Lyophilized lymph nodes for improved delivery of chimeric antigen receptor T cells.

Lymph nodes are crucial organs of the adaptive immune system, orchestrating T cell priming, activation and tolerance. T cell activity and function are highly regulated by lymph nodes, which have a unique structure harbouring distinct cells that work together to detect and respond to pathogen-derived antigens. Here we show that implanted patient-derived freeze-dried lymph nodes loaded with chimeric antigen receptor T cells improve delivery to solid tumours and inhibit tumour recurrence after surgery. Chimeric antigen receptor T cells can be effectively loaded into lyophilized lymph nodes, whose unaltered meshwork and cytokine and chemokine contents promote chimeric antigen receptor T cell viability and activation. In mouse models of cell-line-derived human cervical cancer and patient-derived pancreatic cancer, delivery of chimeric antigen receptor T cells targeting mesothelin via the freeze-dried lymph nodes is more effective in preventing tumour recurrence when compared to hydrogels containing T-cell-supporting cytokines. This tissue-mediated cell delivery strategy holds promise for controlled release of various cells and therapeutics with long-term activity and augmented function.

Selective homing of CAR-CIK cells to the bone marrow niche enhances control of the acute myeloid leukemia burden.

Acute myeloid leukemia (AML) is a hematological malignancy derived from neoplastic myeloid progenitor cells characterized by abnormal clonal proliferation and differentiation. Although novel therapeutic strategies have recently been introduced, the prognosis of AML is still unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)-T-cell therapy in AML has been hampered by several factors, including the poor accumulation of the blood-injected cells in the leukemia bone marrow (BM) niche in which chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within this niche, could improve T-cell homing to the BM and consequently enhance their intimate contact with BM-resident AML cells, facilitating disease eradication. Specifically, we engineered conventional CD33.CAR-cytokine-induced killer cells (CIKs) with the wild-type (wt) CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of patients with warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis syndrome. Overexpression of both CXCR4wt and CXCR4mut in CD33.CAR-CIKs resulted in significant improvement of chemotaxis toward recombinant CXCL12 or BM stromal cell-conditioned medium, with no observed impairment of cytotoxic potential in vitro. Moreover, CXCR4-overexpressing CD33.CAR-CIKs showed enhanced in vivo BM homing, associated with a prolonged retention for the CXCR4R334X variant. However, only CD33.CAR-CIKs coexpressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a noncanonical role for CXCR4 in modulating CAR-CIK functions independent of BM homing. Taken together, these data suggest that arming CAR-CIKs with CXCR4 may represent a promising strategy for increasing their therapeutic potential for AML.

Transdermal cold atmospheric plasma-mediated immune checkpoint blockade therapy.

Despite the promise of immune checkpoint blockade (ICB) therapy against cancer, challenges associated with low objective response rates and severe systemic side effects still remain and limit its clinical applications. Here, we described a cold atmospheric plasma (CAP)-mediated ICB therapy integrated with microneedles (MN) for the transdermal delivery of ICB. We found that a hollow-structured MN (hMN) patch facilitates the transportation of CAP through the skin, causing tumor cell death. The release of tumor-associated antigens then promotes the maturation of dendritic cells in the tumor-draining lymph nodes, subsequently initiating T cell-mediated immune response. Anti-programmed death-ligand 1 antibody (aPDL1), an immune checkpoint inhibitor, released from the MN patch further augments the antitumor immunity. Our findings indicate that the proposed transdermal combined CAP and ICB therapy can inhibit the tumor growth of both primary tumors and distant tumors, prolonging the survival of tumor-bearing mice.

CD16-158-valine chimeric receptor T cells overcome the resistance of KRAS-mutated colorectal carcinoma cells to cetuximab.

KRAS mutations hinder therapeutic efficacy of epidermal growth factor receptor (EGFR)-specific monoclonal antibodies cetuximab and panitumumab-based immunotherapy of EGFR+ cancers. Although cetuximab inhibits KRAS-mutated cancer cell growth in vitro by natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), KRAS-mutated colorectal carcinoma (CRC) cells escape NK cell immunosurveillance in vivo. To overcome this limitation, we used cetuximab and panitumumab to redirect Fcγ chimeric receptor (CR) T cells against KRAS-mutated HCT116 colorectal cancer (CRC) cells. We compared four polymorphic Fcγ-CR constructs including CD16158F -CR, CD16158V -CR, CD32131H -CR, and CD32131R -CR transduced into T cells by retroviral vectors. Percentages of transduced T cells expressing CD32131H -CR (83.5 ± 9.5) and CD32131R -CR (77.7 ± 13.2) were significantly higher than those expressing with CD16158F -CR (30.3 ± 10.2) and CD16158V -CR (51.7 ± 13.7) (p < 0.003). CD32131R -CR T cells specifically bound soluble cetuximab and panitumumab. However, only CD16158V -CR T cells released high levels of interferon gamma (IFNγ = 1,145.5 pg/ml ±16.5 pg/ml, p < 0.001) and tumor necrosis factor alpha (TNFα = 614 pg/ml ± 21 pg/ml, p < 0.001) upon incubation with cetuximab-opsonized HCT116 cells. Moreover, only CD16158V -CR T cells combined with cetuximab killed HCT116 cells and A549 KRAS-mutated cells in vitro. CD16158V -CR T cells also effectively controlled subcutaneous growth of HCT116 cells in CB17-SCID mice in vivo. Thus, CD16158V -CR T cells combined with cetuximab represent useful reagents to develop innovative EGFR+KRAS-mutated CRC immunotherapies.

In vitro elimination of epidermal growth factor receptor-overexpressing cancer cells by CD32A-chimeric receptor T cells in combination with cetuximab or panitumumab.

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). Although the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects the poor binding affinity of human IgG2 Fc for the FcγRIII (CD16) on natural killer (NK) cells. However, both human IgG1 and IgG2 bind the FcγRII (CD32A) to a similar extent. Our study compares the ability of T cells, engineered with a novel low-affinity CD32A131R -chimeric receptor (CR), and those engineered with the low-affinity CD16158F -CR T cells, in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. After T-cell transduction, the percentage of CD32A131R -CR T cells was 74 ± 10%, whereas the percentage of CD16158F -CR T cells was 46 ± 15%. Only CD32A131R -CR T cells bound panitumumab. CD32A131R -CR T cells combined with the mAb 8.26 (anti-CD32) and CD16158F -CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116FcγR+ cells, in a reverse ADCC assay in vitro. Crosslinking of CD32A131R -CR on T cells by cetuximab or panitumumab and CD16158F -CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and the secretion of interferon gamma and tumor necrosis factor alpha. Neither cetuximab nor panitumumab induced Fcγ-CR T antitumor activity against Kirsten rat sarcoma (KRAS)-mutated HCT116, nonsmall-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. The ADCC of Fcγ-CR T cells was associated with the overexpression of EGFR on ECCs. In conclusion, CD32A131R -CR T cells are efficiently redirected by cetuximab or panitumumab against breast cancer cells overexpressing EGFR.

The Role of Immunological Synapse in Predicting the Efficacy of Chimeric Antigen Receptor (CAR) Immunotherapy.

Chimeric Antigen Receptor (CAR) immunotherapy utilizes genetically-engineered immune cells that express a unique cell surface receptor that combines tumor antigen specificity with immune cell activation. In recent clinical trials, the adoptive transfer of CAR-modified immune cells (including CAR-T and CAR-NK cells) into patients has been remarkably successful in treating multiple refractory blood cancers. To improve safety and efficacy, and expand potential applicability to other cancer types, CARs with different target specificities and sequence modifications are being developed and tested by many laboratories. Despite the overall progress in CAR immunotherapy, conventional tools to design and evaluate the efficacy and safety of CAR immunotherapies can be inaccurate, time-consuming, costly, and labor-intensive. Furthermore, existing tools cannot always determine how responsive individual patients will be to a particular CAR immunotherapy. Recent work in our laboratory suggests that the quality of the immunological synapse (IS) can accurately predict CAR-modified cell efficacy (and toxicity) that can correlate with clinical outcomes. Here we review current efforts to develop a Synapse Predicts Efficacy (SPE) system for easy, rapid and cost-effective evaluation of CAR-modified immune cell immunotherapy. Ultimately, we hypothesize the conceptual basis and clinical application of SPE will serve as an important parameter in evaluating CAR immunotherapy and significantly advance precision cancer immunotherapy. Video abstract Graphic abstract for manuscript CCAS-D-20-00136 by Liu, D., et al., 'The Role of Immunological Synapse in Predicting the Efficacy of Chimeric Antigen Receptor (CAR) Immunotherapy". The various branches of evaluating cancer immunotherapy metaphorically represented as a Rubik's cube. The development of a novel approach to predict the effectiveness of Chimeric Antigen Receptor (CAR)-modified cells by quantifying the quality of CAR IS will introduce a new parameter to the rapidly expanding field of cancer immunotherapy. Currently, no single parameter can predict the clinical outcome or efficacy of a specific type of CAR-modified cell. IS quality will serve as a quantifiable measure to evaluate CAR products and can be used in conjunction with other conventional parameters to form a composite clinical predictor. Much like a Rubik's cube has countless configurations, several methods and combinations of clinical metrics have arisen for evaluating the ability of a given immunotherapeutic strategy to treat cancer. The quality of IS depicting cancer immunotherapy is metaphorically expressed as a Rubik's cube. Each face/color represents one aspect of cancer therapy. Each grid in one face indicates one factor within that aspect of cancer therapy. For example, the green color represents the tumor microenvironment, and one out of the nine grids in the green color indicates suppressor cells (suppressors in green). Changes in one factor may completely alter the entire strategy of cancer therapy. However, the quality of IS (illuminated center red grid) makes the effectiveness of CAR immunotherapy predictable.

Chimeric antigen receptor-redirected T cells return to the bench.

While the clinical progress of chimeric antigen receptor T cell (CAR-T) immunotherapy has garnered attention to the field, our understanding of the biology of these chimeric molecules is still emerging. Our aim within this review is to bring to light the mechanistic understanding of these multi-modular receptors and how these individual components confer particular properties to CAR-Ts. In addition, we will discuss extrinsic factors that can be manipulated to influence CAR-T performance such as choice of cellular population, culturing conditions and additional modifications that enhance their activity particularly in solid tumors. Finally, we will also consider the emerging toxicity associated with CAR-Ts. By breaking apart the CAR and examining the role of each piece, we can build a better functioning cellular vehicle for optimized treatment of cancer patients.

Genetically engineered CAR NK cells display selective cytotoxicity against FLT3-positive B-ALL and inhibit in vivo leukemia growth.

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells represent a promising effector cell type for adoptive cancer immunotherapy. Both, genetically modified donor-derived NK cells as well as continuously expanding NK-92 cells are currently under clinical development. To enhance their therapeutic utility for the treatment of pre-B-cell acute lymphoblastic leukemia (B-ALL), we engineered NK-92 cells by lentiviral gene transfer to express a FMS-like tyrosine kinase 3 (FLT3)-specific CAR that contains a composite CD28-CD3ζ signaling domain. FLT3 has primarily been described as a therapeutic target for acute myeloid leukemia, but overexpression of FLT3 has also been reported in B-ALL. Exposure of FLT3-positive targets to CAR NK-92 cells resulted in conjugate formation between NK and leukemia cells, NK-cell degranulation and selective cytotoxicity toward established B-ALL cell lines and primary blasts that were resistant to parental NK-92. In a SEM B-ALL xenograft model in NOD-SCID IL2R γnull mice, treatment with CAR NK-92 but not parental NK-92 cells markedly inhibited disease progression, demonstrating high antileukemic activity in vivo. As FLT3 is known to be also expressed on precursor cells, we assessed the feasibility of incorporating an inducible caspase-9 (iCasp9) suicide switch to enhance safety of our approach. Upon addition of the chemical dimerizer AP20187 to NK-92 cells coexpressing the FLT3-specific CAR and iCasp9, rapid iCasp9 activation was observed, precluding further CAR-mediated cytotoxicity. Our data demonstrate that B-ALL can be effectively targeted by FLT3-specific CAR NK cells which may complement CD19-directed immunotherapies, particularly in cases of inherent or acquired resistance to the latter.

Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody-Activated Chimeric Antigen Receptor-Modified T Cells.

The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO+CCR7- effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28-activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell-derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro-expanded T cells.

Immunological Synapse Predicts Effectiveness of Chimeric Antigen Receptor Cells.

Chimeric antigen receptor (CAR)-modified T cell therapy has the potential to improve the overall survival of patients with malignancies by enhancing the effectiveness of CAR T cells. Precisely predicting the effectiveness of various CAR T cells represents one of today's key unsolved problems in immunotherapy. Here, we predict the effectiveness of CAR-modified cells by evaluating the quality of the CAR-mediated immunological synapse (IS) by quantitation of F-actin, clustering of tumor antigen, polarization of lytic granules (LGs), and distribution of key signaling molecules within the IS. Long-term killing capability, but not secretion of conventional cytokines or standard 4-hr cytotoxicity, correlates positively with the quality of the IS in two different CAR T cells that share identical antigen specificity. Xenograft model data confirm that the quality of the IS in vitro correlates positively with performance of CAR-modified immune cells in vivo. Therefore, we propose that the quality of the IS predicts the effectiveness of CAR-modified immune cells, which provides a novel strategy to guide CAR therapy.

In Vivo Fate and Activity of Second- versus Third-Generation CD19-Specific CAR-T Cells in B Cell Non-Hodgkin's Lymphomas.

Second-generation (2G) chimeric antigen receptors (CARs) targeting CD19 are highly active against B cell malignancies, but it is unknown whether any of the costimulatory domains incorporated in the CAR have superior activity to others. Because CD28 and 4-1BB signaling activate different pathways, combining them in a single third-generation (3G) CAR may overcome the limitations of each individual costimulatory domain. We designed a clinical trial in which two autologous CD19-specific CAR-transduced T cell products (CD19.CARTs), 2G (with CD28 only) and 3G (CD28 and 4-1BB), were infused simultaneously in 16 patients with relapsed or refractory non-Hodgkin's lymphoma. 3G CD19.CARTs had superior expansion and longer persistence than 2G CD19.CARTs. This difference was most striking in the five patients with low disease burden and few circulating normal B cells, in whom 2G CD19.CARTs had limited expansion and persistence and correspondingly reduced area under the curve. Of the 11 patients with measurable disease, three achieved complete responses and three had partial responses. Cytokine release syndrome occurred in six patients but was mild, and no patient required anti-IL-6 therapy. Hence, 3G CD19.CARTs combining 4-1BB with CD28 produce superior CART expansion and may be of particular value when treating low disease burden in patients whose normal B cells are depleted by prior therapy.

Engineering PD-1-Presenting Platelets for Cancer Immunotherapy.

Radical surgery still represents the treatment choice for several malignancies. However, local and distant tumor relapses remain the major causes of treatment failure, indicating that a postsurgery consolidation treatment is necessary. Immunotherapy with checkpoint inhibitors has elicited impressive clinical responses in several types of human malignancies and may represent the ideal consolidation treatment after surgery. Here, we genetically engineered platelets from megakaryocyte (MK) progenitor cells to express the programmed cell death protein 1 (PD-1). The PD-1 platelet and its derived microparticle could accumulate within the tumor surgical wound and revert exhausted CD8+ T cells, leading to the eradication of residual tumor cells. Furthermore, when a low dose of cyclophosphamide (CP) was loaded into PD-1-expressing platelets to deplete regulatory T cells (Tregs), an increased frequency of reinvigorated CD8+ lymphocyte cells was observed within the postsurgery tumor microenvironment, directly preventing tumor relapse.

Utilizing cell-based therapeutics to overcome immune evasion in hematologic malignancies.

Hematologic malignancies provide a suitable testing environment for cell-based immunotherapies, which were pioneered by the development of allogeneic hematopoietic stem cell transplant. All types of cell-based therapies, from donor lymphocyte infusion to dendritic cell vaccines, and adoptive transfer of tumor-specific cytotoxic T cells and natural killer cells, have been clinically translated for hematologic malignancies. The recent success of chimeric antigen receptor-modified T lymphocytes in B-cell malignancies has stimulated the development of this approach toward other hematologic tumors. Similarly, the remarkable activity of checkpoint inhibitors as single agents has created enthusiasm for potential combinations with other cell-based immune therapies. However, tumor cells continuously develop various strategies to evade their immune-mediated elimination. Meanwhile, the recruitment of immunosuppressive cells and the release of inhibitory factors contribute to the development of a tumor microenvironment that hampers the initiation of effective immune responses or blocks the functions of immune effector cells. Understanding how tumor cells escape from immune attack and favor immunosuppression is essential for the improvement of immune cell-based therapies and the development of rational combination approaches.

Inducible Caspase-9 Selectively Modulates the Toxicities of CD19-Specific Chimeric Antigen Receptor-Modified T Cells.

Immunotherapy with T cells expressing the chimeric antigen receptor (CAR) specific for the CD19 antigen (CD19.CAR-Ts) is a very effective treatment in B cell lymphoid malignancies. However, B cell aplasia and cytokine release syndrome (CRS) secondary to the infusion of CD19.CAR-Ts remain significant drawbacks. The inclusion of safety switches into the vector encoding the CAR is seen as the safest method to terminate the effects of CD19.CAR-Ts in case of severe toxicities or after achieving long-term sustained remissions. By contrast, the complete elimination of CD19.CAR-Ts when CRS occurs may jeopardize clinical responses as CRS and antitumor activity seem to concur. We have demonstrated, in a humanized mouse model, that the inducible caspase-9 (iC9) safety switch can eliminate CD19.CAR-Ts in a dose-dependent manner, allowing either a selective containment of CD19.CAR-T expansion in case of CRS or complete deletion on demand granting normal B cell reconstitution.

CAR T Cells Administered in Combination with Lymphodepletion and PD-1 Inhibition to Patients with Neuroblastoma.

Targeting disialoganglioside (GD2) on neuroblastoma (NB) with T cells expressing a first-generation chimeric antigen receptor (CAR) was safe, but the cells had poor expansion and long-term persistence. We developed a third-generation GD2-CAR (GD2-CAR3) and hypothesized that GD2-CAR3 T cells (CARTs) would be safe and effective. This phase 1 study enrolled relapsed or refractory NB patients in three cohorts. Cohort 1 received CART alone, cohort 2 received CARTs plus cyclophosphamide and fludarabine (Cy/Flu), and cohort 3 was treated with CARTs, Cy/Flu, and a programmed death-1 (PD-1) inhibitor. Eleven patients were treated with CARTs. The infusions were safe, and no dose-limiting toxicities occurred. CARTs were detectable in cohort 1, but the lymphodepletion induced by Cy/Flu increased circulating levels of the homeostatic cytokine interleukin (IL)-15 (p = 0.003) and increased CART expansion by up to 3 logs (p = 0.03). PD-1 inhibition did not further enhance expansion or persistence. Antitumor responses at 6 weeks were modest. We observed a striking expansion of CD45/CD33/CD11b/CD163+ myeloid cells (change from baseline, p = 0.0126) in all patients, which may have contributed to the modest early antitumor responses; the effect of these cells merits further study. Thus, CARTs are safe, and Cy/Flu can further increase their expansion.

Design and development of therapies using chimeric antigen receptor-expressing T cells.

Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non-protein antigens) with the persistence, trafficking, and effector functions of a T cell. This article describes how the past two decades have seen a crescendo of research which has now begun to translate these potential benefits into effective treatments for patients with cancer. We describe the basic design of CARs, describe how antigenic targets are selected, and the initial clinical experience with CAR-T cells. Our review then describes our own and other investigators' work aimed at improving the function of CARs and reviews the clinical studies in hematological and solid malignancies that are beginning to exploit these approaches. Finally, we show the value of adding additional engineering features to CAR-T cells, irrespective of their target, to render them better suited to function in the tumor environment, and discuss how the safety of these heavily modified cells may be maintained.

Tumor indoleamine 2,3-dioxygenase (IDO) inhibits CD19-CAR T cells and is downregulated by lymphodepleting drugs.

Although T cells expressing CD19-specific chimeric antigen receptors (CARs) are a promising new therapy for B-cell malignancies, objective responses are observed at lower frequencies in patients with lymphoma than in those with acute B-cell leukemia. We postulated that the tumor microenvironment suppresses CAR-expressing T cells (CARTs) through the activity of indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme that converts tryptophan into metabolites that inhibit T -: cell activity. To investigate the effects of tumor IDO on CD19-CART therapy, we used a xenograft lymphoma model expressing IDO as a transgene. CD19-CARTs inhibited IDO-negative tumor growth but had no effect on IDO-positive tumors. An IDO inhibitor (1-methyl-tryptophan) restored IDO-positive tumor control. Moreover, tryptophan metabolites inhibited interleukin (IL)-2-, IL-7-, and IL-15-dependent expansion of CARTs; diminished their proliferation, cytotoxicity, and cytokine secretion in vitro in response to CD19 recognition; and increased their apoptosis. Inhibition of CD19-CARTs was not mitigated by the incorporation of costimulatory domains, such as 4-1BB, into the CD19-CAR. Finally, we found that fludarabine and cyclophosphamide, frequently used before CART administration, downregulated IDO expression in lymphoma cells and improved the antitumor activity of CD19-CART in vivo. Because tumor IDO inhibits CD19-CARTs, antagonizing this enzyme may benefit CD19-CART therapy.

Inducible caspase-9 suicide gene controls adverse effects from alloreplete T cells after haploidentical stem cell transplantation.

To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103.

Serial Activation of the Inducible Caspase 9 Safety Switch After Human Stem Cell Transplantation.

Activation of the inducible caspase 9 (iC9) safety gene by a dimerizing drug (chemical inducer of dimerization (CID) AP1903) effectively resolves the symptoms and signs of graft-versus-host disease (GvHD) in haploidentical stem cell transplant (HSCT) recipients. However, after CID treatment, 1% of iC9-T cells remain and can regrow over time; although these resurgent T cells do not cause recurrent GvHD, it remains unclear whether repeat CID treatments are a safe and feasible way to further deplete residual gene-modified T cells should any other adverse effects associated with them occur. Here, we report a patient who received an infusion of haploidentical iC9-T cells after HSCT and subsequently received three treatments with AP1903. There was a mild (grade 2) and transient pancytopenia following each AP1903 administration but no non-hematological toxicity. Ninety five percent of circulating iC9-T cells (CD3(+)CD19(+)) were eliminated after the first AP1903 treatment. Three months later, the residual cells had expanded more than eightfold and had a lower level of iC9 expression. Each repeated AP1903 administration eliminated a diminishing percentage of the residual repopulating cells, but elimination could be enhanced by T-cell activation. These data support the safety and efficiency of repeated CID treatments for persistent or recurring toxicity from T-cell therapies.