Cancer gene and immunotherapy: recent developments

Gene and immunotherapeutic approaches to treat human malignant tumors are reviewed. Special attention is given to the different strategies of cancer gene therapy and to recent aspects of cytokine-supported tumor immunotherapy or tumor-specific vaccination. The limitations of these therapy approaches are critically discussed especially with respect to immune escape mechanisms.     

Animal transgenesis: recent data and perspectives

Gene transfer to generate transgenic animals is used more and more to study gene regulation and function. It is also an essential tool to prepare pharmaceuticals or pig organs for transplantation to humans. It is also expected to be a potent way to generate farm animals having traits that cannot emerge by conventional selection. During the last few years, the different techniques to generate transgenic animals and obtain a well-controlled expression of the transgenes have been quite significantly improved. This paper is a brief summary of the most recent relevant data in this field.     

Cancer immunotherapy: exploiting neoepitopes

The efficacy of cancer vaccines has long been hampered by insufficient definition of tumor-specific antigens. A recent study by Kreiter et al. published in Nature has provided a blueprint for a patient-tailored approach to develop individualized RNA vaccines.For the past two decades, the concept of vaccination against cancer has suffered from insufficient definition of relevant target antigens. Consequently, clinical vaccination trials, typically targeting tumor-associated self-antigens, have generally failed to elicit therapeutic immunity in spite of detection of vaccine-induced T-cell responses in blood. In hindsight, these failures can be readily explained by the finding that many of these self-antigens are expressed in the thymus, resulting in deletion of the highly reactive T-cell repertoire and development of suppressive T-regulatory cells¹. Moreover, circumvention of thymic tolerance by infusion of genetically engineered T cells targeting such antigens was found to be associated with severe toxicity in vital somatic tissues, vividly illustrating the physiological importance of immunological tolerance to many tumor-associated antigens².In an almost ironic twist of science, clinical trials employing the two main ''competing'' approaches for cancer immunotherapy, have shown us how to move forward with cancer vaccines. First, in-depth evaluation of the T-cell response in melanoma patients who responded to TIL-therapy, and infusion of ex vivo expanded tumor-infiltrating T cells, revealed that the T-cell clones driving therapeutic efficacy were not directed against lineage-specific and cancer testis antigens, but instead against neo-epitopes encoded by the tumor mutanome³. Essentially the same observation was subsequently made in patients responding to so-called checkpoint inhibitors, antibodies against cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) or the programmed cell death (PD)-1/PD-L1 axis, that can activate T cells by neutralizing inhibitory pathways in T cells⁴.Since checkpoint inhibitors are not uniformly effective in every patient and every cancer type, while durable clinical responses frequently occur in the absence of overt manifestations of autoimmunity, it was fair to postulate that T cells unleashed by checkpoint inhibitors recognize patient- and cancer-specific neoantigens derived from non-synonymous mutations rather than conserved self-antigens. Indeed, whole-exome sequencing of pre- and post-treatment tumor tissue has since revealed a strong association of the clinical response to checkpoint inhibition with the frequency of pre-treatment non-synonymous mutations in mouse models⁵ and in human melanoma⁴ and non-small cell lung cancer⁶, two types of cancer with a particularly high mutational load.These findings have set the stage for coordinated programs to identify patient-specific mutated antigens and target these through specific vaccines or transfer of antigen-specific T cells. Set aside the economic and regulatory challenges resulting from this highly individualized approach, several important questions have to be clarified: How can we predict which of the 50-500 mutations in a given tumor are immunogenic? How can we identify not only HLA class I-restricted epitopes, but also HLA class II-restricted CD4+ T-helper epitopes that are of critical importance to support the immune attack by CD8+ T cells and innate effector cells? How do we implement this information towards the design of patient-tailored vaccines?The recent study by Kreiter and coworkers provided a blueprint for such an approach in the context of syngeneic mouse tumor models⁷. After identification of non-synonymous mutations through RNA and DNA sequencing and prioritization of the mutations based on expression level and prediction of presentation on MHC, animals were vaccinated using synthetic RNA vaccines encoding long peptides. Interestingly, the majority of immunogenic epitopes where found to be MHC class II-restricted⁷. The fact that non-synonymous cancer mutations are preferentially recognized by CD4+ T cells may not be so surprising as the requirements for peptide processing and binding to MHC II are less restrictive than that for MHC I⁸. A downside of this flexibility is that prediction algorithms for MHC class II presentation suffers from inaccuracy compared to class I algorithms. Hence testing in pre-clinical models such as MHC-transgenic mice may be necessary to experimentally identify such epitopes, as exemplified by a recent study characterizing a class II neo-epitope of the glioma driver mutation IDH1R132H⁹. Complementary assays such as in situ proximity ligation assay¹⁰ may aid the selection of neo-epitopes presented on class II molecules by MHC class II+ tumor cells or tumor-infiltrating antigen-presenting cells.The efficacy of neoantigen-specific CD4+ T cells in controlling established tumors raises the question as to what their mechanism of action is. While direct cytotoxicity by CD4+ T cells¹¹ may play a role in tumors expressing MHC class II, more prominent effector mechanism probably include the orchestration of CD8+ T cells and innate effector cells, as well as the anti-tumor impact of cytokines such as interferon-γ or tumor necrosis factor¹². The study by Kreiter et al. suggests that a multimer vaccine targeting CD4 neoepitopes may act by unmasking CD8 epitopes previously not visible or not sufficiently visible to the immune system. The delineation of this complex process of antigen spreading to CD8 epitopes will be important in the future to help further tailor individualized (neo)antigen-specific cancer vaccines.     

Cancer immunotherapy

Cancer is the second leading cause of death in the industrialized world. Most cancer patients are treated by a combination of surgery, radiation and/or chemotherapy. Whereas the primary tumor can, in most cases, be efficiently treated by a combination of these standard therapies, preventing the metastatic spread of the disease through disseminated tumor cells is often not effective. The eradication of disseminated tumor cells present in the blood circulation and micro-metastases in distant organs therefore represents another promising approach in cancer immunotherapy. Main strategies of cancer immunotherapy aim at exploiting the therapeutic potential of tumor-specific antibodies and cellular immune effector mechanisms. Whereas passive antibody therapy relies on the repeated application of large quantities of tumor antigen-specific antibodies, active immunotherapy aims at the generation of a tumor-specific immune response combining both humoral and cytotoxic T cell effector mechanisms by the host's immune system following vaccination. In the first part of this review, concurrent developments in active and passive cancer immunotherapy are discussed. In the second part, the various approaches for the production of optimized monoclonal antibodies used for anti-cancer vaccination are summarized.     

The mirror mechanism: recent findings and perspectives

Mirror neurons are a specific type of visuomotor neuron that discharge both when a monkey executes a motor act and when it observes a similar motor act performed by another individual. In this article, we review first the basic properties of these neurons. We then describe visual features recently investigated which indicate that, besides encoding the goal of motor acts, mirror neurons are modulated by location in space of the observed motor acts, by the perspective from which the others’ motor acts are seen, and by the value associated with the object on which others’ motor acts are performed. In the last part of this article, we discuss the role of the mirror mechanism in planning actions and in understanding the intention underlying the others’ motor acts. We also review some human studies suggesting that motor intention in humans may rely, as in the monkey, on the mirror mechanism.     

Phytochemicals and endothelial dysfunction: recent advances and perspectives

Phytochemistry Reviews - Endothelial dysfunction (impaired vasodilation, prothrombotic, proinflammatory and prooxidant states) is involved in the development of cardiovascular diseases...     

Sturgeon genetics and cytogenetics: recent advancements and perspectives

The aim of this review is to introduce current knowledge in the field of sturgeon genetics. The first section deals with sturgeon cytogenetics, reviewing karyotype organization and polyploidization events during evolution of Acipenseriformes. The second section concerns the results of applications of molecular biology to studies of phylogenetic relationships between extant species, intraspecific analysis of wild populations and stocks for conservation purposes, together with characterization of molecular markers for species identification, relevant to forensic and conservation issues.     

Steady progress and recent breakthroughs in the accuracy of automated genome annotation

The sequencing of large, complex genomes has become routine, but understanding how sequences relate to biological function is less straightforward. Although much attention is focused on how to annotate genomic features such as developmental enhancers and non-coding RNAs, there is still no higher eukaryote for which we know the correct exon-intron structure of at least one ORF for each gene. Despite this uncomfortable truth, genome annotation has made remarkable progress since the first drafts of the human genome were analysed. By combining several computational and experimental methods, we are now closer to producing complete and accurate gene catalogues than ever before.     

Vertebrate evolution: recent perspectives from fish

Recent progress in understanding the evolution of vertebrate genomes has been rapid, and previous notions that all such genomes could be regarded as equivalent in their gene content have been rendered outdated. This notion, often embodied in the representation that vertebrates possess four Hox complexes, still appears in contemporary textbooks of developmental biology. Recent data from the genomes of teleost fish show that this assumption is untrue and suggest that interesting situations might arise from the apparent proliferation of genes among fish.     

Cancer immunotherapy.

Important contributions that stimulated studies in cancer immunotherapy included: (1) the discovery of tumour-associated antigens; (2) the observation that infection with bacille Calmette-Guérin (BCG) in animals was protective against tumour challenge; and (3) the observation that immunodepression due either to malignant disease or to treatment of the disease, was, in some instances, related to prognosis. Immunotherapy trials with microbial agents have involved attempts to obtain a local effect by injecting the agent into the tumour or into the region of the tumour and to obtain a "systemic" effect distant from the site of injection. Trials with active specific immunotherapy involving tumour cells or tumour cell extracts have frequently involved the combination of these specific agents with a nonspecific adjuvant such as BCG. Recent studies with thymosin and levamisole in patients with lung cancer and other types of malignant disease have shown prolonged survival in the groups receiving immunotherapy.     

Current perspectives of bispecific antibody-based immunotherapy

The field of bispecific antibodies is an evolving field of research that has increasing clinical appeal.The fusion of two antibodies or antibody fragments introduced a new way to override natural specificity of T cell and induce effector responses against tumor targets in MHC-unrestricted manner. Initial experiences with bispecific antibodies demonstrate both the promise for and limitations of this anti-cancer strategy. Significant body of work has shown that bispecific antibodies have potential to induce T cell mediated anti-tumor responses in pre-clinical models. However, immunotherapy with bispecific antibodies in humans has yet to prove its value in clinical settings. In addition, the production of high-quality bispecific antibodies for clinical applications, the optimal size and avidity of bispecific antibodies, and in vivo T cell pre-activation remain critical issues. In this review, we summarize recent progress in bispecific antibody-based immunotherapy and address essential aspects of this anti-cancer strategy.     

Cancer immunotherapy: moving beyond current vaccines

Great progress has been made in the field of tumor immunology in the past decade, but optimism about the clinical application of currently available cancer vaccine approaches is based more on surrogate endpoints than on clinical tumor regression. In our cancer vaccine trials of 440 patients, the objective response rate was low (2.6%), and comparable to the results obtained by others. We consider here results in cancer vaccine trials and highlight alternate strategies that mediate cancer regression in preclinical and clinical models.     

Cryopreservation of organs by vitrification: perspectives and recent advances

The cryopreservation of organs became an active area of research in the 1950s as a result of the rediscovery of the cryoprotective properties of glycerol by Polge, Smith, and Parkes in 1949. Over the ensuing four decades of research in this area, the advantages of vitrification, or ice-free cryopreservation, have become apparent. To date, experimental attempts to apply vitrification methods to vascularized whole organs have been confined almost entirely to the rabbit kidney. Using techniques available as of 1997, it was possible to vitrify blood vessels and smaller systems with reasonable success, but not whole organs. Beginning in 1998, a series of novel advances involving the control of cryoprotectant toxicity, nucleation, crystal growth, and chilling injury began to provide the tools needed to achieve success. Based on these new findings, we were first able to show that an 8.4M solution (VMP) designed to prevent chilling injury at -22 degrees C was entirely non-toxic to rabbit kidneys when perfused at -3 degrees C and permitted perfusion-cooling to -22 degrees C with only mild additional damage. We next investigated the ability of the kidney to tolerate a 9.3M solution known as M22, which does not devitrify when warmed from below -150 degrees C at 1 degrees C/min. When M22 was added and removed at -22 degrees C, it was sometimes [corrected] fatal, but when it was perfused for 25min at -22 degrees C and washed out simultaneously with warming, postoperative renal function recovered fully. When kidneys loaded with M22 at -22 degrees C were further cooled to an average intrarenal temperature of about -45 degrees C (about halfway through the putative temperature zone of increasing vulnerability to chilling injury), all kidneys supported life after transplantation and returned creatinine values to baseline, though after a higher transient creatinine peak. However, medullary, papillary, and pelvic biopsies taken from kidneys perfused with M22 for 25min at -22 degrees C were found to devitrify when vitrified and rewarmed at 20 degrees C/min in a differential scanning calorimeter. It remains to be determined whether this devitrification is seriously damaging and whether it can be suppressed by improving cryoprotectant distribution to more weakly perfused regions of the kidney or by rewarming at higher rates. In conclusion, although the goal of organ vitrification remains elusive, the prospects for success have never been more promising.     

Cancer immunotherapy with CpG-ODN

Bacterial DNA and synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODN) are the ligands for the Toll-like receptor 9 (TLR9), which is expressed by B-lymphocytes and a subset of dendritic cells. CpG-ODN are strong activators of both innate and specific immunity, and drive the immune response towards the Th1 phenotype. Given the promising results obtained in several experimental models of allergies or infections, CpG-ODN are now entering clinical trials for these diseases. In cancer, promising approaches combined CpG-ODN with tumor antigens, monoclonal antibodies or dendritic cells. When no relevant tumor antigen is known, CpG-ODN can be used alone to activate locally the innate immunity and trigger a tumor-specific immune response, overcoming the need for the identification of a tumoral antigen. Preclinical models have shown impressive results and several clinical trials are on-going worldwide in melanoma, lymphoma, renal carcinoma, breast cancer and glioblastoma.