Marco Pravetoni, Ph.D. - US grants

DESCRIPTION (provided by applicant): Drug addiction vaccines show encouraging efficacy in pre-clinical studies, yet translation to the clinic has been slowed by the variability of serum antibody concentrations reported in immunized subjects. Establishing early markers of vaccine immunogenicity would help predict efficacy, accelerate screening of vaccine designs, and individualize vaccine design selection in clinical settings. The overall hypothesis of this study i that clinically significant responses to drug addiction vaccines (drug hapten-protein conjugate) can be predicted from the number of hapten-specific B cells in naive (unimmunized) and immunized subjects. Specifically oxycodone (OXY)-specific B cells will be compared between different OXY vaccines and will correlate with OXY-specific serum antibodies and OXY distribution before and after immunization with OXY vaccines in rodents. A recently developed strategy detects rare antigen-specific B cells in naive and immunized subjects. Antigen-specific B cells are selected from the total B cell repertoire present in an unimmunized or immunized host by a fluorescent antigen-based enrichment method and then characterized by flow cytometry. This method can be adapted to drug conjugate vaccines by conjugating drug haptens to a fluorescent carrier protein used to detect hapten-specific B cells. Our lab has developed two OXY vaccines showing a range of effects in reducing brain distribution and behavioral effects of OXY, one of the most commonly abused prescription opioids. These vaccines will be used to test if OXY-specific B cells can be used to explain variability between structurally different vaccines and individual variability between subjects after immunization. We will test the hypotheses that: 1) the numbers of pre- and post-immunization OXY-specific B cells will be higher for the more efficient OXY vaccine in both mice and rats 2) the numbers of pre- and post- immunization OXY-specific B cells will correlate with OXY-specific serum antibody titers and with the effect of immunization on OXY distribution to the brain in rats. This will be tested by: aim 1A) characterizing the time course of OXY-specific B cell and OXY-specific antibody responses to two OXY vaccines in mice to determine B cell responses; aim 1B) assessing the same method in a second species (rats); aim 2A) correlating pre- immunization na¿ve OXY-specific B cells to OXY-specific antibody titers and their effects on OXY distribution to the brain in rats, and aim 2B) correlating early post- immunization activated OXY-specific B cells to OXY- specific serum antibody titers and their effects on OXY distribution to brain in rats PUBLIC HEALTH SIGNIFICANCE. Prescription drug abuse is the fastest-growing drug problem in the US, with oxycodone and oxymorphone among the most abused. Drug addiction vaccines offer a promising advantage to current pharmacotherapies to treat dependence. Early screening of immunogenicity will enable individualized treatment by matching the vaccine to patient and rapid treatment adjustment. This approach could be expanded to other drug-conjugate vaccines.

? DESCRIPTION (provided by applicant): The aim of this application is to enable the submission of IND applications for Oxy(Gly)4-dKLH, a vaccine directed against oxycodone and related prescription opioids, and M(Gly)4-dKLH, a vaccine directed against heroin and morphine. The long-term goal of this work (not included in this proposal but planned to immediately follow) is an IND submission for the co-administration of these two vaccines to treat abuse or addiction from a broad range of prescription or illicit opioids. Opioid addiction and associated deaths have increased markedly in the last 15 years due to increased abuse of prescription opioids and more recently heroin as well. Medications to treat opioid abuse are available but, because of real or perceived limitations, less than 1 in 5 who could benefit opt to use them. Opioid vaccines represent an additional treatment option that could circumvent these limitations. In contrast to agonist medications (methadone, buprenorphine), vaccines are not addictive, pose no diversion risk, and have proven free of significant side effects. In contrast to naltrexone, vaccines don't cause dysphoria, won't preclude the use of certain opioids for legitimate medical needs such as surgery, and would have a longer duration of action than even depot naltrexone. For both agonist and antagonist maintenance therapies, many addicts choose to skip or stop their medication entirely at some point and relapse rates are quite high. Vaccines could be used in addition to these medications and offer protection against relapse during medication noncompliance or discontinuation. Heroin vaccines elicit high titers of drug-specific antibodies in animals that can block a wide range of addiction- relevant behaviors including heroin self-administration and reinstatement. We have developed one such vaccine M(Gly)4-dKLH consisting of morphine conjugated to keyhole limpet hemocyanin subunit dimer (dKLH), a highly immunogenic carrier protein that is suitable for human use. We have also developed an analogous oxycodone vaccine Oxy(Gly)4-dKLH that blocks the effects of the most commonly abused prescription opioids: oxycodone, hydrocodone, and oxymorphone. When co-administered to rats, these two vaccines provide blockade of a wide variety of abused opioids. Specific Aims of the project are Aim 1: Optimization of vaccine formulation and dosing, and confirmation of key aspects of efficacy and safety of the vaccines in animal models, Aim 2: Optimize manufacturing processes, qualify product release assay methods for intermediates and final products, and transfer methods to Contract Manufacturing Organizations, Aim 3: Toxicology testing of cGMP vaccines, and Aim 4: Submission of IND application for Oxy(Gly)4-dKLH and completion of studies to support an IND application for M(Gly)4-dKLH. The main hypothesis of this project is that the M(Gly)4-dKLH and Oxy(Gly)4-dKLH vaccines can be manufactured in formulations suitable for human use, with preservation or enhancement of their efficacy over that previously demonstrated, and no toxicity.

? DESCRIPTION (provided by applicant):This Strategic Alliance seeks funding to develop an immunotherapeutic intervention for prescription opioid abuse that combines a lead oxycodone vaccine with immunomodulators formulated through a novel thermosensitive gelling polymer technology to enhance the post-vaccination antibody (Ab) response. Vaccines for substance use disorders (SUD) have the potential to be a long-lasting, safe, cheap, and effective intervention. Vaccines for SUD stimulate the patient's own immune system to produce anti-drug Ab that prevent drug distribution to the brain and drug-induced behavior. First-generation SUD vaccines did not meet expectations, yet demonstrated proof-of-principle in the subset of immunized subjects that achieved high levels of anti-drug serum Ab. Generation of Ab results from T cell-dependent B cell differentiation in germinal centers (GC) in secondary lymphoid organs. GC-dependent antigen-specific B and T cell differentiation is regulated by specific signaling pathways controlled by cytokines (e.g., interleukins), co- stimulatory molecules, and immune checkpoints. A wealth of pre-clinical studies have tested whether the efficacy of SUD vaccines can be improved by hapten and bioconjugation chemistry, adjuvant, peptides and protein carriers, liposome-protein nanoparticle, or polymer-based nanoparticles. In contrast, no studies have tested the broad range of available immunomodulators, which have revolutionized immunotherapy for cancer and autoimmune diseases. Using the model vaccine 6OXY-KLH, our group has identified immunological mechanisms underlying post-vaccination Ab and vaccine efficacy, and we are currently exploiting these targets for developing more effective SUD vaccines. In a screening study to identify potential lead immunomodulators, we found that co-administration of 6OXY-KLH with IL-4, or a monoclonal Ab (mAb) that blocks IL-2 signaling, increased efficacy against oxycodone. To effectively combine the 6OXY-KLH with IL-4 and/or other immunomodulators in a suitable injectable formulation, we have partnered with i-novion, a start-up company specialized in polymer technology, which has exclusive ownership of novel pentablock co-polymers (PBC). AIM1 will test whether combination of 6OXY-KLH and selected immunomodulators improves post-vaccination anti-oxycodone Ab, 6OXY-specific B cells, and vaccine efficacy in mice. AIM2 will test whether PBC-assisted delivery improves efficacy of 6OXY-KLH and leads from AIM1. AIM3 will test the efficacy of leads from AIM1 and AIM2, in blocking oxycodone-induced striatal dopamine release and conditioned place preference in mice, and oxycodone i.v. self-administration in rats. Completion of these aims will generate an immunotherapeutic intervention for prescription opioid abuse, ready for cGMP production and IND enabling studies. Completion of this project will provide data supporting a novel mechanism-based strategy for enhancing post-vaccination Ab responses, which could be applied to vaccines for other SUD or challenging diseases such as HIV, malaria, or cancer.

Project Summary Currently, abuse of prescription opioid analgesics and heroin is a pervasive problem in the U.S. Although several medications, including methadone, buprenorphine, and naltrexone, are available and effective in treating opioid use disorder (OUD), long-term relapse rates remain high. The current studies are designed to examine a novel approach to treating OUD, namely use of a vaccine (OXY-KLH) targeted against oxycodone, one of the most commonly abused prescription opioids, and a vaccine (M-KLH) targeted against heroin/morphine. Ultimately, a bivalent vaccine (OXY-KLH and M-KLH) will be developed that will target oxycodone and heroin. The long-term goal of this research is to develop a multivalent vaccine directed against oxycodone, heroin, and other relevant opioids. However, during our pre-IND meeting, the FDA/CBER asked that we study each vaccine separately before moving forward with development of the bivalent vaccine. The current application proposes a Phase 1a/1b study designed to evaluate the safety (Aim 1), immunogenicity (Aim 2), and preliminary efficacy (Aim 3) of OXY-KLH during the UG3 phase of the grant period (Study 1) and a similar Phase 1a/1b study of M-KLH during the UH3 phase of the grant period (Study 2). Transition to the UH3 phase will be determined after safety, immunogenicity, and preliminary efficacy analyses have been performed for the OXY-KLH vaccine. Overall, the proposed studies will provide a great deal of information about the safety and potential efficacy of the vaccines in reducing the abuse liability of opioids, which will be administered in a controlled laboratory setting. If the outcomes of the proposed studies with OXY-KLH and M- KLH are favorable, we will proceed with development of the bivalent vaccine (OXY-KLH plus M-KLH).

Project Summary Opioid use disorders (OUD) are a national public health emergency with more than 115 fatal overdoses occurring each day in the USA. Annually, the economic burden of OUD is over US$78 billion. Several medications are available for treating OUD, but their access is limited and efficacy is often sub-optimal. It is thus urgent to develop new and affordable strategies to treat OUD. Immunopharmacotherapy has emerged as a promising treatment approach against OUD. In contrast to traditional pharmacotherapies involving pharmacological agonists and antagonists of the opioid receptors, immunopharmacotherapy relies on drug specific antibodies to bind the circulating drug molecules to reduce their distribution to the brain, and thus reducing opioid-induced behaviors and toxicity. Vaccination is likely the safer and more cost-effective immunopharmacotherapeutic intervention, due to the ability of vaccines to trigger innate and adaptive immune responses in patients to offer long lasting protection against OUD. Due to their selectivity, vaccines are not expected to interfere with endogenous opioids nor with opioids used in pain management or treatment of OUD. Furthermore, it is possible to combine vaccines with current medications for more effective OUD treatment because of the different mechanism of action. Current anti-opioid vaccine candidates are primarily conjugate vaccines (opioid hapten-carrier protein conjugates) delivered in adjuvants for immune recognition. Although these conjugate vaccines have shown promising pre- clinical efficacy and selectivity against OUD, it is critical to test novel immunization platforms that may further improve vaccine efficacy against OUD. Hence, the goal of this project is to fabricate novel nanoparticle-based vaccines against OUD, which will likely lead to an effective immune response against the target opioid by offering these unique features: 1) efficient presentation of B cell and T cell epitopes, 2) improved uptake of vaccine particles by immune cells, and 3) incorporation of molecular adjuvants to promote a synergistic activation of adaptive immune pathways. The innovation of this project involves merging Dr. Zhang's uniquely structured lipid- polymer nanocarriers with Dr. Pravetoni's well-established opioid-based hapten series and pre-clinical platform to identify vaccine candidates. Development will be staggered across UG3/UH3 phases, and we expect to identify lead formulations of nanovaccines that offer protection against either oxycodone, fentanyl, or both at once. The broad impact of this project resides in the rational design of nanoparticle-based vaccines that are safe and effective against opioids. This novel nanoparticle-based immunization strategy can be applied to the development of next-generation vaccines against a range of OUD and other substance use disorders.

ABSTRACT. This UG3/UH3 project will develop vaccines against fentanyl and fentanyl-like compounds as a strategy to reduce illicit opioid use and the incidence of fatal overdoses. Proposed activities will include lead vaccine selection and optimization, manufacturing of GMP vaccines, and IND-enabling GLP toxicology studies. The US has seen dramatic increases in fatal overdoses due to heroin, counterfeit prescription drugs, and cocaine adulterated with fentanyl or fentanyl-like analogs. Current medications may not be sufficient to address the opioid overdose epidemic. As a complementary strategy to current medications, we will develop vaccines against fentanyl and fentanyl-like compounds to reduce their abuse liability and lethality. Our team has already developed vaccines against heroin and oxycodone that induce antibodies effective in reducing opioid distribution to the brain, opioid-induced behaviors, and opioid-induced respiratory depression. Vaccines effectively and selectively target the intended opioid but do not interfere with endogenous opioids nor with approved pharmacotherapies. Opioid vaccines offer a long-lasting, safe and cost-effective intervention complementary to medication assisted treatment (MAT). Vaccines may reduce overdoses in opioid users as well as protect those in professions (e.g., law enforcement, airport security, postal workers) at risk of accidental exposure to fentanyl and fentanyl analogs. Our team has identified a candidate fentanyl vaccine consisting of a fentanyl-based hapten (F0) conjugated to the keyhole limpet hemocyanin (KLH) carrier protein, and adsorbed onto alum adjuvant. Immunization with F0-KLH reduces fentanyl distribution to the brain, and selectively reduces fentanyl-induced antinociception and respiratory depression in rats. We will further optimize the lead F0-KLH by testing alternative carrier proteins. In parallel we will develop other conjugate vaccines containing a new series of haptens (Fn) to target carfentanil, remifentanil, and other fentanyl analogs. Development will be staggered across UG3/UH3 phases, and we expect that the first lead F0-carrier conjugate vaccine will be ready for IND filing by the end of Year 3. AIM1 focuses on optimization of vaccines containing the lead F0 and new Fn haptens conjugated to industry-standard carrier proteins, including two E. coli-expressed carriers obtained from our industry partner FinaBiosolutions. Leads are identified for efficacy in inducing antibodies that will reduce fentanyl (or fentanyl analog) distribution to the brain as well as reducing antinociception, respiratory depression and lethality in rats. AIM1 also characterizes additional immunization regimens and vaccine efficacy in fentanyl self-administration rat models. As a contingency plan, AIM2 tests whether AIM1 leads containing F0 and Fn haptens can be co-administered in a multicomponent vaccine formulation to simultaneously target fentanyl and its analogs. AIM3 focuses on manufacturing of GMP vaccines and evaluate their safety in GLP pre-clinical toxicology studies through CMO and CRO partners. AIM3 will then file an IND for at least one vaccine formulation against fentanyl and/or its analogs.

ABSTRACT. This project will develop monoclonal antibodies (mAb) against toxicity and lethality from accidental or deliberate exposure to fentanyl, carfentanil, acetylfentanil, and alfentanil. The US has seen dramatic increases in fatal opioid poisoning due to the widespread availability of fentanyl and fentanyl-like analogs. Because of the risk for accidental or deliberate mass casualty incidents, the fentanyl chemical family is listed in the DHS Chemical Threat Risk Assessment list. As a complementary adjuvant strategy to current medications for rescue of opioid poisoning (e.g., the opioid receptor antagonist naloxone), our team is developing mAb against fentanyl and its potent analogs to reduce toxicity and lethality in both civilian and defense applications. Our team has shown that antibody-based strategies effectively reduce opioid distribution to the brain as well as opioid-induced respiratory depression and bradycardia in mice and rats. Antibodies selectively target the intended opioid, but do not interfere with endogenous opioids nor with naloxone or other approved medications. Our team has identified lead first-generation mouse anti-fentanyl mAbs using an antigen-based enrichment strategy to isolate opioid-specific B cell lymphocytes from immunized mice to generate hybridomas, and mouse/human chimeric mAb for expression in a suitable mammalian system. The proposed U01 project will further optimize these leads through an iterative developmental plan. AIM1 focuses on optimization of humanized mAb against fentanyl, carfentanil, acetylfentanyl, and alfentanil by integrating humanization of lead mouse mAb and immunization of humanized or transgenic mice. The lead mAb will be used as a benchmark for selection of next-generation candidates. Leads will be identified for their affinity and selectivity in vitro, and for in vivo efficacy in reducing antinociception, respiratory depression and lethality in rats. The humanization process will be guided by biophysical characterization of mAb binding to fentanyl and its analogs by X-ray crystallography. AIM2 focuses on optimization of lead mAb (Fab or F(ab)2 fragments as alternative options) via polymer-based modifications to improve half-life and volume of distribution, and increase efficacy against fentanyl and its analogs. AIM3 will test efficacy of leads co-administered as a multicomponent mAb formulation in combination with opioid antagonists to provide enhanced protection against respiratory depression and lethality in rats challenged with fentanyl and its derivatives. To provide proof of scalability, AIM4 tests the efficacy of the lead humanized mAb in a non-human primate model of opioid-induced respiratory depression. Leads from previous aims will be synthesized at a larger scale at a contractor site. Finally, AIM5 focuses on drafting a regulatory path toward late-stage development and commercialization.