Unveiling the proteome-wide autoreactome enables enhanced evaluation of emerging CAR-T therapies in autoimmunity

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Abstract

Given the global surge in autoimmune diseases, it is critical to evaluate emerging therapeutic interventions. Despite numerous new targeted immunomodulatory therapies, comprehensive approaches to apply and evaluate the effects of these treatments longitudinally are lacking. Here, we leveraged advances in programmable-phage immunoprecipitation (PhIP-Seq) methodology to explore the modulation, or lack thereof, of autoantibody profiles, proteome-wide, in both health and disease. Using a custom set of over 730,000 human derived peptides, we demonstrated that each individual, regardless of disease state, possesses a distinct and complex constellation of autoreactive antibodies. For each individual, the set of resulting autoreactivites constituted a unique immunological fingerprint, or "autoreactome,” that was remarkably stable over years. Using the autoreactome as a primary output, we evaluated the relative effectiveness of various immunomodulatory therapies in altering autoantibody repertoires. We found that therapies targeting B-Cell Maturation Antigen (BCMA) profoundly altered an individual’s autoreactome, while anti-CD19 and CD20 therapies had minimal effects. These data both confirm that the autoreactome is comprised of autoantibodies secreted by plasma cells, and strongly suggest that BCMA or other plasma cell targeting therapies may be highly effective in treating currently refractory autoantibody mediated diseases.

Authors

Aaron Bodansky, David J.L. Yu, Alysa N. Rallistan, Muge Kalaycioglu, Jim Boonyaratanakornkit, Damian J. Green, Jordan Gauthier, Cameron J. Turtle, Kelsey C. Zorn, Brian O'Donovan, Caleigh Mandel-Brehm, James Asaki, Hannah Kortbawi, Andrew F. Kung, Elze Rackaityte, Chung-Yu Wang, Aditi Saxena, Kimberly de Dios, Gianvito Masi, Richard J. Nowak, Kevin C. O'Connor, Hao Li, Valentina E. Diaz, Rowan Saloner, Kaitlin B. Casaletto, Eva Q. Gontrum, Brandon J. Chan, Joel H. Kramer, Michael R. Wilson, Paul J. Utz, Joshua A. Hill, Shaun W. Jackson, Mark S. Anderson, Joseph L. DeRisi

Peripherally targeted analgesia via AAV-mediated sensory neuron-specific inhibition of multiple pronociceptive sodium channels

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Abstract

This study reports that targeting intrinsically disordered regions of NaV1.7 protein facilitates discovery of sodium channel inhibitory peptide aptamers (NaViPA) for adeno-associated virus (AAV)-mediated, sensory neuron-specific analgesia. A multipronged inhibition of INa1.7, INa1.6, INa1.3, and INa1.1. but not INa1.5 and INa1.8 was found for a prototype, named NaViPA1, which was derived from the NaV1.7 intracellular loop 1 and is conserved among the TTXs NaV subtypes. NaViPA1 expression in primary sensory neurons (PSNs) of dorsal root ganglia (DRG) produced significant inhibition of TTXs INa but not TTXr INa. DRG injection of AAV6-encoded NaViPA1 significantly attenuated evoked and spontaneous pain behaviors in both male and female rats with neuropathic pain induced by tibial nerve injury (TNI). Whole-cell current clamp of the PSNs showed that NaViPA1 expression normalized PSN excitability in TNI rats, suggesting that NaViPA1 attenuated pain by reversal of injury-induced neuronal hypersensitivity. Immunohistochemistry revealed efficient NaViPA1 expression restricted in PSNs and their central and peripheral terminals, indicating PSN-restricted AAV biodistribution. Inhibition of sodium channels by NaViPA1 was replicated in the human iPSC-derived sensory neurons. These results summate that NaViPA1 is a promising analgesic lead that, combined with AAV-mediated PSN-specific block of multiple TTXs NaVs, has potential as peripheral nerve-restricted analgesic therapeutics.

Authors

Seung Min Shin, Brandon Itson-Zoske, Fan Fan, Yucheng Xiao, Chensheng Qiu, Theodore R. Cummins, Quinn H. Hogan, Hongwei Yu

Gravi-D peptide disrupts HDAC11 association with an AKAP to stimulate adipocyte thermogenic signaling

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Abstract

Authors

Emma L. Robinson, Charles A. Tharp, Rushita A. Bagchi, Timothy A. McKinsey

Anti–PD-1 chimeric antigen receptor T cells efficiently target SIV-infected CD4⁺ T cells in germinal centers

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Abstract

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti–PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239–infected rhesus macaques (RMs). Adoptive transfer of anti–PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti–PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti–PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

Authors

Karsten Eichholz, Yoshinori Fukazawa, Christopher W. Peterson, Francoise Haeseleer, Manuel Medina, Shelby Hoffmeister, Derick M. Duell, Benjamin D. Varco-Merth, Sandra Dross, Haesun Park, Caralyn S. Labriola, Michael K. Axthelm, Robert D. Murnane, Jeremy V. Smedley, Lei Jin, Jiaxin Gong, Blake J. Rust, Deborah H. Fuller, Hans-Peter Kiem, Louis J. Picker, Afam A. Okoye, Lawrence Corey

Mediator kinase inhibition reverses castration resistance of advanced prostate cancer

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Abstract

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppresses the growth of CRPC xenografts but also induces tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplifies and modulates the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affects stromal gene expression, indicating that Mediator kinase activity in CRPC molds the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulates the MYC pathway, and Mediator kinase inhibition suppresses a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors show efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlates with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediate androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

Authors

Jing Li, Thomas A. Hilimire, Liu Yueying, Lili Wang, Jiaxin Liang, Balázs Győrffy, Vitali Sikirzhytski, Hao Ji, Li Zhang, Chen Cheng, Xiaokai Ding, Kendall R. Kerr, Charles E. Dowling, Alexander A. Chumanevich, Zachary T. Mack, Gary P. Schools, Chang-uk Lim, Leigh Ellis, Xiaolin Zi, Donald C. Porter, Eugenia V. Broude, Campbell McInnes, George Wilding, Michael B. Lilly, Igor B. Roninson, Mengqian Chen

Pharmacological suppression of the OTUD4-CD73 proteolytic axis revives antitumor immunity against immune-suppressive breast cancers

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Abstract

Despite widespread utilization of immunotherapy, challenge to treat immune-cold tumors needs to be resolved. Multiomic analyses and experimental validation identified the OTUD4-CD73 proteolytic axis as a promising target in treating immune-suppressive triple negative breast cancer (TNBC). Mechanistically, deubiquitylation of CD73 by OTUD4 counteracted its ubiquitylation by TRIM21, resulting in CD73 stabilization that inhibits tumor immune responses. We further demonstrated the importance of TGF-β signaling for orchestrating the OTUD4-CD73 proteolytic axis within tumor cells. Spatial transcriptomics profiling discovered spatially resolved features of interacting malignant and immune cells pertaining to expression levels of OTUD4 and CD73. In addition, ST80, a newly developed inhibitor, specifically disrupted proteolytic interaction between CD73 and OTUD4, leading to reinvigoration of cytotoxic CD8+ T cell activities. In preclinical models of TNBC, ST80 treatment sensitized refractory tumors to anti-PD-L1 therapy. Collectively, our findings uncover a novel strategy for targeting immunosuppressive OTUD4-CD73 proteolytic axis in treating immune-suppressive breast cancers with the inhibitor ST80.

Authors

Yueming Zhu, Anupam Banerjee, Ping Xie, Andrey A. Ivanov, Amad Uddin, Qiao Jiao, Junlong J. Chi, Lidan Zeng, Ji Young Lee, Yifan Xue, Xinghua Lu, Massimo Cristofanilli, William J. Gradishar, Curtis J. Henry, Theresa W. Gillespie, Manali Ajay Bhave, Kevin Kalinsky, Haian Fu, Ivet Bahar, Bin Zhang, Yong Wan

Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing

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Abstract

Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable, however designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated, drug product for targeting CD33+ tumors called dimerization agent regulated immunoreceptor complex (DARIC33). T cell products demonstrated target specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following re-exposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for OFF-ON state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase 1 DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and anti-tumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.

Authors

Jacob Appelbaum, April E. Price, Kaori Oda, Joy Zhang, Wai-Hang Leung, Giacomo Tampella, Dong Xia, Pauline P.L. So, Sarah K. Hilton, Claudya Evandy, Semanti Sarkar, Unja Martin, Anne-Rachel Krostag, Marissa Leonardi, Daniel E. Zak, Rachael Logan, Paula Lewis, Secil Franke-Welch, Njabulo Ngwenyama, Michael Fitzgerald, Niklas Tulberg, Stephanie Rawlings-Rhea, Rebecca A. Gardner, Kyle Jones, Angelica Sanabria, William Crago, John Timmer, Andrew Hollands, Brendan Eckelman, Sanela Bilic, Jim Woodworth, Adam Lamble, Philip D. Gregory, Jordan Jarjour, Mark Pogson, Joshua A. Gustafson, Alexander Astrakhan, Michael C. Jensen

STING activation reprograms the microenvironment to sensitize NF1-related malignant peripheral nerve sheath tumors for immunotherapy

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Abstract

Neurofibromatosis Type 1 (NF1) is caused by mutations in the NF1 gene that encodes neurofibromin, a RAS GTPase-Activating Protein. Inactivating NF1 mutations cause hyperactivation of RAS-mediated signaling, resulting in development of multiple neoplasms, including Malignant Peripheral Nerve Sheath Tumors (MPNSTs). MPNSTs are an aggressive tumor and the main cause of mortality in NF1 patients. MPNSTs are difficult to resect and refractory to chemo- and radiotherapy, and no molecular therapies currently exist. Immune Checkpoint Blockade (ICB) is an approach to treat inoperable, undruggable cancers like MPNST, but successful outcomes require an immune cell-rich tumor microenvironment (TME). While MPNSTs are non-inflamed “cold” tumors, here, we turned MPNSTs into T cell-inflamed “hot” tumors by activating “stimulator of interferon genes” (STING) signaling. Mouse genetic and human xenograft MPNST models treated with STING agonist plus ICB exhibited growth delay via increased apoptotic cell death. This strategy offers a potential treatment regimen for MPNST.

Authors

Bandarigoda N. Somatilaka, Laasya Madana, Ali Sadek, Zhiguo Chen, Sanjay Chandrasekaran, Renee M. McKay, Lu Q. Le

Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction

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Abstract

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown, and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed a novel screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we identified therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized anti-tumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations—including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of β2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.

Authors

Kyle Vaccaro, Juliet Allen, Troy W. Whitfield, Asaf Maoz, Sarah Reeves, José Velarde, Dian Yang, Anna Meglan, Juliano Ribeiro, Jasmine Blandin, Nicole Phan, George W. Bell, Aaron Hata, Kipp Weiskopf

A BMP-controlled metabolic-epigenetic signaling cascade directs midfacial morphogenesis

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Abstract

Craniofacial anomalies, especially midline facial defects, are among the most common birth defects in patients associated with increased mortality or require lifelong treatment. During mammalian embryogenesis, specific instructions arising at genetic, signaling, and metabolic levels are important for stem cell behaviors and fate determination, but how these functionally relevant mechanisms are coordinated to regulate craniofacial morphogenesis remain unknown. Here, we report that BMP signaling in cranial neural crest cells (CNCCs) is critical for glycolytic lactate production and subsequent epigenetic histone lactylation, thereby dictating craniofacial morphogenesis. Elevated BMP signaling in CNCCs through constitutively activated ACVR1 (ca-ACVR1) suppressed glycolytic activity and blocked lactate production via a p53-dependent process that resulted in severe midline facial defects. By modulating epigenetic remodeling, BMP signaling-dependent lactate generation drived histone lactylation levels to alter essential genes of Pdgfra thus regulating CNCC behavior in vitro as well as in vivo. These findings define an axis wherein the BMP signaling controls a metabolic-epigenetic cascade to direct craniofacial morphogenesis, thus providing a conceptual framework for understanding the interaction between genetic and metabolic cues operative during embryonic development. These findings indicate potential preventive strategies of congenital craniofacial birth defects via modulating metabolic-driven histone lactylation.

Authors

Jingwen Yang, Lingxin Zhu, Haichun Pan, Hiroki Ueharu, Masako Toda, Qian Yang, Shawn A. Hallett, Lorin E. Olson, Yuji Mishina