Khalid A. Mohamedali

Background: Granzyme B (GrB) is a key effector molecule, delivered by cytotoxic T lymphocytes and natural killer cells during immune surveillance to induce cell death. Fusion proteins and immunoconjugates represent an innovative therapeutic approach to specifically deliver a deadly payload to target cells. Epithelial membrane protein-2 (EMP2) is highly expressed in invasive breast cancer (BC), including triple-negative BC (TNBC), and represents an attractive therapeutic target. Methods: We designed a novel fusion protein (GrB-Fc-KS49) composed of an active GrB fused to an anti-EMP2 single-chain antibody tethered through the immunoglobulin G heavy chain (Fc) domain. We assessed the construct’s GrB enzymatic activity, anti-EMP2 binding affinity, and cytotoxicity against a panel of BC cells. The construct’s pharmacokinetics (PK), toxicity profile, and in vivo efficacy were also evaluated. Results: GrB-Fc-KS49 exhibited comparable GrB enzymatic activity to commercial GrB, as well as high affinity to an EMP2 peptide, with the dissociation constant in the picomolar range. The fusion protein rapidly internalized into EMP2+cancer cells and showed in vitro cytotoxicity to cell lines expressing surface EMP2, with half-maximal cytotoxicity (IC₅₀) values below 100 nM for most positive lines. Ex vivo stability at 37°C indicated a half-life exceeding 96 hours while in vivo PK indicated a biexponential plasma clearance, with a moderate initial clearance (t_1/2α=18.4 hours) and a much slower terminal clearance rate (t_1/2β=73.1 hours). No toxicity was measured in a Chem16 panel between the control and the GrB-Fc-KS49. In vivo, the GrB-Fc-KS49 showed efficacy against a TNBC syngeneic (4T1/_FLuc) mouse model, reducing tumor volume and cell proliferation and increasing cell death compared with controls. Treatment using an EMT6 mouse model confirmed these results. In addition to a significant impact on cell proliferation, GrB-Fc-KS49 treatment also resulted in a dramatic increase of tumor-infiltrating CD45+ cells and redistribution of tumor-associated macrophages. Transcriptomic analysis of tumors post-treatment confirmed the remodeling of the immune tumor microenvironment by the GrB-Fc-KS49 immunotoxin. Conclusions: GrB-Fc-KS49 showed high specificity and cytotoxicity towards EMP2-positive cells. In vivo, it reduced tumor burden and increased the recruitment of immune cells into the tumor, suggesting that GrB-Fc-KS49 is a promising therapeutic candidate against BC.

Supplementary Figures - PDF file 791K, Supplementary Figure 1. Representative image showing selective detection of CD-31+ pixels using MetaMorph 7.7 imaging software. The original image is shown in Figure 6B. Supplementary Figure 2. Original whole gel images of data depicted in Figure 1B. Lanes shown in Figure 1B are depicted by an asterisk. Gel 1: elution profile of Granzyme B/VEGF121 from HEK-293T conditioned media. Purity greater than 95% was obtained. Gel 2: Asterisked SDS-PAGE lanes show GrB/VEGF121 under reducing and non-reducing conditions, and show that the majority of the purified protein is a natural homodimer. Supplementary Figure 3. Total VEGFR-2 expression assessed by Western blot analysis (top) and flow cytometry (bottom). Corresponding western blot lanes for VEGFR-2 and Actin are numerically indicated. VEGFR-2 expression in bEND.3 cells was tested using both methods, and has been previously reported as 2 x 105 receptors per cell1. Supplementary Figure 4. Internalization of GrB/VEGF121 into PAE/VEGFR-1 is much lower than into PAE/VEGFR-2 cells. Cells were treated with 20 nM GrB/VEGF121 for 24 h, followed by removal of cell surface-bound protein as described in Methods. Cells were treated with mouse anti-GrB monoclonal antibody followed by a goat anti-mouse AF488 antibody. 1. Veenendaal LM, Jin H, Ran S, Cheung L, Navone N, Marks JW, Waltenberger J, Thorpe P, Rosenblum MG. In vitro and in vivo studies of a VEGF121/rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors. Proc Natl Acad Sci U S A 2002;99:7866-71.

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.

We previously demonstrated that a human fusion protein construct targeting the Fn14 receptor and containing the cytotoxic granzyme B (GrB) payload (GrB-Fc-IT4) displays impressive in vitro and in vivo cytotoxic effects. Comprehensive mechanism of action studies show that GrB-Fc-IT4 effectively induces cell death in vitro against a broad selection of tumor types with high specificity. The intracellular pathway includes irreversible activation of the caspase cascade and independent mitochondrial depolarization leading to an intense apoptotic cellular damage. This activation occurs quickly and efficiently. Pharmacokinetic studies in mice showed that GrB-Fc-IT4 was cleared bi-exponentially from plasma with a rapid initial clearance (t _½alpha = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t _1/2beta = 35 hours) similar to that of IgGs. Based on our pharmacokinetic study, we employed a QODX5 therapy schedule and demonstrated impressive antitumor activity against several tumor models including PDX-lung, A459 (NSCLC) and MDA-MB231(TNBC) tumor models. In the present study we compared the in vivo antitumor efficacy of GrB-Fc-IT4 against A549 (NSCLC) tumor cell line, using two different dosing schedules (QODx5 vs QWx5). There was a clear dose and schedule dependence and that treating mice bearing A549 tumors with 32mg/kg/dose using a QOWx5 schedule induced a complete tumor growth inhibition (7/10 tumors regressed) with 3/10 tumors showing no growth up to 50 days after the last dose. Preliminary results suggest that GrB-Fc-IT4 may reduce the formation of spontaneous A549 metastasis into lungs. Further studies are planned to examine the potential of the fusion construct to prevent spontaneous metastatic spread in lung tumor models. Although the fusion construct cross-reacts with the murine Fn14 antigen, maximum tolerated dose studies have confirmed that GrB-Fc-IT4 is a safe product, showing no evidence of toxicity (weight loss) in mice treated with up to 500mg/kg. This suggests that the therapeutic index (TI) for this class of agents is >4. Our data suggest that GrB-Fc-IT4 is a novel class of antitumor agents with a unique mechanism of action. This agent seems to be an exceptionally safe and effective drug and appears to be an ideal candidate for advancing to clinical trials. Research conducted, in part, by Clayton Foundation for Research. Citation Format: Ana Alvarez De Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Mihai Gagea, Michael G. Rosenblum. Optimizing antitumor efficacy of granzyme B human fusion constructs targeting the Fn14 antigen in solid tumors. Pre IND pharmacokinetics, schedule, dose optimization and MTD studies [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 5216.

Immunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade. We designed a recombinant fusion construct, GrB-Fc-4D5, composed of a humanized anti-HER2 scFv fused to active GrB for recognition of tumor cells and internal delivery of GrB, simulating T and B cell therapy. We assessed the construct’s antigen-binding specificity and GrB enzymatic activity, as well as in vitro cytotoxicity and internalization into target and control cells. We also assessed pharmacokinetic and toxicology parameters in vivo. GrB-Fc-4D5 was highly cytotoxic to Her2 positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC50 values of 56, 99 and 27 nM, respectively, and against a panel of HER2+ cell lines regardless of endogenous expression levels of the PI-9 inhibitor. Contemporaneous studies with Kadcyla demonstrated similar levels of in vitro activity against virtually all cells tested. GrB-Fc-4D5 internalized rapidly into target SKOV3 cells within 1 h of exposure rapidly delivering GrB to the cytoplasmic compartment. In keeping with its relatively high molecular weight (160 kDa), the construct demonstrated a terminal-phase serum half-life in mice of 39.2 h. Toxicity studies conducted on BALB/c mice demonstrated no statistically significant changes in SGPT, SGOT or serum LDH. Histopathologic analysis of tissues from treated mice demonstrated no drug-related changes in any tissues examined. GrB-Fc-4D5 shows excellent, specific cytotoxicity and demonstrates no significant toxicity in normal, antigen-negative murine models. This construct constitutes a novel approach against HER2-expressing tumors and is an excellent candidate for further development.

Angiogenesis is a critical process in numerous diseases, and intervention in neovascularization has therapeutic value in several disease settings, including ocular diseases, arthritis, and in tumor progression and metastatic spread. Various vascular targeting agents have been developed, including those that inhibit growth factor receptor tyrosine kinases, blocking antibodies that interfere with receptor signal transduction, and strategies that trap growth factor ligands. Limited anti-tumor efficacy studies have suggested that the targeted delivery of the human pro-apoptotic molecule Granzyme B to tumor cells has significant potential for cancer treatment. Here, we review biological vascular targeting agents, and describe a unique vascular targeting agent composed of Granzyme B and the VEGF receptor ligand VEGF₁₂₁. The fusion protein GrB/VEGF₁₂₁ demonstrates cytotoxicity at nanomolar or sub-nanomolar levels, excellent pharmacokinetic and efficacy profiles, and has significant therapeutic potential targeting tumor vasculature.

The cytokine TWEAK and its receptor, Fn14, have emerged as potentially valuable targets for cancer therapy. Granzyme B (GrB)–containing Fn14-targeted constructs were generated containing either the Fn14 ligand TWEAK (GrB-TWEAK) or an anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both constructs showed high affinity and selective cytotoxicity against a panel of Fn14-expressing human tumor cells including triple-negative breast cancer (TNBC) lines. Cellular expression of the GrB inhibitor PI-9 in target cells had no impact on the cytotoxic effect of either construct. Cellular expression of MDR1 showed no cross-resistance to the fusion constructs. GrB-TWEAK and GrB-Fc-IT4 activated intracellular caspase cascades and cytochrome c–related proapoptotic pathways consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing established HT-29 xenografts with GrB-TWEAK showed significant tumor growth inhibition compared with vehicle alone (P < 0.05). Both GrB-TWEAK and GrB-Fc-IT4 displayed significant tumor growth inhibition when administered to mice bearing orthotopic MDA-MB-231 (TNBC) tumor xenografts. The Cancer Genome Atlas analysis revealed that Fn14 mRNA expression was significantly higher in TNBC and in HER2-positive disease (P < 0.0001) compared with hormone receptor–positive breast cancer, and in basal-like 2 tumors (P = 0.01) compared with other TNBC molecular subtypes. IHC analysis of a 101 patient TNBC tumor microarray showed that 55 of 101 (54%) of tumors stained positive for Fn14, suggesting that this may be an excellent potential target for precision therapeutic approaches. Targeting Fn14 using fully human, GrB-containing fusion constructs may form the basis for a new class of novel, potent, and highly effective constructs for targeted therapeutic applications. Mol Cancer Ther; 13(11); 2688–705. ©2014 AACR.

The fusion protein VEGF121/rGel composed of the growth factor VEGF121 and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF121/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo.

Purpose: A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF₁₂₁/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting nontumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Experimental Design: Cells from MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa, were injected into the femurs of mice. Osteoblastic progression was monitored following systemic administration of VEGF₁₂₁/rGel. Results: VEGF₁₂₁/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF₁₂₁/rGel internalization into osteoblasts was VEGF₁₂₁ receptor driven. Furthermore, VEGF₁₂₁/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, VEGF₁₂₁/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomographic analysis revealed that VEGF₁₂₁/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non–tumor-bearing) femurs. VEGF₁₂₁/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF₁₂₁/rGel-treated mice had significantly less tumor burden than control mice. Our results thus indicate that VEGF₁₂₁/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Conclusions: Targeting VEGF receptor (VEGFR)-1- or VEGFR-2–expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa. Clin Cancer Res; 17(8); 2328–38. ©2011 AACR.