The Society for Immunotherapy of Cancer (SITC) is pleased to present highlights of the latest advances in immunotherapy emerging from the 2023 ASGCT Annual Meeting. Below is a recap of highlighted research presented from Tuesday, May 16 through Saturday, May 20, 2023.
Scientific Highlights
CAR signaling domains affect the dynamics of CAR T cell anti-cancer activity
89: CAR Signaling Drives Distinct Immunological Synapse Dynamics That Influence the T Cell Behavior in Killing Cancer
Ahmed Z. Gad (Baylor College of Medicine, Houston, TX) presented a study comparing the dynamics of CD28 and 41BB signaling domains at CAR immunological synapses. Previous studies indicate CAR T cells with CD28 signaling domains exhibit more rapid activation and less persistence while CAR T cells with 41BB signaling domains exhibit substantial proliferation and in some cases, decades-long persistence. CAR T cell products targeting HER2 and CD19 were studied. The CD28 signaling domain polarizes CAR T cells to CD8 cells with highly acute killing behavior, whereas the 41BB signaling domain supports expansion of a balanced population of CD4 and CD8 cells. Time-lapse confocal microscopy indicated CD28 CAR T cells interacted with tumor cells quickly and killed them efficiently, and 41BB CAR T cells exhibited a more prolonged interaction with tumor cells and a more cooperative killing mechanism. proteomic analysis of the CAR immunological synapse (CARIS) lipid raft supported these findings, with CD28 CAR molecules associating with the lipid raft in a more dynamic manner, with association occurring earlier and for shorter periods of time, compared to 41BB CAR molecules. Killing machinery was also different, with CD28 CAR T cells being more efficient at directing lytic granules toward the tumor cell and 41BB killing mediated by the Fas ligand. This study suggests that CAR signaling domains mediate CAR T cell dynamics with tumor cells and distinct killing behaviors. Further understanding of these dynamics and mechanisms has the potential to optimize CAR T cell functionality against cancer cells.
Modulating the tumor microenvironment for CAR T cellular therapy of pancreatic cancer
91: Engineering Optimal CAR T Cells to Overcome Pancreatic Tumors with Secreted Antagonistic Peptides
Heather K. Lin (Emory University School of Medicine, Atlanta, GA) presented a study to develop CAR T cells for pancreatic ductal adenocarcinoma (PDAC), a disease that is largely resistant to current immunotherapies. The CAR T cells targeted the ectodomain of Muc16 (Muc16CD), which is significantly overexpressed in PDAC tissues compared to non-cancerous pancreatic tissues. To overcome the immunosuppressive tumor microenvironment, anti-Muc16CD CARs were engineered to express a novel vasoactive intestinal peptide receptor antagonist (VIPRa). Vasoactive intestinal peptide (VIP) is a neuropeptide that has an immunosuppressive effect on T cells. Previous studies have shown that VIPRa reduces the immunosuppressive effects of VIP. CAR T cells expressing VIPRa (VIPRa CAR T cells) exhibited higher viability and enhanced activation and expansion in presence of Muc16CD antigen, compared to second-generation CAR T cells that did not express VIPRa. Metabolic profiling indicated that VIPRa CAR T cells exhibit a memory-like phenotype. After chronic antigen stimulation, VIPRa CAR T cells exhibited a proliferative advantage and higher cytotoxic function compared to second-generation CAR T cells and stimulated anti-cancer activity of bystander T cells. When mice were engrafted with human Panc1 tumors and treated with VIPRa CAR T cells, tumor burden was reduced compared to mice treated with second-generation CAR T cells. After 100 days, hCD3+ T cells were detected at higher levels in VIPRa CAR T-treated mice, suggesting expression of VIPRa improved persistence of CAR T cells. These results indicate that Muc16CD could be a potential target for cellular therapy for PDAC and that locally secreted VIPR antagonists can modulate the immunosuppressive TME in PDAC.
Investigating CD39 in cellular therapy for metastatic colorectal cancer
92: Harnessing CD39 for the Treatment of Colorectal Cancer and Liver Metastases by Engineered T Cells
Alessia Potenza (IRCCS San Raffaele Scientific Institute, Milan, Italy) reported on a study to address immune cell exhaustion in primary colorectal cancers (CRC) and CRC liver metastases. Analyses of healthy, peritumoral, and neoplastic tissues of primary CRC tumors and of liver metastases indicated extensive transcriptional and spatial remodeling across tumors, mostly in metabolic pathways. Tumor-infiltrating T cells were mainly localized to the front edges of tumors and exhibited unique inhibitory receptor signatures. CD39, a diphosphohydrolase converting ATP into AMP, was an inhibitory receptor that was highly expressed in primary and metastatic tumors, and it served as a potential target for T cell engineering. T cells were triple edited: the endogenous TCR and CD39 were disrupted by CRISPR/Cas9, and a novel T cell receptor targeting the HER-2 antigen was engineered. CD39-disrupted T cells exhibited a functional advantage of killing tumor cells in vitro. Providing further evidence that the ATP/adenosine pathway affects T cell functionality in the TME, addition of adenosine abrogated this advantage. HER2+ patient-derived organoids (PDOs) were injected in livers of mice, and mice were treated with CD39-disrupted HER2-directed T cells. The CD39-disrupted T cells slowed tumor growth and exhibited an advantage in eliminating PDOs. This study indicates the relevance of the ATP/adenosine/CD39 axis to future adoptive T cell therapies to treat CRC.
A knock-in system to generate armored CARs
93: Enhancement of K-Ras Neo-Antigen Targeting CAR-T Cells via Homogenous Knock in of Inducible IL-12
Alex Benton (Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA) presented a novel approach to develop CAR T cells to treat solid tumors with increased efficacy and fewer toxicities. CAR-T cells targeting K-Ras neoantigens presented in MHC molecules (NeoCARs) were developed, but NeoCARs against G12V mutant peptides exhibited low anti-tumor efficacy due to the relatively low abundance of the K-Ras neoantigen expressed on the tumor cells. NeoCARs were armored with the immunostimulatory cytokine IL-12 to stimulate NeoCAR anti-cancer activity and to enhance safety profile. The previously developed Uni-Vect platform was used to design a single lentiviral vector for inducible expression of IL-12 and constitutive expression of the CAR. NeoCARs expressing inducible IL-12 exhibited an increase in IFN-gamma secretion and enhanced killing of tumor spheroids in vitro. When tested in vivo with a mouse model of ovarian cancer, NeoCARs expressing inducible IL-12 exhibited complete solid tumor clearance but elicited lethal T cell receptor (TCR)-mediated toxicity in all mice. To enhance the safety profile and to eliminate aberrant endogenous TCR signaling as a source of inducible IL-12 expression, Uni-Vect was adapted to integrate the inducible IL-12 vector at the TRAC locus by homology directed repair (HDR), generating TCR disrupted, inducible IL-12 armored NeoCARs. When these cells were injected in mice, solid tumors were eliminated, and no lethal toxicities were observed. This novel knock-in Uni-Vect provides a translatable platform to generate the next generation of neo-antigen CAR-T cells that target solid tumors.
Lymphodepletion protocol affects clinical efficacy of CD123-targeting CAR T cell therapy
94: AMELI-01: A Phase I Trial of UCART123v1.2, an Anti-CD123 Allogeneic CAR-T Cell Product, in Adult Patients with Relapsed or Refractory (R/R) CD123+ Acute Myeloid Leukemia (AML)
Daniel J. Lee (Cellectis, Inc, New York, NY) presented results from AMELI-01, a Phase I trial of UCART123v1.2 (UCART123), a CD123-targeting genetically modified allogeneic T cell product, for relapsed or refractory acute myeloid leukemia (AML). UCART123 is manufactured from healthy donor cells, and in addition to expressing an anti-CD123 CAR, the T cell receptor alpha constant (TRAC) and CD52 genes are disrupted by TALEN technology to minimize the risk of graft versus host disease (GvHD) and to enable use of anti-CD52 therapy as part of lymphodepletion (LD). 17 patients in the trial received UCART123. 8 patients received treatment after LD with fludarabine and cyclophosphamide (FC), and 9 patients received treatment after LD with FC and alemtuzumab (FCA). 5 patients experienced dose-limiting toxicities, 3 in the FC arm and 2 in the FCA arm. All patients experienced cytokine release syndrome (CRS). UCART activity was observed in 4 of the 17 patients by Day 28. In the FC arm, one patient achieved stable disease (SD), and one patient achieved a morphologic leukemia free state (MLFS). In the FCA arm, one patient achieved stable disease with greater than 90% bone marrow blast reduction, and one patient achieved a long-term durable (> 12 months) MRD-negative complete response. 7 of 8 patients in the FC arm exhibited host lymphocyte recovery, indicating adequate lymphodepletion did not occur with FC. UCART123 expansion was significantly higher in FCA group, and increased expression of inflammatory markers correlated with CAR T expansion and CRS. These results support further studies of the safety and efficacy of UCART123 after LD with FCA for CD123-positive r/r AML. The study has been amended to include a 2-dose regimen; the second dose is hypothesized to provide additional clinical activity and CAR T expansion without additional LD. Enrollment for the study is ongoing.
Targeting IL-2 and IL-21 to CAR T cells to enhance CAR T cell expansion
57: Cis-Targeted Cytokines for Specific Stimulation of CAR T Cells
Sara Sleiman (University of Pennsylvania, Philadelphia, PA) presented an approach to selectively control expansion of CAR T cells after infusion, as previous studies have established that CAR T cell expansion in vivo correlates with improved patient outcomes. IL-2 family cytokines can be administered to promote CAR T cell expansion, but IL-2 cytokines are also associated with increased risk of severe toxicities and expansion of immunosuppressive cells, such as regulatory T cells (Tregs). A cis-targeted cytokine was developed, consisting of an IL-2 mutein with diminished binding to IL2R alpha/beta or an IL-21 mutein with diminished binding to IL-21R subunit associated with an antibody against EGFRt, a non-signaling receptor expressed with the CAR, to target the IL-2/IL-21 muteins to CAR T cells. The targeted cytokines exhibited more than 100-fold selectivity for CAR+ T cells compared to CAR- T cells in vitro. Mice were injected with a leukemia cell line and treated with CD19-targeting EGFRt CAR T cells and the cis-targeted cytokines. Targeted IL-2 and IL-21 both improved the efficacy of CAR T cells in terms of tumor reduction and survival. Toxicity, measured by weight loss, was not associated with either targeted cytokine. Peripheral blood samples indicated that targeted IL-2 and IL-21 both induced expansion of CAR T cells in vivo, with IL-2 inducing a stronger initial expansion compared to IL-21. These data show promise for antibody-targeted cytokines as a method to specifically enhance CAR T cellular therapies. Future studies to characterize pharmacokinetics and safety in non-human primates are in progress.
Using IL15 and an iC9 safety switch to enhance CAR T cell anti-tumor activity in liver cancer
8: GPC3-CAR T Cells Co-Expressing IL15 Mediate Potent Antitumor Activity in Liver Cancer Patients Associated with Toxicity That Can Be Mitigated Using iC9 Safety Switch
David Steffin (Baylor College of Medicine, Houston, TX) reported results of a phase I clinical trial of CAR T cells targeting Glypican 3 (GPC) co-expressing interleukin 15 (IL15). Glypican 3 (GPC) is expressed in several solid cancers, including liver cancers, and preclinical studies have shown CAR T cells co-expressing IL-15 with the CAR exhibit improved persistence and anti-tumor activity. 18 patients participated in the trial; 6 patients received treatment with GPC-targeting CAR T cells (CAR), and 12 patients received treatment with GPC-targeting CAR T cells that co-expressed IL-15 and an iC9 safety switch (15.CAR). Expression of IL15 was associated with increased adverse events (AEs), specifically cytokine release syndrome (CRS) and CRS-related toxicities. CRS was associated with higher levels of CAR T cell expansion and expression of inflammatory cytokines. Severe toxicities in the 15.CAR group were mitigated with the iC9 safety switch (n=3), which, when activated, provided significant improvement in symptoms and was associated with a rapid decrease in inflammatory cytokines. 15.CAR induced anti-tumor responses at primary tumors in the liver and at metastatic sites in the lungs. 15.CAR induced improved response: 4 patients achieved partial response (3 in the 15.CAR group), and a total of 8 patients achieved a decrease in tumor size. Single cell RNA sequencing identified changes in the transcriptome brought about by IL15 co-expression. 15.CAR cells expressed CD8a, CD8b, and cytolytic markers at higher levels than CAR cells, and tumor samples from the 15.CAR group were enriched for CD8 T cell subsets. IL15 expression was also associated with changes in peripheral blood samples, including increased expansion of CAR T cells, increased expression of cytolytic markers and markers of an activated, differentiated state, as well as increased expression of mitochondrial metabolism-related genes. Based on these results, expression of IL-15 with GPC-CAR T cells leads to increased but manageable toxicity with use of the iC9 safety switch. IL15 also significantly improves expansion of GPC-CAR T cells, resulting in more potent antitumor activity.
Development of novel biosensors to overcome immunosuppression of CAR T cells in the tumor microenvironment
146: Designed Tumor Microenvironment Responsive Biosensors Enhance Chimeric Antigen Receptor T Cell Therapy for Cancer
Jan A. Rath (University of Lausanne, Lausanne, Switzerland) presented a study to enhance CAR T cell persistence in the immunosuppressive tumor microenvironment. Two novel chimeric antigen receptor-based biosensors that were responsive to vascular endothelial growth factor (VEGFA; VMR) or to colony stimulating factor 1 (CSF1; CMR) were designed. Both biosensors included c-MPL signaling domains to produce an output signal. A computational pipeline was used to predict and develop the optimal chimera for both constructs. When expressed in activated T cells, the VMR and CMR biosensors were highly responsive to VEGF and CSF1, respectively, as measured by phosphorylated STAT5 levels. VMR or CMR biosensors were co-expressed with CARs targeting Ephrin type A receptor 2 (EphA2) or BCMA, respectively. VMR+ CAR T cells and CMR+ CAR T cells expanded and persisted better in vitro than their CAR T cell counterparts in the presence of VEGF or CSF1. CMR+ CAR T cells also exhibited significantly higher CSF1-dependent cytotoxicity against the MM.1S multiple myeloma cell line compared to control CAR T cells. In mouse models of metastatic lung cancer or multiple myeloma, VMR and CMR, respectively, enhanced anti-tumor function of CAR T cells. These data indicate that these novel inducible biosensors have potential to overcome challenges posed by solid tumors by enhancing persistence and anti-cancer activity of CAR T cells in the immunosuppressive tumor microenvironment.
Translational analyses of a TCR-engineered T cell product for synovial sarcoma
222: Mechanistic Characterization of Afamitresgene Autoleucel (Afami-cel; Formerly ADP-A2M4)
Laura Hudson (Adaptimmune, Abingdon, Oxfordshire, United Kingdom) reported translational analyses of six patients from the SPEARHEAD-1 trial, which tested afamitresgene autoleucel (afami-cel), a genetically modified autologous T cell product expressing an engineered TCR recognizing an HLA-A*02 restricted MAGE-A4 peptide, for synovial sarcoma and myxoid/round cell liposarcoma. In the Phase 1 trial of afami-cel, there was an overall response rate (ORR) of 44%, a disease control rate of 94%, and a median duration of response of 6.4 months in patients with synovial sarcoma. Proportions of CD4/CD8 amfami-cel cD3+ T cells varied among and between responders (n=3) and non-responders (n=3). High levels of activation marker expression were associated with increases in cytokine release and mitochondrial mass but not patient response. While afami-cel proliferation was not associated with patient response, proliferation was associated with increased Ki67 expression and retention of transduced cells. Retention of CD8+ TCR+ cells after stimulation was linked to cytotoxicity of the cell population and associated with release of granzyme B. These data indicate that multiple phenotypic characteristics of afami-cel, such as cellular expansion, cytotoxicity, cytokine secretion, and metabolic fitness, are likely linked to its anti-tumor functions. Analyses of correlative relationships between these characteristics and clinical endpoints are ongoing.
Preclinical studies of CD19-BCMA dual-targeting CAR T cells against low-CD19 lymphoma
224: CD19/BCMA Dual-Targeting CAR-T Cells Generated by Co-Transduction for the Treatment of Non-Hodgkin Lymphoma
Mireia Bachiller (Fundacio Clinic per la Recerca Biomedica, Barcelona, Spain) reported on the development of CD19/BCMA dual-targeting CAR-T cells to prevent resistance to CD19 CAR T cell therapy in lymphoma. Co-transduction with the ARI-0001 CD19-CAR and the ARI-0002 BCMA-CAR was found to be the most effective method to generate CD19/BCMA dual-targeting CAR T cells (ARI-0003) with the highest effector functions. ARI-003 prolonged animal survival compared to ARI-001 CAR T cells for xenograft models of BCMA-negative Burkitt lymphoma with wild type CD19 expression and low CD19 expression, and survival benefits were significantly improved in the model with low CD19 expression. In mouse models of BCMA-negative CD19-low lymphoma, ARI-003 preserved anti-tumor activity after treatment with CD19 CAR T cells and eradicated tumors in six of seven mice. ARI-0003 exhibited cytotoxicity against both BCMA+ CD19+ and BCMA+ CD19- lymphoma spheroids derived from patients with disease that relapsed after treatment with cD19 CAR T cells. Clinical-grade ARI-0003 CAR-T cells were produced with CliniMACS Prodigy technology, and for all five batches, CD19-BCMA-CAR represented over 20% of the CAR+ population. Studies to test ARI-0003 in patients with non-Hodgkin lymphoma, including those with relapsed disease after CD19 CAR T cellular therapy, are being planned.
Universal blood cancer immunotherapy with epitope editing of CD45
226: Epitope Editing in Hematopoietic Cells Enables Universal Blood Cancer Immune Therapy
Nils Wellhausen (University of Pennsylvania, Philadelphia, PA) presented a study to develop a CD45-targeting universal CAR T cellular therapy for hematopoietic malignancies. As CD45 is expressed on hematopoietic stem cells (HSCs) and their progeny, the use of CD45 CAR T cells would be lethal. To safely and effectively target a shared antigen, the CD45 epitope on both, HSCs and CAR T cells was edited during ex vivo production, so CAR T cells targeting CD45 would not recognize and kill transplanted HSCs as well as each other while retaining their essential functions. Alanine mutagenesis identified the CD45 epitope targeted by the CD45 CAR, and the epitope was mutated by CRISPR-based editing. CD45 CAR T cells expressing the edited CD45 (CD45BE) were able to avoid fratricide but retained CD45 function. Acute myeloid leukemia (AML) cells were grafted on mice, and mouse models of acute myeloid leukemia (AML) were treated with wild type CD19 CAR T cells or with CD45 CD45BE CAR T cells (CAR45). The CAR45 T cells eliminated tumor cells, prolonged survival, and provided long-term immune surveillance in controlling tumors in NSG mice. To prevent on-target/off-tumor toxicities, human HSCs were edited with CD45BE and transferred to mouse models of AML. Mice were treated with CAR45, and tumor cells were destroyed, while base-edited HSCs were spared and remained detectable in peripheral blood and bone marrow. CD45BE HSCs were able to engraft, persist, and differentiate in vivo, and effector immune cells maintained function. This study provides the foundation of a versatile platform that, when coupled with edited HSC transplant, can provide universal treatment of all blood cancers with CAR T cell therapy and beyond.
Next-generation controllable CAR T cells for AML
148: Enhanced Anti-AML Potency of DARIC33 by iSynPro-IL-15*: An IL-15 Expression Module Driven by a Tightly Regulated Synthetic Promoter Activated by Antigen Receptor Signaling
Jacob Appelbaum (University of Washington, Seattle, WA) presented approaches to generate next-generation drug-controllable CAR T cells. A CAR construct specific for CD33 that requires low doses of rapamycin (RAPA) for activity – DARIC33 - was used. In addition, genetic modules were designed in which following antigen signaling a novel inducible synthetic promoter (iSynPro, iSP) transiently drives transcription of human IL-15 (iSP-IL-15) or IL-15 modified to restrict signaling to cells expressing IL-15Ra (iSP-IL-15*). DARIC33 CAR T cells using lentiviral vectors with or without integrated iSP-IL-15 modules were generated and preclinically tested. iSP-IL-15 and iSP-IL-15* augmented products exhibited higher CD8+ T cell proportions. Following in vitro challenge with AML cells, DARIC33.iSP-IL-15 and DARIC33.iSP-IL-15* exhibited RAPA-dependent secretion of IL-15 and other effector cytokines. In vitro cell expansion, vector copy number, and CD33 antigen-binding capacity were similar among T cells transduced with DARIC33, DARIC33.iSP-IL-15 or DARIC33.iSP-IL-15*. However, in IL-2- and IL-15-free media, both iSP-IL-15* and iSP-IL-15 DARIC33 products demonstrated enhanced expansion but normal contraction kinetics. In an AML tumor-bearing immunodeficient animal model, both DARIC33.iSP-IL-15 and DARIC33.iSP-IL-15*showed tumor control upon administration of a low engineered cell dose, whereas DARIC33 could not control tumor growth at the same dose. In summary, features for next-generation CAR T cell products, e.g., iSynPro-regulated expression, IL-15* safety modification, and RAPA controlled DARIC33 activation can increase safety and prevent unrestrained T cell outgrowth.
Cancer vaccination by using a mixed solution of neo-antigens and oncolytic virus
291: A Personalised Neo-Antigen Viro-Immunotherapy Platform for Triple-Negative Breast Cancer
Yaohe Wang (Queen Mary University of London, London, United Kingdom) presented an approach to deliver tumor neo-epitopes through an oncolytic virotherapy using a murine model of triple-negative breast cancer (TNBC). The group previously developed a new generation of tumor-specific oncolytic Vaccinia Virus (VV) which can directly destroy tumor cells and can be used to co-deliver peptides and neo-antigens. A total of 30 HLA-I-restricted neo-epitopes from the murine breast cancer E0771.LMB cell line was identified using bioinformatic tools and cell-based assays, of which nine were found to be immunogenic. E0771.LMB tumor bearing C57BL/6 mice were then vaccinated with selected immunogenic neo-epitopes. The neo-antigens were delivered in a mixed solution with an oncolytic VV in a prime/boost regimen. Delivery of tumor neo-epitopes with oncolytic VV induced the formation of neo-epitope specific CD8+ T cells. However, only mice receiving PD1 checkpoint blockade antibody (aPD1) treatment in addition to neo-antigens and oncolytic VV showed improved tumor control and overall survival. Combined neo-epitope, oncolytic VV, and aPD1 treated mice showed higher infiltration of immune cells into the tumor and induced maturation of tumor-infiltrating dendritic cells. In summary, this approach could be used to co-deliver personalized neo-epitopes with an oncolytic virotherapy to increase anti-tumor responses.
Gene-edited CAR gamma delta T cells as an allogeneic treatment approach
267: Non-Virally Engineered Polyclonal gamma delta T Cells Exhibit Potent Anti-Tumor Activity In Vivo
Jacob Bridge (University of Minnesota, Minneapolis, MN) presented an approach for large-scale production of polyclonal, non-virally engineered and base edited CAR- gamma delta T cells for the use as an allogeneic cancer immunotherapy. Using plate-bound pan- gamma delta TCR and soluble CD28 antibodies, >10,000-fold cell expansion and greater TCR diversity was reached when compared to gamma delta-populations derived from zoledronate-based expansion methods. Cells were then transduced with a CD19-specific CAR construct using non-viral transposon-based integration. In addition, candidate genes linked to gamma delta T cell inhibition, including CISH, PD1, and Fas, were inactivated using Cas9 adenosine base editing to modulate the effector functions. Engineered cells were then tested in vitro and in vivo. Base edited gamma delta CAR T cells exhibited potent in vitro anti-cancer activity in killing assays, and improved mouse survival in an immunodeficient NSG Burkitt’s Lymphoma model. Peripheral V delta 1+ gamma delta T cells were enriched over time in treated mice and exhibited increased persistence when engineered to secrete IL15. Interestingly, a distinct population of αβ TCR-V delta 1-V delta 2- cells representing <7% of injected gamma delta T cells predominated at endpoint in non-IL15-expressing groups. In summary, this proof-of-concept study supports the use of an antibody-based method for large-scale gamma delta T cell expansion, and the combinatorial approach of transposon-based CAR-engineering with base editing to modulate effector functions.
Decitabine enhances CAR T cell efficacy in AML
268: Combining Anti-CD33 Chimeric Antigen-Receptor T Cells with the Hypomethylating Agent Decitabine to Treat Acute Myeloid Leukemia
Kenneth K.H. Ng (City of Hope, Duarte, CA) presented a study using a combinatorial treatment approach to improve CAR T cell efficacy in AML. The group has previously developed a CD33-specific CAR using a donor matched allogeneic product for the treatment of r/r AML, which is currently being tested in a phase I clinical trial. These CAR T cells show potent and specific anti-leukemia activity in vitro and in vivo, and do not target normal hematopoietic stem and progenitor cells. To further increase CAR T cell efficacy, CD33-specific CAR T cells were combined with decitabine (DAC), a hypomethylating agent approved for the use in AML. In vitro, enhanced antileukemia CAR T cell activity was observed when tumor cells were pre-treated with DAC for 3 days, suggesting that decitabine can sensitize AML cells and thereby enhance the efficacy of CD33 CAR T cells. In an in vivo AML-animal model using immunodeficient NSG mice, the combinatorial administration of DAC and CAR T cells controlled tumor growth and improved overall survival. In summary, this study provides a proof-of-concept to combine CAR T cells with DAC as an approach to improve CAR T cell efficacy in AML.
Pairing PERC with AAV for multi-locus cell editing
319: Efficient and Minimally Perturbative CAR-T Cell Engineering Using Peptide-Enabled CRISPR RNP Delivery
Joseph J. Muldoon (UCSF, San Francisco, CA) presented a new method called peptide-enabled RNP delivery for CRISPR engineering (PERC) which allows precise cell editing without the need for electroporation. Thereby, an RNP enzyme is mixed with a particular amphiphilic peptide and then applied to cells in culture, resulting in efficient gene knockout. In vitro experiments showed that PERC produces significantly higher yields of edited cells, preserves the naïve and memory phenotype, and is less perturbative at the transcriptomic level when compared to electroporation. This method was then paired with adeno-associated virus (AAV) bearing a homology-directed repair template for making efficient electroporation-free knock-in of a CAR transgene to the TCR alpha constant (TRAC) locus under endogenous promoter control, which has been previously shown to improve anti-tumor potency compared to virus-mediated pseudorandom integration. With combined PERC and AAV, 75% or 25% knock-in efficiency of single TRAC-CAR or dual knock-in at TRAC and B2M, respectively, was achieved without detectable chromosomal rearrangement. Functionality of single and dual knock-in CAR T cells were tested in vitro as well as in vivo, showing that these cells were at least equal to electroporated cells. In summary, this study showed the possibility of pairing PERC with AAV which enables efficient, convenient, non-toxic, minimally perturbative, and multi-locus editing of cells.
Culture media modifications during manufacturing improves CAR T cell products
323: Development of a GMP-Compatible Manufacturing Process for Highly-Edited, Stem-Like, Metabolically Fit, Virus Free CRISPR CAR T Cells
Dan Cappabianca (University of Wisconsin-Madison, Madison, WI) presented a study to further optimize their GMP-compliant CAR T cell manufacturing protocol. It was previously shown that virus-free CRISPR (VFC) CAR T cell production through CRIPSR/Cas9-induced knockout of the endogenous TRAC gene is feasible and leads to a favorable memory-enriched phenotype. The group has now further improved quality attributes of VFC CAR T cells through the addition of small molecules and a novel media switch post-electroporation (EP) to produce edited and ‘metabolically fit’ cells. Upon post-EP addition of the DNA-PK inhibitor M3814 (Nedisertib), up to 50% transgene positive VFC CAR T cells were produced. Cell functionality and metabolical fitness was further improved when two different basal medias, TexMacs and Immunocult XF, pre- and post-EP, respectively, were used as shown by the group in seahorse assays and within in vivo xenograft neuroblastoma mouse models. In summary, this study shows an approach to optimize CAR T cell manufacturing by optimizing the cell culture media, and the GMP-compliant protocol allows the use of this approach in a clinical setting.