............ 81 Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 17 of 84 LIST OF ABBREVIATIONS 6mDCR 6-month Disease Control Rate 6m-irDCR 6-month Immune -Related Disease Control Rate AE(s) Adverse event(s) ALT Alanine aminotransferase ANA Antinuclear antibodies AST Aspartate aminotransferase AT As treated CBC Complete blood count CFR Code of Federal Regulations cGMP Current Good Manufacturing Practice CI Confidence interval CRF Case report form CT Computed tomography CTCAE Common Terminology Criteria for Adverse Events CTL Cytotoxic T lymphocytes CTLA4 Cytotoxic T -lymphocyte associated antigen 4 CR Complete response CY Cyclophosphamide DC Dendritic cells DCR Disease control rate DMSO Dimethyl sulfoxide EC Ethics Committee ECG Electrocardiograms ECOG Eastern Cooperative Oncology Group ELISA Enzyme -linked immunosorbent assay ELISPOT Enzyme -linked immunosorbent spot EOT End of Treatment ER Endoplasmic reticulum ESR Erythrocyte sedimentation r ate EX Excluded Patients FDA Food and Drug Administration FFPE Formalin -fixed paraffin embedded GCP Good Clinical Practice GLP Good Laboratory Practice G-CSF Granulocyte colony -stimulating factor gzB Granzyme B HIV Human immunodeficiency virus HLA Human leukocyte antigen HSA Human serum albumin IBC Institutional Bioethics Committee ICH International Conference on Harmonization ICS Intracellular Cytokine Staining IFN Interferon gamma IHC Immunohistochemistry Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 18 of 84 IR Immunologic Response IRB Institutional Review Board irCR Immune -Related Complete Response irDCR Immune -Related Overall Disease Control Rate irORR Immune -Related Overall Response irPD Immune -Related Progressive Disease irPFS Immune -Related Progression Free Survival irPR Immun e-Related Partial Response irRC Immune -Related Response Criteria irSD Immune -Related Stable Disease ISR Injection Site Reaction ITT Intention to treat i.v. Intravenous IXRS Interactive Response System LTFU Long -term Follow -up MedRA Medical Dicti onary for Regulatory Activities MHC Major histocompatibility complex MRI Magnetic resonance imaging mRNA Messenger Ribonucleic Acid MSDS Material safety data sheet NCI National Cancer Institute NK Natural killer NSCLC Non-small cell lung cancer ORR Overall Response Rate OS Overall Survival PBMC Peripheral blood mononuclear cell PC Physician’s Choice PCR Polymerase Chain Reaction PD Progressive Disease PD-1 Programmed death 1 receptor PD-L1 Programmed death ligand 1 PFS Progression -Free Survival P.O. Per os PR Partial Response PS Performance Status RBC Red blood cell SAE Serious adverse event SAR Suspected adverse reaction SD Stable Disease SOA Schedule of Assessments T-reg Regulatory T cell TCR T-cell Receptor TEAE Treatment e mergent adverse event TIL Tumor Infiltrating Lymphocyte TKI Tyrosine Kinase Inhibitor Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 19 of 84 ULN Upper limit of normal US United States WBC White blood cell Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 20 of 84 1.0 BACKGROUND 1.1 Non-Small Cell Lung Cancer The annual incidence of non -small cell lung cancer (NSCLC) in the United States is the third highest of all malignancies, with ~200,000 cases being recorded in 2012 .1 More than 50% of patients have local or distant metastatic disease at diagnosis , and in stage IV the disease is almost uniformly fatal with a 5 -year survival of <5%.2 The overall annual mortality from lung cancer worldwide is 1.4 million, which is higher than that from colon, breast, and prostate carcinoma combined. Despite a number of ther apeutic advances in the last decade, response to first line and subsequent therapy for advanced NSCLC remains suboptimal . Patients with metastatic or recurrent NSCLC who lack a relevant mutation are usually treated with platinum -containing doublet chemoth erapy (with or without the anti-VEGF antibody bevacizumab [Avastin]) .3 Patients with EGFR or EML4 -ALK mutations generally do better with appropriate molecularly targeted therapies, compared to wild type patients treated with doublet chemotherapy, but even these patients eventually progress and end up requiring conventional cytotoxic chemotherapy.4,5 With standard contemporary regimens, including maintenance therapy in the first line setting, median survival of patients with advanced NSCLC in 2013 was in the range of 12-16 months .6,7 Despite improvements in the first line management of advanced NSCLC, all patients will eventually progress or remain refractory to initial therapy and at least 50 percent will then subsequently receive second line treatme nt, usually consisting of docetaxel, erlotinib or pemetrexed.8 An increasing proportion of these patients are also becoming candidates for third and fourth line interventions. However, despite a growing tendency to ward more aggressive treatment of later stages of the disease, outcomes are limited as response to currently available therapies significantly diminishes at each successive stage of treatment. One large recent series of Western patients showed that response rates were reduced from a first line figure of 20.9 % to a second line value o f 16.3% , a third line one of 2.3% and a fourth line outcome of 0%. The disease control rate (response plus stable disease) also decreased dramatically from first - to fourth -line treatment .9 A similar recent study in Japanes e patients showed that 69.3% , 38.4 %, 17.7 %, and 6.0 % of patients received second -, third -, fourth -, and fifth -line chemotherapy, respectively , with median survival times of 15.3 months, 12.8 months , 12.0 months and 9.9 months respectively. Overall response ra tes and disease control rates with third - and fourth -line chemotherapy were 17.0 % and 34.4 % and 11.3 % and 24.5 %, respectively .10 The relatively poor responses seen in the third and fourth line setting are due to a combination of progressi ve biological resist ance of the underlying tumor arising from successive prior regimens of chemotherapy11,12 and a decline in the performance status of patients, due to treatment and disease related morbidities.13,14,15 The increasing proportion of patients bei ng delivered to the third line arena, combined with a lack of effective strategies with which to treat them, collectively create a significant clinical need for additional third line and beyond treatment interventions , which are mechanistically novel and o f inherently low toxicity. Vaccine -based immunotherapy is one such candidate to meet the demands of t his particular therapeutic challenge and is the subject of the current trial in patients with advanced NSCLC being treated in the third line setting. Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 21 of 84 1.2 Vaccine -based Immunotherapy for NSCLC 1.2.1 Background While some types of tumor s, such as renal carcinoma and melanoma , are traditionally thought to be immunogenic, as evidenced by documented spontaneous clinical regressions and durable responses to high - dose immunomodulatory agents , such as interleukin -2, evidence for an immune role in NSCLC has historically been less compelling, although substantial clinical evidence is now emerging to support this proposition . In particular, t he observation of objective resp onses following immunotherapy in patients with highly refractory NSCLC validates this approach in a preliminary manner suggesting that additional immunological strategies for tumor control , such as vaccines, are worth pursuing in the clinic.16 Vaccination approaches to immunotherapy focus on the induction of innate (natural killer [NK] cell) responses and tumor -specific T lymphocytes by various means . The first FDA -approved therapeutic cancer vaccine (Provenge, Dendreon Corp) consists of a pati ent autologous preparation of antigen presenting cells that have been pulsed with a single antigen (prostatic acid phosphatase) in vitro . These cells are then re - infused into patients and were shown to increase overall survival in patients with advanced pr ostate cancer. This approach is an example of an autologous vaccine and requires significant processing of patient material for each individualized treatment. This paradigm leads to increased cost of therapy, prolongs the period in which a patient may be o ff-treatment and is encumbered by the potential for manufacturing failure or shortfalls. Another class of vaccine with a range of agents in various phases of clinical development include s allogeneic , cell-based therapy . One of the first in this class cons isted of g ranulocyte -macrophage colony - stimulating factor (GM -CSF) transfected tumor cells used as vaccine s through indirect presentation of tumor antigen to T cells by dendritic cells ( DC).17,18,19,20 Other examples include tumor cells transfected with B7.1 ( CD80) ,21,22,23,24,25,26 human leukocyte antigen (HLA) molecules ,27,28 or anti-sense transforming growth factor β2 ( TGF -β2) DNA constructs or tumor cells decorated with bacterial sugar residues. What all of these approaches have in common, is that the transfer of tumor antigens from the injected vaccine construct to the patient immune system is an unregulated and random event. Upon administration to patients, the allogeneic cells are destroyed by the patient’s immune system and the resulting tumor cell fragments are endocytosed by tissue resident macrophages. Typically, antigens that are interpreted by the patient’s immune sy stem in this manner are subsequently displayed on major histocompatibility complex ( MHC ) class II, resulting predominantly in a CD4+ helper T cell -mediated ( not CD8+ cytotoxic T cell -mediated) immune response. This is a critical distinguishing characterist ic between the current approach with viagenpumatucel -L, which exclusively leads to tumor antigen transfer to MHC I and activation of a CD8+ cytotoxic T cell -mediated response (discussed further below). Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 22 of 84 1.2.2 Vaccines in development for NSCLC There are two vacc ines currently in clinical development for the treatment of potentially curable earlier stage NSCLC. The MAGE -A3 v accine is being evaluated in the treatment of resectable stage I –IIIA NSCLC29 and MUC 1 liposomal -BLP25 in unresectable stage III B NSCLC after definitive chemoradiation .30,31,32 MAGE -A3 is an antigen specifically associated with various soli d tumors, including NSCLC , while MUC1 is a membrane -bound glycoprotein that becomes overexpressed and undergoes aberrant glycosylation with malignant transforma tion of diverse tumor types , including NSCLC. One of the common features of the MAGE -A3 and the MUC -1 vaccines is that they each target only a single antigen. Although up to 50 percent of patient tumors may express either MAGE -A3 and/or MUC -1, the expression of these antigens within an individual tumor is rarely uniform. This observation is representative of the underlying genetic heterogeneity of advanced cancer and predicts that even patients who are ‘positive’ for an individual antigen may have a significant tumor burden that is also antigen ‘negative’. Following treatment with a single -antigen vaccine, it is reasonable to predict that , even in the presence of a robust immune response, MAGE -A3 or MUC -1 negative tumor cells will be resistant to the tumor -specific immune response. Various strategies have therefore been devised to circumvent this limitation, including more diverse antigen targeting with allogeneic tumor cell vaccines , such as belagenpum atucel –L and viagenpumatucel -L. Belagenpumatuce l-L is a tumor cell vaccine made with four irradiated NSCLC cell lines (two adenocarcinoma lines, one squamous and one large -cell carcinoma line) modified with TGF -β2 antisense plasmid. Belagenpumatucel -L is currently being clinically evaluated in NCSLC in the advanced disease setting,33 along with two other vaccine approaches. TG4010 is a recombinant viral vector genetically modified to express MUC1 and interleukin -2,34,35,36 while the EGF vaccine (CIMAvax EGF) consists of human recombinant EGF combined with a Neisseria meningitis -derived carrier protein and an immunoadjuvant .37,38 Overall therefore, a variety of vaccine approaches is being evaluated in NSCLC in a range of clinical contexts, including adjuvant, maintenance and first line treatment settings. While none of these has yet received the validation of a successful phase III trial outcome, the promise of the results demonstrated in phase II trials attests to the growing clinical evidence supporting the utility of vaccine -based treatment strategies in this tumor type . Viagenpumatucel -L is distinct from other competitive approaches in clinical testing as it is an allogeneic cell-based vaccine containing a broad range of NSCLC tumor antigens combined with a specific mechanism for the transport of these antige ns to patient antigen presenting cells for the generation of a specific CD8+ cytotoxic T cell response (described below). In this trial, viagenpumatucel -L is used in conjunction with metronomic low dose cyclopho sphamide . Although it is anticipated that administration of viagenpumatucel -L alone will be associated with a substantial CD8+ response, one important mechanism that tumors use for defense against anti -tumor T cell s is the recruitment of regulatory T cells (T -regs) to the tumor microenvironment. T -regs are the population of T cells that serves to protect from autoimmune disease and are frequently co -opted by tumors for their Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 23 of 84 own defense. Cyclophosphamide specifically inhibit s T-reg function. In studies dating back to the 1970s, it has been shown tha t while treatment with high -dose cyclophosphamide leads to tumor cell killing through direct chemical cytotoxicity, treatment with low -dose cyclophosphamide (<200 mg/m2) leads to tumor cell death via (amongst other mechanisms) inhibition of T -regs and subs equent facilitation of cytotoxic T cell - mediated killing.39,40 1.3 Description of Investigational Agent HS -110 Viagenpumatucel -L is a whole cell vaccine derived from a human lung adenocarcinoma cell line AD100 , containing HLA -A1 and gp96 -Ig that is both species (human) and indication (lung cancer) -specific , that has been irradiated to render cell replication incompetent while maintaining biological activity. Gp96 is a member of the heat shock protein family and has evolved specific characteristics that are adva ntageous for a therapeutic cancer vaccine. Similar to other heat shock proteins, gp96 is an intracellular protein that functions as a protein chaperone to facilitate the proper folding of other cellular proteins. In contrast to other heat shock proteins, g p96 is restricted to the endoplasmic reticulum ( ER) via expression of a KDEL ER retention sequence on its C-terminal end. Within the ER, gp96 has exposure to a majority of proteins and antigens that eventually are displayed on MHC I. This property is likel y to have influenced the evolution of a secondary role for gp96 as a molecular warning system for necrotic cell death. Because gp96 is an ER -restricted protein, it is typically released from cells only upon physical destruction or necrotic cell death . Upon release from a dying cell, extracellular gp96 is able to interact with a series of receptors, including Toll -like receptors (TLR) -2 and -4 on the surface of antigen presenting cells. Engagement of TLR -2 and -4 provides an adjuvant signal to a patient’s antigen presenting cells. Simultaneously, gp96 is able to bind to the endocytic scavenger receptor, CD91, to facilitate internalization of gp96 together with whatever protein cargo it bound within the dying cell. Any antigens brought into an antigen prese nting cell by gp96 are then transferred to MHC I, through the cross -presentation pathway, for induction of a CD8+ T cell -mediated cytotoxic T cell response. Heat Biologic ’s ImPACT technology seeks to replicate this natural mechanism by engineering gp96 t o become a secretable molecule through the replacement of the KDEL retention signal with a secretory sequence from an immunoglobulin molecule. The resulting gp96 -Ig fusion protein can then be transfected into a tumor cell line that contains a profile of an tigens that are specific for a given tumor type and used to stimulate a CD8+ T cell response toward those antigens. In the case of viagenpumatucel -L, Heat Biologics has screened a series of NSCLC cell lines to prioritize antigens that are known to be share d amongst a high proportion of patients with NSCLC (including MAGE A3, NYESO1, etc .) and selected the AD100 cell line based on these shared antigen expression characteristics. 1.4 Prior Human Experience A first -in-humans Phase 1 study to examine the safety and immunogenicity of viagenpumatucel -L has been conducted in patients with stage IV, or recurrent NSCLC who had failed at least one line of standard chemotherapy.41 The study was closed prematurely for administrative reasons . Follow -up continued through April 2011 for the 18 vaccinated patients across 3 different dose -schedule (DS) cohorts and is summarized here. All cohorts had up to 3 6-week courses of treatment. The DS -1 cohort (11 patients) Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 24 of 84 receiv ed > 4 x 107 cells (~ 5.7 x 105 cells/kg) every other week (up to 9 vaccinations), the DS -2 cohort (4 patients) received > 2 x 107 cells (~ 2.9 x 105 cells/kg) weekly (up to 18 vaccinations), and the DS -3 cohort (3 patients) received > 1 x 107 cells (~ 1.4 x 105 cells/kg) twice weekly (up to 36 vaccinations). Two DS -3 patients completed the planned 3 -course treatment. Five patients (2 DS -1 and 3 DS-2) completed 2 courses before being taken off treatment due to progressive disease, and 8 DS-1 patients comp leted 1 course before progression. Three patients, 1 in each cohort, died (2) or progressed (1) before completing the first course of vaccination. Most adverse events were Grade 1 or Grade 2. Five adverse events among 3 treated patients were Grade 3 rectal hemorrhage, dyspnea, chest pain, pneumonia and fatigue. By April 2011 , 14 patients had died and 4 surviving patients had been followed for a median of 10.6 months (range 2.6 to 16.6). The Kaplan -Meier estimate of median survival was 8.0 months (95% co nfidence interval [CI]: 6.7 to 18.2), and the 6, 12, and 24 -month overall survival (OS) rates were 77.8% (95% CI: 51.1 to 91.0%), 41.9% (95% CI: 19.1 to 63.3%), and 11.2% (95% CI: 0.8 to 37.3%), respectively. Although comparison is limited due to incomple te accrual to DS -2 and DS -3, 2 of 4 DS -2 patients and 2 of 3 DS -3 patients have survived longer than the median survival time of 7.1 months for the 11 DS -1 patients. Time to progression for DS -2 and DS -3 patients also appears to compare favorably with the median progression - free survival (PFS) of 1.3 months for DS -1 patients. Patient immune responses were monitored in this trial using peripheral blood samples isolated from each patient before treatment and at 3 additional timepoints throughout the trial (6, 12 and 18 weeks). Using these samples, individual immune responses were measured by ELISPOT assays to determine the presence of an immune response characterized by production of IFNγ by patient CD8+ T cells. Of the 18 patients enrolled in the trial, at least 2 samples (baseline and either week 6, 12 and/or 18) were available for 15 of the 18 patients on the trial. Of the 15 patients with samples that were able to be analyzed and compared to baseline, 11 of the 15 demonstrated a doubling or greater (in some cases upwards of 100 -fold increases) of a CD8+ T cell - mediated , vaccine -specific immune response . Furthermore, there was no increase in CD4+ T cell -mediated immunity, confirming preclinical findings that the effect of the gp96 -Ig approach is limi ted to CD8+ cytotoxic T cell responses . When the survival of the immune responders (11/15 patients) was compared to that of the non -responders (4/15), it was observed that the immune responder overall survival was 16.5 months as compared to 4 months in t he immune non -responder group. These data provide a possible link between increased vaccine - specific cytotoxic T cell responses and increased survival , which will be explored further in the current study. Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 25 of 84 2.0 RATIONALE 2.1 Background Rationale As a dvanced NSCLC is an incurable condition all patients given first and second line therapy will eventually relapse, or remain refractory from the outset. Consequently, treatment in the third and fourth line arena is an important and expanding area of overall medical manage ment, with approximately 40 % of patients who receive initial therapy for advanced disease going on to third line or higher treatment.42 Furthermore, patient perspectives are increasingly exceeding th ose of physicians, in favo r of a greater preference for more prolonged treatment into the latest stages of the disease,43,44 with almost 50 % and 25 % of patients undergoing active therapy within the last 4 and 2 weeks of their lives , respectively .45 Although objective response rates and PFS fall precipitously after second line therapy,9 recently completed randomiz ed trials with a placebo arm (MISSION and ZEPHYR), suggest that the medi an survival of patients in the post -second line setting is now as long as 8 months,46,47 which compares highly favo rably with previous estimates of just 3 -4 months,48 although there is as yet no clear evidence that survival is significantly improved by existi ng therapeutic interventions.49 The fact that a growing proportion of patients are reaching third, four th and even fifth line settings10 and the finding that over 50% of overall median survival may now reside in the post -second line context mean that the third line and beyond space is a significant and evolving area of therape utic potential in the overall management of advanced NSCLC. While current National Comprehensive Cancer Network (NCCN) guidelines recommend several options for first -line and second -line therapies, only erlotinib is endorsed as a third -line therapy in unse lected patients.50 Additionally crizotinib has been approved, essentially for any line of therapy, in patients with an EML4/ALK translocation.51 However, recent clinical results have shown that, at least in the second line setting, erlotinib treatment leads to inferior outcomes, when compared to chemotherapy, in patients with wild type EGFR status.52,53 Furthermore, crizotinib is only suitable for the 4 -5% of NSCLC patients who actually have an EML4/ALK abnormality.54 Therefore, for the vast majority of patients without mutated disease who lack corresponding molecularly targeted therapy, treatment options are significantly limited and substantially sub -optimal in the post - second line setting. A variety of molecularly target approaches55 and chemotherapy strategie s are currently employed empirically in the third and fourth line setting. Chemotherapy -based regimens include platin, or non -platin - based doublet therapy and also mono -therapy with agents , such as paclitaxel, docetaxel, vinorelbine, gemcitabine and pemetr exed.56 However, in practice the range of treatment choices actually available to patients is severely constrained by co-morbidities, often arising from prior first or second line therapy. For example, pemetrexed use may be complicated in patients with ren al impairment57 or uncontrolled effusions,58 while docetaxel may be contra - indicated in patients with residual neuropathy from prior platin -based therapy.59 Many patients also have a significantly high risk of febrile neutropenic complications from chemothera py, a liability compounded by poor performance status and advanced age ,60 which are frequently characteristic features of patients in the more advanced stages of NSCLC. Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 26 of 84 Overall , therefore, the range of clinically useful agents is extremely limited in the third line and further setting. Although no agents have so far shown a survival advantage in the third line context, second line agents, such as docetaxel and pemetrexed , have sh own improved survival despite achieving very low response rates, suggesting that most of the utility derived in the later stages of NSCL C is from disease stabilization,9 an ideal mechanistic setting for a vaccine -based therapeutic approach. It is therefore anticipated that a vaccine , such as viagenpumatucel -L (HS-110), could offer superior efficacy to currently available third line strategies with a comparable or better safety profile. The current trial will examine the clinical utility of a vaccine -based therapy compared to a range of widely used conventional third line agents. 2.2 Study -Specific Rationale Summarizing the background information in the s ections above, the rationale for this study of viagenpumatucel -L in combination with metronomic low -dose cyclophosphamide (CY) is as follows: (1) Recent and paradigm -changing clinical data suggest that effective NSCLC immunotherapy is feasible and that u nder the right circumstances NSCLC cells may, in fact, become susceptible to attack by cytotoxic T lymphocytes (CTL) . (2) Secreted gp96 -Ig from viagenpumatucel -L combines adjuvant activity with polyvalent peptide specificity and activates DC, NK cells, and CTL. As a result tumor cells can be killed by NK cell-specific mechanisms and by MHC -restricted CD8+ CTL activity. The activation of DC and NK cells by viagenpumatucel -L may also counteract the generation of immunosuppressive T-regs. (3) Viagenpuma tucel -L stimulates antigen cross presentation via the CD91 receptor, TLR2 and TRL4 on DC and macrophages (as described above). Allogeneic NSCLC cells are known to share public tumor antigens , including MAGE A3, MUC1, NY -ESO -1 and others. Therefore allogeneic, gp96 -secreting tumor cells used as a vaccine (such as viagenpumatucel -L) are expected to generate NK and CTL activity against the patient’s autologous tumor through common expression of selected shared tumor antigens. (4) The rationale for administering viagenpumatucel -L in combination with low -dose metronomic CY is based on the presumption that a majority of established tumors have developed an active mechanism of immune evasion. For some tumors this may include direct expression or recruitment of cells expressing CTLA -4 or PD -L1. In many cases, this immunosuppression may also be mediated by T -regs. The use of low-dose metronomic CY (50 mg, PO, QD, 1 week on/1 week off) has been demonstrated to prov ide prolonged suppression of both T -reg numbers and function without having direct cytotoxic effects on tumor cells.61 2.3 Justification of Study Drug Administration Strategy 2.3.1 Viagenpumatucel -L The initial Phase 1 study in lung cancer examined 3 dosing schedule s: twice weekly, weekly and once every 2 weeks. The twice weekly and weekly dosing schedule s were determined to produce the most desirable immune response (at least a doubling of CD8+ numbers from baseline). The weekly dosing schedule was Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 27 of 84 selected for thi s trial as it represents the schedule with greatest patient acceptability for frequency of treatment visits. The dose of 1x 107 cells was selected for this trial. The 1x107 cells is half the dose that was used in the Phase 1 trial (DS -2 cohort). Murine data suggested that lower doses may produce sufficient immune response and may even result in superior clinical effects , and, therefore, the lower dose of 1x107 cells was chosen. Additionally, this lower dose is prudent from a safety perspective as this trial is the first testing the combination with metronomic low -dose CY. Vaccine will be administered weekly as 5 x 0.1 mL intradermal injections (see Section 7.1). The selection of the route of administration is based on the hypothesis that split doses stimulate stronger immune responses by allowing vaccine antigens t o reach more regional lymph nodes (where immune responses are generated). The vaccine will be administered intradermally to enhance stimulation of cellular immune responses as compared to subcutaneous or intramuscular immunization. 2.3.2 Cyclophosphamide (CY) When administered at conventional cytotoxic doses (10 -15 mg/kg intravenously) , CY usage is associated with significant clinical toxicity, including myelosuppression (predominantly leu kopenia and neutropenia and less frequently anemia or thrombocytopenia), hemorrhagic cystitis, nausea, vomiting, diarrhea, anorexia, alopecia, skin pigmentation and allergic reactions.62 However, at the dose proposed for the current study (50 mg daily on alternate weeks) the predominant toxicity is expected to be mild neutropeni a or leukopenia, based upon extensive experience in the literature with the use of low -dose metronomic CY in a range of malignancies, including breast, prostate and lung cancer. Typically, grade 3 or higher hematological toxicities have been documented in less than 5% of patients in these studies and grade 3 or higher non -hematological toxicities in less than 2%, supporting the contention that low -dose CY, as proposed for the current study, is a safe and well tolerated intervention63,64,65,66 2.4 Rationale for Perfo rming Tumor Biopsies In order for an allogeneic cell -based vaccine to lead to a clinical response in a treated cancer patient, that vaccine must not just stimulate a tumor -antigen -specific immune response as detected by circulating lymphocytes in the peripheral blood, but those lymphocytes must infiltrate the tumor microenvironment in order to kill tumor cells. Patients who are predicted to respond to viagenpumatucel -L are those whose tumors express antigens that are known to overlap with the shared a ntigens contained within the AD100 cell backbone of viagenpumatucel -L. In addition, it is predicted that those patients whose tumors express a number of antigens that are shared with viagenpumatucel -L will have improved clinical responses as compared to t hose patients whose tumors only share 1 or 2 antigens with viagenpumatucel -L. Further more , it is possible that the likelihood of a patient having a clinical response to treatment with viagenpumatucel -L could be pred icted by the presence and extent of tumor infiltration by CD8+ T cells , determined in turn by the degree of correlation found with shared individual antigens between patient tumors and viagenpumatucel -L. Consequently, acquisition of tumor tissue is essential to histologically examine tumor antigen expression and generate predictive information on which patients are most likely to have a clinical Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 28 of 84 response, and thus inform optimal patient selection for future trials . Archival biopsy tissue , if available, will be ac ceptable for the pre -treatment tumor antigen analysis. 3.0 OBJECTIVES 3.1 Primary Objective To evaluate overall survival 3.2 Secondary Objectives  To evaluate the safety of the combination of viagenpumatucel -L and low -dose CY  To evaluate immune -related overall respon se rate (irORR), (complete response and pa rtial response) and also overall response rate (ORR) by RECIST  To evaluate immune -related disease control rate (irDCR) (complete response, partial response, and stable disease) and also disease control rate (DCR) b y RECIST at 6 months following randomization and overall  To evaluate immune -related progression free survival (irPFS) and also progression -free survival (PFS) by RECIST  To evaluate immune -related time to progression (irTTP) and also time to progression (T TP) by RECIST  To evaluate the proportion of patients who are alive at 6 months following randomization  To evaluate the proportion of patients who a re alive at 12 months following randomiz ation  To characterize the peripheral blood immunologic response via intracellular cytokine staining (ICS) by flow cytometry and/or ELISPOT on IFNγ -positive CD8+ cells following vaccination 3.3 Exploratory Objectives  To evalua te exploratory endpoints , which may incl ude: o Peripheral blood immunologic response by flow cytometry and ELISPOT o Total peripheral blood mononuclear cell ( PBMC ) counts by flow cytometry , including lymphocyte subsets o Evaluation of tumor tissue obtained from pre -treatment biopsy or archival biopsy tissue for shared tumor antigen expression, expression of MHC class I, and expression of immunosuppressive molecules o Evaluation of tumor tissue obtained from post -treatment biopsy for presence of tumor - infiltrating T lymphocytes (TIL) o Evaluation of tumor t issue and PBMCs for T cell receptor (TCR) sequencing to determine correlation between clonally expanded T cell populations and other endpoints Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 29 of 84 4.0 TRIAL DESIGN Patients who have failed 2 or 3 prior line s of therapy for advanced disease will be randomized 2:1 to 1 of the following treatment groups : Experimental Group : Viagenpumatucel -L plus metronomic CY (82 patients) Control Group : Physician’s Choice ( PC) (41 patients) Therapy to be given in nominal 21 day cycles with dose and schedule according to investigator’s standard practice  Vinorelbine  Erlotinib  Gemcitabine  Paclitaxel  Docetaxel  Pemetrexed Patients randomized to the experimental group will be treated during an induction phase with combination metronomic CY and viagenpumatucel -L weekly for 12 weeks followed by monotherapy viagenpumatucel - L in the maintenance phase once every 9 weeks for up to 12 months or until discontinuation from study treatment, at which time patients will be discontinued from treatment and followed for overall survival as defined by irRC , at which time patients will be discontinued from treatment and followed for overall survival . Patients randomized to the control group will be treated with a PC regimen until until discontinuation from study treatment, at which time patients will be followed for overall survival. Patients with progressive disease by irRC at an assessment may continue on treatment until the next assessment if judged by the investigator to be in the patient’s best interest. While treatment may continue beyond irPD, statistical analysis will still follow strict definitions as defined in section 6.5.1 Tumor Assessments. Blood samples will be taken to evaluate the CD8+ immune response in PBMC s and their correlation to overall survival. Patients will have samples drawn prior to first study treatment, at Weeks 4, 10, 13, and 22, and at End of Treatment (EOT) . Where considered appropriate by the investigator, patients randomized to the experimenta l group will be invited to consent for biopsies pre-treatment and at Week 1 3 for exploratory biomarker analysis. Patients will also be asked to consent to provide archival biopsy tissue if available . Examination of tumor samples will enable screening for expression of MHC class I (necessary for any immunologic response), expression of immunosuppressive molecules , including , for example, CTLA 4, PD -L1, and PD -1 (informative for future po ssible combination studies), expression of tumor antigens that are kno wn to be expressed by viagenpumatucel -L, presence of TILs, and TCR sequencing . Patients will be monitored for safety with adverse event (AE) reporting, clinical laboratory parameters (hematology, liver function, electrolytes, renal function, and urinalysis ), 12-lead electrocardiogram (ECG) recording, vital signs, and physical exams. Efficacy assessments will include survival, tumor evaluation, and immune response. Tumor assessments will be done at screening, every 9 weeks until Week 28, and Heat Biologics, Inc. Investigational Product: Viagenpumatucel -L Protocol HS110 -201 CONFIDENTIAL Version 4.0; 29 -Oct-2014 Page 30 of 84 every 12 weeks thereafter until progression, unless clinical signs of progression necessitate earlier assessment . The overall study design is summarized below in Figure 1.