Refer to Appendix 3 for further details. Heat Biologics, Inc. Viagenpumatucel -L Protocol HS110 -102 CONFIDENTIAL Amendment 6, 24 May 2019 Page 63 of 95 The disease response measures will allow for the calculation of the ORR, PFS, and DCR. T he ORR includes CR /iCR, and PR/iPR. PFS is the time between treatment assignment and the date of PD /iPD or death, whichever comes first. The DCR includes responses of PR/iPR, CR/iCR or SD/iSD. 14.2. Immunologic Response (IR) Peripheral blood samples (50 mL) will be collected at Screening ,Week 7, Week 13, and EOT . All samples will be shipped to and processed by Heat Biologics, Inc . or its designee. Forty (40) mL heparinized blood and 10 mL of non -heparinized blood will be drawn at each of the above time point s and used for analysis as outlined below . PBMCs will be isolated from heparinized blood samples by Ficoll separation. Isolated PBMCs will be utilized for immunophenotyping and functional analysis using fluorescence activated cell sorting (FACS) and ELISPOT assays as described below. A subset of isolated PBMCs will by lysed in nucleic acid preservative buffers for downstream analysis of molecular signatures. Serum collected from non-heparinized blood samples will be stored for batch analysis of antibody titers to HLA -A1, in non-HLA -A1 positive patients and serum cytokines/chemokines. In addition to the analyses specified below, subsequent exploratory mechanistic analyses, the nature of which will be determined by emerging PBMC and tumor biopsy data on the immunological environment pre - and post -study treatment, may also be performed. 14.3. Production of Interferon -gamma and Granzyme B from PBMCs (ELISPOT) The immune response will be evaluated by antigen -specific ELISPOT analysis for IFNγ and Granzyme B production by patient cells. These ELISPOT assays will be conducted in our research facility . Previously cryopreserved and thawed PBMCs will be challenged or restimulated with specific tumor vaccine antigens as follows: • PBMC s + PMA + Ionomycin (positive control) . • PBMC s + HS-110 whole cell lysates • PBMC s + a n overlapping cocktail of known shared antigens, which may include Survivin, TTK, NY -ESO -1, CEP55, MAGEA1, MAGEA3, MAGEA6, MAGEA 12, MAGEB1, MAGEC1, MAGEC2, CXORF61, GAGE1, BAGE1, CTCFL, DDX43, SSX1, SSX4, and HORMAD 1. For the ELISPOT assay, the frozen PBMCs will be thawed, antigen challenged, and plated for analysis of IFNγ and GzB production by two color enzymatic ELISPOT assa y and analyzed in duplicate per time point for each patient. Following incubation, samples will be quantitated in an automated ELISPOT reader. The frequency of IFN  producing PBMC is thought to mirror the fr equency of cytotoxic CD8 + cells; h owever, cytotoxicity is primarily mediated by perforin and granzymes following granule exocytosis. In some instances , IFNγ secretion and granule exocytosis are uncoupled. Therefore, the direct measurement of gzB secretion by ELISPOT may provide a more direct readout for Heat Biologics, Inc. Viagenpumatucel -L Protocol HS110 -102 CONFIDENTIAL Amendment 6, 24 May 2019 Page 64 of 95 antigen -specific granule exocytosis and cytotoxicity. Antigen specificit y of the ELISPOT response will be evalua ted by comparing responses generated using the various stimulator cell populations and specific NSCLC shared antigens as indicated. This will also enable separation of the ‘allo-antigen’ specific response with the po tential parent cell line tumor antigen -specific response. Positive responses will be defined as a greater than two -fold increase in the number of IFNγ -positive PBMC compared to baseline. 14.4. Phenotyping Phenotypic Analysis of Blood Lymphocyte Subsets by FACS Flow cytometric analysis of patient peripheral blood samples will quantitatively determine the frequency and number of lymphocytes (CD4 5 +), B-cells (CD4 5 +, CD3 -, CD19 +), helper T -cells (CD4 5 +, CD3 +, CD4 +), T-cells (CD4 5 +, CD3 +, CD8 +), regulatory T -cells (CD 3 +, CD4+, CD25+, FoxP3 +), and NK cells (CD4 5 +, CD56 +, CD3 -) at baseline and over the course of therapy. In addition, a NK/T-cell activation panel (Ki67, CD137, CD27, CD28 ), memory panel (CD27, Cd28, CD45 RA, CD197, CD57) and exhaustion panel (CTLA4, PD -1, Tim3, TIGIT) will be performed. 14.5. Analysis of Infiltrating T -Cells (Post -Treatment Tumor Biopsy) Post-treatment biopsies will be collected at week 10, when feasible , and the tissue will be examined for the presence of TILs. Multicolor fluorescence immunohistochemistry (IHC) will be used for quantification of infiltrating CD3+, CD4+ and FOXP3+ T-cells and measurement o f their activation status , Ki67 and PD -1 expression , and cytotoxic activity ( gzB expression) in situ. These data will be correlated with clinical response.