Chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment of B-cell malignancies, yet challenges including manufacturing delays, T-cell exhaustion, and limited persistence impede broader clinical success. Here, we report the single day production of non-activated CAR T-cells engineered to secrete interleukin-18 (IL-18), a pro-inflammatory cytokine that enhances T-cell function. These non-activated CART-IL18 cells exhibit robust anti-tumor efficacy across xenograft models of lymphoma, leukemia, and pancreatic cancer. IL-18 expression enhances the functional advantages of naïve-like non-activated CAR T-cells, resulting in improved persistence, metabolic fitness, and resistance to exhaustion. Single-cell transcriptomic analysis revealed upregulation of IL7R, KLF2, and MCL1, alongside suppression of inhibitory checkpoint genes such as PDCD1, TOX, and HAVCR2. Metabolomic profiling demonstrated enhanced mitochondrial bioenergetics, with increased spare respiratory capacity and accumulation of α-ketoglutarate, malate, and spermine. Functional in vitro and in vivo profiling demonstrated enhanced per-cell cytotoxicity and in vivo durability. We complemented these studies with single-cell transcriptomic and metabolomic analyses to define CAR T-cell biological states beyond what is captured by xenograft tumor clearance. This IL-18-enhanced, activation-free CAR T product offers a clinically actionable platform with the potential to reduce vein-to-vein time while improving product potency and persistence, providing a rationale for clinical testing in patients with tumors refractory to standard CAR T.

Adoptively transferred T cells require cytokine stimulation, which is achieved using lymphodepleting chemotherapy with or without administration of exogenous interleukin 2 (IL-2). Lymphodepleting chemotherapy (LDC) is associated with cytopenias and attendant complications, while high dose IL-2 causes severe infusion toxicity and can stimulate undesirable cell populations. To address these challenges, we developed cis-targeted IL-2 fusion molecules which are comprised of an IL-2 mutein with attenuated binding to IL-2Rα and IL-2Rβ linked to an antibody that targets a cell-surface molecule expressed specifically on engineered T cells. Using T cells from healthy donors as well as from lymphoma and melanoma patients, we selectively stimulated CAR-T cells or engineered TILs and enhanced their antitumor function in multiple tumor models. We also showed that cis-targeted IL-2 can mediate CART expansion and B cell aplasia in the absence of lymphodepletion in a nonhuman primate model.

Background: Chimeric antigen receptor (CAR) T cell therapies have transformed the treatment of B-cell malignancies, yet challenges including manufacturing delays, T cell exhaustion, and limited persistence impede broader clinical success. Here, we report the development and preclinical evaluation of a 1-day, non-activated CAR T cell product engineered to secrete interleukin-18 (CART19-IL18), a pro-inflammatory cytokine that enhances T cell function, manufactured without T cell activation or expansion, and designed for clinical translation in B cell malignancies. Methods: Human T cells were transduced with a lentiviral vector encoding a CD19- or mesothelin-targeting CARs co-expressing IL-18 or GFP, without prior stimulation and expansion, and harvested after 24 hours of transduction. Phenotypic and functional analyses were conducted in vitro using flow cytometry, cytokine secretion assays, cytotoxicity assays, metabolomics, and Seahorse analysis. In vivo efficacy was evaluated in xenograft models of B cell leukemia (Nalm6), lymphoma (JeKo-1), and pancreatic cancer (AsPC-1). Single-cell RNA-sequencing and metabolomics was performed to profile cell state and exhaustion. Comparison groups included traditionally manufactured activated CAR T cells (3-days), and non-activated CAR T cells without IL-18. Results: IL-18 expression significantly improved tumor control by non-activated CAR T cells across all models tested, despite lower CAR transduction rates (~10%). IL-18 synergized with the naïve-like phenotype of non-activated T cells, promoting expression of memory and survival-associated genes (IL7R, KLF2, and MCL1) while suppressing of inhibitory checkpoint regulators (TOX, PDCD1, and HAVCR2). These cells retained potent cytolytic function despite reduced terminal differentiation. Gene set enrichment revealed enhanced biosynthetic, mitochondrial, and telomere maintenance pathways. Metabolomic profiling of mouse serum revealed increased serum levels of α-ketoglutarate and spermine in non-activated CAR T19-IL18 treated animals, correlating with upregulation of key metabolic enzymes (SAT1, GLS, SLC38A1) in recovered CAR T cells; supporting oxidative metabolism and redox balance. Seahorse analysis confirmed increased spare respiratory capacity and basal respiration in non-activated CART19-IL18 cells, supporting a bioenergetically favorable metabolic profile. In leukemia and lymphoma xenograft models, CART19-IL18 demonstrated significantly enhanced tumor clearance and persistence, even when administered at lower doses. IL-18 expression was critical for in vivo efficacy, particularly in the non-activated manufacturing context. Functional assays confirmed superior cytotoxicity and metabolic fitness of CART19-IL18 cells. These findings were consistent across multiple donors and tumor models. Functionally, these cells preserved cytolytic activity, memory-associated transcriptional programs, and exhaustion resistance. This phenotype emerged in the absence of exogenous stimulation, suggesting that avoiding supraphysiologic TCR signaling preserves intrinsic T cell fitness. The 1-day non-activated manufacturing approach bypasses conventional activation steps, accelerates production, reduces reagent use, and allows for fresh product infusion. These features have the potential to expand access and improve outcomes in patients with rapidly progressing or post-CAR T relapsed malignancies. Conclusion: We have developed a novel, non-activated CART19-IL18 product using a 1-day manufacturing protocol that preserves key T cell features critical for antitumor immunity. This approach builds directly on the clinical success of the activated CART19-IL18 trial. The rapid, activation-free protocol not only simplifies manufacturing logistics but also yields a more potent product. These results support the clinical translation of 1-day CART19-IL18 cells as a next-generation CAR T cell therapy.

CD45-directed CAR-T cells (CART45) are a promising immunotherapy for a broad range of hematologic malignancies, given the pan-leukocyte expression of CD45. However, their clinical use is limited by cytotoxicity against normal hematopoietic cells expressing CD45. Epitope editing of CD45 in human hematopoietic stem and progenitor cells (HSPCs) offers a strategy to protect the normal hematopoietic system from CART45-mediated pancytopenia. While electroporation (EP) is widely used for CRISPR-based gene editing in HSPCs, it involves physical manipulation of cells and can reduce viability and function, particularly in primitive hematopoietic stem cells (HSCs). Lipid nanoparticles (LNPs) offer a non-viral, RNA-based delivery alternative that operates through endocytic uptake and membrane fusion, providing a gentle, scalable, and GMP-compatible platform. Although LNPs have been successfully used in other cell types, their use for ex vivo gene editing of human HSPCs remains limited. This study directly compares the editing efficiency of a Cas9-derived adenine base editor (ABE) targeting the CD45 epitope in human HSPCs delivered via LNP versus EP. To compile a preclinical data package for potential future clinical translation, we evaluated how each delivery method affects HSPC viability and preservation of stem cell properties such as multi-lineage differentiation and engraftment in immunodeficient mice. Human CD34⁺ HSPCs were edited ex vivo with an ABE and a guide RNA targeting the relevant CD45 epitope to install a non-synonymous mutation, delivered via either EP or LNP. Editing efficiency, colony-forming units, and immunophenotypic HSCs showed comparable results between both methods ex vivo. While electroporation achieved slightly higher editing efficiency, LNP delivery preserved cell viability, reaching a 1.5-fold increase in cell recovery relative to EP at 48 hours post-editing. Functional human HSC frequency, as determined by limiting dilution analysis (LDA) in immunodeficient mice (n=45) with equal serially diluted cell doses, was comparable between the two delivery methods. The estimated frequency of functional HSCs among the cells injected was 1 in 81,089 for EP- and 1 in 80,915 for LNP-mediated delivery. These findings indicate that the delivery of the base editing machinery via LNP supports long-term engraftment of HSCs at a similar level to EP. Both groups of cells gave rise to multilineage hematopoiesis in peripheral blood and long-term engraftment in bone marrow, assessed until 28 weeks in primary recipients. Both delivery methods achieved efficient and stable epitope editing, with editing efficiencies of 95% for EP and 81% for LNP before transplantation, which was maintained at 87% for EP and 78% for LNP post-transplantation. Furthermore, donor-matched CART45 cells selectively eliminated unedited cells while preserving edited hematopoietic populations using either EP or LNP delivery, both in ex vivo and in vivo settings. The total number of cells in the bone marrow derived from edited HSPCs was approximately 300-fold higher than that of unedited cells. This demonstrates that epitope-edited hematopoietic cells are effectively shielded from CAR-T cell-mediated cell killing. These findings demonstrate that our developed process using LNP-mediated base editing of CD45 enables highly efficient gene editing in HSPCs, improving cell recovery, preserving long-term HSC function, and simplifying the manufacturing process compared to EP. In addition, CD45-edited HSPCs via LNP are protected from CAR-T cell-mediated cytotoxicity. This pre-clinical LNP delivery process offers a clinically scalable alternative to electroporation for genetic engineering of HSPCs with potential applicability across a broad spectrum of hematologic malignancies and diseases.

Physiologic serum fructose levels range from 20-150. In the context of AML, fructose accumulates in the bone marrow, reaching concentrations of 2mM, 5mM and in some reports 8mM. Implicit in this observation is that fructose is produced by cells present in the bone marrow and diffuses into the larger blood volume of the periphery. Transmembrane flux of glucose and/or fructose is facilitated by glucose transporters (GLUT) that play a vital role in T cell metabolic reprogramming and anti-tumour function. GLUTs display preferential selectivity for carbohydrate macronutrients including glucose, galactose, and fructose. GLUT5, which selectively transports fructose over glucose, has never been explored as a genetic engineering strategy to enhance CAR-T cell serial killing and durable anti-tumor function in fructose-rich tumour environments. Here, we demonstrate that the expression of wild-type GLUT5 restores T cell metabolic fitness in glucose-free, high fructose conditions. We find that GLUT5 supports maximal glycolytic capacity, expedites ATP replenishments, and rescues IL-2 production by using fructose as the primary nutrient source. Using steady state tracer technology, we show that 13C6 fructose supports glycolytic reprogramming and TCA anaplerosis in CAR-T cells undergoing log phase expansion. In cytotoxicity assays, GLUT5 rescues T cell cytolytic function in glucose-free medium. The fructose/GLUT5 metabolic axis also supports maximal migratory velocity, which provides mechanistic insight into why GLUT5-expressing CAR-Ts have superior effector function as they undergo “hit-and-run” serial killing. Our findings have immediate translational relevance as GLUT5 confers a competitive edge in a fructose-enriched milieu, and is a novel approach to overcome glucose depletion in hostile tumour microenvironments (TMEs). Importantly, the source of fructose production has not been described. As sorbitol dehydrogenase (encoded by SORD) synthesizes fructose at the end of the polyol pathway, we profiled SORD abundance in human bone marrow using single cell transcriptomic data generated from the anti-CD123-CAR-T cell clinical trial (NCT04106076) performed at the University of Pennsylvania. UMAP visualization revealed SORD expression in AML blasts (CD33+ and CD34+). This was expected as they account for 20-80% of all the cells in the leukemic bone marrow. We found that the highest SORD transcript levels were mapped to a non-AML cell population. These cells are haematopoietic in origin (CD45/PTPRC+ cells). Interestingly, data from The Human Protein Atlas reveals that naive B cells express high levels of GLUT5 which could facilitate diffusion of fructose across the surface (Fig. S1B). scRNA seq data also indicated high Glut5 and GAPDH as well as LDHA in tumor cells which suggests that Glut5 is fueling glycolysis and the rapid growth of tumor cells These findings imply that Glut5 could be a target for cancer treatment. We recognize that AML blasts and CD123-CAR-T cells engineered to express GLUT5 may compete for fructose in the bone marrow. As GLUT8 also displays high affinity for fructose, it emerges as an important candidate to inspire similar approaches. Intuitively, select inhibitors of GLUT5 such as MSNBA (N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine; Ki of 3.2 ± 0.4 μM) can be combined with GLUT8-expressing CAR-T cells to bypass competition for fructose in AML. In a subset of patients Glut1 is high (SLC2A1) and these tumor cells do not express high levels of GAPDH or LDHA. These data suggest that the complete oxidation of glucose in the mitochondria maybe supporting the growth of AML blasts; positioning the complex 1 inhibitor metformin as an important candidate along with standard therapy. In summary, we show that T cell dependency on glucose can be mitigated by facilitating the metabolism of fructose, a closely related functional isomer of glucose. Our findings provide an important advance in the clinical applications of CAR-T cell therapy against AML, and potentially other tumors where fructose is abundant. Expressing glucose transporters to optimize fuel selection, expedite ATP replenishment, support cytokine production, and bolster anti-tumour function has been fraught with challenges. Here, we show that GLUT5 is an ideal candidate with immediate translational relevance, including CAR-Ts against AML.

Activated T cells undergo a metabolic shift to aerobic glycolysis to support the energetic demands of proliferation, differentiation, and cytolytic function. Transmembrane glucose flux is facilitated by glucose transporters (GLUT) that play a vital role in T cell metabolic reprogramming and anti-tumour function. GLUT isoforms are regulated at the level of expression and subcellular distribution. GLUTs also display preferential selectivity for carbohydrate macronutrients including glucose, galactose, and fructose. GLUT5, which selectively transports fructose over glucose, has never been explored as a genetic engineering strategy to enhance CAR-T cells in fructose-rich tumour environments. Fructose levels are significantly elevated in the bone marrow and the plasma of acute myeloid leukaemia (AML) patients. Here, we demonstrate that the expression of wild-type GLUT5 restores T cell metabolic fitness in glucose-free, high fructose conditions. We find that fructose supports maximal glycolytic capacity and ATP replenishment rates in GLUT5-expressing T cells. Using steady state tracer technology, we show that 13C6 fructose supports glycolytic reprogramming and TCA anaplerosis in CAR-T cells undergoing log phase expansion. In cytotoxicity assays, GLUT5 rescues T cell cytolytic function in glucose-free medium. The fructose/GLUT5 metabolic axis also supports maximal migratory velocity, which provides mechanistic insight into why GLUT5-expressing CAR-Ts have superior effector function as they undergo "hit-and-run" serial killing. These findings translate to superior anti-tumour function in a xenograft model of AML. In fact, we found that GLUT5 enhances CAR-T cell anti-tumour function in vivo without any need for fructose intervention. Accordingly, we hypothesize that GLUT5 is sufficient to enhance CAR-T resilience by increasing the cells' competitiveness for glucose at physiologic metabolite levels. Our findings have immediate translational relevance by providing the first evidence that GLUT5 confers a competitive edge in a fructose-enriched milieu, and is a novel approach to overcome glucose depletion in hostile tumour microenvironments (TMEs).

Cell surface molecules transiently upregulated on activated T cells can play a counter-regulatory role by inhibiting T cell function. Deletion or blockade of such immune checkpoint receptors has been investigated to improve the function of engineered immune effector cells. CD38 is upregulated on activated T cells, and although there have been studies showing that CD38 can play an inhibitory role in T cells, how it does so has not fully been elucidated. In comparison with molecules such as PD1, CTLA4, LAG3, and TIM3, we found that CD38 displays more sustained and intense expression following acute activation. After deleting CD38 from human chimeric antigen receptor (CAR) T cells, we showed relative resistance to exhaustion in vitro and improved anti-tumor function in vivo. CD38 is a multifunctional ectoenzyme with hydrolase and cyclase activities. Reintroduction of CD38 mutants into T cells lacking CD38 provided further evidence supporting the understanding that CD38 plays a crucial role in producing the immunosuppressive metabolite adenosine and utilizing nicotinamide adenine dinucleotide (NAD) in human T cells. Taken together, these results highlight a role for CD38 as an immunometabolic checkpoint in T cells and lead us to propose CD38 deletion as an additional avenue for boosting CAR T cell function.

Abstract Background: In patients with chronic lymphocytic leukemia (CLL), we and others have shown that the addition of the BTK inhibitor ibrutinib to CART cells increases the rates of durable complete responses. The mechanism for this remains unknown. Lymphodepletion (LD) is considered critical for CART cell therapy success, yet emergency myelopoiesis during recovery from LD may induce a surge of myeloid-derived suppressor cells (MDSC), particularly in patients with advanced cancer. BTK inhibition has been shown to inhibit MDSC function in vitro. We therefore hypothesized that administration of ibrutinib during and following LD could reduce MDSC-mediated suppression of CART cells and thereby improve CART cell outcomes. Methods: To explore the effects of ibrutinib on human MDSCs, we engineered suppressive monocytes through in vitro culture of CD14+ cells in the presence of IL-6 and GM-CSF. We added ibrutinib to the culture on Days 0-4 and compared the ability of the monocytes to inhibit proliferation of human CAR-T cells. To model CLL treatment with CART in immunocompetent mice, we used the Em-TCL1 adaptive transfer model of CLL. Mice were treated with ibrutinib (I), cyclophosphamide and fludarabine (FC), or ibrutinib + FC (FC+I). MDSCs were isolated from these mice for in vitro function studies. In subsequent experiments mice were also treated with syngeneic CART19 with or without FC or FC+I. Results: In vitro generated human MDSC suppressed CART19 function, and this was prevented by pre-treatment of MDSC with ibrutinib. Myeloid cells from the spleens of CLL-bearing mice inhibited syngeneic CART19 function ex vivo regardless of treatment with FC or I alone, but myeloid cells harvested from CLL-bearing mice treated with FC+I lost suppressive function. Finally, mice treated with CART19 after a LD regimen resembling that in our recently reported clinical trial (FC+I) showed a survival advantage over all other groups. Conclusions: BTK inhibition with ibrutinib prevents myeloid cell suppression of CART cells in vitro in human and murine systems. When combined with lymphodepleting chemotherapy (FC), ibrutinib enhances CART cell control of CLL in vivo, possibly by preventing MDSC-mediated suppression of CART cells. Citation Format: Benjamin F. Frost, Olga Shestova, Feng Shen, Chia Sharpe, John C. Byrd, Saar I. Gill. Ibrutinib improves chimeric antigen receptor T cell control of leukemia by inhibiting myeloid-derived suppressor cells [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 57.

Limited in vivo persistence and the need for lymphodepleting chemotherapy are ongoing challenges in CAR T cell therapy for hematologic malignancies. In vivo behavior of CAR T cells has shown a strong correlation with improved outcomes, leading to an interest in developing approaches to enhance the expansion and persistence of infused CAR T cells. Exogenous cytokines such as IL-2 can be administered in vivo, but are associated with adverse effects and have the potential to stimulate regulatory T cells, thus impeding the immune response. Lymphodepletion aids CAR T cell function but causes pancytopenia and associated complications. To address these challenges, we developed a CAR T-specific IL-2 fusion molecule that selectively activates CAR T cells by recognizing an extracellular tag. We hypothesized that this method should improve the anti-tumor efficacy and may eliminate the need for lymphodepleting chemotherapy. We developed an IL-2 molecule that signals selectively on CD19 or CD20-specific CAR T cells by fusing a CAR T cell-targeting antibody to an attenuated IL-2 mutein with diminished binding to IL-2R⍺ and IL-2Rβ. CAR T cell targeting antibodies were against the non-signaling truncated EGFR tag co-expressed with the CAR. While wild-type IL-2 signals on cells expressing its receptor, cis-targeted IL-2 fusion molecules targeting the EGFRt (EGFRt-IL2) enable selective IL-2 signaling by binding in cis to EGFRt on CAR T cells. Using STAT5 phosphorylation as a readout, in vitro stimulation of human or rhesus macaque CAR T cells that express EGFRt with EGFRt-IL2 led to a >100-fold increase in selectivity compared with wild-type (WT) IL-2. In vivo activity was demonstrated in NSG mice bearing an acute lymphoblastic leukemia (ALL) cell line treated with a “stress” dose of healthy donor anti-CD19-EGFRt CAR T cells with or without EGFRt-IL2. This was subsequently confirmed in a model where the anti-CD19-EGFRt CAR T cells were generated from a lymphoma patient. Response to treatment was accompanied by CAR T cell expansion, resulting in superior survival in the EGFRt-IL2-treated mice with a median overall survival of 61 days compared to 20 days for mice treated with PBS (p = 0.0015). To test whether IL-2 mutein administration could abrogate the need for lymphodepleting chemotherapy (LD), we treated two rhesus macaques (RM) with a single dose of autologous anti-CD20-EGFRt CAR T cells with (RM#1) or without (RM#2) IL-2 mutein. In the absence of LD, there was no evidence of B cell depletion after CAR T 20 infusion in RM#2, and anti-CAR antibodies were detected from day 11 onwards. In contrast, RM#1 experienced profound B cell depletion in blood and bone marrow lasting approximately 35 days. The animal also experienced cytokine release syndrome (CRS) on day 7 and required dexamethasone and tocilizumab, showing clinical improvement within 12-24 hours. B cell recovery was accompanied by the development of anti-CAR antibodies (which were first detected on day 28). To test whether re-dosing with IL2 mutein at a later time point (day 49) could expand the previously administered CAR T cells and induce B cell aplasia again, we injected RM#1 with the same dose of EGFRt-IL2 on day 49 without re-administering CAR T cells. This time there was no reduction in B cells, and anti-drug antibody (ADA) became detectable on day 62 (14 days post re-dosing). In summary, EGFRt-IL2 enhances in vivo anti-tumor activity and survival in xenografted mice, enhances the activity of lymphoma patient CAR T cells, and may abrogate the need for lymphodepletion prior to CAR T cell infusion. Temporal control of cis-targeted cytokines directed by anti-tag antibodies represents a promising approach to enhance CAR T cell therapies.

The addition of the BTK inhibitor ibrutinib to CART therapy increased complete response rate from 28% (n = 32) to 53% (n = 19) in patients with chronic lymphocytic leukemia (CLL) in two sequential studies at our institution (J Clin Oncol. 2020 Sep 1:2862-71; Blood Advances. 2022 Nov:4774-85). This observation was hypothesized to stem from direct inhibition of the related kinase ITK in patient T cells and/or from reduction of CLL burden with secondary effects on T cell quality (Blood. 2016 Mar:1117-27). However, BTK is also expressed in myeloid cells, and ibrutinib has been shown to deplete myeloid derived suppressor cells (MDSCs) in murine tumor models (Cancer Res. 2016 Apr:2125-36). Here we sought to systematically define the mechanism(s) by which ibrutinib improves CART function. We first compared the function of residual CART19 infusion products biobanked from a clinical trial of monotherapy (NCT01747486) to one in which patients also received ibrutinib starting at least six months prior to infusion (NCT02640209). We used a leukemia cell line (NALM6) as a universal target. There was no difference in killing (p = 0.76, n = 10) or antigen-specific proliferation (p = 0.74, n = 18), suggesting that ibrutinib treatment did not meaningfully affect CART19 quality despite substantial differences in patient responses. We next evaluated patient myeloid cells in peripheral blood (PB) samples taken the day prior to infusion (D-1). Patients not receiving ibrutinib (n=9) had a median of 144 immunophenotypic M-MDSCs per uL of PB (range 3-241 cells/uL) compared to a median of 49/uL (range 11-110 cells/uL) among patients who were (n = 10, p = 0.024). We then tested the ability of these myeloid cells to inhibit autologous CART19 cells. The addition of CD14+ myeloid cells from ibrutinib-untreated patients to CART19 and NALM6 cell co-culture reduced the mean percent proliferating CART19 cells from 92.4% to 64.2% (p = 0.0097), while CD14+ cells from ibrutinib treated patients did not impair T cell proliferation (95.6% vs 97.0%; p = 0.49). MDSCs are thought to form in response to soluble factors elaborated in the context of systemic malignancy. We thus hypothesized that patient serum could induce MDSC formation. Indeed, the presence of healthy donor (HD) CD14+ exposed for five days to serum from ibrutinib-untreated patients reduced mean antigen stimulated CART19 cell proliferation from 93.2% to 59.7% (p = 0.01). This inhibition was reversed if the serum was supplemented with 1 uM ibrutinib (mean 95.3% CART19 cells proliferating; p = 0.002). In contrast, the serum of patients receiving ibrutinib did not convert HD CD14+ cells to MDSC as the presence of these cells in co-culture did not inhibit CART19 proliferation (p = 0.99). This sera is assumed to contain ibrutinib, and we also compared its cytokine prolife to that of ibrutinib-untreated patients. Ibrutinib-treated patient serum had lower levels of the MDSC associated cytokines IL-10 (p = 0.007), IL-13 (p = 0.0008), GM-CSF (p < 0.0001), and IL-4 (p = 0.03), but higher levels of the Th1 secreted cytokines TNFα (p = 0.0009), IL-2 (p = 0.0045), and IFNγ (p < 0.0001). Since ibrutinib inhibits several kinases, we tested a more selective BTK inhibitor (acalabrutinib) and again found that immunophenotypic and functionally defined MDSC formation was prevented (p < 0.0001 for both analyses). To further validate the role of BTK in MDSC development, we deleted BTK in HD CD14+ cells. BTK-sufficient HD CD14+ cells exposed to IL-6 and GM-CSF over 4 days developed into CD33+HLA-DRlow MDSC capable of potently suppressing CART19 proliferation, while BTK-deficient HD CD14 did not (p = 0.04 for phenotypic comparison, p = 0.002 for functional comparison). Finally, we explored if the effect of ibrutinib on MDSC formation was generalizable beyond CLL. Exposure to serum samples collected on D-1 from four multiple myeloma or three pancreatic cancer patients treated on institutional CART trials polarized HD CD14+ cells to potently suppressive MDSC (p = 0.012 and p = 0.008 respectively), and this suppressive effect was abolished in the presence of ibrutinib (p = 0.98 and p = 0.99 respectively). These data suggest that ibrutinib improves CART19 therapy in CLL not by generating functionally superior CART cells per se, but by preventing the formation of MDSC, thus removing a critical counter-regulatory break on CART function. These results provide a mechanism by which ibrutinib can enhance CART therapy in CLL and beyond.

Many hematologic malignancies are not curable with chemotherapy and require novel therapeutic approaches. Chimeric antigen receptor (CAR) T-cell therapy is 1 such approach that involves the transfer of T cells engineered to express CARs for a specific cell-surface antigen. CD38 is a validated tumor antigen in multiple myeloma (MM) and T-cell acute lymphoblastic leukemia (T-ALL) and is also overexpressed in acute myeloid leukemia (AML). Here, we developed human CD38-redirected T cells (CART-38) as a unified approach to treat 3 different hematologic malignancies that occur across the pediatric-to-adult age spectrum. Importantly, CD38 expression on activated T cells did not impair CART-38 cells expansion or in vitro function. In xenografted mice, CART-38 mediated the rejection of AML, T-ALL, and MM cell lines and primary samples and prolonged survival. In a xenograft model of normal human hematopoiesis, CART-38 resulted in the expected reduction of hematopoietic progenitors, which warrants caution and careful monitoring of this potential toxicity when translating this new immunotherapy into the clinic. Deploying CART-38 against multiple CD38-expressing malignancies is significant because it expands the potential for this novel therapy to affect diverse patient populations.

INTRODUCTION Approximately 50% of patients with acute lymphoid leukemia (ALL) or with aggressive lymphoma who receive anti-CD19 CART cells (CART-19) remain relapse-free at 1 year. In vivo behavior of CART-19 correlates with improved outcomes, spurring interest in the development of approaches to selectively control the in vivo expansion of infused CART cells . Administration of exogenous cytokines belonging to the IL-2 family is one such approach. However, the clinical potential of combining IL-2 family cytokines with CART cells is hampered by the pleiotropic nature of the current molecules, which leads to severe toxicities and expansion of multiple endogenous cells (e.g. Tregs) in addition to CAR-T cells. While mutations have been introduced into existing engineered IL-2 variants to alter potency, these molecules are not entirely selective since endogenous cells are still stimulated. Other approaches using orthogonal cytokine/cytokine receptors are interesting but require further genetic modification of the T cells. To address these challenges, we developed CAR-T specific IL-2 or IL-21 fusion molecules that selectively activate CART cells by recognizing an extracellular tag. METHODS Cis-targeted cytokine fusions are comprised of (1) a targeting antibody directed against a tag expressed on the CAR-T surface (truncated non-signaling epidermal growth factor receptor [EGFRt] that is co-expressed with the CAR in at least one clinical-stage CART19 product) and (2) a cytokine mutein with attenuated binding to its cognate cytokine receptor. Specifically, IL-2 muteins exhibit diminished binding to IL2R⍺ and IL2Rβ while the IL-21 mutein has diminished binding to the IL-21R subunit. The targeting arm of the fusion molecule provides avidity to the attenuated cytokines, resulting in selective activation of the associated cytokine receptors on CARTs. We engineered one cis-targeted EGFRt-IL2 fusion molecule and one EGFRt-IL21 fusion molecule. Both molecules were characterized in vitro using primary human CART cells. In vivo activity was tested in a "stress test” model of B-cell ALL by engrafting 1x106 NALM6 cells that expressed luciferase into NSG mice for six days, prior to IV injection with a low dose of human CART19 (0.1x106). Cis-targeted cytokine fusions were administered intraperitoneally once, one day after CAR-T infusion. Anti-tumor activity was measured weekly by bioluminescence imaging (BLI) and analysis of peripheral blood was performed to examine the phenotype of the CAR-Ts. RESULTS The specificity of the EGFRt-IL2 molecule was demonstrated by its ability to selectively induce pSTAT5 signaling and EGFRt-IL21 molecule by pSTAT3, resulting in >100-fold preferential STAT activity in CAR-expressing cells compared to CAR-negative cells. In vivo BLI revealed that when sub-optimal doses of CAR-Ts were injected into leukemia-bearing mice, both EGFRt-IL2 and EGFRt-IL21 induced substantial tumor regression (Figure 1). EGFRt-IL21 induced greater tumor clearance (CR 5/5) with continued tumor regression without relapse through day 35, post CAR-T infusion. Interestingly, while EGFRt-IL2 induced a stronger initial expansion of CART19 in vivo than EGFR2t-IL21 (median 19068 CAR19T cells/ul blood vs 56 CAR19T cells/ul blood, p< 0.0001 on day 16), the EGFRt-IL21 group retained the 1:1 ratio of CD4:CD8 T cells in the infusion product while there was a much greater level of CD4 T cells present in the EGFRt-IL2 group. Both therapies were superior to CART19 control (without exogenous cytokine) and were equally well tolerated in mice. These observations suggest that the IL-2 and IL-21 cytokine fusions mediate their effects through different mechanisms. CONCLUSIONS Cis-targeted IL-2 or IL-21 cytokine fusion molecules selectively augment CAR-Ts in vitro and enhance in vivo anti-tumor activity and survival. Temporal control of the cis-targeted cytokines directed by anti-tag antibodies represent a promising approach to enhance CART cell therapies. DISCLOSURES NDM, KDM, WC, PB, CK, DP, TP, AY, and ID are employees of Asher Biotherapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chimaeric antigen receptor (CAR) T cells can generate durable clinical responses in B-cell haematologic malignancies. The manufacturing of these T cells typically involves their activation, followed by viral transduction and expansion ex vivo for at least 6 days. However, the activation and expansion of CAR T cells leads to their progressive differentiation and the associated loss of anti-leukaemic activity. Here we show that functional CAR T cells can be generated within 24 hours from T cells derived from peripheral blood without the need for T-cell activation or ex vivo expansion, and that the efficiency of viral transduction in this process is substantially influenced by the formulation of the medium and the surface area-to-volume ratio of the culture vessel. In mouse xenograft models of human leukaemias, the rapidly generated non-activated CAR T cells exhibited higher anti-leukaemic in vivo activity per cell than the corresponding activated CAR T cells produced using the standard protocol. The rapid manufacturing of CAR T cells may reduce production costs and broaden their applicability. Potent chimaeric antigen receptor T cells can be generated within one day from T cells derived from peripheral blood without the need for T-cell activation.

Abstract Despite recent advances in T cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Macrophages are usually the most abundant immune cell in the tumor microenvironment (TME) where, as immunosuppressive tumor-associated macrophages (TAMs), they participate in disease progression. The current goals of macrophage-based immunotherapies are to reduce TAM infiltration or enhance TAM phagocytosis. In contrast, we have developed a new paradigm based on the adoptive transfer of genetically engineered chimeric antigen receptor (CAR) macrophages (CAR-M) for the treatment of human cancer. CAR-M can only be produced using a unique adenoviral vector, since human macrophages are highly resistant to other methods of gene transfer. We have previously shown that the primary mechanism of action of CAR-M is phagocytosis, and that a single dose of primary human anti-HER2 CAR-M led to significantly improved overall survival in multiple xenograft models. We now establish that Ad5f35-transduced anti-HER2 CAR-M (CT-0508) adopt a unique proinflammatory and antitumor M1 phenotype. Functional evaluation and RNA sequencing revealed that CT-0508 maintain a proinflammatory M1 phenotype despite challenge with immunosuppressive environments in vitro, highlighting their resistance to subversion. By engrafting immunodeficient mice with human hematopoietic cells and human cancer cells, we established a novel xenografted human TME model. We demonstrate with single-cell resolution that CT-0508 maintain their phenotype within the human TME. Additionally, CT-0508 activated the human TME and generated an activated human dendritic cell signature. To further investigate the potential of CT-0508 for TME activation, we modeled the interaction of CT-0508 with immunosuppressive macrophages, dendritic cells, and T cells. CT-0508 shifted bystander macrophages toward a proinflammatory phenotype, induced activation and maturation markers on DCs, and recruited resting as well as activated T cells in chemotaxis assays. Lastly, CT-0508 demonstrated enhanced antigen presentation when compared to control human macrophages. These results show that in addition to direct antitumor activity, the anti-HER2 CAR macrophage cell product CT-0508 is capable of activating the solid cancer TME and promoting a proinflammatory phenotype. The safety of CT-0508 will be evaluated in an upcoming first-in-human phase I clinical trial. Citation Format: Konrad Gabrusiewicz, Nicholas Anderson, Xueqing Lu, Xinhe Shan, Olga Shestova, Nicholas Petty, Feng Shen, Maggie Schmierer, Andrew Best, Martha Zeeman, Yumi Ohtani, Katherine Cummins, Saar Gill, Michael Klichinsky. CT-0508, a novel CAR macrophage product directed against HER2, promotes a proinflammatory tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B65.

Abstract Despite recent advances in T cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Macrophages are often the most abundant immune cell in the tumor microenvironment (TME) where, as immunosuppressive tumor associated macrophages (TAMs), they participate in disease progression. Currently, most macrophage based immunotherapeutic approaches are focused on the depletion, repolarization, or phagocytic disinhibition of TAMs. We have developed a new paradigm based on the adoptive transfer of genetically engineered CAR macrophages (CAR-M) for the treatment of human cancer. CAR-M can be efficiently produced using the chimeric adenoviral vector Ad5f35. We have previously shown that the primary mechanism of action of CAR-M is antigen dependent phagocytosis, and that a single dose of primary human anti-HER2 CAR-M leads to significantly improved overall survival in multiple solid tumor xenograft models. Given that Ad5f35-transduced anti-HER2 CAR-M (CT-0508) adopt a unique pro-inflammatory M1-like phenotype, we hypothesized that CT-0508 may have the capacity to reprogram the TME toward an activated state. Functional evaluation and transcriptome-wide characterization revealed that CT-0508 maintain a pro-inflammatory phenotype despite challenge with immunosuppressive environments in vitro. By engrafting immunodeficient mice with human hematopoietic cells and human cancer cells we established a novel xenografted human TME model. We demonstrate with single cell resolution that CT-0508 maintain their M1 phenotype within the human TME. Additionally, CT-0508 augmented the human TME by inducing a pro-inflammatory signature in surrounding immune cells, characterized by induction of MHC-II and TNFα. To further investigate the potential of CT-0508 for TME activation, we modeled the interaction of CT-0508 with primary human M2 macrophages, dendritic cells, and T cells in vitro. CT-0508 repolarized bystander M2 macrophages toward a pro-inflammatory phenotype, induced activation and maturation markers on immature dendritic cells, and recruited resting as well as activated T cells in chemotaxis assays. CT-0508 demonstrated enhanced antigen presentation when compared to control human macrophages and cross-presented tumor derived intracellular antigens to CD8 T cells after tumor phagocytosis. Our results show that in addition to direct anti-tumor activity, the anti-HER2 CAR macrophage cell product CT-0508 is capable of promoting a pro-inflammatory tumor microenvironment and has the potential to induce epitope spreading via T cell recruitment and antigen presentation. Citation Format: Michael Klichinsky, Konrad Gabrusiewicz, Nicholas Anderson, Maggie Schmierer, Andrew Best, Martha Zeeman, Sotheavy Chhum, Yumi Ohtani, Olga Shestova, Xueqing Lu, Nicholas Petty, Xinhe Shan, Feng Shen, Saar Gill. CT-0508 is an anti-HER2 chimeric antigen receptor (CAR) macrophage with targeted anti-tumor activity that promotes a pro-inflammatory solid tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3242.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging1–4. Given the unique effector functions of macrophages and their capacity to penetrate tumors5, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity. Primary macrophages engineered to express chimeric antigen receptors have anti-tumor activity in humanized mice.