Please refer to the current Investigator's Brochure for details of these and other studies. 2.3.1 Pharmacodynamics studies In the pre -clinical studies conducted by Heat Biologics in mouse animal models, species specific (mouse) surrogate vaccines (mHS -130 and mHS -110 secreting OX40-Ig and gp96 -Ig respectively) were developed. Human HS -130 and HS -110 c ould not be administered to mice and vice versa due to their xenophilic nature leading to hyperacute xenograft rejection -mediated by innate and adaptive immune responses. 8,9,10 Similarly, mHS -130/mHS -110 was developed, which refers to the mouse version of the combination 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 20 of 60 Version 2.0 - 11 Feb 2020 Confidential - Entire Page vaccine secreting both OX40L -Ig and gp96 -Ig in a cellular background specific for model species being tested . In a study11,12 to determine the T cell costimulatory activit y of 3 agonist antibodies (OX40, ICOS and 4 - 1BB) when co -administered with mHS -110, OX40 agonist antibody showed the best synergy in promoting CD8+ T cell expansion, without the inducti on of CD4+ FoxP 3+ T regulatory cell populations (Tregs). Comparatively, the addition of an ICOS agonist antibody did not increase CD8+ T cell expansion over mHS - 110 alone, while the addition of a 4 -1BB agonist antibody led to a significant increase in Tregs. In an experiment11,12 to explore a strategy to enable combination immunotherapy within a single vaccine product and to measure the efficacy of the gp96-Ig combination cellular vaccines, a cohort of mice was administere d the combination vaccine mHS -130/mHS -110 and their immune response was recorded. The CD8+ T cell mediated immune response, via local OX40L -Ig expression, appeared to be superior to other fusion proteins (ICOSL -Ig, 4 -1BBL -Ig) generating significant increases in both primary and boost immune responses evidenced by the expansion of antigen -specific CD8+ T cells. In a preclinical study11,12 to evaluate the memory and antigen specific response of mHS -130/mHS -110 combination vaccine, the combination vaccine resulted in superior activation and expansion of antigen - specific CD8+ and memory T cell subsets, along with antigen -specific CD8+ cytotoxic and CD4+ helper T cell responses without triggering a systemic induction of proinflammatory cytokines, which were associated with OX40 agonist antibody treatment. The antitumor effect of mHS -130/mHS -110 was also studied in two preclinical mouse tumor models; B16 - F10-melanoma and CT26 -colon carci noma. In both models mHS -110/mHS -130 generated potent antigen - specific T cell expansion and tumor infiltrating lymphocyte (TIL) counts were associated with delays in tumor growth 11,12. This study repeated earlier findings that combination vaccination with mHS -130 (OX40L-Ig) and mHS-110 (gp96-I g) could lead to increased frequency of complete tumor rejection, while concomitantly providing a potent memory response without increases in Treg numbers. 2.3.2 Immuno -toxicity Studies Up until April 2019, over 100 patients with non -small cell lung cancer (NSCLC) have been treated, and over 1000 doses (up to 10 million cells /dose ) of the HS- 110 cellular vaccine have been administered (IND 14814) , without any overt toxicity. No formal GLP toxicology studies for HS -130 have been proposed or conducted to date . Since HS-130 and HS-110 are s pecies (human) -specific, viable, replication-incompetent whole cell vaccines , that are administered intradermally, experimental experience suggests that safety pharmacology and toxicology studies with HS-130 and HS-110 in animals would be unlikely to predict similar effects in humans due to their nature as xenogeneic cellular entities. The mechanism for hyperacute xenograft rejection of discordant cellular entities (e.g., HS -130 in a mouse model) has been well characterized, involving responses from both innate and adaptive arms of the immune system. 8,9,10 These responses are rapidly induced and lead to overwhelming pro -inflammatory states that destroy the graft unless the recipient undergoes a conditioning regimen .9,13,14 Such conditioning regimens are highly t oxic and would modify 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 21 of 60 Version 2.0 - 11 Feb 2020 Confidential - Entire Page the host recipient in ways that would be too confounding for the purposes of studying the safety , pharmacology and toxicology of HS-130. Based on the results from an in-vitro experiment15 to compare the signaling strength of mouse, monkey and human OX40L -Ig in a human OX40 receptor signaling assay, the potency of OX40L from different species was found to be comparable in its magnitude to activate the human OX40 receptor in the Jurkat/OX40 cell based signaling system. It was observed that monkey (rhesus or cynomolgus) OX40L ligand is similar to human as well as compared to mouse OX40L in the human OX40 receptor assay. This suggests that all three species are compatible and share similar aff inity for each receptor allowing for preclinical translation of mouse model results to human. To study the mechanism of action of cancer vaccines, an intact and syngeneic immune system is required. There are no validated cancer cell line models for in vivo experiments in monkeys and the n on-human primate tumor models are not validated or well characterized. Therefore, mouse surrogate cell lines in murine animal models were utilized to demonstrate in vivo proof -of-concept for activity and to study the toxicokinetics in order to estimate a safe clinical starting dose of HS-130 and HS -110. To establish the acute effects in terms of cytokine and chemokine release, a non -GLP study 16 was conducted in mice to establish the differences betwe en xenogeneic (HS -130) vs. same species (mHS -130) vs. a mouse OX40 agonist an tibody . Five single escalating doses of HS-130, mHS- 130, and an agonist OX40 antibody were administered to mice to determine a cell number threshold of HS-130, that can induce a robust xenogeneic rejection response in mice, as measured by systemic cytokine release. Significant responses were seen for the cytokines: IL -17A, ICL10, MCP-3/CCL7, MIP -1-beta/CCL4, TNF -alpha, Gro- alpha/KC/CXCL1, which could be partially attributed to an anti -human, xenotransplantation response and/or related to the expected innate immune response to foreign cellular matter, required to remove foreign protein particles . Significant response was only observed for the highest cellular dose t ested. The No Observed Adverse Effect Level (NOAEL) was determined to be 400 μg (20 mg/kg) for agonist OX40 antibody, 219.5 ng/24-hr per animal (0.0109 mg/kg) and 387.2 ng/24-hr per animal (0.0193 mg/kg) for HS-130 and mHS -130, respectively. 2.3.3 Dose Range Fi nding Studies to Support Clinical Dosing In the clinical study described in this protocol, HS-130 will be administered to the patients along with separate administration of HS-110 at the same injection sites . To justify the selected clinical doses, the minimum anticipated biological effect level (MABEL ) for dose combinations of mHS -130 (OX40L-Ig) and mHS -110 (gp96-Ig) in mice was determined using anti -tumor CD8+ T cell expansion and tumor growth inhibition as a measure of vaccine efficacy 17. Mice were injected with three different dose ratios of mHS-130 to mHS -110, based on the secretion of OX40L-Ig to gp96 -Ig (1.3:1, 2.5:1 and 5:1), in a dose -escalating manner. Based on this study, a ratio of 1.3 - part OX40L- Ig to 1 -part gp96 -Ig provided the greatest expansion of anti -tumor CD8+ T cells, with the optimal expansion found to be 441 ng/24-hr of OX40L-Ig combined with 339 ng/24-hr of gp96-Ig. This dose and ratio combination also resulted in a higher proportion of activated, endogenous CD8+ T cells, marked as CD44 hi, and an increase in the percentage of short -lived effector cells (SLECs), which are responsible for early tumor cell killing. This dose combination also resulted in highly significant (p<0.001) tumor gro wth inhibition which accompanied an increase in CD4+ and CD8+ TIL accumulation in the tumor. The lowest dose combination provided a No Observed Effect Level (NOEL) of 50 ng/24-hr of OX40L-Ig to 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 22 of 60 Version 2.0 - 11 Feb 2020 Confidential - Entire Page 38 ng /24-hr of gp96 -Ig, at a ratio of 1.3 to 1, with a MABEL of 147 ng /24-hr of OX40L-Ig to 113 ng /24-hr of gp96 -Ig. 2.4 Previous Clinical Studies There are no previo us clinical studies with HS-130. HS-110 has been studied in humans and t o 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). The standard dose of HS -110 in the phase 1/2 study NCT02439450 is 1× 10 7 viable cells /0.5 mL , administered as 5 (0.1 mL) intradermal injections weekly for 18 weeks (Arm 5), or weekly for 13 weeks followed by every 3 weeks boost dosing until progressive disease (Arm 6) . 2.5 Rationale for Starting Dose and Dosing Schedule Both HS-130 and HS -110 are viable allogenic tumor cell-based products, based on the human lung adenocarcinoma cell line AD100, which have been rendered replication-incompetent via irradiation. While the tolerability and safety to HS- 110 is relatively well known (IND No. 14814), HS -130 has previously not been a dministered to humans and cannot be reliably studied in rodents due to hyperacute xenograft rejection . The starting dose rationale for this current study is based on the clinical experience for HS-110 and derived from non -clinical studies in mice using syngeneic cell lines as surrogates for HS -130 (mHS - 130) and HS -110 (mHS -110) to calculate a MABEL. The use of MABEL is considered appropriate since the immunological (pharmacodynamic) response (CD8+ T cell clone and total CD8+ population) takes time to develo p and is not related to plasma concentrations. Based on the experimental outcome of the study conducted to determine CD8+ T cell expansion in mice when administered variable ratios of mHS-130 to mHS-110 with escalating doses of OX40L-Ig and gp96 -Ig prot eins within each ratio , a MABEL for selection of first human dose in clinical trials was determined, with no adjustments for weight or body surface area . In this mouse study, a significant minimal effect was detected in a CD8+ T cell clone at an absolute dose of 50 ng /24- hr of OX40-Ig (mHS -130) to 38 ng /24-hr of gp96-Ig (mHS -110) at a 1.3 to 1 dose ratio respectively, compared to 38 ng /24-hr of gp96 -Ig (mHS -110) alone (a dose without any measurable effect). For sub-populations of CD8+ T cells (KLRG1 hi, CD127lo and CD44hi, short lived effector cells, SLEC), a significant increase was not detected until an absolute dose of 441 ng/24-hr of OX40L-Ig to 339 ng /24- hr of gp96 -Ig at a 1.3:1 dose ratio. This also correlated with tumor growth inhibition and increased TIL numbers in tumors. Thus, our suggested starti ng dose in patients is 150 ng/24-hr of HS-130 to 150 ng/24-hr of HS-110 (1:1 ratio ) administered every 2 -weeks . This dos e: • Represents the MABEL dose in mice for a specific clone of CD8+ T cells and the expansion of peripheral CD8+T cell populations (SELC and CD44 activated) using mHS -110 and mHS130 drug product surrogates . No adjustments for weight or BSA are suggested, which gives a safety factor to the huma n dose of > 100-fold. • Represents less than 1/10th of the safe and effective current clinical dose for HS- 110 (IND 14814). At the highest suggested dose in this study, HS -110 is given at approximately 0.8 of the documented HS -110 dose currently administere d in patients. 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 23 of 60 Version 2.0 - 11 Feb 2020 Confidential - Entire Page • More than 2.5 -fold below the absolute dose (ng per injection) NOA EL (no significant cytokine/chemokine release) of mHS -130 alone in mice . To escalat e the dose level, we propose the next dose at a 2 :1 ratio of HS-130 to HS -110 (300 ng /24-hr OX40L-Ig to 150 ng /24-hr gp96 -Ig) and so forth as described in the dose escalation scheme (Section 6.1.1 ). This approach is designed to bracket the HS-130 to HS -110 dose ratio findings in the mouse study. 2.6 Potential Risks and Benefits As this is t he first -in-man study of HS -130, no human data regarding the sa fety or potential benefit of HS- 130 is available. All currently available clinical data for HS -110 (viagenpumatucel -L) suggest that this allogeneic cell therapy is safe and well tolerated in adults with NSCLC. Mild to moderate injection site reactions, especially induration, erythema, pruritus, pain and rash are expected as they indicate the vaccine has an immunogenic effect, but these have been well-tolerated. No significant safety concerns or risks associated with this allogeneic cell therapy have been reported. To date, no clinically signific ant toxicities deemed by study Investigators as possibly, probably or definitely related to HS-110 (viagenpumatucel -L) have been reported, nor have there been any HS-110 related deaths or SARs in the d evelopment program. Please see the current Investigator Brochure for further details about the potential risks and benefits associated with this study.