京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22347号 / 医博第4588号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 江藤 浩之, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In the last decade, six CAR T cell therapies against hematological malignancies have been approved for commercial manufacturing and several clinical trials are underway. This has led to extensive preclinical research focused on optimizing individual manufacturing steps of adoptive T cell therapeutics. Ex vivo expansion of T cells is one of the crucial manufacturing steps, as is necessary to obtain clinically required cell numbers for infusion. However, ex vivo expansion is also a complex step as it involves multiple different variables including culture medium, serum and cytokine supplementation, activation reagent and mode of genetic modification. Consequently, our understanding of changes in T cells during ex vivo expansion and the impact of expansion conditions on the final product; and thus the outcome of the therapy remain mostly elusive. Therefore, this project was designed to understand the changes in T cells at different stages of ex vivo expansion compared to freshly isolated T cells with a focus on understanding the ongoing transcriptional changes. The T cells were isolated from healthy blood donor buffy coats using FABian®-Cell Isolation System based on Fab-TACS technology. T cells were cultured for 7 days in X-VIVO 15 media (supplemented with 5% human serum and 50 IU/ml IL-2) with activation for initial 3 days using anti-CD3/CD28 TranAct. The T cell kinetics during ex vivo expansion was characterized based on cell activation, differentiation and proliferation. For whole transcriptome sequencing, Total RNA was harvested from 6 different time points, freshly isolated cells (0 hr), and cells cultured for 4, 12, 24, 72 and 168 hr (7 days). The RNA sequencing libraries were prepared using “Illumina TruSeq Stranded Total RNA library prep' workflow and whole transcriptome sequencing was performed on Illumina Novaseq 6000. Further, changes in T cell trafficking capabilities, cell size and cell cycle progression were studied in freshly isolated T cells and cultured cells. The changes in T cell trafficking was studied by analyzing the changes in VLA4 mediated T cell adhesion to VCAM1 coated surface under increasing shear stress. The cell size and volume of freshly isolated T cells and cultured cells were analyzed using multisizer instrument. Additionally, an in vitro model was developed to simulate the behavior of cultured T cells upon re-infusion into the blood and changes in cell cycle was analyzed. The components of in vitro reconstituted blood model were pooled human AB serum, erythrocyte concentrates and cultured T cells. The absolute lymphocyte count in buffy coats and total number of T cells isolated per buffy coat were in range compared to cell isolation and enrichment through standard leukapheresis. Thus suggesting that healthy donor-derived buffy coats and enrichment of T cells using Fab-TACS technology can be a suitable starting material and cell enrichment device respectively. The T cell growth kinetics was analyzed based on surface expression of specific markers, which also closely resembled their gene expression. The T cell kinetics observed during ex vivo expansion was similar to T cell kinetics observed in several preclinical CAR T cell expansion studies. The T cell proliferation in terms of increase in cell numbers and gene ontology (GO) terms related to DNA replication and cell division were significantly enriched only after 3 days of ex vivo expansion. The final cell numbers after 7 days of ex vivo expansion were approx. 1.0E+9 T cells, which was well above the clinically required infusion dosage of currently approved CAR T cell therapies. Taken together, the ex vivo expansion protocol followed in this study generates T cells in range required for clinical infusion dose and the growth kinetics of T cells observed were in line with the commercial expansion protocol. Hierarchical clustering of genes based on their expression over time identified 29 different gene-clusters which followed the pattern of mono-, bi- and triphasic modulation. The gene-clusters 11 and 18 were significantly enriched with T cell immune function related GO terms. The GO analysis of differentially expressed genes identified enrichment several bioprocesses, signaling pathways and T cell immune functions including commonly known activation, differentiation and proliferation. The ex vivo expansion of T cells was associated with early (i.e. upto 24 hr time point) enrichment of several GO terms associated with cytokine production such as IL-1, IL-2, IL-5, IL-6, IL-10, IL-13, IL-17, TNF and IFN-γ. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) and Mitogen-activated protein kinase (MAPK) signaling cascades related GO terms were enriched as a result of autocrine signaling mediated by cytokines released during ex vivo expansion. These data demonstrate that cytokine release in T cells is activated during ex vivo manufacturing, and should be considered during future optimization. The gene expression analysis of commonly known exhaustion markers revealed two different patterns of expression. The CTLA4, TIGIT, TBX21 and BATF was upregulated at early time points. Whereas, the expression of TIM3, LAG3 and CX3CR1 was upregulated at later time points. The early expression of exhaustion markers can be attributed to immune check point function to prevent over-activation, and later expression of exhaustion markers may contribute to inhibitory function. In vitro investigation of ex vivo expanded T cells exhibited stronger VLA4 mediated adhesion to VCAM1 coated surface compared to freshly isolated cells under increasing shear stress. This was in contrast to the downregulation of alpha 4/beta 1 integrin gene expression during ex vivo expansion. The cell size analysis revealed cultured T cells were larger in terms of both size and volume at the end of 7-day culture period with doubled cell volume compared to freshly isolated T cells. These results taken together, suggest that increased adhesion capacity and increased cell size, after T cell expansion may be associated with accumulation of T cells in lungs upon infusion. The freshly isolated T cells that closely represent the T cells circulating in peripheral blood were arrested in G0/G1 phase. However, during ex vivo expansion T cells entered cell cycle, and T cells were found to be predominantly in S+G2/M phase on day 3, 5 and 7. Surprisingly, the cultured T cells were still in cell cycle even after 48 h of incubation in reconstituted blood in vitro. This suggests that a prolonged resting phase of ex vivo expanded T cells for more 48 hr before infusion into the patients can be advantageous in minimizing the risks associated with T cell therapy. In conclusion, this study has revealed a number of novel insights into transcriptional regulation and signaling processes occurring during culture expansion. In the study, the different patterns of transcriptional regulation and enrichment of various associated bio-processes and signaling pathways during ex vivo expansion were explored. In addition, an in-depth analysis of genes related to T cell activation and differentiation, adhesion and migration, and exhaustion markers was performed. The protein-protein interaction analysis and transcriptional factor enrichment analysis provide valuable data for further in silico investigations of transcriptional changes in T cells during ex vivo expansion. Additionally, this study provides a comprehensive overview of long non-coding RNAs at different stages of ex vivo expansion of T cells, thus providing a resource for novel understanding of impact of lncRNAs on T cells during ex vivo expansion for adoptive T cell therapies. The complete data of 48 transcriptomes derived from 8 donors over 6 time points is reposited (GEO: GSE250311) to a publicly available database and will allow exploration for future studies which aim at the characterization of alterations in expanded T cells for therapy, and optimization of conditions for their future use in patients.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapy is a new treatment for hematologic malignancies including aggressive B-cell non-Hodgkin’s lymphoma (NHL). Although it has provided an effective treatment option for patients who have few options, CAR T-cell therapy does have many associated toxicities. Prolonged cytopenias are one of the lesser understood toxicities that can affect upwards of 40% of patients. METHODS: In this retrospective study, we reviewed 106 patients who received commercial CAR T-cell therapy between November 2017 and September 2019. Prolonged cytopenias were defined as having absolute neutrophil count (ANC) <1000/mm3, platelets (PLT) <50,000/mm3, and/or hemoglobin (Hgb) <10 g/dL at least once after 30 days post-CAR T-cell infusion. Furthermore, if only one incidence of cytopenia was recorded 30 days post infusion, we required that the patient had to have received either a transfusion or granulocyte-colony stimulating factor (GCSF) after the date of the recorded cytopenic value to be considered a part of the cytopenic cohort. RESULTS: 22 patients met the criteria of having prolonged cytopenias. 64% of the cytopenic cohort had >1 type of prolonged cytopenias. Anemia was the most prevalent affecting 72% of cytopenic patients. The length of time from diagnosis of aggressive B-cell NHL to date of CAR T-cell infusion was found to be positively correlated with an increased risk of developing prolonged cytopenias following CAR T-cell therapy. Additional risk factors associated with an increased risk of delayed cytopenias by univariate analysis included neutropenia on the day of infusion (day 0), a high C-reactive protein (CRP) before lymphodepletion and on day 0, day 0 PLT count, and Hgb before lymphodepletion and on day 0. On multivariate analysis, only high CRP before lymphodepletion was associated with an increased risk of prolonged cytopenias while high ferritin and PLT values on day 0 were associated with not developing prolonged cytopenias. There was no statistical difference between the cytopenic and non-cytopenic cohorts in rates of progression free survival (PFS) and overall survival (OS). Also, no difference was seen in rates or severity of other toxicities between cohorts. 41% of the cytopenic cohort experienced infectious complications post-infusion with one patient dying from their infectious complications. However, there was no association with incidence of infection and prolonged cytopenias when compared to the incidence of infection in the non-cytopenic cohort. CONCLUSIONS: A longer time from diagnosis of aggressive B-cell NHL to time of CAR T-cell infusion was associated with prolonged cytopenias while the number of lines of prior chemotherapy and rate of prior high dose chemotherapy with an autologous stem cell transplant (HD-ASCT) were not associated. It would be valuable to confirm this association and why it is associated since the other two factors were not. We lacked bone marrow biopsies before CAR T-cell infusion and did not have bone marrow biopsies for many patients after CAR T-cell infusion. It would be beneficial to collect data regarding bone marrow biopsies from these time points to highlight any changes that could be related to CAR T-cell therapy. Cytogenetic information of individual patient’s diseases would be worth analyzing to help determine if there are biological factors associated with prolonged cytopenias in response to CAR T-cell therapy. Additional studies should investigate the laboratory values we found to have associations with either cohort to help identify possible predictive values providers could use to identify patients at higher risk of having prolonged cytopenias. There is also a need to see if specific prior chemotherapy regimens increase a patient’s risk of having prolonged cytopenias. Overall, since prolonged cytopenias after CAR T-cell infusions have not been heavily investigated, further investigation is needed to better understand the predictive factors and identify possible mechanisms of prolonged cytopenias seen in CAR T-cell patients.

abstract: The success of genetically-modified T-cells in treating hematological malignancies has accelerated the research timeline for Chimeric Antigen Receptor-T (CAR-T) cell therapy. Since there are only two approved products (Kymriah and Yescarta), the process knowledge is limited. This leads to a low efficiency at manufacturing stage with serious challenges corresponding to high cost and scalability. In addition, the individualized nature of the therapy limits inventory and creates a high risk of product loss due to supply chain failure. The sector needs a new manufacturing paradigm capable of quickly responding to individualized demands while considering complex system dynamics. The research formulates the problem of Chimeric Antigen Receptor-T (CAR-T) manufacturing design, understanding the performance for large scale production of personalized therapies. The solution looks to develop a simulation environment for bio-manufacturing systems with single-use equipment. The result is BioMan: a discrete-event simulation model that considers the role of therapy's individualized nature, type of processing and quality-management policies on process yield and time, while dealing with the available resource constraints simultaneously. The tool will be useful to understand the impact of varying factor inputs on Chimeric Antigen Receptor-T (CAR-T) cell manufacturing and will eventually facilitate the decision-maker to finalize the right strategies achieving better processing, high resource utilization, and less failure rates. / Dissertation/Thesis / Masters Thesis Industrial Engineering 2020

Glioblastoma multiforme (GBM) is the most common malignant brain cancer that invades normal brain tissue and impedes surgical eradication, resulting in early local recurrence and high mortality. In addition, most therapeutic agents lack permeability across the blood brain barrier (BBB), further reducing the efficacy of chemotherapy. Thus, effective treatment against GBM requires tumor specific targets and efficient intracranial drug delivery. With the most recent advances in immunotherapy, genetically engineered T cells with chimeric antigen receptors (CARs) are becoming a promising approach for treating cancer. By transducing T lymphocytes with CAR constructs containing a tumor-associated antigen (TAA) recognition domain linked to the constant regions of a signaling T cell receptor, CAR T cells may recognize a predefined TAA with high specificity in a non-MHC restricted manner, and is independent of antigen processing. Active T cells can travel across the BBB, providing additional advantage for drug delivery and tumor targeting. Here we review the CAR design and technical innovations, the major targets that are in pre-clinical and clinical development with a focus on GBM, and multiple strategies developed to improve CAR T cell efficacy.

Despite the remarkable success of adoptive T cell therapy in the treatment of melanoma and hematological malignancies, therapeutic capacity in a broad range of solid tumours is impaired due to immunosuppressive events that render tumour-specific T cells unable to persist and kill transformed cells. To address some of the limitations of ACT in solid tumours, our laboratory has developed a therapeutic modality utilizing oncolytic virus, which expresses a tumour-associated antigen, known as an oncolytic viral vaccine (OVV), in combination with tumour specific central memory T cells. With this therapeutic approach (ACT), we can achieve robust in vivo expansion of transferred cells resulting in the complete and durable tumour regression in multiple solid murine tumour models. However, we demonstrate that the curative potential is lost when the tumour stage and burden increase as expanded transferred cells differentiate to a dysfunctional state resulting in the progressive decline in the tumour-specific CD8+ T cell response. Thus, we believe that restoring the T cell response in late-stage tumours will lead to enhanced curative potential of ACT in late-stage tumours. We have previously shown that HDACi, MS-275, can enhance the therapeutic capacity of a T cell-based therapy in an aggressive brain tumour model. In addition, concomitant delivery of MS-275 with ACT ensures durable cures through immunomodulatory mechanisms. Strikingly, concomitant delivery of MS-275, a class 1 histone deacetylase inhibitor (HDACi), with ACT in late-stage tumours completely restores the transferred T cell response to similar levels observed in early-stage tumours resulting in the complete regression of advance-stage tumours. Furthermore, MS-275 enhanced the proliferative capacity and tumour-specific cytotoxic function of transferred cells, independently of tumour stage, type and mouse strain. Interestingly, we did not observe a complete reversal of T cell dysfunction, but rather observed that MS-275 conferred unique properties to T cells as the expression of some markers typically associated with T cell dysfunction was enhanced in addition to persistence and proliferation capacity. Moreover, concomitant delivery of MS-275 also restored the therapeutic capacity of endogenously primed tumour-specific CD8+ T cells expanded by an OVV in late-stage tumours, demonstrating the potential for general use for MS-275 in T cell-based therapies. Our data suggests the use of HDACi may potentiate T cell-based immunotherapies to overcome tumour-mediated T cell dysfunction in advanced stage solid tumours. / Thesis / Master of Science in Medical Sciences (MSMS)

CAR T-cellbehandling är en immunterapi som har visat lovande resultat vid behandling av cancer. Trots det riktade immunsvaret som kan uppnås, betonar komplexiteten i tillverkningsprocessen och behandlingsproceduren det utrymme somm finns för förbättringar. Omprogrammerade T-celler har illustrerat en hög persistens hos patienter, som utsätter dem för risken för systemisk toxicitet. In-vivo transienta CAR T-celler som använder självförstärkande mRNA leverade genom affinitetsproteinbelagda LNP, föreslås som ett standardiserat alternativ som möjligör dosering av terapin vid behov.  Med hjälp av fagdisplay utfördes ett urval av affibody molekyler mot de tre immunonkologiska målproteinerna CD5, CD8 och CD19, i fyra cykler. Monoklonal fag-ELISA och DNA-sekvensering identifierade sju förmodade kandidater mot CD5, en förmodad kandidat mot CD8 och tre mot CD19. SPR analys visade specifik binding från CD5 kandidaterna, medan binding till målprotein inte kunde påvisas för CD8- och CD19 kandidaterna. De identifierade CD5-bindarna kan konjugeras till LNP för T-cell inriktad leverans av själv-amplififerande mRNA, med genetisk kod för en valfri CAR. / Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy which has shown promising results in treating patients suffering from oncological malignancies. Despite the targeted immune response that can be achieved, elaborate manufacturing and procedure processes emphasise room for improvement. Engineered T-cells have illustrated a high persistence in patients, exposing them to the risk of systemic toxicity. In-vivo transient CAR T-cells using self-amplifying mRNA by delivery through affinity protein coated lipid nanoparticles (LNP) is proposed as a standardised and reversible alternative, allowing for dosing when needed. Using phage display technology, selection of affibody molecules toward the three immune oncology proteins CD5, CD8 and CD19 was performed in four cycles. Monoclonal phage enzyme-linked immunosorbent assay (ELISA) and DNA sequencing identified seven putative candidates toward CD5, one putative candidate was isolated toward CD8, and three toward CD19. Surface plasmon resonance analysis (SPR) showed specific target binding of the CD5 candidate binders, while target binding could not be demonstrated for the CD8 and CD19 candidates. The identified CD5 binders could be conjugated to LNP for T-cell targeted delivery of self-amplifying mRNA encoding any CAR of interest.

Recombinant oncolytic virus (OV) vaccines that encode tumour-associated antigens are potent boosting agents for adoptive transfer of tumor-specific T cells (adoptive T cell therapy or ACT). Current strategies to exploit boosting vaccines for ACT rely on a priori knowledge of targetable tumour epitopes and isolation of matched epitope-specific T cells. Therefore, booster vaccines must be developed on a patient-by-patient basis, which severely limits clinical feasibility. To overcome the requirement for individualized pairing of vaccines and T cells, we propose a “universal” strategy for boosting tumor-specific T cells where the boost is provided through a synthetic receptor that can be engineered into any T cell and a matched vaccine. To this end, we are employing chimeric antigen receptors (CARs), which confer MHC-independent antigen specificity to engineered T cells, and a paired OV vaccine that encodes the CAR target. As proof-of-concept, we have developed and evaluated a model where murine TCR transgenic T cells are engineered with boosting CARs against a surrogate antigen for studies in immunocompetent hosts. In chapter 3, I optimized a murine CAR-T cell manufacturing protocol that allows for generation of highly-transduced T cells that maintain a predominantly central memory (Tcm) phenotype. This protocol leads to generation of highly functional CAR-T cell products that can be cryopreserved at the end of ex vivo culture for future use in adoptive transfer and vaccination studies. In chapter 4, I evaluated the in vivo boosting potential of our dual-specific CAR-T cells with paired OV vaccines. Adoptive transfer of these CAR-engineered tumor-specific T cells followed by vaccination with paired oncolytic vesicular stomatitis virus (VSV) vaccine leads to robust, but variable and transient, CAR-mediated expansion of tumour-specific CD8+ T-cells, resulting in delayed tumour progression in aggressive syngeneic tumour models. In chapter 5, I investigated the role of OV-induced type I interferon (IFN-I) responses on CAR-T cell boosting. I found that CAR-T cell expansion and anti-tumour function following OV vaccination is limited by the IFN-I response and can be further enhanced by blocking interferon alpha and beta receptor subunit 1 (IFNAR1). This IFN-I-mediated T cell suppression was found to be T cell-extrinsic and related to premature termination of OV infection and antigen expression in vivo. In chapter 6, I investigated the role of CD4+ T cell help in vaccine-mediated T cell boosting and evaluated different genetic engineering strategies to integrate pro-survival STAT5 signaling into the CAR-T cell product in an effort to improve persistence and long-term anti-tumour efficacy. The work presented herein describes a novel and clinically feasible approach to enhancing adoptive T cell therapies and contributes to the basic understanding of T cell biology in the context of CAR-engineering and cancer vaccination. / Thesis / Doctor of Philosophy (PhD) / Despite recent advances in cancer prevention, detection, and treatment, 2 in 5 Canadians are expected to be diagnosed with cancer in their lifetime and approximately 1 in 4 will succumb to their disease. New, more specific therapies are needed to improve responses to treatment and reduce therapy-related side effects. Cell therapy is a new way to treat cancer that uses the patient’s own immune cells as a living drug. The immune cells are taken from a patient’s blood or tumour, trained to attack cancer in the laboratory, and infused back into the patient where they will find and kill cancer cells. A major challenge with this strategy is that the trained immune cells do not always survive in the patient for long enough to get rid of the tumour. To “boost” the immune cells, we are developing a new strategy where the immune cells are genetically modified and combined with a vaccine to enhance their anti-tumor activity. Just like a vaccine against a bacteria or virus, this vaccine will tell the modified immune cells to turn on, make more of themselves, and to find and kill the cancer cells. By delivering this “go” signal through a vaccine, we think that the immune cells will be better able to survive and generate a stronger, longer-lasting immune response against the cancer. This thesis tests this approach in relevant mouse models of cancer and aims to understand how we can best design the immune cells and vaccine to work together in their tumour-killing activities.

Cancer immunotherapy has recently made remarkable clinical progress. Adoptive transfer of T-cells engineered with a chimeric antigen receptor (CAR) against CD19 has been successful in treatment of B-cell leukemia. Patient’s T-cells are isolated, activated, transduced with a vector encoding the CAR molecule and then expanded before being transferred back to the patient. However some obstacles restrict its success in solid tumors. This thesis explores different aspects to improve CAR T-cells therapy of cancer. Ex vivo expanded T-cells are usually sensitive to the harsh tumor microenvironment after reinfusion. We developed a novel expansion method for T-cells, named AEP, by using irradiated and preactivated allo-sensitized allogeneic lymphocytes (ASALs) and allogeneic mature dendritic cells (DCs). AEP-expanded T-cells exhibited better survival and cytotoxic efficacy under oxidative and immunosuppressive stress, compared to T-cells expanded with established procedures. Integrating retro/lentivirus (RV/LV) used for CAR expressions randomly integrate in the T-cell genome and has the potential risk of causing insertional mutagenesis. We developed a non-integrating lentiviral (NILV) vector containing a scaffold matrix attachment region (S/MAR) element (NILV-S/MAR) for T-cells transduction. NILV-S/MAR-engineered CAR T-cells display similar cytotoxicity to LV-engineered CAR T-cells with undetectable level of insertional event, which makes them safer than CAR T-cells used in the clinic today. CD19-CAR T-cells have so far been successful for B-cell leukemia but less successful for B-cell lymphomas, which present semi-solid structure with an immunosuppressive microenvironment. We have developed CAR T-cells armed with H. pylorineutrophil-activating protein (HP-NAP). HP-NAP is a major virulence factor and plays important role in T-helper type 1 (Th1) polarizing. NAP-CAR T-cells showed the ability to mature DCs, attract innate immune cells and increase secretion of Th1 cytokines and chemokines, which presumably leads to better CAR T-cell therapy for B-cell lymphoma. Allogeneic-DCs (alloDCs) were used to further alter tumor microenvironment. The premise relies on initiation of an allo-reactive immune response for cytokine and chemokines secretion, as well as stimulation of T-cell response by bringing in tumor-associated antigen. We demonstrated that alloDCs promote migration and activation of immune cells and prolong the survival of tumor-bearing mice by attracting T-cells to tumors and reverse the immune suppressive tumor microenvironment.

Immunotherapy for cancer is a young research field progressing at high speed. The first chimera of an antibody and a signaling chain was designed by Zelig Eshhar and was later further developed to enhance existing T cell therapy by combining a single-chain fragment of an antibody with the CD3 zeta chain of the TCR complex. T cells expressing these chimeric antigen receptors (CARs) could recognize and specifically kill tumor cells. However the T cells, lacked in persistence and tumor rejection did not occur. Thus, the CAR constructs have been improved by providing the T cell with costimulatory signals promoting activation. The focus of this thesis has been to evaluate second and third generation αCD19-CAR T cells for the treatment of B cell leukemia and lymphoma. B cell tumors commonly upregulate anti-apoptotic proteins such as Bcl-2, which generates therapy resistance. In the first paper a second generation (2G) αCD19-CD28-CAR T cell was combined with the Bcl-2 family inhibitor ABT-737. ABT-737 sensitized tumor cells to CAR T cell therapy and may be an interesting clinical combination treatment. In paper II, the phenotype and function of a third generation (3G) αCD19-CD28-4-1BB-CAR T cell were evaluated. B cell-stimulated CAR T cells showed increased proliferation and an antigen-driven accumulation of CAR+ T cells. 3G CAR T cells had equal cytotoxic capacity, similar lineage, memory and exhaustion profile phenotype compared to 2G CARs. However, 3G CAR T cells proliferated better and had increased activation of intracellular signaling pathways compared to 2G CAR T cells. In paper III, αCD19-CD28-4-1BB-CAR T cells were used to stimulate immature dendritic cells leading to an upregulation of maturation markers on co-cultured dendritic cells. Hence, CAR T cells may not only directly kill the tumor cells, but may induce bystander immunity that indirectly aids tumor control. This thesis also include supplementary information about the development and implementation of protocols for GMP production of CAR T cell batches for a phase I/IIa clinical trial currently ongoing for patients with refractory B cell leukemia and lymphoma. So far, two patients have safely been treated on the lowest dose.