Disputats, Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser. Med populärvetenskaplig sammanfattning på svenska.

Cancer immunotherapy seeks to boost the host’s immune system to respond to tumor antigens. The adaptive immune system comprises of two arms, one that elicits a cellular immune response and one that elicits a humoral immune response. Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides presented to them in the context of class I major histocompatibility complex (MHC) molecules and are capable of killing tumor cells. CTL are educated to discriminate between foreign and self-antigen. Tumors frequently express self-antigen which usually makes them poorly immunogenic. Because tumors are genetically unstable, they may present excess self peptides and/or peptides in a reading frame different from wild type self proteins. These frameshift (FS) peptides, are caused by an insertion or deletion of nucleotides that disrupt translation of the normal reading frame and alters the protein produced such that it is non-self. Binding affinity, dissociation rate and the overall stability of the peptide/MHC/β₂-microglobulin complex are important considerations in determining the immunogenicity of a given peptide. Interaction between the anchor residues in a peptide and binding pockets in MHC are essential, but this interaction is not always strong enough to stimulate T cell responses. This indicates that not all amino acids of the peptide ligand bound to MHC are equally important for the functional outcome of the receptor engagement and that other amino acid residues in the sequence are important for binding. Optimized peptide ligands (OPL) are analogues derived from natural wild type antigenic peptides that contain amino acid substitutions at anchor and auxiliary residues. OPL can be rationally designed to generate a more robust immune response compared to that of the wild-type peptide. Active immunotherapy using OPL of tumor antigen epitopes are designed to elicit tumor-specific CTL that can overcome tolerance and either re-awaken or elicit new T-cell responses to an antigen. The work and principles presented here using brain tumor-derived peptides demonstrates that HLA-A*0201-restricted CTL generated against wild type, frameshift and OPL peptides elicit CTL that were able to recognize and respond to wild type, tumorderived peptides. The response was donor dependent in that not all individuals responded more strongly to OPL; a minority responded better to wild type peptide. This data further suggests that the rational design and testing of multiple peptides for the same epitope should elicit a broader response among different individuals than single peptide immunization.

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<p>Southern Africa is facing one of the most serious HIV epidemics. This project contributes to the HIVNET, Network for Prevention Trials cohort for vaccine development. HIV’s biology and rapid mutation rate have made vaccine design difficult. We examined HIV-1 subtype C diversity and how it relates to CTL epitope location along viral gag sequences. We found a negative correlation between codon sites under positive selection and epitope regions / suggesting epitope regions are evolutionarily conserved. It is possible that epitopes exist in non-conserved regions, yet fail to be detected due to the reference strain diverging from the circulating viral population. To test if CTL clustering is an artifact of the reference strain, we calculated differences between the gag codons and the reference strain. We found a weak negative correlation, suggesting epitopes in less conserved regions maybe evading detection. Locating conserved and optimal epitopes that can be recognized by CTLs is essential for the design of vaccine reagents.</p>

The aims of this thesis were to investigate the utility of in vitro drug sensitivity testing to optimize the use of cancer chemotherapy and to assess the properties of a new cancer drug in a phase I clinical trial. Tumour cells from patients were analysed with the short-term Fluorometric Microculture Cytotoxicity Assay (FMCA). In samples from a wide spectrum of tumour types, the effect of the drug combination FEC (5Fu-epirubicin-cyclophosphamide) was generally appropriately predicted from the effect of the best component drug. However, of samples intermediately sensitive to the best single drug, 45% converted to sensitive when testing the combination. Thus, combination testing may identify advantageous interactions and improve in vitro test performance. In tumour samples from peritoneal carcinomatosis, significant differences in drug sensitivity between diagnoses were observed, cross-resistance between most drugs was modest or absent, and the concentration-effect relationships for two drugs in individual samples varied considerably. Thus, for optimal selection of drugs for intraperitoneal chemotherapy, differences in drug sensitivity at the diagnosis and individual patient level should be considered. In samples from patients with ovarian carcinoma, drug sensitivity was related to tumour grade, histologic subtype and patient treatment status. In a homogeneous subset of patients, the FMCA predicted individual patient tumour response with high sensitivity and specificity. Thus, if carefully interpreted in the context of important clinical variables, in vitro testing could be of value for individualizing chemotherapy in ovarian cancer. Employing a once weekly dosing schedule in a phase I trial, the mechanistically new and preclinically promising NAD depleting drug CHS 828 produced dose limiting thrombocytopenia and gastrointestinal toxicity without clear evidence of anti-tumour efficacy. It is concluded that in vitro drug sensitivity testing could be a way to optimize the use of chemotherapy and that successful development of new cancer drugs needs improved strategies.

Ovarian cancer, with the highest mortality rate amongst gynaecological malignancies in Australia, is the eighth most common cancer and the fifth cause of cancer-related deaths in women. Currently, five-year survival for women diagnosed with ovarian cancer is only 40 % and despite many patients experiencing remission, approximately 80 % of them will relapse due to residual micrometastasis. The limited impact of standard therapies on the prognosis for recurrent chemotherapy-resistant disease and the need to identify less toxic alternatives has motivated the development of strategies to combat the aggressive and life-threatening burden of ovarian cancer. A novel therapy against cancer utilises dendritic cells (DC), potent antigen presenting cells, to deliver tumour antigens to the immune system for the stimulation of cytotoxic T-lymphocyte (CTL) responses. DC immunotherapy has been used for the treatment of patients with ovarian cancer; however, clinical responses after the injection of antigen-loaded DC have been disappointing. Therefore, the identification of additional tumour associated antigens (TAA) is required. A TAA highly expressed in ovarian cancer cells, CA125, is a candidate target for DC-based immunotherapy. Initially, CTL responses to CA125 were studied in the context of HLA-A*0201. CD8+ T-cell responses specific for CA125 peptides (with high affinity for the MHC class I) were generated from cultures initiated with peptide-loaded monocyte-derived DC (Mo-DC). To expand the evaluation of T-cell recognition of CA125 to non-HLA-A*0201 individuals, messenger RNA (mRNA) was investigated as an antigen-loading vehicle. RNA encodes for the repertoire of epitopes presented by the TAA, potentially inducing immune responses in the context of multiple MHC class I and II molecules to known/unknown antigens. One focus of this study was to investigate a novel mRNA transfection system utilising mannan for the delivery of mRNA into DC. Initially the immunomodulating effect of mannan was examined in terms of DC activation. Mannan induced the phenotypic and functional maturation of immature Mo-DC in vitro. Next, the ability of oxidised mannan (OxM) linked to mRNA was investigated for its capacity to deliver enhanced green fluorescent protein (EGFP) mRNA into DC. We observed high transfection efficiencies in the murine and in human DC systems using low mRNA concentrations, in the absence of significant cell viability impairment. Interestingly, upon mRNA delivery via the OxM-PEI complex, DC maturation was induced to considerably higher levels as compared with that achieved with electroporation and non-transfected controls, this was measured by phenotype (CD83) and IL-12 secretion. Within this study, OxM-PEI did not deliver TAA encoding mRNA into DC for the stimulation of CTL. In summary, mannan is a novel strategy to deliver mRNA and a strong maturation signal simultaneously to human Mo-DC. The functional capacity of this system requires further investigation before it can be considered for clinical use. Electroporation has evolved as a superior method for mRNA delivery into DC as reported in the literature. Therefore, a comprehensive study was performed encompassing the critical issues associated with transfection efficiency, in order to standardise an electroporation protocol for use in DC immunotherapy schedules. EGFP was used as a model antigen to optimise mRNA uptake by Mo-DC by monitoring the expression of the reporter gene by FACS analysis. Influenza matrix protein 1 mRNA was, then, utilised as a model antigen for MHC class I restricted antigen presentation, for confirmation of the optimised loading parameters. The efficiency of this delivery system was assessed using CA125 mRNA in stimulating antigen-specific T-cell responses in PBMC of healthy individuals. CD4+ and CD8+ antigen-specific T-cell responses were generated recognising CA125 mRNA loaded Mo-DC and also ovarian cancer cell lines endogenously expressing CA125. This study has identified CA125 specific T-cell responses in healthy donors, allowing further investigation into the potential for its use as a candidate TAA in ovarian cancer immunotherapy. Furthermore, the use of Mo-DC transfected with mRNA encoding TAA is a promising strategy for the delivery of TAA in the generation of antigen-specific T-cell responses. In summary, the results gained from this PhD thesis should be taken into consideration when designing future DC immunotherapy strategies to combat one of the leading causes of cancer mortality in women, ovarian cancer.

The Mater Medical Research Institute proposes to undertake a Phase I clinical trial using CMRF-56 immunoselected blood dendritic cells (BDC) loaded with control and myeloma-associated tumour peptide antigens for the treatment of multiple myeloma (MM) patients with minimal residual disease. This thesis describes some of the fundamental pre-clinical in vitro experiments undertaken in preparation for this trial so as to maximise the potential of this vaccine to induce myeloma-specific immune responses. These experiments involved determining the parameters for optimal activation of the CMRF-56 immunoselected cell preparation and exploring the potential of novel myeloma peptide antigens to induce anti-myeloma cytotoxic T lymphocyte (CTL) responses. CMRF-56 immunoselected cell preparations, containing predominately myeloid BDC, monocytes and B cells, were prepared from both healthy donors and myeloma patients. Activation of this preparation with granulocyte macrophage colony stimulating factor (GM-CSF) was found to increase co-stimulatory molecule expression by and survival of BDC, improve peptide- and lysate-specific CTL induction, and, in combination with prostaglandin E2 (PGE2), improve chemokine-specific migration of BDC. Following optimisation of in vitro CTL generation protocols, GM-CSF activated CMRF-56 immunoselected cells were examined for their ability to induce myeloma-specific immunity. Using lysate from myeloma cell line U266 as an antigen source, a polyclonal T cell pool was generated within which peptide specific CTL recognising myeloma antigens Muc1, HM1.24/BST2, DKK-1 and CT-7/MAGE-C1 could be identified. Furthermore, GM-CSF activated CMRF-56 immunoselected cells pulsed with HLA-A*201 restricted peptides derived from Muc1, HM1.24/BST2 and CT-7/MAGE-C1 could induce CTL capable of lysing both peptide- and myeloma cell line targets in both healthy donors and myeloma patients. These results provide the first evidence of immunogenic HLA-A*201 restricted epitopes of novel myeloma antigen CT-7/MAGE-C1. The data collected in this study supports the application of GM-CSF activated CMRF-56 immunoselected cells loaded with defined myeloma peptide antigens for the therapeutic vaccination of MM patients with minimal residual disease.

Interest in cellular immunotherapy has increased with the recognition of the pivotal role that dendritic cells (DC) play in the adaptive immune system. The preparation of DC to present tumour antigens and subsequent induction of tumour specific T cells have been widely documented. This thesis studied the ability of cord blood (CB) stem cells to differentiate into functional CD34+DC, followed by the optimisation of electroporation of RNA into these cells. Total RNA derived from a leukaemic cell line and a primary human leukaemic sample was electroporated into CD34+DC DC and we were able to generate anti-leukaemic cytotoxic T lymphocytes (CTL). The CTL specifically targeted leukaemia but not normal cells. While the in vitro data showed promising results of the CTL specificity, a NOD-SCID model of human ALL was established to allow the CTL to be tested in vivo. We established a reproducible model of human ALL in NOD-SCID mouse using four primary human ALL samples. The adoptively transferred anti-leukaemic CTL into the ALL bearing NOD-SCID mice showed that ALL engraftment was significantly delayed. However, the addition of total RNA loaded CD34+DC DC did not enhance the in vivo CTL effect. Lastly, by dissecting the CTL response, we found that the polyclonal CTL were targeting survivin, HM1.24 and CT-7 antigens. The CTL clones generated from these polyclonal CTL showed high specificity for leukaemia but not normal cells. In conclusion, these preliminary data support the use of total RNA electroporated CD34+DC as a means of inducing anti-leukaemic CTL, and have demonstrated the efficacy of the CTL in a NOD-SCID model of ALL. This study has also provided insight into the polyclonal CTL response and future studies will likely continue along this path.

The Mater Medical Research Institute proposes to undertake a Phase I clinical trial using CMRF-56 immunoselected blood dendritic cells (BDC) loaded with control and myeloma-associated tumour peptide antigens for the treatment of multiple myeloma (MM) patients with minimal residual disease. This thesis describes some of the fundamental pre-clinical in vitro experiments undertaken in preparation for this trial so as to maximise the potential of this vaccine to induce myeloma-specific immune responses. These experiments involved determining the parameters for optimal activation of the CMRF-56 immunoselected cell preparation and exploring the potential of novel myeloma peptide antigens to induce anti-myeloma cytotoxic T lymphocyte (CTL) responses. CMRF-56 immunoselected cell preparations, containing predominately myeloid BDC, monocytes and B cells, were prepared from both healthy donors and myeloma patients. Activation of this preparation with granulocyte macrophage colony stimulating factor (GM-CSF) was found to increase co-stimulatory molecule expression by and survival of BDC, improve peptide- and lysate-specific CTL induction, and, in combination with prostaglandin E2 (PGE2), improve chemokine-specific migration of BDC. Following optimisation of in vitro CTL generation protocols, GM-CSF activated CMRF-56 immunoselected cells were examined for their ability to induce myeloma-specific immunity. Using lysate from myeloma cell line U266 as an antigen source, a polyclonal T cell pool was generated within which peptide specific CTL recognising myeloma antigens Muc1, HM1.24/BST2, DKK-1 and CT-7/MAGE-C1 could be identified. Furthermore, GM-CSF activated CMRF-56 immunoselected cells pulsed with HLA-A*201 restricted peptides derived from Muc1, HM1.24/BST2 and CT-7/MAGE-C1 could induce CTL capable of lysing both peptide- and myeloma cell line targets in both healthy donors and myeloma patients. These results provide the first evidence of immunogenic HLA-A*201 restricted epitopes of novel myeloma antigen CT-7/MAGE-C1. The data collected in this study supports the application of GM-CSF activated CMRF-56 immunoselected cells loaded with defined myeloma peptide antigens for the therapeutic vaccination of MM patients with minimal residual disease.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.