Services
Cell culture and vaccine preparation
The laboratory cultures and expands a broad variety of hematopoietic and non-hematopoietic cells in flasks, cell factories, or Aastrom Replicell cassettes, as appropriate, generally in serum-free media supplemented or not with cytokines. Cultures of various lymphocytes, lymphoid cell lines, or lymphocyte subsets, dendritic cells (DC), skin fibroblasts, synoviocytes, and various tumor cells are expertly handled and tested for sterility (aerobic and anaerobic culture), mycoplasma, and levels of endotoxin prior to release of these cell products for therapy. To prepare vaccines for patients with cancer, the laboratory performs a broad range of tasks, including mixing of peptide preparations with an adjuvant or pulsing of DC with diluted peptide solutions. Vaccines are prepared and delivered to the clinic or the bedside at times specified in clinical protocols.
Pulsing or antigen-loading of DC
The CPL performs pre-clinical studies to determine optimal conditions for antigen or peptide pulsing of cultured antigen presenting cells (APC). Once the pre-clinical studies are completed, the production process becomes an SOP-driven routine. At present, DC are either pulsed with peptides or fed with apoptotic tumor cells or tumor-cell lysates or electroporated with plasmids or genomic DNA. Tumor lysates in saline are prepared by repeated freeze-thaw cycles followed by sonication. When apoptotic tumor cells (ATC) are to be used as a source of TAA, these cells are first irradiated with 1500 μw/cm2 UVB to induce their apoptosis. When DC are to be pulsed with peptides or tumor lysates, a separate replicate culture of DC is used for each antigen and these are combined prior to a vaccine release for therapy. All procedures, reagents, and assays used in CPL for therapeutic product generation follow SOPs submitted to FDA as a part of IND applications.
Gene therapy laboratory
The CPL has developed extensive experience in transduction of normal tissue or tumor cells with cytokine genes and selection of transduced cells for therapy. The participation of the laboratory in the gene therapy trials ongoing at the UPCI includes:
(a) development of technology for growth of human dermal fibroblasts, cell lines, various tumor cells in primary cultures, and subsets of human hematopoietic cells, including DC and subsets of lymphocytes (T, NK, B cells);
(b) pre-clinical studies to develop and optimize methods for transduction of these cells with retroviral, adenoviral, or plasmid vectors and selection as well as phenotypic and functional evaluation of transduced and selected cells for expression of the transgene; and
(c) clinical-scale generation of allogeneic human fibroblasts nucleofected with tumor-derived genomic DNA for therapy of cancer.
Tissue procurement, processing, and distribution for clinical trials and internal use
This service is offered in support of the UPCI clinical trials and consists of the collection and processing of serial tissue biopsies or tumor tissues body fluids and buffy coats. These specimens are processed for recovery of viable cells and cryopreservaton. Recovered tumor cells are used as a source of antigens for vaccine preparation. The CPL only banks products, cells, or human materials needed for therapy or directly associated with ongoing clinical trials. This activity involves prospective collection, processing, and distribution of samples from the clinical trials. The need for the laboratory support of this aspect of clinical trials is great, as reflected by the following statistics: in FY2006, the CPL collected and processed 185 tissues specimens and 5,056 body fluids; additionally, the CPL processed 130 leukapaks or buffy coats and initiated large scale cultures of two cell lines (NK-92 and MRC-5) to serve as working cell banks for future therapeutic cell products. These services are routinely available for handling, processing, expanding, and testing of cells to be used in clinical trials at the UPCI
Development of new procedures and products
The CPL provides translational research and development services that are essential for the implementation of future clinical trials and thus benefit multiple users. Examples of the recent developmental efforts are as follows:
- Experiments with novel DC maturation cocktails, containing various cytokines to promote more effective antigen presentation to T cells and thus more robust generation of tumor-specific CTL and helper T cells
- Experiments with Astrom Replicell System or Miltenyi Clinimax aimed at a large-scale culture of more active DC (or other cells) with a higher purity in a closed system compatible with GMP regulations
- Manufacture of tolerogenic DC for diabetes
- Clinimax-based separation of T cell subsets
- Expansion of TALL-104 T cells for adoptive therapy of patients with prostate carcinoma
- Expansion of NK-92 cells for immune therapy of patients with ALL
- Generation of EBV-specific CTL for treatment of PTLD
- Isolation of autologous HIV-1 and its inactivation: vaccine development for HIV-1 patients
- Expansion of human neural stem cells