Sanborn, M.D. Providence Cancer Institute Portland, OR , USA 2.0 BACKGROUND INFORM ATION 2.1 Introduction The dramatic clinical success of cancer immunotherapies in a small percentage of patients has highlighted the need to identify combination approaches that increase the frequency of responders. Vaccination strategies using T cell costimul atory agents and/or blockade of immune inhibitory molecules (i.e. checkpoint inhibition) are being tested and developed as combination approaches in many ongoing clinical trials. Agonist antibodies targeting T cell costimulatory receptors such as 4 -1BB, OX40, TNFRSF25, GITR and ICOS are currently in clinical development based on their anti -tumor effects in a variety of pre -clinical model systems 2. These agonist antibodies are typically delivered by intravenous infusion at target -saturating doses and achieve systemic bio -distribution within minutes, often resulting in global pathway activation3. This route of delivery for T cell costimulation is quite different than the physiological context in which the ligands for 4 -1BB, OX40, TNFRSF25, GITR and ICOS, are expressed, which is localized to individual tissue Heat Biologics Protocol No. HS130 -001 A Phase I Study of HS -130 in combination with HS-110 in patients with solid tumors HS130- 001 17 of 60 Version 2.0 - 11 Feb 2020 Confidential - Entire Page microenvironments wherein innate [commonly Toll-like receptor (TLR)] ligands stimulate antigen presenting cells to express these ligands4,5,6. In addition to being localized to tissue microenv ironments, the kinetics of 4 -1BB ligand (L), OX40L, TL1A, GITRL and ICOSL expression demonstrate rapid induction and subsequent down -regulation within a 2 -5 day window4,5. Thus, the transient expression of costimulatory molecules is typically limited to individual tissue microenvironments wherein antigen- specific immune responses are being activated, but do not persist for more than a few days – a pattern that is not reproduced by systemic costimulation with monoclonal antibodies. Recently, it has been shown that micro -injection of monoclonal agonist antibodies into tumor lesions directly leads to anti-tumor benefits over systemic antibody administration, thus underscoring the benefit of employing localized co - stimulation versus systemic costimulation7. Heat Biologics, Inc. has developed v iagenpumatucel -L, also known as HS -110, a novel allogeneic cellular immunotherapy designed to induce cell -mediated immunity for use in treatment of patients with refractory or metastatic non -small cell lung cancer (NSCLC). The active ingredients in viagenpumatucel -L are derived from a human lung adenocarcinoma cell line, AD100, episomally expressed HLA-A1 (the human histocompatibility surface antigen for identity) and gp96 -Ig (transgene constructed from sequences encoding the human gp96 gene), and a robust repertoire of NSCLC associated overexpressed cancer testis antigens (CTAs) . The secreted fusion protein (gp96 -Ig) acts as a chaperone for these cancer antigens and an antigen presenting cell (APC) adjuvant (TLR activation) to induce CD8 biased cellular immune responses to these tumor antigens expressed by the AD100 host cell. To date, over 100 patients with non -small cell lung cancer have been treated with HS -110 with over 1000 doses of HS -110 administered intradermally (IND No. 14814). To evaluate the additional therapeutic role of localized specific costimulation within the context of the HS-110 vaccine, Heat Biologics would like to explore the possibility of co -expression of a T cell costimulatory ligand fusion protein toget her with gp96 -Ig to provide an efficacious combination immunotherapy , ultimately within a single cell-based product. On screening a panel of costimulatory molecules in murine animal models , OX40L co-stimulator was found to provide the most robust activatio n of antigen specific CD8 T cells with superior T cell expansion when secreted locally as a combination vaccine with gp96 -Ig expression as compared to systemically administered OX40 agonist antibodies. To study the effect of OX40L co -stimulator in combin ation with HS -110, Heat Biologics has developed HS- 130, which is a viable, replication-incompetent (as final drug product) cell line genetically engineered from the same human lung adenocarcinoma cell line (AD100) as HS -110 but secreting OX40L-Ig fusion pr otein instead of gp96 -Ig and HLA -A1. In the initial trial to be conducted under this IND, Heat Biologics would like to evaluate the safety and optimal immunologic dose of HS-130 in combination with HS-110 administered in a 1:1 or 2:1 ratio based on expression of OX40L -Ig and gp96 -Ig (fg/24 -hr/cell) respectively, in patients with solid tumors refractory to standard care. Heat Biologics future development plan is to produce a combination therapy incorporating both T cell co - stimulation (OX4 0L-Ig) and vaccination (gp96 -Ig) in one commercialized product using a single vector in a single cell line (AD100). After determining the optimal dose range of co -administered HS -110 and HS -130 from this clinical study, the combination will continue to be developed and evaluated in additional clinical trials of safety and efficacy after selection, manufacturing and clinical development of the combined product on a single vector/single cell line. The quantity of gp96 -Ig and OX40L -Ig fusion proteins secreted by the combined product would be designed to mimic the same optimal dose level determined to be immunologically active from the initial studies conducted with HS-110 and HS -130. Heat Biologics understands that this development change constitutes a new dru g product and will require a new IND Heat Biologics Protocol No. HS130 -001 A Phase I Study of HS -130 in combination with HS-110 in patients with solid tumors HS130- 001 18 of 60 Version 2.0 - 11 Feb 2020 Confidential - Entire Page submission. 2.2 The Investigational Product 2.2.1 HS-130 The drug product HS -130 is a viable whole cell vaccine , made replication-incompetent by radiation, expressing the co -stimulatory fusion protein OX40L- Ig, which is the ligand for the OX40 receptor, a member of the TNF -receptor superfamily. Similar to HS -110 (described below), HS -130 is derived from a human lung adenocarcinoma cell line established from a biopsy of a lung cancer patient and this cell line is designated as AD100. AD100 is transfected with a 7192 bp plasmid cDNA ‘ pcDNA3.4 OX40L -Ig’ stably expressing the cDNA for OX40L- Ig to develop an irradiated drug product, HS -130. The plasmid vector map for pcDNA3.4 OX40L -Ig is presented below in Figure 1. Figure 1: pcDNA3.4 OX40L -Ig Vector Map The HS -130 intermediate drug product is supplied as a frozen product filled under aseptic conditions in 1.2 mL cryovials, containing 0.6 mL suspension of 2 x 10 7 viable cells/mL in a cryopreservative buffer, with a final formulation of 0.567% sodium chlori de, 0.5% HSA, 0.007% sodium bicarbonate, 6% pentastarch, and 5% DMSO. These cryovials are irradiated at Steris to constitute final drug product and stored at Waisman Biomanufacturing prior to the initiation of clinical testing . Heat Biologics Protocol No. HS130 -001 A Phase I Study of HS -130 in combination with HS-110 in patients with solid tumors HS130- 001 19 of 60 Version 2.0 - 11 Feb 2020 Confidential - Entire Page 2.2.2 HS-110 HS-110 (viagenpumatucel -L) is an allogeneic, cell-based immunotherapy containing a broad range of NSCLC tumor antigens (up to 66 known cancer testis antigens) combined with a specific mechanism for the transport of these antigens to patient ’s antigen -presenting cells that results in a specific CD8+ cytotoxic T cell response. Heat Biologics’ HS-110 cell vaccine is engineered to produce a secretable gp96 fusion protein due to presence of a secretory sequence from an immunoglobulin constant reg ion. The resulting gp96 -Ig fusion protein plasmid is then transfected into a lung cancer cell line AD100 based on the shared ant igen expression characteristics amongst a high proportion of patients with non-small cell lung cancer (NSCLC). Figure 2 below illustrates the structure of HS-110 cell -based vaccine. Figure 2: Schematic diagram showing the structure of HS- 110. This vaccine has a cellular backbone (AD100 cell line) which has been modified with a gp96 -Ig expression plasmid. Tumor cell antigen -gp96 -Ig complexes are secreted by the cell in order to deliver a pan- antigenic antitumor response HS-110 (viagenpumatucel -L) is packaged in single -dose vials as fully -diluted frozen liquid filled under aseptic conditions in 1.2 mL cryovials , containing 0.6 mL suspension of 2 x 107 cell/mL (targeted) in a cryopreservative buffer, with a final formulation of 0.567% sodium chlor ide, 0.5% HSA, 0.007% sodium bicarbonate, 6% pentastarch, and 5% DMS O. 2.3 Preclinical Studies Key preclinical findings are summarized below.