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Background Neuroblastoma (NB) is the most common malignant extracranial solid tumor in children. Despite significant survival improvements in multiple pediatric malignancies, children with relapsed neuroblastoma (NB) have dismal outcomes. ROR1 is highly expressed on the majority of NB (Rebagay/Yan Front Oncol, 2012). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (Chu/Cairo, et al, Can Imm Res 2015). N-803 (ALT-803) is a novel IL15 superagonist complex where mutant IL-15N72D is bound to an IL-15RαSu-Fc fusion protein with higher potency and longer half-life compared to IL-15 (Han/Wong, Cytokine, 2011). Objectives To determine if anti-ROR1 CAR engineered NK cells can efficiently target ROR1+ NB and if the cytotoxicity can be further enhanced in combination with N-803. Methods ExPBNK cells were expanded with lethally irradiated K562-mbIL21cells (Denman/Lee Plos One, 2012) and isolated as previously described (Chu/Cairo, Can Imm Res, 2015). ExPBNK cells were electroporated with anti-ROR1-CAR mRNA (ROR1-CAR was generously provided by Riddell S) using maxcyte electroporator. ALT-803 was generously provided by Altor BioScience. CHLA-255 and CHLA-136 were generously provided by Seeger R. In vitro cytotoxicity of anti-ROR1 CAR NK against NB cell lines were examined at different E:T ratios. Granzyme B, Perforin and IFN-γ levels were evaluated by ELISA assays. High dimentionsal CyTOF analysis was performed to character the immunophenotying of anti-ROR1-CAR-NK. NB xenografted NSG mice were generated and tumor burden was monitored as we previously described (Chu/Cairo, Oncoimmunology 2017). Results Anti-ROR1-CAR was efficiently expressed on exPBNK after CAR mRNA-electroporation at 24 hrs. Anti-ROR1-CAR-NK were effective in killing NB cells even at very low E:T ratio compared to mock NK with enhanced granzyme B, perforin and interferon- release (p Conclusion anti-ROR1-CAR NK had significantly enhanced cytotoxicity against ROR1+ NB cells in vitro and in vivo using human NB xenografted NSG mice compared to controls. ALT-803 further enhanced the anti-tumor effect of anti-ROR1 CAR NK (supported by W81XWH-17-1-0420).

The potential of vaccine-elicited anti-HIV envelope antibodies to control HIV-infection was evaluated by immunizing macaques with the HIV envelope protein and transiently depleting them of their CD8+ cells before intravenous challenge with the pathogenic CCR5-tropic SIV/HIV chimeric virus, SHIVSF162P4. Although sterilizing immunity was not achieved, all vaccinated animals effectively controlled infection and remained free of disease for the duration of observation (over 3 years). In contrast, during the same period, the control animals progressed to disease. Both the vaccinees and the controls developed robust cell-mediated antiviral and neutralizing antibody responses following infection. A comparative analysis of these responses suggests that the more effective long-term control of infection by the vaccinated animals is due to the more rapid development of anti-HIV envelope antibodies. These studies suggest that priming by vaccination of B cell anti-HIV envelope responses maybe crucial for the long-term control of HIV infection.

Multiple monoclonal and polyclonal antibody preparations have been shown to neutralize HIV-1 infection in vitro . Upon direct testing in humans, however, many of these have failed to demonstrate clinical efficacy. Hu-PBL-SCID mice offer a model system in which to test the pre-clinical efficacy of antibody preparations. Testing in hu-PBL-SCID mice has shown that some antibodies are able to mediate pre- and post-exposure protection against HIV-1 infection, at concentrations that should be attainable in humans. Despite differences in the route and mode of transmission in humans and in hu-PBL-SCID mice, several aspects of the model make it a favorable model for future testing of antibody protection against HIV-1 infection. These include the architecture of the peritoneal cavity, the mixture of human cells that engraft, the density of human target cells for HIV-1 infection, and the presence of complement and NK cells that can interact with antibody preparations in blocking HIV-1 infection. The use of this model in testing newer antibody preparations for efficacy against primary isolates should enhance our knowledge of the mechanisms of antibody protection against HIV-1 infection in vivo and speed the pre-clinical evaluation of potential immunoprophylactic agents against HIV-1.

A small population of HIV-1-infected individuals remains clinically healthy and immunologically normal for more than ten years. We have studied ten subjects who have been asymptomatic with normal and stable CD4+ lymphocyte counts, despite 12 to 16 years of HIV-1 infection, to gain information on the determinants of nonprogression. Multiple methods were used to determine the viral load in their blood. Plasma cultures were uniformly negative for infectious virus. However, particle-associated HIV-1 RNA was detectable in four subjects using a sensitive branched DNA amplification assay. In peripheral blood mononuclear cells (PBMC), infectious HIV-1 was quantified in three subjects using a standard limiting-dilution culture method. Infectious virus was recovered from another subject using a CD8-depleted culture. In contrast, six subjects had no detectable infectious virus in PBMC. All had detectable viral DNA in PBMC by a quantitative polymerase chain reaction assay, but the copy numbers were low, ranging from 10 to 100 copies per 10(6) PBMC in all but two subjects. Overall, the viral burden in the plasma and PBMC of long-term survivors was orders of magnitude lower than those typically found in progressors. Possible mechanisms for low levels of HIV-1 in vivo were examined experimentally.

In the majority of patients, HIV-1 replication is quickly and efficiently controlled after the initial burst of viremia. Recent studies have begun to elucidate the underlying immune responses that may be responsible for this dramatic reduction in viral load. These responses include those of HIV-specific CD8+ cytotoxic T lymphocytes and antibodies capable of binding the virus.

We have examined four human ovarian tumor lines (A2780, AD10, OVC-8, and SKOV-3) selected for their sensitivity and/or resistance to the recombinant human tumor necrosis factor alpha (TNF-alpha) and the chemotherapeutic drug doxorubicin (DOXO). The tumor lines were either sensitive to both agents, resistant to one or the other, or resistant to both. Of the four lines examined only the DOXO-resistant line AD10 exhibited the multidrug-resistance (MDR) phenotype. Enhanced cytotoxicity was seen with the combination of TNF-alpha and DOXO in each line regardless of their sensitivity or resistance patterns and, thus, demonstrates that drug resistance due to the expression of the MDR phenotype or its absence can be overcome by TNF-alpha and DOXO. We then examined whether TNF-alpha or TNF-alpha and DOXO modulated the MDR phenotype in AD10 as a possible mechanism of overcoming drug resistance. TNF-alpha had no effect on either DOXO intake or efflux as measured by flow cytometry. Further, TNF-alpha treatment showed no effect on the level of MDR-1 mRNA. These results suggest that the enhanced cytotoxicity seen with the combination of TNF-alpha and DOXO is not the result of any modulation of drug influx or efflux levels by TNF-alpha. Overall, these findings suggest that combination treatment with TNF-alpha and DOXO can overcome resistance inflicted by different mechanisms.