[Special considerations for the regulation of biological medicinal products in individualised medicine. More than stratified medicine]. / Besonderheiten bei der Regulierung biologischer Arzneimittel in der individualisierten Medizin. Mehr als stratifizierte Medizin.

The term individualised medicine, also called personalised medicine, is commonly used as an equivalent to stratified medicine. However, this is erroneous since quite often it is forgotten that especially biological medicinal products have other aspects of individualization that go beyond mere stratification. The principles of stratified medicine have been applied for biological medicinal products for many years. A historical example is diphtheria antitoxin made from horse serum, while current examples are transfusion of red blood cells and the administration of factor VIII in haemophilia A. The stratifying aspects of these medicinal products are given by the following considerations: diphtheria antitoxin is only administered after a diagnosis of diphtheria and not in other forms of tonsillitis, red blood cells should only be transfused once blood group compatibility as been established and factor VIII replacement is only administered in haemophilia A as opposed to other acquired or hereditary disease of the coagulation system. The peculiarities of biological medicinal products, in particular the inherent variability of the drug, are especially important for autologous cellular medicinal products. In addition to the expected variability of the biological source material there is interindividual variability of patients as cell donors, which make definition of specifications and determination of criteria for pharmaceutical quality and potency tests difficult. Therapy with modified autologous cells, a common and important application of advanced therapy medicinal products, is exemplary for the special considerations that must be made when evaluating pharmaceutical quality, mode of action and toxicological properties of the biological medicine. The clinical investigation of advanced therapy medicinal products with the intent of demonstrating safety and efficacy is particularly challenging because of the complexity of therapy, which often involves invasive interventions. The development of biomarkers accelerates the process towards stratified or individualised therapies. Increased requirements for companion diagnostics are a possible consequence. Progress in analytical processes and in biotechnology make a higher degree of individualization likely, possibly to the degree that medicinal products will be individually manufactured for each patient. Current principles of medicinal product testing and market authorization may be applicable only with limitations, because the individual medicinal products are not uniform and are not repeatedly manufactured. The assessment of the process, performed on several different medicinal products manufactured by the same process could potentially serve as a basis for the assessment. For the evaluation of risk for the patient in clinical trials new concepts must be considered, which can be facilitated by interaction of regulatory authorities and developers.

RNAi-mediated gene silencing in tumour tissue using replication-competent retroviral vectors.

RNAi represents a powerful technology to specifically downregulate the expression of target genes. For cancer research and therapy, an efficient in vivo delivery system is supposed to distribute RNAi to all tumour cells upon systemic administration. We present replication-competent murine leukaemia virus (MLV) vectors, which deliver RNAi to tumour tissue upon tail vein injection. In HT1080 cells stably expressing GFP or luciferase, GFP expression was suppressed by more than 80% and luciferase (luc) activity by more than 90%, even when only 0.1% of the cells were initially infected with reporter gene specific vectors. To demonstrate its potential, PLK1- and MMP14-specific small hairpin RNA expression cassettes were applied in the system. Upon infection, PLK1 and MMP14 levels were reduced on mRNA and protein level. MLV-shPLK1-infected cells were arrested in the G2-phase and underwent apoptosis. MLV-shMMP14-infected cells showed reduced MMP2 activity, as well as substantially reduced invasion and tumour growth. In vivo, MLV-shLuc silenced luc expression in HT1080-luc tumour tissue by more than 80% and MLV-shPLK1 reduced tumour growth substantially, demonstrating the therapeutic relevance of this system. This RNAi vector system allows long-term downregulation of target gene expression as well as efficient delivery to and distribution throughout tumour tissue in vivo.

Pseudotyping lentiviral vectors with the wild-type measles virus glycoproteins improves titer and selectivity.

We pseudotyped HIV-1 vectors with cytoplasmic tail-truncated envelope glycoproteins of a wild-type (WT) measles virus (MV). The particles entered the lymphatic cells exclusively through the signaling lymphocyte activation molecule (SLAM, CD150), whereas particles pseudotyped with the MV vaccine strain glycoproteins also recognized the ubiquitous membrane cofactor protein (CD46) as receptor and had less specific cell entry. MV(WT)-HIV vectors reached titers of 10(8) t.u. ml(-1), which were up to 10-fold higher than those of MV(Vac)-HIV vectors, and discriminated between SLAM-positive and SLAM-negative cells, also in mixed cell cultures. As these vectors transduce primary human cells more efficiently than vesicular stomatitis virus-G pseudotyped vectors do, they are promising candidates for gene transfer to human lymphocytes and certain epithelial cells.

Cell entry targeting restricts biodistribution of replication-competent retroviruses to tumour tissue.

Virotherapy is currently being developed for many different types of viruses including replication-competent murine leukaemia virus (MLV) as a novel tool in cancer therapy. However, there is the risk of insertional mutagenesis associated with this virus, making careful preclinical studies necessary before its first application in man. We have previously generated conditionally replication-competent MLV variants that require activation by tumour-associated proteases to become infectious. Here we analysed in a comparative study the spreading of non-targeted and of such tumour-targeted MLV variants to tumour and extratumoural organs in immunodeficient mice. Both virus types were able to efficiently infect tumour cells after systemic administration. The non-targeted virus, however, also infected extratumoural organs like bone marrow, spleen and liver efficiently. In contrast, the targeted viruses revealed in a quantitative analysis of virus spreading an up to 500-fold more selective infection of tumour tissue than the non-targeted virus. The data raise serious doubts about a safe clinical use of non-targeted MLV. Engineering the virus to become activatable by tumour-associated proteases can significantly improve the safety of MLV.

Spleen necrosis virus-derived C-type retroviral vectors for gene transfer to quiescent cells.

Gene therapy applications of retroviral vectors derived from C-type retroviruses have been limited to introducing genes into dividing target cells. Here, we report genetically engineered C-type retroviral vectors derived from spleen necrosis virus (SNV), which are capable of infecting nondividing cells. This has been achieved by introducing a nuclear localization signal (NLS) sequence into the matrix protein (MA) of SNV by site-directed mutagenesis. This increased the efficiency of infecting nondividing cells and was sufficient to endow the virus with the capability to efficiently infect growth-arrested human T lymphocytes and quiescent primary monocyte-derived macrophages. We demonstrate that this vector actively penetrates the nucleus of a target cell, and has potential use as a gene therapy vector to transfer genes into nondividing cells.

Productive infection of both CD4+ and CD4- human cell lines with HIV-1, HIV-2 and SIVagm.

Human monolayer cells of various origins were shown to be susceptible to infection by HIV-1, HIV-2 and simian immunodeficiency virus obtained from African green monkeys (SIVagm). Immunoperoxidase staining revealed infection of 2-7% of the monolayer cells, although in order to achieve infection approximately 50-fold more virus was necessary than with CD4(+)-permissive lymphoma cells. No CD4-receptor antigen expression by fibroblastoid cells was detectable by immunofluorescence using several monoclonal antibodies (MAbs), although a low level of CD4-specific messenger RNA expression was revealed by Northern analysis (with the exception of Tera-1 and RD cells). Attempts to block viral infection by anti-CD4 MAbs indicated a CD4 receptor-mediated mechanism for all lines tested except RD cells. We conclude that a low level of CD4-receptor expression is sufficient to allow infection of fibroblastoid cells. The infectability of a CD4-negative cell line indicates a second pathway of cellular infection, possibly mediated by a cellular receptor distinct from the CD4 molecule.

Lack of HIV-1 V3 region sequence diversity in two haemophiliac patients infected with a putative biologic clone of HIV-1.

Peripheral blood mononuclear cells (PBMC) from two haemophilia B patients, who presumably became infected with a putative HIV-1 biological clone following treatment with the same suspected batch of commercial factor, were used to clone and sequence the hypervariable V3 region of the HIV-1 envelope protein. In 10 of 12 clones the V3 region was identical and two (one from each patient) had a single non-synonymous point mutation. Viable reisolates (shown to be authentic by sequencing of V3) currently appear to be monocyte tropic. These results strongly indicate that the patients were infected from a common source with a very low number of infectious particles and indicate that variability under these conditions is limited.

Targeting human T cells by retroviral vectors displaying antibody domains selected from a phage display library.

To generate T cell-specific retroviral vectors an scFv phage display library derived from immunized mice was selected for binding to the human T cell line Molt-4/8. The scFv cDNAs recovered from the selected phages were transiently expressed as an N-terminal fusion of the spleen necrosis virus (SNV) transmembrane protein (TM) subunit of the viral envelope protein (Env) in the cell line DSH-cxl, which packages the beta-galactosidase gene into SNV particles. Screening of supernatants from about 150 transfections resulted in the identification of 5 scFvs that mediated efficient transduction of Molt-4/8 cells. Using stable packaging cell lines vector preparations with titers greater than 10(4) EFU/ml on human T cells were obtained. The scFv 7A5 in particular was able to mediate selective transduction of human T cells with high efficiency. Titers of up to 106 EFU/ml were reached on Molt-4/8, Jurkat, and A301 cells, while titers on HeLa cells, TE671 cells, 293T cells, and HT1080 cells were below 102 EFU/ml. Transduction of stimulated primary human peripheral blood cells, which consisted mainly of T cells, was about fivefold more efficient than transduction of B cells. Western blot analysis of supernatant from the 7A5 packaging cells demonstrated incorporation of 7A5-TM into vector particles and indicated proteolytic processing of the coexpressed unmodified TM during particle formation. Binding of bacterially expressed 7A5-scFv to a panel of cell lines correlated well with the transduction results. These data provide the first proof of concept that a general approach can be taken to obtain scFvs able to mediate selective gene transfer into target cells.

Vaccine protection against SIVmac infection by high- but not low-dose whole inactivated virus immunogen.

Eight rhesus macaques were immunized intramuscularly four times (0, 1, 2, and 4 months) over a period of 4 months with a formalin-inactivated whole Simian immunodeficiency virus (SIV) vaccine in the presence of muramyl dipeptide (MDP) as adjuvant. Four animals received 0.5 mg and the other four received 0.1 mg immunogen per injection. Three weeks after the final immunization, the vaccinated monkeys along with two control monkeys were challenged intravenously with 50 MID50 of SIVmac251-32H. At the time of challenge, one monkey from the high dose group had a high titer (1:761) of antibody able to neutralize in vitro the homologous 32H strain of SIV mac. All other animals had lower but measurable titers (1:57-1:453), although there was no correlation between the levels of neutralizing antibody and subsequent protection. Upon challenge, three of four animals from the low dose group (plus the nonvaccinated control animals) became infected as demonstrated by reisolation of virus from peripheral blood mononuclear cells, by SIVmac-specific polymerase chain reaction, and by the development of a strong anamnestic response. In the sera from these animals the titer of neutralizing antibody rose to over 1:5,100. All other animals (one from low dose group and all four of the high dose group) remained negative by all parameters at 4 months post challenge. These data indicate that when used in conjunction with MDP, the amount of immunogen required per immunization for protection against challenge is between 0.1 and 0.5 mg.

Retroviral cell targeting vectors.

The availability of cell targeting vectors is an unalterable requirement for in vivo gene therapy trials. This review will describe the different strategies developed over the past few years in order to target retroviral vectors to preselected human cell types by genetic modification of the envelope (Env) proteins. Current targeting concepts include the substitution of the complete Env protein as well as the incorporation of new receptor binding domains into the Env protein. These approaches are aimed at altering the host range of vectors with a natural tropism for non-human cells to specific human cell types, or achieving tissue-specificity for vectors that would naturally infect a wide spectrum of human cell types. Targeting concepts and efficient targeting vectors with potential for clinical trials will be described, and their advantages and disadvantages will be discussed.

Obtaining marketing authorization for nucleic acid vaccines in the European Union.

Nucleic acid vaccines are promising candidates for easy-to-handle and cost-effective vaccines that combine the safety of subunit vaccines with the efficacy of live virus vaccines. In order to obtain marketing authorization for a nucleic acid vaccine in all member states of the European Union, a single application dossier has to be filed with the European Agency for the Evaluation of Medicinal Products. Notes for Guidance on the data necessary to support applications are available. The preclinical development of nucleic acid vaccines has to follow procedures of contained use according to the relevant EC directives which were translated into the German Gene Law. Clinical trials in Germany would follow the known procedures defined in the German Drug Law, whereas the Gene Law is not applicable. Clinical trials should be started only after having obtained consent of the Commission for Gene Therapy Trials formed under the auspices of the Federal Chamber of Physicians. Experience in intramuscular nucleic acid inoculation of animals has been gained using expression constructs comprising single and multiple genes of simian immunodeficiency virus. Specific antibodies were induced against multiple antigens. No adverse effects of nucleic acid inoculations were found, but more rigorous testing of specific safety problems will have to be performed.

Induction of antibodies against SIV antigens after intramuscular nucleic acid inoculation using complex expression constructs.

By studying the infection of rhesus macaques with simian immunodeficiency virus (SIVmac) the potential of nucleic acid immunisation against AIDS can be evaluated. As a first step towards the development of suitable expression constructs, the levels and the durations of expression elicited by the house-keeping gene promoters of the murine phospho-glycerate kinase (PGK) gene and rat proto-ras 1Ha, a lentiviral LTR and the CMV-intron A promoter were tested in BALB/c mice intramuscularly inoculated with marker gene constructs encoding luciferase. The expression levels achieved by the CMV-intron A and the lentiviral promoter were comparably high, and also the PGK promoter induced a high level of expression for at least 64 days. Following the inoculation of plasmids comprising single or multiple genes of SIV, the induction of specific antibodies directed against SIV antigens was demonstrated. We previously showed in vitro that int- and nef-defective mutants of SIVmac were able to initiate a limited and self-abortive infection of permissive cells in the absence of chromosomal integration of the viral DNA. Intramuscular inoculations in monkeys using int-defective proviral DNA of SIV will show whether an increased immune response may be induced by expression of viruses undergoing a self-limited replication in vivo.

European Union guidance on the quality, safety and efficacy of DNA vaccines and regulatory requirements.

A European Union note for guidance on gene transfer medicinal products is being developed by the Biotechnology Working Party (BWP) of the Committee for Proprietary Medicinal Products (CPMP) which will include guidance for DNA vaccines. The 'Note for Guidance on the Quality, Preclinical and Clinical Aspects of Gene Transfer Medicinal Products' outlines the information required to assure the quality of the plasmid DNA intended to be used as a vaccine. It also provides guidance on preclinical safety evaluation and on clinical efficacy and safety evaluation with regard to obtaining marketing authorisation according to Council Regulation No. (EEC) 2309/93. Before initiating a clinical trial, it is necessary to obtain an appraisal from the relevant central and/or local ethics committees, and the competent authority within the member state(s) concerned has to authorise, or be notified of, the clinical trial. Harmonisation of gene therapy and DNA vaccine regulations as well as co-operation between relevant ethics committees and authorities in Europe is currently being improved.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

[Approval of clinical trials of immunobiological medicinal products at the Paul Ehrlich Institute]. / Genehmigung der klinischen Prüfung immunbiologischer Arzneimittel am Paul-Ehrlich-Institut.

The GCP Directive 2001/20/EG has been implemented in Germany by the 12th Law Amending the Drug Law of 6 August 2004, thereby introducing new regulations for the performance of clinical trials. The amount of the required documentation has increased, but the assessment and the approval of clinical trials as well as scientific advice procedures (national or by the EMEA) allow the early discussion of many details of the development and the non-clinical and clinical testing of the medicinal product with the experts of the Paul Ehrlich Institute (PEI). This might shorten the times required for later marketing authorisation procedures. To facilitate these new tasks, the PEI has created a new central section "Approval of Clinical Trials", which is responsible for the assessment of the clinical trial applications and will coordinate the procedures within the institute. The main topics of clinical trial applications and the particularities of biological/biotechnological medicinal products such as allergens, blood products, vaccines, sera/mAb and products for cell and gene therapy as well as the differences from chemically defined products are discussed.