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Antigen presentation by chaperon proteins and cd4+ t cell specific help for a new and efficient strategy of cancer vaccination.

Deliverables

Methods were established to purify heat-shock protein 70 (hsp 70) from mouse and human normal tissues and tumours. We have confirmed that hsp 70 isolated from methylcholanthrene (MC) induced tumours can elicit protective immunity against challenge with the autologous tumour. This was paralleled by induction of tumour specific T cells, which could produce IFN-gamma in vitro. Several independent research groups have provided evidence that Hsps are able to stimulate and activate cells of the innate immune system. Extensive efforts were made by us to confirm these findings using endotoxin free highly purified mouse hsp70. When extreme care was taken to avoid LPS contamination, the ability of hsp70 to induce activation of dendritic cells (DC) could not be demonstrated, bringing into question some of the results published by others. Our earlier results have demonstrated that antigens complex to hsp70 have an enhanced capacity to activate protective T cells in vivo. Based on these results, a series of recombinant pDNA constructs were made which are chimeric molecules expressing both hsp70 and a defined tumour antigen (Her-2/neu). We are analyzing if the association between the tumour antigen and the hsp70 protein will render these molecules more antigenic, as measured both by antibody responses and T cell assays. This information is of direct relevance for the production of effective cancer vaccines. The end users will be cancer centres.
New antigens have been characterized both in melanoma and lung cancer patients. These antigens are restricted either by class I or II HLA and can be recognized by patient's T lymphocytes. These proteins, or their peptides containing the epitope, stimulate the patient's immune defence system. The potential applications of these new antigens include vaccination of cancer patients in clinical trials and/or diagnosis of melanoma or lung cancer. This latter development will be helped by the availability of antibodies specifically recognizing the antigen. End users of this products will be hospitals and cancer research centres.
A new mutated protein antigen has been discovered in melanoma cells of a patient who is disease-free after 10 years from surgical resection of her lymph-node metastasis. This antigen has been characterized in its full aminoacid composition and site of the mutation that makes it different from the wild type protein and, therefore, recognizable by the patient's T cells. We showed that, years after surgery, the patient's blood still contains a high frequency of CD4+ T cells directed against the mutated protein. It is likely that this is the reason why the patient remained without disease for such a long time. Moreover, since this protein is physiologically involved in regulating cell-to-cell adhesion, its mutation could favour progression and metastasis of melanoma. Potential applications include the use of the mutated protein or peptide as vaccine in clinical studies (depending on the frequency of expression of this antigen) or as diagnostic tool for melanoma if a monoclonal antigen-specific antibody can be produced.
We have shown that the 4-1BB molecule expressed by antigen-presenting cells can deliver in vivo a powerful co-stimulatory signal to T cells. This signal was found to replace the need for CD4 + T cell help in stimulating tumour-specific cytotoxic T lymphocytes (CTLs). Triggering of 4-1BB showed a powerful effect on the induction, amplification and persistence of CTL responses. Thus this signal permits an increased survival of antigen stimulated CTLs and, therefore, a better activity of vaccines. In addition, we used the bacterial oligonucleotide CpG to increase the effectiveness of peptide-based cancer vaccines. We found that CpG is a strong activator of CTL responses in vivo. Moreover, CpG could replace the need for CD4+ T-helper cells with respect to the induction of strong CTL activity. Importantly, when CpG was mixed with peptide-vaccines and administered to mice with established tumours, all animals showed sharp reductions in tumour-load and 80% was able to eradicate their tumour. Our data show that in mice new powerful generation of therapeutic cancer vaccines could consist of completely synthetic peptide antigens and synthetic CpG. These results are being confirmed in humans. If so, this information will be crucial for manufacturing effective cancer vaccines. End users will be hospitals and cancer centres.
The heat-shock proteins (HSPs) 96 were shown to mediate maturation of mouse and human dendritic cells (DCs) through the binding of the specific receptors, Toll-like 2 and 4. These proteins were shown to bind also to platelets. Experiments in animal models demonstrated that immunization with HSP96 derived from tumour cells could lead to the improvement of the effectiveness of anti-tumour vaccines. This information may allow the exploitation of HSPs purified from tumour cells for constructing new vaccines that contain the full repertoire of tumour antigens. End users of this result will be doctors and scientists involved in experimental and clinical vaccination of cancer patients (see results No. 5 and 9.)
To be effective, a classical vaccine should supply two types of immune signals: an antigen (Ag) and a suitable accessory signal. In fact, recognizing the Agby itself is not sufficient to protect the host; antigen recognition has to elicit a biological response suited to the nature of the pathogen or the tumour cells. We have shown that a soluble LAG-3 molecule (a high affinity MHC class II ligand, see http://www.ncbi.nlm.nih.gov/prow/guide/1656481751_g.htm) can deliver in vitro a powerful accessory signal to human DCs. In vivo, LAG-3 expressed following tumour cell transfection or injection of a murine LAG-3Ig recombinant molecule in cancer vaccines has been shown to induce specific CTLs and tumour regression in syngeneic tumour models such as breast or renal adenocarcinoma, melanoma and sarcoma. LAG-3 co-localizes with the TCR and the two other MHC ligands (i.e. CD4 and CD8) on the surface of activated T cells. LAG-3Ig (a fusion molecule linking the 4 extra cellular domains of LAG-3 and the Fc fraction of an Ig) was identified as a molecule involved in mediating maturation signals for DCs. LAG-3, like CD40L, also contributes to DC maturation and increases signal 2 deliveries. It therefore increases the immunogenicity of Ag-loaded mature DCs reaching the lymph node, leading to efficient Ag presentation and priming of naïve T and B cells. In addition to increased signal 2 delivery, inflammation induction is often associated with increased immunogenicity and inflammatory cytokines, such as IL-1 and IL-12, have been shown to provide a third signal for activation of naïve CD4+ and CD8+ T cells. Our results indicate that LAG-3, like LPS, clearly induced inflammatory cytokines, such as IL-8 and MIP-1α/CCL3, and signal 3 may thus be involved in the elicitation of Th1 type response in mice, when using a soluble mLAG-3Ig molecule as an adjuvant. In addition to signals 2 and 3, the induction of an immune response also critically depends on Ag (signal 1) reaching lymphoid organs. Immune responsiveness that is increased or initiated by adjuvants may simply be a result of enhanced translocation of vaccine antigen from the peripheral site of injection towards the draining local lymph node. Along this line, LAG-3, like CD40L (but not LPS), induces MDC and TARC production (chemokines that are required for DC migration to lymph nodes) that may participate to the adjuvant effect observed in mice by contributing to the delivery of an appropriate TCR signal. Finally, we have characterized the molecules involved in MHC class II signalling following LAG-3 binding on human DC. These include PLCgamma2, p70syk and a substrate for PI3K (Akt). Overall, our data show that MHC class II signalling into DCs induced after their engagement with LAG-3 permits an increased conditioning of DCs and, therefore, a better activity of vaccines. Indeed, we found that LAG-3 is highly effective when given as adjuvant into the same site of the proteinaceous vaccine. Thus, new powerful generation of therapeutic cancer vaccines could consist of recombinant protein Ags and recombinant LAG-3Ig. These results have to be confirmed in humans following the production of GMP batches of LAG-3Ig by Immutep S.A. (http://www.immutep.com/), a start-up company developing the LAG-3 technology. This information can be exploited, to optimize the schedules of immunization for anti-cancer vaccines. End users will be hospitals and cancer centres.
Several new T-helper cell epitopes have been identified in the HPV16 E2, E6 and E7 proteins. Upon HPV16 infection, MHC class II restricted epitopes of the E2 and E6 proteins induce strong T-helper type 1-memory responses. HPV16 E7-specific T-helper cell reactivity is generally seen in the blood of patients with a high antigen load such as patients with progressive lesions as well as with cervical carcinoma. Importantly, candidate vaccines that have been injected in healthy individuals as well as in patients with HPV16-related disease in order to induce HPV16 E6- and E7-specific immunity frequently induced strong T-helper responses against the epitopes found in the HPV16 E6 and/or E7 oncoproteins. Enhancement of such HPV16 E6/E7 T-helper reactivity was paralleled by clinical responses in HPV16+ CINIII patients. Currently, the E2 protein is not incorporated in candidate vaccines against HPV16-induced vaccines. The design of new vaccines that incorporate all three antigens (E2, E6 and E7) may find its potential application in the therapeutic and prophylactic vaccination of individuals that do not, as part of the normal response, induce E2 and E6 T-helper cell reactivity. Moreover, these vaccines may induce E7-specific T-helper reactivity, which normally develops too late as a consequence of progressive lesions. End users will be hospital and cancer centres but international public health organizations may be interested for preventive vaccination in countries with a high incidence of this tumour.

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