Blocking CD38-driven fratricide among T cells enables effective antitumor activity by CD38-specific chimeric antigen receptor T cells

Chimeric antigen receptor T-cell(CAR T) therapy is a kind of effective cancer immunotherapy. However,designing CARs remains a challenge because many targetable antigens are shared by T cells and tumor cells. This shared expression of antigens can cause CAR T cell fratricide. CD38-targeting approaches(e.g.,daratumumab) have been used in clinical therapy and have shown promising results. CD38 is a kind of surface glycoprotein present in a variety of cells, such as T lymphocytes and tumor cells. It was previously reported that CD38-based CAR T cells may undergo apoptosis or T cell-mediated killing(fratricide) during cell manufacturing. In this study, a CAR containing a sequence targeting human CD38 was designed to be functional. To avoid fratricide driven by CD38 and ensure the production of CAR T cells, two distinct strategies based on antibodies(clone MM12 T or clone MM27) or proteins(H02 H or H08 H) were used to block CD38 or the CAR single-chain variable fragment(scFv) domain, respectively, on the T cell surface.The results indicated that the antibodies or proteins, especially the antibody MM27, could affect CAR T cells by inhibiting fratricide while promoting expansion and enrichment. Anti-CD38 CAR T cells exhibited robust and specific cytotoxicity to CD38~+ cell lines and tumor cells. Furthermore, the levels of the proinflammatory factors TNF-a, IFN-g and IL-2 were significantly upregulated in the supernatants of A549~(CD38~+) cells. Finally, significant control of disease progression was demonstrated in xenograft mouse models. In conclusion, these findings will help to further enhance the expansion, persistence and function of anti-CD38 CAR T cells in subsequent clinical trials.     

The long road to the first FDA-approved gene therapy: chimeric antigen receptor T cells targeting CD19

Thirty years after initial publications of the concept of a chimeric antigen receptor (CAR), the U.S. Food and Drug Administration (FDA) approved the first anti-CD19 CAR T-cell therapy. Unlike other immunotherapies, such as immune checkpoint inhibitors and bispecific antibodies, CAR T cells are unique as they are “living drugs,” that is, gene-edited killer cells that can recognize and kill cancer. During these 30 years of development, the CAR construct, T-cell manufacturing process, and clinical patient management have gone through rounds of failures and successes that drove continuous improvement. Tisagenlecleucel was the first gene therapy to receive approval from the FDA for any indication. The initial approval was for relapsed or refractory (r/r) pediatric and young-adult B-cell acute lymphoblastic leukemia in August 2017 and in May 2018 for adult r/r diffuse large B-cell lymphoma. Here we review the preclinical and clinical development of what began as CART19 at the University of Pennsylvania and later developed into tisagenlecleucel.     

The clinical outcomes of fresh versus cryopreserved CD19-directed chimeric antigen receptor T cells in non-Hodgkin lymphoma patients

CD19-directed chimeric antigen receptor T (CAR-T) cells have been widely reported in the therapy of relapsed/refractory non-Hodgkin lymphoma (NHL). Both cryopreserved and fresh formulations of CAR-T have been used in previous studies. However, quite a few studies investigated the effects of cryopreservation on the clinical outcomes of CAR-T cells. Here we retrospectively analyzed a phase I/II clinical trial of CD19-directed CAR-T cells in NHL patients, and compared the safety and efficacy of cryopreserved and fresh CAR-T products. All CAR-T cells were prepared using the same manufacturing process except the formulation step. Fifteen patients were infused with cryopreserved/thawed CAR-T cells, and 8 patients were treated with fresh CAR-T cells. Comparative overall response rates and in vivo expansion kinetics of CAR-T cells were observed between the cryopreserved cohort and fresh cohort. The occurrence rates of cytokine release syndrome and neurotoxicity were also similar in both groups. Patients in the fresh cohort showed higher incidence of acute hematological toxicity including anemia, hypoleukemia, and thrombocytopenia. This study demonstrated that cryopreservation showed negligible effects on the efficacy of CD19-directed CAR-T cells, but endowed CAR-T cells with higher safety in NHL patients, supporting the application of cryopreserved CAR-T products for NHL therapy.     

Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells

BackgroundChimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study.MethodsA third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests.ResultsUp to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process.DiscussionCryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.     

Prostate cancer cells show elevated urokinase receptor in a mouse model of metastasis

Background  

The urokinase receptor (uPAR) governs several functions necessary during invasion and metastasis such as motility, degradation of the extracellular matrix and adhesion. This receptor has been recently associated with clinical prostate cancer progression. Experimentally, inhibition of uPAR reduces colonization of extra-prostatic sites in animal models. Our objective in this study was to compare uPAR expression in orthotopic vs. metastatic foci in vivo and to examine at the cellular level how uPAR might promote early stages of metastasis.     

Bispecific targeting of CD20 and CD19 increases polyfunctionality of chimeric antigen receptor T-cell products in B-cell malignancies

Background aimsSelective immune pressure contributes to relapse due to target antigen downregulation in patients treated with anti-CD19 chimeric antigen receptor (CAR) T cells. Bispecific lentiviral anti-CD20/anti-CD19 (LV20.19) CAR T cells may prevent progression/relapse due to antigen escape. Highly polyfunctional T cells within a CAR T-cell product have been associated with response in single-antigen-targeted anti-CD19 CAR T cells.MethodsThe authors performed a single-cell proteomic analysis to assess polyfunctional cells in our LV20.19 CAR T-cell product. Analysis was limited to those treated at a fixed dose of 2.5 × 10⁶ cells/kg (n = 16). Unused pre-infusion CAR T cells were thawed, sorted into CD4/CD8 subsets and stimulated with K562 cells transduced to express CD19 or CD20. Single-cell production of 32 individual analytes was measured and polyfunctionality and polyfunctional strength index (PSI) were calculated.ResultsFifteen patients had adequate leftover cells for analysis upon stimulation with CD19, and nine patients had adequate leftover cells for analysis upon stimulation with CD20. For LV20.19 CAR T cells, PSI was 866–1109 and polyfunctionality was 40–45%, which were higher than previously reported values for other CAR T-cell products.ConclusionsStimulation with either CD19 or CD20 antigens resulted in similar levels of analyte activation, suggesting that this product may have efficacy in CD19– patient populations.     

Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center

Background aims

Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients.

Developing lisocabtagene maraleucel chimeric antigen receptor T-cell manufacturing for improved process,product quality and consistency across CD19+ hematologic indications

Background aimsAutologous chimeric antigen receptor (CAR) T-cell therapies have demonstrated substantial clinical benefit across several hematologic malignancies. However, patient-to-patient variability and heterogeneity of starting cellular material across patient populations and disease indications pose challenges to manufacturing consistency. Lisocabtagene maraleucel (liso-cel) is an autologous, CD19-directed, defined-composition, 4-1BB CAR T-cell product administered at equal target doses of CD8⁺ and CD4⁺ CAR⁺ T cells. Here the authors describe the optimization of the liso-cel manufacturing platform for product quality and consistency.MethodsLeukapheresis starting materials were collected from patients with large B-cell lymphoma, mantle cell lymphoma or chronic lymphocytic leukemia treated with liso-cel in clinical trials (NCT02631044 and NCT03331198). The liso-cel manufacturing process involves selection of CD8⁺ and CD4⁺ T cells from leukapheresis material followed by independent CD8⁺ and CD4⁺ T-cell activation, transduction, expansion, formulation and cryopreservation. Multivariate design of experimental approaches was utilized to optimize process conditions at both specific unit operations and across the process. Flow cytometry methods were used to assess cellular composition, memory phenotypes and cell proliferation. Antigen-specific functions, including cytokine secretion, cytolytic activity and proliferation, were assessed using endpoint assays after independent stimulation of CD8⁺ and CD4⁺ CAR⁺ T-cell product components.ResultsReductions in process duration time, optimization of drug product container and formulation and activation signal optimization led to significantly increased CAR⁺ T-cell product viability. The heterogeneity of patient-derived starting material, including low absolute lymphocyte counts in some samples, was reduced through early T-cell purification, leading to median T-cell frequencies >95% in selected materials across disease indications and limited non-T-cell impurities. These changes further increased lineage purity in CD8⁺ and CD4⁺ CAR⁺ T-cell drug products. CD8⁺ and CD4⁺ CAR⁺ T-cell component lot functional profiles demonstrated multifunctional mechanisms of action, including differential cytokine release, differential cytolytic kinetics and high frequencies of proliferating cells. Correlative analyses demonstrated strong underlying associations between starting material attributes and final CAR⁺ T-cell product phenotype.ConclusionsDespite substantial heterogeneity of starting leukapheresis material quality/composition between individual patients and across disease indications/histologies, the liso-cel manufacturing platform is robust and capable of generating a consistent drug product from diverse starting materials with a single manufacturing platform.     

Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA⁺ cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.     

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.     

Combined 4-1BB and ICOS co-stimulation improves anti-tumor efficacy and persistence of dual anti-CD19/CD20 chimeric antigen receptor T cells

Chimeric antigen receptor (CAR) T-cell therapy is a promising therapeutic strategy against lymphoma. However, post-treatment relapses due to antigen loss remain a challenge. Here the authors designed a novel bicistronic CAR construct and tested its functions in vitro and in vivo. The CAR construct consisted of individual anti-CD19 and anti-CD20 single-chain fragment variables equipped with ICOS-CD3ζ and 4-1BB-CD3ζ intracellular domains, respectively. The CD19 and CD20 bicistronic CAR T cells exhibited tumor lytic capacities equivalent to corresponding monospecific CAR T cells. Moreover, when stimulated with CD19 and CD20 simultaneously, the bicistronic CAR T cells showed prolonged persistence and enhanced cytokine generation compared with single stimulations. Interestingly, the authors found that the 4-1BB signal was predominant in the signaling profiles of ICOS and 4-1BB doubly activated CAR T cells. In vivo study using a CD19/CD20 double-positive tumor model revealed that the bicistronic CAR T cells were more efficient than monospecific CD19 CAR T cells in eradicating tumors and prolonging mouse survival. The authors’ novel bicistronic CD19/CD20 CAR T cells demonstrate improved anti-tumor efficacy in response to dual antigen stimulations. These data provide optimism that this novel bicistronic CAR construct can improve treatment outcomes in patients with relapsed/refractory B cell malignancy.     

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcγRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3ζ chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Construction and functional characterization of a fully human anti-CD19 chimeric antigen receptor (huCAR)-expressing primary human T cells

Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti-CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti-mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti-mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti-CD19 CAR T cells—thereby reducing the risk of tumor relapse—but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second-generation fully human anti-CD19 CAR (or huCAR19) containing a fully human single-chain variable fragment (ScFv) fused with a CD8a hinge, a 4-1BB transmembrane domain and intracellular T cell signaling domains of 4-1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19⁺ target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen-independent tonic signaling in the absence of cognate antigen. Considering our results, our anti-CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse-derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice.     

CCR7 expression in CD19 chimeric antigen receptor-engineered natural killer cells improves migration toward CCL19-expressing lymphoma cells and increases tumor control in mice with human lymphoma

Background aimsChimeric antigen receptor (CAR) T-cell therapy can be associated with significant toxicities. CAR-engineered natural killer (NK) cells provide a safer alternative while maintaining anti-tumor effects. Activated NK (aNK) cells are a clinical-grade cellular product obtained from the NK-92 cell line that have demonstrated both safety and potent cytotoxicity toward a wide range of cancers in phase 1 trials. Genetically engineered variants of aNK cells expressing a high-affinity Fc receptor (haNK) or co-expressing a CAR (t-haNK) are currently in phase 1/2 clinical trials. A key factor in the efficacy of cellular immunotherapies is biodistribution and tumor infiltration, which affect the local effector:target ratio. The chemokines CCL19 and CCL21 can drive recruitment of CCR7 receptor-expressing immune cells to secondary lymphoid organs.MethodsSince NK-92 cells do not spontaneously express CCR7, clinical-grade aNK cells were transfected with a non-viral vector containing the CCR7 receptor, an anti-CD19 CAR and a high-affinity CD16 Fc receptor.ResultsCCR7-engineered CD19 t-haNK showed significant migration in vitro toward K562 cells engineered to secrete CCL19. This observation was confirmed in a NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mouse model in which subcutaneous tumors of CCL19-expressing K562 cells displayed a higher number of infiltrating CCR7_CD19 t-haNK cells than CCR7-negative CD19 t-haNK cells. In NSG mice inoculated either intravenously or subcutaneously with CCL19-secreting Raji cells, treatment with CCR7_CD19 t-haNK improved survival and tumor control compared with CD19 t-haNK or vehicle.ConclusionsExpression of CCR7 receptor by off-the-shelf t-haNK cells improves their homing toward lymph node chemokines both in vitro and in vivo, resulting in superior tumor control.     

CD4+ T cell-mediated cytotoxicity is associated with MHC class II expression on malignant CD19+ B cells in diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is a common B cell malignancy with approximately 30% of patients present relapsed or refractory disease after first-line therapy. Research of further treatment options is needed. Cytotoxic CD4⁺ T cells express cytolytic molecules and have potential antitumor function. Here, we showed that the CD19⁺ cells from DLBCL patients presented significantly reduced expression of MHC II molecules than those from healthy controls. Three years after the first-line treatment, patients that presented relapsed disease had significantly lower MHC II expression on their CD19⁺ cells than patients who did not show recurrence. Examining cytotoxic CD4⁺ T cells show that DLBCL patients presented significantly elevated frequencies of granzyme A-, granzyme B-, and/or perforin-expressing cytotoxic CD4⁺ T cells. Also, frequency of cytotoxic CD4⁺ T cells in DLBCL patients was positively correlated with the MHC II expression level. Subsequently, the cytotoxic potential of CD4⁺ T cells against autologous CD19⁺ cells was investigated. We found that the cytotoxic potential of CD4⁺ T cells was highest in MHC II-high, intermediate in MHC II-mid, and lowest in MHC II-low patients. The percentage of MHC II-expressing viable CD19⁺ cells presented a significant reduction after longer incubation with cytotoxic CD4⁺ T cells, suggesting that cytotoxic CD4⁺ T cells preferentially eliminated MHC II-expressing CD19⁺ cells. Blocking MHC II on CD19⁺ cells significantly reduced the cytolytic capacity of CD4⁺ T cells. Despite these discoveries, the frequency of cytotoxic CD4⁺ T cells did not predict the clinical outcome of DLBCL patients. Together, these results demonstrated that cytotoxic CD4⁺ T cells presented an MHC II-dependent cytotoxic potential against autologous CD19⁺ cells and could potentially represent a future treatment option for DLBCL.     

Efficacy and toxicity for CD22/CD19 chimeric antigen receptor T-cell therapy in patients with relapsed/refractory aggressive B-cell lymphoma involving the gastrointestinal tract

Gastrointestinal (GI) tract is the most common site of extranodal involvement in non-Hodgkin lymphoma. Life-threatening complications of GI may occur because of tumor or chemotherapy. Chimeric antigen receptor (CAR) T-cell therapy has been successfully used to treat refractory/relapse B-cell lymphoma, however, little is known about the efficacy and safety of CAR-T cell therapy for GI lymphoma. Here, we reported the efficacy and safety of CAR-T cell therapy in 14 patients with relapsed/refractory aggressive B-cell lymphoma involving the GI tract. After a sequential anti-CD22/anti-CD19 CAR-T therapy, 10 patients achieved an objective response, and seven patients achieved a complete response. CAR transgene and B-cell aplasia persisted in the majority of patients irrespective of response status. Six patients with partial response or stable disease developed progressive disease; two patients lost target antigens. Cytokine release syndrome (CRS) and GI adverse events were generally mild and manageable. The most common GI adverse events were diarrhea (4/14), vomiting (3/14) and hemorrhage (2/14). No perforation occurred during follow-up. Infection is a severe complication in GI lymphoma. Two patients were infected with bacteria that are able to colonize at GI; one died of sepsis early after CAR-T cells infusion. In conclusion, our study showed promising efficacy and safety of CAR-T cell therapy in refractory/relapsed B-cell lymphoma involving the GI tract. However, the characteristics of CAR-T–related infection in GI lymphoma should be further clarified to prevent and control infection.     

Myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones include double-positive CD8+CD4+ T cells: evidence from myeloma-bearing mouse model

The graft-versus-myeloma (GVM) effect represents a powerful form of immune attack exerted by alloreactive T cells against multiple myeloma cells, which leads to clinical responses in multiple myeloma transplant recipients. Whether myeloma cells are themselves able to induce alloreactive T cells capable of the GVM effect is not defined. Using adoptive transfer of T naive cells into myeloma-bearing mice (established by transplantation of human RPMI8226-TGL myeloma cells into CD122(+) cell-depleted NOD/SCID hosts), we found that myeloma cells induced alloreactive T cells that suppressed myeloma growth and prolonged survival of T cell recipients. Myeloma-induced alloreactive T cells arising in the myeloma-infiltrated bones exerted cytotoxic activity against resident myeloma cells, but limited activity against control myeloma cells obtained from myeloma-bearing mice that did not receive T naive cells. These myeloma-induced alloreactive T cells were derived through multiple CD8(+) T cell divisions and enriched in double-positive (DP) T cells coexpressing the CD8αα and CD4 coreceptors. MHC class I expression on myeloma cells and contact with T cells were required for CD8(+) T cell divisions and DP-T cell development. DP-T cells present in myeloma-infiltrated bones contained a higher proportion of cells expressing cytotoxic mediators IFN-γ and/or perforin compared with single-positive CD8(+) T cells, acquired the capacity to degranulate as measured by CD107 expression, and contributed to an elevated perforin level seen in the myeloma-infiltrated bones. These observations suggest that myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones are enriched with DP-T cells equipped with cytotoxic effector functions that are likely to be involved in the GVM effect.     

HLA-A*02:01-directed chimeric antigen receptor/forkhead box P3-engineered CD4+ T cells adopt a regulatory phenotype and suppress established graft-versus-host disease

Background aimsTo investigate the feasibility of using CD4 + T cells genetically modified to express an allo-HLA directed CAR and FOXP3 to suppress T cell proliferation and cytokine secretion in GvHD. Methods: Human CD4+ T cells from A*02:01 negative donors were transduced to express A*02 CAR and FOXP3 and co-cultured in mixed lymphocyte reaction assays to demonstrate T cell suppression. A*02- CAR/FOXP CD4+ T cells were then injected into mice engrafted with allogeneic T cells in a GvHD mouse model.ResultsCD4+ T cells genetically modified to express allo-HLA-directed CAR and FOXP3 proliferate rapidly, downregulate CD127 and interferon-γ, express high CD25 and Helios and convert to a stable antigen-dependent suppressive phenotype. In mixed lymphocyte reaction assays, these cells potently suppressed T-cell proliferation and secreted IL-10. In a graft-versus-host disease model, A*02-CAR/FOXP3 CD4+ T cells outperformed polyclonal Tregs by reducing liver and lung inflammation, inhibiting pro-inflammatory cytokine production and limiting grafted CD3+ T-cell expansion.ConclusionsCD4 + T cells expressing allo-antigen directed HLA-specific CAR and FOXP3 act as potent, specific and stable suppressors of inflammation that out-perform their Treg counterparts both in vitro and in vivo.