Michael C. Milone
· M.D., Ph.D.VerifiedUniversity of Pennsylvania · Rehabilitation Medicine
Active 1995–2026
About
Michael C. Milone, M.D., Ph.D., is an Associate Professor of Pathology and Laboratory Medicine at the Hospital of the University of Pennsylvania. He also serves as Staff Physician in the Department of Pathology and Laboratory Medicine and as Associate Director of the Toxicology and Mass Spectrometry Laboratory. Additionally, he holds the position of Associate Vice Chair for Professional Development and Academic Affairs in the same department at the Perelman School of Medicine. His research focuses on T cell biology and the applications of genetically-engineered T cells for disease treatment. His active projects include developing chimeric antigen receptors (CARs) and other engineering approaches to enhance the function and safety of adoptive T cell therapy, creating synthetic immunoreceptors for immune-mediated disease treatment, and exploring the role of co-stimulatory signals in T cell metabolism and their impact on T cell function within tumors. Dr. Milone's work aims to advance cellular immunotherapies and improve therapeutic strategies for cancer and immune-related conditions.
Research topics
- Materials science
- Oncology
- Internal medicine
- Pharmacology
- Medicine
- Immunology
Selected publications
Cancer Research · 2026-04-03
articleAbstract Over half of B-NHL patients (pts) receiving FDA-approved CD19-targeting chimeric antigen receptor (CAR) T experience progressive disease within 1 year1, demonstrating need for more durable therapies. Unlike CD3-based single-chain CAR, killer immunoglobulin-like receptor (KIR)-based CAR have a multi-chain design derived from natural killer (NK) cells2 with separated antigen binding and activation signals, and reduced tonic signaling and off-target activity3. SynKIR-310 is an autologous T cell therapy targeting CD19 with canine-derived single chain variable fragment (scFv), DS191. We previously compared SynKIR-310 to single-chain FMC63-41BBζ (tisagenlecleucel analog) for anti-tumor functionality in NALM6 B-cell leukemia-engrafted NOD-SCID-IL2Rγc-/- (NSG) mice. SynKIR-310 showed faster tumor regression and increased tumor control with reduced systemic cytokines and comparable T cell persistence4. Here we evaluated SynKIR-310 in B-NHL Raji cell Burkitt lymphoma xenograft NSG mice compared with FMC63-41BBζ and single-chain FMC63-CD28ζ (axicabtagene ciloleucel analog). Mice were IV injected with Raji tumors and then IV injected with T cells. Tumor progression was monitored by bioluminescent imaging. In this Raji model, SynKIR-310 and FMC63-41BBζ showed comparable anti-tumor efficacy and increased overall survival, while FMC63-CD28ζ had no impact over negative control animals despite similar T cell persistence across groups. SynKIR-310 and FMC63-41BBζ produced similar levels of cytokines in vivo, while FMC63-CD28ζ produced significantly more cytokines at both early and late timepoints, despite worse tumor control. At the early timepoint, FMC63-CD28ζ produced 11-fold more IL-2 than SynKIR-310 and FMC63-41BBζ, while IFNγ and TNFα were comparable across groups. At the late timepoint, FMC63-CD28ζ produced 11-fold more IFNγ and 9-fold more TNFα, compared to SynKIR-310 and FMC63-41BBζ. SynKIR-310 achieves significantly improved tumor control compared to FMC63-CD28ζ in a Raji B-NHL mouse model, with reduced cytokine production. We have previously shown SynKIR-310 has superior anti-tumor efficacy over FMC63-41BBζ in a leukemia NALM6 model, here FMC63-41BBζ and SynKIR-310 had comparable anti-tumor efficacy against B-NHL Raji, though SynKIR-310 was the only group with 100% survival. These data support a potentially increased benefit-risk profile of SynKIR-310 compared with conventional CD3-based CAR T and merits further investigation in patients with B-NHL. We are enrolling pts in a Phase 1 first-in human multi-site U.S.-based clinical trial for relapsed/refractory B-NHL, including pts with or without prior exposure to CAR T (NCT06544265). Early clinical pt data will be presented. 1 Cappell KM Nat Rev Clin Oncol 2023 2 Wang E Cancer Immunol Res 2015 3 Yucel N JITC Nov 2025 Abst 298 4 Blair M Blood Dec 2025 Abst 4103 Citation Format: Megan C. Blair, Jun Xu, Nora Yucel, Tony Truong, William Stanley, Michael Tees, Olivia Dermody, Susan K. Howard, Andrea Campanile, Michael Milone, Don L. Siegel, Laura A. Johnson. Novel SynKIR-310 outperforms CD3-based second-generation CD28 or 41BB co-stimulated CAR T in B-cell non-Hodgkin lymphoma xenograft mice and shows early clinical signal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5193.
Cancer Research · 2026-04-17
articleAbstract SynKIR-110 is a novel mesothelin-targeting autologous chimeric antigen receptor (CAR) T cell modeled after multichain immune receptors derived from natural killer cells, used in a first in human (FIH) phase 1 clinical trial to treat patients with advanced mesothelin-expressing solid tumors. Mesothelin-targeting single-chain variable fragment SS1 was recombined with killer-immunoglobulin-like receptor (KIR), which interacts with DNAX-activation protein of 12 kDa (DAP12) to induce effector T cell function. Preclinical studies with KIR-CAR showed decreased PD1 and Tim3 T cell exhaustion markers, with increased in vivo antitumor activity and decreased cytokine secretion compared with conventional CAR T. In this study, we assessed the safety and activity of SynKIR-110 in patients. A phase 1 dose-escalation study in patients with advanced ovarian cancer, mesothelioma, or cholangiocarcinoma who were refractory to standard of care treatment was conducted at four medical centers in the United States. Patients in dose cohorts 1-3 received a single intravenous dose of 1, 3, or 10 × 107 transduced SynKIR-110 T cells/m2, respectively, following lymphodepletion (LD) with cyclophosphamide and fludarabine; follow-up was for 12 months or until disease progression. Primary endpoints included safety and determination of a maximum tolerated dose (MTD) for a dose-expansion to evaluate anti-tumor activity. Circulating biomarkers were used to measure SynKIR-110 persistence and T cell activation. Clinical responses were evaluated via computed-tomography imaging starting 28 days after treatment. This ongoing study is registered with ClinicalTrials.gov NCT05568680. In cohorts 1-3, no dose-limiting toxicities or events meeting protocol-defined stopping criteria were reported. Three of 9 (33%) patients treated experienced low-grade cytokine release syndrome (≤grade 2), with no immune effector cell-associated neurologic events (0/9). Five of 9 (56%) patients experienced hematologic adverse events related to LD chemotherapy. SynKIR-110 was quantified in peripheral blood and increased with dosage, reaching peak levels >20,000 copies/µg DNA in cohorts 2 and 3, approximately 7 days after cell infusion. Effector T cell cytokines including interferon-gamma and tumor necrosis factor-alpha, reached peak serum concentration between 7 and 14 days after treatment, consistent with expectations for CAR T class therapies. Tumor responses were seen in 4 of 9 patients, up to 47% reduction for 1 patient each in cohorts 1 and 2, and 2 patients in cohort 3, one of whom maintained an iRECIST partial response through 6 months of follow-up. SynKIR-110 mesothelin-targeted KIR-CAR treatment showed a favorable safety profile, with notable clinical activity in the first 3 of 6 planned dose cohorts, in heavily pretreated patients with refractory mesothelin-expressing advanced solid tumors. MTD was not reached and dose-escalation enrollment is ongoing in this phase 1 FIH trial. Funding: Verismo Therapeutics Citation Format: Janos L. Tanyi, Andrew Haas, Mark O'Hara, Zhubin Gahvari, Raed Al-Rajabi, Mehmet Altan, Daniel Sterman, Emily Winters, Andrea Campanile, Susan Howard, Raymond Luke, Tony Truong, Megan Blair, Nora Yucel, Jun Xu, Don L. Siegel, Carl H. June, Michael C. Milone, Laura A. Johnson. Initial results of a first in human dose-escalation study of KIR-CAR in patients with advanced mesothelin-expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT104.
β-hydroxybutyrate enhances the metabolic fitness of CAR T cells in cancer
Cell · 2026-03-01 · 2 citations
articleOpen accessAbstract 6596: Understanding DAP12-independent antitumor activation in KIR-based CAR T cells
Cancer Research · 2026-04-03
articleSenior authorAbstract We previously described a chimeric killer immunoglobulin (KIR)-like receptor (cKIR) approach to modifying T cell antigenic specificity that mimics the multichain design of natural KIR immunoreceptors (Wang E et al. Cancer Immunol Res 2014). T cells modified to express a cKIR with the natural ITAM-containing adaptor, DAP12 that associates with activating KIRs, show significantly enhanced anti-tumor activity acrossmultiple solid tumor models when compared to traditional CD3z-based chimeric antigen receptors (CARs). However, unlike TCR, where assembly with ITAM-containing CD3 family members is required for plasma membrane expression, natural and chimeric KIR exhibit membrane expression in the absence of DAP12. We show that cKIR engineeredT cells also produce IFN-g and retain cytotoxic activity in the absence of DAP12, revealing previously unrecognized DAP12-independent signaling pathways. This is associated with phosphorylation of ERK, JNK and STAT3 at early time points (30-60 mins) following cKIR engagement with target antigen. Although the cytoplasmic domain of KIR2DS2 used in cKIR is short and lacks canonical signaling motifs, we hypothesized that this domain plays a functional role in T cells by recruiting alternative adaptors to promote noncanonical signaling in the absence of DAP12. Immunoprecipitation of cKIR in T cells lacking DAP12 identified GRB2 as an associated adaptor protein. CRISPR- mediated GRB2 knockout selectively abolished ERK activation, establishing GRB2 as a crucial link to the Ras/MAPK pathway in cKIR-activated T cells. Structure-functionanalysis of the KIR2DS2 cytoplasmic tail, containing one tyrosine, one threonine, and four serine residues, revealed that progressive truncation of these residues had minimal effect on membrane expression, but increasingly impaired cytotoxicity, cytokine production, proliferation, and downstream signaling with a > 25-fold loss of IFN-g andcytotoxicity observed with the truncation of C-terminal to serine residue 3. Collectively, these results suggest that DAP12-independent signaling by cKIR, mediated through GRB2 and likely other yet-to-be-identified adaptors, contribute to diverse signaling in T cells and may contribute to the enhanced anti-tumor function observed with KIR-based CAR T cells compared with conventional CAR designs. Citation Format: Qian Zhang, Kevin Zhou, Zhongze Sun, Chen Zhang, Marta Chebotar, Michael C. Milone. Understanding DAP12-independent antitumor activation in KIR-based CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6596.
Cancer Research · 2026-04-03
articleAbstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by frequent KRAS-G12 oncogenic mutations, low immune cell infiltration, and early metastatic spread, leading to poor outcomes. Selective pressure from systemic therapies promotes the emergence of resistant clones and relapse. Patient-derived models that capture tumor heterogeneity are needed to optimize T cell receptor-engineered T cell (TCR-T) strategies and anticipate mechanisms of resistance. Methods: We developed a high-throughput ex vivo screening platform to test cell-based immunotherapies in patient-derived PDAC spheroids. KRAS-G12V-specific CD8+ TCR-T cells were co-cultured with KRAS-mutant 3D tumor spheroids under graded levels of cytotoxic pressure and monitored using automated image cytometry. Microcavity plates enabled parallel tracking of thousands of spheroids across two sequential TCR-T challenges. Surviving cells were profiled by single-cell RNA sequencing (scRNA-seq), followed by 2D regrowth assays and orthotopic transplantation into NSG mice to assess tumor-initiating capacity. Results: High-throughput resistance screening of over 33,000 microcavities revealed that 6.6% of 3D-PDAC spheroids remained viable after repeated TCR-T cell challenge, enriching for rare resistant subclones. After the first challenge, most cells exhibited an antiviral-like inflammatory response, characterized by increased levels of MX1, MX2, RIG-I, and interferon-stimulated gene expression. scRNA-seq after re-challenge revealed proliferating cells with upregulation of MHC II molecules and TUBA1B-associated aggrephagy pathways, implicating aggrephagy in immune escape. Resistant cells retained tumor-initiating potential and generated distant metastases in orthotopic NSG models (n = 10). In vitro, paclitaxel, a microtubule disassembly inhibitor, reduced colony formation by TCR-T-escaped clones, supporting a rationale for combination therapy. Conclusions: High-throughput ex vivo cytotoxicity screening of patient-derived PDAC models reveals aggrephagy-enriched, therapy-resistant clones and nominates rational combination partners such as paclitaxel. This platform may help anticipate patient-specific resistance to KRAS-targeted TCR-T therapy and guide the design of preventative combination strategies. Citation Format: Masoumeh Eshaghi, Fei Miao, Ali Abdollahzadeh, Sixing Chen, Jacopo Chiaro, Elahe Kamali G, Joshua Glover, Vineeth Koneru, Michael C. Milone, Miren L Baroja, Gerald P. Linette, Beatriz M. Carreno, Emma E. Furth, Vincenzo Cerullo, Joseph Fraietta, Carl H. June, Friederike Herbst-Nowrouzi. High-throughput ex vivo TCR-T cells screening identifies aggrephagy-driven, therapy-resistant clones in KRAS-mutant PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5183.
Rab5 improves CAR T cell efficacy via reducing fratricide and maintaining surface CAR levels
The Journal of Experimental Medicine · 2026-03-27 · 1 citations
articleWe show continuous tumor exposure results in a loss of chimeric antigen receptor (CAR) T cell (CART) endocytic activity due to downregulation of Rab5. Loss of endocytic activity exacerbates the effects of trogocytosis, the bidirectional transfer of tumor target antigens and CARs between malignant cells and CARTs, resulting in CART dysfunction and fratricide. Constitutive expression of Rab5 within the CARTs reduced fratricide by reducing the amount of trogocytosed antigens on the cell surface, while simultaneously enhancing CAR availability through dissociation of CAR from target, recycling unbound CAR back to the plasma membrane, and limiting CAR capture by tumor cells. Rab5-expressing CARTs exhibited superior antitumor activity in both BCMA-CARTs isolated from the bone marrow of treated patients and mesothelin-specific CARTs in a solid tumor model. These studies uncover an unexpected relationship between endocytosis and CART function and suggest that pairing Rab5 with CAR expression could improve the clinical efficacy of CART therapy.
2025 Advancing Cell and Gene Therapies for Cancer post-meeting report
Molecular Therapy · 2026-05-01
articleCancer Research · 2026-04-17
articleAbstract Glioblastoma (GBM) is the most common primary brain cancer in adults, with a prognosis of 15-18 months’ survival despite standard treatment of resection, chemotherapy, and radiotherapy. Recent clinical trials investigating autologous chimeric antigen-receptor (CAR) T cells targeting epidermal growth factor receptor (EGFR) variants in GBM through single-chain variable fragments (scFv) recombined with 41BB-co-stimulation and CD3ζ activation showed encouraging radiographic evidence of early tumor reductions, within days of CAR T treatment. Unfortunately, this anti-tumor function was short-lived, with tumor outgrowth generally occurring within days to weeks, potentially attributed to rapid loss of T cell function due to exhaustion, or target antigen loss. In efforts to bypass CD3-based CAR signaling exhaustion and prolong CAR T anti-tumor function, the recombinant single-chain CD3ζ-based CAR was replaced with a more physiologic split-signal system derived from natural killer (NK) cells, utilizing killer immunoglobulin-like receptor (KIR) and DNAX-activation protein of 12 kDa (DAP12) activation to allow a more natural cellular activation/rest cycle upon target cell engagement and disengagement. Second-generation EGFR/EGFRvIII-targeted single-chain 41BB-CD3ζ CAR was replaced with multi-chain KIR/DAP12 CAR signaling, and CAR and KIR T cells were compared in vitro for recognition specificity, cytolytic function, cytokine secretion, and T cell differentiation phenotype. In vivo efficacy was evaluated in established CAR-resistant immunocompromised NOD/SCID/gamma-chain-/- (NSG) murine GBM xenograft models, where tumor size was evaluated by in vivo imaging and caliper measurements and survival per Kaplan–Meier graph. Following lentiviral transduction and ex vivo expansion, KIR-CAR T displayed an increased naïve-like phenotype, with reduced effector-memory cells compared with CD3-based CAR T. In vitro, both CAR and KIR-signaling formats conferred comparable target specificity, cytotoxicity, T cell activation and cytokine release. In contrast, in vivo KIR-CAR T cells repeatedly outperformed 41BB-CD3ζ CARs, with the most effective treatment delivered by an scFv targeting multiple EGFR tumor-associated variants, demonstrating superior tumor regressions, including complete anti-tumor responses and increased survival. With the same targeting specificity, similar cytokine production in vitro, and significantly increased anti-tumor functional impact and prolonged survival in vivo, EGFR-KIR-CAR has the potential for rapid translation to the clinic to overcome the limitations of CD3-based single-chain CAR T in treating patients with GBM. Funded by NIH grant DP2CA174502 (LAJ). Citation Format: Jun Xu, Radhika Thokala, Yibo Yin, Chong Xu, Alina C. Boesteanu, Alexandria P. Cogdill, Zev A. Binder, Logan Zhang, Jiasi Vicky Zhang, Enxiu Wang, Carl H. June, Donald M. O'Rourke, Michael C. Milone, Laura A. Johnson. Natural killer cell-based signaling in EGFR-targeted KIR-CAR T overcomes CD3-based CAR T functional deficits to eliminate resistant glioblastomas in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB138.
Cancer Chemotherapy and Pharmacology · 2025-02-11
articleOpen accessSenior authorAbstract Purpose Apply population pharmacokinetic modeling to a single institution busulfan therapeutic drug monitoring (TDM) data set from infants and children to refine dosing methods. Methods One-compartment pharmacokinetic model was fit to busulfan TDM data from 328 infants and children with malignant and non-malignant diseases treated with busulfan-containing transplant conditioning regimens. Age-dependence of busulfan clearance scaled to body weight and body surface area (BSA) was compared, and busulfan AUC was simulated for a BSA-scaled dose of 100 mg/m 2 combined with a BSA-banded dosing table for infants and children with a BSA < 0.5 m 2 . Results Busulfan clearance scaled to body weight is age-dependent. Clearance in children ≤ 3 years (0.234 L/[h•kg]) is higher than the typical value for the population, (0.205 L/[h•kg]), and 48% of children < 5 years have subtherapeutic busulfan AUCs after the first dose. Busulfan clearance scaled to BSA (typical value, 5.47 L/[h•m 2 ]) is more uniform across the pediatric age span, except for infants (≤ 1 year, 4.27 L/[h•m 2 ]). Simulated busulfan AUCs with a dose of 100 mg/m 2 for patients with a BSA ≥ 0.5 m 2 combined with a BSA-banded dosing table for patients with a BSA < 0.5 m 2 achieved a therapeutic AUC after the first dose in 49% more patients than body weight scaled doses. Conclusion Our model predicts a greater proportion of children would achieve a therapeutic busulfan AUC after the first dose with a dose of 100 mg/m 2 /d combined with the infant dosing table for patients with a BSA < 0.5 m 2 compared to body weight-scaled dosing.
Clinical advances in gene, cell, and RNA therapies
Molecular Therapy · 2025-05-20
editorial1st authorCorresponding
Recent grants
Enhancing Chimeric Antigen Receptor T Cell Therapies for HematologicMalignancies: Beyond CART 19
NIH · $39.1M · 2017–2028
Frequent coauthors
- 138 shared
Carl H. June
Parker Institute for Cancer Immunotherapy
- 59 shared
Selene Nuñez-Cruz
University of Pennsylvania
- 58 shared
Bruce L. Levine
University of Pennsylvania
- 50 shared
Stephan A. Grupp
Children's Hospital of Philadelphia
- 45 shared
Roddy S. O’Connor
California University of Pennsylvania
- 44 shared
David M. Barrett
Kite (United States)
- 44 shared
Simon F. Lacey
University of Pennsylvania
- 42 shared
Vijay Bhoj
University of Pennsylvania
Labs
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