CD123 CAR T cells for the treatment of myelodysplastic syndrome

Abstract

•Autologous T cells derived from MDS patients can be successfully transduced and are highly active in multiple functional assays.•A xenograft model was developed in which high-risk MDS specimens engraft to high levels and thus provide an in vivo model for characterization of MDS stem cell behavior.•Autologous T cells are highly active in our xenograft model and selectively target the CD123-positive population. Importantly, CD123 CAR T cells did not exhibit appreciable targeting of normal hematopoietic stem and progenitor cells. Myelodysplastic syndrome (MDS) is a group of heterogeneous disorders caused by ineffective hematopoiesis and characterized by bone marrow dysplasia and cytopenia. Current treatment options for MDS are limited to supportive care, hypomethylating agents, and stem cell transplant. Most patients eventually succumb to the disease or progress to leukemia. Previously, we found that CD123 can be used to delineate MDS stem cells in patients at high risk for MDS and that the CD123-positive population is biologically distinct from normal hematopoietic stem cells. Furthermore, selective targeting of MDS stem cells can dramatically reduce tumor burden in preclinical models. On the basis of these findings, we propose CD123 as a candidate target for chimeric antigen receptor (CAR) T-cell therapy in high-risk MDS patients. To test this concept, we employed a CAR lentiviral vector containing a CD123-specific single-chain variable fragment in combination with the CD28 costimulatory domain, CD3ζ signaling domain, and truncated epidermal growth factor. Utilizing this system, we illustrate that CD123 CAR can be expressed on both healthy donor and MDS patient-derived T lymphocytes with high efficiency, leading to the successful elimination of MDS stem cells both in vitro and in patient-derived xenografts. These results provide the concept for the use of CD123-targeted CAR T cells as a therapeutic option for patients with MDS. Myelodysplastic syndrome (MDS) is a group of heterogeneous disorders caused by ineffective hematopoiesis and characterized by bone marrow dysplasia and cytopenia. Current treatment options for MDS are limited to supportive care, hypomethylating agents, and stem cell transplant. Most patients eventually succumb to the disease or progress to leukemia. Previously, we found that CD123 can be used to delineate MDS stem cells in patients at high risk for MDS and that the CD123-positive population is biologically distinct from normal hematopoietic stem cells. Furthermore, selective targeting of MDS stem cells can dramatically reduce tumor burden in preclinical models. On the basis of these findings, we propose CD123 as a candidate target for chimeric antigen receptor (CAR) T-cell therapy in high-risk MDS patients. To test this concept, we employed a CAR lentiviral vector containing a CD123-specific single-chain variable fragment in combination with the CD28 costimulatory domain, CD3ζ signaling domain, and truncated epidermal growth factor. Utilizing this system, we illustrate that CD123 CAR can be expressed on both healthy donor and MDS patient-derived T lymphocytes with high efficiency, leading to the successful elimination of MDS stem cells both in vitro and in patient-derived xenografts. These results provide the concept for the use of CD123-targeted CAR T cells as a therapeutic option for patients with MDS. Myelodysplastic syndrome (MDS) is a hematological disorder resulting from stem cell–driven clonal growth of pathological hematopoietic progenitors and ineffective hematopoiesis [1Heaney M DW DGMyelodysplasia.N Engl J Med. 1999; 340: 1649-1659Crossref PubMed Scopus (428) Google Scholar]. The annual incidence of MDS is in excess of 20 per 100,000 people [2Rollison D Howlader N Smith M et al.Epidemiology of myelodysplastic syndromes and chronic myeloproliferative disorders in the United States, 2001–2004, using data from the NAACCR and SEER programs.Blood. 2008; 112: 452-459Crossref Scopus (523) Google Scholar]. The pathological progression of MDS is described with each distinct stage of disease evolution and characterized by increasingly aberrant biological features. The International Prognostic Scoring System (IPSS-R) is utilized to define life expectancy and leukemic progression [3Greenberg PL Tuechler H Schanz J et al.Revised International Prognostic Scoring System for myelodysplastic syndromes.Blood. 2012; 120: 12Crossref Scopus (1997) Google Scholar, 4Voso MT Fenu S Latagliata R et al.Revised International Prognostic Scoring System (IPSS) predicts survival and leukemic evolution of myelodysplastic syndromes significantly better than IPSS and WHO Prognostic Scoring System: validation by the Gruppo Romano Mielodisplasie Italian Regional Database.J Clin Oncol. 2013; 31: 2671-2677Crossref PubMed Scopus (111) Google Scholar]. It incorporates degree of pancytopenia, cytogenetic abnormalities, and number of blasts. Early stage, also known as very low-risk and low-risk MDS, is characterized by low IPSS-R scores. Intermediate-, high-, and very high-risk MDS patients have high IPSS-R scores with profound pancytopenia, unfavorable cytogenetic abnormalities, and increased blast count. Approximately 25% of high- and very high-risk patients will progress to acute myeloid leukemia (AML) within a year [5Germing U Hildebrandt B Pfeilstöcker M et al.Refinement of the International Prognostic Scoring System (IPSS) by including LDH as an additional prognostic variable to improve risk assessment in patients with primary myelodysplastic syndromes (MDS).Leukemia. 2005; 19: 2223-2231Crossref PubMed Scopus (137) Google Scholar]. Low-risk MDS patients are typically managed with supportive care. Upon acquisition of high-risk features, patients are typically treated with hypomethylating agents (azacitidine or decitabine), but response rates are only about 30% and resistance develops within 2 years. Thus, development of new and improved therapies is critical. Like most myeloid malignancies, MDS is thought to arise from mutations in early hematopoietic stem or progenitor cells. Thus, to investigate more effective MDS therapies, we have focused on properties inherent to malignant hematopoietic stem cells (HSCs). Notably, although malignant stem cells in more advanced diseases such as AML have received significant attention, MDS stem cells remain much less characterized [6Stevens BM Khan N D'Alessandro A et al.Characterization and targeting of malignant stem cells in patients with advanced myelodysplastic syndromes.Nat Commun. 2018; 9: 3694Crossref PubMed Scopus (45) Google Scholar, 7Chung SS Eng WS Hu W et al.CD99 is a therapeutic target on disease stem cells in myeloid malignancies.Sci Transl Med. 2017; 9 (eaaj2025)Crossref Scopus (75) Google Scholar, 8Barreyro L Will B Bartholdy B et al.Overexpression of IL-1 receptor accessory protein in stem and progenitor cells and outcome correlation in AML and MDS.Blood. 2012; 120: 1290-1298Crossref PubMed Scopus (130) Google Scholar]. This is likely due to the lack of good experimental models that adequately mimic human disease, as well as the relatively low abundance of tumor cells that can be isolated from patients. Nonetheless, defining therapies that effectively target MDS stem cells is of undeniable importance, and has been the focus of our efforts. We recently reported a detailed study of high-risk MDS patients in which specific molecular and cellular characteristics were described [6Stevens BM Khan N D'Alessandro A et al.Characterization and targeting of malignant stem cells in patients with advanced myelodysplastic syndromes.Nat Commun. 2018; 9: 3694Crossref PubMed Scopus (45) Google Scholar]. Of particular importance to the present study, we found that the cell surface antigen CD123 is upregulated on MDS stem cells. This is a notable observation because, although CD123 is expressed at low levels on normal HSCs, its expression was also found to be upregulated in AML, implying that the antigen is activated during pathogenesis [9Mardiros A Santos CD McDonald T et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; PubMed Scopus Google Scholar]. have described a of CD123 in and high-risk MDS R et cells in myelodysplastic syndrome malignant to in J PubMed Scopus Google Scholar]. illustrate that expression of CD123 with in the molecular of MDS stem which a in protein and in [6Stevens BM Khan N D'Alessandro A et al.Characterization and targeting of malignant stem cells in patients with advanced myelodysplastic syndromes.Nat Commun. 2018; 9: 3694Crossref PubMed Scopus (45) Google Scholar]. These a progression the of Thus, not only expression of CD123 provide a to and target MDS stem also a for the most stage of the On the basis of the we to therapies that target MDS cells expressing we to the cell of chimeric antigen receptor (CAR) T cells. are T and of target cells in a highly specific and This is an antigen that is typically derived from a in is to an signaling of a T-cell costimulatory dramatically the of CAR T cells S B and development of therapies using chimeric antigen T PubMed Scopus Google Scholar, B et and of chimeric antigen T cells in Clin PubMed Scopus Google Scholar, M M The of chimeric antigen PubMed Scopus Google Scholar, M R The of chimeric antigen receptor 2013; PubMed Scopus Google Scholar]. the present study, we a CD123 CAR of a single-chain variable fragment derived from a specific for CD123 [9Mardiros A Santos CD McDonald T et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; PubMed Scopus Google Scholar]. This fragment is to the human by the CD28 domain, CD3ζ signaling domain, and truncated epidermal growth [9Mardiros A Santos CD McDonald T et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; PubMed Scopus Google Scholar, et cell surface for in vivo and of PubMed Scopus Google Scholar]. CD123 CAR T cells have in AML in preclinical [9Mardiros A Santos CD McDonald T et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; PubMed Scopus Google and are in a to patients with AML and cell L et of acute myeloid leukemia and cell treatment with CD123-specific CAR T a 2017; Scholar]. the expression of CD123 in MDS stem we targeting CD123 as a for patients with high-risk MDS. CD123 CAR and CAR were described [9Mardiros A Santos CD McDonald T et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; PubMed Scopus Google Scholar]. CD123 and lentiviral were in cells. cells were to in with to on the of On the of the of lentiviral and CAR vector in 2 2 were to the was at for at a with a 25% of and at the was at at for and at at for 20 was and the was in of the with and at for The of the and in with the of T cells were from cells using T cells were from from our MDS T cells were from the using human cells were in with and and T cells were with and transduced with CD123 CAR or were from T and CD123 CAR expression in transduced T cells was by with L and To CAR T CD123 CAR T cells were with number of cells or bone marrow cells derived from MDS patients in with for at with cell in expression of and was by to the number of cells and CAR T cells in using the and primary MDS cell of MDS stem cells and CAR T cells were by with the and of each cell population was by the were at and with the To CAR T-cell response to target tumor expression and were in CAR T cells that were with target cells. of CD123 CAR T cells and target tumor cells of cells or MDS bone marrow were in with and for at with The cells were and with and as well as to with the cells were with for was using CD123 CAR T CAR T and T cells were from healthy donor cells and with cells in with for at an of were in cell to human and with per of and were with were with the System with an were by the of and To patient-derived xenograft each was with in of in to xenograft transplant. cells were in with to MDS bone marrow cells cells from MDS or cells from MDS per were used as of was in a that was with high-risk MDS bone marrow cells MDS cell were cells were by using and was as than in bone marrow cells and the of were with the and with were from and of CD123 expression were to the were using were used to significant treatment these we utilized a characterized CD123 CAR that has been reported to be effective in AML cells both in vitro and in vivo [9Mardiros A Santos CD McDonald T et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; PubMed Scopus Google Scholar]. CD123 CAR an that by a CD28 domain, CD3ζ signaling domain, and that as an to successfully transduced T cells. a we used an that [9Mardiros A Santos CD McDonald T et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; PubMed Scopus Google Scholar, et cell surface for in vivo and of PubMed Scopus Google the vector To CAR T we utilized the in cells were isolated from healthy donor by T-cell with and T cells were transduced in the of and the lentiviral vector to in and We of and to were of and were effectively transduced with CD123 and CAR and in healthy To the antitumor of CD123 CAR T we the of healthy CD123 CAR T cells against the cell S H et of receptor MDS cells and of by PubMed Scopus Google Scholar]. was derived from an MDS with a of cells the cells were effectively with CD123 CAR T cells for at an of was cells were with CAR T with T or T cells These results elimination of cells by CD123 CAR T cells. To the antitumor of CD123 CAR T of the was in CD123 CAR T CAR T cells and T cells with at an of or tumor cell for A significant and in cells was in response to the CD123 CAR T-cell with cells. This was not in CAR T cells or T cells an increased number of CD123 CAR T cells surface expression of a for of CD123 CAR T cells in the of cells the of CD123 CAR T cells in targeting cell we the of CD123 CAR T cells against primary MDS specimens We recently reported that CD123 is upregulated during MDS pathogenesis and a stage of disease evolution in which malignant stem cells multiple molecular properties with AML [6Stevens BM Khan N D'Alessandro A et al.Characterization and targeting of malignant stem cells in patients with advanced myelodysplastic syndromes.Nat Commun. 2018; 9: 3694Crossref PubMed Scopus (45) Google Scholar]. MDS stem cells are characterized as were using specimens from patients with high-risk MDS CD123 CAR T cells from healthy were with each for We elimination of primary MDS stem cells by the of MDS stem cells to treatment These data that CD123 CAR T cells exhibit and specific antitumor against cell and primary MDS of from patients with primary high-risk myelodysplastic of of of of of of in a new the of CD123 CAR T cells [9Mardiros A Santos CD McDonald T et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; PubMed Scopus Google the of targeting of cells be from the in and use of T cells significantly we to T cells derived from high-risk MDS T cells can also be used to effective CD123 CAR T cells to MDS cells. To our CAR T cells have been from MDS thus we to test the of T cells were derived from high-risk MDS patients to CD123 CAR T cells as described than was with patients and of and was with T cells from the and T-cell were transduced These are to in T cells from AML patients transduced with CD123 [9Mardiros A Santos CD McDonald T et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; PubMed Scopus Google MDS T cells successfully transduced to CD123 CAR and bone MDS stem cells in T cells from high-risk MDS patients and were transduced with CD123 CD123 CAR T cells or T cells were by was in and derived T cells. CD123 CAR T cells from patients and were for and and expressed CD123 of and in CAR T cells and T cells for The for and of cells in MDS bone marrow and MDS stem cells of patients and with CD123 CAR T cells or T cells for MDS cells from patients and were with T cells or CD123 CAR T cells for at an of MDS stem cells in bone marrow are significantly with CD123 CAR T but not T cells. to T-cell treatment as by cell surface is significantly in CD123 CAR T cells with T cells with an bone marrow at an of for in as by is significantly in CD123 CAR T cells than in T cells with an bone marrow in the T-cell population and both and at an for in patients 2 and of and in of MDS bone marrow with CD123 CAR T cells is significantly with that of T cells for at an of for patients 2 and and for at an of for to test the functional of CAR T we CD123 CAR and T cells with MDS bone marrow for at an of or We elimination of both tumor and MDS stem cells Notably, as in and for MDS stem cells and with cells at in in and in to T-cell T cells did not present significant against target cells with the bone group in of the of CD123 in cells were for A significant in the CD123 was treatment of CD123 CAR T cells in to T cells or T cells. The CD123 CAR T-cell cells CD123 for specimens expressing CD123 a of CD123 CAR T-cell at about CD123 at To the functional of CAR T we and of as by cell surface was in CD123 CAR T but not in T cell of in a with MDS bone marrow levels and of were also increased in both The in was found in both and a of human was from the of the CD123 CAR T cells were with MDS bone marrow for in and were in patient-derived CD123 CAR T-cell with T cells significant in was by these results that CD123 CAR T cells are effective at MDS stem cells in patient-derived specimens in To the of CD123 CAR T cells in the of a preclinical in vivo we a patient-derived xenograft to the of MDS stem cells. we reported that high-risk MDS specimens can successfully engraft the are with and T cells from the to [6Stevens BM Khan N D'Alessandro A et al.Characterization and targeting of malignant stem cells in patients with advanced myelodysplastic syndromes.Nat Commun. 2018; 9: 3694Crossref PubMed Scopus (45) Google Scholar]. this we of with primary MDS marrow derived from patients and Notably, both specimens were to engraft marrow with high as by the expression of human in to the of in the The from MDS of marrow cells at with of cells. of MDS cells and of progenitor cells were The from MDS a of at with 25% of cells expressing CD123 expression was in this with and cells in the and with the MDS CAR T cells were received CD123 CAR T cells or T cells of each CD123 CAR T cells or T cells for each T-cell of human cells in the and of human cells in bone marrow were CD123 CAR T cells as by the expression of the of MDS cells MDS progenitors and MDS stem cells a significantly MDS tumor burden CD123 CAR T-cell treatment The MDS tumor burden was by of bone marrow from MDS CD123 CAR T-cell The a in tumor burden were treated with CD123 CAR T cells with T cells. The of with the high-risk MDS an to that of as in and 2 were to MDS tumor cell and CAR T-cell in xenograft bone of of CAR T CAR T cells and in bone marrow and CD123 CAR T-cell 2 CD123 CAR T-cell was by the of MDS as by significant in the of tumor cells MDS progenitors and MDS stem cells the elimination of MDS cells in treated with CD123 CAR T but not T cells To the of CD123 CAR T-cell treatment of normal hematopoietic stem healthy CD123 CAR T CAR T cells and T cells were with cells for at an of were in human to with cells per of and were and were to CD123 CAR T cells did not have on cell This normal cells are not significantly by treatment with CD123 CAR T of CD123 and CAR T cells. of CD123 and CAR T-cell and of CD123 and healthy donor T cells or T cells from healthy donor and of in healthy are by the and for CD123 CAR T-cell MDS cells from were with T cells or CD123 CAR T cells for at an of MDS stem cells were by cells in bone marrow cells with CD123 CAR T T or T cells were for of of CD123 in cells with CD123 CAR T cells is significantly with that with T cells or T a of CD123 CAR T-cell at about CD123 in CD123 CAR T cells and T cells with MDS bone marrow CD123 CAR or T cells derived from patients 2 and were with MDS at an for for and by was increased in but not in the or the cells in CD123 CAR T cells with T cells derived from significant were found T-cell CD123 CAR T cells and T cells derived from of of CD123 CAR T cells in and bone marrow of to high-risk MDS and progression to acute leukemia a To MDS therapies have been limited to the use of hypomethylating agents, which are not and not progression to MDS is a disease of stem cell and the of good models to study MDS pathogenesis and malignant stem cell has also been a significant relatively have to define the properties of MDS stem cells derived from primary specimens SS Eng WS Hu W et al.CD99 is a therapeutic target on disease stem cells in myeloid malignancies.Sci Transl Med. 2017; 9 (eaaj2025)Crossref Scopus (75) Google Scholar, 8Barreyro L Will B Bartholdy B et al.Overexpression of IL-1 receptor accessory protein in stem and progenitor cells and outcome correlation in AML and MDS.Blood. 2012; 120: 1290-1298Crossref PubMed Scopus (130) Google Scholar, J et is for activated myelodysplastic syndrome stem Commun. PubMed Scopus Google Scholar, M J et in myelodysplastic syndromes and therapy response and PubMed Scopus Google Scholar, U et syndromes are by and distinct human stem cells in PubMed Scopus Google Scholar]. have reported that MDS stem cells can be including the expression of SS Eng WS Hu W et al.CD99 is a therapeutic target on disease stem cells in myeloid malignancies.Sci Transl Med. 2017; 9 (eaaj2025)Crossref Scopus (75) Google and L Will B Bartholdy B et al.Overexpression of IL-1 receptor accessory protein in stem and progenitor cells and outcome correlation in AML and MDS.Blood. 2012; 120: 1290-1298Crossref PubMed Scopus (130) Google on the surface and that these can be Furthermore, MDS stem cells have been found to exhibit and molecular properties including increased and signaling A et is a therapeutic target in acute myeloid leukemia and myelodysplastic PubMed Scopus Google Scholar, W et as a therapeutic against MDS and AML stem PubMed Scopus Google Scholar]. has described specific molecular that of MDS stem cell pathogenesis and of CD123 as a in the evolution of MDS stem cells [6Stevens BM Khan N D'Alessandro A et al.Characterization and targeting of malignant stem cells in patients with advanced myelodysplastic syndromes.Nat Commun. 2018; 9: 3694Crossref PubMed Scopus (45) Google Scholar]. CD123 has been the of in the AML of its selective expression on AML stem cells. Thus, multiple on targeting CD123 are [9Mardiros A Santos CD McDonald T et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; PubMed Scopus Google Scholar, L et targeting of receptor human acute myeloid leukemic stem PubMed Scopus Google Scholar, et al.Characterization of a for acute myeloid 2018; PubMed Scopus Google Scholar, D et receptor is a for human acute leukemia stem PubMed Scopus Google Scholar]. the present study, we utilized a CAR T-cell to target CAR T-cell therapy has in a high with CAR T with and acute leukemia with of J et in and with Engl J Med. 2018; PubMed Scopus Google and with J et antigen receptor T cells in Engl J Med. 2017; PubMed Scopus Google Scholar]. CAR T-cell therapy a better for patients with than to are of that have been with therapy and are as for therapy for patients with The in this study illustrate we found for the that T cells derived from MDS patients can be successfully transduced and are highly active in multiple functional Importantly, we effective of both and T cells from MDS we developed and characterized a xenograft model in which high-risk MDS specimens engraft to high levels and thus provide an in vivo model for characterization of MDS stem cell we found that T cells are highly active in our xenograft model and selectively target the CD123-positive population. CD123 CAR T cells did not exhibit appreciable targeting of normal hematopoietic stem and progenitor cells. at study reported to normal cells S M et targeting of human acute myeloid leukemia and using chimeric antigen T PubMed Scopus Google that the specific and of a CAR can be for a of that to be a of antigen for CAR T cells has been reported S et antigen the of chimeric antigen effector T PubMed Scopus Google is not that malignant cells can be from normal progenitors that CD123 expression is known to be on malignant [6Stevens BM Khan N D'Alessandro A et al.Characterization and targeting of malignant stem cells in patients with advanced myelodysplastic syndromes.Nat Commun. 2018; 9: 3694Crossref PubMed Scopus (45) Google Scholar, L et targeting of receptor human acute myeloid leukemic stem PubMed Scopus Google Scholar, D et receptor is a for human acute leukemia stem PubMed Scopus Google Scholar, T J et cell human acute myeloid PubMed Scopus Google Scholar]. our we cells. we found that CD123 CAR T cells are to target cells with CD123 at about or This is with from and likely the survival of healthy progenitors the of CAR T at the of Scholar]. It is to that although CD123-positive MDS stem cells reduce disease burden and that CD123 MDS stem cells et reported a in CD123 expression as diseases from to to high-risk R et cells in myelodysplastic syndrome malignant to in J PubMed Scopus Google that MDS stem cells in low-risk disease will not be by CD123 CAR T cells. the that most MDS patients are advanced in the risk of progression to AML and with high-risk MDS have highly significant The preclinical provide a for CD123 CAR T cells for the treatment of high-risk MDS patients. of CD123 a target antigen for therapy and also as an to the of CAR T Furthermore, as a less acute disease than AML, the of MDS a in which for CAR T use be