AbstractAdoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38+ fractions of CD34+ hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38+ malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies.
Multiple myeloma (MM), a malignant disorder of antibody-producing clonal plasma cells, is the second most common hematologic neoplasia worldwide.1 Despite four decades of drug innovation, MM remains incurable with chemotherapy. Furthermore, the prognosis of MM patients who become refractory to recently developed novel agents is very poor.2 On the other hand, clinical and experimental data collected over the past decades suggest that MM could be successfully treated through (cellular) immunotherapy.43 The curative potential of cellular immunotherapy in MM is illustrated by the induction of long-term sustained remissions after allogeneic stem cell transplantation or donor lymphocyte infusions in a subset of patients.65 A highly appealing and more specific immunotherapy strategy for cancer is the adoptive transfer of cytotoxic T cells that are genetically engineered to express chimeric antigen receptors (CAR).87 A CAR is an artificial hybrid receptor, in which the antigen-recognizing domain of a tumor-reactive monoclonal antibody is fused with T-cell signaling domains. Upon retroviral or lentiviral transduction of cytotoxic T cells, CAR expressed on the cell surface redirect the cytotoxic T cells toward the original target of the antibody in a non-HLA-restricted manner,87 making it possible to apply the therapy regardless of the patient’s HLA type. Currently the most successful CAR-approaches are based on targeting the CD19 molecule, which is broadly expressed in several B-cell malignancies but not on the malignant plasma cells from patients with MM. Among a few potential CAR candidates for MM,9 the CD38 molecule, with its high and uniform expression on malignant plasma cells, has long been suggested a suitable target for antibody therapy of MM. The utility of CD38 as a suitable target has been supported by the results of recently initiated clinical trials in which MM patients were safely and effectively treated with the CD38-specific human monoclonal antibody daratumumab.10
Encouraged by these clinical results, we started to explore the feasibility of development of a CART-cell therapy based on targeting the CD38 molecule. Using variable heavy and light chain sequences of three different human CD38 antibodies, we generated three different CD38-CAR. We transduced T cells from healthy individuals and MM patients with the CD38-CAR and evaluated them for essential functions such as antigen-specific proliferation and cytokine production, for in vitro and in vivo anti-tumor efficacy and for potential undesired effects such as targeting normal CD38 cell fractions in the peripheral blood and bone marrow. We also evaluated the feasibility of controlling CD38-CART cells by introducing a caspase-9-based suicide gene.
Bone marrow mononuclear cells from patients with multiple myeloma or acute myeloid leukemia
Bone marrow mononuclear cells containing 5–20% malignant plasma cells or ~50% acute myeloid leukemia (AML) blasts were isolated from bone marrow aspirates of MM/AML patients through Ficoll-Paque density centrifugation and cryopreserved in liquid nitrogen until use. All bone marrow and blood sampling from the patients was performed after informed consent and approved by the institutional medical ethical committee.
Peripheral blood mononuclear cells from healthy individuals
Peripheral blood mononuclear cells were isolated from the buffy coats of healthy blood-bank donors by Ficoll-Paque density centrifugation after informed consent and approval by the institutional medical ethical committee.
The sequences of three different human CD38 antibodies, which are distinct from, but display similar affinities to the recently documented daratumumab10 (Online Supplementary Table S1) were kindly provided by Genmab. Cloning methods are described in the Online Supplementary Methods.
Retroviral chimeric antigen receptor transduction into T cells
Transduction methods are described in the Online Supplementary Methods.
Flow cytometry-based cell lysis assays
To detect the lysis of various cell subsets by CART cells in mononuclear cells from whole bone marrow or peripheral blood, serial dilutions of CART cells were incubated with CFSE-labeled bone marrow mononuclear cells or peripheral blood mononuclear cells for 24 h. The cells were then harvested, stained for different CD markers and topro3 or LIVE/DEAD Fixable Near-IR (Life Technologies L10119) and were quantitatively analyzed through volume-equalized measurements using a FACS Canto flow cytometer. For each cell subset identified with a CD marker, CFSE, viable/Topro3 cells were counted as surviving target cells. Percentage cell lysis in a treated sample was calculated as follows and only if the analyzed target cell population contained >500 viable cells in the untreated samples. % lysis cells = 1 − (absolute number of surviving cells in treated wells / absolute number of surviving cells in untreated wells) × 100%.
Bioluminescence imaging-based cell lysis assays
To determine the lysis of Luc-GFP-transduced human malignant cell lines by CD38-CART cells, serial dilutions of mock or CD38-CART cells were co-incubated with the malignant cell lines. The luciferase signal produced by surviving malignant cells was determined after 16–24 h with a SpectraMax luminometer (Molecular Devices) within 15 min after the addition of 125 μg/mL beetle luciferin (Promega).11 The percent lysis was then calculated as in the flow-based cytotoxicity assay above.
RAG2γc mice used in this study were originally obtained from the Amsterdam Medical Center (AMC, Amsterdam, the Netherlands). The mice were bred and maintained in filter top cages under specified pathogen-free conditions at the Central Animal Facility (GDL, Utrecht University, Utrecht, the Netherlands) and received sterile water and radiation-sterilized food pellets ad libitum.
In vivo efficacy of CD38-chimeric antigen receptor-transduced T cells against multiple myeloma tumors growing in a humanized microenvironment
To create a human bone marrow-like environment in mice, hybrid scaffolds were coated in vitro with human mesenchymal stromal cells. After a week of in vitro culture, humanized scaffolds were seeded with CD38 UM9 cells and implanted subcutaneously into the mice, as described previously,1211 and in the Online Supplementary Methods.
Generation of CD38-chimeric antigen receptor-transduced T cells
We used the variable heavy and light chain sequences of three different CD38 antibodies with CD38 binding affinities comparable to that of daratumumab (Online Supplementary Table S1), which is now being tested in clinical trials. T cells from healthy peripheral blood mononuclear cells were transduced with the different CD38-CAR genes or with the empty vector (mock) separately. After selection of transduced cells to high purity by neomycin treatment, the surface expression of CAR was determined by incubating the T cells with biotinylated bacterial protein L, which specifically binds to the variable region of kappa light chains of antibodies.13 Indirect staining with phycoerythrin-conjugated streptavidin revealed the expression of all three CAR on >95% of the T cells, whereas T cells transduced with an empty vector (mock-transduced T cells) displayed only background staining (Figure 1B, left panel). The CAR-transduced cells contained variable levels of both CD4 and CD8 cells (Figure 1B, right panel).
CD38-dependent proliferation and cytokine secretion of CD38-chimeric antigen receptor-transduced T cells
To analyze their proliferative and functional properties, neomycin-selected, highly purified CD38-CART cells were expanded using irradiated feeder cells in the presence of phytohemagglutinin and interleukin-2. While the mock T cells initially expanded better than the CD38-CART cells (Figure 1C, left panel), the growth disadvantage of CD38-CART cells disappeared in the second round of expansion (Figure 1C, right panel), indicating that transduction of the CD38-CAR construct did not affect the proliferative capacity of T cells. We then tested whether CD38-CART cells can be activated by CD38-triggering. To this end, we co-cultured mock- and CD38-CAR-transduced T cells with the irradiated CD38 MM cell line UM9 and used the CD38 MM cell line U266 as a control (Figure 1D, left panel). CD38-CART cells, but not mock T cells, specifically proliferated and produced interferon-γ, tumor necrosis factor-α and interleukin-2 (Figure 1D, right panel), but not interleukin-4, -5 or -10 (data not shown) upon stimulation with UM9 cells. These results indicate that CD38-CART cells had no defects in cytokine production but displayed a typical Th1-like cytokine response upon target recognition. Furthermore, the CD38 cell line U266 was unable to stimulate CD38-CART cells, demonstrating the proper antigen-specific function of CD38-CART cells.
CD38-dependent lysis of multiple myeloma cell lines by CD38-chimeric antigen receptor-transduced T cells
To determine the CD38-dependent lysis of malignant cells by CD38-CART cells, we first used luciferase-transduced MM cell lines with variable CD38 expression levels in bioluminescence imaging-based cytotoxicity assays.1411 As expected, there was no CD38-CAR-specific lysis of the CD38 U266 cell line (Figure 2A). In contrast, all three types of CD38-CART cells, but not mock T cells, effectively lysed the CD38 MM cell line UM9 in a cell-dose dependent manner (Figure 2B), showing the feasibility of generating effective CART cells with any of the CD38 antibody sequences we used. Since there was no functional difference between the three different CD38-CAR (028, 056, 026), we continued our investigation with one type of CD38-CART cell (CAR056). Flow cytometry and bioluminescence imaging-based cytotoxicity assays, performed using other malignant cell lines expressing various levels of CD38 (Online Supplementary Figure S1) as target cells, revealed a good correlation between CD38 expression and CD38-CART cell-mediated lysis (Figure 2C). One AML cell line, the Burkitt lymphoma-derived cell line Daudi as well as normal T cells appeared less sensitive to CD38-CART cell-mediated lysis as compared to MM cell lines with similar levels of CD38 expression (Figure 2C).
Lysis of primary multiple myeloma and acute myeloid leukemia cells by CD38-chimeric antigen receptor-transduced T cells
To test the efficacy of CD38-CART cells against primary MM and AML cells, we used a previously described flow cytometry-based ex vivo cytotoxicity assay, in which the lysis of malignant cells is tested directly in bone marrow mononuclear cells without isolating them from other cells.15 As depicted in Figure 3A, primary CD138CD38 MM cells from three different MM patients, who were refractory to treatment with lenalidomide and bortezomib (left panel), were effectively lysed by CD38-CART cells, but not by mock-transduced T cells. Similarly, in the bone marrow mononuclear cells of two AML patients’ malignant cells, which were identified as CD13 CD45 cells and expressed either low/intermediate (patient 1) or high (patient 2) levels of CD38, were effectively lysed by CD38-CART cells (Figure 3A). Finally, CD38-CART cells that were generated (Figure 3B) from a MM patient were effective towards autologous malignant MM cells in bone marrow mononuclear cells, indicating the feasibility of generating effective CD38-CART cells also from MM patients.
Fully functional CD38-chimeric antigen receptor-transduced T cells are negative for CD38
While CD38-CART cells had no apparent functional deficiencies, a phenotyping assay revealed that, despite a mixed effector/central memory phenotype, they lost the expression of CD38 (Figure 4A). Interestingly, when we co-cultured CD38-CART cells with an autologous CD19-CART cell population, these CD19-CART cells also became largely negative for CD38 expression but fully maintained their capacities to proliferate, secrete cytokines and kill the relevant target cells in a CD19-dependent fashion (Online Supplementary Figure S2), indicating that the loss of CD38 was not associated with detectable T-cell dysfunction. Nonetheless, since the CD38 molecule could also play a role in migration, we evaluated whether CD38 CD38-CART cells could migrate properly through endothelial layers in a transwell migration assay (Figure 4B). These assays revealed no differences between the mock-transduced, CD38 and CD38-CAR-transduced CD38 T cells, ruling out an apparent migratory dysfunction of CD38-CART cells.
In vivo efficacy of CD38-chimeric antigen receptor-transduced T cells against multiple myeloma tumors growing in a humanized microenvironment
To substantiate the in vitro results, we questioned whether the CD38 CART cells could mediate anti-MM effects in vivo after systemic injection in our recently developed model in Rag2γc mice, in which a humanized bone marrow-like niche for MM cells is generated by subcutaneous implantation of ceramic scaffolds coated with human bone marrow stromal cells1211 (Figure 4). Thus, we implanted such scaffolds seeded with luciferase-transduced UM9 MM cells in the back of the mice (6 scaffolds per mouse). Upon detection of the luciferase signal by bioluminescence imaging, we treated the mice with intravenous injections of CD38-CART cells using a previously established treatment scheme.16 Mock-transduced T cells were used as controls. As illustrated in Figure 4B, in the control group treated with mock T cells, tumors showed fast progression. Although not curative, treatment of the tumor-bearing mice with CD38-CART cells induced a significant anti-tumor effect (Figure 4B,C) underscoring the potential of CD38-CART cells to properly infiltrate and lyse MM tumors growing in their natural, protective niche. Post mortem analyses revealed that the remaining CD138 tumors were still positive for CD38 (Figure 4D), thus ruling out tumor escape due to “antigen loss” variants.
Impact of CD38-chimeric antigen receptor-transduced T cells on CD38+ normal hematopoietic cells and hematopoietic progenitor cells
Besides the high levels expressed in MM cells, the CD38 molecule is expressed at intermediate levels on a subset of hematopoietic progenitor cells17 and on a fraction of normal hematopoietic cells including activated T cells, natural killer cells, B cells and monocytes. We, therefore, evaluated the possible negative impact of CART cells on these cell subsets by co-incubating unsorted bone marrow mononuclear cells with CD38-CART cells. CD38-CART cells appeared to eliminate the CD38 fractions of mature T, B, natural killer and monocyte cell subsets (Figure 5A) and the CD38 fraction of CD34 cells (Figure 5B) in a 4 h assay. The lysis of CD34CD38 cells did not, however, have any influence on the development of colony-forming units of monocytes or of granulocytes in a 14-day hematopoietic precursor cell colony-forming assay1918 (Figure 5C,D).
Specific elimination of CD38-chimeric antigen receptor-transduced T cells using a suicide gene (iCasp9)
Although CD38-CART cells did not lyse the CD38 fractions of mature hematopoietic cells and did not inhibit the outgrowth of these cell populations, a cautious approach toward the clinical application of this construct is still required. As a first step towards safer application of CD38-CART cells, we tested the possibility of controlling them with a suicide gene based on the inducible caspase-9 (iCasp9) gene that is activated with a small dimerizer molecule AP20187 (B/B).20 Thus, we inserted an iCasp9 vector containing a green fluorescent protein (GFP) marker gene into the CD38-CART cells by retroviral transduction. Around 50% of the CD38-CART cells were transduced, as detected by GFP expression (Figure 6A, upper panel). When tested without sorting the iCasp9-transduced (GFP) cells, all iCasp9-transduced, GFP, but none of the iCasp9-non-transduced, GFP-CD38-CART cells were eliminated upon incubation with the dimerizer AP20187 (Figure 6A, lower panel). As expected, the dimerizer treatment also resulted in a proportional decrease in the lysis of the MM cell line UM9. (Figure 6B). There was still some remaining lysis due to the surviving iCasp9-negative CD38-CART cells, indicating that triggering the suicide gene did not induce bystander damage to the cells in the close vicinity. When tested after sorting for GFP cells (Figure 6C,D), almost all GFP cells died after treatment with the dimerizer (Figure 6C) and there was no CD38-specific lysis left (Figure 6D), confirming the results obtained in previous studies,2120 and suggesting the possibility of controlling CD38-CART cells using the iCasp9 suicide gene without undesired consequences.
While cellular immunotherapy of hematologic malignancies has been applied for many decades in the most non-specific form as allogeneic stem cell transplantation or donor lymphocyte infusions, it has recently entered a more specific level of innovation with several encouraging strategies, including vaccination with antigen-loaded dendritic cells or adoptive immunotherapy with T-cell receptor-gene transferred T cells, tumor infiltrating T cells and more recently with cytotoxic T cells endowed with tumor-reactive CAR. Among these strategies, CAR-based therapies are perhaps the most appealing, as CART cells recognize their target antigens in an MHC-independent manner. Setting out to develop a CAR-based strategy for MM, we have been encouraged by the highly promising clinical results of therapy with daratumumab, which targets CD38, a type II transmembrane glycoprotein, expressed at high and uniform levels in most, if not all, MM cells in all stages of the disease.10 Daratumumab has recently been administered to several patients at moderate to high doses and for prolonged periods with little or no toxicity, despite the fact that the CD38 molecule is also expressed, albeit at lower levels, on a fraction of hematopoietic cells, cerebellar Purkinje cells, liver and lung smooth muscle cells, and insulin-secreting β cells of pancreas.17 Our study was, therefore, designed to test the feasibility, potential efficacy and pitfalls of a CD38-based CART-cell approach for MM. To investigate the feasibility of generating CD38-CAR, we started the study using three distinct human CD38 antibodies, which showed similar binding affinities to CD38 as that of daratumumab (Online Supplementary Table S1). Based on successful usage of 4-1BB-containing CAR in recent studies2422 we constructed CAR containing 4-1BB (CD137) co-stimulatory and CD3ζ activating domains. Our results demonstrate the successful generation of CD38-CAR and CD38-CART cells regardless of the antibody sequences. T cells transduced with these CD38-CAR are highly proliferative, produce inflammatory Th1-like cytokines and, most importantly, are effective in killing malignant cells and normal hematopoietic cells in a CD38-dependent fashion, with some subtle differences between cell lines or hematopoietic cell types.
More importantly, CD38-CART cells appeared capable of eliminating primary CD38 MM cells from patients who had become resistant to various chemotherapies. This suggests that CD38-CAR therapy could be a viable choice for patients with few or no further chemotherapy options. These in vitro data were substantiated by the results obtained in our in vivo model. Although we did not observe the complete eradication of MM cells in our in vivo assays, we need to note that, since our CD38-CART cells appeared to lose their CD38 expression upon culture, we primarily designed our in vivo assays to determine the anti-tumor efficacy of these CD38, but long-term cultured CD38-CART cells. This may have negatively influenced the anti-tumor efficacy, since it is known that long-term cultured T cells rapidly lose their in vivo persistence capacities.2625 In addition, and perhaps even more importantly, in our model, unlike all previously reported CAR studies, the human MM tumors were grown to larger masses in a fully humanized bone marrow microenvironment. The MM microenvironment is known to provide essential signals for survival, growth and, more importantly, immune resistance of MM cells.28271211 Since our model includes some of the microenvironment-related aspects, our results suggest that the efficacy of CART-cell treatment could be improved if the therapy were to be combined with immune checkpoint inhibitors and/or with survivin and/or MCL-1 inhibitors which are effective modifiers of cell adhesion-mediated immune resistance induced by the tumor microenvironment.11
Unlike a number of earlier reports, which mainly focused on the anti-tumor efficacy of CD38-CART cells,3129 we devoted a considerable part of our investigation to identifying the potential drawbacks and risks of CD38-CART-cell therapy. Although CD38-CART cells eliminated the CD38 fractions of immune cell subsets as well as the CD38 fraction of hematopoietic progenitor cells, we observed no inhibition of the outgrowth of hematopoietic lineages from CD34CD38 progenitor cells. Furthermore, CD38-CART cells did not induce complete depletion of mature hematopoietic cells in the periphery. The CD38-fractions of important immune cells, such as B and T cells, were also unaffected. These results suggest that the therapy will spare sufficient numbers of T and B cells for these to maintain their functions. However, since CD38 is a well-known T-cell activation molecule, and has also been implicated in chemotaxis,32 T-cell development,33 dendritic cell trafficking and humoral immune responses,34 it would be relevant to determine whether an intact immune response would be possible in the absence of CD38. A partial solution to this issue came from the analyses of CD38-CART cells: remarkably, we discovered that the CD38-CART cells, regardless of which single chain variant fragment was used, became completely devoid of CD38 expression on their surface in various independently generated batches of cells. The loss of CD38 was thus unlikely to be caused by a genetic defect, but was most probably due to the “self lysis” of the CD38 fractions, which was also described in another CD38-CAR study.29 Our CD38-CD38-CART cells, however, had no growth disadvantage, had a highly activated status, displayed CD38-dependent proliferation, cytokine production, and cytotoxic activities and showed no other detectable functional aberrancies. This was also the case for CD19-CART cells which became CD38 after co-culture with CD38-CART cells (Online Supplementary Figure S2). Furthermore CD38-CART cells did not show any defects in transmigration assays and they also mediated significant anti-MM effects in vivo, thus indicating their capacity to migrate properly and infiltrate into the MM niches and to kill them. Thus, it seems likely that: (i) not all activated T cells have to be CD38, and (ii) CD38 expression is not essential for T cells to fulfill their functions. This latter conclusion is also supported by the fact that there is still no evidence, even from CD38 knockout mice,32 that CD38-deficient effector T cells are functionally defective.
On the other hand, the relatively broad expression of the target antigen of CD38-CART cells increases the risk of the so-called “cytokine release syndrome” due to massive activation of CART cells, as has been observed in previous trials with ERBB2- and CD19-CART cells.3735 Although the interleukin-6 receptor antagonist tocilizumab appears to reduce cytokine release syndrome38 it would still be desirable to minimize the occurrence of such severe side effects. Furthermore, since we cannot rule out toxicities occurring due to the possible attack of non-hematopoietic CD38 cells, development of an optimal CD38-CART-cell therapy would require the improvement of the target-specificity as well as the in vivo control of CD38-CART cells, and probably also in the case of other CART-cell approaches targeting the kappa light chain,39 CD138,40 Lewis Y antigen,41 BCMA,42 CS1,4443 and CD44v6.45 One future option to improve the target-specificity could be optimization of the target cell affinity of CART cells. In addition, suicide genes may enable the in vivo control of adoptively transferred CART cells. Indeed, in our first attempt to improve the safety profile of CD38-CART cells we observed that the iCasp9 gene4620 can effectively control CART cells. These results, which are in agreement with those of other studies,474520 provide positive prospects for future clinical trials. The safety profile of CART cells could also be improved by the generation of inducible CAR constructs or using the recently developed dual CAR technologies.
Considering all the data together, we conclude that CD38-CART cells are powerful immunotherapeutic tools and can be beneficial, especially for MM patients who have no other chemotherapy options. These results warrant further studies aimed at diminishing the undesired effects of CD38-CART cells against normal CD38 cells through optimizing the formers’ CD38 affinity and improving in vivo controllability.
We thank Dr. C. June for providing the sequence for the 4-1BB-CD3ζ transgene, Dr. D. Spencer for the inducible caspase-9 plasmid (15567), Dr. M. Sadelain for providing viral supernatant for CD19-CAR, Drs G.J. Ossenkoppele, A.A. van de Loosdrecht and S. Zweegman for critically reading the manuscript and suggestions, R. de Jong-Korlaar, M. Emmelot and L. Lubbers for technical assistance with in vivo experiments. The RAG2γc mice used in this study were originally obtained from the Amsterdam Medical Center, Amsterdam, the Netherlands.
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/5/616
- Received October 6, 2015.
- Accepted February 3, 2016.
- Kyle RA, Rajkumar SV. Multiple myeloma. N Engl J Med. 2004; 351(18):1860-1873. PubMedhttps://doi.org/10.1056/NEJMra041875Google Scholar
- Kumar SK, Lee JH, Lahuerta JJ. Risk of progression and survival in multiple myeloma relapsing after therapy with IMiDs and bortezomib: a multicenter International Myeloma Working Group study. Leukemia. 2012; 26(1):149-157. PubMedhttps://doi.org/10.1038/leu.2011.196Google Scholar
- Kröger N, Damon L, Zander AR. Secondary acute leukemia following mitoxantrone-based high-dose chemotherapy for primary breast cancer patients. Bone Marrow Transplant. 2003; 32(12):1153-1157. PubMedhttps://doi.org/10.1038/sj.bmt.1704291Google Scholar
- Bensinger WI, Buckner CD, Anasetti C. Allogeneic marrow transplantation for multiple myeloma: an analysis of risk factors on outcome. Blood. 1996; 88(7):2787-2793. PubMedGoogle Scholar
- Kröger N, Miyamura K, Bishop MR. Minimal residual disease following allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant. 2011; 17(1 Suppl):S94-100. PubMedhttps://doi.org/10.1016/j.bbmt.2010.10.031Google Scholar
- Lokhorst HM, Schattenberg A, Cornelissen JJ. Donor lymphocyte infusions for relapsed multiple myeloma after allogeneic stem-cell transplantation: predictive factors for response and long-term outcome. J Clin Oncol. 2000; 18(16):3031-3037. PubMedGoogle Scholar
- Brentjens RJ, Davila ML, Riviere I. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013; 5(177):177ra38. PubMedhttps://doi.org/10.1126/scitranslmed.3005930Google Scholar
- Grupp SA, Kalos M, Barrett D. Chimeric antigen receptor–modified T cells for acute lymphoid leukemia. N Engl J Med. 2013; 368(16):1509-1518. PubMedhttps://doi.org/10.1056/NEJMoa1215134Google Scholar
- Garfall AL, Fraietta JA, Maus MV. Immunotherapy with chimeric antigen receptors for multiple myeloma. Discov Med. 2014; 17(91):37-46. PubMedGoogle Scholar
- Lokhorst HM, Plesner T, Laubach JP. Targeting CD38 with daratumumab monotherapy in multiple myeloma. N Engl J Med. 2015; 373(13):1207-1219. PubMedhttps://doi.org/10.1056/NEJMoa1506348Google Scholar
- De Haart SJ, van de Donk NWCJ, Minnema MC. Accessory cells of the microenvironment protect multiple myeloma from T-cell cytotoxicity through cell adhesion-mediated immune resistance. Clin Cancer Res. 2013; 19(20):5591-5601. PubMedhttps://doi.org/10.1158/1078-0432.CCR-12-3676Google Scholar
- Groen RWJ, Noort WA, Raymakers RA. Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma. Blood. 2012; 120(3):e9-e16. PubMedhttps://doi.org/10.1182/blood-2012-03-414920Google Scholar
- Zheng Z, Chinnasamy N, Morgan RA. Protein L: a novel reagent for the detection of chimeric antigen receptor (CAR) expression by flow cytometry. J Transl Med. 2012; 10(1):29. PubMedhttps://doi.org/10.1186/1479-5876-10-29Google Scholar
- McMillin DW, Delmore J, Weisberg E. Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity. Nat Med. 2010; 16(4):483-489. PubMedhttps://doi.org/10.1038/nm.2112Google Scholar
- Van der Veer MS, de Weers M, van Kessel B. Towards effective immunotherapy of myeloma enhanced elimination of myeloma cells by combination of lenalidomide with the human CD38 monoclonal antibody daratumumab. Haematologica. 2011; 96(2):284-290. PubMedhttps://doi.org/10.3324/haematol.2010.030759Google Scholar
- Spaapen RM, Groen RWJ, van den Oudenalder K. Eradication of medullary multiple myeloma by CD4+ cytotoxic human T lymphocytes directed at a single minor histocompatibility antigen. Clin Cancer Res. 2010; 16(22):5481-5488. PubMedhttps://doi.org/10.1158/1078-0432.CCR-10-1340Google Scholar
- Malavasi F, Deaglio S, Funaro A. Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology. Physiol Rev. 2008; 88(3):841-886. PubMedhttps://doi.org/10.1152/physrev.00035.2007Google Scholar
- Mutis T, Schrama E, van Luxemburg-Heijs SA. HLA class II restricted T-cell reactivity to a developmentally regulated antigen shared by leukemic cells and CD34+ early progenitor cells. Blood. 1997; 90(3):1083-1090. PubMedGoogle Scholar
- Miller CL, Lai B. Human and mouse hematopoietic colony-forming cell assays. Methods Mol Biol. 2005; 290:71-89. PubMedGoogle Scholar
- Straathof KC, Pulè MA, Yotnda P. An inducible caspase 9 safety switch for T-cell therapy. Blood. 2005; 105(11):4247-4254. PubMedhttps://doi.org/10.1182/blood-2004-11-4564Google Scholar
- Tey S-K, Dotti G, Rooney Cliona M. Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation. Biol Blood Marrow Transplant. 2007; 13(8):913-924. PubMedhttps://doi.org/10.1016/j.bbmt.2007.04.005Google Scholar
- Kalos M, Levine BL, Porter DL. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med. 2011; 3(95):95ra73. PubMedhttps://doi.org/10.1126/scitranslmed.3002842Google Scholar
- Frigault MJ, Lee J, Basil MC. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells. Cancer Immunol Res. 2015; 3(4):356-367. PubMedhttps://doi.org/10.1158/2326-6066.CIR-14-0186Google Scholar
- Imai C, Mihara K, Andreansky M. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia. 2004; 18(4):676-684. PubMedhttps://doi.org/10.1038/sj.leu.2403302Google Scholar
- Spaulding C, Guo W, Effros RB. Resistance to apoptosis in human CD8+ T cells that reach replicative senescence after multiple rounds of antigen-specific proliferation. Exp Gerontol. 1999; 34(5):633-644. PubMedhttps://doi.org/10.1016/S0531-5565(99)00033-9Google Scholar
- Akbar AN, Henson SM. Are senescence and exhaustion intertwined or unrelated processes that compromise immunity?. Nat Rev Immunol. 2011; 11(4):289-295. PubMedhttps://doi.org/10.1038/nri2959Google Scholar
- Mitsiades CS, Mitsiades NS, Richardson PG, Munshi NC, Anderson KC. Multiple myeloma: A prototypic disease model for the characterization and therapeutic targeting of interactions between tumor cells and their local microenvironment. J Cell Biochem. 2007; 101(4):950-968. PubMedhttps://doi.org/10.1002/jcb.21213Google Scholar
- Meads MB, Hazlehurst LA, Dalton WS. The bone marrow microenvironment as a tumor sanctuary and contributor to drug resistance. Clin Cancer Res. 2008; 14(9):2519-2526. PubMedhttps://doi.org/10.1158/1078-0432.CCR-07-2223Google Scholar
- Mihara K, Yanagihara K, Takigahira M. Activated T-cell-mediated immunotherapy with a chimeric receptor against CD38 in B-cell non-Hodgkin lymphoma. J Immnotherapy. 2009; 32(7):737-743. https://doi.org/10.1097/CJI.0b013e3181adaff1Google Scholar
- Bhattacharyya J, Mihara K, Kitanaka A. T-cell immunotherapy with a chimeric receptor against CD38 is effective in eradicating chemotherapy-resistant B-cell lymphoma cells overexpressing survivin induced by BMI-1. Blood Cancer J. 2012; 2(6):e75. PubMedhttps://doi.org/10.1038/bcj.2012.21Google Scholar
- Mihara K, Bhattacharyya J, Kitanaka A. T-cell immunotherapy with a chimeric receptor against CD38 is effective in eliminating myeloma cells. Leukemia. 2012; 32(7):737-743. Google Scholar
- Partida-Sánchez S, Cockayne DA, Monard S. Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo. Nat Med. 2001; 7(11):1209-1216. PubMedhttps://doi.org/10.1038/nm1101-1209Google Scholar
- Bean AG, Godfrey DI, Ferlin WG. CD38 expression on mouse T cells: CD38 defines functionally distinct subsets of alpha beta TCR+CD4-CD8-thymocytes. Int Immunol. 1995; 7(2):213-221. PubMedhttps://doi.org/10.1093/intimm/7.2.213Google Scholar
- Partida-Sánchez S, Goodrich S, Kusser K. Regulation of dendritic cell trafficking by the ADP-ribosyl cyclase CD38: impact on the development of humoral immunity. Immunity. 2004; 20(3):279-291. PubMedhttps://doi.org/10.1016/S1074-7613(04)00048-2Google Scholar
- Morgan RA, Yang JC, Kitano M. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther. 2010; 18(4):843-851. PubMedhttps://doi.org/10.1038/mt.2010.24Google Scholar
- Kochenderfer JN, Dudley ME, Feldman SA. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012; 119(12):2709-2720. PubMedhttps://doi.org/10.1182/blood-2011-10-384388Google Scholar
- Casucci M, Hawkins RE, Dotti G, Bondanza A. Overcoming the toxicity hurdles of genetically targeted T cells. Cancer Immunol Immunother. 2015; 64(1):123-130. PubMedhttps://doi.org/10.1007/s00262-014-1641-9Google Scholar
- Maude SL, Barrett D, Teachey DT, Grupp SA. Managing cytokine release syndrome associated with novel T cell-engaging therapies. Cancer J. 2014; 20(2):119-122. PubMedhttps://doi.org/10.1097/PPO.0000000000000035Google Scholar
- Vera J, Savoldo B, Vigouroux S. T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells. Blood. 2006; 108(12):3890-3897. PubMedhttps://doi.org/10.1182/blood-2006-04-017061Google Scholar
- Jiang H, Zhang W, Shang P. Transfection of chimeric anti-CD138 gene enhances natural killer cell activation and killing of multiple myeloma cells. Mol Oncol. 2014; 8(2):297-310. PubMedhttps://doi.org/10.1016/j.molonc.2013.12.001Google Scholar
- Peinert S, Prince HM, Guru PM. Gene-modified T cells as immunotherapy for multiple myeloma and acute myeloid leukemia expressing the Lewis Y antigen. Gene Ther. 2010; 17(5):678-686. PubMedhttps://doi.org/10.1038/gt.2010.21Google Scholar
- Carpenter RO, Evbuomwan MO, Pittaluga S. B-cell maturation antigen is a promising target for adoptive T-cell therapy of multiple myeloma. Clin Cancer Res. 2013; 19(8):2048-2060. PubMedhttps://doi.org/10.1158/1078-0432.CCR-12-2422Google Scholar
- Chu J, Deng Y, Benson DM. CS1-specific chimeric antigen receptor (CAR)-engineered natural killer cells enhance in vitro and in vivo antitumor activity against human multiple myeloma. Leukemia. 2014; 28(4):917-927. PubMedhttps://doi.org/10.1038/leu.2013.279Google Scholar
- Chu J, He S, Deng Y. Genetic modification of T cells redirected toward CS1 enhances eradication of myeloma cells. Clin Cancer Res. 2014; 20(15):3989-4000. PubMedhttps://doi.org/10.1158/1078-0432.CCR-13-2510Google Scholar
- Casucci M, Nicolis di Robilant B, Falcone L. CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma. Blood. 2013; 122(20):3461-3472. PubMedhttps://doi.org/10.1182/blood-2013-04-493361Google Scholar
- Hoyos V, Savoldo B, Quintarelli C. Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia. 2010; 24(6):1160-1170. PubMedhttps://doi.org/10.1038/leu.2010.75Google Scholar
- Budde LE, Berger C, Lin Y. Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma. PLoS One. 2013; 8(12):e82742. PubMedhttps://doi.org/10.1371/journal.pone.0082742Google Scholar