CD22 CAR Optimization for Improved in-Human Activity Following Inadequate CD22 CAR Activity in Phase 1 Clinical Trial PLAT-04

Background: CD19 targeting chimeric antigen receptor (CAR) T cells have induced unprecedented remission rates in high-risk precursor B Acute Lymphoblastic Leukemia (ALL); however recurrent disease with CD19 antigen escape variants is not uncommon. Therefore, we developed a novel CD22 targeting CAR, and following preclinical validation, tested it in a first-in-human pediatric and young adult phase 1 clinical trial, PLAT-04 (NCT03244306). Four subjects were treated at 2 dose levels (DL) (1x10 6/kg (DL1) and 3x10 6/kg (DL2)). The CD22 CAR T cell product (SCRI-CAR22v1) was successfully manufactured (n=4) and no dose limiting toxicity (DLTs), cytokine release syndrome (CRS) or neurotoxicity was observed. However, all subjects had minimal CAR T cell expansion, with 3 of 4 subjects demonstrating persistent or progressive disease at day 21 evaluation despite continued CD22 expression on leukemic blasts. Based on the poor in vivo expansion and lack of activity, enrollment was voluntarily halted to interrogate and optimize the CAR construct for enhanced performance. Methods: Human T cells were transduced to express one of two CD22 CAR constructs. We designed SCRI-CAR22v2, a CD22 CAR that utilizes the same scFv as SCRI-CAR22v1 but with a shorter linker between M971 VH and VL and a shorter hinge with differing transmembrane region, and both using CD8 alpha (Figure A). This construct maintained the truncated EGFR extracellular tag (EGFRt) for tracking and potential in vivo suicide mechanism. The two transduced CAR T cell products were compared preclinically by flow cytometry, chromium release assay and in an in vivo murine model to understand differing T cell activity between the CAR constructs. Additionally, SCRI-CAR22v2 is currently under investigation in a dose finding phase 1 clinical trial, PLAT-07 (NCT04571138). Results: Following use of cetuximab-APC and biotinylated anti-human Fab antibody for surface EGFRt and CAR detection, the SCRI-CAR22v1 expresses lower levels of EGFRt but similar CAR levels on the cell surface demonstrated by MFI (Figure B). Biotinylated, soluble CD22 antigen was also used to evaluate CD22 CAR receptor activity and, as measured by MFI, a higher affinity is suggested via SCRI-CAR22v2 as compared to SCRI-CAR22v1 (Figure B). K562 cells expressing low, medium or high CD22 were used to evaluate the impact of surface antigen expression on the CAR activity level. SCRI-CAR22v2 demonstrates improved targeted cell lysis at all 3 antigen quantity levels by chromium release assay (Figure C). In NSG mice inoculated with Raji tumor cells expressing ffluc, SCRI-CAR22v2 demonstrated improved survival compared to SCRI-CAR22v1 (Figure D) and clearance of Raji tumor cells (Figure E). Based on this promising preclinical data, we initiated enrollment onto PLAT-07, a phase 1 dose finding trial (2x10 5cells/kg (DL1), 5x10 5cells/kg (DL2) and 1x10 6cells/kg (DL3)) of SCRI-CAR22v2. To date, 3 subjects have been enrolled and successfully infused at DL1. All had prior CD19-CAR therapy and 2 lacked CD19 leukemic expression at the time of SCRI-CAR22v2 infusion. At the time of cell infusion, one subject had only extramedullary disease, one had MRD of <1% and one subject had a larger disease burden of 30% ALL. None experienced a DLT and all were MRD negative in the bone marrow at day 28 and the subject with EMD demonstrated a complete metabolic response by PET scan. Figure F exhibits the improved expansion and engraftment of the SCRI-CAR22v2 cells as compared to SCRI-CAR22v1 DL1 (n=3) and DL2 (n=1), and higher peak levels of CD22 CAR T cells as compared to SCRI-CAR22v1 DL1 and DL2 (Figure G). Conclusions: Despite encouraging preclinical data, SCRI-CAR22v1 demonstrated poor expansion and engraftment in a Phase 1 trial. Notably, minor CAR alterations lead to encouraging in-human activity in early clinical findings. Our experience suggests a shorter linker and hinge as well as incorporation of an CD8 alpha transmembrane region improves the clinical activity of CD22 targeted CAR T cells in subjects with recurrent disease following CD19 CAR T cells. Further evaluation is needed to elucidate the critical CAR components and/or assays at the preclinical level that can best predict which CAR should be brought to the clinic for further evaluation. Figure 1 Figure 1. Disclosures Orentas: Lentigen: Patents & Royalties. Jensen: BMS: Patents & Royalties; Umoja Biopharma: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Research Funding. Gardner: Novartis: Consultancy; BMS: Patents & Royalties.

A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity

The cytotoxic activity of T cells and Natural Killer cells is usually measured with the chromium release assay (CRA), which involves the use of 51Chromium (51Cr), a radioactive substance dangerous to the operator and expensive to handle and dismiss. The accuracy of the measurements depends on how well the target cells incorporate 51Cr during labelling which, in turn, depends on cellular division. Due to bystander metabolism, the target cells spontaneously release 51Cr, producing a high background noise. Alternative radioactive-free methods have been developed. Here, we compare a bioluminescence (BLI)-based and a carboxyfluorescein succinimidyl ester (CFSE)-based cytotoxicity assay to the standard radioactive CRA. In the first assay, the target cells stably express the enzyme luciferase, and vitality is measured by photon emission upon the addition of the substrate d-luciferin. In the second one, the target cells are labelled with CFSE, and the signal is detected by Flow Cytometry. We used these two protocols to measure cytotoxicity induced by treatment with NK cells. The cytotoxicity of NK cells was determined by adding increasing doses of human NK cells. The results obtained with the BLI method were consistent with those obtained with the CRA- or CFSE-based assays 4 hours after adding the NK cells. Most importantly, with the BLI assay, the kinetic of NK cells’ killing was thoroughly traced with multiple time point measurements, in contrast with the single time point measurement the other two methods allow, which unveiled additional information on NK cell killing pathways.

Neuroblastoma and DIPG Organoid Coculture System for Personalized Assessment of Novel Anticancer Immunotherapies

Cancer immunotherapy has transformed the landscape of adult cancer treatment and holds a great promise to treat paediatric malignancies. However, in vitro test coculture systems to evaluate the efficacy of immunotherapies on representative paediatric tumour models are lacking. Here, we describe a detailed procedure for the establishment of an ex vivo test coculture system of paediatric tumour organoids and immune cells that enables assessment of different immunotherapy approaches in paediatric tumour organoids. We provide a step-by-step protocol for an efficient generation of patient-derived diffuse intrinsic pontine glioma (DIPG) and neuroblastoma organoids stably expressing eGFP-ffLuc transgenes using defined serum-free medium. In contrast to the chromium-release assay, the new platform allows for visualization, monitoring and robust quantification of tumour organoid cell cytotoxicity using a non-radioactive assay in real-time. To evaluate the utility of this system for drug testing in the paediatric immuno-oncology field, we tested our in vitro assay using a clinically used immunotherapy strategy for children with high-risk neuroblastoma, dinutuximab (anti-GD2 monoclonal antibody), on GD2 proficient and deficient patient-derived neuroblastoma organoids. We demonstrated the feasibility and sensitivity of our ex vivo coculture system using human immune cells and paediatric tumour organoids as ex vivo tumour models. Our study provides a novel platform for personalized testing of potential anticancer immunotherapies for aggressive paediatric cancers such as neuroblastoma and DIPG.

KIR Gene Haplotype: An Independent Predictor of Clinical Outcome in MDS Patients

Myelodysplastic syndromes (MDS) are a group of hematopoietic disorders affecting the myeloid lineage, characterized by cytopenias and clonal evolution to AML. Immune responses against MDS, partly mediated by NK cells, have the potential to affect disease progression. However, immune evasion remains an important barrier. NK cells express both activating and inhibitory killer immunoglobulin-like receptors (KIRs) that interact to regulate NK effector function. Based on the number and distribution of inherited KIR genes, individuals can be classified in two broad haplotypes. Haplotype A comprises a single activating KIR (aKIR) gene (KIR2DS4), while haplotype B incorporates various combinations of aKIR genes (up to 6). We hypothesized that the aKIR gene repertoire may be useful in refining predictions of clinical outcomes in MDS. Thus, we studied the variations in aKIR gene content and haplotype in MDS and their relationship to the risk of AML transformation and patient outcomes. We first compared the number of aKIR genes in 108 MDS patients treated at MDACC with that in 139 HSC donors. The median number of aKIR genes was significantly lower in MDS than controls: 2 (range 0-6) vs 3 (range 0-6), p=0.001. Functional studies revealed that compared to healthy controls, NK cells from MDS patients demonstrate less Interferon gamma production (p<0.0001) and less degranulation (p=0.0028) in response to K562 cell line, and have lower killing ability evidenced by chromium release assay (p=0.04). We next examined the influence of KIR haplotype on the risk of AML transformation and outcomes in the cohort of 108 MDS patients (28 KIR haplotype A and 80 KIR haplotype B patients). On multivariate analysis, cytogenetic risk group and KIR haplotype were identified as independent predictors of MDS progression to AML. The relative risk of MDS-AML transformation in patients with haplotype A vs. haplotype B was 2.67 (1.13-6.31) (p=0.02). Similarly, cytogenetic risk group, IPSS and KIR haplotype independently predicted survival. MDS patients with KIR haplotype A had worse adjusted PFS (RR with 95%CI 2.96 [1.59-5.52], p=0.001) and OS (2.25 [1.17-4.31], p=0.02), compared to patients with haplotype B. These novel findings may help identify a subgroup of MDS patients with a high risk of disease progression and poor outcomes, who would likely benefit from adoptive NK cell therapy. Disclosures Kantarjian: ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

Haemophagocytic Lymphohistiocytosis (HLH) Associated With a Heterozygous PRF1 Mutation Not Previously Described As Pathogenic

Case Presentation A 16 year old male presented with three weeks of fatigue, nausea, vomiting, anorexia, arthralgias, and pancytopaenia. He was febrile, with mouth ulcers, hepato splenomegaly, and palpable non tender supraclavicular and axillary lymphadenopathy. Neurological examination was normal. Remarkable abnormal tests: Hb 77 g/L, platelets 29 x109/L, WCC 0.7 x109/L (neutrophils 0.33), normal PT and APTT but hypofibrinogenaemia 0.8 g/L. Ferritin was elevated 56 061 ug/L (20– 150). Liver function tests showed a mixed pattern with elevated LDH (2137 U/L), mild hypertriglyceridaemia (4.6 mmol/L) and low haptoglobin (< 0.06 g/L), EBV IgM was equivocal with reactive IgG. Bone marrow showed evidence of haemophagocytosis (Fig 1). All HLH-2004 criteria were fulfilled except elevated sCD25 (unavailable). NK cell chromium release assay revealed severely suppressed NK cell function. In addition, perforin gene (PRF1) sequencing for mutation screening showed a missense mutation Ala437Val (1310 C>T) PRF1 heterozygous, a previously unclassified variant. There was no evidence of an underlying cause of HLH. Our patient received induction therapy with etoposide 150 mg/m2 and dexamethasone 10 mg/m2 with dosing and frequency as suggested by the HLH-94 protocol. Pancytopaenia recurred and an increase in ferritin concentration was interpreted as relapse of the condition. At that point, cyclosporine was added on in conjunction with maintenance etoposide and dexamethasone obtaining partial remission. He underwent allogeneic SCT successfully in another centre.Figure 1Bone marrow aspirate of this patient showing evidence of haemophagocytosis (H & E). A and B show large macrophages undergoing phagocytosis of different cellular elements of the bone marrow.Figure 1. Bone marrow aspirate of this patient showing evidence of haemophagocytosis (H & E). A and B show large macrophages undergoing phagocytosis of different cellular elements of the bone marrow. Discussion Several genes have been implicated in the genesis of primary HLH; all of them have in common impaired cytotoxic function by NK and T cells. It has been reported that mutations of the PRF1 gene comprise approximately 20 to 30% of the cases of primary HLH. This variant has been reported as a polymorphism and was not conclusive for the diagnosis of perforin deficiency as the cause of HLH in this case. In addition, the mutation was heterozygous. Although HLH is usually a recessive disorder, there are reports of potential disease associations in the heterozygous state. There is significant overlap between primary and secondary HLH and the role of some heterozygous mutations remain to be investigated. Disclosures: No relevant conflicts of interest to declare.