Abstract Prostate cancer is the most common type of cancer in men. Immunotherapies such as Sipuleucel-T have shown that stimulating the immune system to target the prostate is a viable therapeutic option. Inovio is a clinical-stage biotechnology company that uses DNA vaccines as a novel immunotherapy strategy. An advantage to plasmid DNA vaccine therapy is the ability to encode adjuvants within the vaccine in order to increase immunogenicity and efficacy. We conducted an adjuvant screen in BALB/c mice with 25 candidate plasmid encoded genetic adjuvants in combination with the prostate cancer specific tumor-associated antigen STEAP1. Antigen-specific T cell responses were measured by IFNg ELISpot. The screen revealed that the addition of a plasmid encoding dendritic cell (DC)-activating Fms-like tyrosine kinase 3 ligand (Flt3L) fused to an Fc domain, significantly increased antigen-specific T cells (p<0.001, 2.8 fold). Mice treated with Flt3L-Fc had an enhanced immune response to STEAP1 vaccination as early as 7 days post dose 1 (p<0.01, 2.4 fold), which developed into an enhanced memory response measured 12 weeks later (p<0.001, 8.5 fold). Flow cytometry showed that Flt3L-Fc increased DC populations at the site of injection and at the draining lymph node 8 days following the initial vaccination. DCs are considered the most potent antigen-presenting cells in the immune system and DCs at the tumor site have been shown to be critical for T cell immunity. Our data shows that enhancing DC populations and function through the use of a genetic adjuvant can enhance the immunogenicity of a DNA cancer vaccine. Future studies will assess the efficacy of Flt3L-Fc in combination with STEAP1 in a mouse tumor model.
The epidermal growth factor receptor (EGFR) is both amplified and frequently mutated in glioblastoma (GBM). While downstream inhibition of EGFR signaling through small molecule tyrosine kinase inhibitors (TKIs) has shown little clinical efficacy, monoclonal antibody targeting is proving to be a promising alternative. The monoclonal antibody 806 (mAb806) specifically binds the most common EGFR mutant, EGFRvIII, as well as amplified WT-EGFR, but not EGFR expressed at low levels. The selectivity of mAb806 is thought to be due to its targeting a tumor-specific epitope of EGFR which is only accessible as the receptor transitions between an inactive tethered conformation to an active ligand-bound dimer. To address this question, we modeled several point mutations of the EGFR ectodomain (ECD) which naturally occur in GBM patients. Using molecular dynamics simulations we show that G63R and R108K expose the mAb806 epitope by way of their extreme flexibility at the domain I-II hinge region. In vitro analyses of these mutants reveals an increased ability to bind mAb806 in glioma cell lines. Furthermore, these point mutations and others were found to increase tumorigenicity in vivo. Examining downstream signaling pathways revealed similarities in signaling between the ECD deletion mutants EGFRvIII and EGFRvII while the individual point mutants R108K, G63R, A289V and G598V signal similarly to WT-EGFR. We go on to show that although treatment with a TKI before mAb806 increases binding to U87 parental and WT-EGFR expressing cells, and thus will likely prove to have increased toxicity in patients, forcing the kinase domain of EGFR into an active vs. inactive state had surprisingly different effects on mAb806 exposure based on its mutational status. Thus, the ECD mutants may be used as a tool to increase the understanding of how EGFR structure affects function and ultimately oncogenicity.
Overcoming tolerance to tumor-associated antigens remains a hurdle for cancer vaccine-based immunotherapy. A strategy to enhance the anti-tumor immune response is the inclusion of adjuvants to cancer vaccine protocols. In this report, we generated and systematically screened over twenty gene-based molecular adjuvants composed of cytokines, chemokines, and T cell co-stimulators for the ability to increase anti-tumor antigen T cell immunity. We identified several robust adjuvants whose addition to vaccine formulations resulted in enhanced T cell responses targeting the cancer antigens STEAP1 and TERT. We further characterized direct T cell stimulation through CD80-Fc and indirect T cell targeting via the dendritic cell activator Flt3L-Fc. Mechanistically, intramuscular delivery of Flt3L-Fc into mice was associated with a significant increase in infiltration of dendritic cells at the site of administration and trafficking of activated dendritic cells to the draining lymph node. Gene expression analysis of the muscle tissue confirmed a significant up-regulation in genes associated with dendritic cell signaling. Addition of CD80-Fc to STEAP1 vaccine formulation mimicked the engagement provided by DCs and increased T cell responses to STEAP1 by 8-fold, significantly increasing the frequency of antigen-specific cells expressing IFNγ, TNFα, and CD107a for both CD8+ and CD4+ T cells. CD80-Fc enhanced T cell responses to multiple tumor-associated antigens including Survivin and HPV, indicating its potential as a universal adjuvant for cancer vaccines. Together, the results of our study highlight the adjuvanting effect of T cell engagement either directly, CD80-Fc, or indirectly, Flt3L-Fc, for cancer vaccines.
Ciro Zanca1, Genaro R. Villa1,2,3, Jorge A. Benitez1, Amy Haseley Thorne1, Tomoyuki Koga1, Matteo D'Antonio4, Shiro Ikegami1, Jianhui Ma1, Antonia D. Boyer1, Afsheen Banisadr1, Nathan M. Jameson1, Alison D. Parisian1, Olesja V. Eliseeva5, Gabriela F. Barnabe1, Feng Liu1,6,7,8, Sihan Wu1, Huijun Yang1, Jill Wykosky1, Kelly A. Frazer4,9,10, Vladislav V. Verkhusha11, Maria G. Isaguliants5,12,13, William A. Weiss14,15,16, Timothy C. Gahman1, Andrew K. Shiau1, Clark C. Chen4, Paul S. Mischel1,4,17, Webster K. Cavenee1,4,18 and Frank B. Furnari1,4,17 1Ludwig Institute for Cancer Research, La Jolla, California 92093, USA; 2Department of Molecular and Medical Pharmacology, School of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA; 3Medical Scientist Training Program, School of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA; 4Moores Cancer Center, University of California at San Diego, La Jolla, California 92093, USA; 5Gamaleya Research Center of Epidemiology and Microbiology, Moscow 123098, Russian Federation; 6National Research Center for Translational Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 7State Key Laboratory of Medical Genomics, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 8Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 9Institute for Genomic Medicine, University of California at San Diego, La Jolla, California 92093, USA; 10Department of Pediatrics, Rady Children's Hospital, Division of Genome Information Sciences, University of California at San Diego, La Jolla, California 92093, USA; 11Department of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA; 12Department of Microbiology, Tumor, and Cell Biology, Karolinska Institutet, Stockholm 17177, Sweden; 13Department of Research, Riga Stradins University, Riga LV-1007, Latvia; 14Department of Neurology, Brain Tumor Research Center, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, San Francisco, California 94159, USA; 15Department of Pediatrics, Brain Tumor Research Center, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, San Francisco, California 94159, USA; 16Department of Neurosurgery, Brain Tumor Research Center, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, San Francisco, California 94159, USA; 17Department of Pathology, School of Medicine, University of California at San Diego, La Jolla, California 92093, USA; 18Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, California 92093, USA Corresponding author: ffurnari{at}ucsd.edu
Significance EGFR cancer mutations display an astonishing tissue-specific asymmetry: in lung cancer, mutations target the intracellular kinase (KD), while in glioblastomas (GBMs), a variety of missense clusters and deletions concentrate at the ectodomain (ECD). Intriguingly, GBM-activating mutations share a paradoxical preference for inhibitors that bind the inactive kinase. By integrating simulations, small-angle X-ray scattering, and GBM models, we demonstrate that ECD mutants converge to a transition state characterized by a cryptic epitope, allosterically coupled to an intermediate kinase, and synergistically blocked by antibodies and inhibitors. Our findings indicate that apparently heterogeneous aberrations remove a similar steric restrain on KD activation. The diversity of structural tricks in ECD mutants to achieve the same conformational state constitutes a potent example of molecular mimicry and convergence.
Abstract ASP-1929 photoimmunotherapy (PIT), an investigational drug-device combination, consists of an epithelial growth factor receptor (EGFR)-targeting drug, cetuximab, conjugated to a light-activatable dye, IRDye® 700DX, and a laser light. Localized illumination results in rapid and selective tumor necrosis in the preclinical setting, and early clinical studies demonstrate a manageable safety profile in patients with head and neck squamous cell carcinoma. In the ongoing clinical trials, however, edema is a commonly reported adverse event and laryngeal edema, requiring prophylactic tracheostomy, has been noted in some patients. Thus, a critical need exists to provide clinicians with tools to manage ASP-1929 PIT-associated edema to ensure optimal patient outcome. To evaluate edema following cancer-targeted PIT, we developed a mouse tumor model and used it to characterize the mechanism of edema development and evaluate various methods for edema reduction. Briefly, cohorts of mice bearing syngeneic LL/2 tumors engineered to express Ephrin A2 (EphA2) were administered an anti-EphA2 antibody conjugated to IR700 (conjugate) or saline control. Light was applied 24h following administration and edema was measured by caliper. In control mice, light alone did not generate edema; however, mice treated with conjugate plus light showed a light-dose dependent increase in edema volume which peaked at 6h post light delivery. We next evaluated a series of inflammatory cytokines and immune cell populations in the blood and tumor region at the onset (2h), peak (6h), and resolution (24h) of PIT-induced edema. We found a striking increase in neutrophils (500-fold greater than control mice, n=10, p<0.0001) in the blood at the peak of edema formation coinciding with a significant increase in IL-6 (n=5, p<0.001) and IL-10 (n=5, p<0.05), indicating the onset of a heightened inflammatory response. To combat the edema formation, we next evaluated the effect of various anti-inflammatory drugs commonly used in the clinical setting. Cohorts of mice were administered conjugate plus light in the presence or absence of steroids or the selective COX-2 inhibitor meloxicam. Results show there is no reduction in edema by steroids regardless of timing of administration. Conversely, the addition of meloxicam resulted in a significant reduction in edema at all time points and light doses evaluated: 40-50% reduction at both 2h and 6h post-PIT when administered prophylactically and 25% reduction at 6h post-PIT when administered post-light (all settings, n=10, p<0.0001). Importantly, we found the reduction in edema with meloxicam was not associated with a loss of therapeutic benefit based on measurement of tumor growth. In conclusion, the use of COX-2 inhibitors may be directly translated to the clinic for the benefit of patients receiving ASP-1929 PIT and should undergo further evaluation. Citation Format: Gina Ma, Myra Gordon, Jason Lapetoda, Abram Lozano, Christopher M. Amantea, Takuya Osada, Amy H. Thorne. Reduction in photoimmunotherapy-induced edema with COX-2 inhibition: Combatting clinically relevant adverse events without compromising efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1415.
Abstract Introduction: Photoimmunotherapy is an investigational anticancer treatment platform that combines the cell surface binding of an antibody conjugated to a light activatable dye (IRDye® 700DX, IR700) with non-thermal red light illumination for selective cell killing. PD-L1, the target of anti-PD-L1 inhibitors, is a suppressive checkpoint marker expressed on tumor cells and immunosuppressive myeloid cells in the tumor microenvironment. Here we examined the anticancer activity and immune activation elicited by anti-PD-L1-IR700 photoimmunotherapy (PD-L1 photoimmunotherapy). Methods: CT26 or LL/2 tumors were used to assess antitumor immune response following PD-L1 photoimmunotherapy. CT26PD-L1-/- tumors were generated by CRISPR/Cas9. Tumor volume was determined by caliper measurements in illuminated and non-illuminated tumors. Intratumoral immune responses were assessed by flow cytometry. Complete responder (CR) mice were challenged with either CT26 or 4T1 tumors to evaluate immune memory. Results: Mice bearing CT26 or LL/2 tumors treated with PD-L1 photoimmunotherapy exhibited a notable reduction of tumor growth compared to mice that received control treatments (saline, anti-PD-L1-IR700 conjugate alone without illumination, or multi-dosing with anti-PD-L1 antibody). Pre-treatment depletion of CD8+ T cells in the mice abrogated the antitumor activity of PD-L1 photoimmunotherapy, demonstrating a key role of CD8+ T-cell effector activity in the responses. PD-L1 photoimmunotherapy induced CRs in 7/15 mice, and all CR mice rejected CT26 tumor growth after re-challenge, indicating the generation of immunological memory. Furthermore, 6/8 mice rejected inoculation of 4T1 tumors, suggesting an enhanced ability to prime new T cells with tumor neoantigens. Intratumoral immune cell analysis showed a reduction of myeloid cells 2 hours following illumination, an increase of CD103+ dendritic cells 2 days following illumination, and an increase of non-exhausted PD-1-CD8+ T effector cells 8 days after illumination. In a bilateral tumor model, PD-L1 photoimmunotherapy resulted in tumor reduction in the non-illuminated tumor. Mice bearing CT26PD-L1-/- tumor cells similarly exhibited tumor reduction after PD-L1 photoimmunotherapy, suggesting that antitumor activity resulted from the elimination of PD-L1+ non-tumor cells. Conclusions: PD-L1photoimmunotherapy induces anticancer responses by killing PD-L1+ myeloid cells and possibly PD-L1+ cancer cells within the tumor, resulting in augmentation of local and systemic antitumor immunity. These results indicate that PD-L1 photoimmunotherapy can elicit anticancer immune responses in target and distal tumors, including syngeneic mouse models refractory to checkpoint inhibition. Citation Format: Amy H. Thorne, Michelle H. Hsu, Daniel Mendoza, Jason Lapetoda, Christopher M. Parry, Jerry J. Fong, Miguel Garcia-Guzman. PD-L1 photoimmunotherapy kills immunosuppressive myeloid cells to activate local and systemic antitumor immunity in syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1796.
SapC-DOPS is a novel nanotherapeutic that has been shown to target and induce cell death in a variety of cancers, including glioblastoma (GBM). GBM is a primary brain tumor known to frequently demonstrate resistance to apoptosis-inducing therapeutics. Here we explore the mode of action for SapC-DOPS in GBM, a treatment being developed by Bexion Pharmaceuticals for clinical testing in patients. SapC-DOPS treatment was observed to induce lysosomal dysfunction of GBM cells characterized by decreased glycosylation of LAMP1 and altered proteolytic processing of cathepsin D independent of apoptosis and autophagic cell death. We observed that SapC-DOPS induced lysosomal membrane permeability (LMP) as shown by LysoTracker Red and Acridine Orange staining along with an increase of sphingosine, a known inducer of LMP. Additionally, SapC-DOPS displayed strong synergistic interactions with the apoptosis-inducing agent TMZ. Collectively our data suggest that SapC-DOPS induces lysosomal cell death in GBM cells, providing a new approach for treating tumors resistant to traditional apoptosis-inducing agents.
