SCIENTIFIC DISCUSSION This module reflects the initial scientific discussion for the approval of Simulect. This scientific discussion has been updated until 1 October 2004. For information on changes after this date please refer to module 8B. 1. Introduction Simulect contains the active substance basiliximab, which is a chimeric murine/human monoclonal antibody (MAb). Basiliximab reacts with the CD25 antigen on T-cells, inhibiting the binding of interleukin-2 (IL-2) to its receptor (IL-2R), and functions as an immunosuppressive agent. In vitro tests with human tissues indicate that basiliximab binds only to activated T-lymphocytes and to monocytes/macrophages. Simulect is a sterile freeze-dried powder for intravenous infusion or injection after reconstitution with the solvent, water for injection. Simulect is marketed in the 20 mg strength. Post-Authorisation, a second strength of 10 mg is authorised. In adult patients, peak basiliximab serum concentration following a 20-30 minute infusion of 20 mg Simulect is 7.1 ± 5.1 mg/ml. The elimination half-life is 7.2 ± 3.2 days, and total body clearance is 41 ± 19 ml/hr. In infants and children (age 1–11 years, n=25), distribution volume and clearance are reduced by about 50 % compared to adult renal transplantation patients whereas disposition in adolescents was similar to that in adult patients. The applied indication of Simulect was first for the prophylaxis of acute organ rejection in de novo allogeneic renal transplantation and to be used concomitantly with cyclosporin for microemulsion- and corticosteroid-based immunosuppression in patients with panel reactive antibodies less than 80%. In the meantime the indication has been extended for paediatric patients and for the prophylaxis in the triple maintenance immunosuppressive regimen containing ciclosporin for microemulsion - and corticosteroids, and either azathioprine or mycophenolate mofetil. The proposed standard total dose in adults is 40 mg, given in two doses of 20 mg each. The first dose should be given within 2 hours prior to transplantation surgery. The second dose should be given 4 days after transplantation. Elderly patients do not require a different dosage from younger adult patients. In paediatric patients weighing less than 35 kg, the recommended total dose is 20 mg, given in two doses of 10 mg each. In paediatric patients weighing 35 kg or more, the recommended dose is the adult dose, i.e. a total dose of 40 mg, given in two doses of 20 mg each. The first dose should be given within 2 hours prior to transplantation surgery. The second dose should be given 4 days after transplantation. The second dose should be withheld if post-operative complications such as graft loss occur. Reconstituted Simulect can be administered as an intravenous bolus injection or as an intravenous infusion over 20–30 minutes. 2. Part II: Chemical, pharmaceutical and biological aspects Basiliximab, the active substance of Simulect, is a chimeric murine/human monoclonal antibody (MAb) of the IgG class. The variable regions of the heavy and light chains are of murine origin, and the constant regions are of human origin. Composition Simulect is a white lyophilisate to be dissolved in the solvent, water for injection, before administration. Each vial contains 10 mg or 20 mg basiliximab. Buffering agents, sodium chloride, sucrose and mannitol are also present and comply with Ph. Eur. Specifications. The qualitative composition for both strengths is identical. The filling weight indicated corresponds to a 4.0 ml theoretical filling volume for the 20 mg strength and to 2.0 ml theoretical filling volume for the 10 mg strength, respectively. The 7.5% overfilling which permits the withdrawal of the nominal dose from the single-dose container is not included. Formulations of 5, 10 and 20 mg lyophilisates were used in clinical studies. 1/14 EMEA 2005 The container is a 6 ml colourless glass vial (borosilicate glass, type I, Ph. Eur.) closed with a rubber stopper and sealed with an aluminum cap with a blue polypropylene flip-off cover. Development pharmaceutics The size of the galenical form, of the composition, manufacturing procedure and packaging material has been described sufficiently and in very detail. Method of preparation The required amounts of excipients are dissolved in water for injections. The frozen solution of the drug substance is thawed in a laminar flow environment and the calculated amount is added to the excipient solution. Samples of the resulting solution are retained for in-process controls (IPC). The bulk solution is prefiltered in a sterilised vessel and transported to the filling area. It is then sterile filtered and aliquots are dispensed into depyrogenised glass vials. After stoppering, the vials are lyophilised using a procedure which is validated by the quality of the batches produced. Drug product vials are stored at 2-8°C pending analytical release. Particular manufacturing precautions like thawing and equilibration time, maximum standing time of ingredients, requirements for tubings and membrane filters, stirring requirements, are specified. Production and control of starting materials The active ingredient in the drug product is a chimeric monoclonal antibody, which recognises the CD25 antigen on the surface of T-cells and inhibits the binding of interleukin-2. The antibody has a molecular weight of ca. 150`000 daltons and comprises light and heavy chains with variable regions of murine origin and constant human regions. The antibody has been developed under the laboratory code, SDZ CHI 621. The proposed International Nonproprietary Name (INN) is basiliximab. Development genetics Simulect originates from a murine hybridoma cell line (RFT5, IgG 2a) producing a monoclonal antibody against the alpha chain of the human IL-2-receptor. In order to retain high avidity to CD25 and reduce immunogenicity in humans, the variable regions of the heavy and light chains are of murine origin, and the constant regions are of human origin. In order to construct and express a monoclonal antibody binding to the CD25 epitope, the genetic information for the heavy and light chain variable regions from an existing murine antibody (RFT5) as well as for the constant parts from other sources had to be isolated. The heavy and light chain variable regions of Simulect were isolated after the construction of genomic libraries from the hybridoma cell line RFT5. Positive clones isolated from the libraries were characterised for the existence of the complete promoter/enhancer/leader/exon sequences and the deduced amino acid sequences from the heavy and light chain variable region exons were compared to the AA-sequence resulting from direct protein sequencing. The heavy and light chain variable region exons including regulatory sequences were cloned in front of human heavy and light chain constant regions. The resulting expression constructs were either the murine variable light chain and human light chain constant region or the murine variable heavy chain and human heavy chain constant region in the background of the pSV2neo plasmid. A mutated DHFR gene was introduced in the light chain construct to allow the amplification of the introduced constructs in transfected cells after selection with methotrexate. Both constructs were transfected by electroporation into a cell line, which was negative for the background expression of immunoglobulins. Transfected cells were selected by the addition of G418, cloned by limiting dilution and screened for their ability to inhibit the binding of iodine labelled hIL-2 to either human PBL or MT4 cells. The clone producing the highest amounts of antibodies were used to construct the MCB. An estimation of the number of integrated copies of the two constructs in the MCB and the MWCB revealed 2 copies for the heavy chain and 4 for the light chain construct. Genetic stability of the cell lines was adequately demonstrated. Stability of the expression of the constructs was determined by Northern blot analysis of the heavy and light chain mRNA and indicated stable expression in the MCB, MWCB and 2 post production lots. Cell bank system The antibody is expressed in murine myeloma cells in cell culture. The cell bank system was tested appropriately according to the CPMP/III/5271/94 and CPMP/ICH/295/95 guidelines. 2/14 EMEA 2005 The MCB was established in 1990. Vial 4 of the primary seed lot was used to prepare a Master Cell Bank (MCB). The cells were cultivated in serum-free medium, but for long term storage were resuspended in a freezing medium which contains foetal calf serum. Thus, FCS was used only during the establishment of the MCB. The supplier for the foetal calf serum (US origin) guarantees that the serum is free of mycoplasmas and viruses. The MCB was shown to be free of viral contamination of bovine origin. Today, after a storage period of 7 years, no significant decrease in the viability after thawing is observed. Vial 14 of the MCB was used to prepare a Working Cell Bank (WCB). In the medium (2057.24.02) bovine-derived insulin was replaced by recombinant human insulin from Escherichia coli. Cells were grown in a T-flask and then expanded to roller bottles. Cells were harvested by centrifugation and resuspended in a new freezing medium (without calf serum) prior to dispensing into vials. The vials were frozen and stored in the vapour phase of liquid nitrogen. Vials were labelled with a laboratory code (CHI 621), the code identifying the primary seed lot, MCB vial number and the vial number of the WCB 1-95. All test results indicate that the cells are well characterised and stable. The absence of detectable adventitious viruses, bovine viruses and murine viruses has been demonstrated with the only exception of a xenotropic retrovirus which is not unexpected for a murine cell line. It could be shown that this retrovirus is an infectious retrovirus derived from the persistently infected SP2/0 murine myeloma cell line.