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Soluble Form of Carbonic Anhydrase IX (s-CA IX), Assays to Detect s-CA IX, CA IX's Coexpression with Her-2/neu/c-erbB-2, and CA IX-Specific Monoclonal Antibodies to Non-Immunodominant Epitopes

a carbonic anhydrase and carbon dioxide technology, applied in the field of medical genetics, can solve problems such as significant production obstacles, and achieve the effects of enlarge the clinical perspective, the therapeutic resource and diagnostic/prognostic parameters, and enhance the effectiveness of several chemotherapeutic agents

Inactive Publication Date: 2008-07-24
BIOMEDICAL RES CENT OF THE SLOVAK ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The patent is about new antibodies that target a protein called CA IX, which is associated with cancer. These antibodies were generated in mice that lacked the gene for CA IX. The antibodies can be used for various purposes such as diagnostics, therapy, and research. They can detect and measure the soluble form of CA IX in body fluids and can be used to predict the outcome of cancer treatment. The patent also describes the use of these antibodies in combination with other antibodies or other methods to improve their effectiveness. Overall, the patent provides new tools to improve the diagnosis and treatment of cancer."

Problems solved by technology

There had been many significant obstacles to producing such antibodies previously.

Method used

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  • Soluble Form of Carbonic Anhydrase IX (s-CA IX), Assays to Detect s-CA IX, CA IX's Coexpression with Her-2/neu/c-erbB-2, and CA IX-Specific Monoclonal Antibodies to Non-Immunodominant Epitopes
  • Soluble Form of Carbonic Anhydrase IX (s-CA IX), Assays to Detect s-CA IX, CA IX's Coexpression with Her-2/neu/c-erbB-2, and CA IX-Specific Monoclonal Antibodies to Non-Immunodominant Epitopes
  • Soluble Form of Carbonic Anhydrase IX (s-CA IX), Assays to Detect s-CA IX, CA IX's Coexpression with Her-2/neu/c-erbB-2, and CA IX-Specific Monoclonal Antibodies to Non-Immunodominant Epitopes

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Ca Ix-Deficient Mice

Materials and Methods

[0571]Cloning of Mouse Car9 cDNA

[0572]Mouse cDNA was obtained by RT PCR using primers derived from a human Car9 cDNA (EMBL Accession No. X66839; SEQ ID NO: 70). The template total RNA was prepared from C57BL / 6J mouse stomach by acid guanidium thiocyanate-phenol / chloroform isolation [Siebert et al. (1993)]. First strand synthesis was carried out under standard conditions using SuperScript II reverse transcriptase (Life Technologies, Germany). The set of the human primers was as follows: sense ATC CAC GTG GTT CAC CTC AG (SEQ ID NO: 71) and antisense CTTTGG TTC CCC TTC TGT GC (SEQ ID NO: 72); corresponding to the human cDNA fragment 760-1345 [SEQ ID NO: 73] of FIG. 1A-1C. PCR reactions were carried out in 30 cycles (94° C., 30 sec, 55° C., 40 sec, and 72° C., 60 sec). Entire cDNA including open reading frame, 3′- and 5′-untranslated regions, was obtained by the 3′ and 5′ RACE (rapid amplification of cDNA ends). The 3′ and 5′ RACE ...

example 2

CA IX-Specific Monoclonal Antibodies Generated from CA IX—Deficient Mice

Materials and Methods

[0627]Cell Culture

[0628]Hybridoma cell lines were grown in DMEM medium supplemented with 10% FCS (BioWhittaker, Verviers, Belgium), 2 mM glutamine and 40 μg / ml gentamicin (Lek, Slovenia) at 37° C. in 5% CO2 in air. The same cultivation conditions were applied to following cell lines that were used either as a source of CA IX antigen or as negative controls: mouse NIH 3T3 fibroblasts permanently transfected with the full-length human CA9 cDNA (NIH 3T3-flCA IX) and corresponding mock transfected NIH 3T3-neo controls [Pastorek et al. (1994)], MDCK cells transfected with the full-length CA9 cDNA (MDCK-flCA IX) and corresponding MDCK-neo controls, human HT 29 colon carcinoma cells as well as human HeLa cervical carcinoma cells naturally expressing CA IX, and C33a cervical carcinoma cells negative for CA IX.

[0629]For the immunization purposes, the cells grown for 2448 h were washed twice in PBS, s...

examples 3-10

[0675]The following materials and methods were used in the Examples 3-10 below.

Materials and Methods

Cell and Tumor Cultures and Media

[0676]The cell line HT29 (DSMZ ACC299), derived from colorectal carcinoma, and A498 (ATCC No. HTB-44) from renal clear cell carcinoma, were grown in D-MEM supplied with 10% FCS (Gibco), unless otherwise stated. In Example 8, the HT29 cells were cultivated in Dulbecco's MEM medium (Biowhiftaker, Verviers, Belgium) supplemented with 10% FCS (BioWhittaker) and 160 g / ml gentamicin (Lek, Slovenia). The A549 lung carcinoma cells were grown in DMEM (Sigma) supplemented with 10% FCS (Globepharm), L-glutamine (2 μM), penicillin (50 IU / ml), and streptomycin sulphate (50 μg / ml). Cell cultures were maintained in a humidified atmosphere with 5% CO2 at 37° C. For short-term cultures of tumors, fresh tumor excisions were cut in 1 mm pieces, rinsed with PBS, suspended in D-MEM with 10% FCS (about 50-100 mg of fresh weight of tumor fragments in 5 ml of the medium) and ...

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Abstract

Disclosed herein is the discovery of a soluble MN / CA IX (s-CA IX) in body fluids, such as, urine and serum. Said s-CA IX comprises the extracellular domain of CA IX or portions thereof. The Predominant s-CA IX species is the extracellular domain comprising a proteoglycan-like (PG) domain and carbonic anhydrase (CA) domain, and having a molecular weight of about 50 / 54 kilodaltons (kd) upon Western blot. A smaller s-CA IX form of about 20 to about 30 kd comprising the CA domain or parts thereof, not linked to the PG domain, has also been found in body fluids. Diagnostic / prognostic methods for precancer and cancer that detect or detect and quantitate said s-CA IX in body fluids, are described. Also disclosed herein is the coexpression of CA IX and HER-2 / neu / c-erbB-2 that provides parallel, alternative and potentially synergistic diagnostic / prognostic and therapeutic strategies for precancer and cancer. Further disclosed are new MN / CA IX-specific antibodies generated from MN / CA IX-deficient mice, preferably monoclonal antibodies and immunoreactive fragments and engineered variants thereof. Such new MN / CA IX-specific antibodies, fragments and variants are useful diagnostically / prognostically and therapeutically for cancer and precancer. Particularly preferred are the new monoclonal antibodies, fragments and variants that are specific for the non-immunodominant epitopes of MN / CA IX, which antibodies are, among other uses, useful to detect soluble MN / CA IX (s-CA IX) in body fluids, alone but preferably in combination with antibodies specific to the immunodominant epitopes of MN / CA IX, for example, in a sandwich assay.

Description

FIELD OF THE INVENTION[0001]The present invention is in the general area of medical genetics and in the fields of biochemical engineering, immunochemistry and oncology. More specifically, it relates to the MN gene—a cellular gene considered to be an oncogene, known alternatively as MN / CA9, CA9, or carbonic anhydrase 9, which gene encodes the oncoprotein now known alternatively as the MN protein, the MN / CA IX isoenzyme, MN / CA IX, carbonic anhydrase IX, CA IX or the MN / G250 protein.[0002]More specifically, the instant invention is directed to new MN / CA IX-specific antibodies prepared in MN / CA IX-deficient mice (knock out mice) and to uses of those new antibodies. Significantly, some of the new MN / CA IX-specific antibodies, preferably monoclonal antibodies and immunoreactive fragments thereof are directed to non-immunodominant epitopes on CA IX proteins and polypeptides, rendering such antibodies and immunoreactive fragments, among many diagnostic / prognostic and therapeutic uses, usefu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/18C12N15/09C07K16/32C07K16/40C12N5/10C12N5/20C12P21/08
CPCC07K14/82C07K16/32C12N9/88G01N2333/82C07K16/40A61P35/00A61P43/00C07K2317/14C07K2317/30C07K2317/56C07K2317/622
Inventor PASTOREK, JAROMIRPASTOREKOVA, SILVIAZATOVICOVA, MIRIAMZAVADA, JANGUT, MARTA ORTOVAZAVADOVA, ZUZANNA
Owner BIOMEDICAL RES CENT OF THE SLOVAK ACADEMY OF SCI
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