We have previously shown that Salmonella immunotherapy is effective to treat B-cell non-Hodgkin lymphoma (B-NHL) in mice. However, this model involves animals with high tumor burden, whereas in the clinics B-NHL patients are usually treated with chemotherapy (CHOP: cyclophosphamide, doxorubicin, vincristine, and prednisone) as first-line therapy prior to immunotherapy. Recently, we have described a NHL-B preclinical model using CHOP chemotherapy to achieve MRD in immunocompetent animals that closely resemble patients’ conditions. In this work, we assessed the efficacy of Salmonella immunotherapy in B-NHL-bearing mice undergoing chemotherapy. Salmonella administration significantly delayed tumor growth and prolonged survival of chemotherapy-treated NHL-bearing animals. Mice receiving the CHOP–Salmonella combined therapy showed increased numbers of tumor-infiltrating leukocytes and a different profile of cytokines and chemokines expressed in the tumor microenvironment. Further, Salmonella immunotherapy in CHOP-treated animals also enhanced NK cells cytotoxic activity as well as induced systemic lymphoma-specific humoral and cellular responses. Chemotherapy treatment profoundly impacted on the general health status of recipient animals, but those receiving Salmonella showed significantly better overall body condition. Altogether, the results clearly demonstrated that Salmonella immunotherapy could be safely used in individuals under CHOP treatment, resulting in a better prognosis. These results give strong support to consider Salmonella as a neoadjuvant therapy in a clinical setting.

Genomic islands are DNA regions containing variable genetic information related to secondary metabolism. Frequently, they have the ability to excise from and integrate into replicons through site-specific recombination. Thus, they are usually flanked by short direct repeats that act as attachment sites, and contain genes for an integrase and an excisionase which carry out the genetic exchange. These mobility events would be at the basis of the horizontal transfer of genomic islands among bacteria. Microcin H47 is a ribosomally-synthesized antibacterial peptide that belongs to the group of chromosome-encoded microcins. The 13 kb-genetic system responsible for its production resides in the chromosome of the Escherichia coli H47 strain and is flanked by extensive and imperfect direct repeats. In this work, both excision and integration of the microcin H47 system were experimentally detected. The analyses were mainly performed in E. coli K12 cells carrying the microcin system cloned in a multicopy plasmid. As expected of a site-specific recombination event, the genetic exchange also occurred in a context deficient for homologous recombination. The DNA sequence of the attachment sites resulting from excision were hybrid between the sequences of the direct repeats. Unexpectedly, different hybrid attachment sites appeared which resulted from recombination in four segments of identity between the direct repeats. Genes encoding the trans-acting proteins responsible for the site-specific recombination were shown to be absent in the microcin H47 system. Therefore, they should be provided by the remaining genetic context, not only in the H47 strain but also in E. coli K12 cells, where both excision and integration occurred. Moreover, a survey of the attachment sites in data banks revealed that they are widely spread among E. coli strains. It is concluded that the microcin system is a small island –H47 genomic island- that would employ a parasitic strategy for its mobility.

Aim: We evaluated a novel approach combining the use of attenuated Salmonella immunotherapy with a Toll-like receptor agonist, imiquimod, in B16F1 melanoma-bearing mice. Materials & methods: B16F1 melanoma-bearing mice were daily treated with topical imiquimod in combination with one intratumoral injection of attenuated Salmonella enterica serovar Typhimurium LVR01. Results: The combined therapy resulted in retarded tumor growth and prolonged survival. Combination treatment led to an enhancement in the expression of pro-inflammatory cytokines and chemokines in the tumor microenvironment, with a Th1-skewed profile, resulting in a broad antitumor response. The induced immunity was effective in controlling the occurrence of metastasis. Conclusion: Salmonella LVR01 immunotherapy in combination with imiquimod is a novel approach that could be considered as an effective antimelanoma therapy.

Non-Hodgkin lymphomas (NHL) are the most frequent hemato-oncological malignancies. Despite recent major advances in treatment, a substantial proportion of patients relapses highlighting the need for new therapeutic modalities. Promissory results obtained in pre-clinical studies are usually not translated when moving into clinical trials. Pre-clinical studies are mainly conducted in animals with high tumor burden; instead patients undergo chemotherapy as first line of treatment and most likely are under remission when immunotherapies are applied. Thus, an animal model that more closely resembles patients’ conditions would be a valuable tool. BALB/c mice were injected subcutaneously with A20 lymphoma cells and after tumor development different doses of chemotherapy were assessed to find optimal conditions for minimal residual disease (MRD) establishment. Tumor growth and survival, as well as drugs side effects, were all evaluated. Complete lymphoma remission was monitored in vivo using positron emission tomography (PET), and the results were correlated with histology. Immunological status was assessed by splenocytes proliferation assays in NHL-complete remission mice and by analyzing tumor cell infiltrates and chemokines/cytokines gene expression in the tumor microenvironment of animals with residual lymphoma. Two cycles of CHOP chemotherapy at days 25 and 35 post-tumor implantation induced complete remission for around 20 days. PET showed to be a suitable follow-up technique for MRD condition with 85.7 and 75% of sensibility and specificity respectively. Proliferative responses upon mitogen stimulation were similar in animals that received chemotherapy and wild type mice. Tumors from animals with residual lymphoma showed higher numbers of CD4+ and CD8+ and similar numbers of NK, neutrophils and Tregs infiltrating cells as compared with non-treated animals. Gene expression of several cytokines as well as an array of chemokines associated with migration of activated T cells to tumor sites was upregulated in the tumor microenvironment of animals that received chemotherapy treatment. We established a NHL-B pre-clinical model using standard chemotherapy to achieve MRD in immunocompetent animals. The MRD condition is maintained for approximately 20 days providing a therapeutic window of time where new immunotherapies can be tested in conditions closer to the clinics.

The introduction of new therapeutics requires validation of Good Manufacturing Practice (GMP)-grade manufacturing including suitable quality controls. This is challenging for Advanced Therapy Medicinal Products (ATMP) with personalized batches. We have developed a person-alized, cell-based gene therapy to treat age-related macular degeneration and established a vali-dation strategy of the GMP-grade manufacture for the ATMP; manufacturing and quality control were challenging due to a low cell number, batch-to-batch variability and short production duration. Instead of patient iris pigment epithelial cells, human donor tissue was used to produce the transfected cell product (“tIPE”). We implemented an extended validation of 104 tIPE productions. Procedure, operators and devices have been validated and qualified by determining cell number, viability, extracellular DNA, sterility, duration, temperature and volume. Transfected autologous cells were transplanted to rabbits verifying feasibility of the treatment. A container has been engineered to ensure a safe transport from the production to the surgery site. Criteria for successful validation and qualification were based on tIPE’s Critical Quality Attributes and Process Parameters, its manufacture and release criteria. The validated process and qualified operators are essential to bring the ATMP into clinic and offer a general strategy for the transfer to other manufacture centers and personalized ATMPs.