ALBERT

Description: Introduction CTL019 is a novel, investigational, chimeric antigen receptor (CAR) immunotherapy whereby autologous T cells are genetically modified with a chimeric antigen receptor to target CD19 on the surface of malignant as well as healthy B cells. The cellular kinetics of CTL019 have been evaluated in several trials for patients with relapsed/refractory CD19+ leukemias, including pediatric acute lymphoblastic leukemia (pALL), adult ALL (aALL), and chronic lymphocytic leukemia (CLL) (Maude 2014, Porter 2015). Methods The cellular kinetic profile of CTL019 was determined in peripheral blood (PB) and bone marrow (BM) through serial measurements using flow cytometry and quantitative real-time polymerase-chain-reaction (qPCR) assay in 3 studies comprised of (i) 55 pALL patients (NCT01626495), (ii) 28 adult CLL patients from a dose selection study (NCT01747486), and (iii) 14 CLL and 6 adult ALL patients (NCT01029366). The flow cytometry assay used a CAR19-specific anti-idiotype antibody to enumerate CTL019 T cells as a % of CD3+ T cell (Porter 2015). Cellular kinetic parameters included: maximal extent of expansion as measured by peak copies of CTL019 DNA and peak % by flow cytometry (Cmax), area under the curve at day 28 (AUC0-28d) describing expansion and persistence in the first month, and time to reach Cmax (Tmax). Parameters were derived by non-compartmental methods. Where estimable, persistence was described by the half-life (T1/2) based on the slope of the terminal phase. Results Following infusion, CTL019, expansion and persistence was evident in the patients who responded to CTL019 as measured by both PK assays across all 3 studies. Table 1 summarizes (arithmetic mean (SD)) the CTL019 kinetic parameters. With complete remission (CR/CRi), CTL019 cells undergo rapid in vivo expansion beyond the original CTL019 dose with maximal expansion at a mean of 11 days in pALL and aALL and approximately 14-18 days in adult CLL as determined by qPCR and flow cytometry (Table 1). In CR/CRi patients the transgene level-profiles in PB reveal a kinetic profile with an initial rapid expansion followed by a slower decay function with some fluctuations of transgene over time resulting in higher AUC0-28d and Cmax, while non-responder (NR) patients tend to have a lower expansion and faster decay (shorter T1/2)of CAR positive T-cells resulting in lower AUC0-28d and Cmax by, leaving the mechanism to be further explored. In pALL, significantly higher AUC0-28d and Cmax were observed in CR/CRi patients compared to NR patients by flow cytometry; however, a wide range of mean AUC0-28d and Cmax was observed in NR patients (n=3) resulting from significant expansion in one NR patient as determined by qPCR. In CLL, the exposure metrics AUC0-28d and Cmax were approximately 12 times higher in CR/CRi patients compared with PRi/NR/PD in NCT01747486; a similar trend was observed in NCT01029366. Similar findings were captured by the flow cytometry based measurements as summarized in Table 1. In pALL and CLL, CR/CRi patients tend to maintain higher levels of CTL019 transgene over longer periods of time (〉6 months) compared to NR patients as demonstrated by the longer T1/2 value. Cellular kinetic parameters were not summarized by response category for aALL due to the small sample size (n=5 CR/CRi; n=1 NR). CTL019 transgene levels ranged from below the limit of quantification (BLQ) to 178,000 copies/ug in aALL patients with CR/CRi and BLQ to 21,900 copies/ug in the NR. CTL019 positive cells were also shown to traffic to BM at 1 month in responders (CR/CRi), irrespective of the disease. Conclusions Overall, significantly higher levels of in vivo proliferation and persistence were observed in patients who successfully responded to CTL019 (i.e. CR/CRi/PR) compared to NRs in both CLL and (adult and pediatric) ALL patients, as captured by both analytical measures, indicating that the kinetics of CTL019 T cells and that proliferation and persistence of CTL019 reasonably predicts response to therapy. These are the first three studies to demonstrate that cellular kinetics may predict responses to CAR based cellular therapy. These results imply that measures to increase proliferation and persistence of CAR T cells may enhance responses in resistant patients. Figure. CTL019 concentration-time profiles for %CD3+/CTL019+ measured by flow cytometry and cellular kinetic parameters for qPCR and flow cytometry for p-ALL and adult CLL Figure. CTL019 concentration-time profiles for %CD3+/CTL019+ measured by flow cytometry and cellular kinetic parameters for qPCR and flow cytometry for p-ALL and adult CLL Disclosures Mueller: Novartis Pharmaceuticals: Employment. Chakraborty:Novartis Pharmaceuticals: Employment, Equity Ownership. Wood:Novartis Pharmaceuticals: Employment, Other: Stock. Awasthi:Novartis Pharmaceuticals: Employment. Quintas-Cardama:Novartis Pharmaceuticals: Employment, Equity Ownership. Han:Novartis Pharmaceuticals: Employment, Equity Ownership. Maude:Novartis: Consultancy. Grupp:Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding; Genentech: Employment. Frey:Novartis: Research Funding; Amgen: Consultancy. Marcucci:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. June:Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership; Pfizer: Honoraria; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Tmunity: Equity Ownership, Other: Founder, stockholder ; Johnson & Johnson: Research Funding. Lacey:Novartis: Research Funding.

Description: We recently conducted a clinical trial of CD22-directed chimeric antigen receptor (CAR) T cells in children and adults with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL). While we did observe some transient responses, overall outcomes were inferior to another recent trial of CD22 CAR T cells in ALL performed at the NCI (Fry, T.J. et al. Nat Med, 2018). Intriguingly, these trials used a CAR that employed the same antigen-binding and intracellular signaling domains, and differed only in the length of linker connecting the variable regions of the single chain variable fragment (scFv). Based on these clinical observations, we sought to identify how the scFv linker impacts CAR biology and regulates CAR-driven T cell activity. The University of Pennsylvania's CD22 CAR contained a long 20 amino acid scFv linker ("CAR22-L") while the NCI's CAR had a 5 amino acid linker ("CAR22-S"). We began by investigating the impact of linker length on CAR biochemistry. Both CAR22-L and CAR22-S had similar antigen-binding affinities (KD of 1.67nM and 6.05nM, respectively). Chromatography revealed that CAR22-L remained monomeric in solution while CAR22-S formed homodimers. To explore how dimerization influenced surface-membrane biology, we developed GFP-tagged versions of each CAR and performed confocal microscopy on CAR+ T cells. CAR22-L exhibited homogenous surface membrane expression, while CAR22-S appeared to self-aggregate and cluster (Fig. 1a). We investigated the impact of this clustering on receptor signaling and found that CAR22-S demonstrated high levels of signaling molecule activation (i.e. Akt, p70-S6 and STAT3) in the absence of antigen engagement. This is consistent with previous reports establishing that CAR clustering can lead to tonic signaling (Long, A.H. et al. Nat Med, 2015). Importantly, this tonic signaling did not lead to autonomous T cell proliferation. We proceeded to evaluate how clustering and tonic signaling impacted CAR function upon antigen engagement. Microscopic evaluation of CAR T cells combined with CD22+ Nalm6 cells revealed greater actin and microtubule organizing complex polarization (P = 0.02 and 0.01, respectively) in CAR22-S cells, consistent with superior immune synapse formation. This was accompanied by increased phosphorylation of PI3K, MAPK and calcium signaling proteins (Fig. 1b) after CAR engagement. RNA sequencing revealed significantly greater activation of immune response gene programs in CAR22-S cells as compared to CAR22-L after overnight exposure to Nalm6. We next investigated the impact that this enhanced receptor-driven activity had on CAR T cell anti-tumor function. CAR T cells were combined with Nalm6 in vitro and residual Nalm6 was serially quantified, revealing that CAR22-S mediated greater tumor control than CAR22-L, particularly at later time periods (P 〈 0.001). This was associated with greater secretion of IFNg, IL-2 and TNFa (all P 〈 0.001). Finally, we compared anti-tumor efficacy in xenograft models of systemic Nalm6. NOD/SCID/cg-/- mice were engrafted with Nalm6 and received 1x106 CAR T cells 7 days later. CAR22-S demonstrated greater in vivo expansion (P 〈 0.0001) and enhanced control of systemic disease (Fig. 1c,P = 0.017), resulting in prolongation of animal survival (Fig. 1d,P = 0.013). Based on these observations, we have designed a novel, affinity-enhanced CD22 CAR and confirmed that shorter linker length improves anti-tumor activity of this CAR. T cells expressing this CAR are currently undergoing evaluation in a phase I clinical trial (ClinicalTrials.org Identifiers NCT03620058 and NCT02650414). Thus far, 4 children and 2 adults have been infused with manageable toxicity. Early outcomes are promising, with 67% achieving complete remission at day 28, compared to 50% in our original CART22 trials. In summary, by investigating the potential mechanisms for an apparent discrepancy in outcomes between two different clinical trials, we demonstrate that reducing the length of the scFv linker results in significant changes to CAR biochemistry that directly lead to antigen-independent receptor activity. In contrast to previously published data demonstrating that tonic signaling of CD28-costimulated CARs is detrimental to T cell function (Long, A.H. et al. Nat Med, 2015), we found that tonic signaling of 4-1BB-costimulated CARs may be beneficial, possibly by priming T cells for rapid response to antigen. Disclosures Singh: University of Pennsylvania: Patents & Royalties. Frey:Novartis: Research Funding. Engels:Novartis: Employment. Zhao:Novartis: Employment. Peng:Novartis: Employment. Granda:Novartis: Employment. Ramones:Novartis: Employment. Lacey:Novartis: Research Funding; Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding. Young:novartis: Research Funding. Brogdon:Novartis: Employment. Grupp:Roche: Consultancy; GSK: Consultancy; Novartis: Consultancy, Research Funding; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards; Cure Genetics: Consultancy. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Maude:Novartis: Consultancy; Kite: Consultancy. Gill:Novartis: Research Funding; Tmunity Therapeutics: Research Funding; Carisma Therapeutics: Research Funding; Amphivena: Consultancy; Aro: Consultancy; Intellia: Consultancy; Sensei Bio: Consultancy; Carisma Therapeutics: Equity Ownership. Ruella:AbClon: Membership on an entity's Board of Directors or advisory committees; Nanostring: Consultancy, Speakers Bureau; Novartis: Patents & Royalties: CART for cancer.

Description: Recent studies using direct stimulation of PBMC from CMV-positive individuals with optimal CTL epitope peptides have identified new antigens that are targets of the host immune response. The growing number of targets of the cellular immune response has prompted an evaluation of which antigens are potentially associated with protective immunity. A panel of seven human cytomegalovirus (CMV) epitope peptides and the corresponding MHC-I tetramers was used to evaluate cellular immunity in six healthy seropositive donors and in six hematopoietic stem cell transplant recipients (HSCT). Broad CMV-specific responses were found to epitopes within several CMV polypeptides that were restricted by multiple HLA alleles in several individuals. The results document the simultaneous expansion of several of these CD8+ T-lymphocyte populations following allogeneic HSCT. The combined levels of CMV epitope-specific T-cells exceeded 20% of CD8+ T-lymphocytes in some individuals. The cytotoxic functionality of 26 different populations of CMV-specific T-lymphocytes detected within this group of 12 individuals was addressed by utilizing a recently described assay that measures transient surface levels of the lysosomal membrane proteins LAMP-1 (CD107a) and LAMP 2 (CD107b) after peptide stimulation. This degranulation/mobilization assay can be combined with tetramer staining of antigen-specific CD8+ T-lymphocytes, and has potential as a surrogate marker for cytotoxic function. We found that a significant proportion of CD8+ T-lymphocytes specific for epitopes within the CMV pp65 and pp50 gene products had functional potential as measured by this assay (median percentage of cells within 14 T-cell populations staining with pp65 or pp50 tetramers that degranulated on stimulation with cognate peptide = 26.0% and 19.8%). By contrast, CD8+ T-lymphocytes specific for epitopes within the CMV IE-1 gene product had markedly reduced functionality (median percentage of cells within 12 T-cell populations staining with IE-1 tetramers that degranulated on stimulation with cognate peptide = 5.6%). This difference was significant (p= 0.003 by an F-test after adjusting for HLA and using interaction of subjects and epitopes as the error). This reduced degranulation efficiency of IE-1-specific T cells is consistent with their inefficient cytotoxic recognition of CMV-infected autologous fibroblasts. Further characterization of this functional dichotomy includes comparison of cell surface marker phenotype and cytokine release in response to antigenic presentation. These functional differences between T-lymphocyte populations within the same individual have implications for choosing antigens that are both protective and necessary to include in a CMV vaccine for transplant patients at risk for infectious complications after viral reactivation.

Description: Neurologic toxicity has been observed with anti-CD19 chimeric antigen receptor (CAR) T cells and the anti-CD19 BiTE blinatumomab. Both focal (e.g., cranial nerve palsy) and global (e.g., generalized seizures) abnormalities have been reported, often associated with systemic cytokine release syndrome (CRS) but also observed after recovery from or in absence of CRS. CART-BCMA consists of expanded autologous T cells transduced with a 4-1BB:CD3-zeta-based CAR specific for B Cell Maturation Antigen. Here, we report clinical features and management of a severe neurotoxicity observed on a phase 1 trial of CART-BCMA for multiple myeloma (MM) (NCT02546167). The subject is a 55-year-old female with high-risk IgA lambda MM. At time of CART-BCMA infusion, her MM manifestations included cytopenias and plasmacytomas of the pleura and paravertebral muscles. Bone marrow (BM) was 〉95% BCMA+ plasma cells. Pre-treatment brain MRI showed pachymeningeal thickening and enhancement over the left cerebral convexity, possibly due to extension of calvarial MM lesions. There was no evidence of CNS MM on a neurologist's exam or by CSF cytology. The subject received 2x108 CART-BCMA cells, 40% of the planned dose, over two consecutive days, without lymphodepleting chemotherapy; a third planned infusion was held due to fevers. Over the next 4 days, fevers persisted, hypoxia and delirium developed, and cytopenias worsened. Brain MRI and lumbar puncture on day 4 showed no new abnormalities. Evaluation for infection was negative. These symptoms coincided with rise in serum IL-6 (nl range

Description: CTLO19 cells are CAR-modified T cells which recognize CD19 and produce high durable remission rates for pts with relapsed or refractory acute lymphoblastic leukemia (ALL). Cytokine Release Syndrome (CRS) has emerged as the major treatment related effect from CTL019, with symptoms that include high fevers and malaise but can progress to capillary leak, hypoxia and hypotension. CRS occurs hours to days after CTL019 infusion and correlates with rapid in vivo CTL019 expansion and marked elevation of serum IL6. In most cases, CRS is self-limited or rapidly reversed with anti-cytokine directed therapies. Here we report 3 cases of refractory CRS in adult pts with ALL. Our experience offers insight into clinical and investigational parameters describing this syndrome; highlights the variance of CRS across disease types and illustrates complexities of CRS management during concurrent infectious illness. As of 7/1/14, 97 pts (30 pediatric ALL, 12 adult ALL, 41 CLL, 14 NHL) have been treated with CTLO19. To capture clinical manifestations of CRS across protocols, we developed a novel CRS grading scale which will be described. Severe CRS (Gr 3-5) occurred in 27 (64%) of ALL pts and only 16 (29%) of CLL/NHL pts (p=0.001). 12 adults with ALL received CTL019; 8/9 evaluable pts achieved CR (MRD negative) at 1 month and 1 pt with extramedullary disease had marked reduction of PET avid disease which is maintained at 1 yr. Severe CRS occurred in 11 of 12 adult ALL pts. CRS was self-limited in 2 pts, rapidly reversed with anti-IL6 directed therapy in 6 pts and was refractory to therapy, contributing to death in 3 pts who were not evaluable for disease response. No baseline attributes differentiate these 3 pts from the 9 adult ALL pts with manageable Gr1-4 CRS. We have shown however that ALL disease burden correlates with CRS severity (in press) and all 3 pts had significant disease burden at baseline. All received lymphodepleting chemotherapy with cyclophosphamide 300 mg/m2 q12h x 6 followed by infusion of CTLO19 cells. These 3 pts each received 6.50E+06, 6.70E+06 and 8.45E+06 CTLO19 cells/kg compared to median CTL019 dose of 3.62E+06 in the 9 adult ALL pts with manageable CRS. Pt 21413-03 developed CRS 12 hrs after infusion and tested positive for influenza B on D3. Despite broad spectrum antimicrobials (including oseltamivir) and anticytokine directed therapy with tocilizumab (4mg/kg x 2) and steroids, he died with refractory hypotension on D5. Pt 21413-06 had extensive disease after 2 prior allogeneic SCTs and developed CRS within 12 hrs of infusion. In addition to broad spectrum antibiotics, she received tocilizumab 8mg/kg (D 3, 6 and 12); intermittent high dose steroids (D 4-15) and etanercept (D14). She died D15 with hypotension, hypoxic respiratory failure and concurrent MDR pseudomonas sepsis and pneumonia. Pt 21413-11 developed CRS within 24 hrs of infusion. He received tocilizumab 8mg/kg (D3&4); siltuximab (D5&15) and intermittent high dose steroids (D 4-15). After an initial response, he developed recurrent fever, pulmonary infiltrates and blood cultures positive for stenotrophomonas. He died D15 with refractory hypoxia and hypotension. All 3 pts’ clinical CRS correlated with marked in vivo CTL019 expansion and progressive serum cytokine elevations (data to be shown). CONCLUSIONS: CRS is the major toxicity of CTL019 therapy and its clinical course varies depending on disease type (more frequent and severe in ALL) and disease burden (in ALL). The 3 refractory CRS cases described here (of 97 total pts treated) have all occurred in adult ALL pts with significant disease burden who received relatively high doses of CTL019 cells. In addition, all 3 had significant infectious complications which potentially fueled underlying CRS and/or were made more virulent due to impairment of immunity with administration of anti-cytokine directed therapies. Future protocol modifications will be made goal of limiting severity of CRS while maintaining high durable remission rates. Further exploration is planned to better correlate timing and choice of anticytokine directed therapy in relation to clinical and investigation parameters of CRS. Disclosures Frey: Novartis: Research Funding. Off Label Use: USe of CART19 cells to treat CLL. Levine:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding. Schuster:Novartis: Research Funding. Hwang:NVS: Research Funding. Leung:Novartis: Employment. Shen:Novartis: Employment. Ericson:Novartis: Employment. Melenhorst:Novartis: Research Funding. June:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding.

Description: BACKGROUND: Patients (pts) with follicular lymphoma (FL) who have progression of disease within 2 years of immunochemotherapy have poor outcomes and represent a distinct group for whom development of new therapies is warranted (Casulo et al. J Clin Oncol 2015). Autologous T cells genetically modified to express a chimeric antigen receptor consisting of an external anti-CD19 single chain murine antibody domain with CD3ζ and 4-1BB signaling domains (CTL019 cells) can mediate potent anti-tumor effects in pts with relapsed or refractory chronic lymphocytic leukemia, acute lymphoblastic leukemia, and B cell lymphomas. We evaluated the safety and efficacy of CTL019 cells in pts with relapsed or refractory FL as part of an ongoing phase IIa clinical trial (NCT02030834). METHODS: Eligible pts have CD19+ FL with progression of lymphoma