Evidence of clinical efficacy of a first generation CD19 CAR T cell in B cell malignancies.

The persistence and reactivity of CAR T cells were enhanced by adding co-stimulatory domains, which is the basis of currently approved CAR-T cell therapies. However, this comes at the expense of increasing toxicities from the strong cytokine release effect. This is the first report from anti-CD19 CAR-T cell therapy with a single activation domain to show a favourable safety profile and clinical efficacy with two patients who achieved durable responses up to 28 months in a cohort with heavily pretreated B cell malignancies.

CYAD-01, an autologous NKG2D-based CAR T-cell therapy, in relapsed or refractory acute myeloid leukaemia and myelodysplastic syndromes or multiple myeloma (THINK): haematological cohorts of the dose escalation segment of a phase 1 trial.

BACKGROUND: CYAD-01 is an autologous chimeric antigen receptor (CAR) T-cell product based on the natural killer (NK) group 2D (NKG2D) receptor, which binds eight ligands that are overexpressed in a wide range of haematological malignancies but are largely absent on non-neoplastic cells. Initial clinical evaluation of a single infusion of CYAD-01 at a low dose in patients with relapsed or refractory acute myeloid leukaemia, myelodysplastic syndromes, and multiple myeloma supported the feasibility of the approach and prompted further evaluation of CYAD-01. The aim of the present study was to determine the safety and recommended phase 2 dosing of CYAD-01 administered without preconditioning or bridging chemotherapy. METHODS: The multicentre THINK study was an open-label, dose-escalation, phase 1 study for patients with relapsed or refractory acute myeloid leukaemia, myelodysplastic syndromes, or multiple myeloma, after at least one previous line of therapy. Patients were recruited from five hospitals in the USA and Belgium. The dose-escalation segment evaluated three dose levels: 3 × 108 (dose level one), 1 × 109 (dose level two), and 3 × 109 (dose level three) cells per infusion with a 3 + 3 Fibonacci study design using a schedule of three infusions at 2-week intervals followed by potential consolidation treatment consisting of three additional infusions. The occurrence of dose-limiting toxicities post-CYAD-01 infusion was assessed as the primary endpoint in the total treated patient population. The trial was registered with ClinicalTrials.gov, NCT03018405, and EudraCT, 2016-003312-12, and has been completed. FINDINGS: Between Feb 6, 2017, and Oct 9, 2018, 25 patients were registered in the haematological dose-escalation segment. Seven patients had manufacturing failure for insufficient yield and two had screening failure. 16 patients were treated with CYAD-01 (three with multiple myeloma and three with acute myeloid leukaemia at dose level one; three with acute myeloid leukaemia at dose level two; and six with acute myeloid leukaemia and one with myelodysplastic syndromes at dose level three). Median follow-up was 118 days (IQR 46-180). Seven patients (44%) had grade 3 or 4 treatment-related adverse events. In total, five patients (31%) had grade 3 or 4 cytokine release syndrome across all dose levels. One dose-limiting toxicity of cytokine release syndrome was reported at dose level three. No treatment-related deaths occurred, and the maximum tolerated dose was not reached. Three (25%) of 12 evaluable patients with relapsed or refractory acute myeloid leukaemia or myelodysplastic syndromes had an objective response. Among responders, two patients with acute myeloid leukaemia proceeded to allogeneic haematopoietic stem-cell transplantation (HSCT) after CYAD-01 treatment, with durable ongoing remissions (5 and 61 months). INTERPRETATION: Treatment with a multiple CYAD-01 infusion schedule without preconditioning is well tolerated and shows anti-leukaemic activity, although without durability outside of patients bridged to allogeneic HSCT. These phase 1 data support the proof-of-concept of targeting NKG2D ligands by CAR T-cell therapy. Further clinical studies with NKG2D-based CAR T-cells are warranted, potentially via combinatorial antigen targeted approaches, to improve anti-tumour activity. FUNDING: Celyad Oncology.

Clinical Grade Manufacture of CYAD-101, a NKG2D-based, First in Class, Non-Gene-edited Allogeneic CAR T-Cell Therapy.

Allogeneic chimeric antigen receptor (CAR) T holds the promise of taking this therapeutic approach to broader patient populations while avoiding the intensive manufacturing demands of autologous cell products. One limitation to delivering an allogeneic CAR T is T-cell receptor (TCR) driven toxicity. In this work, the expression of a peptide to interfere with TCR signaling was assessed for the generation of allogeneic CAR T cells. The expression of a truncated CD3ζ peptide was shown to incorporate into the TCR complex and to result in blunted TCR responses. When coexpressed with a natural killer group 2D (NKG2D) CAR, the allogeneic T cells (called CYAD-101) failed to induce graft-versus-host disease in mouse models while maintaining antitumor activity driven by the CAR in vitro and in vivo. Two clinical grade discrete batches of CYAD-101 cells were produced of single donor apheresis resulting in 48 billion CAR T cells sufficient for the entire dose-escalation phase of the proposed clinical trial. The 2 batches showed high consistency producing a predominantly CD4+ T-cell population that displayed an effector/central memory phenotype with no evidence of exhaustion markers expression. These clinical grade CYAD-101 cells secreted cytokines and chemokines in response to ligands expressing target cells in vitro, demonstrating effector function through the CAR. Moreover, CYAD-101 cells failed to respond to TCR stimulation, indicating a lack of allogeneic potential. This bank of clinical grade, non-gene-edited, allogeneic CYAD-101 cells are used in the alloSHRINK clinical trial (NCT03692429).

CAR T cells targeting tumor endothelial marker CLEC14A inhibit tumor growth.

Engineering T cells to express chimeric antigen receptors (CARs) specific for antigens on hematological cancers has yielded remarkable clinical responses, but with solid tumors, benefit has been more limited. This may reflect lack of suitable target antigens, immune evasion mechanisms in malignant cells, and/or lack of T cell infiltration into tumors. An alternative approach, to circumvent these problems, is targeting the tumor vasculature rather than the malignant cells directly. CLEC14A is a glycoprotein selectively overexpressed on the vasculature of many solid human cancers and is, therefore, of considerable interest as a target antigen. Here, we generated CARs from 2 CLEC14A-specific antibodies and expressed them in T cells. In vitro studies demonstrated that, when exposed to their target antigen, these engineered T cells proliferate, release IFN-γ, and mediate cytotoxicity. Infusing CAR engineered T cells into healthy mice showed no signs of toxicity, yet these T cells targeted tumor tissue and significantly inhibited tumor growth in 3 mouse models of cancer (Rip-Tag2, mPDAC, and Lewis lung carcinoma). Reduced tumor burden also correlated with significant loss of CLEC14A expression and reduced vascular density within malignant tissues. These data suggest the tumor vasculature can be safely and effectively targeted with CLEC14A-specific CAR T cells, offering a potent and widely applicable therapy for cancer.

The integrated stress response promotes B7H6 expression.

The B7 family member, B7H6, is a ligand for the natural killer cell receptor NKp30. B7H6 is hardly expressed on normal tissues, but undergoes upregulation on different types of tumors, implicating it as an attractive target for cancer immunotherapy. The molecular mechanisms that control B7H6 expression are poorly understood. We report that in contrast to other NK cell ligands, endoplasmic reticulum (ER) stress upregulates B7H6 mRNA levels and surface expression. B7H6 induction by ER stress requires protein kinase R-like ER kinase (PERK), one of the three canonical sensors of the unfolded protein response. PERK phosphorylates eIF2α, which regulates protein synthesis and gene expression. Because eIF2α is phosphorylated by several kinases following different stress conditions, the program downstream to eIF2α phosphorylation is called the integrated stress response (ISR). Several drugs were reported to promote the ISR. Nelfinavir and lopinavir, two clinically approved HIV protease inhibitors, promote eIF2α phosphorylation by different mechanisms. We show that nelfinavir and lopinavir sustainably instigate B7H6 expression at their pharmacologically relevant concentrations. As such, ER stress and ISR conditions sensitize melanoma targets to CAR-T cells directed against B7H6. Our study highlights a novel mechanism to induce B7H6 expression and suggests a pharmacological approach to improve B7H6-directed immunotherapy. KEY MESSAGES: B7H6 is induced by ER stress in a PERK-dependent mechanism. Induction of B7H6 is obtained pharmacologically by HIV protease inhibitors. Exposure of tumor cells to the HIV protease inhibitor nelfinavir improves the recognition by B7H6-directed CAR-T.

Chimeric Antigen Receptor-T Cells for Targeting Solid Tumors: Current Challenges and Existing Strategies.

Chimeric antigen receptor-T cells (CAR-Ts) are an exciting new cancer treatment modality exemplified by the recent regulatory approval of two CD19-targeted CAR-T therapies for certain B cell malignancies. However, this success in the hematological setting has yet to translate to a significant level of objective clinical responses in the solid tumor setting. The reason for this lack of translation undoubtedly lies in the substantial challenges raised by solid tumors to all therapies, including CAR-T, that differ from B cell malignancies. For instance, intravenously infused CAR-Ts are likely to make rapid contact with cancerous B cells since both tend to reside in the same vascular compartments within the body. By contrast, solid cancers tend to form discrete tumor masses with an immune-suppressive tumor microenvironment composed of tumor cells and non-tumor stromal cells served by abnormal vasculature that restricts lymphocyte infiltration and suppresses immune function, expansion, and persistence. Moreover, the paucity of uniquely and homogeneously expressed tumor antigens and inherent plasticity of cancer cells provide major challenges to the specificity, potency, and overall effectiveness of CAR-T therapies. This review focuses on the major preclinical and clinical strategies currently being pursued to tackle these challenges in order to drive the success of CAR-T therapy against solid tumors.

Phase I Trial of Autologous CAR T Cells Targeting NKG2D Ligands in Patients with AML/MDS and Multiple Myeloma.

NKG2D ligands are widely expressed in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. We conducted a phase I dose-escalation study to evaluate the safety and feasibility of a single infusion of NKG2D-chimeric antigen receptor (CAR) T cells, without lymphodepleting conditioning in subjects with acute myeloid leukemia/myelodysplastic syndrome or relapsed/refractory multiple myeloma. Autologous T cells were transfected with a γ-retroviral vector encoding a CAR fusing human NKG2D with the CD3ζ signaling domain. Four dose levels (1 × 106-3 × 107 total viable T cells) were evaluated. Twelve subjects were infused [7 acute myeloid leukemia (AML) and 5 multiple myeloma]. NKG2D-CAR products demonstrated a median 75% vector-driven NKG2D expression on CD3+ T cells. No dose-limiting toxicities, cytokine release syndrome, or CAR T cell-related neurotoxicity was observed. No significant autoimmune reactions were noted, and none of the ≥ grade 3 adverse events were attributable to NKG2D-CAR T cells. At the single injection of low cell doses used in this trial, no objective tumor responses were observed. However, hematologic parameters transiently improved in one subject with AML at the highest dose, and cases of disease stability without further therapy or on subsequent treatments were noted. At 24 hours, the cytokine RANTES increased a median of 1.9-fold among all subjects and 5.8-fold among six AML patients. Consistent with preclinical studies, NKG2D-CAR T cell-expansion and persistence were limited. Manufactured NKG2D-CAR T cells exhibited functional activity against autologous tumor cells in vitro, but modifications to enhance CAR T-cell expansion and target density may be needed to boost clinical activity.

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings. We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general. This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described. Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice. These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Manufacturing development and clinical production of NKG2D chimeric antigen receptor-expressing T cells for autologous adoptive cell therapy.

BACKGROUND AIMS: Adoptive cell therapy employing natural killer group 2D (NKG2D) chimeric antigen receptor (CAR)-modified T cells has demonstrated preclinical efficacy in several model systems, including hematological and solid tumors. We present comprehensive data on manufacturing development and clinical production of autologous NKG2D CAR T cells for treatment of acute myeloid leukemia and multiple myeloma (ClinicalTrials.gov Identifier: NCT02203825). An NKG2D CAR was generated by fusing native full-length human NKG2D to the human CD3ζ cytoplasmic signaling domain. NKG2D naturally associates with native costimulatory molecule DAP10, effectively generating a second-generation CAR against multiple ligands upregulated during malignant transformation including MIC-A, MIC-B and the UL-16 binding proteins. METHODS: CAR T cells were infused fresh after a 9-day process wherein OKT3-activated T cells were genetically modified with replication-defective gamma-retroviral vector and expanded ex vivo for 5 days with recombinant human interleukin-2. RESULTS: Despite sizable interpatient variation in originally collected cells, release criteria, including T-cell expansion and purity (median 98%), T-cell transduction (median 66% CD8+ T cells), and functional activity against NKG2D ligand-positive cells, were met for 100% of healthy donors and patients enrolled and collected. There was minimal carryover of non-T cells, particularly malignant cells; both effector memory and central memory cells were generated, and inflammatory cytokines such as granulocyte colony-stimulating factor, RANTES, interferon-γ and tumor necrosis factor-α were selectively up-regulated. CONCLUSIONS: The process resulted in production of required cell doses for the first-in-human phase I NKG2D CAR T clinical trial and provides a robust, flexible base for further optimization of NKG2D CAR T-cell manufacturing.

Ex vivo expanded tumour-infiltrating lymphocytes from ovarian cancer patients release anti-tumour cytokines in response to autologous primary ovarian cancer cells.

Epithelial ovarian cancer (EOC) is the leading cause of gynaecological cancer-related death in Europe. Although most patients achieve an initial complete response with first-line treatment, recurrence occurs in more than 80% of cases. Thus, there is a clear unmet need for novel second-line treatments. EOC is frequently infiltrated with T lymphocytes, the presence of which has been shown to be associated with improved clinical outcomes. Adoptive T-cell therapy (ACT) using ex vivo-expanded tumour-infiltrating lymphocytes (TILs) has shown remarkable efficacy in other immunogenic tumours, and may represent a promising therapeutic strategy for EOC. In this preclinical study, we investigated the efficacy of using anti-CD3/anti-CD28 magnetic beads and IL-2 to expand TILs from freshly resected ovarian tumours. TILs were expanded for up to 3 weeks, and then subjected to a rapid-expansion protocol (REP) using irradiated feeder cells. Tumours were collected from 45 patients with EOC and TILs were successfully expanded from 89.7% of biopsies. Expanded CD4+ and CD8+ subsets demonstrated features associated with memory phenotypes, and had significantly higher expression of key activation and functional markers than unexpanded TILs. Expanded TILs produced anti-tumour cytokines when co-cultured with autologous tumour cells, inferring tumour cytotoxicity. Our findings demonstrate that it is possible to re-activate and expand tumour-reactive T cells from ovarian tumours. This presents a promising immunotherapy that could be used sequentially or in combination with current therapeutic strategies.

Celyad's novel CAR T-cell therapy for solid malignancies.

Celyad recently initiated several clinical trials with the CYAD-01 product, a natural killer group 2D (NKG2D)-based chimeric antigen receptor (CAR), in both solid and hematologic tumor types. This review discusses the unique properties of CYAD-01, expecting to provide a new paradigm to fight against solid tumors.

CD19 CAR T Cells Expressing IL-12 Eradicate Lymphoma in Fully Lymphoreplete Mice through Induction of Host Immunity.

Chimeric antigen receptor (CAR) T cell therapy represents a significant advancement in cancer therapy. Larger studies have shown ∼90% complete remission rates against chemoresistant and/or refractory CD19+ leukemia or lymphoma. Effective CAR T cell therapy is highly dependent on lymphodepleting preconditioning, which is achieved through chemotherapy or radiotherapy that carries with it significant toxicities. These can exclude patients of low performance status. In order to overcome the need for preconditioning, we constructed fully mouse first and second generation anti-murine CD19 CARs with or without interleukin-12 (IL-12) secretion. To test these CARs, we established a mouse model to reflect the human situation without preconditioning. Murine second generation CAR T cells expressing IL-12 were capable of eradicating established B cell lymphoma with a long-term survival rate of ∼25%. We believe this to be the first study in a truly lymphoreplete model. We provide evidence that IL-12-expressing CAR T cells not only directly kill target CD19+ cells, but also recruit host immune cells to an anti-cancer immune response. This finding is critical because lymphodepletion regimens required for the success of current CAR T cell technology eliminate host immune cells whose anti-cancer activity could otherwise be harnessed by strategies such as IL-12-secreting CAR T cells.

Overcoming Target Driven Fratricide for T Cell Therapy.

Chimeric Antigen Receptor (CAR) T cells expressing the fusion of the NKG2D protein with CD3ζ (NKG2D-CAR T Cells) acquire a specificity for stress-induced ligands expressed on hematological and solid cancers. However, these stress ligands are also transiently expressed by activated T cells implying that NKG2D-based T cells may undergo self-killing (fratricide) during cell manufacturing or during the freeze thaw cycle prior to infusion in patients. To avoid target-driven fratricide and enable the production of NKG2D-CAR T cells for clinical application, two distinct approaches were investigated. The first focused upon the inclusion of a Phosphoinositol-3-Kinase inhibitor (LY294002) into the production process. A second strategy involved the inclusion of antibody blockade of NKG2D itself. Both processes impacted T cell fratricide, albeit at different levels with the antibody process being the most effective in terms of cell yield. While both approaches generated comparable NKG2D-CAR T cells, there were subtle differences, for example in differentiation status, that were fine-tuned through the phasing of the inhibitor and antibody during culture in order to generate a highly potent NKG2D-CAR T cell product. By means of targeted inhibition of NKG2D expression or generic inhibition of enzyme function, target-driven CAR T fratricide can be overcome. These strategies have been incorporated into on-going clinical trials to enable a highly efficient and reproducible manufacturing process for NKG2D-CAR T cells.

Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer.

Chimeric antigen receptor (CAR) T cells represent a novel targeted approach to overcome both quantitative and qualitative shortfalls of the host immune system relating to the detection and subsequent destruction of tumors. The identification of antigens expressed specifically on the surface of tumor cells is a critical first step in the ability to utilize CAR T cells for the treatment of cancer. The 5T4 is a tumor-associated antigen which is expressed on the cell surface of most solid tumors including ovarian cancer. Matched blood and tumor samples were collected from 12 patients with ovarian cancer; all tumors were positive for 5T4 expression by immunohistochemistry. Patient T cells were effectively transduced with 2 different anti-5T4 CAR constructs which differed in their affinity for the target antigen. Co-culture of CAR T cells with matched autologous tumor disaggregates resulted in antigen-specific secretion of IFN-gamma. Furthermore, assessment of the efficacy of anti-5T4 CAR T cells in a mouse model resulted in therapeutic benefit against established ovarian tumors. These results demonstrate proof of principle that 5T4 is an attractive target for immune intervention in ovarian cancer and that patient T cells engineered to express a 5T4-specific CAR can recognize and respond physiologically to autologous tumor cells.

Study protocol for THINK: a multinational open-label phase I study to assess the safety and clinical activity of multiple administrations of NKR-2 in patients with different metastatic tumour types.

INTRODUCTION: NKR-2 are autologous T cells genetically modified to express a chimeric antigen receptor (CAR) comprising a fusion of the natural killer group 2D (NKG2D) receptor with the CD3ζ signalling domain, which associates with the adaptor molecule DNAX-activating protein of 10 kDa (DAP10) to provide co-stimulatory signal upon ligand binding. NKG2D binds eight different ligands expressed on the cell surface of many tumour cells and which are normally absent on non-neoplastic cells. In preclinical studies, NKR-2 demonstrated long-term antitumour activity towards a breadth of tumour indications, with maximum efficacy observed after multiple NKR-2 administrations. Importantly, NKR-2 targeted tumour cells and tumour neovasculature and the local tumour immunosuppressive microenvironment and this mechanism of action of NKR-2 was established in the absence of preconditioning. METHODS AND ANALYSIS: This open-label phase I study will assess the safety and clinical activity of NKR-2 treatment administered three times, with a 2-week interval between each administration in different tumour types. The study will contain two consecutive segments: a dose escalation phase followed by an expansion phase. The dose escalation study involves two arms, one in solid tumours (five specific indications) and one in haematological tumours (two specific indications) and will include three dose levels in each arm: 3×108, 1×109 and 3×109 NKR-2 per injection. On the identification of the recommended dose in the first segment, based on dose-limiting toxicity occurrences, the study will expand to seven different cohorts examining the seven different tumour types separately. Clinical responses will be determined according to standard Response Evaluation Criteria In Solid Tumors (RECIST) criteria for solid tumours or international working group response criteria in haematological tumours. ETHICS APPROVAL AND DISSEMINATION: Ethical approval has been obtained at all sites. Written informed consent will be taken from all participants. The results of this study will be disseminated through presentation at international scientific conferences and reported in peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: NCT03018405, EudraCT 2016-003312-12; Pre-result.

'Atypical' CAR T cells: NKG2D and Erb-B as examples of natural receptor/ligands to target recalcitrant solid tumors.

Chimeric antigen receptor (CAR) T-cell therapy has recently been recommended for approval for certain B-cell malignancies bringing the approach closer to mainstream cancer treatment. This rapid rise to prominence has been driven by impressive clinical results and the means to successfully commercialize the approach now being actively pursued. The current success of CAR T cells in B-cell malignancies relies upon the absolute lineage specificity of the CD19 antigen. CARs can also be targeted using non-antibody approaches, including the use of receptors and ligands to provide target specificity that have different specificities and binding kinetics. The specific examples of NKG2D and Erb-B are used that provide different characteristics and target profiles for CAR T-cell therapy of cancer.

Exploiting natural killer group 2D receptors for CAR T-cell therapy.

Chimeric antigen receptors (CARs) are genetically engineered proteins that combine an extracellular antigen-specific recognition domain with one or several intracellular T-cell signaling domains. When expressed in T cells, these CARs specifically trigger T-cell activation upon antigen recognition. While the clinical proof of principle of CAR T-cell therapy has been established in hematological cancers, CAR T cells are only at the early stages of being explored to tackle solid cancers. This special report discusses the concept of exploiting natural killer cell receptors as an approach that could broaden the specificity of CAR T cells and potentially enhance the efficacy of this therapy against solid tumors. New data demonstrating feasibility of this approach in humans and supporting the ongoing clinical trial are also presented.

The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity.

The primary aim of this clinical trial was to determine the feasibility of delivering first-generation CAR T cell therapy to patients with advanced, CEACAM5+ malignancy. Secondary aims were to assess clinical efficacy, immune effector function and optimal dose of CAR T cells. Three cohorts of patients received increasing doses of CEACAM5+-specific CAR T cells after fludarabine pre-conditioning plus systemic IL2 support post T cell infusion. Patients in cohort 4 received increased intensity pre-conditioning (cyclophosphamide and fludarabine), systemic IL2 support and CAR T cells. No objective clinical responses were observed. CAR T cell engraftment in patients within cohort 4 was significantly higher. However, engraftment was short-lived with a rapid decline of systemic CAR T cells within 14 days. Patients in cohort 4 had transient, acute respiratory toxicity which, in combination with lack of prolonged CAR T cell persistence, resulted in the premature closure of the trial. Elevated levels of systemic IFNγ and IL-6 implied that the CEACAM5-specific T cells had undergone immune activation in vivo but only in patients receiving high-intensity pre-conditioning. Expression of CEACAM5 on lung epithelium may have resulted in this transient toxicity. Raised levels of serum cytokines including IL-6 in these patients implicate cytokine release as one of several potential factors exacerbating the observed respiratory toxicity. Whilst improved CAR designs and T cell production methods could improve the systemic persistence and activity, methods to control CAR T 'on-target, off-tissue' toxicity are required to enable a clinical impact of this approach in solid malignancies.

CCR7+ selected gene-modified T cells maintain a central memory phenotype and display enhanced persistence in peripheral blood in vivo.

BACKGROUND: Adoptive T cell immunotherapy (ATCT) for cancer entails infusing patients with T cells that recognise and destroy tumour cells. Efficient engraftment of T cells and persistence in the circulation correlate with favourable clinical outcomes. T cells of early differentiation possess an increased capacity for proliferation and therefore persistence, using these cells for ATCT could therefore lead to improved clinical outcomes. METHOD: We describe a method to enrich T cells of early differentiation status using paramagnetic beads and antibodies targeting cells expressing C-C motif chemokine receptor 7 (CCR7). RESULTS: Selection of cells expressing CCR7 enriches T cells of bearing markers of early differentiation status. This was validated through analysis of an array of surface markers and an observed reduction in effector cell functions ex vivo. CCR7 selection resulted in dramatic 83.6 and 137 fold increases in circulating levels of CD4 and CD8 T cells respectively compared to non-sorted T cells 3 weeks after adoptive transfer to NSG mice. We observed no significant difference in the engraftment levels of CCR7 or CD62L selected cells in the NSG mouse model. Comparison of cells ex vivo, however, suggests CCR7 selection is superior to CD62L selection in enriching T cells of early differentiation status. CONCLUSIONS: CCR7 selection offers a means to enrich T cells of early differentiation status for ACTC. Together our data suggests that these T cells are likely to display enhanced engraftment and persistence in patients in vivo and could therefore improve therapeutic efficacy of ACTC.