Patent Application
20080145875
The present invention concerns immunoassays and kits for the analysis of tissue samples and the detection and diagnosis of tumors and cancers.
Tenascin is a polymorphic extracellular matrix glycoprotein that is over-expressed in a variety of tumors including gliomas, melanomas and breast carcinomas. See Bourdon et al., Cancer Res. 43: 2796-2805 (1983); Howeedy et al., Lab. Invest. 63: 798-806 (1990); and Mackie et al., Proc. Natl. Acad. Sci. USA. 84: 4621-4625 (1987).
Bigner et al., U.S. Pat. No. 5,624,659, describes methods of treating solid and cystic tumors with monoclonal antibody 81C6. See also D. Bigner et al., J. Clin. Oncol. 16:2202-2212 (1998).
Rizzieri et al., U.S. patent application Ser. No. 10/008,062 (Publication No. US-2002-0187100-A1) describes anti-tenascin monoclonal antibody therapy for the treatment of lymphoma. See also D. Rizzieri et al., Blood 104, 642-648 (2004) (prepublished online Apr. 20, 2004); G. Akabani, G. et al., Int. J. Radiat. Oncol. Biol. Phys. 46:947-958 (2000).
Abrams et al., U.S. Pat. No. RE 38,008, concerns methods of improved cell targeting of antibody, antibody fragments, hormones and other targeting agents, and conjugates thereof.
There is, however, a need for specific, immunoassays and diagnostic kits for the analysis of tissue samples and the detection and diagnosis of tumors and cancers as well methods that provide an indication of potential patient response to therapy for the treatment of tumors and cancers.
A first aspect of the invention relates to an immunoassay for detecting a tumor in a subject, comprising producing an antibody that specifically binds to tenascin, contacting the antibody with a biological sample suspected of containing tumor cells and determining the binding of the antibody to the biological sample. The antibody that binds to tenascin can be selected from the group consisting of monoclonal antibody 81C6 and an antibody that binds to the epitope bound by monoclonal antibody 81C6. The antibody that binds to tenascin can further be an antibody that specifically binds to tenascin domain TNfn C-Dhis.
A further aspect of the invention relates to a method of identifying a subject for treatment of a tumor comprising contacting an antibody that specifically binds to tenascin with a biological sample suspected of containing tumor cells, determining the binding of the antibody to the biological sample and assessing the overexpression of tenascin, wherein assessment of tenascin overexpression indicates that the subject is a candidate for treatment of a tumor comprising administering an antibody selected from the group consisting of monoclonal antibody 81C6 and an antibody that binds to the epitope bound by monoclonal antibody 81C6.
Additional aspects of the present invention relate to kits for a direct immunohistochemical or immunocytochemical assay comprising (a) an antibody that specifically binds to tenascin, the antibody labeled with a detectable group, and (b) instructions for use thereof in the immunohistochemical or immunocytochemical assay.
Further aspects of the present invention relate to kits for an indirect immunohistochemical or immunocytochemical assay comprising (a) a primary antibody that specifically binds to tenascin, (b) a secondary antibody that specifically binds to the primary antibody, wherein the secondary antibody is labeled with a detectable group, and (c) instructions for use thereof in the indirect immunohistochemical or immunocytochemical assay.
In still further aspects of the present invention, for the kits described herein, the extent of binding of the antibody to tenascin can be used to detect the presence of a tumor in a subject or identify subjects for treatment of a tumor comprising administering an antibody selected from the group consisting of monoclonal antibody 81C6 and antibodies that bind to the epitope bound by monoclonal antibody 81C6. Moreover, the kits can be packaged in a container and can also comprise control samples, wherein the control samples are positive, negative or both.
Additional aspects of the present invention relate to a novel antibody that specifically binds to tenascin domain TNfn C-Dhis.
The foregoing and other objects and aspects of the present invention are explained in detail in the drawings herein and the specification set forth below.
It should be noted that as used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the invention.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
The term “biological sample” as used herein refers to a fluid (blood, serum, urine, semen), intact cells or extracts thereof, or tissue samples. The biological sample may be a clinical cytology specimen (e.g., fine needle breast biopsy or pulmonary cytology specimen) or a human tissue specimen from, for example, stomach, lung, breast, ovarian, pancreatic, prostate or brain tumors. The tissue specimen may be fresh or frozen.
The terms “monoclonal antibody 81C6”, “antibody 81C6”, or similar terms encompass both the murine monoclonal antibody 81C6 and the humanized chimeric antibody 81C6, both of which are described in U.S. Pat. No. 6,624,659. Such monoclonal antibodies are produced in accordance with known techniques.
The term “antibodies” as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The term “immunoglobulin” includes the subtypes of these immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, etc. Of these immunoglobulins, IgM and IgG are preferred, and IgG is particularly preferred. The antibodies may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26, 403-11 (1989). The term “antibody” as used herein includes antibody fragments which retain the capability of binding to a target antigen, for example, Fab, F(ab′)2, and Fv fragments, and the corresponding fragments obtained from antibodies other than IgG. Such fragments are also produced by known techniques.
The term “polyclonal antibody” as used herein refers to multiple immunoglobulins in antiserum produced to an antigen following immunization, and which may recognize and bind to one or more epitopes to that antigen. Polyclonal antibodies used to carry out the present invention can be produced by immunizing a suitable subject of any species of origin, including (for example) mouse, rat, rabbit, goat, sheep, chicken, donkey, horse or human, with an antigen to which a monoclonal antibody to the target binds, collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, in accordance with known procedures.
The term “primary antibody” as used herein refers to an antibody which binds specifically to the target protein antigen in a tissue sample. A primary antibody is generally the first antibody used in an immunohistochemical procedure. The primary antibody can be the only antibody used in an immunohistochemical procedure.
The term “secondary antibody” as used herein refers to an antibody which binds specifically to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent, if any. The secondary antibody is generally the second antibody used in an immunohistochemical procedure.
Subjects of the present invention include both human subjects for medical purposes and animal subjects for veterinary and drug screening and development purposes. Suitable animal subjects include both avians and mammals, with mammals being preferred. The term “avian” as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys and pheasants. The term “mammal” as used herein includes, but is not limited to, primates, bovines, ovines, caprines, porcines, equines, felines, canines, lagomorphs, rodents (e.g., rats and mice), etc. Human subjects are the most preferred. Human subjects include fetal, neonatal, infant, juvenile and adult subjects.
Moreover, subjects described herein include subjects afflicted with or suspected of being afflicted with lymphoma, as well as subjects afflicted with or suspected of being afflicted with solid tumors or cancers such as lung, colon, breast, brain, liver, prostate, spleen, muscle, ovary, pancreas, skin (including melanoma), etc.
The monoclonal antibodies of the present invention may be recombinant monoclonal antibodies produced according to the methods disclosed in Reading, U.S. Pat. No. 4,474,893, or Cabilly et al., U.S. Pat. No. 4,816,567. The antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in Segel et al., U.S. Pat. No. 4,676,980. Applicants specifically intend that the disclosure of all U.S. patent references cited herein be incorporated herein by reference in their entirety.
Monoclonal antibodies may be chimeric antibodies produced in accordance with known techniques. For example, chimeric monoclonal antibodies may be complementarily determining region-grafted antibodies (or “CDR-grafted antibodies”) produced in accordance with known techniques.
Monoclonal Fab fragments may be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246, 1275-81 (1989).
As noted above, antibodies employed in carrying out the present invention are those which bind to tenascin. In some embodiments of the present invention, the antibody can be monoclonal antibody 81C6 or an antibody that binds to the epitope bound by monoclonal antibody 81C6 (i.e., antibodies that cross-react with, or block the binding of, monoclonal antibody 81C6). The monoclonal antibody 81C6 is a murine IgG2b monoclonal antibody raised from a hybridoma fusion following immunization of BALB/c mice with the glial fibrillary acidic protein (GFAP)-expressing permanent human glioma line U-251 MG, as known and described in M. Bourdon et al., Cancer Res. 43, 2796 (1983). In other embodiments of the present invention, the antibody can be a polyclonal antibody against a spliced variant of tenascin.
Particularly preferred for carrying out the present invention is a mouse-human chimeric monoclonal antibody 81C6, as described in U.S. Pat. No. 5,624,659 to Bigner and Zalutsky, or a rabbit polyclonal antibody, anti-TNfn C-D, as further described in the examples section below.
Antibodies for use in the present invention specifically bind to tenascin with a relatively high binding affinity, for example, with a dissociation constant of about 10−4 to 10−13. In embodiments of the invention, the dissociation constant of the antibody-tenascin complex is at least 10−4, preferably at least 10−6, and more preferably at least 10−9.
Antibodies of the present invention may be coupled to a radioisotope. The antibody can be coupled to a radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991). Examples of radioisotopes which may be coupled to the antibody include, but are not limited to, 227Ac, 211At, 131Ba, 77Br, 14C, 109Cd, 51Cr, 67Cu, 165Dy, 155Eu, 153 Gd, 198Au, 3H, 166Ho, 113mIn, 115mIn, 123I, 125I, 131I, 189Ir, 191Ir, 192Ir, 194Ir, 52Fe, 55Fe, 59Fe, 177Lu, 109Pd, 32P, 226Ra, 186Re, 188Re, 153Sm, 46Sc, 47Sc, 72Se, 75Se, 105Ag, 89Sr, 35S, 177Ta, 117mSn, 121Sn, 166Yb, 169Yb, 90Yt, 212Bi, 119Sb, 197Hg, 97Ru, 100Pd, 101mRh, and 212Pb.
It will be appreciated that monoclonal antibodies as used herein incorporate those portions of the constant region of an antibody necessary to evoke the useful immunological response in the subject being affected.
Examples of tumors, cancers, and neoplastic tissue that can be detected and/or diagnosed according to the present invention include, but are not limited to, malignant disorders such as breast cancers; osteosarcomas; angiosarcomas; fibrosarcomas and other sarcomas; leukemias; lymphomas (Hodgkin's lymphoma and Non-Hodgkin's lymphoma), and other blood cancers; myelodysplasia, myeloproliferative disorders; sinus tumors; ovarian, uretal, bladder, prostate and other genitourinary cancers; colon, esophageal and stomach cancers and other gastrointestinal cancers; lung cancers; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; and brain or central and peripheral nervous system tumors, malignant or benign, including gliomas and neuroblastomas.
Immunohistochemical (IHC) methods are well known by those skilled in the art. See, for example, U.S. Pat. No. 6,441,143 to Koski et al., U.S. Pat. No. 6,376,201 to Miron et al., U.S. Pat. No. 5,876,712 to Cheever et al., U.S. Pat. No. 5,854,009 to Klug, and U.S. Pat. No. 5,843,684 to Levine et al., U.S. Pat. No. 4,968,603 to Slamon et al. and “DAKO anti-Her2 IHC System for Immunoenzymatic Staining” (Package Insert) DAKO Corporation. As described in U.S. Pat. No. 6,573,043 to Cohen et al., two general methods of IHC are available: direct and indirect assays. According to the first assay, binding of an antibody to the target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction. The fluorescent tag or label can be fluorescein. The enzymatic label can be horseradish peroxidase or alkaline phosphatase.
In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. Where the secondary antibody is conjugated to an enzymatic label, a chromagenic or fluorogenic substrate can be added to provide visualization of the antigen. Such are described above. Signal amplification may occur because several secondary antibodies may react with different epitopes on the primary antibody. The primary and/or secondary antibody used for immunohistochemistry typically can be labeled with a detectable moiety. IHC techniques are further described in Immunohistochemical Staining Methods. Thomas Boenisch, ed. (3rd ed. 2001).
The present invention will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.
An 81C6 column was prepared according to the following protocol.
Weigh out CNBr activated Sepharose-4B, place into plastic centrifuge tube and swell in deionized water. One gram of dry Sepharose is 2-3 ml of swollen gel. Use 1 ml of gel for every 5 mg of 81C6 used. When gel is swollen remove water by centrifuging at 500×g. Discard supernatant and add 81C6 (1-2 mg/ml) in 115 mM Phosphate buffer, pH 7.4. Rock for two hours at room temperature and then overnight at 4° C. Remove non-bound 81C6 by centrifuging at 500×g. Save supernatant and read A280 Calculate percent bound to Sepharose so you know total 81C6 bound. About 10 mg 81C6 bound is desired to bind 1 mg of Tenascin later. Wash Sepharose two more times and then add 1 M ethanolamine in 115 mM phosphate buffer and react for one hour at room temperature. Pour Sepharose into column and wash column with pH 11 Caps buffer and then with pH 3.5 citrate buffer (removes charged bound 81C6). Equilibrate column with 115 mM phosphate buffer and 0.5% Na Azide and store at 4° C.
A tenascin column was prepared according to the following protocol.
Weigh out CNBr activated Sepharose-4B, place into plastic centrifuge tube and swell in deionized water. One gram of dry Sepharose is 2-3 ml of swollen gel. Use 1 ml of gel for every milligram of tenascin used. When gel is swollen remove water by centrifuging at 500×g. Discard supernatant and add tenascin (0.1-1 mg/ml) in 0.1 M borate buffer, pH 8.5. Rock for two hours at room temperature and then overnight at 4° C. Remove non-bound tenascin by centrifuging at 500×g. Save supernatant and read A280. Calculate percent bound to Sepharose so you know total tenascin bound. Wash Sepharose two more times and then add 1 M ethanolamine in 115 mM phosphate buffer and react for one hour at room temperature. Pour Sepharose into column and wash column with pH 11 Caps buffer and then equilibrate column with 115 mM phosphate buffer and 0.5% Na Azide and store at 4° C. It is preferred that acid pH buffer is not used on the tenascin column.
Tenascin immunoaffinity purification was carried out according to the procedures set forth below.
Rabbit anti-tenascin immuno-affinity purification was carried out according to the following procedure.
Tenascin was initially purified from U-251 MG-C13 supernatant by immunoaffinity chromatography using the murine anti-tenascin MAb 81C6 (Bourdon et al., 1983). Culture supernatant was passed over an 81C6-Sepharose 4B affinity column at room temperature, the column was washed with 10 mM Tris plus 500 mM NaCl (pH 8.0), and the tenascin was eluted with 0.1 mM CAPS in 500 mM NaCl (pH 11.0) into tubes containing 30 ng of glycine per ml of eluate to neutralize the pH to approximately 8.3. Tenascin used for polyclonal antibody preparation was subjected to an additional glycerol gradient-sedimentation purification step (Erickson and Taylor, 1987).
Polyclonal antiserum to tenascin was prepared against affinity purified tenascin. 5 μg of tenascin in Freund's complete adjuvant was injected s.c. into rabbits; nine subsequent monthly i.v. boosts of 5 p.g were administered, with high titers (1:50,000 against purified human tenascin) noted after the second boost. Antiserum from a bleed drawn 11 days after the second boost was used for all studies. No reactivity of this antiserum to ZO +10% FBS or to purified human fibronectin was noted on immunoblots (data not shown). See Ventimiglia J. B. et al., Journal of Neuroimmunology, 36 (1992) 41-55.
2.213 gms CAPS.
2.922 gms NaCl.
Dissolve in 100 ml of deionized water and adjust pH to 11.0 with HCl.
Glycine-HCl buffer pH 3.0.
41.83 gms glycine.
8.5 gms NaCl.
8.3 ml concentrated (12 N)HCl.
Dissolve in 1000 ml deionized H20, check pH and adjust to 3.0.
60.55 gms Tris base.
Q.S. to 500 ml with deionized H2O.
Adjust pH with HCl to pH 8.0 or 9.0.
Tris-0.15M NaCl buffer.
8.5 gms NaCl.
10 ml 1.0 M Tris-buffer pH 8.0.
Q.S. to 1000 ml with deionized H2O.
Tris-0.5M NaCl buffer.
22.13 gms NaCl.
10 ml 1.0 M Tris-buffer pH 8.0. Q.S. to 1000 ml with deionized H2O
Phosphate Buffer (0.115M phosphate). 5× stock concentrate.
90.65 gms NaH2PO4
373.05 gms Na2HPO4
Q.S. to 6 liters with deionized H2O and pH should be between 7.3 and 7.4
Dilute 1 part with 4 parts deionized H2O for working solution.
0.1 M sodium citrate 29.4 gms/liter deionized H2O.
0.1 M citric acid 21 gms/liter deionized H2O.
Adjust pH of 0.1 M citric acid solution to pH 3.5 with 0.1M sodium citrate solution.
0.1M Na Borate
0.5M NaCl
Adjust pH to 8.5
Tenascin domain TNfn C-Dhis expressing E. coli were grown in superbroth in an orbital shaker at 37° C. until it reached an optical density at A600 of 1.5 to 2.0. Proteins were then expressed by the addition of IPTG to a concentration of 1 mM. Cultures were incubated for another 90 minutes and centrifuged at 13,000×g for 10 minutes. Bacterial pellets were resuspended in 50 mM Tris and 0.5 M NaCl; pH 8.0 buffer (TBS). Thirty milliliters TBS was added per 10 gms of bacteria and pellet resuspended by homogenizing with a Virtis VirTishear homogenizer. Bacteria was frozen and thawed twice and then completely lysed by the addition of 0.2 g lysozyme per ml of bacteria suspension. After agitating for one hour at room temperature, DNase 1 (Sigma Chemical Co.) was added to the lysed bacteria at a concentration 2000 units per 10 gms to break up DNA. After incubating for 30 minutes at room temperature, the preparation was centrifuged for 30 minutes at 13,000×g. Pellets were resuspended in TBS with 1% Triton X-100 and agitated for one hour at room temperature and then centrifuged for 40 minutes at 22.000×g. Pellet was resuspended in TBS with 1% Triton X-100 and centrifugation repeated. Resuspensions and centrifugations were repeated until supernatant contained only trace amounts of protein. Pellets were resuspended in TBS with 6 M urea and agitated. TNfn C-Dhis was purified on a nickel-NTA silica HPLC column (Qiagen, cat #30710) both from the 1% Triton X-100 extract and the 6 M urea extract. The TNfn C-Dhis in 6 M urea was refolded on the nickel column by decreasing the urea concentration form 6 M to TBS without urea with a 2 hour linear gradient. TNfn C-Dhis was eluted from column with a 1 hour linear gradient from TBS to TBS plus 300 mM imidazole. Eluted protein was dialyzed against 115 mM phosphate buffer pH 7.4. Protein concentration determined by Lowry method and then aliquoted and stored frozen at −135° C. until needed.
Rabbits were immunized with 100 μg of purified TNfn C-Dhis in complete Freund's adjuvant and a test bleed performed at days 21, 39 and 53. Since titer at day 53 had decreased they were boosted at day 60 with 50 μg TNfn C-Dhis in incomplete Freund's Adjuvant and were bled every 14 days until the titer starts to drop. The rabbits were then boosted with 50 μg TNfn C-Dhis in incomplete Freund's Adjuvant and bled every 14 days until titer started to drop. This procedure was repeated as needed. Titers were measured by ELISA against TNfn C-Dhis coated plates and response from primary immunization is shown in
An affinity column was made by coupling purified TNfn C-D through amine groups to a NHS activated-Sepharose 4 Fast Flow resin (Amersham, Cat # 17-0906-01). Rabbit antiserum was passed through a column, and the column was then rinsed with 10 column volumes of equilibrated buffer. Anti-TNfn C-D was eluted with 0.1 M CAPS buffer, pH 11.0. Eluted fraction were neutralized by the addition of powered glycine (5 mg/ml) to collection tube. Antibody was dialyzed against 115 mM phosphate buffer pH 7.4 and then filtered through a 0.22 μg filter into sterile vials. Protein concentration was determined by Lowry and vials were stored at 4° C. until used.
TNfnA-D bacterial expression plasmid vector was graciously provided by H. P. Erickson, Duke University, Durham, N.C. An NdeI site and ATG translation initiation codon were introduced at the 5′-end and a tag of six-Histidine cDNA sequence as well as a stop codon with EcoRI site were introduced at the 3′-end of the TNfn C-D cDNA fragment respectively by PCR using the TNfnA-D as the template. The resulting PCR fragment was used to clone into an expression vector pMR1scFv (Kuan et al., 1999), pre-cut by NdeI and EcoRI enzymes, to produce an expression plasmid. The cDNA and deduced amino acid sequence are shown in
The following protocol can be employed to determine if rabbit anti-tenascin polyvalent/polyclonal antibody reacts with tenascin in patient samples, wherein tenascin is a large extracellular matrix protein in gliomas often associated with blood vessels.
Formalin fixed patient brain tumor cut at 5-10 microns on slides, provided by Histology.
Needed: 6 slides, one section per slide, at 5-10 microns.
Formalin fixed D245 (human glioma tissue positive for tenascin, grown as rat xenograft).
Needed: 6 slides, one section per slide, at 5-10 microns.
Quality Control The following antibody must be run with anti-tenascin polyvalent serum in every assay:
1-Normal Rabbit IgG: Source: DAKO, #X0936. Beef liver powder and agarose absorbed as necessary; quantitation of rabbit IgG by Quantitative Capture ELISA required after absorption for determination of IgG concentration.
Glass staining dishes and trays (VWR 25445004)
Fume hood (when using xylenes)
To remove paraffin:
Giemsa Counter Staining (changes melanin from brown to green):
Positive Control Tissue: known glioma (D245MG rat xenograft)
Negative Reagent Control: DPBS, irrelevant murine IgG2b (M45.6), IgG1 (P588)
Negative Assay Controls: DPBS as 1° reagent
The present invention is not to be limited in scope 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 the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
It is further to be understood that all values are approximate, and are provided for description.
Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.
This application is a continuation application of U.S. patent application Ser. No. 11/282,117, filed Nov. 16, 2005 which claims the benefit of U.S. Provisional Patent Application No. 60/628,940, filed Nov. 17, 2004, which are hereby incorporated by reference in their entirety.
This invention was made with Government support under grant numbers MO1-RR 30, NS20023, CA11898, CA70164, CA42324, 1P50CA108786-01, 5P20CA96890 and PDT-414 from the National Center for Research Resources General Clinical Research Centers Program and National Institutes of Health. The Government has certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60628940 | Nov 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11282117 | Nov 2005 | US |
| Child | 12035328 | US |
