Phase I trial investigating the safety and efficacy of donor-derived CAR T cells for relapsed B-cell malignancies
Allogeneic Donor-derived CD19-Chimeric Antigen Receptor (CAR) T-cells for Relapsed B-cell Malignancies After Hematopoietic Stem Cell Transplantation
Ibrahim N. Muhsen, MD (Baylor College of Medicine) reported on results of the phase 1 CARPASCIO clinical trial to investigate the efficacy of donor-derived allogenic CD19 CAR T cells on patients with relapsed B-cell malignancies after allogenic hematopoietic stem cell transplant (allo-HSCT). The trial consisted of 7 patients who were post-allo-HSCT with relapsed malignancies or at high risk of relapse: 5 patients had B-cell acute lymphoblastic leukemia (ALL) and 2 had non-Hodgkin lymphoma (NHL). All patients had received at least four lines of therapy, including allo-HSCT, prior to the trial. 4 of 7 patients received pre-infusion lymphodepletion and all patients were infused with second generation CD19-CD28 CAR-T cells manufactured from their allo-HSCT donors. Primary outcomes were CAR T cell safety and persistence, and secondary outcome was patient response to therapy at 6 weeks. CAR T cell expansion and persistence, measured by qPCR of peripheral blood mononuclear cells, peaked 1-2 weeks post-infusion, and detectable signal persisted for 3.5 to 6 years in long-term survivors. qPCR signal was higher in patients who received pre-infusion lymphodepletion chemotherapy. No patients developed graft-versus-host disease (GvHD) or ICANS. 2 patients developed grade 1 or 2 cytokine release syndrome, and 4 patients developed grade 3 or 4 lymphopenias. Overall response rate was 5/7 (71%), and one-year overall survival was 4/7 (57%). 3 patients achieved complete remission after infusion, and 2 patients (one with ALL and one with NHL) have maintained remission more than five years with no GvHD or long-term effects. The remaining 5 patients died of disease 3 to 19 months after infusion. These results indicate that second-generation allogenic donor-derived CD19 CAR T cells are safe and can induce remission in patients with relapsed B-cell malignancies. Lymphodepletion may be necessary for long-term CAR T cell persistence. Additional approaches are being investigated to improve efficacy and persistence of the donor CAR T cells.
Engineering CAR T cells to reduce rejection and increase persistence
Engineering Stealth CAR T Cells to Evade the Host Immune Responses
Korneel Grauwet, PhD (Massachusetts General Hospital/Harvard Medical School) reported on efforts to reduce host rejection of autologous or allogeneic chimeric antigen receptor (CAR) T cells by reducing surface expression of human leukocyte antigens. Expression of viral transporter associated with antigen processing (TAP) inhibitors reduced expression of HLA class I on primary T cells. T cells with reduced expression of surface HLA I had slightly reduced lysis by NK cells in comparison to Beta2M knockout. Short hairpin RNAs (shRNA) were expressed to silence CIITA to reduce HLA Class II expression, which has the advantage over CRISPR Cas9/sgRNA of avoiding double strand breaks and gene editing. This data was used to generate a single vector design approach to downregulate both HLA I and II in CAR T cells targeting CD19. These anti-CD19 CAR T cells expressing viral TAP inhibitors and shRNA (stealth CAR T cells) showed reduced HLA I and II expression and similar levels of anti-tumor activity against lymphoblastic leukemia and lymphoma lines in vitro and in NSG mouse models of lymphoblastic leukemia (NALM6) compared to control anti-CD19 CAR T cells. PBMC from patients who received control anti-CD19 CAR T cells exhibited a cellular immune response against autologous anti-CD19 CAR T cells, but no immune response was observed with stealth anti-CD19 CAR T cells. These data suggest that stealth CAR T cells may be advantageous for preventing rejection and increasing persistence in patients receiving cell therapy.
Novel ligand-independent cytokine receptors to augment CAR T cell therapy
Leucine Zipper-based Cytokine Receptors Augments CAR T-cell Immunotherapy for Solid Tumors
Matthew R. Bell (St. Jude Children’s Research Hospital) reported on efforts to activate cytokine signaling in a cell-intrinsic manner to enhance CAR T cell therapy. Systemic administration of cytokines is associated with elevated CAR T expansion and persistence, but this practice is associated with toxicities. The extracellular domain of IL2R was replaced with two leucine zipper domains (Zip2R) to generate ligand-independent, constitutively active IL-2 receptors. Zip2R, when expressed in primary human T cells, activated STAT5 and improved T cell survival in the absence of exogenous cytokines. B7-H3 targeted CAR T cells expressing Zip2R (CAR.Zip2R) exhibited improved antitumor activity during chronic antigen exposure and improved antitumor activity in mice and against the human lung cancer cell line A549. A ZipReceptor (ZipR) for IL-7 was also engineered (Zip7R), and it exhibits improved antigen-specific expansion and anti-tumor activity in vitro, but CAR-Zip7R cells induce toxicities at high levels in mice due to massive antigen-dependent expansion at the tumor site. Ruxolitinib effectively inhibits ZipR JAK/STAT signaling, serving as a safety switch and a potential tool for management of toxicities. ZipRs are modular, and ZipRs for IL-21, IL-10, and IL-22 have been engineered and are in various phases of testing. These novel leucine zipper-based cytokine receptors improve the effector function of CAR T cells in preclinical models of solid tumors and could potentially enhance existing CAR T cell therapies.
Engineering CAR T cells to secrete a bispecific T cell engager for treatment of leukemia
Optimized CD70-Targeted CAR Secreting a CD33-Targeted Bispecific T-cell Engager Overcomes Antigen Heterogeneity for Acute Myeloid Leukemia
Mark Leick (Massachusetts General Hospital Cancer Center) reported on the development of CD70-targeted CAR that secretes a CD33-targeted bispecific T cell engager antibody (TEAM). This construct was developed to prevent antigen-escape observed in preclinical studies of CD70-targeting CAR T cells to treat patient derived xenograft (PDX) models of acute myeloid leukemia (AML) as well as leverage untransduced (UTD) bystander T cells (which are approximately 80% of clinical products) to also be directed against the tumor. A vector containing the CD70-CAR-CD33-TEAM construct was transduced in healthy donor T cells and utilizing a transwell cytotoxicity assay which is permeable by TEAM but not cells observed UTD killing of CD33+ target cells. CD70-CAR-CD33-TEAM cells eliminated Molm13 wild type, Cd33 knockout, and CD70 knockout tumors in vitro. In mixed in vivo models of wildtype and antigen knockout Molm13, CD70-CAR-CD33-TEAM cells exhibited superior expansion and destruction of AML tumor compared to CD33-targeted and optimized CD70-targeted CAR T cells. In the PDX AML model which previously showed CD70- relapse when treated with CD70 CAR, CD70-CAR-CD33-TEAM had superior efficacy with continued survival while all control groups succumbed to disease. These results support future studies of CD70-CAR-CD33-TEAM cells as a potential treatment for AML.
Surveying the cell surface and extracellular matrix for new CAR T cell targets
Targeting the C Domain of Tenascin C with CAR T Cells for the Immunotherapy of Pediatric Brain and Solid Tumors
Elizabeth Wickman (St. Jude Graduate School) reported results from analysis of over 1500 RNAseq pediatric samples including fifteen different tumor types and analyzing the surfaceome and matrisome across tumors and normal tissues to increase the repertoire of potential CAR targets for solid tumors. The C domain of Tenascin C (C.TNC), a component of the extracellular matrix and contributor to malignancy was identified as a potential CAR T cell target for treating pediatric glioma and osteosarcoma (OS). A C.TNC-CAR with a C.TNC-specific scFv and CD28 zeta signaling domain was constructed and transduced into CD3/CD28 activated peripheral blood T cells. C.TNC-CAR T cells produced cytokines in response to C.TNC-expressing OS cell lines and exhibited cytotoxic activity against these cells in vitro. C.TNC-CAR T cells exhibited transient anti-tumor activity in locoregional models of OS. C.TNC-CARs with different hinge, transmembrane, and signaling domains have been constructed, and constructs associated with improved cytotoxicity have been identified. Assays to characterize these new C.TNC CAR T cell constructs in vivo are in progress. These findings warrant further investigation of TNC and other secreted ECM proteins as potential CAR T cell targets in solid pediatric tumors.
Use of a switch receptor and combination therapy to improve efficacy of CAR T cells
aPDL1/41BB Switch-Receptor Enhanced CAR-T Cell Cytotoxicity and Synergize with Radiotherapy and aPD-1 Therapy in Solid Tumors
You Lu (West China Hospital, Sichuan University) reported on the development of CAR-T cells that express a novel anti-PDL1/41BB switch-receptor to improve the function and persistence of CAR-T cells in the solid tumor microenvironment. The anti-PDL1/41BB switch-receptor construct turns the inhibitory signal of PD-L1 into an activating signal through downstream PI3K signaling. T cells expressing the switch-receptor showed enhanced cytokine release (IL-2, TNF alpha, IFN gamma) and degranulation in response to PD-L1 and superior cytotoxicity in vitro. Anti-PDL1/41BB CAR-T cells exhibited higher anti-tumor activity in solid tumors in mice compared to conventional CAR-T cells. Transcriptional analyses revealed the switch receptor CARs had lower levels of apoptosis and exhaustion signatures compared to control CARs. Radiotherapy promoted CAR-T cell migration and increased T cell recruiting chemokines in vitro. A triple combination of radiotherapy, anti-PDL1/41BB CAR-T cells, and anti-PD-1 therapy exhibited high levels of anti-tumor activity in mice with increased tumor infiltration compared to single or dual-combination therapy. These data suggest that the anti-PDL1/41BB switch-receptor can increase T cell persistence and function and that combining anti-PDL1/41BB CAR-T cells with radiotherapy and immune checkpoint blockade can further augment their anti-cancer activity against solid tumors.
Results from the Phase 1/2a clinical trial of Temferon for glioblastoma
Autologous Cell & Gene Therapy for the Therapeutic Targeting of Immune Payloads to the Solid Tumor Microenvironment: Preliminary Results of the TEM-GBM Study
Bernard Gentner, MD (San Raffaele Telethon Institute for Gene Therapy) reported results from a Phase 1/2a clinical trial of Temferon, a platform of genetically modified cell-based autologous hematopoietic stem cells that selectively deliver interferon alpha (IFNa) to the TME. Preclinical studies have suggested that Temferon can reprogram tumor-associate macrophages from playing a role in cancer progression to playing a role in stimulating immunity and suppressing tumor growth. Survivors are resistant to rechallenge with tumor cells, and Temferon is synergistic with other T cell therapies. 15 patients with newly diagnosed unmethylated MGMT Glioblastoma (GBM), assigned to 5 dose cohorts, received 3 escalating doses of Temferon (from 0.5 to 2.0 x 10^6 cells/kg). No dose-limiting toxicities were observed. Temferon-derived cells were identified in peripheral blood and bone marrow at proportions up to 30% at one month in the cohort receiving the highest dose and persisted up to 18 months after treatment. Median overall survival is 15 months from surgery, and progression free survival is 8.3 months, higher than what is typically observed in GBM. Serious adverse events have been attributed to chemotherapy or to disease progression. 4 of the 15 patients in low dose cohorts have undergone a second surgery. Recurrent tumor samples from 3 of 4 patients analyzed show evidence of relocation of transduced cells from the bone marrow to the tumor. Single cell RNA seq of CD45+ myeloid cells purified from the TME of Temferon-treated patients vs patients receiving standard of care (rituximab + temozolomide) reveals that cells in the TME of Temferon-treated patients show induction of IFNa responses and macrophage repolarization. TCR sequencing reveals tumor-specific expansion. Preliminary data indicate an over-representation of inflammatory macrophages in the tumors of Temferon-treated patients, suggesting the occurrence of interferon-mediated inflammatory reprogramming in the TME of human GBMs, which was previously observed in preclinical mouse studies. Longer follow-up data from higher dose cohorts is in progress, but these early data suggest Temferon has the potential to modulate the TME, making the tumor more hospitable to immuno- and cellular therapies.
CD4 and CD8 CAR T cells in cytokine release syndrome
CD4 CAR T Cells Drive Extensive CD8 CAR T Cell Expansion, Leading to Severe Cytokine Release Syndrome
Camilla Bove (IRCCS San Raffaele Scientific Institute) reported on an investigation into the roles of CD4 and CD8 CAR T cell populations interact during CAR T cell therapy and the different roles of these cells in the development of cytokine release syndrome (CRS). CD4 CAR T cells exhibit a reduced killing capacity in vitro compared to CD8 CAR T cells, but they exhibit higher levels of proliferation, degranulation, and activation after exposure to antigen. Increased activation triggers increased activation of myeloid cells, potentially causing CRS. CD4 CAR T cells are the main contributors to CRS in HSPC-humanized mouse models, and CRS is less frequent when CD4 CARs are constructed with the 4-1BB co-stimulatory domain instead of the CD28 domain. CD4 CAR T cells guided expansion kinetics of CD8 CAR T cells in vivo, indicating CD8 CAR T cells require CD4 cells to function properly. Combining CD4.BBz with CD8.28z results in the lowest toxicity levels. These results suggest that CD4 CAR T cells affect CAR T cell responses and play a major role in the development of toxicities. Targeted engineering of CD4- and CD8-specific CARs may result in more effective and safer versions of cell therapies.
Identifying novel T cell receptors from patient responders to immunotherapy
Discovery of a Novel C07:02-Restricted Epitope on MAGE-A1 and Pre-Clinical Development of an Enhanced TCR-T Cell Therapy Candidate for the Treatment of Solid Tumors
Gavin MacBeath, PhD (TScan Therapeutics) reported results from a TargetScan genome-wide screen to identify patient-derived T cell receptors (TCR) that underwent clonal expansion during therapy with immune checkpoint inhibitors (ICIs). A screen of expanded TCRs from a patient with head and neck cancer who responded to ICIs identified the cancer testis antigen MAGEA1. Two different TCRs recognized a novel C*07:02 epitope. T cells expressing each TCR exhibited activation, cytokine production, and cytotoxicity against HLA-C*07:02-positive cell lines that expressed MAGEA1. TCR-41 was identified as a specific TCR that did not exhibit off-target recognition of other HLA-I molecules or non-cancerous C*07:02-positive cells. A vector that delivers the TCR-41 gene, CD8alpha/beta genes and a dominant-negative form of TGFbR2 has been introduced into CD4+ and CD8+ T cells, and these novel TCR-T cells are being investigated in Independent New Drug-enabling studies. This study validates the use of the TargetScan screen to identify tumor antigens and T cell receptors from expanded T cell populations from patients who responded to immunotherapy.
Disrupting gene silencing to combat T cell exhaustion
Disruption of H3K9me3-Mediated Gene Silencing Augments CAR T Cell Functional Persistence
Nayan Jain, Btech (Memorial Sloan Kettering Cancer Center) reported on efforts to increase the persistence of T cells expressing anti-CD19 chimeric antigen receptors (CARs) with CD28 costimulatory signaling (1928z). Loss of function in T and CAR T cells is associated with extensive chromatin remodeling due to chronic CAR signaling associated with histone methylation. In order to limit the transcriptional and epigenetic changes that occur in T cells during T cell dysfunction, the histone methyltransferase gene SUV39H1 was disrupted in 1928z CAR T cells. Immune-deficient mouse models of ALL were treated with SUV39H1-edited (SUV39H1ed) CAR T cells. 9/10 mice treated with SUV39H1ed CAR T cells survived over the 90-day observation period, compared to 1/12 mice treated with wild type 1928z CAR T cells. SUV39H1ed CAR T cells were also associated with greater initial T cell expansion, higher levels of CAR T cells in bone marrow and spleen, and a sustained memory phenotype. SUV39H1ed CAR T cells also outperformed wild type 1928z CAR T cells in eliminating tumors upon multiple rechallenges with leukemia cell lines. While control 1928z CARs exhibited an exhaustion phenotype from these animals, SUV39H1 edited CAR T cells had an improved memory phenotype with increased expression of LEF1, TCF7, CCR7, and IL7R. These data illustrate that loss of SUV39H1 in CAR-T cells improve anti-tumor efficacy and T cell persistence, providing proof-of-concept for combining CAR and epigenome engineering.
Engineering dual-targeting CAR T cells for acute meyloid leukemia
Dual CD33/CLL-1 Targeted CAR T Cells for Treatment of Acute Myeloid Leukemia
Kevin Kowal (Washington University School of Medicine) reported on the development of CAR T cells that target both CD33 and CLL-1(CD371), which are expressed on most acute myeloid leukemia cells. A variety of dual-targeting constructs including tandem, loop and dual designs in multiple orientations were screened for binding of both targets by flow cytometry. Based on their protein binding in vitro, lead candidates were screened in vitro sing cytotoxicity luciferase assays against AML cell line THP-1 and against CD33/CD371 negative, single-antigen positive, and dual positive Ramos cells. The three best-performing CAR constructs were then selected for in vivo experiments with the double positive Ramos cell line. The Dual2 construct, consisting of a single promoter and two full-length CAR constructs separated by a self-cleaving P2A peptide, provided complete tumor clearance and long-term tumor-free survival. NSG mice were injected with a mixture of Ramos cells expressing only CD33 and only CD371 and treated with either single antigen targeting CAR, pooled single antigen targeting CAR or Dual2 CAR T cells. The single-targeting CD33- and CD371-CART cells alone were not effective at controlling mixed tumor growth, and the Dual 2 CAR T cells had the highest anti-tumor activity against mixed tumors. These data support the potential effectiveness of dual-targeting CD33/CLL-1 CAR T cells in controlling tumors expressing both CD33 and CLL-1 and tumors with mixed populations of cells expressing CD33 or CLL-1.
Modifying the CAR T cell epigenome to increase CAR T cell persistence
Epigenome Editing Enables PD1 Silencing in CAR T-cells
Maria Silvia Roman Azcona (University of Freiburg) presented primary results from a study to disrupt interaction of PD1 with PDL1 in CAR T cells to reduce CAR T cell exhaustion. PDCD1-specific Designer Epigenome Modifiers (DEMs), which have the advantage of not requiring double stranded breaks in DNA, were designed to silence the PD1 encoding gene (PDCD1) via cytosine methylation. DEMs caused a significant reduction in transcript and protein levels of PD-1 in primary T cells. The DEM was expressed in a PSMA-directed CAR T cell, and the epigenetically edited CAR T cells (epiCART) exhibited no differences in cytotoxicity, cytokine release (IFNg, TNF, IL2), or proliferation, compared to unmodified CAR T cells. In a chronic stimulation assay with 4 antigen encounters over 12 days, no appreciative differences in CAR T cell proliferation were observed, but upon day 12 of the experiment there was decreased PD-1 at the transcript and protein level. The data suggest that epigenetic modifications of the PD1-encoding gene in CAR T cells can potentially increase CAR T cell persistence without compromising anti-tumor activity.
An off-the-shelf genetically engineered CAR-NK cell for pan-tumor targeting
FT536: A First-of-Kind, Off-the-Shelf CAR-iNK Cell Product Candidate for Solid Tumors Designed to Specifically Target MICA/B Stress Proteins and Overcome Mechanisms of Tumor Evasion
John Goulding, PhD (Fate Therapeutics) reported on FT536, a novel induced pluripotent stem cell (iPSC)-derived NK (iNK) cell product as a potential pan-tumor targeting agent. FT536 expresses a novel chimeric antigen receptor (CAR) that targets the conserved alpha3 domain of MICA and MICB stress proteins, which are expressed on a variety of solid and hematological tumors. FT536 is derived from a clonal master bank of genetically modified iPSCs, making off-the-shelf production of FT536 cost-effective and available on-demand to a broad range of patients. The genetic modifications to FT536 include a high-affinity non-cleavable CD16 (hnCD16) Fc receptor for targeting multiple tumor antigens in combination with the MICA/B CAR, an IL-15 receptor fusion to promote cell persistence and anti-tumor activity, and knockout of CD38 to enhance cellular function and fitness. FT536 has exhibited significant antigen-specific cell expansion and cytotoxicity in vitro and significant inhibition of solid tumor growth, including lung and liver metastases, in xenographic models in vivo. An Investigational New Drug application has been allowed by the FDA, and in-human clinical studies of FT536 monotherapy and FT536 in combination with tumor-specific monoclonal antibody therapy (trastuzumab) for treatment of multiple solid tumors will begin later in 2022.
Amored CAR T cells for treatment of solid tumors
A High Expression of IL15 Receptor Alpha (IL15Rα) in Glioblastoma Microenvironment Enables IL15-Armored CAR T-cells to Modulate Tumor Immunosuppression and Improve Survival in Syngeneic Models
Irina V. Balyasnikova, PhD (Northwestern University Feinberg School of Medicine) reported on a study to identify cells in the glioblastoma (GBM) tumor microenvironment (TME) that express interleukin 15 receptor alpha (IL15Ra) and contribute to the persistence and fitness of CAR T cells within the TME. Paired samples of peripheral and tumor-infiltrating immune cells from GBM patients revealed a several-fold increase in IL15Ra expression in tumor-associated macrophages, B cells and dendritic cells from tumor-infiltrating cells compared to peripheral cells. Murine IL-15-armored CAR T cells expressing IL-15 or IL-15 fused to IL13Ra2 single-chain antibody were co-cultured with myeloid-derived suppressor cells (MDSCs), and both IL-15 armored CAR T cells depleted the MDSCs, with cells expressing the IL15 fusion being more effective. CAR T cells expressing the IL-15 fusion improved survival in tumor-bearing mice, and the TME of these mice exhibited higher frequencies of CD8+ T cells, NK cells, and B cells. CD45+ cells in the TME exhibited decreased expression of genes involved in immunosuppression and monocyte migration, compared to control groups. These data suggest that CAR T cells armored with IL15 are capable of TME modulation and increase survival in mice compared to control CAR T cells. Early phase trials of IL15-armored CAR T cells in patients with GBM are in progress.
Increasing affinity of PD1-CD28 with PD-L1 to improve TCR and CAR T cell efficacy
High-affinity PD1-CD28 Chimeric Switch Receptors Enhance Costimulatory Signaling and Improve TCR and CAR T-cell Antitumor Activity
Brooke Prinzing (St. Jude Children’s Research Hospital) presented data from regarding a high-affinity chimeric switch receptor (CSR) combining the extracellular domain of PD1 with the transmembrane domain of CD28 (PD1-CD28). Previous studies have shown that interaction of PDL1 with PD1-CD28 converts the inhibitory T cell signal of PDL1-PD1 to a costimulatory T cell signal, and increasing affinity of PD1-CD28 with PDL1 could increase T cell function. T cells expressing PD1-CD28 or the high affinity PD1-CD28 (HPD1-CD28) expressed the CSRs at similar levels, and HPD1-CD28 bound to both recombinant PD-L1 protein and cells expressing PD-L1 at higher levels than PD1-CD28. When stimulated with CD3 and PD-L1, T cells expressing CSRs exhibited higher levels of phosphorylated ERK (pERK) and IL-2 compared to control T cells, with HPD1-CD28 cells expressing pERK and IL-2 at the highest levels. Engineered TCR or CAR T cells expressing either CSR showed increased antigen-dependent cytokine production and expansion in the presence of tumor cells compared to control cells, with cells expressing HPD1-CD28 exhibiting a markedly greater increase. CAR T cells expressing the CSRs had higher anti-tumor activity in vivo in an A549 lung cancer xenograph model. HPD1-CD28 showed the most benefit when mice were re-challenged with a second dose of tumor cells. Studies investigating the anti-tumor activity of CSR-modified TCR cells are in progress. These preliminary data provide further evidence supporting the use of PD1-CD28 CSRs, specifically HPD1-CD28, to improve persistence and anti-tumor activity of CAR T cells.
Inhibition of N-glycosylation makes the tumor microenvironment more hospitable to CAR T cell
N-Glycosylation Inhibition Hinders Immunosuppressive Activity of Tumor Microenvironment Cells and Improves CAR T Cell Efficacy
Camilla Sirini (San Raffaele Scientific Institute) described studies investigating the effects of N-glycosylation blockade on the immunosuppressive cells in the tumor microenvironment (TME) in CRC/PDAC-derived liver metastases and on CEA CAR T cell activity. Recent research suggests a role for surface N-glycans in protecting tumor cells from T cell-mediated destruction. Blocking N-glycosylation with the glucose/mannose analogue 2DG blocked the suppression of T cell proliferation by M2 macrophages (M2-M) and hepatic stellate cells (HepSC). Specifically, 2DG inhibited M2-polarization in macrophages, and upregulated M1 gene expression and secretion of TNF-alpha such that M2 macrophages became more like anti-tumor M1 macrophages. huSGM3 mouse models of liver cancer treated with 2DG exhibited significantly reduced frequencies of M2-M in liver tumors. 2DG treatment also corresponded with blocked PD-1/PD-L1 interaction and reduced expression of PDGFR-beta. 2DG also increased the proliferation and the cell-killing activity of CEA CAR T cells against cell lines and patient-derived organoids in vitro, even when co-cultured with TME cells. In addition, huSGM3 bearing patient derived organoids derived from liver metastases had superior response to CEA CAR T therapy when treated in combination with 2DG. These results indicate that treatment with 2DG blocks N-glycosylation and reduces cell-mediated immunosuppression in the TME, which could lead to increased efficacy of CAR T cells against solid tumors.
Results from the phase 3 ZUMA-7 trial of axi-cel for large B-cell lymphoma
Primary Analysis of ZUMA 7: A Phase 3 Randomized Trial of Axicabtagene Ciloleucel (Axi-Cel) versus Standard of Care (SOC) Therapy in Patients with Relapsed/Refractory (R/R) Large B-Cell Lymphoma (LBCL)
Armin Ghobadi, MD (Washington University) presented an analysis of results from the multicenter, Phase 3 ZUMA-7 study to investigate the effects of axi-cel, an autologous ant-CD19 CAR T cell therapy, in patients with LBCL refractory to or relapsed after less than year of first-line chemoimmunotherapy. The primary endpoint was event-free survival (EFS), and the secondary endpoints were objective response rate (ORR) and overall survival (OS; interim analysis). 359 patients were randomized 1:1 to axi-cel or SOC, chemoimmunotherapy followed by high-dose chemotherapy with autologous stem cell transplant (HDT-ASCT) if responsive. 94% of axi-cell patients were infused, and 36% of patients on SOC received HDT-ASCT. The primary endpoint, EFS, was met (hazard ratio 0.398): median EFS at the 24.9 median follow-up was 8.3 months for patients receiving axi-cel, versus 2 months for patients receiving SOC. ORR was 83% for axi-cel and 50% for SOC, and CR rate was 65% for axi-cel versus 32% for SOC. Median OS was not reached for the axi-cel cohort and was 25.7 months for the SOC cohort. 100 (56%) patients in the SOC cohort received immunotherapy off protocol as subsequent therapy. Cytokine release syndrome (CRS) greater than Grade 3 occurred in 11 (6%) axi-cel patients, and neurological events greater than grade 3 occurred in 36 (21%) axi-cel patients. Treatment-related deaths occurred in 1 axi-cel patient and 2 SOC patients. Safety was manageable and like previous trials of third-line use of axi-cel. Preliminary data from the ZUMA-7 trial suggest that axi-cel significantly improves OS, EFS, and the CR rate and enables more patients to receive therapy, providing a new alternative to second-line treatment of relapsed/refractory LBCL.
The role of T helper cells in preventing post-CAR T cell therapy relapse
Single-cell Antigen-specific Activation Profile of CAR-T Infusion Product Identifies Th2 Deficiency in CD19-Positive Relapsed ALL Patients
Zhiliang Bai (Yale University) reported on a study to investigating the functional capacity CAR T cells in the infusion product to address mechanisms behind CD19 positive relapse in patients. 101,326 single cell transcriptomic landscape profiles were developed from CAR T infusion products of 12 pediatric patients with acute lymphocytic leukemia. Patients were subdivided in those with complete, durable remission >54 months (CR), with CD19-positive relapse (RL), and non-responders (NR, n = 2). CAR+ cells from CR, RL, and NR patients exhibited distinct cytokine module expression profiles. Cells from CR patients were enriched for the Th2 module, and cells expressing the Th2 module were depleted in RL patients compared to CR patients. CAR T cells with transcriptional profiles similar to Th2 helpers (IL4, IL5, IL13, and GATA3) were enriched in CR patients compared to RL subjects. Independent intracellular flow cytometry and multiplexed secretomic assays validated these findings, and Th2 cells from CR patients exhibited a higher frequency of cytokine secretion (particularly IL-5) compared to RL patients. In addition, a lower abundance of Tscm and Tcm cells significantly associated with RL. There was no difference in the frequency of Tem CAR but a higher proportion of cells in the RL group differentiated into a Teff state. These findings suggest that engineering CAR T cells with increased Th2 signaling or boosting CAR T Th2 function post-infusion may lead to more durable, long-term remissions in patients.
Engineering CAR T cells to target aberrant cell surface carbohydrates
Targeting Tumors and the Tumor Microenvironment with Banana Lectin Expressing T Cells
Mary Kathryn McKenna, PhD (Baylor College of Medicine) reported on the construction of a chimeric receptor based on modified lectin from banana, H84T BanLec, which recognizes high-mannose, an aberrant glycan that is often expressed in high levels on tumor cells but not normal cells. H84T BanLec was attached to a chimeric antigen receptor (H84T-CAR) and tested against pancreatic ductal adenocarcinoma (PDAC), a tumor with high levels of aberrant glycosylation and a dense stroma. In 3D tumor culture models, H84T-CAR T cells disrupt the tumor stroma and promote tumor infiltration by CAR T cells. This effect is not seen with conventional HER-2-targeted CAR T cells, suggesting the H84T CAR has a unique ability to target tumor stroma. Dual targeting H24T and HER.2 CAR T cells exhibit stronger anti-tumor activity in a xenograft model of PDAC consisting of CFPAC tumor cells and pancreatic stellate cells. These data suggest CARs targeting altered carbohydrate structures may enable CAR T cells to persist and maintain functionality in the heterogeneous solid tumor microenvironment.
Tumor necrosis factor alpha receptor 2 and CAR T cell dysfunction
TNFR2 as a Target to Improve CD19-Directed CAR T-cell Fitness and Antitumor Activity in Large B-cell Lymphoma
Claudia Manriquez Roman, MSc (Mayo Clinic) presented data on the correlation of CAR-T cell surface death receptors and ligands with CAR T cell fitness and patient response to therapy. Flow cytometry studies of ex-vivo stimulated, and not resting, CAR T products of patients from the ZUMA-1 trial (axi-cel) showed that death receptors and ligands were upregulated in CAR T cells from non-responders, compared to responders. In an in vitro co-culture assay, CAR T cells were stimulated, and the tumor necrosis factor alpha receptor 2 (TNFR2) was upregulated, suggesting a role for TNFR2 in antigen-dependent CAR T cell dysfunction. CD19-specific activation upregulated TNFR2 expression but not TNFR1. TNFR2 expression was inversely correlated with naïve T cell marker CCR7 in healthy donors. TNFR2 knockout (TNFR2 k/o) CAR T cells were generated and demonstrated early activation of CD25 and CD69, reduced apoptosis, and enhanced antigen-specific proliferation and cytotoxicity. In a xenograft model of lymphoma, TNFR2 k/o CAR T cells exhibited enhanced CAR T cell expansion and anti-tumor activity. These results suggest a role for TNFR2 in CAR T cell dysfunction following antigen-specific activation and knocking out expression of TNFR2 as an approach to increase CAR T cell fitness.
Engineering T Cell Receptors to recognize mutant KRAS peptides
Preclinical Development of Safe and Effective T Cell Receptors Specific for Mutant KRAS G12V and G12D Peptides
Tijana Martinov, PhD (Fred Hutchinson Cancer Research Center) provided preliminary data on the development of TCR engineered T cells specific for mutant KRAS (mKRAS) G12V for use against solid tumors. KRAS is the most frequently mutated oncogene in solid tumors, and it is mutated in over 80% of pancreatic cancers, thus mutant KRAS are attractive targets for CAR T cell therapy. CD8+ T cells from healthy donors were stimulated antigen presenting cells pulsed with mKRAS peptides predicted to bind to HLA-A*11:01. Cells were sorted, and T cell receptors (TCRs) were sequenced, synthesized, and compared. TCRs were screened for function via avidity, tumor recognition and tumor killing and safety was assessed using alanine, peptide motif, and alloreactivity scans. Lead TCRs were expressed in CD8+ and CD4+ T cells and exhibited significant anti-tumor activity against several tumor cell lines, including colon carcinoma and breast cancer carcinoma, in vitro. This work provides a TCR gene therapy approach to target KRAS-mutant solid tumors via a high-throughput in vitro platform.
Phase I trial testing the safety and efficacy of CAR-NKT cells against solid tumors
Anti-GD2 CAR NKT Cells Are Safe and Produce Antitumor Responses in Patients with Relapsed/Refractory Neuroblastoma
Andras Heczey (Baylor College of Medicine) reported results from phase I trials assessing safety, persistence, and efficacy of second-generation chimeric antigen receptor V alpha 24 natural killer T cells (CAR-NKTs) co-expressing a GD2-CAR and IL-15. NKT cells are attractive lymphocytes for cellular therapy of solid tumors because they are trafficked to neuroblastoma (NB) in a CCL2/CCL20-dependent manner, and NKT infiltration in NB tissues is associated with improved patient outcomes. These CAR-NKTs were previously shown to have anti-tumor activity in mouse models of NB. 12 patients with relapsed/refractory NB (median age 7.6 years) were infused with cells on four different dose levels. No dose-limiting toxicities or toxicities over grade 2 were observed. All patients exhibited expansion of CAR-NKTs in peripheral blood, and peaks appeared two to four weeks after infusion. CAR-NKTs were also detected in neuroblastomas, bone metastases, and bone marrow. Of the 5 responders, one had partial response, one had complete response, and 3 had stable disease. Responders exhibited higher levels of CAR-NKT expansion and persistence, and this correlated with CD62L+ CAR NKT frequencies, suggesting CAR-NKT anti-tumor activity is associated with increased expansion rate and with CD62L expression. Durable complete remission was observed in one patient. The data support the use of CAR-NKTs to treat solid tumors safely and produce objective responses in patients with NB.