Manuel L. Penichet - US grants

DESCRIPTION: (provided by Applicant) My immediate career goal is continue my research in the fields of cancer research focusing mainly in the development of new drugs for cancer therapy. To achieve this goal I propose to develop universal delivery systems that can be used to deliver DNA or cytotoxic molecules to tumor cells expressing on their surface the interleukin-2 (IL-2) or transferrin (Tf) receptor (IL-2R or Tf-R). The delivery system will be antibody fusion proteins that either have the ligand (e.g., IL-2 or Tf) covalently associated or have a variable region that recognizes a growth factor receptor (e.g., anti-TfR). The antibody fusion proteins containing IL- 2 or Tf will be specific for the hapten dansyl (DNS) and will be used for the delivery of dansylated agents. The anti-TfR antibody will be covalently fused to avidin and will be used for the delivery of biotinylated agents. To produce and characterize the delivery vehicles I propose the following specific aims: 1. Produce the recombinant antibody fusion proteins to be used as universal delivery vehicles; 2. Evaluate the ability of the Ab fusion proteins to deliver proteins or DNA to cells growing in vitro; 3. Determine the properties of the Ab fusion proteins in vivo and their ability to target and eliminate tumors in animal models. I anticipate that the vector systems will provide powerful tools for treating a wide variety of important malignancies. The universal vectors will make it easier to treat tumors with multiple anti-cancer agents and will facilitate the rapid evaluation of potential new therapeutic agents. Although initial principles will be established using IL-2R and/or TfR as targets, future universal vectors can target other tumor associated molecules. Having been trained as an M.D./Ph.D., I have a strong grounding in both basic and clinical science. The training that I will receive during the time of this grant will enhance both my research skills and my facility with English. This additional training is necessary if I am to assume an independent position doing research at a major university. UCLA provides an excellent, supportive environment for the proposed research. There is a large faculty with expertise in virtually every discipline. Excellent seminar series and technical training sessions are available. The University is committed to my career development and will provide resources without requiring any teaching or clinical activities. The training period will also allow me to attend national and international meetings, both expanding my expertise and my network of collaborators and colleagues.

DESCRIPTION (provided by applicant): We have developed an antibody fusion protein composed of avidin fused to a human IgG3 specific for the transferrin receptor (TfR). Our initial goal was to use this molecule (anti-TfR IgG3-Av) as a universal vector to deliver biotinylated agents into cancer cells. We have found that anti-TfR IgG3-Av effectively delivers biotinylated molecules into cancer cells by receptor mediated endocytosis and that these molecules remain active after their internalization. Furthermore, we have unexpectedly discovered that anti-TfR IgG3-Av, but not a recombinant anti-TfR IgG3 or a non-specific IgG3-Av, possesses a strong intrinsic antiproliferative/pro-apoptotic activity against hematopoietic malignant cell lines. Importantly, this cytotoxic activity may be further enhanced by the addition of biotinylated compounds. We now hypothesize that anti-TfR IgG3-Av can be used alone or in combination with other agents as a novel drug against an incurable plasma cell malignancy: multiple myeloma (MM). We propose to determine the effect of anti-TfR IgG3-Av on both proliferation and apoptosis of selected human MM cell lines and to define the mechanism responsible for the inhibition of proliferation and induction of apoptosis. We will also examine the additive/synergistic effect of combining anti-TfR IgG3-Av with other cytotoxic/sensitizing agents, such as biotinylated Pseudomonas exotoxin A (PE) and drugs currently used in the treatment of MM such as Thalidomide and Dexamethasone. Based on our findings we will test the efficacy of anti-TfR IgG3-Av alone or combined with other anti-cancer drugs against primary myeloma cells obtained from MM patients and against human MM tumors growing in immunodeficient mice. Thus, the proposed experiments will result in a better understanding of the mechanism of action of anti-TfR IgG3-Av and will indicate if this novel therapeutic has potential for use in the treatment of MM. We anticipate that the utility of this therapeutic will not be restricted to the elimination of myeloma cells in vivo but can also be used for in vitro approaches including the efficient purging of myeloma cells during ex vivo expansion of hematopoietic progenitor-cells for use in autologous transplantation in MM patients. We would like to stress that the impact of the results obtained from the present studies is not restricted to MM. Similar approaches can be applied to other hematopoietic malignancies such as leukemias and lymphomas.

DESCRIPTION (provided by applicant): We propose to develop novel anobioconjugates for the treatment of breast cancer by inhibiting angiogenesis and stimulating the host immune system simultaneously. The nanobioconjugates are based on a biodegradable and non-toxic polymalic acid nanoplatform with covalently attached anti-transferrin receptor (TfR) monoclonal antibody for delivery through the endothelial system of the tumor vasculature, antisense oligonucleotides (AON) to inhibit angiogenesis, and/or potent immunostimulatory antibody-cytokine fusion proteins specific for the breast cancer antigen HER2/neu. We hypothesize that the new nanobiopolymers when used alone or in combination would be capable of a multi-pronged attack against cancer cells by inhibiting tumor angiogenesis with the subsequent induction of apoptosis and by activating innate and adaptive immune responses resulting in long-term anti-tumor immunity not only against HER2/neu but also against other tumor antigens. In addition, the nanobioconjugates would exhibit superior tumor targeting as a consequence of the combination of transcytosis mediated by TfR overexpressed on the endothelial cells of the tumor neovasculature, targeting of HER2/neu or TfR overexpressed by the tumor, and the enhanced permeability and retention (EPR) effect exhibited by nanopolymers. The anti-angiogenic effect would be achieved using AON to inhibit the expression of vascular protein laminin-411 and/or vascular endothelial growth factor (VEGF), critical factors produced by the cancer cells needed to develop new vasculature that supports the tumor. In addition, to achieve HER2/neu targeting and immunoactivation we will use the potent immunostimulatory cytokines interleukin-2 (IL-2) or interleukin-12 (IL-12) that would be delivered into the tumor by an anti-HER2/neu IgG3-cytokine fusion protein as part of the nanobiopolymer. We will also explore the effect of combining free antibody-cytokine fusion proteins with the anti-angiogenic nanopolymer. Importantly, IL-12 is also an anti-angiogenic cytokine increasing the anti-angiogenic effect of AON. The proposed nanobiopolymers represent novel and unique molecules in terms of structure and mechanisms of action. To the best of our knowledge, neither the proposed therapeutic nor other molecules with similar mechanisms of action have either been described or are under development. The specific aims of this project are: Aim 1. Synthesis and in vitro characterization of nanobiopolymers, Aim 2. Initial pharmacokinetic and toxicological studies of the nanobiopolymers, and Aim 3. Examine the ability of nanobiopolymers to inhibit tumor growth and investigate the mechanism responsible for anti-tumor protection and immunological memory. The proposed nanobiopolymers represent the starting point for a new generation of cancer therapeutics and are expected to be effective against aggressive breast cancer tumors such as those overexpressing HER2/neu. A novel nanobiopolymer-based therapy against HER2/neu expressing cancer cells with sufficient capacity to inhibit angiogenesis and orchestrate an anti-tumor immune response should make a significant clinical impact.

DESCRIPTION (provided by applicant): Breast cancer is the most diagnosed malignancy and the second cause of cancer death in women in the United States. The prognosis for breast cancer patients is particularly poor in those bearing tumors that overexpress the oncoprotein HER2/neu. Although, in general, antibodies used against cancer are of the IgG class, antibodies of the IgE class have several properties that make them advantageous for cancer therapy: 1) higher affinity of IgE for its Fc?Rs compared to IgG for its Fc?Rs, 2) expression of Fc?Rs by key cells responsible for antibody effector functions and antigen presentation, 3) low serum levels of endogenous IgE, resulting in less competition for FcR occupancy, and 4) unlike IgG, IgE does not have an inhibitory FcR. The present proposal seeks to develop a novel anti-HER2/neu antibody of the IgE class as a possible therapy of breast cancer. We hypothesize that the proposed anti-HER2/neu IgE will target HER2/neu expressing tumors and, in so doing, create a local IgE-targeted immediate hypersensitivity (anaphylactic) reaction resulting in acute inflammation in the tumor microenvironment and in rapid tumor destruction, with no escape of cancer cells. The dead cells will be taken up by antigen presenting cells (APC), such as dendritic cells, a process that can be enhanced when dead cancer cells are coated by IgE, resulting in an adaptive immune response not only against HER2/neu, but also against other tumor antigens due to epitope spreading. We also hypothesize that immunocomplexes composed of anti-HER2/neu IgE and soluble extracellular domain of HER2/neu (ECDHER2) will target APC, resulting in efficient antigen uptake and presentation with subsequent anti-tumor immune activation. We have already developed a totally human anti-HER2/neu IgE (C6MH3-B1 IgE) that does not compete with trastuzumab (Herceptin(R)) for its antigen-binding site. This novel IgE exhibits the expected biological properties and is capable of triggering immune activation and anti-tumor protection, while being well tolerated in relevant animal models. The goals of this proposal are: 1) Fill the gaps in the knowledge accrued from the previous studies, 2) open new lines of studies, and 3) create the basis to start moving the anti-HER2/neu IgE into the clinic. We have four independent specific aims: Aim 1: Define the interaction of anti-HER2/neu IgE and antigen in the context of effector cell functions and antigen presentation in vitro; Aim 2: Define the anti-tumor activity of anti-HER2/neu IgE in huFc?RI mice; Aim 3: Define the anti-tumor activity of anti-HER2/neu IgE in SCID mice; and Aim 4: Define the toxicity of anti-HER2/neu IgE in huFc?RI mice and cynomolgus monkeys. The present proposal has a mechanistic (basic science) component and a strong translational potential, which can contribute, potentially, to the cure of breast cancer and perhaps other HER2/neu overexpressing malignancies. The project is expected to open a new dimension in the nascent field of AllergoOncology, which aims to reveal the function of IgE-mediated immune responses against cancer and develop novel IgE-based cancer therapies such as the proposed therapeutic.

? DESCRIPTION (provided by applicant): Multiple myeloma (MM) is an incurable B-cell proliferative disorder of malignant plasma cells. MM is an example of a cancer health disparity with one of the highest African American/European American (AA/EA) incidence rate ratios of all cancers. The explanation for this race-related difference needs to be found in the etiology of the MM premalignant condition known as monoclonal gammopathy of undetermined significance (MGUS), since the prevalence of MGUS in AA is much higher than that observed in EA, while the probability for transition from MGUS to MM is the same in both races. We postulate that this health disparity can be explained by differences in the IgE-(mast cell) system, which are the mediators of the allergic immune response. This response is due to the presence of mast cells (MC) in tissue that are sensitized by IgE antibodies, which when bound to a multivalent antigen (allergen) lead to the cross-linking of the high affinity IgE receptor I (FceRI) and degranulation f MC, resulting in the release of histamine and other mediators of the acute inflammatory (allergic) response. However, in addition to this well-known degranulating response, when bound to MC in the absence of antigens (monomeric IgE), IgE activates MC and prolongs their survival without inducing degranulation. Activated MC secrete the cytokine interleukin-6 (IL-6) and express CD40L on their surface, both of which provide strong signals that activate B cells and trigger their differentiation into plasma cells. Since B-cell activation involves the expressio of the DNA mutating enzyme activation-induced cytidine deaminase (AID), this would increase the probability of mutations that lead to abnormal plasma cells (MGUS). It is well known that the total IgE blood level is higher in AA compared to EA, with and without other pathological conditions associated with high IgE levels. Thus, we hypothesize that the higher IgE levels in AA result in a higher incidence of MGUS through enhanced chronic activation of the MC-(B-cell) axis. Interestingly, there are also differences in MC structure and enzymatic content between AA and EA, with unknown functional implications. Therefore, we also hypothesize that MC from AA have stronger stimulatory activity, compared to those from EA, when exposed to the same amount of monomeric IgE, or even without IgE. However, both hypotheses are not mutually exclusive. We have two specific aims: Aim 1: Define the differences in MC in AA and EA in the absence of IgE and Aim 2: Define the differences of MC in AA and EA in the presence of IgE. In addition to using antigen free (monomeric) IgE to monitor its efficacy to activate the MC:B-cell axis, we will also explore the use of a therapeutic IgE targeting an antigen on MM cells to trigger degranulation and anti-tumor activity, a process known as the re-education of MC to fight cancer. This artificially induced event is possible because degranulating MC release pro-apoptotic molecules with anti-tumor effects and should not be confused with that induced by total levels of IgE activating the MC:B-cell axis. Thus, our proposal has relevant implications in both the prevention and therapy of MM.

? DESCRIPTION (provided by applicant): HIV infection-associated B-cell hyperactivation plays a central role in the genesis of AIDS-associated non- Hodgkin lymphoma (AIDS-NHL), as it is associated with molecular processes that lead to the seminal molecular lesions for NHL. In our prior work, we observed evidence for HIV-associated B-cell activation, including elevated numbers of B cells overexpressing transferrin receptor 1 (TfR1), a marker of B-cell activation, for several years preceding NHL diagnosis. We have developed an antibody (ch128.1) and an antibody-avidin fusion protein (ch128.1Av), specific for human TfR1. ch128.1Av shows enhanced in vitro cytotoxicity against human malignant B cells overexpressing TfR1, including multiple myeloma (MM) and NHL cells, when compared to the parental antibody without avidin (ch128.1). Cytotoxicity is due to the ability of ch128.1Av, and to a lesser extent ch128.1, to decrease cell-surface TfR1 leading to lethal iron deprivation. ch128.1Av is also a universal delivery system and its cytotoxicity can be enhanced by conjugating it to biotinylated drugs such as the plant toxin saporin; making it a unique molecule capable of a two-pronged attack against malignant cells. However, ch128.1Av alone, or complexed with biotinylated saporin, is not toxic to normal human hematopoietic stem cells due to their lack of TfR1 expression. In addition, the combination of ch128.1Av with non-biotinylated drugs, such as the HOX protein inhibitor peptide HXR9 or the natural chemical compound gambogic acid, results in an additive or synergistic anti-cancer activity against malignant B cells. Importantly, in two disseminated human MM xenograft mouse models, a single low dose of ch128.1Av alone, and even ch128.1, resulted in significant anti-tumor activity. Our central hypothesis is that the overexpressed TfR1 on circulating activated B cells in HIV infection and on malignant AIDS-NHL cells represents a meaningful target for the use of the proposed antibody-based therapeutics, which can be used as a potential treatment for AIDS-NHL, as well as for removing activated B-cells in HIV+ persons in order to prevent the development of AIDS-NHL by resetting the B-cell clock. Importantly, we have already obtained preliminary results demonstrating that human AIDS-NHL cell lines, and activated (but not resting) human B cells, express high levels of TfR1 and that our anti-TfR1 Abs are efficacious against both malignant and activated B cells in vitro and in meaningful mouse models. We have four specific aims: Aim 1: Define the ability of ch128.1Av and ch128.1 to inhibit/eliminate HIV- and/or EBV-activated B cells in vitro; Aim 2: Define the ability of ch128.1Av and ch128.1 to inhibit/eliminate AIDS-NHL in vitro; Aim 3: Define the toxicity of ch128.1Av and ch128.1 on hematopoiesis, and in general, as well as their ability to inhibit HIV- or EBV-driven B-cell activation in vivo; Aim 4: Define the potential of ch128.1Av and ch128.1 in passive immunotherapy for AIDS-NHL. This project will develop the scientific basis for the use of ch128.1Av and ch128.1 alone, or combined with other drugs, for the prevention and treatment of AIDS-NHL.