WO2007095748A1 - Cancerous disease modifying antibody 141205-05 - Google Patents
Cancerous disease modifying antibody 141205-05 Download PDFInfo
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- WO2007095748A1 WO2007095748A1 PCT/CA2007/000282 CA2007000282W WO2007095748A1 WO 2007095748 A1 WO2007095748 A1 WO 2007095748A1 CA 2007000282 W CA2007000282 W CA 2007000282W WO 2007095748 A1 WO2007095748 A1 WO 2007095748A1
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
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- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
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- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
Definitions
- This invention relates to the isolation and production of cancerous disease modifying antibodies (CDMAB) and to the use of these CDMAB alone or in combination with one or more CDMAB/chemotherapeutic agents in therapeutic and diagnostic processes.
- the invention further relates to binding assays which utilize the CDMAB of the instant invention.
- TROP-2 is a cell surface glycoprotein expressed on most carcinomas, as well as some normal human tissues. It was initially defined as a molecule recognized by two murine monoclonal antibodies raised to a human choriocarcinoma cell line BeWo that recognized an antigen on human trophoblast cells (Faulk 1978). The same molecule was independently discovered by other investigators which led to multiple names describing the same antigen. Hence, TROP-2 was also referred to as GA733-1 and epithelial glycoprotein- 1 (EGP-I) (Basu 1995, Fornaro 1995).
- the TROP-2 gene is an intronless gene that was thought to have been formed through the retroposition of a homologous gene GA733-2 (also known as epithelial glycoprotein-2, EpCAM and Trop-1) via an RNA intermediate.
- GA733-2 also known as epithelial glycoprotein-2, EpCAM and Trop-1
- the TROP-2 gene has been mapped to chromosome Ip32 (Calabrese 2001 ).
- the protein component of TROP-2 has a molecular mass of approximately 35 kilodaltons. Its mass may be increased by 1 1 -13 kilodaltons with heterogeneous N-linked glycosylation of its extracellular domain. There are many cysteine residues in the extracellular domain which could form disulfide bridge sites.
- TROP-2 is a substrate for protein kinase C, a Ca 2+ dependent protein kinase and the intracellular serine 303 residue has been shown to be phosphorylated (Basu 1995). It has also been shown that crossing-linking of TROP-2 with anti-TROP-2 antibodies transduced a calcium signal as shown by a rise in cytoplasmic Ca" + (Ripani 1998). These data support signal transduction as a physiological function of TROP-2, although to date no physiological ligand has been identified.
- TROP-2 was identified as a member of a group of genes reported to be the most highly overexpressed in ovarian serous papillary carcinoma compared to normal ovarian epithelium in a large-scale gene expression analysis using cDNA microarray technology (Santin 2004).
- the expression profile of TROP-2 has been elucidated through immunohistochemistry (IHC) and flow cytometery studies using many different TROP- 2 antibodies.
- Anti-TROP-2 antibodies 162-25.3 and 162-46.2 were produced through immunization of mice with the human choriocarcinoma cell line BeWo, and were investigated for their reactivity to a series of tumor and lymphoid cell lines and peripheral blood mononuclear cells.
- both antibodies appeared to be trophoblast specific, staining 3 of the 4 choriocarcinoma cell lines tested, while none of the other lymphoid or tumor cell lines (representing fibrosarcoma, cervical sarcoma, colon carcinoma, melanoma, neuroblastoma, erythroleukemia) were stained in an indirect immunofluorescence FACS assay. In addition, none of the normal peripheral blood cells were stained.
- the antibodies were tested for staining of formalin-fixed paraffin-embedded placenta tissue sections and frozen normal sections of liver, kidney, spleen, thymus and lymph node tissues.
- MOvI 6 was generated through the immunization of mice with a crude membrane preparation of poorly differentiated ovarian carcinoma OvCa4343/83. MOv 16 was tested for reactivity to a series of frozen tissue sections of benign and malignant ovarian tumors. MOvI 6 reacted with 31 of 54 malignant ovarian tumors and 2 of 16 benign ovarian tumors. Of the 5 mucinous ovarian tumors that were tested, MOv 16 was completely unreactive.
- MOvI 6 was also tested for reactivity to frozen sections of non-ovarian malignant tumors where it was found to bind 1 17 of 189 breast carcinoma sections and 12 of 18 lung carcinoma sections.
- MOv 16 was completely unreactive on 16 non-epithelial tumors that were tested (including liposarcomas, chondrosarcomas, endotheliomas, histiocytomas and dysgerminomas). When tested on frozen normal tissue sections, MOv- 16 was reactive with breast, pancreas, kidney and prostate sections.
- MOvI 6 reactivity was reported to be negative on lung, spleen, skin, ovary, thyroid, parotid gland, stomach, larynx, uterus and colon sections, though the number of tissue sections that were used was not reported. The authors noted that frozen tissue sections were used because MOv 16 was unreactive to paraffin embedded tissues (Miotti 1987). This antibody has also only been reported for use in in vitro diagnositic studies.
- Anti-TROP-2 antibody Rs7-3G1 1 was generated through the immunization of mice with a crude membrane preparation derived from a surgically removed human primary squamous cell carcinoma of the lung. IHC was used to examine the staining of RS7 on frozen sections of human tumor and normal tissues. RS7 bound to 33 of the 40 sections representing tumors of the breast, colon, kidney, lung, prostate and squamous cell cancer. Of the normal tissues RS7 bound to 16 of 20 sections of breast, colon, kidney, liver, lung and prostate tissues while none of the five sections of spleen tissue were stained. In this study the authors noted that it appeared that antigen density in tumors was higher than in normal epithelial tissues (Stein 1990).
- RS7 tissue specificity of RS7 was tested on a panel of frozen tumor sections and bound to 65 of the 77 sections representing tumors of the lung, stomach, kidney, bladder, colon, breast, ovary, uterus and prostate. There was no binding to the 5 lymphomas tested.
- RS7 was tested on a panel of 85 frozen human normal tissue sections composed of a total of 24 tissue types. 39 sections of 13 normal tissues (lung, bronchus, trachea, esophagus, colon, liver, pancreas, kidney, bladder, skin, thyroid, breast and prostate) were stained by RS7.
- Polyclonal anti-TROP-2 antibodies were prepared by immunizing mice with a synthetic peptide corresponding to amino acid positions between 169 and 182 of the cytoplasmic domain of human TRO P-2.
- the polyclonal antibodies were tested on a tissue array slide that contained formalin-fixed human esophageal hyperplasia and carcinoma tissues. Ten of the 55 carcinoma specimens displayed heavy staining with the polyclonal antibodies, while the mild hyperplasia tissue stained very weakly, indicating expression levels may be related to malignant transformation (Nakashima 2004).
- Radiolabeled RS7 was tested in in vivo models with initial experiments consisting of tumor targeting studies in nude mouse xenograft models. Radiolabeled RS7 injected Lv. was shown to accumulate specifically in the tumor of mice bearing either Calu-3 (lung adenocarcinoma) or GW- 39 (colon carcinoma) tumors (Stein 1990). Further studies were done to investigate the biodistribution of radiolabeled RS7 in a xenograft system and to study the therapeutic potential of RS7 as an immunoconjugate.
- the single dose of 1.0 mCi 131 I-RS7-F(ab') 2 resulted in tumor growth suppression for approximately 5 weeks, while the single dose of 1.5 mCi 131 I-RS7-F(ab') 2 resulted in tumor regression, and the mean tumor size did not exceed the pre-therapy size until the eighth week after radioantibody injection.
- Mice receiving the 1.5 mCi 131 I-RS7-F(ab') 2 dose experienced a mean body weight loss of 18.7 percent, indicating there was toxicity associated with the treatment.
- effects of treatment with naked RS7 or the F(ab') 2 fragment of RS7 were not tested (Stein 1994a).
- mice bearing MDA-MB-468 tumors of approximately 0.1 cm 3 were treated with a single dose Lv. of either 250 microcuries 131 I-RS7 or 250 microcuries 131 I-Ag8 (an isotype matched control antibody).
- Groups of six mice were treated with a single dose /.v. of 30 micrograms of either unlabeled RS7 or Ag8.
- Complete regression of the tumors was seen in the animals treated with 131 I-RS7, which lasted for the duration of the 1 1 week observation period.
- Tumor regression was also seen in 131 I-Ag8 treated mice, though was only observed between 2 weeks and 5 weeks with tumors either persisting or continuing to grow for the remainder of the study. Tumor growth of mice that received unlabeled RS7 or Ag8 was not inhibited and there did not appear to be any differences in the mean tumor volume of RS7 treated mice compared to the Ag8 treated mice.
- Two additional groups of 10 mice bearing larger MDA-MB-468 tumors of approximately 0.2-0.3 cm 3 were treated with a slightly higher single dose of either 275 microcuries 131 1-RS7 or 275 microcuries 131 Ag8 and compared to a similar group of untreated mice. Tumor volume was measured weekly for 15 weeks.
- Immunizing mice with neuramindase pre -treated H3922 human breast carcinoma cells produced the anti-TROP-2 monoclonal antibody BRl 10 (as disclosed in US patent No. 5,850,854, refer to Prior Patents section).
- BRl 10 was shown to react with a wide range of human carcinoma specimens including those of the lung, colon, breast, ovarian, kidney, esophagus, pancreas, skin, lung and tonsil. No human normal tissue sections were tested.
- TROP-2 such as MR54, MR6 and MR23 which were generated from immunization of mice with the ovarian cancer cell line Colo 316 (Stein 1994b) and antibody Tl 6 which was generated by immunization of mice with the T24 bladder cancer cell line (Fradet 1984).
- the use of these antibodies has been limited to biochemical characterization of the TROP-2 antigen and cell line and tissue expression studies. There have been no reports of anti-cancer efficacy of these antibodies, either in vitro or in vivo.
- RS7 was the only antibody that was tested for therapeutic efficacy in preclinical cancer models, with its use being limited to a carrier of radioisotope. There are no reports of any naked TROP-2 antibodies exhibiting therapeutic efficacy in preclinical cancer models either in vitro or in vivo.
- Monoclonal Antibodies as Cancer Therapy Each individual who presents with cancer is unique and has a cancer that is as different from other cancers as that person's identity. Despite this, current therapy treats all patients with the same type of cancer, at the same stage, in the same way. At least 30 percent of these patients will fail the first line therapy, thus leading to further rounds of treatment and the increased probability of treatment failure, metastases, and ultimately, death. A superior approach to treatment would be the customization of therapy for the particular individual. The only current therapy which lends itself to customization is surgery. Chemotherapy and radiation treatment cannot be tailored to the patient, and surgery by itself, in most cases is inadequate for producing cures.
- cancerous cells contain antigens that are specific to transformed cells
- monoclonal antibodies can be designed to specifically target transformed cells by binding specifically to these cancer antigens; thus giving rise to the belief that monoclonal antibodies can serve as "Magic Bullets" to eliminate cancer cells.
- monoclonal antibodies can serve as "Magic Bullets" to eliminate cancer cells.
- no single monoclonal antibody can serve in all instances of cancer, and that monoclonal antibodies can be deployed, as a class, as targeted cancer treatments.
- CDMAB cancerous disease modifying antibodies
- the cancer patient usually has few options of treatment.
- the regimented approach to cancer therapy has produced improvements in global survival and morbidity rates.
- these improved statistics do not necessarily correlate with an improvement in their personal situation.
- Herceptin® was approved in 1998 for first line use in combination with Taxol®. Clinical study results showed an increase in the median time to disease progression for those who received antibody therapy plus Taxol® (6.9 months) in comparison to the group that received Taxol® alone (3.0 months). There was also a slight increase in median survival; 22 versus 18 months for the Herceptin® plus Taxol ® treatment arm versus the Taxol® treatment alone arm. In addition, there was an increase in the number of both complete (8 versus 2 percent) and partial responders (34 versus 15 percent) in the antibody plus Taxol® combination group in comparison to Taxol® alone.
- Herceptin® and Taxol ® led to a higher incidence of cardiotoxicity in comparison to Taxol ® treatment alone (13 versus 1 percent respectively).
- Herceptin® therapy was only effective for patients who over express (as determined through immunohistochemistry (IHC) analysis) the human epidermal growth factor receptor 2 (Her2/neu), a receptor, which currently has no known function or biologically important ligand; approximately 25 percent of patients who have metastatic breast cancer. Therefore, there is still a large unmet need for patients with breast cancer. Even those who can benefit from Herceptin® treatment would still require chemotherapy and consequently would still have to deal with, at least to some degree, the side effects of this kind of treatment.
- the clinical trials investigating colorectal cancer involve antibodies against both glycoprotein and glycolipid targets.
- Antibodies such as 17-1A which has some specificity for adenocarcinomas, has undergone Phase 2 clinical trials in over 60 patients with only 1 patient having a partial response. In other trials, use of 17-1 A produced only 1 complete response and 2 minor responses among 52 patients in protocols using additional cyclophosphamide. To date, Phase III clinical trials of 17-1 A have not demonstrated improved efficacy as adjuvant therapy for stage III colon cancer. The use of a humanized murine monoclonal antibody initially approved for imaging also did not produce tumor regression. Only recently have there been any positive results from colorectal cancer clinical studies with the use of monoclonal antibodies.
- ERBITUX® was approved for the second line treatment of patients with EGFR-expressing metastatic colorectal cancer who are refractory to irinotecan-based chemotherapy.
- Results from both a two-arm Phase II clinical study and a single arm study showed that ERBITUX® in combination with irinotecan had a response rate of 23 and 15 percent respectively with a median time to disease progression of 4.1 and 6.5 months respectively.
- Results from the same two-arm Phase II clinical study and another single arm study showed that treatment with ERBITUXcE ) alone resulted in an 1 1 and 9 percent response rate respectively with a median time to disease progression of 1.5 and 4.2 months respectively.
- ERBITUX® treatment in combination with irinotecan has been approved as a second line treatment of colon cancer patients who have failed first line irinotecan therapy. Therefore, like Herceptin®, treatment in Switzerland is only approved as a combination of monoclonal antibody and chemotherapy. In addition, treatment in both Switzerland and the US is only approved for patients as a second line therapy. Also, in 2004, AVASTIN® was approved for use in combination with intravenous 5-fluorouracil-based chemotherapy as a first line treatment of metastatic colorectal cancer.
- Phase III clinical study results demonstrated a prolongation in the median survival of patients treated with AVASTIN® plus 5- fluorouracil compared to patients treated with 5-fluourouracil alone (20 months versus 16 months respectively).
- treatment is only approved as a combination of monoclonal antibody and chemotherapy.
- lung, brain, ovarian, pancreatic, prostate, and stomach cancer are also known.
- the resulting hybridomas are screened and selected for secretion of antibodies which bind most avidly to the target.
- Many therapeutic and diagnostic antibodies directed against cancer cells including Herceptin® and RITUXIMAB, have been produced using these methods and selected on the basis of their affinity.
- the flaws in this strategy are two-fold. Firstly, the choice of appropriate targets for therapeutic or diagnostic antibody binding is limited by the paucity of knowledge surrounding tissue specific carcinogenic processes and the resulting simplistic methods, such as selection by overexpression, by which these targets are identified. Secondly, the assumption that the drug molecule that binds to the receptor with the greatest affinity usually has the highest probability for initiating or inhibiting a signal may not always be the case.
- U.S. Patent No. 5,750,102 discloses a process wherein cells from a patient's tumor are transfected with MHC genes which may be cloned from cells or tissue from the patient. These transfected cells are then used to vaccinate the patient.
- U.S. Patent No. 5,750,102 discloses a process wherein cells from a patient's tumor are transfected with MHC genes which may be cloned from cells or tissue from the patient. These transfected cells are then used to vaccinate the patient.
- 4,861 ,581 discloses a process comprising the steps of obtaining monoclonal antibodies that are specific to an internal cellular component of neoplastic and normal cells of the mammal but not to external components, labeling the monoclonal antibody, contacting the labeled antibody with tissue of a mammal that has received therapy to kill neoplastic cells, and determining the effectiveness of therapy by measuring the binding of the labeled antibody to the internal cellular component of the degenerating neoplastic cells.
- the patentee recognizes that malignant cells represent a convenient source of such antigens.
- U.S. Patent No. 5,171 ,665 provides a novel antibody and method for its production. Specifically, the patent teaches formation of a monoclonal antibody which has the property of binding strongly to a protein antigen associated with human tumors, e.g. those of the colon and lung, while binding to normal cells to a much lesser degree.
- U.S. Patent No. 5,484,596 provides a method of cancer therapy comprising surgically removing tumor tissue from a human cancer patient, treating the tumor tissue to obtain tumor cells, irradiating the tumor cells to be viable but non- tumorigenic, and using these cells to prepare a vaccine for the patient capable of inhibiting recurrence of the primary tumor while simultaneously inhibiting metastases.
- the patent teaches the development of monoclonal antibodies which are reactive with surface antigens of tumor cells. As set forth at col. 4, lines 45 et seq., the patentees utilize autochthonous tumor cells in the development of monoclonal antibodies expressing active specific immunotherapy in human neoplasia.
- U.S. Patent No. 5,693,763 teaches a glycoprotein antigen characteristic of human carcinomas and not dependent upon the epithelial tissue of origin.
- U.S. Patent No. 5,783,186 is drawn to Anti-Her2 antibodies which induce apoptosis in Her2 expressing cells, hybridoma cell lines producing the antibodies, methods of treating cancer using the antibodies and pharmaceutical compositions including said antibodies.
- U.S. Patent No. 5,849,876 describes new hybridoma cell lines for the production of monoclonal antibodies to mucin antigens purified from tumor and non- tumor tissue sources.
- U.S. Patent No. 5,869,268 is drawn to a method for generating a human lymphocyte producing an antibody specific to a desired antigen, a method for producing a monoclonal antibody, as well as monoclonal antibodies produced by the method.
- the patent is particularly drawn to the production of an anti-HD human monoclonal antibody useful for the diagnosis and treatment of cancers.
- U.S. Patent No. 5,869,045 relates to antibodies, antibody fragments, antibody conjugates and single-chain immunotoxins reactive with human carcinoma cells.
- the mechanism by which these antibodies function is two-fold, in that the molecules are reactive with cell membrane antigens present on the surface of human carcinomas, and further in that the antibodies have the ability to internalize within the carcinoma cells, subsequent to binding, making them especially useful for forming antibody-drug and antibody-toxin conjugates. In their unmodified form the antibodies also manifest cytotoxic properties at specific concentrations.
- U.S. Patent No. 5,780,033 discloses the use of autoantibodies for tumor therapy and prophylaxis. However, this antibody is an antinuclear autoantibody from an aged mammal.
- the autoantibody is said to be one type of natural antibody found in the immune system. Because the autoantibody comes from "an aged mammal", there is no requirement that the autoantibody actually comes from the patient being treated.
- the patent discloses natural and monoclonal antinuclear autoantibody from an aged mammal, and a hybridoma cell line producing a monoclonal antinuclear autoantibody.
- U.S. Patent No. 5,850,854 discloses a specific antibody, BRl 10 directed against GA733- 1. This patent discloses in vitro function for BR 1 10 as an immunotoxin conjugate. There was no in vitro function as a naked antibody disclosed for this antibody. There was also no in vivo function disclosed for this antibody.
- U.S. Patent No. 6,653,104 claims immunotoxin-conjugated antibodies, including but not limited to RS7, directed against a host of antigens, including but not limited to EGP-I .
- the immunotoxin is limited to those possessing ribonucleolytic activity.
- the examples disclose only a specific immunotoxin-conjugated antibody, LL2, directed against CD22. There was no in vitro or in vivo function for RS7 disclosed in this application.
- U.S. Application No. 20040001825Al discloses a specific antibody, RS7 directed against EGP-I .
- This application discloses in vitro function for RS7 as a radiolabeled conjugate. There was no in vitro function as a naked antibody disclosed for this antibody.
- This application also discloses in vivo function for RS7 resulting from radiolabled and unlabeled conjugate administered sequentially. However, this study was limited to one patient and it is unknown whether any of the observed function was due to the unlabeled antibody. There was no in vivo function for RS7 resulting from the administration of the naked antibody.
- This application utilizes methodology for producing patient specific anticancer antibodies taught in the U.S. 6,180,357 patent for isolating hybridoma cell lines which encode for cancerous disease modifying monoclonal antibodies. These antibodies can be made specifically for one tumor and thus make possible the customization of cancer therapy.
- anti-cancer antibodies having either cell-killing (cytotoxic) or cell-growth inhibiting (cytostatic) properties will hereafter be referred to as cytotoxic.
- cytotoxic cell-killing
- cytostatic cell-growth inhibiting
- antibodies generated in this way may target molecules and pathways not previously shown to be integral to the growth and/or survival of malignant tissue. Furthermore, the binding affinities of these antibodies are suited to requirements for initiation of the cytotoxic events that may not be amenable to stronger affinity interactions. Also, it is within the purview of this invention to conjugate standard chemotherapeutic modalities, e.g. radionuclides, with the CDMAB of the instant invention, thereby focusing the use of said chemotherapeutics.
- the CDMAB can also be conjugated to toxins, cytotoxic moieties, enzymes e.g.
- the CDMAB can be used alone or in combination with one or more CDMAB/chemotherapeutic agents.
- a likely clinical scenario is that a tumor sample is obtained at the time of presentation, and banked. From this sample, the tumor can be typed from a panel of pre-existing cancerous disease modifying antibodies.
- the patient will be conventionally staged but the available antibodies can be of use in further staging the patient.
- the patient can be treated immediately with the existing antibodies, and a panel of antibodies specific to the tumor can be produced either using the methods outlined herein or through the use of phage display libraries in conjunction with the screening methods herein disclosed. All the antibodies generated will be added to the library of anti-cancer antibodies since there is a possibility that other tumors can bear some of the same epitopes as the one that is being treated.
- the antibodies produced according to this method may be useful to treat cancerous disease in any number of patients who have cancers that bind to these antibodies.
- the patient can elect to receive the currently recommended therapies as part of a multi-modal regimen of treatment.
- the fact that the antibodies isolated via the present methodology are relatively non-toxic to non-cancerous cells allows for combinations of antibodies at high doses to be used, either alone, or in conjunction with conventional therapy.
- the high therapeutic index will also permit re-treatment on a short time scale that should decrease the likelihood of emergence of treatment resistant cells.
- the process of generating specific antibodies to the tumor can be repeated for re-treatment.
- the anti-cancer antibodies can be conjugated to red blood cells obtained from that patient and re-infused for treatment of metastases.
- metastatic cancers There have been few effective treatments for metastatic cancer and metastases usually portend a poor outcome resulting in death.
- metastatic cancers are usually well vascularized and the delivery of anti-cancer antibodies by red blood cells can have the effect of concentrating the antibodies at the site of the tumor.
- Even prior to metastases most cancer cells are dependent on the host's blood supply for their survival and an anti-cancer antibody conjugated to red blood cells can be effective against in situ tumors as well.
- the antibodies may be conjugated to other hematogenous cells, e.g. lymphocytes, macrophages, monocytes, natural killer cells, etc.
- hematogenous cells e.g. lymphocytes, macrophages, monocytes, natural killer cells, etc.
- lymphocytes e.g. lymphocytes, macrophages, monocytes, natural killer cells, etc.
- CDC complement dependent cytotoxicity
- murine IgM and IgG2a antibodies can activate human complement by binding the C-I component of the complement system thereby activating the classical pathway of complement activation which can lead to tumor lysis.
- human antibodies the most effective complement activating antibodies are generally IgM and IgGl.
- Murine antibodies of the IgG2a and IgG3 isotype are effective at recruiting cytotoxic cells that have Fc receptors which will lead to cell killing by monocytes, macrophages, granulocytes and certain lymphocytes.
- Human antibodies of both the IgGl and IgG3 isotype mediate ADCC.
- the cytotoxicity mediated through the Fc region requires the presence of effector cells, their corresponding receptors, or proteins e.g. NK cells, T-cells and complement. In the absence of these effector mechanisms, the Fc portion of an antibody is inert.
- the Fc portion of an antibody may confer properties that affect the pharmacokinetics of an antibody in vivo, but in vitro this is not operative.
- the cytotoxicity assays under which we test the antibodies do not have any of the effector mechanisms present, and are carried out in vitro. These assays do not have effector cells (NK, Macrophages, or T-cells) or complement present. Since these assays are completely defined by what is added together, each component can be characterized.
- the assays used herein contain only target cells, media and sera.
- the target cells do not have effector functions since they are cancer cells or fibroblasts. Without exogenous cells which have effector function properties there is no cellular elements that have this function.
- the media does not contain complement or any cells.
- the sera used to support the growth of the target cells do not have complement activity as disclosed by the vendors. Furthermore, in our own labs we have verified the absence of complement activity in the sera used.
- the first is the use of antibodies as a vaccine to induce the body to produce an immune response against the putative antigen that resides on the cancer cell.
- the second is the use of antibodies to target growth receptors and interfere with their function or to down regulate that receptor so that its function is effectively lost.
- the third is the effect of such antibodies on direct ligation of cell surface moieties that may lead to direct cell death, such as ligation of death receptors such as TRAIL Rl or TRAIL R2, or integrin molecules such as alpha V beta 3 and the like.
- RECIST criteria Clinical criteria for such evaluation have been promulgated by Response Evaluation Criteria in Solid Tumors Working Group, a group of international experts in cancer. Drugs with a demonstrated effect on tumor burden, as shown by objective responses according to RECIST criteria, in comparison to the appropriate control group tend to, ultimately, produce direct patient benefit.
- tumor burden is generally more straightforward to assess and document.
- pre-clinical studies can be translated to the clinical setting, drugs that produce prolonged survival in pre-clinical models have the greatest anticipated clinical utility. Analogous to producing positive responses to clinical treatment, drugs that reduce tumor burden in the pre-clinical setting may also have significant direct impact on the disease.
- This invention represents an advance in the field of cancer treatment in that it describes, for the first time, reagents that bind specifically to an epitope or epitopes present on the target molecule, TROP-2, and that also have in vitro cytotoxic properties, as a naked antibody, against malignant tumor cells but not normal cells, and which also directly mediate, as a naked antibody, inhibition of tumor growth and extension of survival in in vivo models of human cancer.
- TROP-2 an advance in relation to any other previously described anti-TROP-2 antibody, since none have been shown to have similar properties. It also provides an advance in the field since it clearly demonstrates, and for the first time, the direct involvement of TROP-2 in events associated with growth and development of certain types of tumors.
- this invention teaches the use of the AR47A6.4.2 antigen as a target for a therapeutic agent, that when administered can reduce the tumor burden of a cancer expressing the antigen in a mammal, and can also lead to a prolonged survival of the treated mammal.
- This invention also teaches the use of CDMAB (AR47A6.4.2), and its derivatives, and antigen binding fragments thereof, and cellular cytotoxicity inducing ligands thereof to target their antigen to reduce the tumor burden of a cancer expressing the antigen in a mammal, and lead to prolonged survival of the treated mammal.
- this invention also teaches the use of detecting the AR47A6.4.2 antigen in cancerous cells that can be useful for the diagnosis, prediction of therapy, and prognosis of mammals bearing tumors that express this antigen.
- CDMAB cancerous disease modifying antibodies
- a method for producing cancerous disease modifying antibodies CDMAB raised against cancerous cells derived from a particular individual, or one or more particular cancer cell lines, which CDMAB are cytotoxic with respect to cancer cells while simultaneously being relatively non-toxic to non-cancerous cells, in order to isolate hybridoma cell lines and the corresponding isolated monoclonal antibodies and antigen binding fragments thereof for which said hybridoma cell lines are encoded.
- CDMAB cancerous disease modifying antibodies
- a still further objective of the instant invention is to produce cancerous disease modifying antibodies which are useful for in a binding assay for diagnosis, prognosis, and monitoring of cancer.
- Figure 1 compares the percentage cytotoxicity and binding levels of the hybridoma supernatants against cell lines OCC- 1 , OVCAR-3 and CCD-27sk.
- Figure 2 tabulates binding of AR47A6.4.2 and the anti-EGFR antibody control to cancer and normal cell lines. The data is presented as the mean fluorescence intensity as a fold increase above isotype control.
- Figure 3 includes representative FACS histograms of AR47A6.4.2 and anti-EGFR antibodies directed against several cancer and non-cancer cell lines.
- Figure 4 demonstrates the effect of AR47A6.4.2 on tumor growth in a prophylactic BxPC-3 pancreatic cancer model.
- the vertical lines indicate the period during which the antibody was administered.
- Data points represent the mean +/- SEM.
- Figure 5 demonstrates the effect of AR47A6.4.2 on body weight in a prophylactic BxPC-3 pancreatic cancer model. Data points represent the mean +/- SEM.
- Figure 6 demonstrates the effect of AR47A6.4.2 on tumor growth in an established BxPC-3 pancreatic cancer model.
- the vertical lines indicate the period during which the antibody was administered. Data points represent the mean +/- SEM.
- Figure 7 demonstrates the effect of AR47A6.4.2 on body weight in an established BxPC-3 pancreatic cancer model. Data points represent the mean +/- SEM.
- Figure 8 demonstrates the effect of AR47A6.4.2 on tumor growth in a prophylactic PL45 pancreatic cancer model.
- the vertical lines indicate the period during which the antibody was administered. Data points represent the mean +/- SEM.
- Figure 9 demonstrates the effect of AR47A6.4.2 on survival in a prophylactic PL45 pancreatic cancer model.
- Figure 10 demonstrates the effect of AR47A6.4.2 on body weight in a prophylactic PL45 pancreatic cancer model. Data points represent the mean +/- SEM.
- Figure 1 1 demonstrates the effect of AR47A6.4.2 on tumor growth in a prophylactic PC-3 prostate cancer model. The vertical lines indicate the period during which the antibody was administered. Data points represent the mean +/- SEM.
- Figure 12 demonstrates the effect of AR47A6.4.2 on survival in a prophylactic PC-3 prostate cancer model.
- Figure 13 demonstrates the effect of AR47A6.4.2 on body weight in a prophylactic PC-3 prostate cancer model. Data points represent the mean +/- SEM.
- Figure 14 demonstrates the effect of AR47A6.4.2 on tumor growth in a prophylactic MCF-7 breast cancer model. The vertical lines indicate the period during which the antibody was administered. Data points represent the median +/- SEM.
- Figure 15 demonstrates the effect of AR47A6.4.2 on body weight in a prophylactic MCF-7 breast cancer model. Data points represent the mean +/- SEM.
- Figure 16 demonstrates the effect of AR47A6.4.2 on survival in a prophylactic MCF-7 breast cancer model.
- Figure 17 demonstrates the effect of AR47A6.4.2 on tumor growth in a prophylactic Colo 205 colon cancer model.
- the vertical lines indicate the period during which the antibody was administered.
- Data points represent the median +/- SEM.
- Figure 18 demonstrates the effect of AR47A6.4.2 on body weight in a prophylactic Colo 205 colon cancer model.
- Data points represent the mean +/- SEM.
- FIG. 19 Western blot of samples from the total membrane fraction of MDA-MB-231 cells (lane 1) and from whole cell lysates of PC-3 (lane 2) and CCD- 27sk (lane 3) cell lines, probed with AR47A6.4.2. Molecular weight markers are indicated on the left.
- Figure 20 Western blot of an immunocomplex prepared by immunoprecipitation with AR47A6.4.2 (lane 1) and with an isotype control (lane 2), from the total membrane fraction of the MDA-MB-231 cell line.
- Antibody-conjugated Protein G-Sepharose beads not incubated with total membrane fraction of MDA-MB- 231 were also used as negative controls (lanes 3 and 4).
- Three replicate blots were probed either with the antibodies AR47A6.4.2, lgG2a isotype control or without primary antibody. Molecular weight markers are indicated on the left.
- FIG. 21 Western blot of immunocomplexes prepared by immunoprecipitation with AR47A6.4.2 (lanes 2 and 3) or with an isotype control (lanes 1 and 4), from the total membrane fraction of the MDA-MB-231 cell line. Replicate aliquots were incubated in the presence (lanes 3 and 4) and absence (buffer only, lanes 1 and 2) of a mixture of glycosidase enzymes. Molecular weight markers are indicated on the left.
- FIG 22 Alignment of images from Western blotting (center panel) and colloidal staining (left panel) of an immunocomplex prepared by a large-scale immunoprecipitation with AR47A6.4.2 (lane 1) and with an isotype control antibody (lane 2), from the total membrane fraction of the MDA-MB-231 cell line.
- AR47A6.4.2- conjugated protein G Sepharose beads only were also used as a negative control (lane 3). Molecular weight markers are indicated on the left.
- Figure 23 Images of the Colloidal Blue stained gel prior to and after coring to isolate protein samples, to be analyzed by Mass Spectrometry after trypsin digestion. Lane description is the same as in Figure 20. Molecular weight markers are indicated on the left.
- Figure 24 Mass spectrogram obtained after trypsin digestion of the samples obtained from coring the Colloidal Blue-stained gel. Below the mass spectograms is the Profound search result summary.
- FIG. 25 MASCOT search summary result from the MS/MS analysis of one of the unique peptides obtained after trypsin digestion of the AR47A6.4.2 immunoprecipitate. SEQ ID NO:9 is shown.
- Figure 26 Western blot of immunocomplexes obtained, with AR47A6.4.2 (lane 1) and with an isotype control antibody (lane 3), from the total membrane fraction of the MDA-MB-231 cell line. AR47A6.4.2-conjugated protein G Sepharose beads only ('mock IP' were also used as a negative control (lane 2)). Replicate blots were probed with the AR47A6.4.2, anti-human TROP-2 and isotype control. Molecular weight markers are indicated on the left.
- FIG. 27 Western blot of purified human recombinant proteins TROP-2 (lane 1) and CD63 large extracellular domain (lane 2). Replicate blots were probed with either AR47A6.4.2, anti-human TROP-2, 1 A245.6 or isotype control. Molecular weight markers are indicated on the left.
- FIG 28 MDA-MB-231 membrane proteins (MB-231) and recombinant human TROP-2 (rh TROP-2) glycosylated (G) and deglycosylated (D) under denaturing and non-denaturing conditions, probed with AR47A6.4.2.
- Figure 29 MDA-MB-231 membrane proteins (MB-231) and recombinant human TROP-2 (rhTROP-2) glycosylated (G) and deglycosylated (D) under denaturing and non-denaturing conditions, probed with anti-human TROP-2.
- FIG 30 MDA-MB-231 membrane proteins (MB-231) and recombinant human TROP-2 (rhTROP-2) glycosylated (G) and deglycosylated (D) under denaturing and non-denaturing conditions, probed with IgG isotype control.
- Figure 31 Western blot of recombinant human TROP-2 probed with different primary antibody solutions. Lanes 3 to 7 were probed with biotinylated AR52A301.5 mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500 microgram/mL and 1000 microgram/mL of non-biotinylated AR52A301.5 respectively.
- Lanes 9 to 13 were probed with biotinylated AR52A301.5 mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500 microgram/mL and 1000 microgram/mL of non-biotinylated AR47A6.4.2 respectively.
- Lanes 15 to 19 were probed with biotinylated AR52A301.5 mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500 microgram/mL and 1000 microgram/mL of non-biotinylated 8A3B.6 respectively.
- Lanes 8 and 14 were incubated with negative control solution and lane 8 was not incubated in secondary solution.
- Lanes 1 , 2 and 20 were incubated with TBST only.
- Figure 32 Western blot of recombinant human TROP-2 probed with different primary antibody solutions.
- Lanes 3 to 7 were probed with biotinylated AR47A6.4.2 mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500 microgram/mL and 1000 microgram/mL of non-biotinylated AR52A301.5 respectively.
- Lanes 9 to 13 were probed with biotinylated AR47A6.4.2 mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500 microgram/mL and 1000 microgram/mL of non-biotinylated AR47A6.4.2 respectively.
- Lanes 15 to 19 were probed with biotinylated AR47A6.4.2 mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500 microgram/mL and 1000 microgram/mL of non-biotinylated 1B7.11 respectively. Lanes 8 and 14 were incubated with negative control solution and lane 8 was not incubated in secondary solution. Lanes 1, 2 and 20 were incubated with TBST only.
- Figure 33 tabulates an IHC comparison of AR47A6.4.2 versus positive and negative controls on a normal human tissue micro array.
- Figure 34 Representative micrographs showing the binding pattern on spleen tissue obtained with AR47A6.4.2 (A) or the isotype control antibody (B) and on brain tissue obtained with AR47A6.4.2 (C) or the isotype control antibody (D) from a normal human tissue microarray. Magnification is 200X.
- Figure 35 tabulates an IHC comparison of AR47A6.4.2 on various human tumor and normal tissue sections from different tissue micro arrays.
- Figure 36 Representative micrographs showing the binding pattern on breast tumor tissue obtained with AR47A6.4.2 (A) or the isotype control antibody (B) and on prostate tumor tissue obtained with AR47A6.4.2 (C) or the isotype control antibody (D) and on pancreatic tumor tissue obtained with AR47A6.4.2 (E) or the isotype control antibody (F) from various human tumor tissue microarrays.
- Magnification is 400X for the breast and pancreatic tumor tissue and 200X for the prostate tumor tissue.
- Figure 37 tabulates an IHC comparison of AR47A6.4.2 on various human and other species normal tissue sections from different normal species tissue micro arrays.
- Figure 38 Representative micrographs showing the binding pattern on normal human kidney tissue obtained with AR47A6.4.2 (A) or the isotype control antibody (B) and on normal cynomolgus kidney tissue obtained with AR47A6.4.2 (C) or the isotype control antibody (D) and on normal rhesus tissue obtained with AR47A6.4.2 (E) or the isotype control antibody (F) from various multi-species tissue microarrays. Magnification is 200X.
- Figure 39 Sequences of all oligonucleotide primers (SEQ ID NOS: 10- 47) used in the murine sequence determination of AR47A6.4.2.
- FIG. 40 Agarose gel of the RT/PCR amplification of AR47A6.4.2 V H and V L regions.
- FIG. 41 Agarose gel of the PCR colony screen of AR47A6.4.2 V H -C and V n -E.
- Figure 42 Agarose gel of the PCR colony screen of AR47A6.4.2 V L A and V L G.
- Figure 43 AR47A6.4.2 V L amino acid sequence (SEQ ID NO:8).
- FIG. 44 AR47A6.4.2 V H amino acid sequence (SEQ ID NO:7).
- antibody is used in the broadest sense and specifically covers, for example, single monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies, de-immunized, murine, chimeric or humanized antibodies), antibody compositions with polyepitopic specificity, single-chain antibodies, diabodies, triabodies, immunoconjugates and antibody fragments (see below).
- monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
- the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
- the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma (murine or human) method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567).
- the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624- 628 (1991) and Marks et al, J. MoI. Biol, 222:581 -597 (1991), for example.
- Antibody fragments comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof.
- antibody fragments include less than full length antibodies, Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; single-chain antibodies, single domain antibody molecules, fusion proteins, recombinant proteins and multispecific antibodies formed from antibody fragment(s).
- an “intact” antibody is one which comprises an antigen-binding variable region as well as a light chain constant domain (C L ) and heavy chain constant domains, C H I , C H 2 and C H 3.
- the constant domains may be native sequence constant domains
- the intact antibody has one or more effector functions.
- intact antibodies can be assigned to different "classes". There are five- major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl , IgG2, IgG3, IgG4, IgA, and IgA2.
- the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
- the subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.
- Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include CIq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc.
- ADCC antibody-dependent cell-mediated cytotoxicity
- ADCC phagocytosis
- down regulation of cell surface receptors e.g. B cell receptor; BCR
- “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
- FcRs nonspecific cytotoxic cells that express Fc receptors (F
- NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
- FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991 ).
- an in vitro ADCC assay such as that described in U.S. Pat. No. 5,500,362 or 5,821 ,337 may be performed.
- Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
- PBMC peripheral blood mononuclear cells
- NK Natural Killer
- ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
- “Effector cells” are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc ⁇ RIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.
- the effector cells may be isolated from a native source thereof, e.g. from blood or PBMCs as described herein.
- the terms "Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
- the preferred FcR is a native sequence human FcR.
- a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
- Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor") and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
- Activating receptor Fc ⁇ RIlA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
- ITAM immunoreceptor tyrosine-based activation motif
- Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.
- ITIM immunoreceptor tyrosine-based inhibition motif
- the term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 1 17:587 (1976) and Kim et al., Eur. J. Immunol. 24:2429 (1994)).
- FcRn neonatal receptor
- “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement.
- the complement activation pathway is initiated by the binding of the first component of the complement system (CI q) to a molecule (e.g. an antibody) complexed with a cognate antigen.
- a CDC assay e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
- variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs).
- the variable domains of native heavy and light chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
- the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
- the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
- hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
- the hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (e.g.
- "Framework Region” or "FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
- Fv is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-V L dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody.
- variable domain or half of an Fv comprising only three hypervariable regions specific for an antigen
- the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH I) of the heavy chain.
- Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
- Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group.
- F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
- the "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
- Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
- the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen binding.
- diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain (V H ) connected to a variable light domain (V L ) in the same polypeptide chain (V H -V L ).
- V H variable heavy domain
- V L variable light domain
- the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
- Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161 ; and Hollinger et a!., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
- triabodies or “trivalent trimers” refers to the combination of three single chain antibodies. Triabodies are constructed with the amino acid terminus of a V L or V H domain, i.e., without any linker sequence. A triabody has three Fv heads with the polypeptides arranged in a cyclic, head-to-tail fashion. A possible conformation of the triabody is planar with the three binding sites located in a plane at an angle of 120 degrees from one another. Triabodies can be monospecific, bispecific or trispecific. An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment.
- Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
- an antibody "which binds" an antigen of interest e.g. TROP-2 antigen
- an antigen of interest e.g. TROP-2 antigen
- TROP-2 antigen an antigen of interest
- an antigen of interest e.g. TROP-2 antigen
- an antibody which binds TROP-2 it will usually preferentially bind
- TROP-2 as opposed to other receptors, and does not include incidental binding such as non-specific Fc contact, or binding to post-translational modifications common to other antigens and may be one which does not significantly cross-react with other proteins.
- Methods, for the detection of an antibody that binds an antigen of interest are well known in the art and can include but are not limited to assays such as FACS, cell ELISA and Western blot.
- progeny As used herein, the expressions "cell”, “cell line”, and “cell culture” are used interchangeably, and all such designations include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. It will be clear from the context where distinct designations are intended.
- Treatment or treating refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
- Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
- the mammal to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.
- cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth or death. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
- cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
- lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including
- chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
- examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, ura
- paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE®, Aventis, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difiuoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids
- anti-hormonal agents that act to regulate or inhibit hormone action on tumors
- anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
- “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, mice, SCID or nude mice or strains of mice, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, etc.
- the mammal herein is human.
- Oligonucleotides are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (such as phosphotriester, phosphite, or phosphoramidite chemistry, using solid phase techniques such as described in EP 266,032, published 4 May 1988, or via deoxynucleoside H- phosphonate intermediates as described by Froehler et ah, Nucl. Acids Res., 14:5399- 5407, 1986. They are then purified on polyacrylamide gels.
- "humanized” and/or “chimeric" forms of non-human e.g.
- immunoglobulins refer to antibodies which contain specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which results in the decrease of a human anti-mouse antibody (HAMA), human anti-chimeric antibody (HACA) or a human anti-human antibody (HAHA) response, compared to the original antibody, and contain the requisite portions (e.g. CDR(s), antigen binding region(s), variable domain(s) and so on) derived from said non-human immunoglobulin, necessary to reproduce the desired effect, while simultaneously retaining binding characteristics which are comparable to said non-human immunoglobulin.
- HAMA human anti-mouse antibody
- HACA human anti-chimeric antibody
- HAHA human anti-human antibody
- humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient antibody are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
- CDRs complementarity determining regions
- donor antibody non-human species
- Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human FR residues.
- the humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or FR sequences.
- the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin consensus sequence.
- the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
- Fc immunoglobulin constant region
- an antibody which induces "apoptosis” is one which induces programmed cell death by any means, illustrated by but not limited to binding of annexin V, caspase activity, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).
- antibody induced cytotoxicity is understood to mean the cytotoxic effect derived from the hybridoma supernatant or antibody produced by the hybridoma deposited with the IDAC as accession number 141205-05 which effect is not necessarily related to the degree of binding.
- hybridoma cell lines as well as the isolated monoclonal antibodies which are produced therefrom, are alternatively referred to by their internal designation, AR47A6.4.2 or Depository Designation, IDAC 141205- 05.
- antibody-ligand includes a moiety which exhibits binding specificity for at least one epitope of the target antigen, and which may be an intact antibody molecule, antibody fragments, and any molecule having at least an antigen-binding region or portion thereof (i.e., the variable portion of an antibody molecule), e.g., an Fv molecule, Fab molecule, Fab' molecule, F(ab').sub.2 molecule, a bispecific antibody, a fusion protein, or any genetically engineered molecule which specifically recognizes and binds at least one epitope of the antigen bound by the isolated monoclonal antibody produced by the hybridoma cell line designated as IDAC 141205-05 (the IDAC 141205-05 antigen).
- CDMAB cancerous disease modifying antibodies
- a “CDMAB related binding agent”, in its broadest sense, is understood to include, but is not limited to, any form of human or non-human antibodies, antibody fragments, antibody ligands, or the like, which competitively bind to at least one CDMAB target epitope.
- a “competitive binder” is understood to include any form of human or non-human antibodies, antibody fragments, antibody ligands, or the like which has binding affinity for at least one CDMAB target epitope.
- Tumors to be treated include primary tumors and metastatic tumors, as well as refractory tumors.
- Refractory tumors include tumors that fail to respond or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone or combinations thereof.
- Refractory tumors also encompass tumors that appear to be inhibited by treatment with such agents but recur up to five years, sometimes up to ten years or longer after treatment is discontinued.
- Tumors that can be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors.
- solid tumors which can be accordingly treated, include breast carcinoma, lung carcinoma, colorectal carcinoma, pancreatic carcinoma, glioma and lymphoma.
- Some examples of such tumors include epidermoid tumors, squamous tumors, such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors, including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors.
- Kaposi's sarcoma CNS neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas and cerebral metastases, melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma.
- antigen-binding region means a portion of the molecule which recognizes the target antigen.
- target antigen is the IDAC 141205-05 antigen or portions thereof.
- an “immunoconjugate” means any molecule or CDMAB such as an antibody chemically or biologically linked to cytotoxins, radioactive agents, cytokines, interferons, target or reporter moieties, enzymes, toxins, anti-tumor drugs or therapeutic agents.
- the antibody or CDMAB may be linked to the cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, anti- tumor drug or therapeutic agent at any location along the molecule so long as it is able to bind its target.
- immunoconjugates include antibody toxin chemical conjugates and antibody-toxin fusion proteins.
- Radioactive agents suitable for use as anti-tumor agents are known to those skilled in the art. For example, 1311 or 21 IAt is used. These isotopes are attached to the antibody using conventional techniques (e.g. Pedley et al., Br. J. Cancer 68, 69-73 (1993)).
- the anti-tumor agent which is attached to the antibody is an enzyme which activates a prodrug.
- a prodrug may be administered which will remain in its inactive form until it reaches the tumor site where it is converted to its cytotoxin form once the antibody complex is administered.
- the antibody- enzyme conjugate is administered to the patient and allowed to localize in the region of the tissue to be treated.
- the anti-tumor agent conjugated to the antibody is a cytokine such as interleukin-2 (IL-2), interleukin-4 (IL-4) or tumor necrosis factor alpha (TNF- ⁇ ).
- IL-2 interleukin-2
- IL-4 interleukin-4
- TNF- ⁇ tumor necrosis factor alpha
- the antibody targets the cytokine to the tumor so that the cytokine mediates damage to or destruction of the tumor without affecting other tissues.
- the cytokine is fused to the antibody at the DNA level using conventional recombinant DNA techniques. Interferons may also be used.
- fusion protein means any chimeric protein wherein an antigen binding region is connected to a biologically active molecule, e.g., toxin, enzyme, fluorescent proteins, luminescent marker, polypeptide tag, cytokine, interferon, target or reporter moiety or protein drug.
- a biologically active molecule e.g., toxin, enzyme, fluorescent proteins, luminescent marker, polypeptide tag, cytokine, interferon, target or reporter moiety or protein drug.
- the invention further contemplates CDMAB of the present invention to which target or reporter moieties are linked.
- Target moieties are first members of binding pairs.
- Anti-tumor agents for example, are conjugated to second members of such pairs and are thereby directed to the site where the antigen-binding protein is bound.
- a common example of such a binding pair is avidin and biotin.
- biotin is conjugated to the target antigen of the CDMAB of the present invention, and thereby provides a target for an anti-tumor agent or other moiety which is conjugated to avidin or streptavidin.
- biotin or another such moiety is linked to the target antigen of the CDMAB of the present invention and used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin.
- Detectable signal-producing agents are useful in vivo and in vitro for diagnostic purposes.
- the signal producing agent produces a measurable signal which is detectable by external means, usually the measurement of electromagnetic radiation.
- the signal producing agent is an enzyme or chromophore, or emits light by fluorescence, phosphorescence or chemiluminescence.
- Chromophores include dyes which absorb light in the ultraviolet or visible region, and can be substrates or degradation products of enzyme catalyzed reactions.
- kits which contain CDMAB of the present invention. Such kits will be useful for identification of individuals at risk for certain type of cancers by detecting over-expression of the CDMAB 's target antigen on cells of such individuals.
- the CDMAB of the present invention in the form of a diagnostic assay kit for determining the presence of a tumor.
- the tumor will generally be detected in a patient based on the presence of one or more tumor-specific antigens, e.g. proteins and/or polynucleotides which encode such proteins in a biological sample, such as blood, sera, urine and/or tumor biopsies, which samples will have been obtained from the patient.
- tumor-specific antigens e.g. proteins and/or polynucleotides which encode such proteins in a biological sample, such as blood, sera, urine and/or tumor biopsies, which samples will have been obtained from the patient.
- the proteins function as markers which indicate the presence or absence of a particular tumor, for example a colon, breast, lung or prostate tumor. It is further contemplated that the antigen will have utility for the detection of other cancerous tumors.
- Inclusion in the diagnostic assay kits of binding agents comprised of CDMABs of the present invention, or CDMAB related binding agents, enables detection of the level of antigen that binds to the agent in the biological sample.
- Polynucleotide primers and probes may be used to detect the level of mRNA encoding a tumor protein, which is also indicative of the presence or absence of a cancer.
- binding assay In order for the binding assay to be diagnostic, data will have been generated which correlates statistically significant levels of antigen, in relation to that present in normal tissue, so as to render the recognition of binding definitively diagnostic for the presence of a cancerous tumor. It is contemplated that a plurality of formats will be useful for the diagnostic assay of the present invention, as are known to those of ordinary skill in the art, for using a binding agent to detect polypeptide markers in a sample. For example, as illustrated in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Further contemplated are any and all combinations, permutations or modifications of the afore-described diagnostic assay formats.
- the presence or absence of a cancer in a patient will typically be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.
- the assay will involve the use of a CDMAB based binding agent immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample.
- the bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex.
- Illustrative detection reagents may include a CDMAB based binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an antiimmunoglobulin, protein G, protein A or a lectin.
- a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. Indicative of the reactivity of the sample with the immobilized binding agent, is the extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent.
- Suitable polypeptides for use within such assays include full length tumor-specific proteins and/or portions thereof, to which the binding agent has binding affinity.
- the diagnostic kit will be provided with a solid support which may be in the form of any material known to those of ordinary skill in the art to which the protein may be attached. Suitable examples may include a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride.
- the support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681.
- the binding agent will be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature.
- immobilization refers to both noncovalent association, such as adsorption, and covalent attachment, which, in the context of the present invention, may be a direct linkage between the agent and functional groups on the support, or may be a linkage by way of a cross-linking agent.
- immobilization by adsorption to a well in a microtiter plate or to a membrane is preferable.
- Adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time may vary with temperature, and will generally be within a range of between about 1 hour and about 1 day.
- binding agent Covalent attachment of binding agent to a solid support would ordinarily be accomplished by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
- the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at Al 2 A13).
- the diagnostic assay kit will take the form of a two-antibody sandwich assay. This assay may be performed by first contacting an antibody, e.g.
- the instantly disclosed CDMAB that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
- a detection reagent preferably a second antibody capable of binding to a different site on the polypeptide
- the immobilized antibody would then be incubated with the sample, and polypeptide would be allowed to bind to the antibody.
- the sample could be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation.
- PBS phosphate-buffered saline
- an appropriate contact time i.e., incubation time
- the contact time is sufficient to achieve a level of binding that is at least about 95 percent of that achieved at equilibrium between bound and unbound polypeptide.
- the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. It is further contemplated that unbound sample would then be removed by washing the solid support with an appropriate buffer. The second antibody, which contains a reporter group, would then be added to the solid support.
- reporter group Incubation of the detection reagent with the immobilized antibody-polypeptide complex would then be carried out for an amount of time sufficient to detect the bound polypeptide. Subsequently, unbound detection reagent would then be removed and bound detection reagent would be detected using the reporter group.
- the method employed for detecting the reporter group is necessarily specific to the type of reporter group selected, for example for radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
- the signal detected from the reporter group that remains bound to the solid support would generally be compared to a signal that corresponds to a predetermined cut-off value.
- a predetermined cut-off value for the detection of a cancer may be the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer.
- a sample generating a signal that is about three standard deviations above the predetermined cut-off value would be considered positive for the cancer.
- the cut-off value might be determined by using a Receiver Operator Curve, according to the method of Sackett et ah, Clinical Epidemiology.
- the cut-off value could be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100 percent- specificity) that correspond to each possible cut-off value for the diagnostic test result.
- the cut-off value on the plot that is the closest to the upper left-hand corner i.e., the value that encloses the largest area
- a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive.
- the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate.
- a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer.
- the diagnostic assay enabled by the kit will be performed in either a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose.
- a membrane such as nitrocellulose.
- polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane.
- a second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane.
- the detection of bound second binding agent may then be performed as described above.
- the strip test format one end of the membrane to which binding agent is bound will be immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent. Concentration of the second binding agent at the area of immobilized antibody indicates the presence of a cancer.
- the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two- antibody sandwich assay, in the format discussed above.
- Preferred binding agents for use in the instant diagnostic assay are the instantly disclosed antibodies, antigen-binding fragments thereof, and any CDMAB related binding agents as herein described.
- the amount of antibody immobilized on the membrane will be any amount effective to produce a diagnostic assay, and may range from about 25 nanograms to about 1 microgram. Typically such tests may be performed with a very small amount of biological sample.
- CDMAB of the present invention may be used in the laboratory for research due to its ability to identify its target antigen.
- CDMAB i.e., IDAC 141205-05 CDMAB
- IDAC 141205-05 CDMAB specifically recognize and bind the IDAC 141205-05 antigen.
- the CDMAB of the isolated monoclonal antibody produced by the hybridoma deposited with the IDAC as accession number 141205-05 may be in any form as long as it has an antigen-binding region which competitively inhibits the immunospecific binding of the isolated monoclonal antibody produced by hybridoma IDAC 141205-05 to its target antigen.
- any recombinant proteins e.g., fusion proteins wherein the antibody is combined with a second protein such as a lymphokine or a tumor inhibitory growth factor
- a second protein such as a lymphokine or a tumor inhibitory growth factor
- the CDMAB is the IDAC 141205- 05 antibody.
- the CDMAB is an antigen binding fragment which may be a Fv molecule (such as a single-chain Fv molecule), a Fab molecule, a Fab' molecule, a F(ab')2 molecule, a fusion protein, a bispecific antibody, a heteroantibody or any recombinant molecule having the antigen-binding region of the IDAC 141205-05 antibody.
- the CDMAB of the invention is directed to the epitope to which the IDAC 141205-05 monoclonal antibody is directed.
- the CDMAB of the invention may be modified, i.e., by amino acid modifications within the molecule, so as to produce derivative molecules. Chemical modification may also be possible. Modification by direct mutation, methods of affinity maturation, phage display or chain shuffling may also be possible.
- Affinity and specificity can be modified or improved by mutating CDR and/or phenylalanine tryptophan (FW) residues and screening for antigen binding sites having the desired characteristics (e.g., Yang et ah, J. MoI. Biol., (1995) 254: 392-403).
- One way is to randomize individual residues or combinations of residues so that in a population of otherwise identical antigen binding sites, subsets of from two to twenty amino acids are found at particular positions.
- mutations can be induced over a range of residues by error prone PCR methods (e.g., Hawkins et ah, J. MoI. Biol., (1992) 226: 889-96).
- phage display vectors containing heavy and light chain variable region genes can be propagated in mutator strains of E. coli (e.g., Low et ah, J. MoI. Biol., (1996) 250: 359-68). These methods of mutagenesis are illustrative of the many methods known to one of skill in the art.
- Another manner for increasing affinity of the antibodies of the present invention is to carry out chain shuffling, where the heavy or light chain are randomly paired with other heavy or light chains to prepare an antibody with higher affinity.
- the various CDRs of the antibodies may also be shuffled with the corresponding CDRs in other antibodies.
- Derivative molecules would retain the functional property of the polypeptide, namely, the molecule having such substitutions will still permit the binding of the polypeptide to the IDAC 141205-05 antigen or portions thereof.
- amino acid substitutions include, but are not necessarily limited to, amino acid substitutions known in the art as "conservative”.
- Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa.
- Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine and valine (V).
- Methionine (M) which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in particular environments.
- the hybridoma cell line AR47A6.4.2 was deposited, in accordance with the Budapest Treaty, with the International Depository Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada, R3E 3R2, on December 14, 2005, under Accession Number 141205-05.
- IDAC International Depository Authority of Canada
- the depositors assure that all restrictions imposed on the availability to the public of the deposited materials will be irrevocably removed upon the granting of a patent. The deposit will be replaced if the depository cannot dispense viable samples.
- a single cell suspension of frozen human ovarian tumor tissue (endometroid adenocarcinoma; Genomics Collaborative, Cambridge, MA) was prepared in PBS.
- IMMUNEASYTM Qiagen, Venlo, Netherlands adjuvant was prepared for use by gentle mixing.
- Five to seven week old BALB/c mice were immunized by injecting subcutaneously 2 million cells in 50 microliters of the antigen- adjuvant.
- Recently prepared antigen-adjuvant was used to boost the AR47A6.4.2 immunized mice intraperitoneally, 2 and 5 weeks after the initial immunization, with approximately 2 million cells in 50-60 microliters.
- a spleen was used for fusion three days after the last immunization.
- the hybridomas were prepared by fusing the isolated splenocytes with NSO-I myeloma partners. The supernatants from the fusions were tested from subclones of the hybridomas.
- an ELISA assay was employed. 100 microliters/well of goat anti-mouse IgG + IgM (H+L) at a concentration of 2.4 micrograms/mL in coating buffer (0.1 M carbonate/bicarbonate buffer, pH 9.2-9.6) at 4 0 C was added to the ELISA plates overnight. The plates were washed thrice in washing buffer (PBS + 0.05 percent Tween-20). 100 microliters/well blocking buffer (5 percent milk in wash buffer) was added to the plate for 1 hour at room temperature and then washed thrice in washing buffer.
- hybridoma supernatants were tested for antibodies that bound to target cells in a cell ELISA assay.
- Two human ovarian cancer cell lines, and 1 human normal skin cell line were tested: OCC-I , OVCAR-3 and CCD-27sk respectively. All cell lines, except for OCC-I, were obtained from the American Type Tissue Collection (ATCC, Manassas, VA). The OCC-I ovarian cancer cell line was obtained from the Ottawa Regional Cancer Center (Ottawa, ON).
- the plated cells were fixed prior to use.
- the plates were washed thrice with PBS containing MgCl 2 and CaCl 2 at room temperature.
- 100 microliters of 2 percent paraformaldehyde diluted in PBS was added to each well for 10 minutes at room temperature and then discarded.
- the plates were again washed with PBS containing MgCl 2 and CaCl 2 three times at room temperature. Blocking was done with 100 microliters/well of 5 percent milk in wash buffer (PBS + 0.05 percent Tween-20) for 1 hour at room temperature.
- the plates were washed thrice with wash buffer and the hybridoma supernatant was added at 75 microliters/well for 1 hour at room temperature.
- the plates were washed 3 times with wash buffer and 100 microliters/well of 1/25,000 dilution of goat anti-mouse IgG antibody conjugated to horseradish peroxidase (diluted in PBS containing 1 percent milk) was added. After 1 hour incubation at room temperature the plates were washed 3 times with wash buffer and 100 microliter/well of TMB substrate was incubated for 1-3 minutes at room temperature. The reaction was terminated with 50 microliters/well 2M H 2 SO 4 and the plate read at 450 nm with a Perkin-Elmer HTS7000 plate reader.
- the cytotoxic effect of the hybridoma supernatants was tested in the cell lines: OCC-I, OVCAR-3 and CCD- 27sk.
- Calcein AM was obtained from Molecular Probes (Eugene, OR). The assays were performed according to the manufacturer's instructions with the changes outlined below. Cells were plated before the assay at the predetermined appropriate density. After 2 days, 75 microliters of supernatant from the hybridoma microtiter plates were transferred to the cell plates and incubated in a 5 percent CO 2 incubator for 5 days. The wells that served as the positive controls were aspirated until empty and 100 microliters of sodium azide (NaN3) or cycloheximide was added.
- NaN3 sodium azide
- cycloheximide was added.
- FACS flow cytometry
- Cells were prepared for FACS by initially washing the cell monolayer with DPBS (without Ca ++ and Mg ++ ). Cell dissociation buffer (INVITROGEN, Burlington, ON) was then used to dislodge the cells from their cell culture plates at 37°C.
- the cells were resuspended in DPBS containing MgCl 2, CaCl 2 and 2 percent fetal bovine serum at 4°C (staining media) and counted, aliquoted to appropriate cell density, spun down to pellet the cells and resuspended in staining media at 4°C in the presence of test antibody (AR47A6.4.2) or control antibodies (isotype control, anti-EGFR) at 20 micrograms/mL on ice for 30 minutes. Prior to the addition of Alexa Fluor 546-conjugated secondary antibody the cells were washed once with staining media. The Alexa Fluor 546-conjugated antibody in staining media was then added for 30 minutes at 4 0 C.
- test antibody AR47A6.4.2
- control antibodies isotype control, anti-EGFR
- the cells were then washed for the final time and resuspended in fixing media (staining media containing 1.5 percent paraformaldehyde).
- Flow cytometric acquisition of the cells was assessed by running samples on a FACSarrayTM using the FACSarrayTM System Software (BD Biosciences, Oakville, ON).
- the forward (FSC) and side scatter (SSC) of the cells were set by adjusting the voltage and amplitude gains on the FSC and SSC detectors.
- the detectors for the fluorescence (Alexa-546) channel was adjusted by running unstained cells such that cells had a uniform peak with a median fluorescent intensity of approximately 1 -5 units. For each sample, approximately 10,000 gated events (stained fixed cells) were acquired for analysis and the results are presented in Figure 3.
- Figure 2 presents the mean fluorescence intensity fold increase above isotype control.
- Representative histograms of AR47A6.4.2 antibodies were compiled for Figure 3.
- AR47A6.4.2 showed strong binding to the pancreatic cancer cell lines BxPC-3 and PL45 (31.6-fold and 26.4-fold respectively), colon cancer cell lines DLD- 1 , HT-29 and Colo-205 (91.5-fold, 22.1-fold and 44.9-fold respectively), breast cancer cell line MDA-MB-468 (35.9-fold), head and neck cancer cell line FaDu (77.1 -fold), esophageal cancer cell line T.Tn (26.9-fold) and ovarian cancer cell lines OV2008, OVCAR-3 and C-13 (78.4-fold, 28.4-fold and 43.6-fold respectively).
- Binding was also observed on the breast cancer cell line MCF-7 (5.4-fold), prostate cancer cell lines PC-3 and DU-145 (3.3-fold and 5.1-fold respectively), lung cancer cell line NCI-H520 (10.7- fold), colon cancer cell line SWl 1 16 (1.8-fold) and ovarian cancer cell lines Hey, Sk- OV-3, OCC-I and OVCAR-429 (6.6-fold, 1.9-fold, 10-fold and 4.2-fold respectively). Binding to the non-cancer cell lines from skin (CCD-27sk) and lung (Hs888.Lu) was not detectable under these conditions. These data demonstrate that AR47A6.4.2 exhibited functional specificity in that although there was clear binding to a variety of cancer cell lines there was only associated cytotoxicity with some of the lines tested. EXAMPLE 3
- Examples 1 and 2 demonstrated that AR47A6.4.2 had anti-cancer properties against a human cancer cell line with detectable binding across several different cancer indications.
- 6 to 8 week old female SCID mice were implanted with 5 million human pancreatic cancer cells (BxPC-3) in 100 microliters saline injected subcutaneously in the scruff of the neck. The mice were randomly divided into 3 treatment groups of 5.
- AR47A6.4.2 prevented tumor growth and reduced tumor burden in an in vivo prophylactic model of human pancreatic cancer.
- the mean tumor volume in the AR47A6.4.2 treated group was 53 percent less than that of the buffer control-treated group ( ⁇ 0.05; Figure 4).
- Body weight shown in Figure 5
- body weight was used as a surrogate for well-being and failure to thrive.
- body weight was used as a surrogate for well-being and failure to thrive.
- AR47A6.4.2 test antibody or buffer control was administered intraperitoneally to each cohort, with dosing at 20 mg/kg of antibody in a volume of 300 microliters after dilution from the stock concentration with a diluent that contained 2.7 mM KCl, 1 mM KH 2 PO 4 , 137 mM NaCl and 20 mM Na 2 HPO 4 .
- the antibodies were then administered 3 times per week for a total of 10 doses in the same fashion until day 53 post-implantation. Tumor growth was measured about every seventh day with calipers until day 63 post-implantation or until individual animals reached the CCAC end-points. Body weights of the animals were recorded once per week for the duration of the study.
- AR47A6.4.2 significantly reduced tumor burden in an established model of human pancreatic cancer.
- AR47A6.4.2-treated animals had a mean tumor volume that was 40 percent of the mean tumor volume in control-treated animals (pO.OOOl ; Figure 6).
- pO.OOOl a mean tumor volume in control-treated animals
- Body weight measured at weekly intervals was used as a surrogate for well-being and failure to thrive.
- body weight in all groups did not vary significantly over the course of the study.
- AR47A6.4.2 was well-tolerated and decreased the tumor burden in this established human pancreatic cancer xenograft model.
- AR47A6.4.2 has demonstrated efficacy in both a preventative and established model of human pancreatic cancer.
- AR47A6.4.2 completely inhibited tumor growth in the PL45 in vivo prophylactic model of human pancreatic cancer.
- the study was still ongoing at day 102, 45 days after last dose, at which point all mice in the control group had been removed from the study due to tumor volume.
- AR47A6.4.2 still demonstrated almost complete inhibition of tumor growth and 4 mice in that group were still alive (Figure 9).
- Body weight measured at weekly intervals was a surrogate for well-being and failure to thrive.
- the mean body weight increased in all groups over the duration of the study ( Figure 10).
- the mean weight gain between day 13 and day 77 was 3.39 g (17.4 percent) in the control group and 1.7 g (8.6 percent) in the AR47A6.4.2-treated group. There were no significant differences between the groups during the treatment period and at day 77, 20 days after the last dose.
- AR47A6.4.2 was well-tolerated and almost completely inhibited the tumor growth in this human pancreatic cancer xenograft model.
- AR47A6.4.2 treatment also demonstrated increased survival in comparison to buffer treatment.
- AR47A6.4.2 therefore has demonstrated efficacy in two different models of human pancreatic cancer.
- Examples 3, 4 and 5 demonstrated that AR47A6.4.2 had anti-cancer properties against two different human pancreatic cancer xenograft models.
- the antibody was tested on a PC-3 prostate cancer xenograft model.
- PC-3 prostate cancer xenograft model With reference to Figures 1 1, 12, and 13, 8 to 10 week old female SCID mice were implanted with 5 million human prostate cancer cells (PC-3) in 100 microliters PBS solution injected subcutaneously in the scruff of the neck. The mice were randomly divided into 2 treatment groups of 10.
- AR47A6.4.2 inhibited tumor growth in the PC-3 in vivo prophylactic model of human prostate adenocarcinoma cells.
- AR47A6.4.2 was well-tolerated and significantly inhibited the tumor growth in this human prostate cancer xenograft model. Treatment with antibody also demonstrated a survival benefit in comparison to the control group. AR47A6.4.2 has demonstrated efficacy against two different human cancer indications; pancreatic and prostate.
- Examples 3, 4, 5 and 6 demonstrated that AR47A6.4.2 had anti-cancer properties against two different human pancreatic and a prostate cancer xenograft model.
- the antibody was tested on a MCF-7 cancer xenograft model.
- MCF-7 human breast cancer xenograft model.
- RPMI 1640 human breast cancer cells
- AR47A6.4.2 reduced tumor growth in the MCF-7 in vivo prophylactic model of human breast cancer.
- Treatment with ARIUS antibody AR47A6.4.2 resulted in a marked tumor growth delay.
- AR47A6.4.2 induced T/C percent values that were lower than 42 percent from day 18 to day 35 of treatment and close to 42 percent up to day 49 (optimal T/C percent value of 10.9 percent at day 18) ( Figure 14).
- T/C percent value 10.9 percent at day 18
- a post-treatment survival benefit (Figure 16) was associated with AR47A6.4.2 administration.
- the buffer control group reached 100 percent mortality by day 85 post-treatment while 33.3 percent of the AR47A6.4.2 mice were still alive at day 91 post-treatment.
- AR47A6.4.2 was well-tolerated, reduced tumor growth and provided a survival benefit in this human breast cancer xenograft model.
- AR47A6.4.2 has demonstrated efficacy against three different human cancer indications; pancreatic, prostate and breast.
- AR47A6.4.2 inhibited tumor growth in the Colo 205 in vivo prophylactic model of human colorectal adenocarcinoma cells.
- AR47A6.4.2 was well-tolerated and significantly inhibited the tumor growth in this human colon cancer xenograft model.
- AR47A6.4.2 has demonstrated efficacy against four different human cancer indications; pancreatic, prostate, breast and colon. Treatment benefits were observed in several well-recognized models of human cancer disease suggesting pharmacologic and pharmaceutical benefits of this antibody for therapy in other mammals, including man.
- Total cell membranes were prepared from confluent cultures of MDA- MB-231 (MB-231 ) breast cancer cells. Media was removed from cell stacks and the cells were washed with phosphate buffered saline (PBS). Cells were dissociated with dissociation buffer (Gibco-BRL; Grand Island, NY) for 20 minutes at 37°C on a platform shaker. Cells were collected and centrifuged at 90Og for 10 minutes at 4°C. After centrifugation, cell pellets were washed by resuspending in PBS and centrifuging again at 90Og for 10 minutes at 4°C. Pellets were then stored at -8O 0 C until required.
- PBS phosphate buffered saline
- cell pellets were thawed and resuspended in homogenization buffer containing 1 tablet per 50 mL of complete protease inhibitor cocktail (Roche; Laval QC) at a ratio of 3 mL buffer per gram of cells.
- the cell suspension was subjected to homogenization using a polytron homogenizer on ice in order to lyse the cells.
- the cell homogenate was centrifuged at 15,000g for 10 minutes at 4°C to remove the nuclear particulate. Supernatant was harvested, divided into tubes and then centrifuged at 75,60Og for 90 minutes at 4 0 C. Supernatant was carefully removed and each membrane pellet was resuspended in approximately 5 mL of homogenization buffer.
- the membrane pellets from all tubes were combined, divided one more time, and centrifuged at 75,60Og for 90 minutes at 4°C. Supernatant was carefully removed and the pellets were weighed. Solubilization buffer containing 1 percent Triton X-IOO was added to the pellets at a ratio of 3 mL buffer per gram of membrane pellet. Membranes were solubilized by shaking on a platform shaker at 300 rpm, for 1 hour on ice. The membrane suspension was centrifuged at 75,60Og to pellet insoluble material. The supernatant, containing the solubilized membrane proteins, was carefully removed from the tubes, assayed for protein concentration, and stored at -80 0 C.
- Immunoprecipitation of AR47A6.4.2 antigen was carried out as follows: total membrane fraction was diluted to a 1 mg/mL final protein concentration, with IX RIPA buffer containing protease inhibitors. Protein G Sepharose beads chemically conjugated to 8A3B.6 isotype control antibody (conjugated at a ratio of 2 micrograms of antibody per 1 microliters of drained beads), were added to the total membrane fraction and incubated at 4°C for 3 hour, with rotation. After, the sample was centrifuged at 20000 x g for 8 sec. The supernatant (unbound fraction) was removed and the beads were stored on ice.
- the membranes were blocked with 5 percent (w/v) skim milk in TBST, for 1 hour at room temperature (RT), and two replicate blots were then probed as follows: one blot was probed with the antibody AR47A6.4.2 (5 mg/mL, in 5 percent skim milk in TBST) and the replicate blot was probed with an IgG2a isotype control (5 mg/mL, in 5 percent skim milk in TBST). Blots were washed 3 times for 10 minutes in TBST and then incubated with horseradish HRP-conjugated goat anti-mouse IgG (Fc) (Bio-Rad Laboratories; Hercules, CA), for 1 hour at RT.
- Fc horseradish HRP-conjugated goat anti-mouse IgG
- the blots were developed with the ECL PlusTM kit (GE Healthcare, Life Sciences formerly Amersham Biosciences; Piscataway, NJ) following the manufacturers' instructions. The blots were rinsed with water and images were acquired with a gel documentation system (Bio-Rad; Hercules, CA). Blots were imaged under the same conditions of camera focus, aperture and image acquisition time. In Figure 19, when used as a probe on a Western blot, the antibody
- AR47A6.4.2 clearly bound to a protein with an apparent molecular weight of approximately 50 kDa in the total membrane fraction from MB-231 cells, but not to the whole cell lysate of either PC-3 or CCD-27sk cells.
- the antibody AR47A6.4.2 specifically recognized a protein of apparent molecular weight of around 50 kDa that was immunoprecipitated, by AR47A6.4.2-conjugated Protein G Sepharose beads, from MB-231 total membrane fraction. It can be observed that the band recognized by the probe AR47A6.4.2 is very distinct from those observed by cross reactivity of the secondary antibody alone, which represent IgG and IgG heavy chain that leaked off the Protein G beads.
- FIG. 21 shows that glycosidase treatment of immunocomplexes obtained from MB-231 TM fraction resulted in a significant decrease in the mass of the recognized antigen(s). This indicated that the antigen recognized by the AR47A6.4.2 antibody was comprised of at least one glycoprotein.
- Total membrane fraction extract from MB-231 cells (9.4 mg) was prepared by dilution, 1 mg/mL final concentration, with IX RIPA buffer containing a protease inhibitors cocktail.
- Total membrane fraction extract was pre-cleared by incubation with protein G Sepharose beads (5 mL drained beads) for 2 hour, at 4°C with rotation. After centrifugation the beads were removed and stock bovine serum albumin (BSA) (10 mg/mL) was added to a 0.5 mg/mL final BSA concentration.
- BSA bovine serum albumin
- AR47A6.4.2 and 8A3B.6 isotype control antibody-conjugated protein G-Sepharose beads 120 micrograms of antibody chemically cross-linked to 60 microliters of protein G Sepharose
- 1 mL of 0.5 mg/mL BSA by incubation at 4°C, also for 2 hours.
- the antibody-conjugated beads were washed twice for 5 minutes with 1 X RIPA buffer.
- the total membrane extract was then incubated with the isotype control (8A3B.6)-conjugated protein G Sepharose beads (60 microliters of drained beads, 120 micrograms of IgG) at 4°C for 2 hours, with rotation, on an end-over-end rotator. After centrifugation at 20,00Og, for 10 seconds, at 4°C, the supernatant (unbound fraction) was removed and saved, and the beads were washed 3 times for 5 minutes, with 1 mL of RIPA buffer in each wash step. The beads were then rinsed once with 1.5 mL of PBS and then were stored on ice.
- the isotype control 8A3B.6-conjugated protein G Sepharose beads (60 microliters of drained beads, 120 micrograms of IgG) at 4°C for 2 hours, with rotation, on an end-over-end rotator. After centrifugation at 20,00Og, for 10 seconds, at 4°C, the supernatant (unbound fraction
- the saved supernatant (unbound fraction) was then incubated with the AR47A6.4.2-conjugated protein G Sepharose beads (60 microliters of drained beads, 120 micrograms of IgG) at 4 0 C, for 2 hours, with rotation. After centrifugation at 20,00Og, for 10 seconds, at 4°C, the supernatant (unbound fraction) was removed and saved, and the beads were washed 3 times for 5 minutes, with 1 mL of RIPA buffer in each wash step. The beads were then rinsed once with 1.5 mL of PBS and TM fraction extract was saved at -8O 0 C and the beads were stored on ice.
- the isotype control beads and two aliquots containing AR47A6.4.2-protein G Sepharose conjugated beads (one being that used in the immunoprecipitation step and a second aliquot containing identical volume of beads, but not used in any IP (designated as 'mock' IP)).
- the beads were then stored overnight at -85 0 C.
- each sample containing antibody- conjugated Protein G Sepharose beads (samples AR47A6.4.2 IP, AR47A6.4.2 'mock' IP and 8A3B.6 isotype control IP) were divided in two 30 microliter aliquots.
- each sample was added 60 microliters of IX non-reducing SDS- PAGE sample buffer. After boiling for 4 minutes the sample buffer was removed and transferred into the tube containing the second aliquot from the same sample. This pooled sample was then boiled for 4 minutes. After cooling down on ice, each sample was loaded onto two separate gels (1/1 Oth of the sample in one gel, for detection by Western blotting, the remaining 9/1 Oth on the other gel, for detection by staining with Colloidal Blue).
- the gel designated for Western blotting was transferred onto a PVDF membrane for 2 hours at 320 mA, rinsed with deionized water, blocked for 1 hour at RT with 5 percent milk in TBST and then incubated for 2 hours in 5 percent milk in TBST, also at RT. Blots were washed 3 times for 10 minutes in TBST and incubated with an HRP-conjugated Fc-specific goat anti-mouse IgG (1 :50000) in 5 percent milk in TBST, for 1 hour at room temperature. Blots were then washed 3 times for 10 minutes and were developed by using an enhanced chemiluminescence detection system, following the manufaturer's recommendations. The gel designated for protein staining was incubated overnight with the Coomassie Colloidal Blue stain and destained with ultrapure water, for 48 hour.
- Coomassie Colloidal Blue stained gel were lined up using the bands from the molecular weight markers lanes as reference ( Figure 22).
- a specific band from the lane, on the Coomasie Colloidal Blue-stained gel, containing the AR47A6.4.2 immunoprecipitate was cored using a glass pasteur pipette.
- the equivalent regions of all the control lanes (AR47A6.4.2 'mock IP' and 8A3B.6 isotype control IP) and from a region of the gel that did not contain any sample were also cored as shown in Figure 23, where left and right panels are the images of the gel before and after coring, respectively.
- Gel plugs were divided in two aliquots containing similar amounts of gel plugs.
- CHCA matrix a-cyano 4- hydroxy cinnaminic acid; Ciphergen Biosystems Inc., Fremont, CA
- MS/MS data was then analyzed with MASCOT, a publicly accessible online tool for searching protein databases using information from MS/MS spectra.
- the only protein that was suggested as a putative candidate, with a significant degree of confidence was TROP-2.
- Figure 25 is a summary from the MASCOT search. SEQ ID NO:9 is shown. The only protein that was identified with a high degree of probability was TROP-2, supporting the previous identification by MS peptide mass fingerprinting.
- Immunoprecipitates from an MB-231 total membrane extract in IX RIPA buffer prepared with the monoclonal antibodies AR47A6.4.2 and 8A3B.6 IgG2a isotype control, and the AR47A6.4.2 'mock IP' negative control (described above) were analyzed by 1 D SDS-PAGE followed by Western immunoblotting. Equal volume fractions from each immunocomplex sample were analyzed on replicate gels. After electroblotting onto PVDF membranes, the blots from the replicate gels were probed in parallel with the monoclonal antibodies AR47A6.4.2, anti-human TROP-2 and with the IgG2a isotype control.
- Figure 26 demonstrates the result from the cross-IP experiments, in which the material immunoprecipitated by the test monoclonal antibodies AR47A6.4.2 was analyzed by Western immunoblotting.
- Each of the monoclonal antibodies AR47A6.4.2 and anti-human TROP-2 clone 7220.1 1 specifically cross-reacted with similar antigen(s) immunoprecipitated by AR47A6.4.2.
- the isotype control antibody 8A3B.6 did not cross-react with any specific band.
- the antibodies used to probe the Western blots cross-reacted with no bands on the negative control immunocomplexes. This data indicated that the epitope recognized by the AR47A6.4.2 antibody was contained within the TROP-2 antigen.
- AR47A6.4.2 was directly binding to the human TROP-2 antigen, its reactivity was assessed, by Western immunoblotting against recombinant fusion polypeptides containing the extracellular domain of human TROP-2 and the Fc region of human IgGl , and expressed by the mouse myeloma cell line NSO (R&D Systems, Minneapolis, MN).
- Control reactions were included which contained 5 microliters of water instead of deglycosylation enzymes.
- 0.4 micrograms rhTROP-2 or 100 micrograms of MDA-MB-231 membrane proteins were diluted to 35 microliters with water. 10 microliters of incubation buffer was added, along with 1 microliter of each enzyme listed above. Control reactions were included which contained 5 microliters of water instead of deglycosylation enzymes. All reactions were incubated at 37°C for 24 hours. Following deglycosylation, reactions were prepared for SDS-PAGE.
- 16.7 microliters of reducing or non-reducing sample loading buffer (4 X) was added to the denatured and non-denatured reactions, respectively. Samples were boiled for 5 minutes, then cooled to room temperature. 16.7 microliters of each reaction was loaded onto quadruplicate 12 percent SDS-PAGE gels. Gels were run at 150 V until the dye front ran off. Proteins were transferred to PVDF membranes overnight at 40 V. Membranes were blocked with 5 percent milk prepared with TBST (Tris-buffered saline with 0.05 percent Tween-20) for 1 hour, followed by incubation with primary antibodies for 2 hours.
- TBST Tris-buffered saline with 0.05 percent Tween-20
- Each primary antibody was diluted to 5 micrograms/niL in 5 percent milk, except anti-human TROP-2, which was diluted to 2 micrograms/mL. Blots were incubated with one of AR47A6.4.2, anti-human TROP-2 (R&D Systems, Minneapolis, MN) or IgG isotype control. Following primary antibody incubation, blots were washed 3 times, 10 minutes each, with TBST. Blots were incubated with goat anti-mouse IgG Fc HRP secondary antibody diluted to 1 :50,000 in 5 percent milk for 1 hour, then washed 3 times, 10 minutes each, with TBST. Blots were developed with ECL Plus Western Blotting Detection Reagents (GE Healthcare, Life Sciences formerly Amersham Biosciences; Piscataway, NJ) and an X-ray developer.
- ECL Plus Western Blotting Detection Reagents GE Healthcare, Life Sciences formerly Amersham Biosciences; Piscataway, NJ
- Figures 28-30 show the results of the 3 blots probed with AR47A6.4.2, anti-human TROP-2 and IgG isotype control respectively.
- Figure 28 (blot probed with AR47A6.4.2) shows a weak band in lane 1 (non-denatured and non-deglycosylated MB-231 total membrane fraction) at -52 kDa. This band is not detectable in Figure 29 (blot probed with anti -human TROP-2).
- the reactive band in the sample from lane 2 shows a shift to around 37 kDa and was recognized by AR47A6.4.2 (Figure 28) and by the commercial anti-TROP-2 antibody (Figure 29) blot, but not by the isotype control ( Figure 30). No bands were detected in lanes 5 or 6 (denatured and non-deglycosylated and deglycosylated MB-231 total membrane fraction) in any of the blots, indicating that the antibodies did not detectably bind to the target protein in the total membrane fraction under reducing conditions.
- Recombinant human TROP-2 appears as a very intense, very high molecular weight band (apparent molecular weight larger than 22OkDa) in lanes 3 and 4 (non-denatured non-deglycosylated and deglycosylated rhTROP-2, respectively) in Figures 28 and 29 (AR47A6.4.2 and anti-human TROP-2, respectively) correspond to disulfide-bond linked multimers of rhTROP-2.
- This result suggests that the antibodies may recognize a non-carbohydrate epitope, possibly a polypeptide epitope, although it is not possible to rule out that binding may be occurring to a carbohydrate group that was not removed by the particular mixture of glycosidases used in this experiment.
- AR47A6.4.2 and AR52A301.5 recognize similar or distinct epitopes of TROP-2.
- Two micrograms of recombinant fusion polypeptides containing the extracellular domain of human TROP-2 and the Fc region of human IgG 1 , and expressed by the mouse myeloma cell line NSO (R&D Systems, Minneapolis, MN) were subjected to SDS- PAGE under non-reducing conditions using preparative well combs that spanned the entire length of each of two 10 percent polyacrylamide gels.
- the proteins from the gels were transferred to PVDF membranes at 40V for approximately 17 hours at 4°C.
- the membranes were blocked with 5 percent skim milk in TBST for one hour at room temperature on a rotating platform.
- biotinylated AR47A6.4.2 and AR52A301.5 had been prepared using EZ- Link NHS-PEO Solid Phase Biotinylation Kit (Pierce, Rockford, IL).
- Primary antibody solutions were prepared by mixing biotinylated AR47A6.4.2 or biotinylated AR52A301.5 with varying concentrations of non-biotinylated antibodies.
- solutions were prepared containing 0.05 micrograms/mL of biotinylated AR52A301.5 in 3 percent skim milk in TBST plus 0.5 micrograms/mL, 5 micrograms/mL, 50 micrograms/mL, 500 micrograms/mL or 1000 micrograms/mL of non-biotinylated antibody.
- the non-biotinylated antibodies that were used were AR52A301.5, AR47A6.4.2 and control antibody 8A3B.6 (anti-bluetongue virus; IgG2a, kappa, purified in-house). Solutions containing 0.05 micrograms/mL of biotinylated
- AR47A6.4.2 were prepared with the same concentrations listed above of the non- biotinylated antibodies AR52A301.5, AR47A6.4.2 and control antibody 1 B7.1 1 (anti- TNP; IgGl , kappa, 20 micrograms/mL, purified in-house).
- a negative control solution consisting of three percent skim milk in TBST was added to two channels on each membrane.
- the primary antibody solutions were incubated in separate channels on the membranes for 2 hours at room temperature on a rocking platform. Each channel was washed 3 times with TBST for ten minutes on a rocking platform. Secondary solution consisting of 0.01 micrograms/mL peroxidase conjugated streptavidin (Jackson Immunoresearch, West Grove PA) in 3 percent skim milk in TBST was applied to each channel on the membrane, except for one channel on each membrane to which 3 percent milk in TBST alone was applied as a negative control. The membranes were incubated in secondary solution for 1 hour at room temperature on a rocking platform. Each channel was washed 3 times with TBST for ten minutes on a rocking platform.
- the membranes were removed from the multiscreen apparatus and incubated with an enhanced chemiluminescence detection solution (GE Healthcare, Life Sciences formerly Amersham Biosciences; Piscataway, NJ) according to manufacturer's directions. The membranes were then exposed to film and developed.
- an enhanced chemiluminescence detection solution GE Healthcare, Life Sciences formerly Amersham Biosciences; Piscataway, NJ
- Figures 31 and 32 show the results of the antibody competition experiments. Binding of the biotinylated AR52A301.5 was completely inhibited at a concentration of 50 micrograms/mL and greater of non-biotinylated AR52A301.5 (100OX excess; Figure 31 lanes 3-7) while the binding of AR47A6.4.2 was completely inhibited at a concentration of 500 micrograms/mL and greater of non-biotinylated AR47A6.4.2 (10000X excess; Figure 32 lanes 9-13).
- Binding of antibodies to 12 human normal organs, ovary, pancreas, thyroid, brain (cerebrum, cerebellum), lung, spleen, uterus, cervix, heart, skin, and skeletal muscle was performed using a human normal tissue screening array (Biochain, CA, USA).
- the array contained 20 normal human organs; however, only 12 of the organs were interpretable after staining.
- Figure 33 presents a summary of the results of AR47A6.4.2 staining of an array of human normal tissues.
- the AR47A6.4.2 antibody showed binding predominantly to epithelial tissues (endothelium of blood vessels, follicular epithelium of thyroid, acinar and ductal epithelium of pancreas, alveolar epithelium of lung, and epidermal keratinocytes of skin).
- the antibody also showed equivocal binding to lymphoid tissue of the spleen and binding to neural tissue of the brain ( Figure 34). Cellular localization was cytoplasmic and membranous with diffuse staining pattern.
- AR47A6.4.2 showed a similar binding pattern when compared to the commercial anti-TROP-2 (clone 77220.1 1).
- AR47A6.4.2 anti-human muscle actin (Clone HHF35, Dako, Toronto, Ontario), anti- cytokeratin 7 clone OV-TL 12/30 (Dako, Toronto, Ontario) or isotype control antibody (directed towards Aspergillus niger glucose oxidase, an enzyme which is neither present nor inducible in mammalian tissues; Dako, Toronto, Ontario) were diluted in antibody dilution buffer (Dako, Toronto, Ontario) to its working concentration of 5 micrograms/mL for each antibody except for anti-actin which was 0.5 micrograms/mL and anti-cytokeratin 7 which was ready to use. Primary antibody and slides were incubated together for 1 hour at room temperature.
- the slides were washed with PBS 3 times for 2 minutes each. Immunoreactivity of the primary antibodies was detected/visualized with HRP conjugated secondary antibodies as supplied (Dako Envision System, Toronto, Ontario) for 30 minutes at room temperature. Following this step the slides were washed with PBS 3 times for 5 minutes each and a color reaction developed by adding DAB (3,3'-diaminobenzidine tetrahydrachloride, Dako, Toronto, Ontario) chromogen substrate solution for immunoperoxidase staining for 10 minutes at room temperature. Washing the slides in tap water terminated the chromogenic reaction.
- DAB diaminobenzidine tetrahydrachloride
- the slides were dehydrated with graded ethanol (75-100 percent) and cleared with xylene. Using mounting media (Dako Faramount, Toronto, Ontario) the slides were coverslipped.
- pancreatic array Tri Star, Rockville, MD
- the tissue sections were initially air dried at room temperature for 2 hours and air dried again for 30 minutes after fixation with acetone. The endogenous hydrogen peroxide was blocked using 3 percent hydrogen peroxide in methanol for 15 minutes; this step was done after the primary antibody incubation.
- Figure 35 presents a summary of the results of AR47A6.4.2 staining of various human tumors and their corresponding normal tissue sections (10 colon cancers and 1 normal colon, 7 ovarian cancers and 1 normal ovary, 1 1 breast cancers and 3 normal breast, 14 lung cancers and 3 normal lung, 13 prostate cancers and 3 normal prostate, and 13 pancreatic cancers and 4 normal pancreas).
- the tissues were distributed on three different tissue microarrays (Tri Star, Rockville, MD).
- the antibody showed moderate to strong binding to 5/10 (50 percent), 6/7 (86 percent), 10/1 1 (91 percent), 1 1/14 (79 percent), 13/13 (100 percent) and 2/13 (15 percent) of colon, ovarian, breast, lung, prostate and pancreatic cancers, respectively.
- Immunoreactivity of the primary antibodies was detected/visualized with HRP conjugated secondary antibodies as supplied (Dako Envision System, Toronto, Ontario) for 30 minutes at room temperature. Following this step the slides were washed with PBS 3 times for 5 minutes each and a color reaction developed by adding DAB (3,3'-diaminobenzidine tetrahydrachloride, Dako, Toronto, Ontario) chromogen substrate solution for immunoperoxidase staining for 10 minutes at room temperature. Washing the slides in tap water terminated the chromogenic reaction. Following counterstaining with Meyer's Hematoxylin (Sigma Diagnostics, Oakville, ON), the slides were dehydrated with graded ethanols (75-100 percent) and cleared with xylene.
- DAB diaminobenzidine tetrahydrachloride
- the slides were coverslipped.
- mounting media Dermatamount, Toronto, Ontario
- the same protocol was followed with the following modifications.
- the tissue sections were air dried at room temperature for 30 minutes and then washed with cold PBS without acetone fixation (the sections were acetone fixed from the manufacturer).
- the endogenous hydrogen peroxide was blocked using 3 percent hydrogen peroxide in methanol for 20 minutes; this step was done after the primary antibody incubation.
- AR47A6.4.2 showed no detectable binding to the tested mouse, rat, guinea pig, goat, sheep, hamster, chicken, cow, horse or pig normal tissues. For the normal rabbit and dog tissues, there was dissimilar binding to that observed in the corresponding human tissues. For the cynomolgus normal tissues, AR47A6.4.2 showed similar tissue specificity as observed in the corresponding human normal tissues ( Figure 38) for all of the tested organs except for the ovary and testis in which no detectable binding was observed for the cynomolgus sections. For the rhesus normal tissues, AR47A6.4.2 showed similar tissue specificity as observed in the corresponding human normal tissues ( Figure 38).
- rhesus normal tissue panel was smaller than what was tested for the cynomolgus. Based on the staining profiles, both the cynomolgus and rhesus monkey are considered to be suitable toxicology models for AR47A6.4.2.
- mRNA Messenger ribonucleic acid
- AR47A6.4.2 confluent Master Cell Bank
- Poly A Tract System 1000 mRNA extraction kit Promega Corp., Madison, WI.
- mRNA was stored at -80 0 C until required for further use.
- RT-PCR Reverse transcriptase polymerase chain reaction
- 5.0 microliters of mRNA was mixed with 1.0 microliter of 20 pmol/microliter MuIgG ⁇ V L -3' primer OL040 and 5.5 microliters nuclease free water (Promega Corp., Madison, WI).
- 5.0 microliters of mRNA was mixed with 1.0 microliter of 20 pmol/microliter MuIgG ⁇ V L - 3' primer OL042 and 5.5 microliters nuclease free water (Promega Corp., Madison, WI).
- gamma heavy chain 5 microliters of mRNA was mixed with 1.0 microliter of 20 pmol/microliter MuIgGV H -3' primer OL023 and 5.5 microliters nuclease free water (Promega Corp., Madison, WI). All three reaction mixes were placed in the pre- heated block of the thermal cycler set at 70 0 C for 5 minutes.
- reaction mixes were then chilled on ice for 5 minutes before adding to each 4.0 microliters ImPromII 5x reaction buffer (Promega Corp., Madison, WI), 0.5 microliters RNasin ribonuclease inhibitor (Promega Corp., Madison, WI), 2.0 microliters 25mM MgCl 2 (Promega Corp., Madison, WI), 1.0 microliter 10 mM dNTP mix (Invitrogen, Paisley, UK) and 1.0 microliter Improm II reverse transcriptase (Promega Corp., Madison, WI).
- These reaction mixes were incubated at room temperature for 5 minutes before being transferred to a pre-heated PCR block set at 42 0 C for 1 hour. After this time the reverse transcriptase was heat inactivated by incubating at 70 0 C in a PCR block for fifteen minutes.
- 5' single primer (MuIgV H 5'-A and B; MuIg ⁇ V L h5'-A, B and C; MuIg ⁇ V L 5'- A) contained each primer at a concentration of 20 micromolar;
- 5' primer pools (MuIgV H 5'-C to F; MuIg ⁇ V L h5'-D to G) contained each constituent primer at a concentration of 5 micromolar.
- Heavy and light chain sequences were amplified from cDNA.
- a PCR master mix was prepared by adding 37.5 microliters 10x Hi-Fi Expand PCR buffer (Roche, Mannheim, Germany), 7.5 microliters 1OmM dNTP mix (Invitrogen, Paisley, UK) and 3.75 microliters Hi-Fi Expand DNA polymerase (Roche, Mannheim, Germany) to 273.75 microliters nuclease free water. This master mix was dispensed in 21.5 microliter aliquots into 15 thin walled PCR reaction tubes, on ice. Into six of these tubes was added 2.5 microliters of MuIgV H -3' reverse transcription reaction mix and 1.0 microliter of heavy chain 5' primer mix A to F.
- Figure 40 shows the result of the RT-PCR reactions.
- the heavy chain reactions (lanes 2-7) demonstrate a strong band at 500bp amplified using MuIgV H 5'-C (lane 4) and MuIgV H 5'-E (lane 6).
- Light chain reactions (lanes 8-15) demonstrate a strong 450bp product band amplified using primers MuIg ⁇ V L 5'-A (lane 8) and MuIg ⁇ V L 5'-G (lane 14) forward primer.
- PCR products from these reactions were purified using QIAquick PCR Purification Kit (QIAGEN, Crawley, UK).
- Light chain A and G and heavy chain C and E purified PCR products were separately cloned into pGEM-T easy vector using the pGEM-T easy Vector System I (Promega Corp., Madison, WI). Both the light and heavy chain reactions were prepared by adding 3.0 microliters of purified PCR product to 5.0 microliters of 2x ligation buffer, 1.0 microliter pGEM-T easy vector and 1.0 microliter T4 DNA ligase. Plasmids were transformed into sub-cloning grade XLl -blue competent E. coli (Stratgene, La Jolla, CA) as per manufacturer's instructions. For both the light and heavy chain transformations, 2.0 microliters of the ligation reaction was used.
- a PCR master mix was prepared by mixing 513.6 microliters sterile water, 34.0 microliters Dimethyl Sulphoximide (Sigma, Poole, UK), 68.0 microliters 10x Taq buffer (Invitrogen, Paisley, UK), 13.6 microliters 1OmM dNTP mix (Invitrogen, Paisley, UK), 6.8 microliters of 50 pmol/microliter primer OLOOl, 6.8 microliters of 50 pmol/microliter primer OL002 and 3.4 microliters Taq DNA polymerase (Invitrogen, Paisley, UK).
- V H E-2 (lane 1 1)
- V H E-4 (lane 14)
- V H E-7 (lane 17) were positive for a 650bp product band indicating the successful cloning of a 500bp product into pGEM-T easy vector.
- the remaining five V H E and all eight of the VH-C reactions produced bands of a lower molecular weight indicating a negative result for a 500bp insert.
- Figure 42 shows the PCR screening reactions from eight colonies of AR47A6.4.2 V L -A and eight colonies of AR47A6.4.2 V L -G. All eight of the V L A colonies (lanes 2-9) and 3 of the eight V L G colonies, V L G-1 (lane 10), V L G-3 (lane 12) and V L G-5 (lane 14) were positive for a 600bp product band indicating the successful cloning of a 450bp product into pGEM-T easy vector. The remaining five V L -G reactions produced bands of a different molecular weight indicating a negative result for a 450bp insert.
- a maximum of 4 positive colonies from each ligation were chosen to inoculate 5 mL 2YT (Sigma, Poole, UK) broth containing 50 mg/L ampicillin (Sigma, Poole, UK). Cultures were incubated at 37 0 C with shaking overnight. Plasmid DNA was extracted from each culture using Qiagen, QIAprep Spin Miniprep Kit (Qiagen, Crawley, UK).
- V L and V H clones (V L A-I, V L A- 2, V L A-5, V L A-6, V L G-I , V L G-3, V H E-2, V H E-4, and V H E-7) were sequenced at
- Figure 43 shows SEQ ID NO:8 with the underlined CDRs designated SEQ ID NOS:4-6.
- Figure 44 shows SEQ ID NO:7 with the underlined CDRs designated SEQ ID NOS: 1-3.
- CDR definitions and amino acid sequence numbering is done according to Kabat et al. (1991). The Kabat numbering is listed above the amino acid sequence in Figures 43 and 44.
- the correct AR47A6.4.2 V L sequence was found in all 4 of the clones amplified with 5' primer MuIg ⁇ V L -A.
- the two V L clones amplified using 5' primer MuIg ⁇ V L -G contained an aberrant immunoglobulin gene.
- the correct AR47A6.4.2 V H sequence was found in all 3 clones amplified with 5' primer MulgV H -E.
- an individual ordinarily skilled in the art can generate a competitively inhibiting CDMAB, for example a competing antibody, which is one that recognizes the same epitope (Belanger L et al. Clinica Chimica Acta 48: 15- 18 (1973)).
- One method entails immunizing with an immunogen that expresses the antigen recognized by the antibody.
- the sample may include but is not limited to tissues, isolated protein(s) or cell Iine(s).
- Resulting hybridomas could be screened using a competition assay, which is one that identifies antibodies that inhibit the binding of the test antibody, such as ELISA, FACS or Western blotting.
- Another method could make use of phage display antibody libraries and panning for antibodies that recognize at least one epitope of said antigen (Rubinstein JL et al. Anal Biochem 314:294-300 (2003)).
- antibodies are selected based on their ability to displace the binding of the original labeled antibody to at least one epitope of its target antigen.
- Such antibodies would therefore possess the characteristic of recognizing at least one epitope of the antigen as the original antibody.
- variable regions from the heavy (V H ) and light (V L ) chains of monoclonal antibody produced by the AR47A6.4.2 hybridoma cell line were determined (Example 16).
- V H heavy
- V L light
- the variable light and variable heavy domains can be subcloned into an appropriate vector for expression.
- AR47A6.4.2 or its de-immunized, chimeric or humanized version is produced by expressing a nucleic acid encoding the antibody in a transgenic animal, such that the antibody is expressed and can be recovered.
- the antibody can be expressed in a tissue specific manner that facilitates recovery and purification.
- an antibody of the invention is expressed in the mammary gland for secretion during lactation.
- Transgenic animals include but are not limited to mice, goat and rabbit.
- DNA encoding the monoclonal antibody (as outlined in Example 1) is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
- the hybridoma cell serves as a preferred source of such DNA.
- the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
- the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences.
- Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
- immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
- a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be performed the method of Winter and co-workers by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al, Science 239: 1534-1536 (1988); reviewed in Clark, Immunol. Today 21 :397-402 (2000)).
- a humanized antibody can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
- Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
- Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
- FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
- the CDR residues are directly and most substantially involved in influencing antigen binding.
- Antibody Fragments Various techniques have been developed for the production of antibody fragments. These fragments can be produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 1 1 :548-557 (1999); Little et al., Immunol. Today 21 :364-370 (2000)). For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2 fragments (Carter et al., Biotechnology 10: 163-167 (1992)). In another embodiment, the F(ab') 2 is formed using the leucine zipper GCN4 to promote assembly of the F(ab') 2 molecule. According to another approach, Fv, Fab or F(ab') 2 fragments can be isolated directly from recombinant host cell culture.
- the antibody of the present invention can be used as a composition for preventing/treating cancer.
- the composition for preventing/treating cancer which comprises the antibody of the present invention, are low-toxic and can be administered as they are in the form of liquid preparations, or as pharmaceutical compositions of suitable preparations to human or mammals (e.g., rats, rabbits, sheep, swine, bovine, feline, canine, simian, etc.) orally or parenterally (e.g., intravascularly, intraperitoneally, subcutaneously, etc.).
- the antibody of the present invention may be administered in itself, or may be administered as an appropriate composition.
- the composition used for the administration may contain a pharmacologically acceptable carrier with the antibody of the present invention or its salt, a diluent or excipient. Such a composition is provided in the form of pharmaceutical preparations suitable for oral or parenteral administration.
- injectable preparations examples include injectable preparations, suppositories, etc.
- the injectable preparations may include dosage forms such as intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, intraarticular injections, etc.
- These injectable preparations may be prepared by methods publicly known.
- the injectable preparations may be prepared by dissolving, suspending or emulsifying the antibody of the present invention or its salt in a sterile aqueous medium or an oily medium conventionally used for injections.
- aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mols) adduct of hydrogenated castor oil)), etc.
- an alcohol e.g., ethanol
- a polyalcohol e.g., propylene glycol, polyethylene glycol
- a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mols) adduct of hydrogenated castor oil)
- the oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
- a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
- the injection thus prepared is usually filled in an appropriate ampoule.
- the suppository used for rectal administration may be prepared by blending the antibody of the present invention or its salt with conventional bases for suppositories.
- the composition for oral administration includes solid or liquid preparations, specifically, tablets (including dragees and film-coated tablets), pills, granules, powdery preparations, capsules (including soft capsules), syrup, emulsions, suspensions, etc.
- compositions for oral or parenteral use described above are prepared into pharmaceutical preparations with a unit dose suited to fit a dose of the active ingredients.
- unit dose preparations include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
- the amount of the aforesaid compound contained is generally 5 to 500 mg per dosage unit form; it is preferred that the antibody described above is contained in about 5 to about 100 mg especially in the form of injection, and in 10 to 250 mg for the other forms.
- the dose of the aforesaid prophylactic/therapeutic agent or regulator comprising the antibody of the present invention may vary depending upon subject to be administered, target disease, conditions, route of administration, etc.
- the agent can be administered in a dose corresponding to the dose given above. When the condition is especially severe, the dose may be increased according to the condition.
- the antibody of the present invention may be administered as it stands or in the form of an appropriate composition.
- the composition used for the administration may contain a pharmacologically acceptable carrier with the aforesaid antibody or its salts, a diluent or excipient.
- a composition is provided in the form of pharmaceutical preparations suitable for oral or parenteral administration (e.g., intravascular injection, subcutaneous injection, etc.).
- Each composition described above may further contain other active ingredients.
- the antibody of the present invention may be used in combination with other drugs, for example, alkylating agents (e.g., cyclophosphamide, ifosfamide, etc.), metabolic antagonists (e.g., methotrexate, 5- fluorouracil, etc.), anti-tumor antibiotics (e.g., mitomycin, adriamycin, etc.), plant- derived anti-tumor agents (e.g., vincristine, vindesine, Taxol, etc.), cisplatin, carboplatin, etoposide, irinotecan, etc.
- alkylating agents e.g., cyclophosphamide, ifosfamide, etc.
- metabolic antagonists e.g., methotrexate, 5- fluorouracil, etc.
- anti-tumor antibiotics e.g., mitomycin, adriamycin, etc.
- the method of treatment described herein, particularly for cancers, may also be carried out with administration of other antibodies or chemotherapeutic agents.
- an antibody against EGFR such as ERBITUX® (cetuximab)
- ERBITUX® cetuximab
- ERBITUX® has also been shown to be effective for treatment of psoriasis.
- Other antibodies for combination use include Herceptin® (trastuzumab) particularly when treating breast cancer, AVASTIN ® particularly when treating colon cancer and SGN-15 particularly when treating non- small cell lung cancer.
- the administration of the antibody of the present invention with other antibodies/chemotherapeutic agents may occur simultaneously, or separately, via the same or different route.
- chemotherapeutic agent/other antibody regimens utilized include any regimen believed to be optimally suitable for the treatment of the patient's condition. Different malignancies can require use of specific anti-tumor antibodies and specific chemotherapeutic agents, which will be determined on a patient to patient basis.
- chemotherapy is administered concurrently with or, more preferably, subsequent to antibody therapy. It should be emphasized, however, that the present invention is not limited to any particular method or route of administration.
- AR47A6.4.2 mediates anticancer effects and prolongs survival through ligation of epitopes present on TROP-2. It has been shown, in Examples 9, 10 and 1 1, AR47A6.4.2 antibodies can be used to immunoprecipitate the cognate antigen from expressing cells such as MDA-MB-231 cells. Further it has been shown, in Examples 1 , 2 and 13-15, that the AR47A6.4.2 antibody can be used in detection of cells and/or tissues which express a TROP-2 antigenic moiety which specifically binds thereto, utilizing techniques illustrated by, but not limited to FACS, cell ELISA or IHC.
- the immunoprecipitated AR47A6.4.2 antigen can inhibit the binding of AR47A6.4.2 to such cells or tissues using FACS, cell ELISA or IHC assays.
- other anti-TROP-2 antibodies could be used to immunoprecipitate and isolate other forms of the TROP-2 antigen, and the antigen can also be used to inhibit the binding of those antibodies to the cells or tissues that express the antigen using the same types of assays.
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Abstract
Description
Claims
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JP2008555588A JP2009528995A (en) | 2006-02-24 | 2007-02-23 | Cancerous disease modifying antibody 141205-05 |
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CA002643561A CA2643561A1 (en) | 2006-02-24 | 2007-02-23 | Cancerous disease modifying antibody 141205-05 |
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2478047A1 (en) * | 2002-03-01 | 2003-09-12 | Immunomedics, Inc. | Rs7 antibodies |
Family Cites Families (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4828991A (en) | 1984-01-31 | 1989-05-09 | Akzo N.V. | Tumor specific monoclonal antibodies |
AU613590B2 (en) | 1986-11-19 | 1991-08-08 | Bristol-Myers Squibb Company | Hybridomas producing monoclonal antibodies to new mucin epitopes |
US4861581A (en) | 1986-12-05 | 1989-08-29 | Cancer Biologics, Inc. | Detection of necrotic malignant tissue and associated therapy |
JPH0219324A (en) * | 1988-06-23 | 1990-01-23 | Pastan Ira | Monoclonal antibody to ovarian cell |
US5171665A (en) | 1989-04-17 | 1992-12-15 | Oncogen | Monoclonal antibody to novel antigen associated with human tumors |
US6020145A (en) | 1989-06-30 | 2000-02-01 | Bristol-Myers Squibb Company | Methods for determining the presence of carcinoma using the antigen binding region of monoclonal antibody BR96 |
US5366866A (en) * | 1991-07-25 | 1994-11-22 | Duke University | Method of diagnosing ovarian and endometrial cancer with monoclonal antibodies OXA and OXB |
EP0539970B1 (en) | 1991-10-30 | 1999-05-26 | Idemitsu Kosan Company Limited | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
IL105008A0 (en) | 1992-03-13 | 1993-07-08 | Yeda Res & Dev | Double transfectants of mhc genes as cellular vaccines for immunoprevention of tumor metastasis |
US5545532A (en) | 1993-02-05 | 1996-08-13 | Epigen, Inc. | Human carcinoma antigen (HCA), HCA antibodies, HCA immunoassays and methods of imaging |
JPH10505819A (en) | 1994-06-24 | 1998-06-09 | ヴラディミール ピー. トーチリン | Use of autoantibodies for treatment and prevention of tumors |
ES2219699T3 (en) * | 1995-10-19 | 2004-12-01 | Bristol-Myers Squibb Company | BR110 MONOCLONAL ANTIBODY AND USES OF THE SAME. |
US5783186A (en) | 1995-12-05 | 1998-07-21 | Amgen Inc. | Antibody-induced apoptosis |
US6653104B2 (en) | 1996-10-17 | 2003-11-25 | Immunomedics, Inc. | Immunotoxins, comprising an internalizing antibody, directed against malignant and normal cells |
US6180357B1 (en) | 1999-10-08 | 2001-01-30 | Arius Research, Inc. | Individualized patient-specific anti-cancer antibodies |
KR101388611B1 (en) * | 2002-10-16 | 2014-04-23 | 퍼듀 퍼머 엘피 | Antibodies that bind cell-associated ca 125/o772p and methods of use thereof |
US20050027106A1 (en) * | 2003-07-28 | 2005-02-03 | Young David S. F. | Cancerous disease modifying antibodies |
US7348413B2 (en) * | 2004-02-26 | 2008-03-25 | Arius Research Inc. | Cancerous disease modifying antibodies |
US7420041B2 (en) * | 2006-02-24 | 2008-09-02 | Arius Research Inc. | Cytotoxicity mediation of cells evidencing surface expression of TROP-2 |
-
2007
- 2007-02-22 US US11/709,898 patent/US7420041B2/en not_active Expired - Fee Related
- 2007-02-22 US US11/709,676 patent/US7420040B2/en not_active Expired - Fee Related
- 2007-02-23 AU AU2007218961A patent/AU2007218961A1/en not_active Abandoned
- 2007-02-23 EP EP07701822A patent/EP1996700A4/en not_active Withdrawn
- 2007-02-23 EP EP07719386A patent/EP1996701A4/en not_active Withdrawn
- 2007-02-23 WO PCT/CA2007/000282 patent/WO2007095748A1/en active Application Filing
- 2007-02-23 JP JP2008555589A patent/JP2009527230A/en active Pending
- 2007-02-23 CN CNA2007800143605A patent/CN101426904A/en active Pending
- 2007-02-23 CN CNA2007800147771A patent/CN101432422A/en active Pending
- 2007-02-23 CA CA002643603A patent/CA2643603A1/en not_active Withdrawn
- 2007-02-23 CA CA002643561A patent/CA2643561A1/en not_active Withdrawn
- 2007-02-23 AU AU2007218962A patent/AU2007218962A1/en not_active Abandoned
- 2007-02-23 JP JP2008555588A patent/JP2009528995A/en active Pending
- 2007-02-23 WO PCT/CA2007/000283 patent/WO2007095749A1/en active Application Filing
-
2008
- 2008-08-20 US US12/229,187 patent/US20090142263A1/en not_active Abandoned
- 2008-08-20 US US12/229,203 patent/US20090191118A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2478047A1 (en) * | 2002-03-01 | 2003-09-12 | Immunomedics, Inc. | Rs7 antibodies |
Non-Patent Citations (4)
Title |
---|
RIPANI E. ET AL.: "Human Trop-2 is a tumour-associated calcium signal transducer", INTERNATIONAL JOURNAL OF CANCER, vol. 76, 1998, pages 671 - 676, XP002971819 * |
See also references of EP1996700A4 * |
STEIN R. ET AL.: "Radioimmunotherapy of a human lung cancer xenograft with monoclonal antibody RS7: evaluation of 177Lu and comparison of its efficacy with that of 90Y and residualising 131I", JOURNAL OF NUCLEAR MEDICINE, vol. 42, 2001, pages 967 - 974, XP008128955 * |
STEIN R. ET AL.: "Specificity and properties of monoclonal antibody RS7-3G11 and the antigen defined by this pancarcinoma monoclonal antibody", INTERNATIONAL JOURNAL OF CANCER, vol. 55, 1993, pages 938 - 946, XP000616251 * |
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Also Published As
Publication number | Publication date |
---|---|
EP1996701A1 (en) | 2008-12-03 |
US7420041B2 (en) | 2008-09-02 |
WO2007095749A1 (en) | 2007-08-30 |
AU2007218962A1 (en) | 2007-08-30 |
US20070202113A1 (en) | 2007-08-30 |
EP1996700A4 (en) | 2010-04-14 |
US20090142263A1 (en) | 2009-06-04 |
CA2643603A1 (en) | 2007-08-30 |
US20090191118A1 (en) | 2009-07-30 |
CA2643561A1 (en) | 2007-08-30 |
US7420040B2 (en) | 2008-09-02 |
AU2007218961A1 (en) | 2007-08-30 |
US20070202043A1 (en) | 2007-08-30 |
JP2009527230A (en) | 2009-07-30 |
EP1996701A4 (en) | 2010-04-07 |
CN101432422A (en) | 2009-05-13 |
WO2007095749A8 (en) | 2009-01-29 |
JP2009528995A (en) | 2009-08-13 |
CN101426904A (en) | 2009-05-06 |
EP1996700A1 (en) | 2008-12-03 |
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