SBIR-STTR Award

We hypothesize that Protein Kinase C (PKC) agonists such as the non-tumorigenic Bryoids combined with HDAC inhibitors (HDACi) represent a valuable pharmacological approach to purge latent HIV-1 from cellular reservoirs. We also hypothesize that a combination of a Bryoid and an HDACi co-encapsulated in a long- circulation pegylated immunonanosomes coated with anti-PD-L1 nanobodies will provide efficient HIV latency activation and immunological depletion of latent reservoirs. This combination nanosomal approach has the added benefit of concomitantly and significantly reducing systemic toxicities of both PKC and HDACi. Our Phase I Specific Aims are: (1) construct and characterize nanosomes containing the Bryoid, Bryostatin-1 in the lipid membrane and nanosomes of HDAC inhibitors SAHA, Romidepsin and Panobinostat in the aqueous core; (2) evaluate the cytotoxicity of Bryostatin-1, HDACi and of their corresponding nanosomes in cell culture models, and in vivo cytotoxicity and biodistribution in BALB/c mouse model to select most efficacious combination; and (3) evaluate the synergistic and anti-HIV-1 latency antagonistic effects of two nanosomes of different combination of Bryostatin-1 and the alternate HDAC inhibitors in cell culture and in humanized mice models of HIV latency. We plan to conduct this Proof-of-Concept study over a 24-month period with a multidisciplinary team of 1.10 full-time equivalents (FTE) of an engineer, virologist, and manufacturing technician. Additionally, our lean but experienced research team will be advised and supported by Dr. Robert F. Siliciano, Professor of Medicine, Johns Hopkins University School of Medicine and Investigator, Howard Hughes Medical Institute who will also provide support with respect to the ex vivo evaluation of the combination PKC-HDACi nanosomes; Dr. Eduardo Munoz, University of Cordoba, Spain, an immunologist, an HIV latency researcher and Scientific Advisor to Aphios Corporation; Dr. Santiago Moreno, Head, Infectious Diseases, Ramón y Cajal Hospital and Professor of Infectious Diseases, University of Alcalá, Madrid, Spain, an HIV clinician and Principal Investigator of an ongoing Phase I/IIa clinical trial of Bryostatin-1 for HIV latency in HIV patients on cART; and Dr. Joseph L. Bryant, D.V.M at the Institute of Human Virology, University of Maryland Medical School, an HIV animal model expert, who will lead the in vivo toxicity and efficacy animal studies planned. We will establish a Scientific Advisory Review Panel with Drs. Siliciano, Bryant, Munoz and Santiago for the research. Should we be successful in achieving our Phase I milestones, we will prepare and propose a comprehensive Phase II research and development program which will: (1) systematically construct immunonanosomes by coating the most promising nanosomes identified in Phase I anti-PD-L1 nanobodies, and physically, chemically and biologically characterize the immunonanosomes; (2) evaluate most potent HIV latency drug combination in an induction/activation therapy protocol in the SHIV/macaque or alternative model of viral persistence and latency; and (3) determine the potential effects of treatment on selected immunological functions during induction therapy. The end-goal of our research program is to develop a combination therapeutic to clear and free HIV patients from latent viruses, curing a chronic disease and reducing the burden of personal toxicities and ending the economic burden of this disease in both developing and developed countries. In order to reach these objectives, we plan to conduct rigorous clinical studies in a Phase III clinical research program with a commercial partner/investor in which we will: (1) perform cGMP manufacturing of HIV latency combination therapeutic at the pilot-scale level; (2) establish a Drug Master File; (3) design IND-enabling preclinical studies and Phase I/II clinical trials; (4) prepare pre-IND package; and (5) establish and conduct pre-IND meeting with FDA and file an IND with the FDA. We will then conduct Phase I and II clinical trials, and license the therapeutic to a multinational pharmaceutical company such as Roche, J&J, Pfizer, Merck or GSK. In summary, we will utilize Aphios’ proprietary critical fluid nanosomes (CFN) process for the formation of small, uniform liposomes for the co-encapsulation of Bryoids and HDAC inhibitors [US Patent, Castor, 2014], and take advantage of promising data we have generated from preliminary studies [Perez et al., 2010], to develop responsive protocols for Phase I and II clinical studies in HIV-1 patients being treated with cART and having a suppressed viral load [Gutiérrez et al., 2016]. The outcomes of the proposed studies will inform product development for HIV latency, and fast-track the field to complete elimination of HIV infection and sterilizing cure.

Public Health Relevance Statement:
PROJECT NARRATIVE Currently, over 22 million people have died from AIDS and there are over 42 million people living with HIV/AIDS worldwide. In the United States, an estimated 1 million people are currently living with HIV and approximately 40,000 infections occur each year. There is no vaccine against HIV, and AIDS, if untreated, will lead to the death of over 95% of infected individuals 10 years post-infection. HIV infects several cell types during the course of infection and progression to acquired immune deficiency syndrome (AIDS). The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication with anti-retroviral therapy (ART), since latently infected cells remain a permanent source of viral reactivation. It has been hypothesized that intensification of ART could reduce the residual viremia but recent studies strongly suggest that this is not the likely scenario. Moreover, ART is problematic because of long-term toxicity, drug resistance, and the inability to target and eliminate persistent viral reservoirs. Therefore, other pharmacological approaches targeting the HIV-1 reservoir have been suggested by several investigators as a promising strategy to develop new drugs able to activate latent HIV-1 without inducing global T cell-activation. We propose to develop a unique combination therapy consisting of a PKC modulator and an HDAC inhibitor to reactivate these latent HIV reservoirs so that HIV-1 can be eliminated by ART and eradicated from the patient’s body. Our approach has the potential for advancing HIV therapy towards sterilizing cure, which currently is out of reach.

Project Terms:
Acquired Immunodeficiency Syndrome; Agonist; animal efficacy; Animal Model; Anti-Retroviral Agents; Antibody Binding Sites; aqueous; Biodistribution; Blood Circulation; bryostatin; burden of illness; CD4 Positive T Lymphocytes; Cell Culture Techniques; Cell Death; cell type; Cells; Cessation of life; Chronic Disease; Clinical Research; Clinical Trials; Combined Modality Therapy; Communicable Diseases; Cyclic GMP; cytotoxicity; Data; design; Developed Countries; Disease; Drug Combinations; Drug resistance; Economic Burden; Encapsulated; Engineering; Evaluation; experience; Generations; Genetic Transcription; Goals; Head; Histone Deacetylase Inhibitor; HIV; HIV Infections; HIV therapy; HIV-1; Hospitals; Human; humanized mouse; Immune system; Immunologist; Immunotherapy; in vivo; in vivo Model; Inbred BALB C Mice; Individual; Infection; Institutes; Latent Virus; Lead; Legal patent; Licensing; Life; Liposomes; Liquid substance; Lytic; Macaca; Maryland; Medical; medical schools; Medicine; meetings; Membrane Lipids; Modeling; mouse model; multidisciplinary; nanobodies; Neoadjuvant Therapy; neutralizing antibody; novel therapeutics; Outcome; Patients; PDCD1LG1 gene; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phase I Clinical Trials; preclinical study; Principal Investigator; Process; product development; professor; programs; Protein Kinase C; Protocols documentation; purge; Research; research and development; Research Personnel; residence; Residual state; simian human immunodeficiency virus; Source; Spain; systemic toxicity; T-Cell Activation; Therapeutic; Time; Toxic effect; Track and Field; treatment effect; United States; Universities; Vaccines; Viral; Viral Load result; Viral reservoir; Viremia; virology; Virus; Vorinostat

We hypothesize that Protein Kinase C (PKC) agonists such as the non-tumorigenic Bryoids combined with HDAC inhibitors (HDACi) represent a valuable pharmacological approach to purge latent HIV-1 from cellular reservoirs. We also hypothesize that a combination of a Bryoid and an HDACi co-encapsulated in a long- circulation pegylated immunonanosomes coated with anti-PD-L1 nanobodies will provide efficient HIV latency activation and immunological depletion of latent reservoirs. This combination nanosomal approach has the added benefit of concomitantly and significantly reducing systemic toxicities of both PKC and HDACi. Our Phase I Specific Aims are: (1) construct and characterize nanosomes containing the Bryoid, Bryostatin-1 in the lipid membrane and nanosomes of HDAC inhibitors SAHA, Romidepsin and Panobinostat in the aqueous core; (2) evaluate the cytotoxicity of Bryostatin-1, HDACi and of their corresponding nanosomes in cell culture models, and in vivo cytotoxicity and biodistribution in BALB/c mouse model to select most efficacious combination; and (3) evaluate the synergistic and anti-HIV-1 latency antagonistic effects of two nanosomes of different combination of Bryostatin-1 and the alternate HDAC inhibitors in cell culture and in humanized mice models of HIV latency. We plan to conduct this Proof-of-Concept study over a 24-month period with a multidisciplinary team of 1.10 full-time equivalents (FTE) of an engineer, virologist, and manufacturing technician. Additionally, our lean but experienced research team will be advised and supported by Dr. Robert F. Siliciano, Professor of Medicine, Johns Hopkins University School of Medicine and Investigator, Howard Hughes Medical Institute who will also provide support with respect to the ex vivo evaluation of the combination PKC-HDACi nanosomes; Dr. Eduardo Munoz, University of Cordoba, Spain, an immunologist, an HIV latency researcher and Scientific Advisor to Aphios Corporation; Dr. Santiago Moreno, Head, Infectious Diseases, Ramón y Cajal Hospital and Professor of Infectious Diseases, University of Alcalá, Madrid, Spain, an HIV clinician and Principal Investigator of an ongoing Phase I/IIa clinical trial of Bryostatin-1 for HIV latency in HIV patients on cART; and Dr. Joseph L. Bryant, D.V.M at the Institute of Human Virology, University of Maryland Medical School, an HIV animal model expert, who will lead the in vivo toxicity and efficacy animal studies planned. We will establish a Scientific Advisory Review Panel with Drs. Siliciano, Bryant, Munoz and Santiago for the research. Should we be successful in achieving our Phase I milestones, we will prepare and propose a comprehensive Phase II research and development program which will: (1) systematically construct immunonanosomes by coating the most promising nanosomes identified in Phase I anti-PD-L1 nanobodies, and physically, chemically and biologically characterize the immunonanosomes; (2) evaluate most potent HIV latency drug combination in an induction/activation therapy protocol in the SHIV/macaque or alternative model of viral persistence and latency; and (3) determine the potential effects of treatment on selected immunological functions during induction therapy. The end-goal of our research program is to develop a combination therapeutic to clear and free HIV patients from latent viruses, curing a chronic disease and reducing the burden of personal toxicities and ending the economic burden of this disease in both developing and developed countries. In order to reach these objectives, we plan to conduct rigorous clinical studies in a Phase III clinical research program with a commercial partner/investor in which we will: (1) perform cGMP manufacturing of HIV latency combination therapeutic at the pilot-scale level; (2) establish a Drug Master File; (3) design IND-enabling preclinical studies and Phase I/II clinical trials; (4) prepare pre-IND package; and (5) establish and conduct pre-IND meeting with FDA and file an IND with the FDA. We will then conduct Phase I and II clinical trials, and license the therapeutic to a multinational pharmaceutical company such as Roche, J&J, Pfizer, Merck or GSK. In summary, we will utilize Aphios’ proprietary critical fluid nanosomes (CFN) process for the formation of small, uniform liposomes for the co-encapsulation of Bryoids and HDAC inhibitors [US Patent, Castor, 2014], and take advantage of promising data we have generated from preliminary studies [Perez et al., 2010], to develop responsive protocols for Phase I and II clinical studies in HIV-1 patients being treated with cART and having a suppressed viral load [Gutiérrez et al., 2016]. The outcomes of the proposed studies will inform product development for HIV latency, and fast-track the field to complete elimination of HIV infection and sterilizing cure.

Public Health Relevance Statement:
PROJECT NARRATIVE Currently, over 22 million people have died from AIDS and there are over 42 million people living with HIV/AIDS worldwide. In the United States, an estimated 1 million people are currently living with HIV and approximately 40,000 infections occur each year. There is no vaccine against HIV, and AIDS, if untreated, will lead to the death of over 95% of infected individuals 10 years post-infection. HIV infects several cell types during the course of infection and progression to acquired immune deficiency syndrome (AIDS). The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication with anti-retroviral therapy (ART), since latently infected cells remain a permanent source of viral reactivation. It has been hypothesized that intensification of ART could reduce the residual viremia but recent studies strongly suggest that this is not the likely scenario. Moreover, ART is problematic because of long-term toxicity, drug resistance, and the inability to target and eliminate persistent viral reservoirs. Therefore, other pharmacological approaches targeting the HIV-1 reservoir have been suggested by several investigators as a promising strategy to develop new drugs able to activate latent HIV-1 without inducing global T cell-activation. We propose to develop a unique combination therapy consisting of a PKC modulator and an HDAC inhibitor to reactivate these latent HIV reservoirs so that HIV-1 can be eliminated by ART and eradicated from the patient’s body. Our approach has the potential for advancing HIV therapy towards sterilizing cure, which currently is out of reach.

Project Terms:
Acquired Immunodeficiency Syndrome; Agonist; animal efficacy; Animal Model; Anti-Retroviral Agents; Antibody Binding Sites; aqueous; Biodistribution; Biological; Blood Circulation; bryostatin; burden of illness; CD4 Positive T Lymphocytes; Cell Culture Techniques; Cell Death; cell type; Cells; Cessation of life; Chemicals; Chronic Disease; Clinical Research; Clinical Trials; Combined Modality Therapy; Communicable Diseases; Cyclic GMP; cytotoxicity; Data; design; Developed Countries; Developing Countries; Disease; Drug Combinations; Drug resistance; Economic Burden; Encapsulated; Engineering; Evaluation; experience; Generations; Genetic Transcription; Goals; HDAC4 gene; Head; Histone Deacetylase Inhibitor; HIV; HIV Infections; HIV therapy; HIV-1; Hospitals; Human; humanized mouse; immune clearance; immune function; Immune system; Immunologics; Immunologist; Immunotherapy; in vivo; Inbred BALB C Mice; Individual; Infection; Institutes; Latent Virus; Lead; Legal patent; Licensing; Life; Liposomes; Liquid substance; Lytic; Macaca; Maryland; Medical; medical schools; Medicine; meetings; Membrane Lipids; Modeling; mouse model; multidisciplinary; nanobodies; Neoadjuvant Therapy; neutralizing antibody; novel therapeutics; Outcome; Patients; PDCD1LG1 gene; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Phase I Clinical Trials; preclinical study; Principal Investigator; PRKCA gene; Process; product development; professor; Program Development; programs; Protein Kinase C; Protocols documentation; purge; reactivation from latency; Research; research and development; Research Personnel; residence; Residual state; Savings; simian human immunodeficiency virus; Source; Spain; systemic toxicity; T-Cell Activation; Therapeutic; Thinness; Time; Toxic effect; Track and Field; treatment effect; United States; Universities; Vaccines; Viral; Viral Load result; Viral reservoir; Viremia; virology; Virus; Virus Latency; Vorinostat