Granzyme B Research Articles

Orvacabtagene Autoleucel (orva-cel; JCARH125): A Fully Human BCMA-Targeted Second-Generation CAR T Cell Product Characterized By a Predominant Central Memory Phenotype with High in Vitro and In Vivo Proliferative Potential and Sustained In Vivo Persistence

Background: Orva-cel is an investigational B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cell product genetically modified with a lentiviral vector to express a CAR construct with a unique fully human single-chain variable fragment, optimized spacer, and 4-1BB costimulatory and CD3ζ activation domains. Orva-cel is currently being evaluated for efficacy and safety in the ongoing phase 1/2 EVOLVE study (NCT03430011) in heavily pretreated patients with relapsed/refractory multiple myeloma. We characterized orva-cel drug products, manufactured using the process in place for the phase 2 portion of the study and intended for commercial manufacturing, for CAR+ T cell purity, phenotype, and function. Methods: Immunophenotyping was performed by flow cytometry of both surface and intracellular markers, including CD3, CD4, CD8, CD45, CCR7, CD45RA, CD28, CD27, and active caspase 3. Cytokine production after challenge with BCMA+ target cells was assessed by intracellular cytokine staining and Luminex multiplex assay of secreted cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrB). CAR-mediated in vitro proliferative capacity was measured after anti-idiotypic antibody stimulation using the IncuCyte Live-Cell Analysis System (Sartorius, Göttingen, Germany). In vivo CAR+ T cell proliferation and persistence were assessed by quantitative polymerase chain reaction (qPCR). Results: The orva-cel manufacturing process was designed to enable consistent production of highly pure CD3+ cell products (median frequency of CD3+ T cells, 99.96%; quartiles 1-3 interquartile range, 99.9%-100.0%; n = 81). Orva-cel drug products were characterized by high frequencies of less-differentiated CAR+ T cells, leading to a dominant central memory-like population (CCR7+CD45RA- CAR+ T cells) and substantial frequencies of naïve-like cells (CCR7+CD45RA+ CAR+ T cells) (Figure). When assayed for in vitro functional activity, orva-cel drug products showed robust antigen-specific cytokine and effector molecule production (IFN-γ, TNF-α, IL-2, and GrB) upon challenge with BCMA+ tumor cells, as well as vigorous proliferation in response to CAR stimulation. Preliminary correlative analysis suggested that the early memory phenotype may be linked to increased CAR+ T cell proliferative capacity, as determined by in vitro experiments and in vivo PK parameters (ie, maximum CAR+ T cell concentration observed in the blood [Cmax], time to Cmax, area under the curve from Day 0 to 28 [AUC0-28], and CAR+ T cell persistence at Month 3 and Month 6). Consistent with the early CAR T cell memory phenotype, qPCR analysis showed robust in vivo proliferation of CAR+ T cells after infusion, with a median Cmax of 1.54 × 105 transgene copies/µg DNA and median AUC0-28 of 1.61 × 106 transgene copies/µg DNA*day, as well as long-term in vivo persistence, with CAR+ T cells detected in 69% of patients at 6 months postinfusion. Conclusions: The orva-cel manufacturing process results in drug products characterized by highly pure T cells, with high frequencies of early memory and polyfunctional CAR+ T cells. Orva-cel drug products showed robust antigen-specific degranulation, production of multiple cytokines, sustained in vitro and in vivo proliferation, and in vivo persistence. Disclosures Colonna: Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Navarro:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Devries:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Beckett:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Amsberry:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Radhakrishnan:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Piasecki:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Heipel:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Li:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Kavita:Bristol Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Works:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Mujacic:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment.

Blinatumomab Consolidation Post Autologous Hematopoietic Stem Cell Transplantation in Patients with Diffuse Large B Cell Lymphoma

Introduction: Autologous hematopoietic stem cell transplantation (auto-HCT) is the standard treatment for patients with chemo-sensitive relapsed/refractory diffuse large B cell lymphoma (DLBCL). However, post-auto-HCT outcomes are still poor in this population, with 5-year progression free survival (PFS) of 40%. We hypothesize that in patients with DLBCL, blinatumomab consolidation post auto-HCT will eradicate remaining tumor cells, leading to decreased relapse and increased overall survival. Therefore we conducted a pilot study to test blinatumomab as consolidation therapy post auto-HCT for patients with DLBCL. Methods: Adult patients with chemosensitive DLBCL or transformed FL who underwent auto-HCT were included. All patients received one cycle of blinatumomab consolidation starting 42 days post auto-HCT (9 mcg daily as continuous infusion for 7 days, followed by 28 mcg daily for 21 days). Response evaluation was done at day 100 post auto-HCT. Minimal residual disease (MRD) was quantified by immunoglobulin high-throughput sequencing (Ig-HTS) of plasma cell-free DNA on days 42 post auto-HCT (pre-blinatumomab) and on day 100 post auto-HSCT (one month post completion of blinatumomab). Immunophenotyping of T cells in cryopreserved peripheral blood mononuclear cells collected on day 42 (pre-blinatumomab), day 56 (midpoint of blinatumomab treatment cycle), and day 100 (1 month post blinatumomab) was performed using 18-color flow cytometry panels for extracellular and intracellular antigens. Results: As of August 2020, ten patients have been treated with at least 100 days follow up. Patient characteristics and outcomes are summarized in Table 1. Three out of 10 patients (30%) were in partial remission (PR) as determined by CT or PET/CT imaging before auto-HCT. All subjects completed the planned cycle of blinatumomab consolidation. Blinatumomab was well tolerated. Two patients developed grade 1 CRS, with no grade 2 and higher CRS. Immune effector cell-associated neurotoxicity syndrome (ICANS) was not observed. Six patients developed transient tremor (four grade 1, one grade 2, and one grade 3). One subjects developed BCNU pneumonitis and CMV viremia that resolved with steroid and ganciclovir. One hundred days post auto-HCT (one month post blinatumomab consolidation) 10/10 (100%) of patient were in complete remission (CR) as determined by both MRD testing and by CT or PET/CT imaging. Plasma cell free based MRD was positive on day 42 (post auto-HCT and pre-blinatumomab), in two out of ten patients (20%). These two patients achieved MRD negative status after receiving blinatumomab consolidation. With median follow up of 14.5 months (range: 7-34 months), all 10 patients are alive and 6/10 remain in remission.. Interestingly, the 4 patients with disease relapse had lower CD8/CD4 T cell ratio before starting blinatumomab compared with patients who remained in remission (Figure 1A). However, there were no significant differences in the distribution of the major T cell subtypes (naïve, memory, effector and Treg), and expression of markers of T cell activation, proliferation, or exhaustion (Figure 1B-1E). High dimensional analysis with t-stochastic neighbor embedding (tSNE) revealed a cluster of CD8+ and CD4+ T cells characterized by high expression of granzyme B (GB) and perforin that was present in the DLBCL patients before and after blinatumomab treatment but not in a healthy untreated control (Figure 1F-1H). Although further analysis of healthy untreated controls and pre-transplant samples is needed, CD8+ T cells from these DLBCL patients pre-blinatumomab contained very few naïve cells and were enriched for terminally differentiated effector cells. Conclusion: This pilot study shows that blinatumomab consolidation post auto-HCT is safe and well tolerated. MRD response to blinatumomab in all patients with MRD positive disease post auto-HCT is encouraging. Strategies to increase CD8/CD4 ratio and more cycles of consolidation in a larger randomized trial are needed to confirm the efficacy of consolidation with blinatumomab post auto-HCT. Finally, the unusually "high activation" immunophenotype (Teff/GB+) seen in CD8 T cells of DLBCL patients after auto-HCT (compared to those seen in resting peripheral blood) may both impact the response to blinatumomab and provide key insights into optimal timing for administration after auto-HCT. Disclosures Ghobadi: WuGen: Consultancy; Bristol Myers Squibb: Consultancy; Kite: Consultancy, Research Funding; EUSA: Consultancy; Amgen: Consultancy, Research Funding. Mehta-Shah:Bristol Myers-Squibb: Research Funding; Karyopharm Therapeutics: Consultancy; Corvus: Research Funding; Genetech/Roche: Research Funding; Innate Pharmaceuticals: Research Funding; Kyowa Hakko Kirin: Consultancy; Celgene: Research Funding; Verastem: Research Funding; C4 Therapeutics: Consultancy. Kahl:Celgene Corporation: Consultancy; AstraZeneca Pharmaceuticals LP: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics LLC: Consultancy; Roche Laboratories Inc: Consultancy; BeiGene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy; AbbVie: Consultancy. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Granzyme B in peripheral blood mononuclear cells as a measure of cell-mediated immune response in paraneoplastic neurological syndromes and malignancy

BackgroundParaneoplastic neurological syndromes (PNS) may coexist with ovarian or lung cancers. Some tumors coexisting with PNS are smaller and have a better prognosis than tumors without PNS. PNS may constitute an opportunity to observe a natural immune antitumor response. We aimed to investigate a cytotoxic immune response by measuring granzyme B (GrB) in peripheral blood mononuclear cells (PBMC) in patients affected with ovarian or lung malignancy, with and without accompanying PNS.MethodsWe enrolled patients with: nonmalignant lesions (n = 21), ovarian cancer (n = 19), lung cancer (n = 57), and PNS (n = 30). PBMC were isolated by density gradient centrifugation with Ficoll–Paque. We evaluated the expression of GrB in PBMC lysates by ELISA and normalized to protein content as measured by the Lowry method.ResultsGrB levels in PBMC in the group with malignant tumors—median 1650 pg/mg protein (interquartile range 663–3260 pg/mg) and in patients with PNS—median 1890 pg/mg protein (range 1290–2640 pg/mg) was lower than in control group with nonmalignant lesions—median 5240 pg/mg protein (range 2160–7440 pg/mg), p = 0.0003 and p = 0.0038, respectively. The differences in GrB levels in PBMC between these groups were independent of epidemiological factors—age, sex, body mass index (BMI), and the number of immune cells, as confirmed by multiple regression analysis. Within the group of patients with malignancy and PNS, GrB levels in PBMC were elevated if onconeural antibodies were detected (2610; 2390–3700 pg/mg protein) as compared to patients without antibodies (1680; 970–1880 pg/mg protein, p = 0.035). GrB in PBMC was higher if the malignancy was diagnosed at the low (3060; 2120–5220 pg/mg protein) as compared to the high stage (1330; 348–2140, p = 0.00048). In patients with lung cancer, the expression of GrB in PBMC was lower (1430; 635–2660 pg/mg protein) than in the group with ovarian cancer (2580; 1730–3730, p = 0.02).ConclusionThe cytotoxic response measured in peripheral blood by GrB in PBMC is impaired both in the course of malignancy and PNS. Levels of GrB in PBMC were higher if onconeural antibodies were detected. Tracking reactive immune responses, such as GrB in PBMC may have diagnostic and monitoring value in malignancy and PNS.

Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective Tcell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

Aberrantly Methylated and Expressed Genes as Prognostic Epigenetic Biomarkers for Colon Cancer.

This study aimed to identify prognostic epigenetic biomarkers for colon cancer (CC). Methylation and mRNA expression in CC samples with clinical characteristics that corresponded to those in The Cancer Genome Atlas were analyzed. Differentially methylated genes (DMGs) and differentially expressed genes (DEGs) were screened between matched tumor and nontumor tissues. Among the 415 DEGs and DMGs that significantly correlated between cytosine-phosphate-guanine (CpG) methylation and gene expression, unc-5 netrin receptor C (UNC5C), solute carrier family 35 member F (SLC35F)1, Ly6/Neurotoxin (LYNX)1, stathmin (STMN)2, slit guidance ligand (SLIT)3, cell adhesion molecule L1 like (CHL1), CAP-Gly domain containing linker protein family member 4 (CLIP4), transmembrane protein (TMEM) 255A, granzyme B (GZMB), and brain expressed X-Linked (BEX)1 were promising epigenetic biomarkers. Prediction was more accurate when models were based on the expression and/or methylation of GZMB rather than clinical stage. Comparisons of tissues with high or low GZMB expression significantly associated the DEGs with natural killer-mediated cytotoxicity, cytokine-cytokine receptor interactions, and chemokine signaling pathways. From among the 10 epigenetic biomarkers, GZMB might serve as a tumor suppressor and function in several immune-related pathways in CC. Prognostic models based on GZMB expression and/or methylation would be significant for patients with CC.

Immune-Related Gene Expression and Cytokine Secretion Is Reduced Among African American Colon Cancer Patients.

Background: Colorectal cancer is the third most deadly cancer among African Americans (AA). When compared to Caucasian Americans (CA), AA present with more advanced disease and lower survival rates. Here, we investigated if differences in tumor immunology could be contributive to disparities observed between these populations.Methods: We examined gene expression of tumor and non-tumor adjacent tissues from AA and CA by whole transcriptome sequencing, and generated scores for immune cell populations by NanoString. In addition, we utilized “The Cancer Genome Atlas” (TCGA) database from AA and CA as a validation cohort. Finally, we measured the secretion of cytokines characteristic of effector T helper cell (Th) subsets by ELISA using plasma from each AA and CA participant.Results: Colon tumors from AA patients showed significant fold-change increase in gene expression when compared to CA for FOXP3 (6.22 vs. 3.22), IL1B (103 vs. 11.4) and IL8 (220 vs. 28.9) (p < 0.05). In contrast, among CA we observed statistically higher gene expression of markers associated with antitumor activity such as GZMB (Granzyme B), IFNG and the immunotherapy targets PDL1 (CD274) and CTLA4 (p < 0.05). TCGA data validated our observed higher gene expression of GZMB and PDL1 in CA patients when compared to AA. Notably, our observations on immune cell populations show that AA tumors have significantly higher number of exhausted CD8+ cells (p < 0.01), mast cells (p < 0.02) and increased T regulatory cells when compared to CA. AA colon cancer patients differed from CA in cytokine production patterns in plasma (i.e., reduced IL-12).Conclusions: Our study demonstrates significant differences of the immunological profiles of colon tumors from AA compared to CA that suggest a deficiency of appropriate immune defense mechanisms in terms of gene expression, recruitment of immune cells and systemic secretion of cytokines. As such, these immune differences could be mitigated through population-specific therapeutic approaches.

Therapeutic efficacy and safety of a human fusion construct targeting the TWEAK receptor Fn14 and containing a modified granzyme B

BackgroundAntibody-drug conjugates are an exceptional and useful therapeutic tool for multiple diseases, particularly for cancer treatment. We previously showed that the fusion of the serine protease granzyme B (GrB), the effector molecule or T and B cells, to a binding domain allows the controlled and effective delivery of the cytotoxic payload into the target cell. The production of these constructs induced the formation of high molecular aggregates with a potential impact on the efficacy and safety of the protein.MethodsOur laboratory designed a new Fn14 targeted fusion construct designated GrB(C210A)-Fc-IT4 which contains a modified GrB payload for improved protein production and preserved biological activity. We assessed the construct’s enzymatic activity, as well as in vitro cytotoxicity and internalization into target cells. We also assessed pharmacokinetics, efficacy and toxicology parameters in vivo.ResultsGrB(C210A)-Fc-IT4 protein exhibited high affinity and selective cytotoxicity within the nanomolar range when tested against a panel of Fn14-positive human cancer cell lines. The construct rapidly internalized into target cells, activating the caspase cascade and causing mitochondrial membrane depolarization. Pharmacokinetic studies in mice revealed that GrB(C210A)-Fc-IT4 displayed a bi-exponential clearance from plasma with a fast initial clearance (t1/2α=0.36 hour) followed by a prolonged terminal-phase plasma half-life (t1/2β=35 hours). Mice bearing MDA-MB-231 orthotopic tumor xenografts treated with vehicle or GrB(C210A)-Fc-IT4 construct (QODx5) demonstrated tumor regression and long-term (>80 days) suppression of tumor growth. Treatment of mice bearing established, subcutaneous A549 lung tumors showed impressive, long-term tumor suppression compared with a control group treated with vehicle alone. Administration of GrB(C210A)-Fc-IT4 (100 mg/kg total dose) was well-tolerated by mice and resulted in significant reduction of tumor burden in a lung cancer patient-derived xenograft model. Toxicity studies revealed no statistically significant changes in aspartate transferase, alanine transferase or lactate dehydrogenase in treated mice. Histopathological analysis of tissues from treated mice did not demonstrate any specific drug-related changes.ConclusionGrB(C210A)-Fc-IT4 demonstrated excellent, specific cytotoxicity in vitro and impressive in vivo efficacy with no significant toxicity in normal murine models. These studies show GrB(C210A)-Fc-IT4 is an excellent candidate for further preclinical development.

A Potential Mechanism of Anticancer Immune Response Coincident With Immune-related Adverse Events in Patients With Renal Cell Carcinoma.

Some reports showed encouraging efficacy of immune checkpoint inhibitors among patients who experienced immune-related adverse events (irAEs). Thus, characterization of T-cell repertoire and immune signatures in peripheral blood mononuclear cells (PBMCs) and tumors before and after immune checkpoint inhibitors treatment should contribute to better understanding of irAE-provoked anticancer immune responses. We applied expression analysis of immune-related genes and T-cell receptor sequencing in tumor and PBMCs from five patients with renal cell carcinoma before combined immunotherapy and at the onset of severe irAEs. We found that the cluster of differentiation 8 (CD8)/forkhead box P3(FOXP3), granzyme B(GZMB)/CD3, perforin 1(PRF1)/CD3 and programmed cell death 1(PD1)/CD8 expression ratios were significantly elevated in PBMCs at the onset of irAEs. In addition, we found expansion of certain T-cell clones in metastatic tissue after irAEs, which were already increased in peripheral blood at the onset of irAEs. irAE-provoked T-cells may also circulate and attack distant tumors, leading to durable response in patients with irAEs.

GATA1/SP1 and miR-874 mediate enterovirus-71-induced apoptosis in a granzyme-B-dependent manner in Jurkat cells.

Enterovirus 71 (EV71)-induced T lymphocyte apoptosis plays an important role in hand, foot, and mouth disease (HFMD), and granzyme B (GZMB) has been shown to be critical for this process. However, the mechanisms underlying GZMB-mediated apoptosis of T lymphocytes remain unknown. In this study, we investigated whether transcription factors and microRNAs (miRNAs) are involved in GZMB-mediated apoptosis of T lymphocytes in response to EV71 infection. Our findings indicated that EV71 infection significantly induced apoptosis in Jurkat cells, a human T lymphocytes cell line, as revealed in flow cytometric analysis. Furthermore, EV71 increased the expression of pro-apoptosis Bcl-2-associated X (Bax) and cleaved caspase 3 but decreased the expression of anti-apoptosis B-cell lymphoma protein 2 (Bcl2). GZMB knockdown decreased cell apoptosis and prevented EV71-induced changes in the expression of Bax, cleaved caspase 3, and Bcl2 in Jurkat cells, highlighting the role of GZMB as a key factor in EV71-induced apoptosis. Our study also indicated that overexpression of the transcription factors GATA binding factor 1 (GATA1) and specificity protein 1 (SP1) significantly increased luciferase activity when this gene was inserted in the GZMB 3' untranslated region (3'UTR). GATA1/SP1 overexpression induced cell apoptosis, increased the expression of Bax and cleaved caspase 3, and decreased the expression of Bcl2. Finally, our results suggested that miR-874 plays an essential role in GZMB-mediated cell apoptosis, since an miR-874 mimic decreases the expression of GZMB by targeting its 3'UTR. Collectively, these data indicated that GATA1/SP1 and miR-874 mediate EV71-induced apoptosis in a granzyme B-dependent manner. This signaling pathway may provide a new pharmacological target for the prevention and treatment of HFMD.

Abstract 4479: Immune exhaustion of tumor microenvironment mediates crizotinib resistance in ALK rearranged advanced non small cell lung cancer

Abstract Introduction: Previous study showed the correlation between immune tumor microenvironment and the efficacy of checkpoint inhibitor in non-small cell lung cancer (NSCLC). This study evaluated whether biomarkers for immune tumor microenvironment including programmed death ligand 1 (PD-L1), cluster of differentiation 8 (CD8), Granzyme B (GZMB) and T cell receptor (TCR) could predict the treatment response to the first-line crizotinib of patients with ALK-rearranged non-small cell lung cancer (NSCLC). Methods: We retrospectively evaluated patients with advanced NSCLC who received crizotinib at Hunan Cancer Hospital between January 2016 and December 2018. The patients' baseline expression of PD-L1, CD8 and GZMB was evaluated in one slide using immunofluorescence. TCR was also evaluated with paired blood samples simultaneously. Results: All the 108 eligible patients were stratified according to their expression profile into PD-L1+/CD8+/GZMB+ (n=21), PD-L1-/CD8+/GZMB+ (n=42), PD-L1+/CD8-/GZMB- (n=23), and PD-L1-/CD8-/GZMB- (n=22) group. Patients with strong PD-L1 expression have a significantly inferior objective response rate (36.5% versus 72.2%, p = 0.002), and shorter progression-free survival (PFS) (5.6 versus 16.5 months, p &amp;lt; 0.001) as compared to patients with weak or negative PD-L1 expression. Among the four groups, we found patients with PD-L1-/CD8+/GZMB+ had the best response (85.7% versus 26%, p=?), while PD-L1+/CD8-/GZMB- showed primary resistance to crizotinib had the worse outcomes (4.6 versus 18 months, p &amp;lt; 0.001). Furthermore, for TCR analysis, the counts of TCR and Shannon index was strongly correlated with longer PFS for crizotinib treatment (20.6 versus 8.2months, p &amp;lt; 0.001; 19.3 versus 6.1 months, p &amp;lt; 0.001, respectively). However, the clonality acts as poor predictive marker with worse PFS (6.8 versus 18.2 months, p &amp;lt; 0.001). Finally, three patients with de novo resistance to crizotinib experienced a favorable response to anti-PD-1 therapy. Conclusions: This study revealed immune exhaustion for tumor microenvironment as a poor predictive marker in first-line crizotinib-treated ALK-rearranged non-small cell lung cancer. The findings indicate the reshaping of an inflamed immune phenotype characterized by PD-L1, CD8, GZMB, and TCR expression and suggest potential therapeutic sensitivity to PD-1 blockade. Citation Format: Yongchang Zhang. Immune exhaustion of tumor microenvironment mediates crizotinib resistance in ALK rearranged advanced non small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4479.

Abstract 2878: Granzyme B functionalized nanocarriers targeting membrane-bound Hsp70 for multimodal cancer therapies

Abstract Functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as potential clinical tools for cancer theranostics. Membrane-bound 70 kDa heat shock protein (mHsp70) is ubiquitously expressed on the cell membrane of various tumor types but not on normal cells and therefore provides a tumor-specific target. The serine protease granzyme B (GrB) that is produced as an effector molecule by activated T and NK cells has been shown to specifically target mHsp70 on tumor cells. Following binding to Hsp70, GrB is rapidly internalized into tumor cells inducing the process of cellular apoptosis. Decoration of the nanoparticles surface with GrB increased the targeting properties of the nanocarriers as was shown by confocal and electron microscopies. In series of in vivo studies it was demonstrated that GrB functionalized SPIONs act simultaneously as a contrast enhancement agent when being intravenously administered for magnetic resonance imaging and induce specific tumor cell apoptosis (human U87 glioblastoma in NMRI nu/nu mice, rat C6 glioma in Wistar rats, B16 melanoma in C57/Bl6 mice). Continuous prolonged administration of GrB-SPIONs via programmable subcutaneously implanted mini-pump system iPrecio further enhanced the therapeutic benefit of magnetic conjugates. Moreover, significant therapeutic response was achieved in immunocompromised mice models (advanced U87 brain tumor, small lung cancer H1339 with brain metastasis) with a high tumor burden. Combinatorial regimens employing stereotactic radiotherapy and/or magnetic targeting with immune checkpoint inhibitors (anti-CTLA-4 monoclonal antibodies) are found to further enhance the therapeutic efficacy of GrB-SPIONs in different tumor mouse models. The study was funded by RFBR, project № 19-08-00024; DFG grant SFB824/3, Germany, TaGoNaX DFG project № 336532926, STA1520/1-1. Citation Format: Maxim Shevtsov, Susanne Kaesler, Stefan Stangl, Luidmila Yakovleva, Ruslana Tagaeva, Yaroslav Marchenko, Boris Nikolaev, William Khachatryan, Oleg Galibin, Emil Pitkin, Gabriele Multhoff. Granzyme B functionalized nanocarriers targeting membrane-bound Hsp70 for multimodal cancer therapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2878.

Abstract 3091: Liquid biopsy reflecting a T-cell permissive tumor microenvironment identifies metastatic melanoma patients responding to immune checkpoint inhibitor therapy

Abstract Background: Non-invasive biomarkers for predicting response to immune checkpoint inhibitors (ICI) are urgently needed. Crucial for response to ICI's is a T-cell permissive tumor microenvironment (hot tumor), characterized by presence of activated T-cells and low fibrotic activity. Activated T-cells release the protease granzyme B (GzB), which can cleave type IV collagen during tumor infiltration. We evaluated the potential of measuring GzB generated type IV collagen degradation fragments in a liquid biopsy for identifying metastatic melanoma (MM) patients responding to ipilimumab. Methods: A monoclonal antibody was raised against a GzB generated neo-epitope on type IV collagen (C4G), and used to develop a technically robust competitive electro-chemiluminescence immunoassay (ECLIA). C4G was measured in serum from MM patients (n=54) before initiation of ipilimumab treatment. C4G was combined with the fibrosis biomarker PRO-C3, measuring type III collagen formation. Biomarker levels were associated with objective response rate (ORR) and overall survival (OS) outcomes. Results: The C4G assay was specific for a neo-epitope on type IV collagen degradation fragments generated by GzB. The ORR was 2.6 fold higher (18% vs 7%) in patients with high C4G levels (&amp;gt;25th percentile) vs low levels (≤25th percentile). Likewise, high C4G levels at baseline were associated with longer OS, with a median OS of 646 days vs 290 days for low C4G levels (HR=0.48, 95%CI: 0.24-0.98, p=0.045). When combining high C4G with low PRO-C3, the HR dropped to 0.30 (95%CI: 0.15-0.60, p=0.0006), and remained significant when adjusted for the covariates age, lactate dehydrogenase levels and prior treatment (HR: 0.35, 95%CI: 0.18-0.72, p=0.004). Conclusions: A liquid biopsy measuring granzyme B degraded type IV collagen (C4G) as a surrogate of active immune infiltration into the tumor microenvironment is associated with response to the ICI therapy ipilimumab. When combining C4G with the fibrosis biomarker PRO-C3, patients with this special phenotype - low fibrosis and high immune infiltration - have an even better chance of responding compared to high C4G levels alone. If validated, this suggests that specific collagen remodeling biomarkers (C4G+PRO-C3) have potential for predicting response to ICI's in clinical cancer trials. Citation Format: Christina Jensen, Dovile Sinkeviciute, Daniel H. Madsen, Patrik Önnerfjord, Morten Hansen, Henrik Schmidt, Inge Marie Svane, Morten A. Karsdal, Nicholas Willumsen. Liquid biopsy reflecting a T-cell permissive tumor microenvironment identifies metastatic melanoma patients responding to immune checkpoint inhibitor therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3091.

Abstract 5176: A novel, completely human fusion construct containing active Granzyme B efficiently kills Folate Receptor alpha positive tumor cells

Abstract Folate receptor alpha (FRα) is an attractive therapeutic target because it is highly overexpressed on several tumors such as ovarian, breast and lung cancers and demonstrates low or restricted distribution on normal tissues. In addition, FRα has been shown to play a tumor promoting role with its expression associated with poor prognosis of cancer patients. In our laboratory, we have generated two new molecules targeting FRα and containing active granzyme B (GrB) as the cytotoxic payload. We used as binding domains two different scFvs targeting the FRα from antibodies used in clinical trials. These human scFvs (designated mov003 and mov018) were fused to human GrB through an engineered IgG heavy-chain fragment (designated as Fc) which contains a dimerization domain. The constructs were cloned into the pSECTag vector containing an upstream 5 kDa purification tag with an enterokinase (EK) cleavage site for facile removal. The molecules were expressed in transiently-transfected HEK-293E suspension cells over 3 days at 37°C in 5% CO2. The molecules were isolated from culture media and purified by ion-exchange chromatography followed by EK cleavage to remove the free tag. The yields were 12-18 mg/L for both molecules. The assessment of the specific binding of each molecule to the antigen target is ongoing. The cytotoxicity of the constructs against a panel of cell lines expressing various levels of FRα showed a specific cytotoxic effect with an IC50 in the low nanomolar range (5-100 nM), particularly exhibiting very low IC50 values against breast (MDA-MB-231) and ovarian cell lines (IGROV-1). Receptor negative cells demonstrated IC50 levels in the micromolar range. Based on IC50 values, these data demonstrate suggest the fusion constructs targeting FRα are efficiently internalized and effectively deliver the active serine protease to the cytosol, activating the cytotoxic cascade. In vitro stability assays over 96 hours showed that both constructs are highly stable in mouse serum. Immunofluorescence and confocal studies to examine the time course of internalization and the pro-apoptotic events triggered by these agents are under examination and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez de Cienfuegos, Laurence H. Cheung, Khalid A. Mohamedali, Walter N. Hittelman, Michael G. Rosenblum. A novel, completely human fusion construct containing active Granzyme B efficiently kills Folate Receptor alpha positive tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5176.

Abstract 6616: Functional hotness score generated by representative functional cytotoxic T-lymphocytes predicts long-term survival of triple-negative breast cancer independently to the tumor mutational burden

Abstract Background: Cytotoxic T-lymphocytes (CTLs) infiltration in a bulk tumor is a positive prognostic factor in breast cancer. CTL recognition is believed to react to mutation-induced neoantigens, thus, higher tumor mutation burden (TMB) is considered as an important predictor of tumor immunogenicity and response to immunotherapy. Although majority of breast cancer have low tumor mutation burden (TMB), triple-negative breast cancer (TNBC) has higher TMB compared to other breast cancer subtypes. However, it is unclear if the prognostic value high CTL infiltration depends on the TMB as the primary driver of enhanced anti-cancer immunity or represents an independent prognostic marker which can be used as a complement to TMB to predict patients' outcomes. CTLs are identified by CD8 marker, is encoded by CD8A. Granzyme B (GZMB) is a serine protease that is secreted by functionally-active CTLs to induce apoptosis of the target cells. CXCL10 is a chemokine which selectively attracts activated CTLs into subsets of other cancers; but its role in breast cancer remains unknown. We investigated if TNBC infiltrated with high levels of functional CTLs, so called “hot tumors”, have improved survival independently of their TMB. Methods: Utilizing The Cancer Genome Atlas (TCGA) breast cancer cohort, we established Functional Hotness Score (FHS), based on the CD8A, GZMB and CXCL10 gene expression levels of bulk tumors. Total of 4149 breast cancer patients from publicly available multiple cohorts were analyzed to assess FHS and breast cancer patient prognosis as well as distinct immunity markers. Results: FHS predicted the breast cancer patient survival better than each consisting gene. The breast cancer patients with high FHS tumors showed significantly better survival in the testing cohort (TCGA), which was further confirmed in the validation cohort (METABRIC). FHS was higher in the primary breast cancer tissues with metastasis compared to that without metastasis in TCGA. Further, FHS was higher in the metastatic tumors compared to the primary tumors in GSE110590. Among breast cancer subtypes, TNBC showed highest FHS compared to other subtypes in testing TCGA, which was validated in the METABRIC cohort. The patients with high FHS tumor demonstrated significantly better long-term survival than that with low FHS only in the hormone receptor (HR)-negative breast cancers, but not in the HR-positive tumors. The high FHS TNBCs showed higher not only CD8-positive T cell infiltration, but also a broader type-1 anti-cancer immunity. To investigate if FHS predicts patient survival independent of TMB, the patients were divided into high and low TMB groups. Interestingly, the higher FHS patients showed better prognosis not only in the high TMB group but also in the low TMB among TNBC patients. The combination of high TMB with high FHS identified the unique subset of the patients who did not recur over time. Conclusion: In conclusion, TNBC with higher FHS based on the expression levels of CD8A, GZMB and CXCL10 showed improved prognosis with higher anti-cancer immunity regardless of TMB, and constituting an independent prognostic marker of survival, particularly when combined with TMB. Citation Format: Eriko Katsuta, Li Yan, Mateusz Opyrchal, Pawel Kalinski, Kazuaki Takabe. Functional hotness score generated by representative functional cytotoxic T-lymphocytes predicts long-term survival of triple-negative breast cancer independently to the tumor mutational burden [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6616.

Abstract 5194: Development of a cytotoxic fusion protein targeting VEGF receptors with improved cytotoxicity, stability and pharmacokinetics

Abstract The serine protease granzyme B (GrB), a key effector in immune mediated cell killing is capable of inducing apoptosis through both caspase-dependent and caspase-independent multiple-cascade mechanisms. VEGF121 is a naturally-occurring isoform of the VEGF-A family and binds to VEGF receptors R-1 and R-2, which are over-expressed on tumor cells as well as the endothelium of tumor vasculature but not normal vasculature. We have previously reported the expression of a fusion protein composed of Granzyme B and VEGF121 (GrB/VEGF121) and its subsequent in vitro and in vivo characterization. We hypothesized that incorporation of an IgG heavy chain Fc-fragment, resulting in a relatively higher molecular weight (133 kDa vs 80 kDa), would improve in vivo circulation and efficacy. Accordingly, the VEGF inter-chain dimerization domains were mutated so that dimerization occurred only through the Fc domain. Transient expression of GrB-Fc-VEGF121 in HEK-293E cells under serum-free conditions, followed by purification by immobilized metal affinity chromatography, resulted in yields in the range of 20-25 mg/L. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK293E cells. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. Cytotoxicity studies against a panel of tumor and endothelial cells indicated improved cytotoxicity against cell lines expressing high levels of VEGFR-1 or VEGFR-2, unlike GrB/VEGF121, which targeted VEGFR-2+ cells more efficiently. Control (receptor-negative) cells demonstrated IC50 levels in the high micromolar range. Comparison of ex vivo stability of GrB-Fc-VEGF121 to GrB/VEGF121 over 96 hours indicated GrB/VEGF121 signal reduced by approximately 50% over the first 24 hours of incubation, but minimal loss of protein was observed thereafter for the duration of the study. On the other hand, the protein loss of GrB-Fc-VEGF121 was more gradual, while the overall loss of protein over 96 hours was similar to that of GrB/VEGF121. Thus, GrB-Fc-VEGF121 is very stable in mouse serum and further pharmacokinetic analysis is warranted to determine its stability in vivo. In vivo pharmacokinetics and efficacy against various tumor models are currently ongoing and will be presented. In summary, the GrB-Fc-VEGF121 produced in HEK293E cells is active and has significant potential as a targeted therapeutic. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana A. Alvarez-Cienfuegos, Michael G. Rosenblum. Development of a cytotoxic fusion protein targeting VEGF receptors with improved cytotoxicity, stability and pharmacokinetics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5194.

Abstract 2891: Targeting breast cancer stem cells with anti-EMP2 immunotoxins delivering granzyme b

Abstract Despite new therapies, breast cancer remains one of the leading causes of cancer-related death in women worldwide. Part of the issue remains the inability of these therapies to target cancer stem cells (CSCs). These cells show unique resistance to chemotherapy and have enhanced capacity for self-renewal. Recently, our group has shown that Epithelial membrane protein-2, or EMP2, is expressed on CSCs defined by increased aldehyde dehydrogenase activity (ALDH+). Clinical data supports the role of EMP2 in advanced breast cancer as increased expression of the protein is observed in metastatic lesions compared to primary tumors and its expression is enhanced in chemotherapy resistant tumors. In parallel, EMP2 expression correlated with enhanced mammosphere formation, an accepted surrogate for cells with enriched stem cell capacity. We have recently developed a novel IgG1 monoclonal antibody to EMP2, which has been shown to reduce tumor load. As the antibody-antigen complex rapidly internalizes, we hypothesized that its anti-tumor effects could be enhanced through the creation of a fusion protein. Granzyme B (GrB) is a serine protease that plays a critical role in the body's defense against viral infection and tumor development by initiating the apoptotic cascade via both caspase-dependent and -independent mechanisms. We developed novel fusion proteins composed of the anti-EMP2 backbone as the targeting moiety and granzyme B as the cytotoxic payload. Our initial design comprised of the active GrB fused to the IgG heavy chain (Fc) domain containing 2 single-chain antibodies against EMP2, a format that results in a longer plasma retention time. This new construct showed high and specific affinity for EMP2 (10−10M) using both flow cytometry and ELISA based formats. Moreover, treatment with these agents induced cell death to EMP2 positive cells, including those characterized by high ALDEFLUOR activity. Preclinical assessment of the anti-EMP2 immunotoxins showed no acute toxicity and acceptable plasma retention. Preliminary results using a 4T1 model system produced a significant reduction in tumor burden. Our results suggest that continued development of these agents may be successful at targeting advanced disease. Research supported, in part, by NCI R21 CA234642 and the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Kok Su-yin, Lawrence H. Cheung, Naina Dhawan, Michael G. Rosenblum, Madhuri Wadehra. Targeting breast cancer stem cells with anti-EMP2 immunotoxins delivering granzyme b [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2891.

Abstract 3618: Mutational analysis of colon tumors from African American patients and potential association with cancer disparities

Abstract Background: Colorectal cancer is the third most deadly cancer among African Americans (AA). When compared to Caucasian Americans (CA), AA present with more advanced disease and lower survival rates. We have previously demonstrated that colon tumors from AA and CA differ in their immune cell recruitment and their systemic cytokines secretion profiles. Therefore, in this study we investigated if differences in gene expression and mutational profiles could relate to the disparities observed between these populations. Methods: We examined gene expression of colon tumor and non-tumor adjacent tissues from AA (n=20) and CA (n=20) by whole transcriptome sequencing (Illumina); and determined the differential expression of the 170 genes of the Illumina's TruSight Tumor 170 panel (TST170) in AA (n=12) and CA (n=17) colon tumor and non-tumor adjacent tissues. Results: From our gene expression studies, we found that colon tumors from AA patients showed significant fold-change increases in gene expression when compared to CA for FOXP3 (6.97 vs. 3.15), IL1B (122 vs. 14) and IL8 (262 vs. 28) (p &amp;lt; 0.05). In contrast, among CA we observed statistically higher gene expression of markers associated with antitumor activity such as GZMB (Granzyme B), IFNG and immunotherapy targets PDL1 (CD274) and CTLA4 (p &amp;lt; 0.05). Through our mutational profiling we identified 38 genes in AA and 6 in CA differentially expressed between tumor and non-tumor tissues (p &amp;lt; 0.01). Of those, 5 genes associated with several roles in cancer ETV4 (poor prognosis), CCND1 (tumorigenesis), FGFR2 (activation of the RAS-MAPK and the PI3K-AKT pathways) BRCA2 (genome stability), BCL2 (apoptosis regulator) were commonly altered in both cohorts. Conclusions: Our study demonstrates significant differences in the genetic characteristics of colon tumors from AA compared to CA that suggest a deficiency of appropriate immune defense features in terms of gene expression. We also observed divergence in the mutational profiling between the groups, including numerous genes that are indicative of prognosis, treatment and outcomes in colon cancer. As such, these deficits could be mitigated through population-specific therapeutic approaches. Citation Format: Jenny Paredes, Jone Garai, Li Li, Melody Baddoo, Ping Ji, Sayed Imtiaz, Marzia Spagnardi, Mubarak Akadri, Raavi Gupta, Mohamed Alshal, Maksim Agaronov, Henry Talus, Jennie L. Williams, Laura Martello-Rooney, Jovanny Zabaleta. Mutational analysis of colon tumors from African American patients and potential association with cancer disparities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3618.

Secretome analysis reveals upregulated granzyme B in human androgen-repressed prostate cancer cells with mesenchymal and invasive phenotype.

Epithelial-mesenchymal transition (EMT) is a critical early step in cancer metastasis and a complex process that involves multiple factors. In this study, we used proteomics approaches to investigate the secreted proteins (secretome) of paired human androgen-repressed prostate cancer (ARCaP) cell lines, representing the epithelial (ARCaP-E) and mesenchymal (ARCaP-M) phenotypes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses showed high levels of proteins involved in bone remodeling and extracellular matrix degradation in the ARCaP-M cells, consistent with the bone metastasis phenotype. Furthermore, LC-MS/MS showed a significantly higher level of the serine protease granzyme B (GZMB) in ARCaP-M conditioned media (CM) compared to that of ARCaP-E. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to detect mRNA and Western blot to detect protein expression, we further demonstrated that the GZMB gene was expressed by ARCaP-M and the protein was secreted extracellularly. ARCaP-M cells with GZMB gene knockdown using small interfering RNA (siRNA) have markedly reduced invasiveness as demonstrated by the Matrigel invasion assay in comparison with the scrambled siRNA negative control. This study reports that GZMB secretion by mesenchymal-like androgen-repressed human prostate cancer cells promotes invasion, suggesting a possible extracellular role for GZMB in addition to its classic role in immune cell-mediated cytotoxicity.

HBV-specific CD8 T cells present higher TNF-α expression but lower cytotoxicity in hepatocellular carcinoma.

Tumor necrosis factor (TNF)-α is largely regarded as a proinflammatory cytokine, but several recent researches have demonstrated that TNF-α could possess immunoregulatory roles with potential to suppress anti-tumor immunity. Chronic hepatitis B virus (HBV) infection is a major risk factor of hepatocellular carcinoma (HCC), and HBV-specific CD8 T cells could exert anti-tumor roles in HCC patients. Here, we found that HBV-specific CD8 T cells, both in the peripheral blood and in the tumor microenvironment, were more enriched with TNF-α-expressing cells than interferon (IFN)-γ-expressing cells. Compared to IFN-γ-expressing HBV-specific CD8 T cells, TNF-α-expressing HBV-specific CD8 T cells presented lower expression of inhibitory checkpoint molecules, including programmed cell death (PD)-1, T cell immunoglobulin mucin-3 (TIM-3) and cytotoxic T lymphocyte antigen (CTLA)-4. HBV-specific CD8 T cells could mediate the lysis of autologous primary tumor cells, and the inhibition of TNF-α could further elevate their cytotoxic capacity. Subsequently, we demonstrated that TNF-α inhibition in HBV-specific CD8 T cells could significantly increase granzyme B (GZMB) and perforin 1 (PRF1) expression while having no effect towards granzyme A (GZMA) expression. The addition of exogenous TNF-α at low levels had no consistent effect on the expression of GZMA, GZMB and PRF1, but at higher levels, exogenous TNF-α significantly reduced GZMA, GZMB and PRF1 expression. Overall, these results suggested that TNF-α-expressing cells probably presented a deleterious role in HCC but were enriched in HBV-specific CD8 T cells.

Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes

Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.