Methods, Tecnics, Lab

​Development of optimal conditions for generation and activation of clinical-grade dendritic cells (DC)

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One rate-limiting step at present in DC-based cancer vaccination is the inability to fully recapitulate ex vivo the development of immunocompetent DCs, in particular the process of DC activation. Although the basic approach to DC preparation from monocytes is well established, the specific methods used for cell purification, culture, and maturation vary widely. DC differentiation is most commonly induced using GM-CSF and interleukin IL-4 but the doses of each reagent, the culture conditions, the composition of the culture medium, and the cocktail of reagents used to induce maturation all vary substantially. In addition to variability in reagents, investigators preparing clinical grade DCs also employ a variety of different culture schedules. In particular, monocytes have been cultured in GM-CSF/IL-4 to induce a DC phenotype for as short as 2 days or as long as 7 days. We have employed a systematic approach in order to uncover these variables in the manufacturing process for clinical-grade DCs. Our focus will be on short-term protocols that potentially will reduce the cost and labor associated with the process and, when used in combination with closed culture systems, facilitate the handling and reduce the risk of microbial contamination. Moreover, we are combining Toll-like receptor agonists with standard stimuli in order to generate multifunctional, Th1-polarizing DCs capable of IL-12 secretion and lymph node migration.

RNA transfection

The rationale behind DC-based vaccination approaches is to stimulate effective cytotoxic T-cell responses in cancer patients by isolating DCs from the patient, load them with tumor associated antigens (TAAs) and inject the antigen-loaded cells back into the patient. In this regard, the efficacy of antigen loading and delivery into DCs is pivotal for optimal induction of T-cell mediated immune responses. Comparative studies suggest that mRNA electroporation is superior to other antigen-loading techniques in generating immunopotent DCs. With this approach DCs are engineered to synthesize tumor epitopes which are subjected to the full antigen processing and presentation cascade, in a manner indistinguishable from endogenous gene products. In addition, the genetic engineering of DCs is not limited to a single mRNA or a predefined mixture. In fact, the full cohort of mRNA expressed by a given tumor can be used to transfect DCs, for example, as a pool of in-vitro amplified mRNA. With this method, we have assessed the ability of mRNA-transfected DCs to stimulate tumor-specific T cells in patients with primary breast cancer. In this respect, we have shown T-cell reactivity against p53, survivin and hTERT – three universal TAAs that are expressed in different tumor types and appear to play a critical role in tumorigenesis - in more than 40 % of tested patients (9). The observed T-cell responses were also found to be highly associated with the presence of cancer as TAA-specific T-cell responses were only detected in one out of 10 healthy donors at the most.

These data demonstrate that mRNA-transfected DCs are capable of inducing TAA-specific T-cell responses ex vivo in cancer patients. Consequently, these data form the basis of ongoing clinical phase I and II trials at the Department of Oncology, Herlev Hospital in which mRNA-transfected DCs are evaluated in patients with metastatic breast cancer, malignant melanoma or metastatic prostate cancer.

Tumor infiltrating lymphocytes (TILs)

Adoptive cell therapy is a highly personalized form of immunotherapy based on the isolation of tumor-specific T cells from tumor biopsies, i.e. autologous TILs. These cells can be obtained by a surgical resection of a metastasis from the patient. The tumor tissue is then dissected into fragments, and infiltrating lymphocytes are isolated, activated and expanded to large numbers with a two-step protocol consisting of a pre-REP (rapid expansion protocol) and a REP phase (see http://www.ncbi.nlm.nih.gov/pubmed/21955245 for further information). Different culture conditions are used during various steps of the process, including culture in a WAVE Bioreactor™ 2/10 system (GE Healthcare Life Sciences).

Large numbers of activated antitumor lymphocytes are re-infused into the patients upon chemotherapy induced lymphodepletion. This therapy is then followed by intravenous infusion of Interleukin-2 in order to further stimulate the proliferation of the T-cells in vivo.

At CCIT, several patients with otherwise unresponsive metastatic melanoma obtaining disease regression after Adoptive T cell therapy were observed, including long-term (>1 year) complete responses – disappearance of all tumor lesions.

Expansion of blood derived tumor antigen-specific T cells

Adoptive cell therapy with in vitro expanded T cells, derived from surgically excised tumor material (TIL), has proven to be an effective and promising approach in the treatment of metastatic cancer e.g. metastatic melanoma. Objective response in more than 50 % of treated patients and frequent complete responses has been demonstrated in several clinical trials. The modality, however, is completely dependent on the availability of sufficient tumor material, for the generation of TILs.

A similar approach dealing with this limitation is the generation and expansion of tumor-reactive T cells derived from patients blood after vaccination with e.g. DCs presenting TAAs or peptide vaccination.

Peripheral blood mononuclear cells is harvested from blood during leukapheresis, and cells are enriched using specific antigen stimulation followed by an unspecific expansion of T cells in order to obtain high numbers of tumor-specific cells potentially capable of tumor cell killing upon adoptive transfer.

Current experiments focus on optimization of in vitro stimulation and enrichment of antigen specific T cells and prior to expansion using the rapid expansion protocol.​



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