A phase Ib/II dose expansion study of subcutaneous sasanlimab in patients with locally advanced or metastatic non-small-cell lung cancer and urothelial carcinoma.

BACKGROUND: Sasanlimab is an antibody to the programmed cell death protein 1 receptor. We report updated data of subcutaneous sasanlimab in non-small-cell lung cancer (NSCLC) and urothelial carcinoma dose expansion cohorts from a first-in-human phase Ib/II study. PATIENTS AND METHODS: Patients were ≥18 years of age with NSCLC or urothelial carcinoma, and no prior immunotherapies, who progressed on or were intolerant to systemic therapy, or for whom systemic therapy was refused or unavailable. Patients received subcutaneous sasanlimab at 300 mg every 4 weeks (q4w). Primary objectives were to evaluate safety, tolerability, and clinical efficacy by objective response rate (ORR). RESULTS: Sixty-eight and 38 patients with NSCLC and urothelial carcinoma, respectively, received subcutaneous sasanlimab. Overall, sasanlimab was well tolerated; 13.2% of patients experienced grade ≥3 treatment-related adverse events. Confirmed ORR was 16.4% and 18.4% in the NSCLC and urothelial carcinoma cohorts, respectively. ORR was generally higher in patients with high programmed death-ligand 1 (PD-L1) expression (≥25%) and high tumor mutational burden (TMB; >75%). In the NSCLC and urothelial carcinoma cohorts, median progression-free survival (PFS) was 3.7 and 2.9 months, respectively; corresponding median overall survival (OS) was 14.7 and 10.9 months. Overall, longer median PFS and OS correlated with high PD-L1 expression and high TMB. Longer median PFS and OS were also associated with T-cell inflamed gene signature in the urothelial carcinoma cohort. CONCLUSIONS: Subcutaneous sasanlimab at 300 mg q4w was well tolerated with promising clinical efficacy observed. Phase II and III clinical trials of sasanlimab are ongoing to validate clinical benefit. Subcutaneous sasanlimab may be a potential treatment option for patients with NSCLC or urothelial carcinoma.

Improvement of the resistance against early Mycobacterium tuberculosis-infection in the absence of PI3Kγ enzyme is associated with increase of CD4+IL-17+ cells and neutrophils.

Given the impossibility to study the lung immune response during Mycobacterium tuberculosis-latent infection, and consequently, the mechanisms that control the bacterial load, it is reasonable to determine the activation of local immunity in the early phase of the infection. The phosphatidylinositol-3-kinase gamma enzyme (PI3Kγ) is involved in the leukocyte recruitment, phagocytosis and cellular differentiation, and therefore, it is considered a promising target for the development of immunotherapies for chronic inflammatory diseases. Mice genetically deficient in PI3Kγ (PI3Kγ-/-) or WT (Wild Type) were evaluated 15 days post-infection. The enzyme deficiency improved the resistance against infection, increased the frequency of CD4+IL-17+ cells, the production of IL-17 as well as the gene and protein expression of molecules associated with Th17 cell differentiation and neutrophil recruitment. Our findings show, for the first time, the participation of the PI3Kγ in vivo in the M. tuberculosis-infection, and suggest an association of Th17 cells with protection in the early phase of tuberculosis.

M2 macrophages or IL-33 treatment attenuate ongoing Mycobacterium tuberculosis infection.

The protective effects of mycobacterial infections on lung allergy are well documented. However, the inverse relationship between tuberculosis and type 2 immunity is still elusive. Although type 1 immunity is essential to protection against Mycobacterium tuberculosis it might be also detrimental to the host due to the induction of extensive tissue damage. Here, we determined whether lung type 2 immunity induced by allergen sensitization and challenge could affect the outcome of M. tuberculosis infection. We used two different protocols in which sensitization and allergen challenge were performed before or after M. tuberculosis infection. We found an increased resistance to M. tuberculosis only when allergen exposure was given after, but not before infection. Infected mice exposed to allergen exhibited lower bacterial load and cellular infiltrates in the lungs. Enhanced resistance to infection after allergen challenge was associated with increased gene expression of alternatively activated macrophages (M2 macrophages) and IL-33 levels. Accordingly, either adoptive transfer of M2 macrophages or systemic IL-33 treatment was effective in attenuating M. tuberculosis infection. Notably, the enhanced resistance induced by allergen exposure was dependent on IL-33 receptor ST2. Our work indicates that IL-33 might be an alternative therapeutic treatment for severe tuberculosis.

Requirement of MyD88 and Fas pathways for the efficacy of allergen-free immunotherapy.

BACKGROUND: We have shown that mycobacterial antigens and CpG oligodeoxynucleotides downmodulate airway allergic inflammation by mechanisms dependent on T-cell activation. Here, we investigated the participation of the innate response, particularly the role of MyD88 adaptor, and Fas molecules in the effectiveness of DNA-HSP65 or CpG/culture filtrated proteins (CFP) immunotherapy. METHODS: Mice sensitized and challenged with Der p 1 allergen were treated with DNA-HSP65, CpG/CFP, or with adoptively transferred cells from immunized mice. The treatment efficacy was assessed by evaluating eosinophil recruitment, antibody, and cytokine production. RESULTS: In addition to downregulating the Th2 response, DNA-HSP65 and CpG/CFP promoted IL-10 and IFN-γ production. Adoptive transfer of cells from mice immunized with DNA-HSP65 or CpG/CFP to allergic recipients downmodulated the allergic response. Notably, transfer of cells from DNA-HSP65- or CpG/CFP-immunized MyD88(-/-) mice failed to reduce allergy. Additionally, for effective reduction of allergy by cells from CpG/CFP-immunized mice, Fas molecules were required. Although DNA-HSP65 or CpG/CFP immunization stimulated antigen-specific production of IFN-γ and IL-10, the effect of DNA-HSP65 was associated with IL-10 while CpG/CFP was associated with IFN-γ. Moreover, after stimulation with mycobacterial antigens plus Der p 1 allergen, cells from mite-allergic patients with asthma exhibited similar patterns of cytokine production as those found in the lung of treated mice. CONCLUSIONS: This study provides new insights on the mechanisms of allergen-free immunotherapy by showing that both DNA-HSP65 and CpG/CFP downregulated house dust mite-induced allergic airway inflammation via distinct pathways that involve not only induction of mycobacterial-specific adaptive responses but also signaling via MyD88 and Fas molecules.

Estimating Shared Copy Number Aberrations for Array CGH Data: The Linear-Median Method.

MOTIVATION: Existing methods for estimating copy number variations in array comparative genomic hybridization (aCGH) data are limited to estimations of the gain/loss of chromosome regions for single sample analysis. We propose the linear-median method for estimating shared copy numbers in DNA sequences across multiple samples, demonstrate its operating characteristics through simulations and applications to real cancer data, and compare it to two existing methods. RESULTS: Our proposed linear-median method has the power to estimate common changes that appear at isolated single probe positions or very short regions. Such changes are hard to detect by current methods. This new method shows a higher rate of true positives and a lower rate of false positives. The linear-median method is non-parametric and hence is more robust in estimating copy number. Additionally the linear-median method is easily computable for practical aCGH data sets compared to other copy number estimation methods.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.

Serum ghrelin levels in growth hormone-sufficient and growth hormone-deficient patients during growth hormone-releasing hormone plus arginine test.

BACKGROUND AND AIMS: Ghrelin is an orexigenic hormone produced in the stomach and in other organs, exerting a wide range of metabolic functions, including stimulation of GH secretion. Ghrelin secretion is decreased by iv or oral glucose load as well as during euglycemic-hyperinsulinemic clamp and hypoglycemia. We evaluated the circulating ghrelin levels in GH-deficient (GHD) and in GH-sufficient (GHS) patients during GHRH plus arginine test. MATERIALS AND METHODS: The study group comprised 35 patients, including 20 with pituitary tumors, 12 with empty sella, 2 with short stature, and 1 with post-traumatic isolated GH deficiency. According to the results of GHRH plus arginine test, 14 patients were defined as GHD and 21 as GHS. Patients with central hypothyroidism, hypocorticism, and hypogonadism had been on replacement therapy for at least 3 months at the moment of the study. Blood samples were collected every 20 min up to 60 min after GHRH and arginine administration. RESULTS: By definition, GH response to GHRH plus arginine was higher in GHS than GHD group (p<0.0001). Basal serum ghrelin levels were not different in the two groups and did not correlate with body mass index, GH, IGFI and insulin concentrations. After GHRH plus arginine, serum ghrelin decreased significantly in both groups, with percent decreases ranging 13.3-66.6% in GHD patients (p=0.001) and 7.2-42.2% in GHS patients (p=0.004), with no significant difference in the two groups (p=0.12). CONCLUSION: Our results show that ghrelin secretion is not modulated by acute GH increase observed in GHS subjects during GHRH plus arginine infusion. The similar decrease of serum ghrelin after GHRH plus arginine stimulation in both GHS and GHD subjects demonstrated that there is no negative feedback of GH on ghrelin secretion.

Th1 polarized response induced by intramuscular DNA-HSP65 immunization is preserved in experimental atherosclerosis

We previously reported that a DNA vaccine constructed with the heat shock protein (HSP65) gene from Mycobacterium leprae (DNA-HSP65) was protective and also therapeutic in experimental tuberculosis. By the intramuscular route, this vaccine elicited a predominant Th1 response that was consistent with its protective efficacy against tuberculosis. It has been suggested that the immune response to Hsp60/65 may be the link between exposure to microorganisms and increased cardiovascular risk. Additionally, the high cholesterol levels found in atherosclerosis could modulate host immunity. In this context, we evaluated if an atherogenic diet could modulate the immune response induced by the DNA-HSP65 vaccine. C57BL/6 mice (4-6 animals per group) were initially submitted to a protocol of atherosclerosis induction and then immunized by the intramuscular or intradermal route with 4 doses of 100 mug DNA-HSP65. On day 150 (15 days after the last immunization), the animals were sacrificed and antibodies and cytokines were determined. Vaccination by the intramuscular route induced high levels of anti-Hsp65 IgG2a antibodies, but not anti-Hsp65 IgG1 antibodies and a significant production of IL-6, IFN-g and IL-10, but not IL-5, indicating a Th1 profile. Immunization by the intradermal route triggered a mixed pattern (Th1/Th2) characterized by synthesis of anti-Hsp65 IgG2a and IgG1 antibodies and production of high levels of IL-5, IL-6, IL-10, and IFN-g. These results indicate that experimentally induced atherosclerosis did not affect the ability of DNA-HSP65 to induce a predominant Th1 response that is potentially protective against tuberculosis.

Immune modulation induced by tuberculosis DNA vaccine protects non-obese diabetic mice from diabetes progression.

We have described previously the prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in experimental murine tuberculosis. However, the high homology of this protein to the corresponding mammalian 60 kDa heat shock protein (Hsp60), together with the CpG motifs in the plasmid vector, could trigger or exacerbate the development of autoimmune diseases. The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) spontaneously as a consequence of an autoimmune process that leads to destruction of the insulin-producing beta cells of the pancreas. IDDM is characterized by increased T helper 1 (Th1) cell responses toward several autoantigens, including Hsp60, glutamic acid decarboxylase and insulin. In the present study, we evaluated the potential of DNA-HSP65 injection to modulate diabetes in NOD mice. Our results show that DNA-HSP65 or DNA empty vector had no diabetogenic effect and actually protected NOD mice against the development of severe diabetes. However, this effect was more pronounced in DNA-HSP65-injected mice. The protective effect of DNA-HSP65 injection was associated with a clear shift in the cellular infiltration pattern in the pancreas. This change included reduction of CD4(+) and CD8(+) T cells infiltration, appearance of CD25(+) cells influx and an increased staining for interleukin (IL)-10 in the islets. These results show that DNA-HSP65 can protect NOD mice against diabetes and can therefore be considered in the development of new immunotherapeutic strategies.

Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse.

AIMS/HYPOTHESIS: Recent observations suggest the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural history of the disease. Oral administration of probiotic bacteria can modulate local and systemic immune responses; consequently, we investigated the effects of oral administration of the probiotic compound VSL#3 on the occurrence of diabetes in non-obese diabetic (NOD) mice. METHODS: VSL#3 was administered to female NOD mice three times a week starting from 4 weeks of age. A control group received PBS. Whole blood glucose was measured twice a week. IFN-gamma and IL-10 production/expression was evaluated by ELISA in culture supernatants of mononuclear cells isolated from Peyer's patches and the spleen, and by real-time PCR in the pancreas. Insulitis was characterised by immunohistochemistry and histomorphometric studies. RESULTS: Early oral administration of VSL#3 prevented diabetes development in NOD mice. Protected mice showed reduced insulitis and a decreased rate of beta cell destruction. Prevention was associated with an increased production of IL-10 from Peyer's patches and the spleen and with increased IL-10 expression in the pancreas, where IL-10-positive islet-infiltrating mononuclear cells were detected. The protective effect of VSL#3 was transferable to irradiated mice receiving diabetogenic cells and splenocytes from VSL#3-treated mice. CONCLUSIONS/INTERPRETATION: Orally administered VSL#3 prevents autoimmune diabetes and induces immunomodulation by a reduction in insulitis severity. Our results provide a sound rationale for future clinical trials of the primary prevention of type 1 diabetes by oral VSL#3 administration.

A multi-neighbor-joining approach for phylogenetic tree reconstruction and visualization

The computationally challenging problem of reconstructing the phylogeny of a set of contemporary data, such as DNA sequences or morphological attributes, was treated by an extended version of the neighbor-joining (NJ) algorithm. The original NJ algorithm provides a single-tree topology, after a cascade of greedy pairing decisions that tries to simultaneously optimize the minimum evolution and the least squares criteria. Given that some sub-trees are more stable than others, and that the minimum evolution tree may not be achieved by the original NJ algorithm, we propose a multi-neighbor-joining (MNJ) algorithm capable of performing multiple pairing decisions at each level of the tree reconstruction, keeping various partial solutions along the recursive execution of the NJ algorithm. The main advantages of the new reconstruction procedure are: 1) as is the case for the original NJ algorithm, the MNJ algorithm is still a low-cost reconstruction method; 2) a further investigation of the alternative topologies may reveal stable and unstable sub-trees; 3) the chance of achieving the minimum evolution tree is greater; 4) tree topologies with very similar performances will be simultaneously presented at the output. When there are multiple unrooted tree topologies to be compared, a visualization tool is also proposed, using a radial layout to uniformly distribute the branches with the help of well-known metaheuristics used in computer science.

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of CD8+ cells, interferon-gamma recovery and reduction of lung injury.

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-gamma recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-alpha. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-gamma and to restrict the growth of bacilli.

Role of trehalose dimycolate in recruitment of cells and modulation of production of cytokines and NO in tuberculosis.

Mice treated with viable Mycobacterium tuberculosis with no glycolipid trehalose dimycolate (TDM) on the outer cell wall (delipidated M. tuberculosis) by intraperitoneal or intratracheal inoculation presented an intense recruitment of polymorphonuclear cells into the peritoneal cavity and an acute inflammatory reaction in the lungs, respectively. In addition, lung lesions were resolved around the 32nd day after intratracheal inoculation. TDM-loaded biodegradable poly-DL-lactide-coglycolide microspheres as well as TDM-coated charcoal particles induced an intense inflammatory reaction. In addition, high levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), IL-12, IL-10, gamma interferon (IFN-gamma), and IL-4 production were detected in lung cells, and nitric oxide (NO) production was high in culture supernatants of bronchoalveolar lavage cells. These in vivo data were confirmed by in vitro experiments using peritoneal macrophages cultured in the presence of TDM adsorbed onto coverslips. High levels of IFN-gamma, IL-6, TNF-alpha, IL-12, IL-10, and NO were detected in the culture supernatants. Our results suggest that TDM contributes to persistence of infection through production of cytokines, which are important for the recruitment of inflammatory cells and maintenance of a granulomatous reaction. In addition, our findings are important for a better understanding of the immunostimulatory activity of TDM and its possible use as an adjuvant in experiments using DNA vaccine or gene therapy against tuberculosis.

DNA Vaccine for the Prevention and Treatment fo Tuberculosis

Tradicional systems for developing drugs and vaccines are failing spectaculary to deliver the goods in the fight against tuberculosis (TB). The disease that afflicts the developing world defies the imagination in its scale. One third of the world's population - 2 billion people - is infected with Mycobacterium tuberculosis, and 16 million have active TB. Shockingly, TB hit an all-time high in 1999 with 8 million new cases - 95 per cent of them in developing countries - and 2 million deaths. The disease is spreading rapidly throughout the world. The toll is set to rise; AIDS activates the dormant form of the disease, while multidrug resistance is spreading across the planet. The last new drug for TB was introduced over thirty years ago and industry has been reluctant to invest in discovering new families of drugs because of the financial risks in investing in products destined largely for developing country markets. If global health is left to market forces, historians will remember this era as one in which humanity stood idly by while half the planet languished in sickness. Fortunately some researchers have realized this, and are driving forward new models for TB therapy and vaccine discovery. One of the latest sign of this trend is the development of a DNA vaccine for the prevention and treatment of TB by our research group. Over the last few years, some of our experiments in wich mycobacterial antigens were presented to the immune system, as of they were viral antigens (DNA vaccine), have had a significant impact on our understanding of protective immunity against tuberculosis. They also markedly enhanced the prospects for new vaccines. We now know that individual mycobacterial-protein antigens expressed from DNA-vaccine constructs can confer protection equal to that from live BCG vaccine in mice. A critical determinant of the outcome of immunization appears to be the degree to which antigen-specific cytotoxic T cells are generated by the immune response. We have demonstrated that DNA vaccination is an affective way of establishing long lasting cytotoxic T-cell memory and protection against tuberculosis. Moreover, our new preclinical work shows that DNA vaccines, initially designed to prevent infection, can also have a dramatic therapeutic action. In infected mice, the immune response can be caused to switch from one that is relatively inefficient and gives bacterial stasis to one that kills the bacteria, eliminating...

Thimet oligopeptidase (EC 3.4.24.15), a novel protein on the route of MHC class I antigen presentation.

The initial processing of antigens leading to major histocompatibility complex (MHC) class I antigenic peptides is carried out by the proteasome. However, how the final epitopes are generated and protected from degradation by cytosolic peptidases remains unknown. Coincidentally, peptides associated with the MHC class I molecules range from 8 to 13 amino acid residues, similarly to the optimum substrate size required for the cytosolic thimet oligopeptidase. Here we have investigated the putative intracellular function of thimet oligopeptidase related to antigen presentation. Using a well-characterized antigen-presenting cell system, we were able to demonstrate either inhibition or stimulation of CD8 T cell proliferation and cytotoxicity, manipulating intracellular thimet oligopeptidase levels with its specific inhibitor cFP-Ala-Ala-Tyr-pAb or loading the enzyme itself into the antigen-presenting cells. Our results suggest that thimet oligopeptidase should take an important function in the pathway of antigen presentation via MHC class I through a mechanism yet unknown.

DNA encoding individual mycobacterial antigens protects mice against tuberculosis

Over the last few years, some of our experiments in which mycobacterial antigens were presented to the immune system as if they were viral antigens have had a significant impact on our understanding of protective immunity against tuberculosis. They have also markedly enhanced the prospects for new vaccines. We now know that individual mycobacterial protein antigens can confer protection equal to that from live BCG vaccine in mice. A critical determinant of the outcome of immunization appears to be the degree to which antigen-specific cytotoxic T cells are generated by the immune response. Our most recent studies indicate that DNA vaccination is an effective way to establish long-lasting cytotoxic T cell memory and protection against tuberculosis

Protection against tuberculosis by bone marrow cells expressing mycobacterial hsp65.

Although mice acquire only a slight degree of protection against tuberculosis by immunization with Mycobacterium leprae (M. leprae) hsp65 in incomplete Freund's adjuvant, protection is substantial following immunization by injection with J774 macrophage-like tumour cells that express the protein from the mycobacterial gene via a retroviral vector. We here took the same vector, used it to transfect the gene into normal murine bone marrow cells in vitro, and then used the transfected cells to reconstitute haematopoiesis in lethally irradiated mice. Bone marrow-cell clonal expansion and production of the protein in vivo resulted in specific delayed-type hypersensitivity and protection against challenge with Mycobacterium tuberculosis (M. tuberculosis) in about half of recipients. Counts of live bacteria in liver at 3 weeks were fivefold lower in delayed-type hypersensitivity (DTH)-positive than in DTH-negative mice. Other mice acquired neither DTH nor protection despite the presence of the protein in peripheral blood.