: 1, residues 14-452.
2. The isolated antibody of claim 1, wherein the antibody exhibits less than 25% inhibition of binding to the peptide of figure 1 in an ELISA competition assay with a 25-fold excess (wt/wt) of shed CA125/O772P over the peptide of figure 1.
3. The isolated antibody of claim 2, wherein the antibody exhibits less than 20% inhibition of binding to the peptide of figure 1 in an ELISA competition assay with a 25-fold excess (wt/wt) of shed CA125/O772P over the peptide of figure 1.
4. The isolated antibody of claim 3, wherein the antibody exhibits less than 15% inhibition of binding to the peptide of figure 1 in an ELISA competition assay with a 25-fold excess (wt/wt) of shed CA125/O772P over the peptide of figure 1.
5. The isolated antibody of claim 4, wherein the antibody exhibits less than 10% inhibition of binding to the peptide of figure 1 in an ELISA competition assay with a 25-fold excess (wt/wt) of shed CA125/O772P over the peptide of figure 1.
6. The isolated antibody of claim 5, wherein the antibody exhibits less than 5% inhibition of binding to the peptide of figure 1 in an ELISA competition assay with a 25-fold excess (wt/wt) of shed CA125/O772P over the peptide of figure 1.
7. The isolated antibody of claim 1, wherein the antibody exhibits an IC as measured by percent positive cells in a flow cytometry assay50Is at least 0.05mg/ml shed CA 125/O772P.
8. The isolated antibody of claim 7, wherein the antibody exhibits an IC as measured by the percentage of positive cells in a flow cytometry assay50Is at least 0.25mg/ml shed CA 125/O772P.
9. The isolated antibody of claim 8, wherein the antibody exhibits an IC as measured by the percentage of positive cells in a flow cytometry assay50Is at least 0.5mg/ml shed CA 125/O772P.
10. The isolated antibody of claim 9, wherein the antibody exhibits an IC as measured by the percentage of positive cells in a flow cytometry assay50Is at least 0.75mg/ml shed CA 125/O772P.
11. The isolated antibody of claim 10, wherein the antibody exhibits an IC as measured by the percentage of positive cells in a flow cytometry assay50Is at least 1.0mg/ml shed CA 125/O772P.
12. The isolated antibody of claim 1, wherein the antibody binds to the peptide of figure 1, but does not detect binding to shed CA 125/O772P.
13. The isolated antibody of claim 1, wherein the antibody is an IgG class antibody.
14. The isolated antibody of claim 13, wherein the antibody is an IgG1Isoforms.
15. The isolated antibody of claim 1, wherein the antibody is a monoclonal antibody.
16. The isolated antibody of claim 15, wherein the antibody is a chimeric monoclonal antibody.
17. The isolated antibody of claim 16, wherein the chimeric monoclonal antibody comprises a C γ 1 constant region.
18. The isolated antibody of claim 16, wherein the chimeric monoclonal antibody comprises a C γ 4 constant region.
19. The isolated antibody of claim 1, wherein the antibody is a humanized monoclonal antibody.
20. The isolated antibody of claim 15, wherein the antibody is a human monoclonal antibody.
21. The isolated antibody of claim 1, wherein the antibody is a bispecific antibody.
22. The isolated antibody of claim 1, wherein the antibody is a multispecific antibody.
23. The isolated antibody of claim 1, wherein the antibody is a chimeric antibody.
24. The isolated antibody of claim 1, wherein the antibody is a single chain antibody, a disulfide linked Fvs, a single chain Fvs, or an anti-idiotypic antibody.
25.Hybridoma 4E7 with accession number PTA-5109,Hybridoma 7A11 having accession number PTA-5110,Hybridoma 7C6 having accession number PTA-5111, Hybridoma 7F10 having accession number PTA-5112,Hybridoma 7G10 having accession number PTA-5245,Hybridoma 7H1 having accession number PTA-5114,Hybridoma 8A1 having accession number PTA-5115,Hybridoma 8B5 having accession number PTA-5116,Hybridoma 8C3 having accession number PTA-5246,Hybridoma 8E3 having accession number PTA-5118,Hybridoma 8G9 having accession number PTA-5119,Hybridoma 15C9 with accession number PTA-5106,Hybridoma 16C7 with accession number PTA-5107,Hybridoma 16H9 with accession number PTA-5108,Hybridoma 117.1 of accession number PTA-4567,325.1 part of hybridoma with accession number PTA-5120,Hybridoma 368.1 having accession number PTA-4568,The 446.1 portions of the hybridoma with the record number PTA-5549,Hybridoma 501.1 of accession number PTA-4569,621.1 portion of hybridoma having accession number PTA-5121,Hybridoma 633.1, accession number PTA-5122,Hybridoma 654.1 of accession number PTA-5247,Hybridoma 725.1, accession number PTA-5124, orThe monoclonal antibody produced by hybridoma 776.1 having accession number PTA-4570.
26. Is preserved asHybridoma 4E7 with accession number PTA-5109,Hybridoma 7A11 having accession number PTA-5110,Hybridoma 7C6 having accession number PTA-5111,Hybridoma 7F10 having accession number PTA-5112,Hybridoma 7G10 having accession number PTA-5245,Hybridoma 7H1 having accession number PTA-5114, Hybridoma 8A1 having accession number PTA-5115,Hybridoma 8B5 having accession number PTA-5116,Hybridoma 8C3 having accession number PTA-5246,Hybridoma 8E 3 with accession number PTA-5118,Hybridoma 8G9 having accession number PTA-5119,Hybridoma 15C9 with accession number PTA-5106,Hybridoma 16C7 with accession number PTA-5107,Hybridoma 16H9 with accession number PTA-5108,Hybridoma 117.1 of accession number PTA-4567,325.1 part of hybridoma with accession number PTA-5120,Hybridoma 368.1 having accession number PTA-4568,The 446.1 portions of the hybridoma with the record number PTA-5549,Hybridoma 501.1 of accession number PTA-4569,621.1 portion of hybridoma having accession number PTA-5121,Hybridoma 633.1, accession number PTA-5122,Hybridoma 654.1 of accession number PTA-5247,Hybridoma 725.1, accession number PTA-5124, orHybridoma of hybridoma 776.1, accession number PTA-4570.
27. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 27, or a light chain variable region of the amino acid sequence depicted in seq id No. 27.
28. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 29, or a light chain variable region of the amino acid sequence depicted in seq id No. 29.
29. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 31, or a light chain variable region of the amino acid sequence depicted in seq id No. 31.
30. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 33, or a light chain variable region of the amino acid sequence depicted in seq id no.
31. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 54, or a light chain variable region of the amino acid sequence depicted in seq id no.
32. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 56, or a light chain variable region of the amino acid sequence depicted in seq id no.
33. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 28, or a heavy chain variable region of the amino acid sequence depicted in seq id no.
34. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 30, or a heavy chain variable region of the amino acid sequence depicted in seq id no.
35. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 32, or a heavy chain variable region of the amino acid sequence depicted in seq id no.
36. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 34, or a heavy chain variable region of the amino acid sequence depicted in seq id no.
37. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 53, the heavy chain variable region of the amino acid sequence depicted in seq id no.
38. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 55, or a heavy chain variable region of the amino acid sequence depicted in seq id no.
39. The isolated antibody of claim 27, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 28, or a heavy chain variable region of the amino acid sequence depicted in seq id no.
40. The isolated antibody of claim 28, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 30, or a heavy chain variable region of the amino acid sequence depicted in seq id no.
41. The isolated antibody of claim 29, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 32, or a heavy chain variable region of the amino acid sequence depicted in seq id no.
42. The isolated antibody of claim 30, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 34, or a heavy chain variable region of the amino acid sequence depicted in seq id no.
43. The isolated antibody of claim 31, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 53, the heavy chain variable region of the amino acid sequence depicted in seq id no.
44. The isolated antibody of claim 32, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO: 55, or a heavy chain variable region of the amino acid sequence depicted in seq id no.
45. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody of any one of claims 27 to 38.
46. The nucleic acid molecule of claim 45, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 35. 36, 37, 38, 39, 40, 41, 42, 52, 57, 58, or 59.
47. The isolated antibody of claim 1, wherein the K for the antibody to bind the peptide of figure 1 is measured in a BIAcore affinity assaydLess than 100 nM.
48. The isolated antibody of claim 47, wherein the K for the antibody to bind to the peptide of figure 1 is measured in a BIAcore affinity assaydLess than 10 nM.
49. The isolated antibody of claim 48, wherein the K for the antibody to bind to the peptide of figure 1 is measured in a BIAcore affinity assaydLess than 1 nM.
50. The isolated antibody of claim 49, wherein the K for the antibody to bind to the peptide of figure 1 is measured in a BIAcore affinity assaydLess than 100 pM.
51. The isolated antibody of claim 50, wherein the K for the antibody to bind to the peptide of figure 1 is measured in a BIAcore affinity assaydLess than 10 pM.
52. The antibody of claim 1, wherein the antibody is modified by amino acid substitutions, deletions, or additions, or a combination thereof, and has the same or greater affinity for cell-associated CA125/O772P relative to a corresponding unmodified antibody, and wherein the antibody binds to the amino acid sequence of SEQ ID NO: 1, residues 14-452.
53. The isolated antibody of claim 1, wherein the antibody is modified by amino acid substitutions, deletions, or additions, or a combination thereof, and wherein the antibody exhibits the same or greater serum half-life as a corresponding unmodified antibody, and wherein the antibody binds to the amino acid sequence of SEQ ID NO: 1, residues 14-452.
54. The isolated antibody of claim 1, wherein the antibody mediates lysis of CA 125/O772P-positive tumor cells in an ADCC assay.
55. The isolated antibody of claim 54, wherein the antibody mediates at least 10% lysis of CA 125/O772P-positive tumor cells in an ADCC assay at an effector to target ratio of 50: 1 and a concentration of 5.0 μ g/ml of antibody.
56. The isolated antibody of claim 54, wherein the antibody mediates at least 10% lysis of CA 125/O772P-positive tumor cells in an ADCC assay at an effector to target ratio of 25: 1 and a concentration of 5.0 μ g/ml of antibody.
57. The isolated antibody of claim 54, wherein the antibody mediates at least 10% lysis of CA 125/O772P-positive tumor cells in an ADCC assay at an effector to target ratio of 12.5: 1 and a concentration of the antibody of 5.0 μ g/ml.
58. The isolated antibody of claim 54, wherein the antibody mediates at least 10% lysis of CA 125/O772P-positive tumor cells in an ADCC assay at an effector to target ratio of 12.5: 1 at a concentration of 50ng/ml of antibody.
59. The isolated antibody of claim 1, wherein the antibody mediates lysis of CA 125/O772P-positive tumor cells in a Complement Dependent Cytotoxicity (CDC) assay.
60. The isolated antibody of claim 59, wherein the antibody mediates from 15% lysis at 5 μ g/ml antibody to 95% lysis at 0.1 μ g/ml antibody.
61. The isolated antibody of claim 1, wherein the antibody inhibits CA 125/O772P-positive tumor growth.
62. A pharmaceutical composition comprising an antibody that preferentially binds to a cell associated CA125/O772P polypeptide over an shed CA125/O772P polypeptide, and a pharmaceutically acceptable carrier, wherein the antibody binds to the amino acid sequence of SEQ ID NO: 1, residues 14-452.
63. A pharmaceutical composition comprising a monoclonal antibody that preferentially binds to a cell associated CA125/O772P polypeptide over an shed CA125/O772P polypeptide, and a pharmaceutically acceptable carrier, wherein the antibody binds to the amino acid sequence of SEQ ID NO: 1, residues 14-452.
64. A product comprising a packaging material and a pharmaceutical composition contained in the packaging material, wherein the pharmaceutical composition comprises an antibody that preferentially binds to a cell-associated CA125/O772P polypeptide over an shed CA125/O772P polypeptide, wherein the antibody binds to the amino acid sequence of seq id NO: 1, and residues 14-452, in a form suitable for administration to a subject.
65. The product of claim 64, further comprising printed instructions for use of the pharmaceutical composition or administration.
66. The product of claim 65 wherein the instructions for use recommend a dosing regimen for preventing or treating one or more symptoms of a CA 125/O772P-related disorder.
67. The article of manufacture of claim 65, wherein the instructions for use recommend a dosing regimen for preventing or treating one or more symptoms of a cell proliferative disorder.
68. The product of claim 67, wherein the instructions for use recommend a dosing regimen for preventing or treating one or more symptoms of cancer.
69. The product of claim 68 wherein the instructions for use recommend a dosing regimen for preventing or treating one or more symptoms of ovarian cancer.
70. The product of claim 64, further comprising a label for use or administration of the pharmaceutical composition.
71. The product of claim 70 wherein the label recommends a dosing regimen for preventing or treating one or more symptoms of a CA 125/O772P-related disorder.
72. The article of manufacture of claim 70, wherein the label recommends a dosing regimen for preventing or treating one or more symptoms of a cell proliferative disorder.
73. The product of claim 72 wherein the label recommends a dosing regimen for preventing or treating one or more symptoms of cancer.
74. The product of claim 73 wherein the label recommends a dosing regimen for preventing or treating one or more symptoms of ovarian cancer.
75. A fusion polypeptide comprising an antibody that preferentially binds to cell associated CA125/O772P relative to shed CA125/O772P operably linked to a heterologous agent, wherein the antibody binds to the amino acid sequence of SEQ ID NO: 1, residues 14-452.
76. Use of an antibody in the manufacture of a medicament for ameliorating a symptom of a CA 125/O772P-related disorder, wherein the antibody preferentially binds to cell associated CA125/O772P relative to shed CA125/O772P, and wherein the antibody binds to the amino acid sequence of SEQ ID NO: 1, residues 14-452.
77. The use of claim 76, wherein the CA 125/O772P-related disorder is a cell proliferative disorder.
78. The use of claim 77, wherein the cell proliferative disorder is cancer.
79. The use of claim 78, wherein the cancer is cervical or uterine cancer.
80. The use of claim 78, wherein the cancer is breast or lung cancer.
81. The use of claim 78, wherein the cancer is ovarian cancer.
82. The use of claim 76, wherein the antibody is a monoclonal antibody.
83. The use of claim 76, wherein the medicament is for use in combination cancer therapy.
84. The use of claim 83, wherein the medicament is used in combination with a chemotherapeutic agent.
85. The use of claim 84, wherein the chemotherapeutic agent is paclitaxel or cisplatin.
86. The use of claim 83, wherein the medicament is used in combination with radiation therapy.
87. The use of claim 76, wherein the antibody is selected from the group consisting of325.1 part of hybridoma with accession number PTA-5120,621.1 portion of hybridoma having accession number PTA-5121,Hybridoma 633.1, accession number PTA-5122,Hybridoma 654.1 of accession number PTA-5247,Hybridoma 725.1 with accession number PTA-5124,Hybridoma 8G9 having accession number PTA-5119,Hybridoma 7F10 having accession number PTA-5112,Hybridoma 8A1 having accession number PTA-5115,Hybridoma 8C3 having accession number PTA-5246,Hybridoma 15C9 with accession number PTA-5106,Hybridoma 8E3 having accession number PTA-5118,Hybridoma 8B5 having accession number PTA-5116,Hybridoma 7G10 having accession number PTA-5245,Hybridoma 16C7 with accession number PTA-5107,Hybridoma 7C6 having accession number PTA-5111,Hybridoma 7H1 having accession number PTA-5114,Hybridoma 16H9 with accession number PTA-5108,Hybridoma 7A11 having accession number PTA-5110,Hybridoma 4E7 with accession number PTA-5109,Hybridoma 117.1 of accession number PTA-4567,Hybridoma 368.1 having accession number PTA-4568, The 446.1 portions of the hybridoma with the record number PTA-5549,Hybridoma 501.1 having accession number PTA-4569 andantibody produced by hybridoma 776.1, accession number PTA-4570.
88. A method of aiding in the identification of an antibody of claim 1 that preferentially binds to cell associated CA125/O772P, the method comprising:
(a) incubating the antibody with the peptide comprising cell-associated CA125/O772P in the presence of shed CA125/O772P under conditions that allow the antibody to bind to the peptide comprising cell-associated CA125/O772P or shed CA 125/O772P;
(b) removing the antibody that does not bind to the cell associated CA125/O772P containing peptide and shed CA 125/O772P;
(c) measuring the amount of antibody bound to the cell-associated CA 125/O772P-containing peptide; and
(d) comparing the amount in (c) to the amount of antibody that binds to the cell-associated CA 125/O772P-containing peptide in the absence of shed CA 125/O772P.
89. The method of claim 88, wherein the peptide comprising cell-associated CA125/O772P is immobilized on a solid surface.
90. The method of claim 89, wherein the method is performed in an ELISA format.
91. The method of claim 88, wherein the shed CA125/O772P and the peptide comprising cell-associated CA125/O772P are present in a ratio of about 25: 1(wt/wt) shed CA125/O772P to peptide comprising cell-associated CA 125/O772P.
92. A method of facilitating the identification of an antibody of claim 1 that preferentially binds to cell associated CA125/O772P, the method comprising:
(a) contacting the antibody with the peptide comprising cell-associated CA125/O772P in the presence of shed CA125/O772P under conditions that allow the peptide comprising cell-associated CA125/O772P to bind to the antibody;
(b) removing unbound said peptide comprising cell-associated CA 125/O772P;
(c) measuring the amount of peptide containing cell-associated CA125/O772P bound by the antibody; and
(d) comparing the amount measured in (c) to the amount of peptide containing cell-associated CA125/O772P bound by the antibody in the absence of shed CA 125/O772P.
93. The method of claim 92, wherein the amount of shed CA125/O772P is about a 25-fold (weight/weight) excess.
94. The method of claim 92, wherein the antibody is immobilized on a solid surface.
95. The method of claim 94, wherein the method is performed in an ELISA format.
96. A method of facilitating the identification of an antibody of claim 1 that preferentially binds to cell associated CA125/O772P, the method comprising:
(a) contacting the antibody with a cell expressing CA125/O772P in the presence of an amount of shed CA125/O772P under conditions that allow CA125/O772P to bind to the antibody;
(b) Removing unbound cells;
(c) measuring the amount of CA 125/O772P-expressing cells bound by the antibody;
(d) comparing the amount measured in (c) to the amount of CA 125/O772P-expressing cells that bound the antibody in the absence of the amount of shed CA 125/O772P.
97. The method of claim 96, wherein the amount of shed CA125/O772P is at least about 0.5 mg/ml.
98. The method of claim 96, wherein the measuring is performed by flow cytometry.
99. The method of claim 96, wherein the measuring is performed by fluorescence activated cell sorting.
100. A hybridoma capable of secreting an antibody according to claim 1.
101. The isolated antibody of claim 1 conjugated to a cytotoxic agent.
102. The isolated antibody of claim 101, wherein the cytotoxic agent is a radioisotope.
103. The isolated antibody of claim 102, wherein the radioisotope is selected from the group consisting of125I、131I、111In、99mTc and90Y。
104. the monoclonal antibody of claim 25 conjugated to a cytotoxic agent.
105. The monoclonal antibody of claim 104, wherein the cytotoxic agent is a radioisotope.
106. The monoclonal antibody of claim 105, wherein the radioisotope is selected from the group consisting of125I、131I、111In、99mTc and90Y。
107. the pharmaceutical composition of claim 62 or 63, wherein the antibody is conjugated to a cytotoxic agent.
108. The pharmaceutical composition of claim 107, wherein the cytotoxic agent is a radioisotope.
109. The pharmaceutical composition of claim 108, wherein the radioisotope is selected from the group consisting of125I、131I、111In、99mTc and90Y。
110. the use of claim 76, wherein the antibody is conjugated to a cytotoxic agent.
111. The use of claim 110, wherein the cytotoxic agent is a radioisotope.
112. The use of claim 111, wherein the radioisotope is selected from the group consisting of125I、131I、111In、99mTc and90Y。
113. pharmaceutical composition comprising a compound selected from the group consisting of325.1 part of hybridoma with accession number PTA-5120,621.1 portion of hybridoma having accession number PTA-5121,Hybridoma 633.1, accession number PTA-5122,Hybridoma 654.1 of accession number PTA-5247,Hybridoma 725.1 with accession number PTA-5124,Hybridoma 8G9 having accession number PTA-5119,Hybridoma 7F10 having accession number PTA-5112,Hybridoma 8A1 having accession number PTA-5115,Hybridoma 8C3 having accession number PTA-5246,Hybridoma 15C9 with accession number PTA-5106,Hybridoma 8E3 having accession number PTA-5118,Hybridoma 8B5 having accession number PTA-5116,Hybridoma 7G10 having accession number PTA-5245,Hybridoma 16C7 with accession number PTA-5107,Hybridoma 7C6 having accession number PTA-5111,Hybridoma 7H1 having accession number PTA-5114,Hybridoma 16H9 with accession number PTA-5108, Hybridoma 7A11 having accession number PTA-5110,Hybridoma 4E7 with accession number PTA-5109,Impurities of accession number PTA-4567117.1 portions of tumor,Hybridoma 368.1 having accession number PTA-4568,The 446.1 portions of the hybridoma with the record number PTA-5549,Hybridoma 501.1 having accession number PTA-4569 anda monoclonal antibody to the antibody produced by hybridoma 776.1, accession number PTA-4570, and a pharmaceutically acceptable carrier.
HK06106277.5A 2002-10-16 2003-10-15 Antibodies that bind cell-associated ca 125/0722p and methods of use thereof HK1086283B (en)

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US41882802P 2002-10-16 2002-10-16
US60/418,828 2002-10-16
US48598603P 2003-07-10 2003-07-10
US60/485,986 2003-07-10
PCT/US2003/032945 WO2004035537A2 (en) 2002-10-16 2003-10-15 Antibodies that bind cell-associated ca 125/o772p and methods of use thereof

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