Development of a cultivation process for the enhancement of human interferon alpha 2b production in the oleaginous yeast, Yarrowia lipolytica

<p>Abstract</p> <p>Background</p> <p>As an oleaginous yeast, <it>Yarrowia lipolytica </it>is able to assimilate hydrophobic substrates. This led to the isolation of several promoters of key enzymes of this catabolic pathway. Less is known about the behavior of <it>Y. lipolytica </it>in large bioreactors using these substrates. There is therefore a lack of established know-how concerning high cell density culture protocols of this yeast. Consequently, the establishment of suitable induction conditions is required, to maximize recombinant protein production under the control of these promoters.</p> <p>Results</p> <p>Human interferon α2b (huIFN α2b) production in <it>Yarrowia lipolytica </it>was used as a model for the enhancement of recombinant protein production under the control of the oleic acid (OA)-inducible promoter POX2. Cell viability and heterologous protein production were enhanced by exponential glucose feeding, to generate biomass before OA induction. The optimal biomass level before induction was determined (73 g L^-1), and glucose was added with oleic acid during the induction phase. Several oleic acid feeding strategies were assessed. Continuous feeding with OA at a ratio of 0.02 g OA per g dry cell weight increased huIFNα2b production by a factor of 1.88 (425 mg L^-1) and decreased the induction time (by a factor of 2.6, 21 h). huIFN α2b degradation by an aspartic protease secreted by <it>Y. lipolytica </it>was prevented by adding pepstatin (10 μM), leading to produce a 19-fold more active huIFN α2b (26.2 × 10⁷IU mg^-1).</p> <p>Conclusion</p> <p><it>Y. lipolytica</it>, a generally regarded as safe (GRAS) microorganism is one of the most promising non conventional yeasts for the production of biologically active therapeutic proteins under the control of hydrophobic substrate-inducible promoter.</p

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Tumor-Expressed IDO Recruits and Activates MDSCs in a Treg-Dependent Manner

Indoleamine 2,3-dioxygenase (IDO) has been described as a major mechanism of immunosuppression in tumors, though the mechanisms of this are poorly understood. Here, we find that expression of IDO by tumor cells results in aggressive tumor growth and resistance to T-cell-targeting immunotherapies. We demonstrate that IDO orchestrates local and systemic immunosuppressive effects through recruitment and activation of myeloid-derived suppressor cells (MDSCs), through a mechanism dependent on regulatory T cells (Tregs). Supporting these findings, we find that IDO expression in human melanoma tumors is strongly associated with MDSC infiltration. Treatment with a selective IDO inhibitor in vivo reversed tumor-associated immunosuppression by decreasing numbers of tumor-infiltrating MDSCs and Tregs and abolishing their suppressive function. These findings establish an important link between IDO and multiple immunosuppressive mechanisms active in the tumor microenvironment, providing a strong rationale for therapeutic targeting of IDO as one of the central regulators of immune suppression

Specific recognition of an FGFR2 fusion by tumor infiltrating lymphocytes from a patient with metastatic cholangiocarcinoma

Background Metastatic cholangiocarcinoma (CC), a form of gastrointestinal cancer that originates from the bile ducts, cannot be cured by currently available therapies, and is associated with dismal prognosis. In a previous case report, adoptive transfer of autologous tumor infiltrating lymphocytes (TILs), the majority of which recognized a tumor-specific point mutation, led to a profound and durable cancer regression in a patient with metastatic CC. Thus, more effective treatment for patients with this disease may be developed by using TILs that target cancer-specific mutations, but also other genetic aberrations such as gene fusions. In this context, fusions that involve fibroblast growth factor receptor 2 (FGFR2) and function as oncogenes in a subset of patients with intrahepatic CC (ICC) represent particularly attractive targets for adoptive cell therapy. However, no study to date has explored whether FGFR2 fusions can be recognized by patients’ T cells.Method To address whether FGFR2 fusions can be recognized by patients’ T cells, we tested TILs from four patients with FGFR2 fusion-positive ICC for recognition of peptides and minigenes that represented the breakpoint regions of these fusions, which were unique to each of the four patients.Results We found that CD4+ TILs from one patient specifically recognized the breakpoint region of a unique FGFR2-TDRD1 (tudor domain-containing 1) fusion, and we isolated a T-cell receptor responsible for its recognition.Conclusions This finding suggests that FGFR2 fusion-reactive TILs can be isolated from some patients with metastatic ICC, and thus provides a rationale for future exploration of T cell-based therapy targeting FGFR2 fusions in patients with cancer. Furthermore, it augments the rationale for extending such efforts to other types of solid tumors hallmarked by oncogenic gene fusions

The New Era of Cancer Immunotherapy: Manipulating T-Cell Activity to Overcome Malignancy

Using the immune system to control cancer has been investigated for over a century. Yet it is only over the last several years that therapeutic agents acting directly on the immune system have demonstrated improved overall survival for cancer patients in phase III clinical trials. Furthermore, it appears that some patients treated with such agents have been cured of metastatic cancer. This has led to increased interest and acceleration in the rate of progress in cancer immunotherapy. Most of the current immunotherapeutic success in cancer treatment is based on the use of immune-modulating antibodies targeting critical checkpoints (CTLA-4 and PD-1/PD-L1). Several other immune-modulating molecules targeting inhibitory or stimulatory pathways are being developed. The combined use of these medicines is the subject of intense investigation and holds important promise. Combination regimens include those that incorporate targeted therapies that act on growth signaling pathways, as well as standard chemotherapy and radiation therapy. In fact, these standard therapies have intrinsic immune-modulating properties that can support antitumor immunity. In the years ahead, adoptive T-cell therapy will also be an important part of treatment for some cancer patients. Other areas which are regaining interest are the use of oncolytic viruses that immunize patients against their own tumors and the use of vaccines against tumor antigens. Immunotherapy has demonstrated unprecedented durability in controlling multiple types of cancer and we expect its use to continue expanding rapidly.SCOPUS: ar.kinfo:eu-repo/semantics/publishe

Megakaryocytes and platelet-fibrin thrombi characterize multi-organ thrombosis at autopsy in COVID-19: A case series

© 2020 Background: There is increasing recognition of a prothrombotic state in COVID-19. Post-mortem examination can provide important mechanistic insights. Methods: We present a COVID-19 autopsy series including findings in lungs, heart, kidneys, liver, and bone, from a New York academic medical center. Findings: In seven patients (four female), regardless of anticoagulation status, all autopsies demonstrated platelet-rich thrombi in the pulmonary, hepatic, renal, and cardiac microvasculature. Megakaryocytes were seen in higher than usual numbers in the lungs and heart. Two cases had thrombi in the large pulmonary arteries, where casts conformed to the anatomic location. Thrombi in the IVC were not found, but the deep leg veins were not dissected. Two cases had cardiac venous thrombosis with one case exhibiting septal myocardial infarction associated with intramyocardial venous thrombosis, without atherosclerosis. One case had focal acute lymphocyte-predominant inflammation in the myocardium with no virions found in cardiomyocytes. Otherwise, cardiac histopathological changes were limited to minimal epicardial inflammation (n = 1), early ischemic injury (n = 3), and mural fibrin thrombi (n = 2). Platelet-rich peri‑tubular fibrin microthrombi were a prominent renal feature. Acute tubular necrosis, and red blood cell and granular casts were seen in multiple cases. Significant glomerular pathology was notably absent. Numerous platelet-fibrin microthrombi were identified in hepatic sinusoids. All lungs exhibited diffuse alveolar damage (DAD) with a spectrum of exudative and proliferative phases including hyaline membranes, and pneumocyte hyperplasia, with viral inclusions in epithelial cells and macrophages. Three cases had superimposed acute bronchopneumonia, focally necrotizing. Interpretation: In this series of seven COVID-19 autopsies, thrombosis was a prominent feature in multiple organs, in some cases despite full anticoagulation and regardless of timing of the disease course, suggesting that thrombosis plays a role very early in the disease process. The finding of megakaryocytes and platelet-rich thrombi in the lungs, heart and kidneys suggests a role in thrombosis. Funding: None

Presence of PD-1 Expressing T Cells Predicts for Inferior Overall Survival in Newly Diagnosed Multiple Myeloma

Abstract Background: Programmed cell death 1 (PD-1) protein downregulates T cell activation and is related to immune tolerance. PDL1 up regulation, T cell infiltration, and T cell exhaustion are features, which suggest susceptibility to PD-1 blockade antibodies. Blockade of PD-1 or its ligand PD-L1 has shown promising responses in several malignancies. Although little clinical activity has been seen in patients with relapsed multiple myeloma (MM), the role of the PD-1 pathway and T cell exhaustion in newly diagnosed MM has not been explored. Objective: To determine whether T-cell infiltrate or expression of PD-1 correlates with clinical features and prognosis among patients with newly diagnosed multiple myeloma. Methods: We screened a clinically annotated database of 341 patients seen at MSKCC between 1998 and 2012 that had Multiple Myeloma, received a bone marrow transplant and were consented to a biospecimen research protocol for availability of pre-treatment bone marrow specimens. A total of 64 bone marrow biopsy specimens were identified. Immunohistochemistry (IHC) was performed in formalin-fixed paraffin-embedded specimens using an anti human CD3 monoclonal antibody (mAb) (Dako, Clone F7.2.38) and an anti human PD-1 mAb (Cell Marque, catalog #315M-95). CD3 and PD1 IHC staining were graded as negative (<5% for CD3, < 1% for PD1), or positive (≥5% for CD3, ≥1% for PD1). Correlative analyses were performed between CD3/PD1 expression and clinical outcome using the following parameters: International Staging System (ISS), cytogenetic risk, progression free survival (PFS), overall survival (OS), and response to treatment. Groups were compared by Fisher's exact test. OS and PFS were assessed by Cox regression and estimated by Kaplan-Meier methods. Results: 23 specimens (36%) were CD3 positive and 10 specimens (16%) were PD-1 positive. All PD-1 positive specimens were CD3 positive. 41 specimens (64%) were CD3 negative (<5%) and PD1 negative (<1%). Based on these results, specimens were divided into three groups: Exhausted T-cell infiltrate (CD3+/PD1+), non exhausted T-cell infiltrate (CD3+/PD1-) and no T-Cell infiltrate (CD3-/PD1-). In the exhausted T-cell infiltrate group 30% of patients had ISS stage 3 and 40% had high risk cytogenetics. In the non-exhausted T-cell group 15% of patients had ISS stage 3 and 15% high cytogenetic risk. In the no T-cell infiltrate group 10% had ISS stage 3 disease and 22% high cytogenetic risk. These proportions were not significantly different across the 3 groups. Median OS from 1st auto infusion was 7 years while median PFS was 2.3 years. On univariate analysis, there was no significant difference in PFS between the 3 groups. The presence of CD3 and PD1 T-cells were significantly associated with OS (p-value = 0.04). Median OS from 1st auto infusion was 43 months for the exhausted T-Cell infiltrate group followed by 83 months for the no T-Cell infiltrate group. The non exhausted T-cell group had the highest OS, median not reached; OS by 7-years was 75%. Cytogenetic risk at diagnosis was significantly associated with OS (p-value = 0.03). In a multivariable model, CD3/PD1 staining continued to trend toward an association with OS (p-value = 0.08) and cytogenetic risk remained significant (p-value = 0.05). Conclusions: The presence of T-cells with PD-1 expression was not associated with higher risk disease at MM diagnosis based on cytogenetics and ISS stage. The presence of PD-1 expressing CD3+ T cells trends toward an association with poorer overall survival in newly diagnosed MM, especially compared to non exhausted T-cell infiltrate, suggesting the possibility that T cell exhaustion represents a novel high risk disease characteristic. Further investigation is necessary to assess if the presence of CD3+PD-1+ T cells is an independent prognostic feature in newly diagnosed MM. Figure 1. Overall survival by CD3/PD1 Staining in 64 newly diagnosed myeloma patients. Figure 1. Overall survival by CD3/PD1 Staining in 64 newly diagnosed myeloma patients. Disclosures Giralt: JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; TAKEDA: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding. Landgren:International Myeloma Foundation: Research Funding; Onyx: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Medscape: Honoraria; Bristol-Myers Squibb: Honoraria; Medscape: Consultancy; BMJ Publishing: Consultancy; Celgene: Honoraria; Onyx: Honoraria; BMJ Publishing: Honoraria; Onyx: Research Funding. Hassoun:Novartis: Consultancy; Takeda: Research Funding; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Lesokhin:Genentech: Research Funding; Aduro: Consultancy; Janssen: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Efranat: Consultancy

T CELL IMMUNOTHERAPIES TRIGGER NEUTROPHIL ACTIVATION TO ELIMINATE TUMOR ANTIGEN ESCAPE VARIANTS

BackgroundTargeted immune-based therapies such as adoptive T cell transfer (ACT) are often ineffective because tumors evolve over time and under selective pressure display antigen loss variant clones. A classic example in melanoma is de-differentiation and loss of expression of antigenic proteins. Therapies that activate multiple branches of the immune system may eliminate such escape variantsMethodsHere we show that melanoma-specific CD4+ ACT therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate large melanoma tumors with clonal escape variants.ResultsEarly on-target recognition of melanoma antigens by adoptively transferred tumor-specific CD4+ T cells was required. Surprisingly, however, complete tumor eradication was partially dependent on neutrophils. Supporting these findings, extensive neutrophil activation and neutrophil extracellular traps were found in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade.ConclusionsOur findings uncover a novel interplay between T cells mediating the initial tumor- and tissue-specific immune response, and neutrophils mediating tumor destruction of antigen loss variants.Ethics ApprovalAll tissues were collected at MSKCC following study protocol approval by the MSKCC Institutional Review Board. All mouse procedures were performed in accordance with institutional protocol guidelines at Memorial Sloan-Kettering Cancer Center (MSKCC) under an approved protocol.</jats:sec