Hannah Karlsson

Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

CAR T-Cell Therapy: The Role of Physical Barriers and Immunosuppression in Lymphoma

Chimeric antigen receptor (CAR) T-cells have shown remarkable results in patients with B-cell leukemia and lymphoma. However, while CAR T-cells have shown complete responses in a majority of patients with acute lymphoblastic leukemia (ALL), lymphomas are more difficult to treat. Different CAR designs and conditioning protocols seem to affect the persistence of patient responses. However, factors that determine if patients receiving the same CARs will respond or not remain obscure. In Sweden, a phase I/IIa trial using third-generation CAR T-cells is ongoing in which we intend to compare tumor biology and immunology, in each patient, to treatment response. CAR T-cell therapy is a powerful tool to add to the treatment options for this patient group but we need to perform the necessary basic research on the multifactorial mechanisms of action to give patients the best possible option of survival. Such studies are also crucial to expand the success of CAR T-cells beyond CD19+ B-cell malignancy. This review will focus on possible barriers of treating lymphoma to define factors that need to be investigated to develop the next generation of CAR T-cell therapy.

Activation of myeloid and endothelial cells by CD40L gene therapy supports T-cell expansion and migration into the tumor microenvironment

CD40 is an interesting target in cancer immunotherapy due to its ability to stimulate T-helper 1 immunity via maturation of dendritic cells and to drive M2 to M1 macrophage differentiation. Pancreatic cancer has a high M2 content that has shown responsive to anti-CD40 agonist therapy and CD40 may thus be a suitable target for immune activation in these patients. In this study, a novel oncolytic adenovirus armed with a trimerized membrane-bound extracellular CD40L (TMZ-CD40L) was evaluated as a treatment of pancreatic cancer. Further, the CD40L mechanisms of action were elucidated in cancer models. The results demonstrated that the virus transferring TMZ-CD40L had oncolytic capacity in pancreatic cancer cells and could control tumor progression. TMZ-CD40L was a potent stimulator of human myeloid cells and T-cell responses. Further, CD40L-mediated stimulation increased tumor-infiltrating T cells in vivo, which may be due to a direct activation of endothelial cells to upregulate receptors for lymphocyte attachment and transmigration. In conclusion, CD40L-mediated gene therapy is an interesting concept for the treatment of tumors with high levels of M2 macrophages, such as pancreatic cancer, and an oncolytic virus as carrier of CD40L may further boost tumor killing and immune activation.

Abstract A041: CD19-targeting third generation CAR T cells for relapsed and refractory lymphoma and leukemia – report from the Swedish phase I/IIa trial

Chimeric antigen receptor (CAR) T cells have shown promising results in patients with B cell malignancy. In preclinical studies we showed that CD19-targeting third generation (3G) CAR T cells containing signaling domains from both CD28 and 4-1BB as co-stimulatory molecules have a higher activation status and greater proliferation in response to antigens. Herein we report initial results from a phase I/IIa study (NCT:02132624) using these 3G CAR T cells. Patients with relapsed or refractory CD19+ B-cell malignancy were eligible, provided there was no other curative treatment available. Of the first eleven patients reported, nine had lymphoma and two patients had acute lymphoblastic leukemia (ALL). Autologous CAR T cells were manufactured using a gamma retrovirus encoding the CAR and expanded by αCD3/αCD28/IL2. During CAR T cell production, all lymphoma patients received treatment to control tumor burden (-90 to -3 days before T cell infusion). Type of treatment depended on the type of lymphoma and previous treatments. In addition, prior to T cell infusion (day -2 to -1) patients #6-11 received cyclophosphamide (500mg/m2) and fludarabine (25mg/m2) as preconditioning to decrease immunosuppressive cells. The patients received one infusion of CAR T cells starting at a dose of 2x107 cells/m2 (patients 1 and 2), 1x108 (patients 4, 5, 7, 8, 9) and 2x108 (patients 6, 10, 11, 12). Patient #1 (DLBCL) had a mild cytokine release syndrome (CRS) after four weeks (never requiring treatment), followed by a complete response of his lymphoma. A relapse and a second CRS occurred after six weeks and he was treated with prednisone with good symptomatic effect and reduction of tumor size. The patient progressed after three months. Patients #2, 4, 5 (CLL, MCL, MCL) all progressed after 2, 1, and 3 months, respectively. Patient #6 (DLBCL) responded to treatment (CR) prior to T cell infusion and remained in complete remission for 6 months post T cell infusion. Patient #7 (CLL) and #9 (DLBCL) also responded to treatment prior to T cell infusion and remains in complete remission (>3 months). The CLL patient has a tumor negative bone marrow. Patient #8 (FL-DLBCL) progressed after 1 month but had a mild CRS. Patient #10 (ALL) experienced transient CNS toxicity followed by a complete response. However, at 3 months the patient relapsed with a CD19 negative ALL, accompanied by increased levels of immunosuppressive cells. Patient #11 (ALL) is in complete remission after a CRS (>1 month) and patient #12 (FL/Burkitt) had a major CRS requiring intensive care but is too early to evaluate. The CAR transgene could be detected in blood at the time of patient response. In summary, eleven patients have been treated with increasing doses of CAR T cells in Sweden. All treatments were given as out-patients. The conditioning has been relatively mild as compared to previous published studies. Six patients had complete remission, or complete clinical responses, at the time of evaluation of which two relapsed later. Four patients did not respond to treatment and progressed early; and 1 is still too early to evaluate. Correlations between the levels of immunosuppressive cells and patient response are currently under investigation. Citation Format: Gunilla Enblad, Hannah Karlsson, Kristina I. Wikstrom, Magnus Essand, Barbara Savoldo, Malcolm K. Brenner, Gianpietro Dotti, Martin Hoglund, Hans Hagberg, Angelica Loskog. CD19-targeting third generation CAR T cells for relapsed and refractory lymphoma and leukemia – report from the Swedish phase I/IIa trial. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A041.

Approaches to augment CAR T-cell therapy by targeting the apoptotic machinery.

Chimaeric antigen receptor (CAR) T-cells have shown impressive results in patients with B-cell leukaemia. Yet, in patients with lymphoma durable responses are still rare and heavy preconditioning required. Apoptosis resistance is considered a hallmark of cancer, often conveyed by a halted apoptosis signalling. Tumours regularly skew the balance of the components of the apoptotic machinery either through up-regulating anti-apoptotic proteins or silencing pro-apoptotic ones. Malignant B-cells frequently up-regulate anti-apoptotic B-cell lymphoma 2 (Bcl-2) family proteins leading to therapy resistance. CAR T-cells kill tumour cells via apoptosis induction and their efficacy may be affected by the level of Bcl-2 family proteins. Hence, there is an interesting possibility to increase the effect of CAR T-cell therapy by combining it with apoptosis inhibitor blockade agents. Compounds that inhibit Bcl-2, B-cell lymphoma extra large (Bcl-xL) and Bcl-2-like protein 2 (Bcl-w), can restore execution of apoptosis in tumour cells or sensitize them to other apoptosis-dependent treatments. Hence, there is a great interest to combine such agents with CAR T-cell therapy to potentiate the effect of CAR T-cell killing. This review will focus on the potential of targeting the apoptotic machinery to sensitize tumour cells to CAR T-cell killing.

A Phase I/IIa Trial Using CD19-Targeted Third-Generation CAR T Cells for Lymphoma and Leukemia

Purpose: The chimeric antigen receptor (CAR) T-cell therapy has been effective for patients with CD19+ B-cell malignancies. Most studies have investigated the second-generation CARs with either CD28 or 4-1BB costimulatory domains in the CAR receptor. Here, we describe the first clinical phase I/IIa trial using third-generation CAR T cells targeting CD19 to evaluate safety and efficacy. Patients and Methods: Fifteen patients with B-cell lymphoma or leukemia were treated with CAR T cells. The patients with lymphoma received chemotherapy during CAR manufacture and 11 of 15 were given low-dose cyclophosphamide and fludarabine conditioning prior to CAR infusion. Peripheral blood was sampled before and at multiple time points after CAR infusion to evaluate the persistence of CAR T cells and for immune profiling, using quantitative PCR, flow cytometry, and a proteomic array. Results: Treatment with third-generation CAR T cells was generally safe with 4 patients requiring hospitalization due to adverse reactions. Six of the 15 patients had initial complete responses [4/11 lymphoma and 2/4 acute lymphoblastic leukemia (ALL)], and 3 of the patients with lymphoma were in remission at 3 months. Two patients are still alive. Best predictor of response was a good immune status prior to CAR infusion with high IL12, DC-Lamp, Fas ligand, and TRAIL. Responding patients had low monocytic myeloid-derived suppressor cells (MDSCs; CD14+CD33+HLA−DR−) and low levels of IL6, IL8, NAP3, sPDL1, and sPDL2. Conclusions: Third-generation CARs may be efficient in patients with advanced B-cell lymphoproliferative malignancy with only modest toxicity. Immune profiling pre- and posttreatment can be used to find response biomarkers.

748. A Clinical Trial Using Third Generation CD19 Targeting CAR T Cells for Relapsed Lymphoma and Leukemia

A Clinical Trial Using Third Generation CD19 Targeting CAR T Cells for Relapsed Lymphoma and Leukemia