ALBERT

Description: Anti-CD19 chimeric antigen receptor (CAR) T cells have powerful activity against B-cell lymphoma, but improvement is clearly needed. Toxicity, including cytokine-release syndrome (CRS) and neurologic toxicity, occurs after anti-CD19 CAR T cell infusions. Most CAR T-cell toxicity is caused, either directly or indirectly, by cytokines or other proteins that are secreted from CAR T cells. The structure of a CAR is an extracellular antigen-recognition domain connected by hinge and transmembrane (TM) domains to intracellular T-cell signaling moieties. In vitro, T cells expressing CARs with hinge and TM domains from the CD8-alpha molecule released significantly lower levels of cytokines compared with T cells expressing CARs with hinge and TM domains from CD28; however, T cells expressing CARs with hinge and TM domains from CD8-alpha retained sufficient functional capability to eradicate tumors from mice (Alabanza et al. Molecular Therapy. 2017. 25(11) 2452). To reduce cytokine production with a goal of reducing clinical toxicity, we incorporated CD8-alpha hinge and TM domains into an anti-CD19 CAR. The CAR also had a human antigen-recognition domain, a CD28 costimulatory domain, and a CD3-zeta domain. This CAR was designated Hu19-CD828Z and was encoded by a lentiviral vector. Hu19-CD828Z was different from the FMC63-28Z CAR that we used in prior studies. FMC63-28Z had hinge and TM domains from CD28 along with a CD28 costimulatory domain, a CD3-zeta domain, and murine-derived antigen-recognition domains. Twenty patients with B-cell lymphoma were treated on a phase I dose-escalation clinical trial of Hu19-CD828Z T cells (Table). Patients received low-dose cyclophosphamide and fludarabine daily for 3 days on days -5 to -3. Two days later, on day 0, CAR T cells were infused. The overall response rate (ORR) after 1st treatments with Hu19-CD828Z T cells was 70%, and the complete response (CR) rate 55%; the 6-month event-free survival was 55%. The anti-lymphoma activity of Hu19-CD828Z T cells in the current trial was comparable to the anti-lymphoma activity of FMC63-28Z T cells in a similar prior trial that also enrolled patients with advanced B-cell lymphoma. In the prior trial, we observed a 73% ORR, a 55% CR rate, and a 6-month event-free survival of 64% in 22 patients treated with FMC63-28Z T cells (Kochenderfer et al. Journ. Clin. Oncology. 2017 35(16) 1803). In our previous clinical trial of FMC63-28Z T cells, the rate of Grade 3 or 4 neurologic toxicity among 22 patients treated was 55%. Strikingly, in our trial of Hu19-CD828Z T cells, the rate of Grade 3 or 4 neurologic toxicity was only 5% (1/20 patients). In addition, the rate of Grade 2 or greater neurologic toxicity with FMC63-28Z T cells was 77.3% while the rate of Grade 2 or greater neurologic toxicity with Hu19-CD828Z T cells was 15%. To explore the mechanism for the difference in neurologic toxicity in patients receiving FMC63-28Z T cells versus Hu19-CD828Z T cells, we assessed serum levels of 41 proteins in patients treated with these CAR T-cells. This comparison is valid because the same Luminex methodology was used for the serum protein analysis for both trials, and controls of known amounts of each protein were assayed to ensure that protein levels were comparable on the different trials. Lower levels of several serum proteins that might be important in CAR toxicity were found in patients treated with Hu19-CD828Z T cells versus patients treated with FMC63-28Z T cells: Granzyme A (P

Description: Chimeric antigen receptor (CAR) T cells expressing B-cell maturation antigen (BCMA) can target and kill multiple myeloma (MM). BCMA was chosen as a target for MM because it is expressed by almost all cases of MM but has a restricted expression pattern on normal cells. CAR antigen-recognition domains made up of monoclonal antibody-derived, single-chain-variable fragments (scFv) are potentially immunogenic. To reduce the risk of recipient immune responses against CAR T cells, we used the sequence of a novel anti-BCMA, fully-human, heavy-chain-only binding domain designated FHVH33. The FHVH33 binding domain sequence was from TeneoBio, Inc. FHVH33 is smaller than a scFv. FHVH33 lacks the light chain, artificial linker sequence, and 2 associated junctions of a scFv, so it is predicted to be less immunogenic than a scFv, especially murine-derived scFvs. We constructed a CAR incorporating FHVH33, CD8α hinge and transmembrane domains, a 4-1BB costimulatory domain, and a CD3ζ T-cell activation domain. The CAR, FHVH33-CD8BBZ, is encoded by a γ-retroviral vector. FHVH33-CD8BBZ-expressing T cells (FHVH-BCMA-T) exhibited a full range of T-cell functions in vitro and eliminated tumors and disseminated malignancy in mice (Lam et al, Blood (ASH abstract) 2017 vol 130: 504). We are conducting the first clinical trial of FHVH-BCMA-T. Patients receive conditioning chemotherapy on days -5 to -3 with 300 mg/m2 of cyclophosphamide and 30 mg/m2 of fludarabine followed by infusion of FHVH-BCMA-T on day 0. This dose-escalation trial has 5 planned dose levels (DL). Twelve patients have received FHVH-BCMA-T on 3 DLs, 0.75x106, 1.5x106 and 3x106 CAR+ T cells/kg of bodyweight. Three patients were enrolled on the trial but not treated. The median age of patients enrolled was 63 (range 52-70); patients received a median of 6 lines of anti-myeloma therapy (range 3-10) prior to treatment with FHVH-BCMA-T. Ten patients out of 12 patients have achieved objective responses (OR). Five patients have obtained CRs or VGPRs to date. One patient achieved a partial remission (PR) 26 weeks after FHVH-BCMA-T infusion through a continued decrease in a measurable plasmacytoma. Five out of 7 patients who had myeloma with high-risk cytogenetics had an OR (Table). ORs occurred in patients with large soft-tissue plasmacytomas. Loss of BCMA expression on myeloma cells after treatment was documented in 2 patients. Two patients who developed progressive MM after CAR T-cell infusion had evidence of minimal residual disease in bone marrow 1-2 months post infusion of CAR T cells (patients 7,8). Eleven out of 12 patients had cytokine release syndrome (CRS); CRS grades ranged from 1-3 (Lee et al. Biol Blood Marrow Transplant 25 (2019) 625-638). The median peak C reactive protein (CRP) of the patients with CRS was 156.3 mg/L. Of 12 patients, 1 received the interleukin-6-receptor antagonist tocilizumab on day +6 to treat grade 3 CRS with hypotension requiring low-dose pressor therapy, grade 2 ejection fraction (EF) decrease and elevation of creatinine kinase (CK). All parameters returned to baseline by day +10. Patient 12 had a grade 3 decrease in EF which resolved by day +29. Two patients had grade 2 neurotoxicity that resolved without intervention: patient 3 had headaches, dysarthria and word-finding difficulties that resolved after 6 days while patient 6 had headaches on day +4. Patient 12 had grade 3 neurotoxicity with confusion on day +2; she was given dexamethasone with improvement in mental status the same day. After attaining a response, patient 6 died from influenza complications 6 weeks after FHVH-BCMA-T infusion. A median of 10.6% (range 1.1-46) of bone marrow T cells were CAR+ when assessed 14 days after FHVH-BCMA-T infusion. We assessed blood CAR+ cells by quantitative PCR. The median peak level of CAR+ cells was 76.5 cells/µl (range 3-347 cells/µl) and the median day post-infusion of peak blood CAR+ cell levels was 13 (range 9-14). The results from this phase 1 trial demonstrate that FHVH-BCMA-T cells can induce responses at low dose levels. Patients who had no CRS or low-grade CRS achieved objective responses. Toxicity was limited and reversible. Accrual to this trial continues. A maximum tolerated dose has not been determined yet. These results encourage further development of FHVH CAR-T. Table Disclosures Manasanch: Janssen: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Merck: Research Funding; Skyline Diagnostics: Research Funding; Sanofi: Research Funding; Quest Diagnostics: Research Funding; Celgene: Honoraria. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Kochenderfer:Kite and Celgene: Research Funding; Bluebird and CRISPR Therapeutics: Other: received royalties on licensing of his inventions. OffLabel Disclosure: Cyclophosphamide and fludarabine are used in combination for conditioning chemotherapy prior to CAR T-cell infusion

Description: Background: Chimeric antigen receptors (CARs) are fusion proteins that combine antigen-recognition domains and T-cell signaling domains. T cells genetically modified to express CARs directed against the B-cell antigen CD19 can cause remissions of B-cell malignancies. Most CARs in clinical use contain components derived from murine antibodies. Immune responses have been reported to eliminate CAR T cells in clinical trials, especially after second infusions of CAR T cells (C. Turtle et al., Journal of Clinical Investigation, 2016). These immune responses could be directed at the murine components of CARs. Such immune responses might limit the persistence of the CAR T cells, and anti-CAR immune responses might be an especially important problem if multiple infusions of CAR T cells are administered. Development of fully-human CARs could reduce recipient immune responses against CAR T cells. Methods: We designed the first fully-human anti-CD19 CAR (HuCAR-19). The CAR is encoded by a lentiviral vector. This CAR has a fully-human single-chain variable fragment, hinge and transmembrane regions from CD8-alpha, a CD28 costimulatory domain, and a CD3-zeta T-cell activation domain. We conducted a phase I dose-escalation trial with a primary objective of investigating the safety of HuCAR-19 T cells and a secondary objective of assessing anti-lymphoma efficacy. Low-dose chemotherapy was administered before HuCAR-19 T-cell infusions to enhance CAR T-cell activity. The low-dose chemotherapy consisted of cyclophosphamide 300 mg/m2 daily for 3 days and fludarabine 30 mg/m2 daily for 3 days on the same days as cyclophosphamide. HuCAR-19 T cells were infused 2 days after the end of the chemotherapy regimen. Patients with residual lymphoma after a first treatment were potentially eligible for repeat treatments if dose-limiting toxicities did not occur with the first treatment. Repeat infusions were given at the same dose level as the first infusion or 1 dose level higher than the first infusion. Findings: A total of 11 HuCAR-19 T-cell infusions have been administered to 9 patients; 2 patients received 2 infusions each. So far, there is an 86% overall response rate (Table). Grade 3 adverse events (AEs) included expected cytokine-release syndrome toxicities such as fever, tachycardia, and hypotension. Corticosteroids were used to treat toxicity in Patient 3. The interleukin-(IL)-6 receptor antagonist tocilizumab was used to treat toxicity in Patient 4, and both tocilizumab and corticosteroids were used to treat toxicity in Patient 8. Only 1 of 8 evaluable patients, Patient 3, has experienced significant neurological toxicity to date. This patient experienced encephalopathy that was associated with a cerebrospinal fluid (CSF) white blood cell count of 165/mm3. Almost all of the CSF white cells were CAR T cells, and the CSF IL-6 level was elevated. All toxicities have resolved fully in all patients. In Patient 1, tumor biopsies revealed a complete loss of CD19 expression by lymphoma cells after 2 HuCAR-19 T-cell infusions, which to our knowledge is the first documented complete loss of CD19 expression by lymphoma after anti-CD19 CAR T-cell therapy. This loss of CD19 expression was associated with lymphoma progression. After first CAR-19 T-cell infusions, HuCAR-19 cells were detectable in the blood of every patient. The median peak number of blood CAR+ cells was 26/microliter (range 3 to 1005 cells/microliter). Blood HuCAR-19 cells were detected after second infusions in the blood of both patients who received second infusions. Patient 1 obtained a partial response after a second infusion after only obtaining stable disease after a first infusion. We detected elevations of inflammatory cytokines including IL-6, interferon gamma, and IL-8 in the serum of patients experiencing clinical toxicities consistent with cytokine-release syndrome. Interpretation: T cells expressing HuCAR-19 have substantial activity against advanced lymphoma, and infusions of HuCAR-19 T cells caused reversible toxicities attributable to cytokine-release syndrome. Disclosures Kochenderfer: Kite Pharma: Patents & Royalties, Research Funding; bluebird bio: Patents & Royalties, Research Funding.

Description: T cells expressing chimeric antigen receptors (CAR) that target B-cell maturation antigen (BCMA) recognize and eliminate multiple myeloma (MM). BCMA is expressed by nearly all cases of MM. BCMA has a restricted expression pattern on normal cells. To reduce the risk of recipient immune responses against CAR T cells, we used a novel, fully-human, heavy-chain-only anti-BCMA binding domain designated FHVH33 instead of a traditional single-chain variable fragment (scFv). The FHVH33 binding domain lacks the light chain, artificial linker sequence, and 2 associated junctions of a scFv. We constructed a CAR designated FHVH33-CD8BBZ. FHVH33-CD8BBZ was encoded by a γ-retroviral vector and incorporated FHVH33, CD8α hinge and transmembrane domains, a 4-1BB costimulatory domain, and a CD3ζ domain. T cells expressing FHVH33-CD8BBZ are designated FHVH-BCMA-T. On this clinical trial, patients received 300 mg/m2 of cyclophosphamide and 30 mg/m2 of fludarabine on days -5 to -3 followed by infusion of FHVH-BCMA-T on day 0. Twenty-one FHVH-BCMA-T infusions have been administered on 5 dose levels (DL), 0.75x106, 1.5x106, 3x106, 6x106 and 12 x106 CAR+ T cells/kg of bodyweight. DL4 (6 x 106 CAR+ T cells/kg) was identified as the maximum feasible dose (MFD) after weighing toxicity, efficacy and manufacturing factors. Patients are now being enrolled on an expansion phase to test the MFD. One patient (Patient 11) received 2 treatments. Four patients have been enrolled who were not ultimately treated. The median age of the patients enrolled is 64 (range 41-72). Patients received a median of 6 prior lines of therapy (range 3-12). Of the 20 FHVH-BCMA-T treatments evaluable for response, 18 (90%) resulted in objective responses (OR). Twelve treatments resulted in VGPR, complete remission (CR) or stringent complete remission (sCR). Ten patients (50%) have ongoing responses that range between 0-80 weeks (6 sCR/CRs, 3 VGPRs, 1 PR). At the highest two DLs (8 patients), 7 patients (88%) have ongoing responses (median duration 20 weeks, range 0+ to 35+ weeks); progressive MM occurred in only 1 patient who had evidence of spinal cord compression on day +5 due to a rapidly expanding plasmacytoma, which required early intervention with high-dose corticosteroid and radiation therapy. Of the 8 patients evaluated for response who had high-risk cytogenetics at baseline, 7 had ORs. Responses are ongoing in 2 patients with TP53 mutations and 1 patient with t(4;14) translocation. Ten treated patients came off study due to progressive MM (9 patients) or death from other causes (1 patient, influenza). Two of 4 patients who had plasmacytomas evaluated for BCMA expression at relapse had evidence of BCMA-negative MM. Four patients had bone marrow aspirates evaluated for BCMA-expression before treatment and at the time of relapse; 3 of these patients had evidence of loss of BCMA expression at relapse. Of 21 FHVH-BCMA-T treatments administered, 20 (95%) were followed by cytokine release syndrome (CRS) with 16 (76%) cases of grade 1 or 2 CRS, 4 cases (19%) of grade 3 CRS, and no cases of grade 4 CRS. Three patients received tocilizumab. The median peak C-reactive protein after all 21 treatments was 196.9 mg/L. Of 21 total treatments, 8 (38%) were followed by neurologic toxicity; there were 5 cases of grade 1-2 neurologic toxicity (headache, dysarthria, confusion, delirium), 2 cases of grade 3 neurologic toxicity (confusion), and 1 patient with grade 4 spinal cord compression due to progressive MM. Two patients received corticosteroids to manage neurologic toxicities. A median of 3.0% (range 0-95%) of bone marrow T cells were CAR+ when assessed by flow cytometry 14 days after FHVH-BCMA-T infusion. We assessed blood CAR+ cells by quantitative PCR. The median peak level of CAR+ cells was 121 cells/µl (range 3-359 cells/µl) and the median day post-infusion of peak blood CAR+ cell levels was 12 (range 7-14). The results from this phase 1 trial demonstrate that FHVH-BCMA-T cells can induce deep and durable responses of relapsed MM with manageable toxicities. Assessment of durability of responses at the maximum feasible dose is a critical future plan. Accrual to the expansion cohort continues. Table Disclosures Manasanch: Novartis: Research Funding; Adaptive Biotechnologies: Honoraria; GSK: Honoraria; JW Pharma: Research Funding; Merck: Research Funding; Quest Diagnostics: Research Funding; Takeda: Honoraria; Sanofi: Honoraria; BMS: Honoraria; Sanofi: Research Funding. Rosenberg:Kite, A Gilead Company: Consultancy, Patents & Royalties, Research Funding. Kochenderfer:Kite, a Gilead company: Patents & Royalties, Research Funding; Celgene: Patents & Royalties, Research Funding; bluebird, bio: Patents & Royalties. OffLabel Disclosure: cyclophosphamide 300 mg/m2 fludarabine 30 mg/m2 Conditioning chemotherapy prior to CAR T-cell infusion