Maurizio Zanetti - US grants

This research intends to investigate at the immunochemical, genetic, structural and functional level the variable (V) region of self-reactive antibodies bearing a defined idiotype proven to be immunoregulatory. It is based on the hypothesis that self recognition and autoimmunity are physiological events in the immune system -- autoimmune network -- and V regions of some self-reactive antibodies are pivotal immunoregulatory elements. This study will utilize, as a model system, a putative germline idiotype (Id62) borne independently on the heavy and light chains of murine monoclonal autoantibodies from either adult or neonatal mice. Two major issues will be addressed. The genetic relatedness and the structural correlate for a number of adult and neonatal Id62-positive autoantibodies. The functional impact such V regions may have on ontogenic development and reactivity of a defined autoimmune network. The specific aims to this research are as follows: 1) Detection and Immunochemical Analysis of Id62-Positive Antibodies within a large pool of hybridoma self-reactive antibodies. 2) Molecular Genetic Analysis of all Id62-positive V regions and selection by specific VH gene probing of putative Id62-positive antibodies. 3) Amino Acid Sequence Analysis of V regions identified and retained through the preceding two approaches and comparisons with prototype sequence. 4) Functional studies of the autoimmune network based on perturbation of the neonatal repertoire by Id62 or anti-ID62 antibodies. 5) Functional studies of the autoimmune network based on immunization of adult mice with Id62 or anti-Id62 antibodies, including anti-single chain or anti-peptide antibodies. It is hoped that the approach will help considerably to elucidate the contribution that inheritable genetic elements and functional network interactions may have on the ability of the immune system to respond to self components. Undoubtedly, new light will be shed onto the fundamental laws of regulation of autoimmunity and invaluable new knowledge will be generated in understanding autoimmune diseases in general. Ultimately, this study may constitute the basis for rational therapeutic approaches to immunologically-mediated disease process.

Neonates born from human immunodeficiency virus (HIV)-infected mothers are currently identified as a high risk group in Western and Third World countries. It is the aim of this proposal to engineer antibody molecules that can be used for passive immunotherapy to prevent neonatal HIV infection. This project is based on a) the notion that the interaction between the CD4 receptor/molecule on T lymphocytes and HIV is a central feature in HIV infection, and b) a plausible prediction that interference with this interaction could guarantee protection. Protein engineering techniques will be used to construct antibodies that possess the HIV-binding domain of the CD4 receptor/molecule on immune cells, receptor (CD4)-like antibodies. This goal will be pursued by inserting discrete CD4 peptides from the putative HIV-binding site in the form of synthetic oligonucleotides into the immunoglobulin heavy- or light-chain gene, or both genes. These constructs will be expressed in mammalian cells by electroporation. The obtained CD4-like antibodies will be tested in vitro, immunochemically and biologically, for a) expression of the engineered HIV-binding domain, b) binding to HIV, and c) inhibition of viral infection of susceptible cells. The work proposed may constitute the basis for a new rational strategy to passive immunotherapy in the prevention of neonatal HIV infection and its consequences.

This Program Project will investigate the in vivo regulation of the immune response including peripheral T cell tolerance and immune memory, and strategies for the control of autoimmune diseases by induction of anti-TcR immunity. Project 1 "Mechanisms of Peripheral Tolerance and Resistance to Toxic Shock" studies the in vivo response to bacterial superantigen in mice transgenic for a single TcR to elucidate clonal deletion and anergy with respect to: 1) susceptibility of T cell subsets, 2) antigen presenting requirements, and 3) lymphokines involved. The investigations will include experiments on a fatal bacterial superantigen-induced toxic shock syndrome. Project 2 "Immunoregulation at the Maternal-fetal Interface" analyzes pregnancy in transgenic mice engineered to express class I MHC antigens on the trophoblast as a model to investigate tolerance and autoimmunity and their regulation in peripheral organs. Project 3 "Immunomodulation in alphaB Transgenic Chimeric Mice" examines the immune response of mice transgenic for a single TcR before and after vaccination with T cells clones or oligopeptides matched to sequences found in various portions of the alpha- and B- chain of the transgenic receptor. The overall goal is to understand how active anti-TcR vaccination works. Project 4 "Vaccination to TcR structures and autoimmune diseases" proposes a new strategy, antigenized antibodies (AgAbs), to induce active anti-TcR immunity in the prevention and/or treatment of two autoimmune diseases, experimental allergic encephalomyelitis and myasthenia gravis. The levels of specific humoral and cellular immunity will be correlated with the in vivo effects of anti-TcR vaccination by AgAbs. Project 5 "Strategies for Inducing Helper T Cell Memory" studies regulation of immunologic memory, the rapid induction of a quantitatively effective immune response following reexposure to antigen. In vivo and in vitro models will be used to study the development, recirculation, and function of memory TH cells. The goal is to elucidate the requirements for the activation of memory versus naive TH cells, and to devise a methodology for inducing and manipulating T cell memory for the purpose of vaccination.

The overall goal of this revised application is to test immunogens and strategies of new conception for vaccination against MUC-1. MUC-1, a highly glycosylated mucin expressed on the luminal side of glandular epithelial, consists of 30-90 tandem repeats of 20 amino acids within which there exists an immunodominant epitope. During malignant transformation mucin is over-expressed, and mucin-producing cells lose luminal polarity with mucin becoming expressed all around the cell surface rendering tumor cells less susceptible to immune effector mechanisms and favoring apoptosis of T cells. Vaccination against MUC-1 is intended at generating two effects: (1) antibodies of sufficient specificity and avidity could cause redistribution (via cross-linking and capping) of the MUC-1 antigen at the surface of tumor cells hence reinstating the full immunological potential of the host against tumor cells expressing MUC-1; (2) T cells with cytotoxic activity that kill mucin-expressing tumor cells, particularly micro-metastasis. The vaccination experiments proposed herein are based on methods and principled developed in the laboratory and designed at maximizing the immunogenicity of MUC-1 oligopeptides by expressing them in the hyper-variable loops of antibodies, i.e., structures with limited flexibility anchored into the conserved immunoglobulin fold. This approach allows for their expression as conformationally-constrained units, a necessary prerequisite for the induction of biologically active B cell responses. Heterologous epitopes expressed in hyper-variable loops of antibodies are also site of preferential intracellular cleavage so that peptides for the activation of T cells are easily generated. Combined with the advantages of this pro protein engineering approach will be the use of a newly-developed method of DNA-based vaccination (Somatic Transgene Immunization), a method through which the genes of antibodies coding for MUC-1 epitopes are themselves utilized in vivo as vaccines to induce both antibodies and T cells. Studies will be done in normal C57B16 mice, in human MUC-1 transgenic mice to evaluate the limitations (if any) proposed by self tolerance, and in mice knock-out for the alpha (1->3) galactosyl transferase to determine if naturally-occurring antibodies (which are present in these KO mice) limit the extent to which specific cellular responses can be induced by vaccination. The efficacy of this type of vaccination will be evaluated in experiments of tumor protection (prevention or down-regulation of growth) in normal as well as transgenic mice.

DESCRIPTION: (Applicant's Abstract) The general idea of this revised application is to develop a prostate cancer vaccine targeted to the reverse transcriptase of telomerase (TRT), a unique ribonucleoprotein that mediates RNA-dependent synthesis of telomeric DNA. During the past year, the applicant has obtained compelling evidence that this idea is valid and worth pursuing. The main goals are to demonstrate that peptides of human telomerase are immunogenic 1) in vitro for lymphocytes of prostate cancer patients, and 2) in vivo in mice transgenic for the human HLA-A2.1 molecule and in mice bearing a model prostate cancer. These objectives will be pursued in four aims. (1) Identification and testing HLA-A2.1 restricted human (h)TRT peptides. (2) Optimization of peptide immunogenicity in vitro of fusion (hTRT) peptides with natural or artificial signal sequences and targeted point mutations to increase the avidity of MHC binding. (3) Induction of CTL in prostate cancer patients, in which the applicant will assess whether exposure to cancer modifies the available peripheral repertoire (e.g., by tolerance or clonal anergy) and diminishes the precursor frequency for hTRT peptides and their ability to undergo expansion upon immunization. The applicant will study HLA-A2.1+ individuals with clinical/histological diagnosis of prostate cancer. And (4) In vivo immunogenicity of telomerase peptides using two transgenic mouse models, one for the human HLA-A2.1 molecule and the other for the simian virus (SV) 40 large T antigen where SV40 Tag is responsible for prostate cancer. Using the first transgenic model he will test the ability of hTRT peptides to induce HLA-A2.1-restricted CTL responses. Using the second transgenic model, and using murine TRT peptides, he will answer the question "Does presence of prostate tumor affect immunization against telomerase peptides?"

DESCRIPTION: (Adapted from the Applicant's Abstract): In this Innovation Grant we propose a new vaccine approach to induce anti-virus CTL. The new approach is based on a new concept, the use of transgenic lymphocytes to program the immune response as the substrate in which endogenous synthesis of antigen and antigen presenting function are ideally combined. In pilot experiments we have obtained initial evidence that transgenic lymphocyte immunization lends itself to a robust CTL response with a single injection of just 103 cells. This application intends to expand on this exciting observation. Our goal is to determine that B lymphocytes are the pivot driving immunization, and understand the biology of the system in relation to protection in vivo. We will consider parameters such as the extent 01 lyses prior and after in vitro culture with peptide, the enumeration of effector CTL by tetramer staining, and the affinity of their receptor for the antigen/MHC complex. These parameters will be assessed during priming and after in vivo challenge with virus. Finally, CTL responses will be induced with and without Th cell help. Central to our studies will be a comparison between our new approach with some of the approaches currently used to induce CTL, in particular peptide-pulsed dendritic cell vaccines. Once proof of principle is obtained and results will show that transgenic lymphocyte immunization is an effective method to induce protective anti-viral CTL responses, cost effectiveness considerations will make transgenic lymphocyte immunization a very desirable new approach for vaccination against HIV infection.

[unreadable] DESCRIPTION (provided by the applicant): There are still few reliable and simple methods of vaccination in which the resulting immunity is operationally programmable, restricted to possibly one cell type functioning as antigen producing and antigen presenting cell, and endowed with self-amplifying/self-renewing capacity. This type of vaccine could be of extreme value against cancer.For the past years this laboratory has worked towards developing such a new approach. We established that an adult immunocompetent animal can readily be immunized with a single injection of syngeneic lymphocytes made transgenic in vitro for immunoglobulin (Ig) genes controlled by a B cell specific promoter for targeted expression in B lymphocytes. The Ig genes are themselves engineered to code for heterologous epitopes to confer antigen specificity. Since B lymphocytes are excellent Ig producers and present Ig peptides to CD4 and CD8 T cells very efficiently, transgenic lymphocytes are a novel form of cell vaccine in which endogenous synthesis of antigen and antigen presenting cell (APC) function are ideally combined. We term this process Transgenic Lymphocyte Immunization (TLI). Our preliminary data indicate that these events occur with high fidelity and at high immunological efficiency. TLI lends itself to a robust antigen-specific CD4 and CTL responses with a single injection of as few as 70 transgenic cells. New data provided in the revised application also demonstrate that TLI generates T cell immunity against sub-immungenic epitopes and that the ensuing immunity is highly effective (>85 percent) in tumor protection. [unreadable] [unreadable] The work proposed is built on the hypothesis that B lymphocytes are key to TLI. It is our goal to: (1) elucidate the basic mechanisms of TLI for the induction of antigen-specific CD4 and CD8 responses; (2) compare TLI to vaccination with dendritic cells (DC); and (3) test TLI ability to protect in animal models of tumor. [unreadable] [unreadable] Current vaccines need to be improved with respect to their ability to generate the type of immunity needed to (a) enhance natural defenses or train the immune system to develop new ones, and (b) keep the balance between the tumor growth kinetics and the host immune response in favor of the immune response. We believe that TLI is a simple and effective form of APC vaccine that can meet this need and objectives.

DESCRIPTION (provided by applicant): Bacillus anthracis, the agent that causes anthrax, has several characteristics that make it a formidable bioterrorist threat. Presently efforts to develop an effective anthrax vaccine can be categorized into the following groups: (1) Protein vaccines; (2) Live attenuated vaccines; (3) DNA and replicon vaccines: and (4) Identification of new antigens. The steps in host-cell intoxication have been recently clarified. An 83-kDa form of protective antigen (PA83) is secreted from rapidly growing B. Anthracis cells and binds via a 19aa solvent-exposed loop in domain 4 between strand 4A-4B of the protective antigen (PA) to a specific host cell surface receptor termed ATR. X-ray crystallography studies characterized this loop as having the structure of the complementarity-determining region (CDR) of an immunoglobulin (Ig). It is the goal of this application to develop and test as a proof-of-principle a series of Ig molecules expressing a conformationally-constrained 4B9-4B10 PA loop, antigenized antibodies. Furthermore, we will ascertain whether immunization with antibodies antigenized to express 4B9-4B 10 PA loop will result in the induction of site specific antibodies (i.e., directed at the 4B9-4B 10 PA loop) also able to neutralize the internalization of B. anthracis toxin and its cytopathicity. It is hoped that the idea and experiments proposed in this application can speed up the development of a safe and effective method to vaccinate against B. anthracis.

Under the tenure of grant RO1-AI062894 our laboratory developed a new concept and vaccine approach to induce protective CD8 T cell responses against viruses using influenza A virus as model system. The new cell-based vaccine is a unique form of immunization best described as transgenic antigen-presenting cells (APCs) vaccine. Using this system we established that central memory CD8 T cells mediate protection against disease caused by influenza virus. In this revised competing renewal application we will capitalize on the body of information generated in past years and ask new questions designed to better understand the requirements for the induction of central memory CD8 T cells in vivo by vaccination. Three aims are directed at this question. In AIM 1, Direct vs. cross-priming memory CD8 T cell induction by transgenic APCs, using different vaccination modalities we will investigate and distinguish the role of direct presentation, cross-priming by an [unreadable]APC feeding the APC[unreadable] cross-priming mechanism, and crosspriming by antigen synthesized and secreted by the APC, on the generation of central memory CD8 T cells. In Aim 2, Reactivation of TCM cells: Correlation with the modality of vaccination, we will analyze the expansion of already established memory T cells to understand which mechanisms is best in the reactivation of resting memory T cells. In Aim 3, MicroRNAs manipulation to facilitate central memory T cell fate determination, we will investigate a new approach to program from the outset central memory CD8 T cell fate: microRNAs modulation. This revised competing renewal application has been refocused on two main areas with one main common goal: test vaccines and vaccination strategies to selectively generate and expand anti-virus central memory CD8 T cells. It is expected that these studies will broaden our understanding on the principles for optimal generation of protective anti-virus CD8 T cell responses by cell-based vaccination. The experimenst may lead to the development of new safe vaccines against influenza A virus and viruses in general.

[unreadable] DESCRIPTION (provided by applicant): This second revision of a competitive renewal application is based on an in-depth analysis of the mechanism(s) of tumor protection in tolerant mice where protection is initiated by CD4 T cell immunity against MUC.1. At the present time there are no reports showing that CD4 T cell tolerance against MUC.1 can be broken by specific vaccination, nor that CD4 T cell immunity generates tumor protection. Therefore, our model is new. Our studies are based on the induction of CD4 T cell responses against a sub-immunogenic T cell determinant of MUC,1 that exploits a new method to "jump-start" weak Th cell responses, Th-Th cooperation. The method of immunization is also new lymphocytes transgenic for a gene engineered with the two Th cell determinants involved in Th-Th cooperation. Working in MUC.1 transgenic mice during the past two and half years we have been able to show that Th-Th cooperation combined with transgenic lymphocyte immunization (TLI) effectively breaks self tolerance and induce protective immunity. In the experiments proposed in this competitive renewal our goal is to elucidate and understand the mechanism(s) of tumor protection in vivo. Specifically, we will revisit the role of MUC.1 reactive CD4 T cell and assess a potential contribution of CD8 T cells (Aim 1), including the role of central memory and effective memory CD4 and CD8 T cells (Aim 2). In a systematic step-wise approach, we will also study the contribution of bone marrow-derived dendritic cells (DC) and plamacytoid DC (pDC) (Aim 3), and see if activation of NK and NKT cells is part of the overall mechanism of protection (Aim 4). It is hoped that these studies will shed light on the relationship between immunity and protection, and will establish the ground rules for successful intervention against cancer expressing MUC.1 in humans. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This laboratory recently discovered that primary B-lymphocytes can be programmed for the synthesis and delivery of short, non-coding RNAs in vitro and in vivo. In this revised R21 application we intend to rapidly adapt this new concept and test it in the context of tumor therapy to limit or control cancer cell growth and metastasis. miRNA signatures have been identified in both hematological malignancies and solid tumors, distinguishing tumor cells from normal cells. In some instances miRNAs are associated with the prognosis and the progression of cancer. This can happen because of one of two conditions: loss of suppressor miRNAs or overexpression of oncogenic miRNAs. miRNA-based therapy can then be geared at either restore the loss of a particular miRNA or suppress an oncogenic miRNA, respectively. The present proposal aims at gathering first line proof-of-concept that primary B lymphocytes synthesizing and secreting short, non-coding miRNAs can be used to treat cancer in vivo, a new form of therapy that we have termed immunogenomic therapy to typify its hybrid (genomic and immunological) nature. The following three Aims are proposed: (1) Engineer and characterize therapeutic plasmid vectors for miRNA replacement or inhibition; (2) miRNA inhibition: Targeting miR-155 in B leukemic cells; and (3) miRNA replacement: Restoring miR-335 in metastatic breast cancer cells. It is hoped that the work proposed in this high risk/high pay off revised proposal will provide us with the answers needed for a proof-of-principle validation of the new idea. We anticipate that success in obtaining proof-of-concept validation for immunogenomic therapy will open new horizons for the treatment of cancer and metastases.

DESCRIPTION (provided by applicant): Glioblastoma (GBM) is an intractable disease with high mortality. In the U.S, the incidence of GBM is approximately 9,600 new cases/year. Even with current standard-of-care, surgical debulking followed by concurrent radiotherapy and chemotherapy with temozolomide, survival remains modest. There is a clear, major unmet medical need for novel approaches to treat patients with GBM. The goal of this four-year application is to evaluate the innovative approach of targeted prodrug therapy for GBM. In this investigator-initiated Phase 2 clinical study, the applicant will test a vascular-targeted prodrug, G-202 in patients with recurrent GBM. G-202 is a thapsigargin analog (12ADT) coupled with a masking/targeting peptide that renders the prodrug inactive and highly soluble (expected to cross blood-brain barrier). This peptide also targets the prodrug to a protein - prostate-specific membrane antigen (PSMA), expressed selectively on the tumor vasculature. On binding PSMA, the prodrug is cleaved and 12ADT is released, which in turn produces endoplasmic reticulum (ER) stress, apoptosis and cell death. The central hypothesis for this proposal is that G-202 will be clinically active against GBM. The preliminary data show that PSMA is selectively and highly expressed in GBM tumor vasculature. Further, patient-derived GBM cell lines are highly sensitive to thapsigargin in vitro. Studies on mouse xenograft models show renal, breast and prostate cancers to be highly sensitive to G-202. The objective of this study is to evaluate the clinical efficacy of G-202 against recurrent GBM and to investigate the effect of 12ADT on preclinical models of GBM. The applicant proposes three specific aims to test the hypothesis and achieve their objective. Aim 1 will determine the efficacy of G-202 in patients with recurrent GBM in a single-arm, open-label, Phase 2 clinical trial. Aim 2 will involve correlative studies - cerebrospinal (CSF) pharmacokinetics to confirm optimal dosing of G-202 and detailed blood/CSF and tissue biomarker analyses in GBM patients treated with G-202. These studies will attempt to identify markers predictive of response to G-202 and will provide guidelines to stratify patients for future trials. Aim 3, using patient-derive GBM cell lines, will seek to identify molecular predictors of responsiveness to 12ADT, the thapsigargin analog in G-202. In preliminary studies, the applicant found a differential sensitiviy of different GBM lines to thapsigargin that correlated with expression levels of GRP78, the master regulator of the ER stress response.

This new R01 application is submitted in response to Funding Opportunity RFA-CA-15-008 ?Research Answers to NCI's Provocative Questions?. The hypothesis to be tested is in response to PQ3. We hypothesize that the benefit afforded by immune surveillance to the individual and populations of cancer patients may depend on the levels of aneuploidy and expression of the Major Histocompatibility Complex (MHC) complex Class II (MHCII) in the tumor. We further predict that a population of individuals defined by tumors with high aneuploidy and low MHCII expression is at higher risk of progression and poor outcome, and unresponsiveness to immunotherapy by regulating the size and composition of intra-tumor immune infiltrates. Within this new paradigm, we also propose to investigate the possible causative mechanism of aneuploidy- induced proteotoxic stress. The study will be a combination of genomic analyses and immunological interrogation in UCSD Moores Cancer Center patients, and will ultimately seek validation in through TCGA data as well as data from immunotherapy trials. We propose four Aims. (1) To validate aneuploidy/MHC II as predictors of progression and clinical outcome. TCGA data will be interrogated to relate an association between aneuploidy and levels of MHCII expression to overall survival. and response in immunotherapy (ICPi) trials. (2) To measure markers of natural immune response in cancer patients grouped according to aneuploidy/MHC II status. We will study the size and clonality of tumor-infiltrating T cells by profiling TCR reactive with TERT, an antigen expressed by cancer cells at every stage of differentiation, as a proxy of the autochthonous anti-cancer response. By comparing tumor-infiltrating T cells to circulating T cells, we expect to determine if (a) tumor the aneuploidy/MHCII signature predicts T cell infiltration, and (b) lower immune T cell infiltration displays high TCR heterogeneity. (3) To determine whether aneuploidy/MHCII status interferes with response to ICPi therapy. We will determine if high aneuploidy/low MHCII expression impairs immune reactivation after ICPi therapy, and use immunoscore from responder and nonresponder patients. (4) To determine whether proteotoxic stress is the mechanism by which aneuploidy interferes with immune response. Because aneuploidy induces proteotoxic stress, the ensuing UPR provides a mechanistic link between cancer cell biology and immune surveillance. In vitro and in vivo experiments using cells with pharmacologically-induced aneuploidy will be used to determine the extent to which UPR cell nonautonomous effects may account for a disruption of local immunity, i.e., number and characteristics of tumor infiltrating myeloid cells. We believe that an analysis at the interface between cancer genomic and immune surveillance may reveal general rules for immune-mediated control of cancer. This may help stratify patients and their clinical trajectory, and predict clinical responses to immunotherapy more accurately. !