Activity Of Granzyme Research Articles

Granzyme mRNA-miRNA interaction and its implication to functional impact.

Granzyme activity can affect the processing and stability of miRNAs within target cells. They also could induce changes in miRNA expression that impact apoptotic signaling. Granzyme-induced apoptosis might result in changes to the miRNA profile, which can further influence the apoptosis and inflammation processes. The aim of this study was to bioinformatically analyze which miRNAs and transcription factors bind to the CDS and UTR regions of the granzyme family to regulate gene expression in relation to granzyme evolution and their association with human cancer diseases. The expression patterns of granzyme genes were analyzed in various human tissues. MiRNAs binding to the CDS and UTR of the granzyme family were examined, and the transcription factors binding to these miRNAs binding sites were also analyzed. Cytoscape program was used to visualize and analyze the networks of interactions between granzyme mRNA and miRNAs. Additionally, the evolutionary patterns of the granzyme family in relation to miRNAs and transcription factors binding were investigated. Analysis of the expression patterns of the granzyme family in various human tissues shows that GZMA and GZMK are strongly expressed in lymph nodes. GZMB exhibits strong expression in the bone marrow, while GZMA is prominently expressed in the spleen. Twenty-two miRNAs bind to both GZMK and GZMB mRNA, while six miRNAs bind to both GZMK and GZMM mRNA. The only miRNA that binds to GZMK, GZMB, GZMM, and GZMA mRNA is hsa-miR-146a-5p. Transcription factors JUND, FOS, and JUN are distinctly interconnected with has-miR-5696 and GZMK. Association data between the granzyme family and cancers showed that various miRNAs were consistently implicated and exhibited either upregulation or downregulation. Although the granzyme family possesses distinct genetic information, it shows relatively high expression levels in the lymph node, spleen, and bone marrow. Many miRNAs specifically regulate granzyme gene expression, and various transcription factors are involved. Analyzing the granzyme genes-miRNAs-transcription factors-related network will provide crucial insights into the mechanisms of cancer development and suppression.

Selective Detection of Active Extracellular Granzyme A by Using a Novel Fluorescent Immunoprobe with Application to Inflammatory Diseases.

Granzymes (Gzms), a family of serine proteases, expressed by immune and nonimmune cells, present perforin-dependent and independent intracellular and extracellular functions. When released in the extracellular space, GzmA, with trypsin-like activity, is involved in the pathophysiology of different inflammatory diseases. However, there are no validated specific systems to detect active forms of extracellular GzmA, making it difficult to assess its biological relevance and potential use as a biomarker. Here, we have developed fluorescence-energy resonance-transfer (FRET)-based peptide probes (FAM-peptide-DABCYL) to specifically detect GzmA activity in tissue samples and biological fluids in both mouse and human samples during inflammatory diseases. An initial probe was developed and incubated with GzmA and different proteases like GzmB and others with similar cleavage specificity as GzmA like GzmK, thrombin, trypsin, kallikrein, or plasmin. After measuring fluorescence, the probe showed very good specificity and sensitivity for human and mouse GzmA when compared to GzmB, its closest homologue GzmK, and with thrombin. The specificity of this probe was further refined by incubating the samples in a coated plate with a GzmA-specific antibody before adding the probe. The results show a high specific detection of soluble GzmA even when compared with other soluble proteases with very similar cleavage specificity like thrombin, GzmK, trypsin, kallikrein, or plasmin, which shows nearly no fluorescence signal. The high specific detection of GzmA was validated, showing that using pure proteins and serum and tissue samples from GzmA-deficient mice presented a significant reduction in the signal compared with WT mice. The utility of this system in humans was confirmed, showing that GzmA activity was significantly higher in serum samples from septic patients in comparison with healthy donors. Our results present a new immunoprobe with utility to detect extracellular GzmA activity in different biological fluids, confirming the presence of active forms of the soluble protease in vivo during inflammatory and infectious diseases.

Granzymes in health and diseases: the good, the bad and the ugly.

Granzymes are a family of serine proteases, composed of five human members: GA, B, H, M and K. They were first discovered in the 1980s within cytotoxic granules released during NK cell- and T cell-mediated killing. Through their various proteolytic activities, granzymes can trigger different pathways within cells, all of which ultimately lead to the same result, cell death. Over the years, the initial consideration of granzymes as mere cytotoxic mediators has changed due to surprising findings demonstrating their expression in cells other than immune effectors as well as new intracellular and extracellular activities. Additional roles have been identified in the extracellular milieu, following granzyme escape from the immunological synapse or their release by specific cell types. Outside the cell, granzyme activities mediate extracellular matrix alteration via the degradation of matrix proteins or surface receptors. In certain contexts, these processes are essential for tissue homeostasis; in others, excessive matrix degradation and extensive cell death contribute to the onset of chronic diseases, inflammation, and autoimmunity. Here, we provide an overview of both the physiological and pathological roles of granzymes, highlighting their utility while also recognizing how their unregulated presence can trigger the development and/or worsening of diseases.

P017 An activity-based chemiluminescence assay targeting granzyme A for the detection of Inflammatory Bowel Diseases

Abstract Background Inflammatory Bowel Diseases (IBD) represent a pressing global health concern lacking precise diagnostic tools for accurate patient stratification. Conventional methods, such as colonoscopy are extremely invasive whilst generic inflammatory markers (such as CRP or Calprotectin), are not specific enough and pose limitations in assessing disease activity, urging the need for novel, non-invasive diagnostic approaches which can rapidly inform clinicians on the inflammatory status of a patients bowels. Methods Our study focused on developing a non-invasive chemiluminescence assay targeting active granzyme A (GzmA) in stool supernatants. Employing a multifaceted approach encompassing functional, chemical, and imaging assays on human cells and gut tissue biopsies, we discerned elevated GzmA levels predominantly secreted by CD8+ T cells in inflamed gut tissues. Results The design of GzmA-INH, a specific inhibitor for GzmA, underscored the prevalence of active extracellular GzmA and it’s role in driving inflammation in the guts - revealing promising potential as a novel IBD biomarker. Further, we synthesized GzmA-CL, a highly reactive and selective chemiluminescent reporter for GzmA. Integrating GzmA-CL into a robust antibody-coated 96-well plate assay (Figure 1a), we analyzed 30 IBD biosamples along with healthy controls. Notably, substantially higher GzmA levels were observed in IBD patients, with discernible distinctions between ulcerative colitis (UC) and Crohn’s disease (CD) (Figure 1b). These levels correlated positively with established biomarkers, including faecal calprotectin and immune cell counts. Conclusion Our investigation highlighted active extracellular GzmA as a noteworthy biomarker in gut inflammation. The rapid, selective, and sensitive nature of our chemiluminescence assay presents promising prospects for precise IBD diagnosis and advancements in personalized medicine.

Immunomodulatory Effects of Modified Colostrum, Whey, and Their Combination With Other Natural Products: Effects on Natural Killer Cells

ObjectivesNatural killer (NK) cells are important immune system effector cells providing innate defenses against intracellular infections, including viral infections, immune surveillance, and cancer immunoediting. The primary purpose of this study was to investigate whether modified ultra-filtrated colostrum (UC) and hydrolyzed whey (W) products or their combinations with other natural products with reported immunomodulatory properties will stimulate NK cell cytotoxic activity by activation of granzyme B and IFN-γ production. MethodsThe ability of study products to stimulate the cytotoxic activity of human-purified CD56+ NK cells and the production of granzyme B and IFN-γ by activated NK cells was evaluated in the cytotoxic assay. ResultsAll study products significantly increased NK-cell cytotoxic activity at an E: T ratio of 20:1. Treatment of cells with UC had a maximal cytotoxic effect at the minimal dose of 10 µg/ml, which exceeded the cytotoxic activity of IL-2. In contrast, the addition of egg yolk (CE) or CE + botanical blend (CEB) to UC resulted in a dose-dependent cytotoxic response with a maximal response at 1000 µg/ml. The maximal activity of blend products was comparable to UC activity. W exerted minimal stimulatory activity on NK cells. The magnitude of granzyme B and IFN-γ production was closely associated with the cytotoxic activity of NK cells stimulated with the study products. ConclusionsAll study products demonstrated stimulatory activity on NK cells, with UC having a maximal effect on NK cell cytotoxicity. The study products can be used as dietary supplements to support NK cell activity in healthy individuals.

Phorbol-12-myristate-13-acetate is a potent enhancer of B cells with a granzyme B+ regulatory phenotype.

The infusion of ex-vivo-generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including graft-versus-host disease. Previously, we developed a protocol for the generation of a novel population of regulatory B cells, which are characterized by secretion of enzymatically active granzyme B (GraB cells). This protocol uses recombinant interleukin 21 (IL-21) and goat-derived F(ab)'2 fragments against the human B cell receptor (anti-BCR). Generally, the use of xenogeneic material for the manufacturing of advanced therapy medicinal products should be avoided to prevent adverse immune reactions as well as potential transmission of so far unknown diseases. In the present work we demonstrated that phorbol-12-myristate-13-acetate (PMA/TPA), a phorbol ester with a particular analogy to the second messenger diacylglycerol (DAG), is a potent enhancer of IL-21-induced differentiation of pre-activated B cells into GraB cells. The percentage of GraB cells after stimulation of pre-activated B cells with IL-21 and PMA/TPA was not significantly lower compared to stimulation with IL-21 and anti-BCR. Given that PMA/TPA has already undergone encouraging clinical testing in patients with certain haematological diseases, our results suggest that PMA/TPA may be a safe and feasible alternative for ex-vivo manufacturing of GraB cells.

Abstract 6314: Novel granzyme B-based fusion constructs targeting FRα on liquid and solid tumors

Abstract Folate receptor α (FRα) has been an attractive therapeutic target for many decades because it is highly overexpressed on several solid tumors and demonstrates low or restricted distribution on healthy tissues. Additionally, high expression of FRα has been associated with tumor progression and thus poor prognosis of cancer patients. Therefore, utilizing FRα to effectively locate tumor cells for targeted therapy using antibody drug conjugates, small molecule drug conjugates, radioimmunoconjugates or chimeric antigen receptor T cells, has been extensively investigated. Although some of the mentioned targeted therapies have shown promising preclinical results, their clinical utility has been limited due to associated toxicities in healthy tissues. We generated two new fusion constructs targeting FRα and containing active human granzyme B (GrB) as the cytotoxic payload. We used as binding domains two different scFvs targeting the FRα from antibodies previously used in clinical trials. These human scFvs (mov003 and mov018) were fused to the human serine protease GrB through an engineered human IgG heavy-chain fragment (designated as Fc) which contains a dimerization domain for longer half-life. 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 and ovarian cell lines. FRα negative cells demonstrated IC50 levels in the micromolar range. Immunofluorescence studies demonstrated that the fusion constructs targeting FRα are efficiently internalized and effectively delivered the active serine protease to the cytosol. In vitro stability assays over 96 hours showed that both constructs are highly stable in mouse serum. Additionally, recent publications suggested that FRα is significantly associated with acute myeloid leukemia (AML) and might serve as a therapeutic target. We assessed the cytotoxic efficacy of GrB-based constructs on a panel of FRα+ AML cell lines and have found that these cell lines were very sensitive (IC50 in the low nanomolar range). Furthermore, evaluation of GrB fusion efficacy in two AML cell lines resistant to Bcl-2 inhibitor Venetoclax showed IC50s to be similar or lower than their parental cell lines. These results, therefore, demonstrate no cross-resistance to GrB. The safe and effective profile of these drugs together with their unique mechanism of action makes them ideal candidates for advancing pre-clinical development and may offer a potential new strategy for treatment of solid tumors and particularly Venetoclax relapsed AML patients, ineligible for intensive chemotherapy. In vivo efficacy studies to compare the two constructs are warranted and will help to identify the optimal therapeutic for further clinical consideration. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Natalia Baran, Marina Konopleva, Michael G. Rosenblum. Novel granzyme B-based fusion constructs targeting FRα on liquid and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6314.

Abstract 6316: In vitro and in vivo studies of human granzyme B fusion constructs targeting mesothelin

Abstract Mesothelin is highly expressed on tumor cells and promotes tumor growth and drug resistance. Pancreatic, ovarian, mesothelioma, and lung cancers highly express mesothelin and are cancers of unmet needs with ≤16% relative 5-year survival using standard of care. When delivered to tumor cells by immune effector cells, granzyme B (GrB) is a highly cytotoxic serine protease which induces apoptosis through both caspase-dependent and -independent mechanisms. We developed a fusion protein (GrB-Fc-SD1) composed of human GrB and containing SD1, a novel human single-domain antibody that uniquely targets Region III of the glycoprotein, mesothelin. The SD1 and GrB domains are connected by a human IgG heavy chain fragment which enables dimerization. Thus, the increase in total construct molecular weight leads to an increased in vivo circulation time and anti-tumor efficacy. GrB-Fc-SD1 was transiently expressed in HEK-293E cells under serum-free conditions and purified to homogeneity to a final yield of approximately 5 mg/L. Anti-GrB western blot analysis confirmed the expression of GrB-Fc-SD1 exclusively as a dimer. Comparison of GrB-Fc-SD1 with its expected molecular weight suggests significant glycosylation when produced in HEK-293E cells. The enzymatic activity of granzyme B in GrB-Fc-SD1 was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against a limited panel of lung, pancreatic and ovarian cancer cell lines expressing surface mesothelin indicate cytotoxicity in the nanomolar range, while cytotoxicity against mesothelin-negative cells was at micromolar levels. GrB-Fc-SD1 bound specifically to mesothelin extra-cellular domain compared to a non-specific control, as determined by ELISA. Biacore analysis confirmed binding with a dissociation constant between 10−7 to 10−8 M. These data warrant continued investigation of the protein as a therapeutic candidate. Accordingly, ex vivo serum stability studies are underway. In addition, screening of an expanded panel of tumor cell lines for in vitro cytotoxicity is currently in progress and will be reported. In vivo efficacy studies against nude mice bearing Capan-2 xenograft models are currently underway and will also be reported. Research supported by H2Biologics. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Louis R. DePalatis, Claire Thuning-Roberson, Michael G. Rosenblum. In vitro and in vivo studies of human granzyme B fusion constructs targeting mesothelin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6316.

Neuronal Serpina3n is an endogenous protector against blood brain barrier damage following cerebral ischemic stroke

Ischemic stroke results in blood-brain barrier (BBB) disruption, during which the reciprocal interaction between ischemic neurons and components of the BBB appears to play a critical role. However, the underlying mechanisms for BBB protection remain largely unknown. In this study, we found that Serpina3n, a serine protease inhibitor, was significantly upregulated in the ischemic brain, predominantly in ischemic neurons from 6 hours to 3 days after stroke. Using neuron-specific adeno-associated virus (AAV), intranasal delivery of recombinant protein, and immune-deficient Rag1−/− mice, we demonstrated that Serpina3n attenuated BBB disruption and immune cell infiltration following stroke by inhibiting the activity of granzyme B (GZMB) and neutrophil elastase (NE) secreted by T cells and neutrophils. Furthermore, we found that intranasal delivery of rSerpina3n significantly attenuated the neurologic deficits after stroke. In conclusion, Serpina3n is a novel ischemic neuron-derived proteinase inhibitor that counterbalances BBB disruption induced by peripheral T cell and neutrophil infiltration after ischemic stroke. These findings reveal a novel endogenous protective mechanism against BBB damage with Serpina3n being a potential therapeutic target in ischemic stroke.

Decoding the Immune Landscape of Pathogenic T Cells in Acute Graft-Versus-Host Disease By Single-Cell RNA-Seq

Introduction Graft-versus-host disease (GvHD) is the major cause of non-relapse mortality in allogeneic hematopoietic cell transplantation (alloHCT), and about 40-60% cases would progress into steroid refractory (SR) aGvHD resulting in dismal outcomes. The pathophysiology of aGvHD is complex, and donor derived T cells are identified as the culprits of aGvHD. However, the relationships among distinct T cell subsets and molecular mechanisms underlying aGvHD remain incompletely understood. Methods We employed a single-cell RNA sequencing (scRNA-seq) and T cell receptor (TCR) sequencing (scTCR-seq) to profile 49288 T cells in peripheral blood isolated from alloHCT recipients developed into aGvHD (n=9) or not (as control, n=5). Then, samples from SR-aGvHD patients were further investigated to explore the molecular mechanism of pathogenic T cell subsets in SR-aGvHD. Results A total of thirteen unique T cell subsets with distinct molecular properties were identified, including four clusters of CD8+ T cells (CD8+ TN, CD8+ TCM, CD8+ TEM and CD8+ TEFF), five clusters of CD4+ T cells (CD4+ TN, CD4+ TCM, Th1, Th17 and Treg), NK cell cluster and three clusters of other T cells (IFN-T, γδT and MAIT). The composition shifted towards more-developed CD8+ TCM, CD8+ TEM, CD4+ TCM and Th1 cells in aGvHD group due to rapid cell cycling. The expansion of CD8+ TEM subpopulation was confirmed by multiparameter flow cytometry. The CD8+ TEFF and Th1 were considered to be pathogenic in aGvHD as they exhibited aGvHD-related cytokine/granzyme expression and signaling pathway activation. Upregulated genes in aGvHD-derived-Th1 cells were related to chemotaxis (CCL3, CXCR6), cytokine production (type I interferon, IL-23) and JAK-STAT signaling activation. CD8+ TEFF cells showed activity of chemotaxis (CCL3, CXCR6), granzyme and cytokine expression (GZMB, GZMA, TNF-α, IL-6, IL-10, IL-12) and MAPK/MTOR/NF-kappaB activation. The different composition of cytokine expression and activated signaling pathways suggested distinct functions of each subset. Furthermore, CD8+ TEM represented four traits related to a range of T-cell differentiation stages, spanning rapid cell cycling (transcription factor E2F family), chemotaxis (CCL3, CXCR6), cytotoxicity (GZMA, GZMB, and GZMK) and exhaustion (PDCD1, LAG3, HAVCR2 and TIMD4), suggesting CD8+ TEM as a transient stage which might further develop into CD8+ TEFF for aGvHD pathogenesis (Figure 1). To elucidate the molecular mechanism of SR-aGvHD, we dissected the composition and functional heterogeneity of T-cell subsets between SR- and steroid sensitive (SS)-aGvHD. CD8+ TEM expanded to be the largest subset which resulted in the increase of CD8+ TEFF proportion in SR-aGvHD. CD8+ TEM and CD8+ TEFF showed enrichment of GvHD pathogenic scores and up-regulated expression of inflammation transcription factors STAT1 and STAT3 in SR-aGvHD. Of note, the expression of steroid receptor NR3C1 was downregulated in CD8+ TEM subset, as well as the steroid target gene ANXA1, compared with counterparts. Furthermore, both CD8+ TEM and CD8+ TEFF cells exhibited enrichment of JAK-STAT, MAPK, NF-kappaB, and PI3K-AKT signaling activation (Figure 2). Conclusions The effector T-cell subsets CD8+ TEFF and Th1 were co-activated and undertook distinct expression profiles and signaling pathways, which suggests their different roles in aGvHD pathogenesis. CD8+ TEM, characterized by proliferative, cytotoxicity, chemotaxis and exhaustion capacities, provided functional repertoire for CD8+ TEFF. Reduced NR3C1 expression and activation of by-pass survival pathways of CD8+ TEM might provide persistent differentiation origin of CD8+ TEFF to mediate resistance to steroid therapy. Collectively, our data described the immune landscape of aGvHD and provided novel therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD8+ TEMRA Clones Cause Platelet Lysis in Immune Thrombocytopenia

Background Immune thrombocytopenia (ITP) is traditionally thought of as an antibody-mediated disease. However, there are a number of features to suggest alternative mechanisms of platelet destruction. In this study we use a multi-dimensional approach to explore the role of terminally differentiated effector memory CD8+ T cells (TEMRA). Methods We characterised 83 adults with chronic ITP and 30 age-matched controls using immunophenotyping, next generation sequencing of T cell receptor (TCR), single cell (sc) RNA sequencing and functional assays. Next generation DNA sequencing of TCRβ was carried out using the Illumina MiSeq platform. Analysis of the raw TCR sequences was performed using MiXCR. Single cell immune profiling (Gene expression and TCR) was done using Chromium Single Cell V(D)J and 5' Library kits from 10x Genomics. The constructed library was sequenced on the HiSeq platform (Illumina). Seurat v.2.3.4 was used for gene expression analysis. Interactions between CD8+ T cells and platelets were measured by co-culturing CD8+ T cells with autologous platelets overnight and by perfusing them on the autologous platelet covered VenaFluoro8+ microchip channel. Results We found an expansion of CD8+ CD45RA+ CD62L- TEMRA cells expressing IFN gamma, TNF alpha and granzyme B, with no evidence of physiological exhaustion (failure to upregulate TIM-3 or PD-1 expression) in ITP patients which correlated with disease activity. DNA TCR sequencing revealed that expansion of TEMRA cells in ITP was associated with reduced T cell repertoire diversity (p ≤ 0.0001) and lower Simpson's diversity index (p ≤ 0.05). Patients with ITP had a higher number of T cell clones occupying more than 5% of the T cell repertoire when compared to age matched controls (p ≤ 0.05). Expanded clones were not shared across ITP patients and displayed no known antigen specificities when compared to available databases of TCR sequences, providing evidence for the lack of virally driven clonal expansion. Clonally expanded CD8+ T cells persisted over many years in ITP patients and characterised disease severity. Longitudinal samples from 9 patients showed expanded clones and reduced T cell repertoire diversity during active disease (platelet count of less than 30 x 109/L) compared to stable disease (platelet count greater or equal to 30 x 109/L) (p ≤ 0.01; r = 0.56 - Figure 1 shows an example patient). To better characterise CD8+ T cells, we performed paired scRNA and scTCR sequencing on isolated whole CD8+ T cells from patients with active and stable disease. Paired sequencing enables transcriptomic characterisation of T cell clones. ScRNA sequencing revealed four distinct clusters in CD8+ T cells: naïve, TEMRA, central memory (CM) and natural killer T cells (NKT). By mapping scTCR CD8+ sequencing to single cell gene expression, we found that the largest expanded clones showed an aggregative distribution, indicating transcriptional homogeneity and were almost exclusively comprised of TEMRA cells (Figure 2). Consistent with immunophenotyping and DNA TCR sequencing, expanded TEMRA cell clones were expanded in active disease compared to stable disease. To assess the impact of these CD8+ T cells on platelets in ITP, PBMCs from patients with ITP and HC were passed across a platelet layer at flow rates consistent with venous blood flow. CD8+ T cells in ITP patients interacted with platelets 4x more frequently than HCs. Isolated CD8+ T cells were cultured over night with autologous platelets to further explore these interactions: CD8+ T cell:platelet aggregates were identified more frequently in the co-cultures from patients with ITP (p ≤ 0.05) and were inhibited by blocking MHC Class I (p ≤ 0.01). CD8+ T cells within the aggregates had increased CD107a expression compared to non-aggregated CD8+ T cells (p ≤ 0.01), indicating cytolytic activity and release of Granzyme and Perforin. Correspondingly, platelets in the aggregates showed increased CD62P expression, a marker of activation and impending death (p ≤ 0.001). Conclusions Here we demonstrate clonal expansion of disease-associated TEMRA CD8+ T cells in ITP. These cells bind to platelets and cause TCR-mediated platelet activation and death providing an antibody-independent mechanism of platelet destruction. This could serve as biomarker to direct treatment in patients with refractory ITP and targeting specific T-cell clones could be a novel therapeutic option in refractory ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

346 (PB126) - Preventing cystatin F proteolytic activation by cathepsin V increases cytotoxicity of effector immune cells

Background: Granule-dependent cytotoxicity of natural killer cells and cytotoxic T lymphocytes is regulated by the proteolytic network. Cysteine cathepsins C, H and L are needed for the activation of granzymes and perforin, which initiate the killing of target cancer cells. Activity of cathepsins C, H and L is inhibited by cystatin F. Therefore, cystatin F can negatively impact the cytotoxicity of immune cells. Activity of cystatin F is regulated by expression levels, N-glycosylation and proteolytic activation.

Mechanistic and pharmacodynamic studies of DuoBody-CD3x5T4 in preclinical tumor models.

CD3 bispecific antibodies (bsAbs) show great promise as anticancer therapeutics. Here, we show in-depth mechanistic studies of a CD3 bsAb in solid cancer, using DuoBody-CD3x5T4. Cross-linking T cells with tumor cells expressing the oncofetal antigen 5T4 was required to induce cytotoxicity. Naive and memory CD4+ and CD8+ T cells were equally effective at mediating cytotoxicity, and DuoBody-CD3x5T4 induced partial differentiation of naive T-cell subsets into memory-like cells. Tumor cell kill was associated with T-cell activation, proliferation, and production of cytokines, granzyme B, and perforin. Genetic knockout of FAS or IFNGR1 in 5T4+ tumor cells abrogated tumor cell kill. In the presence of 5T4+ tumor cells, bystander kill of 5T4- but not of 5T4-IFNGR1- tumor cells was observed. In humanized xenograft models, DuoBody-CD3x5T4 antitumor activity was associated with intratumoral and peripheral blood T-cell activation. Lastly, in dissociated patient-derived tumor samples, DuoBody-CD3x5T4 activated tumor-infiltrating lymphocytes and induced tumor-cell cytotoxicity, even when most tumor-infiltrating lymphocytes expressed PD-1. These data provide an in-depth view on the mechanism of action of a CD3 bsAb in preclinical models of solid cancer.

Abstract 337: Development of an immune-oncology agent containing granzyme B to target VEGFR+ ovarian cancer cells and activated tumor vasculature

Abstract Granzyme B is a key effector in immune-mediated cell killing by inducing apoptosis through both caspase-dependent and -independent mechanisms. We developed a fusion protein (GrB-Fc-VEGF121) composed of granzyme B, VEGF121 (a naturally-occurring isoform of the VEGF-A family that binds to VEGF receptors R-1 and R-2), and a human IgG heavy chain fragment (for dimerization and to augment in vivo circulation and anti-tumor efficacy). GrB-Fc-VEGF121 was transiently expressed in HEK-293E cells under serum-free conditions purified with a final yield of approximately 40 mg/L. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK-293E cells. The enzymatic activity of granzyme B in GrB-Fc-VEGF121 was comparable to that of commercially available human granzyme B. GrB-Fc-VEGF121 readily internalized into VEGFR-2+ cells within 2 hours of treatment while untargeted granzyme B was not internalized, demonstrating that internalization was receptor-mediated. Cytotoxicity studies against a panel of tumor and endothelial cells indicated cytotoxicity in the nanomolar range against cell lines expressing high levels of VEGFR-1 or VEGFR-2 while control (receptor-negative) cells demonstrated IC50 levels in the high micromolar range. Treatment of VEGFR-2+ endothelial and tumor cells with GrB-Fc-VEGF121 resulted in robust cell death via apoptosis and/or necrosis. Ex vivo serum stability of GrB-Fc-VEGF121 indicated a gradual protein loss of GrB-Fc-VEGF121, with an overall loss of about 50% over 96 hours. The maximum tolerated dose of GrB-Fc-VEGF121 was not reached when BALB/c mice were treated with a total 475 mg/kg given every other day over 5 doses. Treatment of mice bearing OVCAR8 tumor xenografts with GrB-Fc-VEGF121 (100 mg/kg over 5 doses) resulted in significant growth inhibition of established tumors compared to vehicle controls. Treated tumors showed a significant decrease in the number of CD31+ blood vessels and Ki-67+ proliferating tumor cells compared to controls, when assessed at the end of the study. These studies clearly indicate that GrB-Fc-VEGF121 has significant anti-tumor effects in vivo with virtually no toxicity against normal tissues at doses 4-5 times higher than therapeutic levels. The tumor- and vascular-targeted agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Development of an immune-oncology agent containing granzyme B to target VEGFR+ ovarian cancer cells and activated tumor vasculature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 337.

Abstract 3439: An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts

Abstract Carcinoembyronic antigen (CEA) is a cell surface protein normally found at low levels in adult tissues but over-expressed in numerous solid tumors. CEA is constitutively released from tumor cells and reaches detectable levels in peripheral blood, making CEA targeting particularly challenging. Granzyme B is a key effector in immune-mediated killing of target cells, with cell death through both caspase-dependent and -independent mechanisms. We developed a fusion protein composed of granzyme B and a human scFv targeting the juxta-membrane epitope of CEA. The construct also incorporates a fragment of human IgG heavy chain to cause dimerization and provide increased in vivo circulation and antitumor efficacy. The GrB-Fc-CEA construct was cloned into a mammalian expression vector, transiently expressed by HEK-293E suspension cells, and secreted into the culture media. The final yield after purification and activation was 10-15 mg/L. The enzymatic activity of granzyme B in GrB-Fc-CEA was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against CEA-positive cell lines demonstrated cytotoxicity in the nanomolar range, compared to micromolar range cytotoxicity against control (CEA-negative) cell lines. Importantly, GrB-Fc-CEA cytotoxicity against tumor cells in vitro was unaffected by exogenously-added CEA (200 ng/ml) extracellular domain, suggesting that the construct is not competitively absorbed by soluble CEA released by tumors and present in the circulation of patients. GrB-Fc-CEA internalized rapidly (within one hour) into the target cell lines A-431 and HT-1080/CEA whereas internalization was not observed with untargeted granzyme B, clearly demonstrating receptor-mediated internalization. GrB-Fc-CEA induced mitochondrial depolarization and caspase activation, consistent with the well-described granzyme B mechanism of action. Treatment of female nude mice bearing A-549 tumors with GrB-Fc-CEA (80 mg/kg over 5 doses) resulted in a significant growth inhibition of the established tumors compared to vehicle controls (1200 mm3 vs <40 mm3). Complete tumor regression was observed in seven of ten tumors in mice treated with GrB-Fc-CEA. Mouse weight was unaffected in both treated and control groups for the duration of the study, suggesting no apparent toxicity of the construct. Thus, GrB-Fc-CEA appears to have significant potential in targeting a large category of CEA-expressing solid tumors and is a promising candidate for further pre-clinical development. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Lawrence H. Cheung, Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3439.

Multiparameter Longitudinal Imaging of Immune Cell Activity in Chimeric Antigen Receptor T Cell and Checkpoint Blockade Therapies.

Longitudinal multimodal imaging presents unique opportunities for noninvasive surveillance and prediction of treatment response to cancer immunotherapy. In this work we first designed a novel granzyme B activated self-assembly small molecule, G-SNAT, for the assessment of cytotoxic T lymphocyte mediated cancer cell killing. G-SNAT was found to specifically detect the activity of granzyme B within the cytotoxic granules of activated T cells and engaged cancer cells in vitro. In lymphoma tumor-bearing mice, the retention of cyanine 5 labeled G-SNAT-Cy5 correlated to CAR T cell mediated granzyme B exocytosis and tumor eradication. In colorectal tumor-bearing transgenic mice with hematopoietic cells expressing firefly luciferase, longitudinal bioluminescence and fluorescence imaging revealed that after combination treatment of anti-PD-1 and anti-CTLA-4, the dynamics of immune cell trafficking, tumor infiltration, and cytotoxic activity predicted the therapeutic outcome before tumor shrinkage was evident. These results support further development of G-SNAT for imaging early immune response to checkpoint blockade and CAR T-cell therapy in patients and highlight the utility of multimodality imaging for improved mechanistic insights into cancer immunotherapy.

A fluorogenic probe for granzyme B enables in-biopsy evaluation and screening of response to anticancer immunotherapies

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies.

Hypoxia-driven immunosuppression by Treg and type-2 conventional dendritic cells in HCC.

Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated. We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light, immunohistochemistry, and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets, including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DRlo ) type 2 conventional dendritic cell (cDC2), were found to be significantly enriched in hypoxia-high tumor regions. On the other hand, the abundance of active granzyme Bhi PD-1lo CD8+ T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DRlo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2. We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular, we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies.

Human CD4+ T-Cell Clone Expansion Leads to the Expression of the Cysteine Peptidase Inhibitor Cystatin F.

The existence of CD4+ cytotoxic T cells (CTLs) at relatively high levels under different pathological conditions in vivo suggests their role in protective and/or pathogenic immune functions. CD4+ CTLs utilize the fundamental cytotoxic effector mechanisms also utilized by CD8+ CTLs and natural killer cells. During long-term cultivation, CD4+ T cells were also shown to acquire cytotoxic functions. In this study, CD4+ human T-cell clones derived from activated peripheral blood lymphocytes of healthy young adults were examined for the expression of cytotoxic machinery components. Cystatin F is a protein inhibitor of cysteine cathepsins, synthesized by CD8+ CTLs and natural killer cells. Cystatin F affects the cytotoxic efficacy of these cells by inhibiting the major progranzyme convertases cathepsins C and H as well as cathepsin L, which is involved in perforin activation. Here, we show that human CD4+ T-cell clones express the cysteine cathepsins that are involved in the activation of granzymes and perforin. CD4+ T-cell clones contained both the inactive, dimeric form as well as the active, monomeric form of cystatin F. As in CD8+ CTLs, cysteine cathepsins C and H were the major targets of cystatin F in CD4+ T-cell clones. Furthermore, CD4+ T-cell clones expressed the active forms of perforin and granzymes A and B. The levels of the cystatin F decreased with time in culture concomitantly with an increase in the activities of granzymes A and B. Therefore, our results suggest that cystatin F plays a role in regulating CD4+ T cell cytotoxicity. Since cystatin F can be secreted and taken up by bystander cells, our results suggest that CD4+ CTLs may also be involved in regulating immune responses through cystatin F secretion.

Gold Nanoparticles and Graphene Oxide Flakes Enhance Cancer Cells' Phagocytosis through Granzyme-Perforin-Dependent Biomechanism.

The study aimed to investigate the roles of gold nanoparticles (GNPs) and graphene oxide flakes (GOFs) as phagocytosis enhancers against cancer cells. The nanomaterials were characterized through SEM and UV-VIS absorptions. The GNPs and GOFs increased the macrophages’ phagocytosis ability in engulfing, thereby annihilating the cancer cells in both in vitro and in vivo conditions. The GNPs and GOFs augmented serine protease class apoptotic protein, granzyme, passing through the aquaporin class protein, perforin, with mediated delivery through the cell membrane site for the programmed, calibrated, and conditioned cancer cells killing. Additionally, protease inhibitor 3,4-dichloroisocoumarin (DCI) significantly reduced granzyme and perforin activities of macrophages. The results demonstrated that the GOFs and GNPs increased the activation of phagocytic cells as a promising strategy for controlling cancer cells by augmenting the cell mortality through the granzyme-perforin-dependent mechanism.