Mukhopadhyay, Debabrata

Worldwide, more than 4 million patients with end-stage kidney disease require hemodialysis through an arteriovenous fistula (AVF). AVFs fail because of venous neointimal hyperplasia (VNH) resulting in venous stenosis formation. A phase 1 randomized trial in patients undergoing upper extremity AVF placement was performed to evaluate the safety and efficacy of autologous adipose–derived mesenchymal stem cells (MSCs) in improving AVF function. The mechanism of action by which MSCs exert their beneficial effects was investigated using AVFs created in mice and pigs treated with allogenic MSCs and xenotransplant using patient MSCs in CD1- Foxn1nu mice. At a median follow-up of 42 months, patients with MSC-treated AVFs had reduced time to maturation with an increase in the vein diameter compared with controls. AVFs treated with allogenic MSCs in mice and pigs had an increase in M2- and M1-like macrophages with a decrease in VNH. Transcriptomic analysis of AVFs treated with allogenic MSCs in mouse and MSC xenotransplants of patients with successful AVFs showed decreased peroxisome proliferator–activated receptor gamma ( Ppar γ) and leptin receptor ( Lepr ) and increased latent transforming growth factor–beta–binding protein 2 ( Ltbp2 ). PPARγ was reduced in CD68 (+) cells from successful AVFs and allogenic MSC-treated mouse AVFs. Venous stenosis and VNH formation were mitigated in AVFs of mice with Ppar γ ablation in immune cells. Conditioned medium from MSCs of responders versus nonresponders had decreased proinflammatory genes, senescence-associated proteins, and monocyte-to-macrophage differentiation induced by phorbol 12-myristate-13-acetate. Periadventitial delivery of MSCs to AVFs promoted positive vascular remodeling through decreased inflammatory responses.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating cancers, and therapies to treat this malignancy have not evolved rapidly to fulfill the need of affected patients. PDAC, like many solid malignancies, is considered a “cold immunological tumor” where the lack of targetable tumor-associated antigens has hampered the development of successful immunotherapies. Despite newer treatment approaches, PDAC patients with advanced disease have a dismal prognosis. Therefore, there is an urgent need to develop new therapies. Plectin-1 is a high molecular weight protein (∼500 kDa) that participates in cytoskeleton network organization linking intermediate filaments to microtubules and microfilaments. Plectin-1 is aberrantly expressed in the PDAC cellular membrane, contributes to PDAC pathogenesis, and confers a poor prognosis. Plectin-1-targeting peptides (PTP) in mice and humans have been successfully explored for their use in PDAC imaging and targeted payload delivery (small molecules, radioisotopes, and chemotherapy). We sought to take advantage of the specific binding of PTP and develop a peptide-based plectin-1 chimeric antigen receptor (CAR), and test T-cell transduced with this construct in vitro against PDAC cell lines. Methods: Using in silico tools, we optimized different pepto-CAR constructs and selected one that showed the highest levels of cytotoxicity against plectin-1-expressing PDAC cell lines. A myc tag was incorporated in the hinge region for flow cytometry or western blot detection. The intracellular signaling domain of these CAR sequences included 4-1BB and the zeta chain of the CD3 molecule ( Figure 1A). Plasmids containing the CAR sequence were packaged on lentivirus particles using HEK293T cells. Purified plectin-1 CAR lentiviruses were used to transduce T-cells derived from human peripheral blood T-cells obtained and purified from healthy donor whole blood. The expanded CAR T-cells were used for cytotoxic experiments against different cell lines. Results: Expression of plectin-1 CAR was confirmed by flow cytometry using an anti-myc tag antibody ( Figure 1B). Plectin-1 expression was analyzed in different pancreatic cancer cell lines, and the BxPC-3 cells showed the highest expression level ( Figure 1B). Using a luciferase-based assay, we plated 50,000 BxPC-3 cells per well in duplicates with different effector-to-target (E:T) ratios. After 24 hours, luciferin was added, and the plate was read using a luminometer. As a negative control for effector cells, we use T-cells without a CAR ( Figure 1B), and for the target cells, we use the plectin-1 negative lung cancer cell line A549 ( Figure 1B). Plectin-1 CAR T-cells effectively killed BxPC-3 cells at different E:T ratios in a specific manner compared with controls using unmodified T-cells. Furthermore, no cytotoxicity was observed against plectin-1 negative A549 cells ( Figure 1A). Conclusion: Our results show that a novel peptide-based CAR construct design against plectin-1 can induce specific cytotoxicity on plectin-1 expressing PDAC cell line BxPC-3. Furthermore, these data demonstrate the feasibility of using a CAR with a peptide-based antigen binding domain instead of the traditional antibody-derived scFv. This novel peptide-based CAR design offers alternatives for cellular therapy bioengineering. Moreover, our results can significantly impact immune-oncology therapeutic applications, particularly in challenging diseases like pancreatic cancer. Figure 1 . A) In the above section, the Plectin-1 CAR construct is depicted. Cytotoxicity of unmodified T-cells and Plectin-1 CAR T-cells against BxPC-3 and A549 is shown below. B) Expression of Plectin-1 CAR in unmodified T-cells and Plectin-1 CAR T-cells is shown on the left. The expression of plectin-1 antigen on the surface of BxPC-3 cells and A549 cells is shown on the right. PTP: plectin-1 targeting peptide.

Abstract INTRODUCTION: Intratumoral phenylephrine (α1 vasoconstrictor) combined with chemotherapy has been used for cutaneous malignancies with modest local response. The purpose of this study was to improve the efficacy of intratumoral melphalan using sustained-release, liposomal nanoparticles containing phenylephrine in a mouse model of melanoma. METHODS: A modified ethanol injection method was used to develop the liposomal phenylephrine nanoparticles. These were injected peri-tumorally to generate a local vasoconstrictive effect intended to “entrap” intratumorally injected melphalan. Drug exposure/dwell time was measured with fluorescent imaging. Mice bearing B16 (subclone F10) melanoma tumors were treated with intratumoral melphalan (20 ug/50 ul) with or without peri-tumoral liposomal phenylephrine (10 ug/50-100 ul x 4-5 injections around the tumor periphery) twice weekly x 3 weeks. RESULTS: UV-Absorbance spectroscopy confirmed encapsulation of phenylephrine within the liposomal nanoparticles with an expected absorbance of 275 nm. The modified ethanol injection method produced liposomal phenylephrine encapsulation of approximately 25%. Peri-tumoral, liposomal phenylephrine caused vasoconstriction of normal surrounding vessels with an approximate 10% decrease in vessel diameter. Our liposomal phenylephrine nanoparticles also led to increased retention of intratumorally injected fluorescently-labeled (FITC) liposomes 24-48 hours after inoculation, using both melanoma (B16) and breast (TUBO/4T1) cell lines orthotopically grown within mice (C57Bl/6 and BALB/c, respectively). The combination of intratumoral melphalan with peri-tumoral phenylephrine resulted in more effective control of tumor growth and a complete tumor response rate of 44.4%, compared to phenylephrine/melphalan only controls (p < 0.01, n=18 per group). However, there was significant adverse reaction with the combination therapy, resulting in ulceration of the injection/tumor site. Ulcers healed about 14 days after completion of treatment. Immunohistochemistry demonstrated increased infiltration of CD11b+ cells into the tumors, suggesting an immune-mediated response in addition to direct chemotoxicity. CONCLUSIONS: Liposomal nanoparticles containing phenylephrine increased retention/dwell time of intratumorally administered melphalan and resulted in superior anti-tumor response. Our preclinical model holds innovative, translational promise for the clinical advancement of intralesional therapies in cutaneous/subcutaneous malignancies. Citation Format: Emmanuel M. Gabriel, Vijay Madamsetty, Deborah Bahr, Jamie Kaplan, Kristopher Attwood, Sanjay Bagaria, Keith Knutson, Debabrata Mukhopadhyay. Novel approach to intra- and peri-tumoral delivery of sustained-release chemotherapeutics [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 5071.