Michael Klichinsky

CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia

Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML.

Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies.

The Addition of the BTK Inhibitor Ibrutinib to Anti-CD19 Chimeric Antigen Receptor T Cells (CART19) Improves Responses against Mantle Cell Lymphoma

Purpose: Responses to therapy with chimeric antigen receptor T cells recognizing CD19 (CART19, CTL019) may vary by histology. Mantle cell lymphoma (MCL) represents a B-cell malignancy that remains incurable despite novel therapies such as the BTK inhibitor ibrutinib, and where data from CTL019 therapy are scant. Using MCL as a model, we sought to build upon the outcomes from CTL019 and from ibrutinib therapy by combining these in a rational manner. Experimental Design: MCL cell lines and primary MCL samples were combined with autologous or normal donor-derived anti-CD19 CAR T cells along with ibrutinib. The effect of the combination was studied in vitro and in mouse xenograft models. Results: MCL cells strongly activated multiple CTL019 effector functions, and MCL killing by CTL019 was further enhanced in the presence of ibrutinib. In a xenograft MCL model, we showed superior disease control in the CTL019- as compared with ibrutinib-treated mice (median survival not reached vs. 95 days, P < 0.005) but most mice receiving CTL019 monotherapy eventually relapsed. Therefore, we added ibrutinib to CTL019 and showed that 80% to 100% of mice in the CTL019 + ibrutinib arm and 0% to 20% of mice in the CTL019 arm, respectively, remained in long-term remission (P < 0.05). Conclusions: Combining CTL019 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma. Clin Cancer Res; 22(11); 2684–96. ©2016 AACR.

A fungal protease allergen provokes airway hyper-responsiveness in asthma

Asthma, a common disorder that affects more than 250 million people worldwide, is defined by exaggerated bronchoconstriction to inflammatory mediators including acetylcholine, bradykinin, and histamine—also termed airway hyper-responsiveness Nearly 10% of people with asthma have severe, treatment-resistant disease, which is frequently associated with IgE sensitization to ubiquitous fungi, typically Aspergillus fumigatus. Here we show that a major Aspergillus fumigatus allergen, Asp f13, which is a serine protease, alkaline protease 1 (Alp 1), promotes airway hyper-responsiveness by infiltrating the bronchial submucosa and disrupting airway smooth muscle cell-extracellular matrix interactions. Alp 1-mediated extracellular matrix degradation evokes pathophysiological RhoA-dependent Ca2+ sensitivity and bronchoconstriction. These findings support a pathogenic mechanism in asthma and other lung diseases associated with epithelial barrier impairment, whereby airway smooth muscle cells respond directly to inhaled environmental allergens to generate airway hyper-responsiveness.

Kinase inhibitor ibrutinib to prevent cytokine-release syndrome after anti-CD19 chimeric antigen receptor T cells for B-cell neoplasms

Kinase inhibitor ibrutinib to prevent cytokine-release syndrome after anti-CD19 chimeric antigen receptor T cells for B-cell neoplasms

Overcoming the Immunosuppressive Tumor Microenvironment of Hodgkin Lymphoma Using Chimeric Antigen Receptor T Cells

Patients with otherwise treatment-resistant Hodgkin lymphoma could benefit from chimeric antigen receptor T-cell (CART) therapy. However, Hodgkin lymphoma lacks CD19 and contains a highly immunosuppressive tumor microenvironment (TME). We hypothesized that in Hodgkin lymphoma, CART should target both malignant cells and the TME. We demonstrated CD123 on both Hodgkin lymphoma cells and TME, including tumor-associated macrophages (TAM). In vitro, Hodgkin lymphoma cells convert macrophages toward immunosuppressive TAMs that inhibit T-cell proliferation. In contrast, anti-CD123 CART recognized and killed TAMs, thus overcoming immunosuppression. Finally, we showed in immunodeficient mouse models that CART123 eradicated Hodgkin lymphoma and established long-term immune memory. A novel platform that targets malignant cells and the microenvironment may be needed to successfully treat malignancies with an immunosuppressive milieu.Significance: Anti-CD123 chimeric antigen receptor T cells target both the malignant cells and TAMs in Hodgkin lymphoma, thereby eliminating an important immunosuppressive component of the tumor microenvironment. Cancer Discov; 7(10); 1154-67. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.

Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19– leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.A CAR gene unintentionally introduced in a contaminating leukemia cell during the manufacturing of CAR T cells caused a patient to relapse after therapy.

273. Genome Editing Using CRISPR-Cas9 to Increase the Therapeutic Index of Antigen-Specific Immunotherapy in Acute Myeloid Leukemia

Antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells or bispecific T cell engaging (BITE) antibodies directed against CD19 leads to elimination of malignant and normal B cells. These approaches have a broad therapeutic index in acute lymphoid leukemia (ALL) due to a combination of high anti-tumor activity and tolerability of prolonged B cell depletion. In contrast, in acute myeloid leukemia (AML) the absence of surface antigens specific to leukemia implies that potent myeloid-directed immunotherapy will eradicate normal as well as malignant cells, leading to protracted myeloablation and bone marrow failure, as has been shown in several preclinical studies of CD33 or CD123 directed CART cell therapy. We developed a novel approach to circumvent this problem by genetically modifying normal hematopoietic stem cells (HSCs) so that they do not express the antigen recognized by CART cells, thus generating normal myeloid progeny that is resistant to CART cell therapy.CD33 is expressed on the majority of AML as well on normal myeloid progenitors and its function is poorly characterized. We hypothesized that CD33 knockout HSCs and their myeloid progeny would develop normally yet be resistant to treatment with anti-CD33 CART cells (CART33). Electroporation of human CD34+ cells with Cas9 protein and in-vitro transcribed sgRNA generated deleterious mutations in the CD33 gene with 70-80% efficacy as measured by flow cytometry and DNA sequencing. Addition of a single-stranded oligonucleotide template with a short insertional mutation at the sgRNA cut site increased the knockout efficacy up to 90%. In vitro cytotoxicity assays showed CD33 KO HSC progeny are resistant to killing by CART33 compared to wild-type HSC controls (% live cells after CART33 co-culture: 60% vs. 18%, p=0.005). CD33 KO HSC progeny showed a similar growth and differentiation profile in Methocult semi-solid media compared to control cells that were electroporated with an unrelated sgRNA. The differentiated cells had normal neutrophil and macrophage morphology and immunophenotype. We also found that phagocytosis and cytokine secretion capabilities of the CD33 KO progeny were identical to WT control. NOD-SCID-g-/- (NSG) mice injected with CD33 KO HSCs showed normal engraftment with differentiation of both myeloid and lymphoid lineages. As expected, the percentage of CD33+ myeloid cells was lower in CD33 KO recipients than WT control (24% vs 63%, p<0.0001), yet the percentage of CD14+CD11b+ monocytes (45% vs 47%, p=0.77) and CD14-CD11b+ neutrophils (34% vs. 33%, p=0.87) were unaltered.This approach yields a potential strategy to treat AML with potent CD33-specific immunotherapy, followed by infusion of gene-modified CD33 KO HSCs, which will allow persistent antigen-specific immune attack along with reconstitution of effective hematopoiesis.

Abstract 4575: Chimeric antigen receptor macrophages (CARMA) for adoptive cellular immunotherapy of solid tumors

Chimeric antigen receptor (CAR) T cell immunotherapy has demonstrated profound results in hematologic malignancies but clinical efficacy in the solid tumor setting has not been observed. Barriers to T cell entry and function may partially explain this observation. As solid tumors actively recruit myeloid cells, we hypothesized that macrophages have the potential to be a powerful cellular immunotherapeutic agent in this setting if properly activated and redirected. We here describe the development of CAR macrophages (CARMA), demonstrating the feasibility, mechanism, and efficacy of this platform. To examine the function of CARs in macrophages, first generation anti-CD19, anti-mesothelin, or anti-HER2 CARs with a CD3ζ intracellular domain were introduced into the THP1 macrophage model. In vitro function was assessed via quantitative phagocytosis and luciferase-based specific killing assays. CARMA selectively phagocytosed and cleared cognate antigen-bearing tumor cells. To demonstrate the requirement for CAR-mediated intracellular signaling for activity, a CD3ζ-null CAR construct was tested in vitro. The deletion of CD3ζ significantly reduced the phagocytic and killing capacity (p 70% CAR expression). Ad5f35 transduction polarized human macrophages toward a durable immunostimulatory M1 phenotype and rendered CARMA resistant to subversion toward the immunosuppressive M2 phenotype, as defined by surface markers and metabolomics. CARMA enhanced the proliferative capacity of CD8+ T cells in phytohemagglutinin activation assays and secreted factors that activated by-stander macrophages. Primary human anti-HER2 CARMA demonstrated targeted phagocytosis and killing of HER2 expressing ovarian and breast cancer cell lines, and exhibited a six-fold higher luciferase-based killing capacity of SKOV3 cells compared to trastuzumab in vitro (p=0.002). Anti-HER2 CARMA was evaluated in vivo in an intraperitoneal (IP) SKOV3 ovarian cancer xenograft model. Mice that received IP CARMA had a decrease in tumor burden of approximately two orders of magnitude and had a 30-day survival benefit relative to untreated or control macrophage treated mice (p=0.018). In a systemically disseminated SKOV3 model, a single dose of IV CARMA led to a durable anti-tumor response (38-fold reduction relative to control on day 31 post-treatment; p=0.016) Lastly, we demonstrated that the blockade of the anti-phagocytic CD47/SIRPα axis enhanced the phagocytic capacity of CARMA. In summary, we here demonstrate that human macrophages engineered with a CAR exhibit targeted anti-tumor function in both in vitro and in vivo preclinical models. This novel cellular immunotherapeutic approach has a clear translational potential for the treatment of solid tumors. Citation Format: Michael Klichinsky, Marco Ruella, Olga Shestova, Saad S. Kenderian, Miriam Y. Kim, Roddy O9Connor, John Scholler, Carl June, Saar Gill. Chimeric antigen receptor macrophages (CARMA) for adoptive cellular immunotherapy of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4575. doi:10.1158/1538-7445.AM2017-4575

Abstract 3139: CD33 directed chimeric antigen receptor T cell therapy as a novel regimen prior to allogeneic stem cell transplantation in acute myeloid leukemia

Allogeneic stem cell transplantation is the only option in relapsed acute myeloid leukemia (AML). However, patients are often excluded due to refractory disease. The successful translation of Chimeric Antigen Receptor (CAR) T cell therapy to AML would constitute a vertical advance in the field. We developed a 2nd generation CAR using the anti CD33 scFv of Gemtuzumab ozogamicin, 41BB costimulation, CD3ζ signaling domain, and a lentiviral (LV) vector. In vitro, CART33 resulted in robust functions when incubated with the CD33+ cell line MOLM14. In NSGS mice (NOD SCID γc-/-(NSG), transgenic for stem cell factor, IL3 and GM-CSF) engrafted with primary AML, CART33 eradicated AML and led to a long term disease free survival (Table 1). Since CD33 is also expressed on normal myeloid progenitors, we assessed the potential myelotoxicity of CART33 in humanized immune system (HIS) mice (NSG mice engrafted with human fetal liver CD34+ cells). CART33 resulted in human lineage cytopenias and in a significant reduction of CD34+CD38- hematopoietic stem cells and CD34+CD38+ myeloid progenitors. Both the anti-leukemic activity and myeloablative potential of CART33 could be used as a novel conditioning regimen in refractory AML. However, the effect of the CAR T cells needs to be terminated to avoid engraftment failure. We therefore designed a “biodegradable” mRNA modified CAR33. T cells were electroporated with this construct and expressed CAR for up to six days. When compared with LV-CART33, RNA-CART33 resulted in comparable in vitro functions that declined over time post electroporation. In NSG mice engrafted with MOLM14, treatment with cyclophosphamide (for lymphodepletion) plus RNA-CART33 resulted in deeper and longer leukemic response compared to cyclophosphamide plus control T cells. In conclusion, our preclinical studies show potent activity of CART33 in AML. Biodegradable CART33 could be used as a novel anti-leukemic cellular conditioning in patients with relapsed AML Note: This abstract was not presented at the meeting. Citation Format: Saad S. Kenderian, Marco Ruella, Olga Shestova, Michael Klichinsky, John Scholler, Decheng Song, David L. Porter, Martin Carroll, Carl H. June, Saar Gill. CD33 directed chimeric antigen receptor T cell therapy as a novel regimen prior to allogeneic stem cell transplantation in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3139. doi:10.1158/1538-7445.AM2015-3139