Status: formerly approved in U.S. and EU

Organizations involved:

Centocor B.V. – Manuf.; Former

Centocor, Inc. – R&D; Tech.; USA mark.; Former

Johnson & Johnson Co. (J&J) – Parent; Former

Mallinckrodt Inc. – Manuf. other; Europe mark.; Former

Tyco Healthcare – Parent; Former

Cross ref: See Monoclonal Antibodies (entry #300).

Description: Imciromab pentetate or Myoscint is an enzymatically-derived Fab fragment of a murine hybridoma cell cultured IgG2kappa monoclonal antibody with specificity for myosin (a protein in heart muscle) produced by in vitro hybridoma culture covalently linked to diethylenetriaminepentaacetic acid (DTPA). When reacted by an on-site or local nuclear pharmacy with the radioisotope Indium In 111, this forms the radioimmune conjugate – Indium In 111 imciromab pentetate (In 111 Myoscint), which is used for radiodiagnostic imaging of dead heart tissue. Imciromab has a molecular weight of ~50,000 Daltons (~50 kDa).

Myoscint was approved for sale in the U.S. in July 1996 for cardiac radiodiagnostic imaging in the setting of chest pain suspected to be caused by myocardial infarction (heart attack). Marketing approvals for Myoscint were also granted in several European countries. However, manufacture and marketing were halted in 1997, and FDA approval was voluntarily withdrawn in February 1999. The product was too expensive to manufacture and provided insufficient profit.

Imciromab pentetate (Myoscint) was packaged as a kit containing one 1 ml vial of Myoscint solution (containing sodium dihydrogen phosphate) and 1 ml vial of citric acid (citrate-buffer) solution diluent, and a Millipore filter. The Myoscint vial contained 0.5 mg of lmciromab Pentetate in 1.0 mL of 10 mM sodium phosphate buffer, 145 mM sodium chloride and 10% (w/v) maltose (pH 6.5), with no preservatives. One (1.0) mL of the solution from the lmciromab Pentetate vial was to added to the Citrate buffer vial, which contained 1.0 mL of 0.2 M sodium citrate buffer solution (pH 5.0). lmciromab Pentetate was further radiolabeled in the nuclear pharmacy by reacting (mixing) it with a sterile, non-pyrogenic solution of high purity Indium In 111 Chloride manufactured by Mallinckrodt Medical.

After reconstitution, the solution contains radioactive Indium as Indium 111 imciromab pentetate at a concentration 37 MBq/ml. Radionuclide purity is 114 mIn < 0.5kBq/MBq 111-In. Radio-chemical purity is > 90%. Mallinckrodt reported that it manufactured fresh kits every Friday. The dating period for Myoscint when stored at 2-8˚C (refrigerated) was 18 months from the date of manufacture, defined as the date of the final sterile filtration of the formulated finished product.

Biological.: The source imciromab monoclonal antibody, its Fab fragment, the Fab conjugate with the DTPA linker-chelator molecule (Myoscint), and In 111 Myoscint bind with high affinity and specificity to myosin, a protein found in human cardiac myocyte cells. Myosin is expressed exclusively in the intracellular compartment, and is only exposed on the cell surface following a loss of integrity of the myocyte cell membrane, e.g., death caused by myocardial infarction (heart attack). This forms the basis for use of the radioimmune conjugate to image infarct-damaged heart tissue. Cardiac myosin protein was chosen as the intracellular target for antibody localization because of its abundance in myocytes, high molecular weight (500 kDa), and low plasma solubility. Heavy chain myosin is not found free in the circulation. In 111 Myoscint contains the Fab fragment of the parent antibody/immunoglobulin, rather than the whole molecule. The smaller Fab fragment localizes to the injured myocardium to a greater degree and is cleared more rapidly from the circulation.

Nomenclature: Myosin Mab Fab–In 111 radioconj. [BIO]; Myoscint [TR]; imciromab pentetate [FDA USAN BAN]; Indium In 111 imciromab pentetate [USAN CAS]; imciromab [INN for monoclonal antibody]; immunoglobulin G 2a (mouse mo-no-clonal R1D10 cell Fab fragment anti-human cardiac myosin heavy chain), disulfide with mouse monoclonal R11D10 cell kappa-chain, N,N-bis[2-bis(carboxymethyl)-amino]ethyl]-glycine conjugate [CAS]; 138660-99-8 [CAS RN for antibody]; 138660-99-8 [CAS RN for conjugate]; myosin mono-clonal antibody Fab fragment-diethylenetriamine-penta-acetic acid [SY]; myosin Mab Fab-DTPA [SY]; C-4 [SY]; R11D10 [SY]; DRN 4925 [SY; Mallinckrodt no. for imciromab pentetate]; NDC 00045--0310-09 [NDC]

Biological.: Intravenous administration of imciromab pentetate radiolabeled with lndium In 111, a gamma emitting radionuclide, provides a means of identifying regions of myocardial infarction (Ml) using conventional scintigraphic techniques. Following cardiac myocyte injury, the cell membrane undergoes a loss of integrity and becomes permeable to macromolecules, allowing lndium In 111 imciromab pentetate to bind to intracellular myosin. In vivo, radiolabeled imciromab pentetate localizes in infarcted myocardial tissues. Cross-reactivity studies performed in vitro on a broad spectrum of human tissues demonstrated that imciromab pentetate is highly specific for myocardial and skeletal muscle and does not cross-react with any other examined tissues.

Companies.: Myoscint was manufactured by Centocor B.V. (Leiden, Netherlands), CBER/FDA est. no. 1178, a subsidiary of Centocor, Inc., (Malvern, PA), prior to the acquisition of Centocor by Johnson & Johnson (J&J). It was marketed by Centocor, Inc. in the U.S. Mallinckrodt Nuclear Medicine Div. held exclusive marketing rights in Europe, Middle East, and Africa. Development of Myoscint was at least partially funded through Centocor Cardiovascular Imaging Partners, L.P.

Manufacture: The source imciromab monoclonal antibody was produced by standard murine hybridoma technology. The original immunogen used was purified and well-characterized human cardiac myosin. Using conventional hybridoma technology, the immunization of mice with myosin was followed by isolation of spleen B-cells secreting desired antibody and fusion of the selected B-cells with a myeloma cell line. The resulting hybridomas were screening by radioimmunoassay (RIA) with human cardiac myosin, and an optimal hybridoma cell line was selected.

All of the cell banks (MCB, MWCB) were tested for microbial and viral contaminants in accordance with FDA’s Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993) and Draft Points to Consider in the manufacture and Testing of Monoclonal Antibody Products for Human Use (1994). The cell banks were shown to be free of microbial contaminants, adventitious murine viruses and bovine viruses, and adventitious viruses other than retroviruses. A sample taken at the end of fermentation from the first five fermentors from each new Working Cell Bank (WCB) was tested for endogenous retroviruses (ERV) by the Dunni Cell assay. If the results are negative, testing of fermentors was stopped. If any results were positive, all fermentors were tested for ERV. All preformulated bulk from fermentors positive for ERV were required to be tested and found negative by the extended Dunni Cell assay.

The hybridoma cells are cultured (unspecified bioreactor), apparently in medium containing bovine serum albumin (BSA). The imciromab monoclonal antibody was purified from the medium by processing including Protein A affinity chromatography steps, anion exchange chromatography, and gel-filtration chromatography. The imciromab was enzymatically digested with papain to obtain the Fab fragment. Diethyl-enetriamine-penta-acetic acid (DTPA) ligand was covalently bound to the Fab fragments, forming imciromab pentetate, and purified by gel-filtration chromatography. This imciromab pentetate was reacted (radiolabeled) with lndium In 111 chloride in the local nuclear pharmacy to form the final radioimmune conjugate, lndium In 111 imciromab pentetate.

In process controls and specifications for the final bulk imciromab pentetate included sterility, pH, protein identity [immunoelectrophoresis, IEF], and 111-Indium incorporation by instantaneous thin layer chromatography (ITLC). Final bulk could be stored for up to 14 days at 2-8˚C prior to filling. Potency of the product was determined by immunoaffinity chromatography on canine heart myosin, and must exceed 94% of maximal expected incorporation. Biochemical characterization included mass spectroscopy, amino acid analysis, tryptic mapping, monosaccharide analysis, and circular dichroism spectra, in addition to standard biochemical analyses for QA and QC. Process validation studies demonstrated that the manufacturing process yields a product meeting its specifications and quality attributes. These studies included validation of fermentation and downstream processing – virus removal and/or inactivation, Protein A affinity chromatography steps, anion exchange and gel-filtration chromatography, papain digestion, DTPA coupling reactions, and product consistency. The purification process was also validated for the removal of various low molecular weight contaminants and reagents. The manufacturing processes substantially inactivated various viruses, e.g., ≥ 15.2 logs for MuLV; ≥ 10.4 logs for SV40; and ≥ 6.1 logs for poliovirus type I.

Stability of imciromab and imciromab pentetate was demonstrated by chemical, physical, and immunological tests (radioactivity incorporation, IEF, particle formation, immunoreactivity most relevant). Tests were done at elevated (22-35˚C), sub-ambient (-20˚C), and recommended (2-8˚C; refrigerated) storage temperatures. Cell culture harvests are stable at 2-8X for two weeks. Concentrates may be stored at 2-8˚C for up to 9 weeks. Downstream processing intermediates may be stored for fewer than 3 days at 2-8˚C; the final bulk may be stored for up to 14 days at 2-8˚C, and the final diluted preparation is stable for at least 18 months at 2-8˚C. Final bulk imciromab Fab-DTPA product must be shown to be sterile, have pH of 6.4-6.6, protein concentration (by OD at 280) of 0.45 to 0.55 mg/mL, conform to Identity by IEF, and In l 11 incorporation by citrate ITLC must be 90% within 10 minutes. Tests on final containers included color, appearance, pH, particulates, fill volume, protein concentration by optical density (OD; 0.45 to 0.55 mg/ml), purity, and identity by GF-HPLC, purity of Fab-DTPA by SDS-PAGE (non-reduced) of over 97%, charge distribution by IEF, In-l11 incorporation by citrate and WEA ITLC (over 90% within 10 minutes), immunoreactive fraction by myosin affinity column (over 90%), endotoxin (520 EU/mg), sterility, and general safety. All lots must show equivalent spectra with near UV minima of 262, 269 and 277 nm, and a maximum of 292 nm, while far UV has a 216 nm minimum.

FDA class: Biologic BLA

Approvals: Date = 19960703; first approval, BLA ref. no. 95-1210 and 95-1190; orphan designation (expired 7/3/2003)

Date = 19990224; BLA for imciromab pentetate voluntarily withdrawn

Date = 20020219; approval for Indium In-111 Chloride Sterile Solution Use for radiolabeling voluntarily withdrawn

Indications: [excerpt from the Summary Basis of Approval]:

for preparation of Indium (In111) Imciromab pentetate to be used as a cardiac imaging agent for detecting the presence and/or identifying the location of myocardial injury in patients with suspected myocardial infarction

Status: The product received approval in the U.S., several European countries, and Japan by mid-1998. manufacture and marketing were subsequently halted. However, the product was too expensive to manufacture and provided insufficient profit. FDA approval was withdrawn in Feb. 1999.

Trials: Of the ten clinical studies submitted with the original application, seven described the use of lndium In 111 Myoscint in the setting of ischemic heart disease. The pivotal Phase III trial (Protocol CO4l-OOl/CO4I-002) examined use in patients with suspected acute myocardial infarction. The remaining three trials used lndium In 111 Myoscint in other settings (i.e., blunt chest trauma, AIDS, and doxorubicin toxicity). Overall, a total of 1,329 subjects were studied in the clinical trials; 1,287 of these subjects were included in the assessment for ischemic heart disease.

Medical: Myoscint is a cardiac imaging agent previously approved and used for detecting the presence and location of myocardial injury in patients with suspected myocardial infarction, including situations where electrocardiography and cardiac enzymes are nondiag-nostic. The effective radiation dose equivalent (EDE) for 2 mCi lndium In 111 imciromab Pentetate is 1.92 rem (19.2 mSv), which includes 0.3 rem (3.0 mSv) from maximal impurity levels (0.16% of lndium In 111 m/l). The recommended intravenous dose of lndium In 111 imciromab Pentetate was 2 mCi (74 MBq) with a range of 1.8 to 2.2 mCi (67 to 81 MBq). The activity of each dose was measured by a radiation calibration system just prior to administration. Indium In 111 imciromab pentetate was administered as an intravenous bolus over less than one minute. The recom-mended imaging time was approximately 48 hours after intravenous injection. Within the first 24 hours after administration, clearance of radioactivity from the blood pool may not be sufficient for image interpretation, i.e., circulating In 111 imciro-mab pentetate provides too much background radiation noise. Imaging was performed with a gamma scintillation camera. Typically, anterior, approximately anterior oblique, and left lateral (or steep left anterior oblique) views of the chest were obtained for at least 10 minutes per view.