Final Report Summary - ATECT (Advanced T-cell Engineered for Cancer Therapy)
Executive Summary:
The ATECT consortium aimed to improve Chimeric Antigen Receptor (CAR) T-cell cancer therapy.
Work-page 1: The main aim of work-package was to address the cost and complexity of CAR T-cell manufacturing processes. One aspect of this was to explore the use of allogeneic CD19 CAR T-cells in two clinical studies. One clinical study (“CARD”) used matched allogeneic CAR T-cells. In addition, this study explored the use of automated manufacture of CD19 CAR T-cells. Manufactur-ing has been successful. Clinical data from this study will be presented late 2019 or early 2020.
A second more complex allogeneic approach was explored in the “UCART19” study. This ap-proach uses fully mismatched donor CD19 CAR T-cells. To overcome graft-versus-host disease (GvHD), concomitant TALEN mediated disruption of the TCR gene was performed; to overcome rejection, the CD52 gene was similarly disrupted allowing use of the powerful immunosuppressant CD52 targeting therapeutic antibody Alemtuzumab as conditioning. Two patients with infant B-ALL (in whom autologous CAR T-cell production) received UCART19 and outcome from these patients was reported as the first-in-man experience with genome-edited CAR T-cells. A key partner for this work, Cellectis, left the consortium and UCART19 studies continued in a phase I study in children and adults outside the consortium.
A third approach was developed in work-package 1: targeting of AML with an allogeneic approach. Based on what we learned from clinical exploration of UCART19, a protein-based allogeneic ap-proach was developed which simplified manufacture. Since AML stem-cells are heterogeneous in terms of antigen expression between and even within patients, a multi-targeting strategy was de-vised. The consortium has taken this project close to the point of clinical translation.
Work-package 2 focused on exploiting neovascularization which is an intrinsic part of the biology of most cancers. L19 is an antibody which recognizes the extracellular B domain of fibronectin. L19-IL2 (Darleukin) is an immunocytokine which is directed to neovascularization. We explored whether co-administration of L19-IL2 would enhance CAR T-cell engraftment at the tumour site. In addition, we asked whether co-expression of an L19 CAR along with the cancer antigen CAR would enhance CAR T-cell activity by disrupting the microenvironment. We found that combina-tion of L19-IL2 with CAR enhanced activity, but the L19-CAR did not.
Work-package 3 focused on a number of different ways to overcome the hostile microenviron-ment. One approach was to use genome editing to disrupt inhibitory receptors on adoptively trans-ferred T-cells. Targeting PD1 as an example inhibitory receptor, we showed increased immune ac-tivity. Other approaches included engineering T-cells to release immune blockade inhibitory anti-bodies or immune-activatory cytokines.
Overall, the ATECT consortium achieved several landmarks: the first-in-man genome edited al-logeneic CAR T-cells, the first CAR T-cells manufactured by automated production and the first demonstration in vivo of enhancing T-cell activity by disruption of the PD1 gene by genome edit-ing. In addition, ATECT explored several ways of enhancing activity of adoptively transferred T-cells.
Project Context and Objectives:
2.1 Introduction:
Chimeric Antigen Receptors (CARs), generated by fusing the antigen-binding region of a monoclo-nal antibody (mAb) to intracellular T-cell signalling domains1, hold the promise to revolutionize cancer treatment. Introduction of genes coding for CARs into T-cells using integrating vectors en-dow antigen recognition independent of MHC restriction. Recent clinical data leave no doubt that this new form of cancer therapy can be remarkably effective, engendering long-lived remissions in patients with refractory disease.
There are, however, considerable barriers to be overcome to take this new form of therapy. Some of these barriers are practical – for instance can we develop ways of making such products cheaper and easier. Other barriers relate to engraftment of engineered T-cells in the face of hostile microen-vironment. To allow CAR T-cell therapy to be more widely applicable, advanced cellular engineer-ing approaches need to be applied to overcome these barriers.
2.2 Technological Background:
The ATECT consortium proposed to address these limitations through advanced cellular engineer-ing. The central technological theme of this consortium is the application of gene editing strategies alongside advanced standard methods of genetic modification with insertional vectors, which we describe as a combination of advanced “positive” and “negative” engineering (figure F2.1).
T-cell engineering strategies for Cancer therapy, either Chimeric Antigen Receptors (CARs) or TCR transfer holds promise to revolutionize cancer treatment. There are, however, considerable barriers to be overcome to take this form of therapy to a format that can benefit all EU citizens with a wide range of common cancers. The aim of this consortium is to exploit advances in T-cell engineering to allow the full potential of CAR therapy to be unleashed.
At present, CAR therapy requires a bespoke autologous therapeutic product for each patient. This greatly limits practicality, scalability and commercialisation. The development of a strategy for creation of universal engineered T-cells is the first key aim of this consortium. There is an in-creased appreciation of the immunological hostilities (CAR) T-cells face in the tumour microenvi-ronment, and prevention of this local immune suppressive effect will likely be critical in permitting effective tumour control.
The second main aim of this proposal is therefore to engineer CAR T-cells to be resistant to the hostile microenvironment. CAR T-cells can only be effective if they can access the tumour site. Exploiting the fact that neo-angiogenesis is a hallmark of neoplastic progression, the third aim of the consortium is to utilise endothelial cues of neo-angiogenesis to direct CAR T-cell migration and activity.
We planned to develop strategies to engineer T-cells to be highly effective anti-cancer agents. A key part of this is to engineer them to be resistant to the hostile environment that tumour cells gen-erate around them. A key part of this is to engineer the T-cell to secrete cytokines into the tumour bed.
2.3 Partner Organizations:
Partner 1: UCL | University College London : UCL is Europe’s leading health research multidis-ciplinary university with the one of the world's major concentrations of biomedical researchers grouping 8,000 staff and 22,000 studentst.. UCL has a particular expertise in cell and Gene Thera-py. Much of the work of the ATECT consortium will be executed at the UCL Cancer Institute.
Partner 2: CTX | Cellectis Therapeutics: Founded in France in 1999, the Cellectis Group is based on highly specific DNA engineering technologies. Cellectis is today one of the world leading com-panies in the field of genome engineering. The Group has a workforce of 230 employees working on 5 sites worldwide: Paris & Evry in France. Cellectis Therapeutics is a subsidiary of Cellectis. Its aim is innovative strategies for the development of wide-reaching therapeutic treatments based on core cellectis technology.
Partner 3: PHL | Philogen: Philogen was founded in 1996 with the mission to develop new bio-pharmaceuticals for the treatment of angiogenesis-related disorders, such as cancer and rheumatoid arthritis. The company has been a pioneer in the isolation, engineering and clinical development of lead products capable of targeting angiogenesis in vivo and has been the first in the world to demonstrate that human monoclonal antibodies can efficiently and selectively target the tumor neo- vasculatur.
Partner 4: NKI | Netherlands Cancer Institute: The Netherlands Cancer Institute was established in 1913. The Institute accommodates approximately 550 scientists and scientific support personnel, 53 medical specialists, 180 beds, an out-patient clinic that receives 183,000 patients each year, 5 operating theatres and 9 irradiation units. It is the only dedicated cancer centre in The Netherlands and maintains an important role as a national and international centre of scientific and clinical ex-pertise, development and training.
Partner 5: UZH | University of Zurich: Founded in 1833, the University of Zurich is Switzer-land’s largest university, with a current enrolment of over 26,000 students. UZH is made up of sev-en faculties covering approximately 100 different subject areas. As a member of the “League of European Research Universities” (LERU), UZH is one of Europe’s most prestigious research insti-tutions.. Most of the ATECT will be carried out at the Institute of experimental Immunology at the University of Zurich. Read more about the the [UZH], or the [Institute of experimental Immunology].
Project Results:
3. DESCRIPTION OF FINDINGS
3.1 Findings from work-package 1
3.1.1 Introduction for WP1
WP1 is the clinical study work-package for ATECT. In this WP, we aimed to explore the use of al-logeneic CAR T-cell therapy in two clinical studies in B-cell malignancies and to prepare for a fur-ther clinical study in acute myeloid leukaemia (AML).
The two allogeneic CAR T-cell studies tested two different approaches to allogeneic CAR T-cells. With the first study (CARD), matched donors were used as the source of CAR T-cells in the setting of allogeneic haematopoietic stem cell transplant (allo HSCT). In the second study (UCART19), completely mismatched donors were used, and allogeneic effects abrogated by genome editing.
The third aim of this work-package was to develop an allogeneic CAR T-cell strategy for AML.
3.1.2 Automated manufacture and allogeneic 2nd party CAR T-cells for B-cell malignancies
The CARD study aimed to test 2nd party CD19-specific CAR T-cells in the setting of patients with with B-cell malignancies who had relapsed after allo-HSCT. The CAR T-cells would be derived from the HSCT donors. The main clinical question to be asked was the incidence of graft-versus host disease (GvHD). In addition, CARD was the first study to open which used an automated manufacturing process since we could be guaranteed of excellent starting material from the allo-HSCT donors.
The transgene used for the CARD study is shown in figure F3.1 below and incorporated a suicide gene called RQR8 along with a novel CD19 CAR based on the 4G7 binder (figure F3.1).
An automated manufacturing process was developed for this clinical study. This was based on the Miltenyi Prodigy device. At the time this study was initiated, all CAR T-cell products were generat-ed using an open process. This requires very specialized clean-rooms and skilled operators. The Miltenyi Prodigy device is an automated closed fluid handling system with integrated culture sys-tem. Much lower stringency of environment is required for its operation and far less technician time.
The CARD study obtained regulatory approval from the UK regulator in 2017. At the time of writ-ing of this report, we have manufactured donor-derived CD19 CAR T-cell products for 12 patients. This demonstrates feasibility of the concept of approaching, consenting and harvesting matched donors for the manufacture of CAR T-cell products where autologous collections are not possible.
The details of the products are outlined in the table 4 below. All met release criteria (one QP release is pending). Using the CliniMACS Prodigy, the median transduction efficiency of the products at a MOI of 5 was 41.5% (range 13.0- 56.1%) and the CD3 viability was uniformly excellent at >90% for all products. Using this device there was enrichment for naïve and central memory phenotypes within the products. PD-1 expression demonstrated that the final products did not exhibit and ex-hausted phenotype.
CARD clinical data has not yet been presented or published so cannot be included in this final re-port. While we have recruited the number we planned to on original writing of the ATECT proposal (9 patients), given the data are interesting and we have altered conditioning on the study, CARD has now been extended to 14 patients and will proceed beyond the end of ATECT.
A clinical update for CARD is scheduled for the American Society for Haematology meeting in 2019. Any updates or clinical publications will be posted to the ATECT website.
3.1.2 Allogeneic 3rd party CAR T-cells for B-cell malignancies
The UCART19 study aimed to test fully allogeneic “off-the-shelf” CAR T-cells. At time of writing this proposal, CAR T-cell products were autologous. There are considerable limitations to autolo-gous CAR T-cell production. The main limitation is the time taken to produce a CAR T-cell prod-uct. A patient with a rapidly advancing malignancy may not have 3-4 weeks a typical production / release. Further, a patient who has had treatment with multiple rounds of combination chemothera-py, or other treatments such as radiotherapy and allo-HSCT may be T-cell lymphopenic or may have low quality T-cells. Finally, autologous manufacture is “bespoke” and hence cannot make use of economies of scale for manufacture.
Allogeneic approaches are complicated by two immunologic phenomena. Firstly, CAR T-cells ex-pressing a native TCR can cause graft-versus-host disease (GvHD) if the native TCR reacts against the recipient. GvHD is very well understood in allogeneic HSCT, particularly with significant mis-match such as in haploidentical HSCT. Further, transfusion associated GvHD (taGvHD) gives per-haps the best indication of the possibility of GvHD in a completely mismatched setting. In the transfusion era before leukodepletion, packed red cells occasionally caused ta GvHD due to pas-senger lymphocytes often in recipients who were immunosuppressed. As few as 10,000 lympho-cytes were considered sufficient to cause fatal taGvHD.
The other immunological consideration is that of immune rejection of CAR T-cells by the recipient. It was known that a reasonable period of engraftment is required for activity, and in B-ALL a long period of engraftment appeared needed for long-term disease control. From experience with alloge-neic HSCT, the usual conditioning with Fludarabine and Cyclophosphamide seemed unlikely to prevent rejection. Strategies to disrupt the HLA locus would result in the now “null” CAR T-cells being vulnerable to NK cell depletion.
The strategy we devised used TALEN directed genome editing of two loci: the TCR alpha locus and the CD52 locus. Disruption of the TCR alpha locus would lead to disruption of TCR and hence CD3 expression and thus prevent GvHD. CD52 is a densely expressed small GPI anchored protein whose precise function is unknown. CD52 is a pan-lymphoid marker. Alemtuzumab (Campath 1H) is a highly lytic therapeutic antibody currently marketed by Sanofi which targets CD52. Disruption of CD52 expression would allow addition of Alemtuzumab to the standard Fludarabine / Cyclo-phosphamide conditioning which would deplete the recipient’s T-cells but would not target the CAR T-cells. The manufacturing strategy is detailed in the figure below.
Given that this was a highly experimental approach, a phase 0 approach was taken. A considerable unmet need for therapeutic options is infant ALL. CAR T-cells cannot be currently generated from infants. Universal CAR19 (UCART19) T cells by lentiviral transduction of non–human leukocyte antigen–matched donor cells and simultaneous transcription activator-like effector nuclease (TALEN)–mediated gene editing of T cell receptor a chain and CD52 gene loci from two health do-nors. Two infants with relapsed refractory CD19 + B cell acute lymphoblastic leukemia received lymphodepleting chemotherapy and anti-CD52 serotherapy, followed by a single-dose infusion of UCART19 cells. These findings were reported by Qasim et al2 and key findings are summarized in figures F3.4 and F3.5 below.
Molecular remissions were achieved within 28 days in both infants, and UCART19 cells persisted until conditioning ahead of successful allogeneic stem cell transplantation. Both children remain in remission now over 2 years following this procedure. Some aplasia and some signs of possible GvHD were noted but resolved following transplant. This phase 0 experiment demonstrated the therapeutic potential of gene-editing technology and the UCART19 approach.
Cellectis left the ATECT consortium however, and consequently the UCART19 as planned was not continued within the consortium. However, two UCART19 studies did start outside the consortium in adult and paediatric B-ALL respectively (NCT02746952 and NCT02808442). This has recently been reported on at the American Society of Haematology
American Society for Haematology, 2019, #896
Preliminary Data on Safety, Cellular Kinetics and Anti-Leukemic Activity of UCART19, an Al-logeneic Anti-CD19 CAR T-Cell Product, in a Pool of Adult and Pediatric Patients with High-Risk CD19+ Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia.
Presented by: Reuben Benjamin
UCART19 is an allogeneic, genetically modified CAR T-cell product (anti-CD19 scFv- 41BB-CD3ζ) manufactured from healthy donor T cells, in which TRAC and CD52 genes have been knocked out to allow its administration in non-HLA matched patients (pts).
Aims: Preliminary safety/anti leukemic and cellular kinetics data of UCART19 administered to pediatric and adult patients with R/R B-ALL are reported.
Methods: Data of patients included in the ongoing CALM study (adult) and PALL study (pediat-ric) have been pooled. CALM is a dose-escalation study (approximate dose level [DL] 1: 1x105 cells/kg, DL2: 1x106 cells/kg, DL3: 3x106 cells/kg) and PALL is testing a unique dose of 1.1 to 2.3x106 cells/kg. Eligible patients presented with a morphological disease (>5%) or MRD load ≥1x10-3. A lymphodepleting regimen (LD) combining cyclophosphamide (1500 mg/m² in CALM, 120 mg/kg in PALL) and fludarabine (90 mg/m2 in CALM, 150 mg/m2 in PALL) without (FC) or with alemtuzumab (FCA) (1 mg/kg) was administered one week before UCART19 infu-sion on Day 0 (D0). Cellular kinetics of UCART19 was assessed by flow cytometry (flow) in CALM and by vector copy number in PALL.
Results: As of 15 July 2018, 20 pts had received at least one UCART19 infusion, including 13 pts in CALM (6 at DL1; 6 at DL2 and 1 at DL3) and 7 pts in PALL. Seventeen pts had completed D28 evaluation (1 pt died at D15, 2 pts have not reached D28 yet). Prior to LD, blasts % in the bone marrow (median [range]) was 6% [0-68%] in PALL and 25% [0-96%] in CALM. Seventeen pts received LD with FCA and 3 pts received LD with FC.
Safety was evaluable in 18/20 pts. Cytokine release syndrome (CRS) was reported in 17/18 pts and was mild and reversible in the majority of cases (2 G1, 12 G2, 2 G3, 1 G4). One pt died in context of CRS G4 and neutropenic sepsis. According to data available to date, the severity of CRS does not seem to follow the levels of serum cytokines (IL-6, IL-10 and IFNγ). Mild self-limited neurotoxicity events were reported in 6 pts (5 G1 and 1 G2). Two pts (1 infant and 1 adult) developed G1 acute skin GvHD (non-biopsy proven for 1 pt) that was reversible with steroids.
Grade 1-4 viral infections were reported in 8/18 pts. The majority of events recovered, except in 2 pts who died after allo-SCT in context of prolonged cytopenia (defined as persistent G4 beyond D42 post UCART19 infusion): 1 child with BK virus infection and 1 adult with adenovirus infection.
A further 4 pts developed prolonged cytopenia and all recovered after allo-SCT.
Cellular kinetic data was available for 18/20 pts. UCART19 was detectable in blood from D7. Peak expansion was observed in 72% (13/18) pts between D10 and D17, mostly at D14, with a median persistence duration of 28 days. Expansion occurred at each dose tested; without consistent relationship between administered dose and magnitude of expansion. One patient treated at DL2 presented a high expansion and a long persistence (very low detection by flow at D120). UCART19 persistence could not be assessed beyond D42-D56 in 3 pts because the remaining CARs were ablated by the transplant conditioning regimen. No expansion was observed in 5/18 pts in whom early lymphocyte recovery was detected from D14. Of these 5 pts, 3 did not receive alemtuzumab. The role of clinical status, tumor burden and lymphodepleting regimen on UCART19 expansion is under investigation.
Anti-leukemic activity was evaluable in 16/20 pts (1 pt died at D15, 1 pt was not assessed at D28, 2 pts have not yet reached D28). After UCART19 infusion, 88% of evaluable pts (14/16) achieved CR or CRi by D28 or D42 and 86% (12/14) of these pts were MRD negative (MRD- stands for < 1x10-4 copies) by flow or qPCR. Two out of 16 pts had no expansion and showed refractory disease. Among 12 pts achieving MRD-, 5 pts remain in molecular remission 4.5 to 16.4 months post UCART19. In total, 11 pts underwent allo-SCT (5 in PALL and 6 in CALM). Preliminary data suggests that in the majority of pts, anti-leukemic activity is linked with CAR expansion.
Conclusion: Pooled data of 20 pts show an acceptable and manageable safety profile of UCART19. Severe CRS was reported in 15%. Only 2 G1 cutaneous acute GvHD were observed and no severe neurotoxicity was reported. In this heavily pre-treated population, 88% pts of evaluable pts (14/16) achieved CR or CRi of which 86% (12/14) achieved MRD-. All pts who achieved MRD- had evidence of UCART19 expansion. Updated data, including data for the highest dose level in CALM study, will be presented (NCT 02746952, NCT02808442).
3.1.3 Allogeneic CAR T-cells for AML using a sekdel platform
WP1.1 in ATECT concerned itself with an allogeneic platform that relied on genome-editing of the TCR receptor locus. During this work, which included some early clinical experience, we deter-mined several limitations of that approach: (1) Genome editing and CAR T-cell expression was un-coupled which made sorting difficult; (2) Genome editing resulted in translocations and also, (3) a very low level of contaminating TCR positive CAR T-cells was needed to prevent graft-versus-host disease (GvHD).
We have developed an alternative approach termed “TCR-KDEL”. The TCR/CD3 complex assem-bles together in the endoplasmic reticulum aligning several complementary trans-membrane polar residues together. If one of these protein components is missing, the entire TCR/CD3 complex fails to assemble and unassembled components are ubiquitinated. We propose to direct one of these components to the Golgi apparatus using a scFv-KDEL.
To test this, we have incorporated CD3-kdel into a tri-cistronic cassette with a CD19 CAR. We used a CD19 CAR since this is the gold-standard targeting approach and allows us to compare activity with control (CD19 CAR alone).
The CD19 CAR/KDEL was tested in vitro in comparison with CD19 CAR alone and no difference in function was observed (not shown). Next CD19 CAR was compared with CD19 CAR/KDEL in a stress model of efficacy and also a long-term model of xeno-GvHD.
We have validated the TCR/CD3 KDEL approach with CD19. In addition, we have developed a targeting array for AML. This is not yet published, so cannot be disclosed in this report but once published will be posted on the ATECT website.
3.2 Work-package 2
3.2.1 Introduction
Work-package 2 focused on the application of a remarkable molecule L19-IL2 (Darleukin). devel-oped by partner Philogen, to CAR T-cell therapy. Darleukin targets IL2 to the extracellular domain B of fibronectin (EDF-B). The fully human version of this immunocytokine (Darleukin®) has been tested in a randomized Phase IIb study in patients with metastatic melanoma, in combination with dacarbazine versus dacarbazine monotherapy. A registrational Phase II clinical trial for the intra-lesional administration in melanoma skin lesions is also ongoing.
Since L19-IL2 localizes precisely at sites of disseminated lymphoma we wished to explore whether combining CAR T-cells with Darleukin would allow superior CAR T-cell expansion and persistence given that cytokine would be delivered to the tumour bed
In addition, we considered the possibility of exploiting L19 for direct CAR targeting – could direct destruction of the tumour microenvironment enhance CAR T-cell activity?
3.2.2 Combining neovascularization targeting and CAR T-cell therapy
Neovascularization is a hallmark of many cancers. Partner Philogen has developed an antibody “L19” which targets the ED-B domain which is a selective marked of neovascularization. Notably, L19 crosses the species barrier to mice which allows for immunocompetent animal models.
We tested two approaches for combining the targeting of neovascularization with CAR tested in a GD2 CAR model: (a) CAR in combination with an L19-IL2 fusion (immunocytokine, Darleukin) and (b) Co-expressing L19 as a CAR with the GD2 CAR. Early on we identified potential synergy between GD2 CAR and immunocytokine but not between CAR and L19 (figure F3.10 below).
3.2.1 Validating the L19 CAR
To test the L19 CAR, we cloned the ED-B domain as a membrane anchored fragment to allow functional L19 CAR function without having to recapitulate a basement membrane in vitro. The ED-B domain was cloned as a trans-membrane protein by cloning in frame with the CD8 stalk; the ED-B was also cloned as a GPI anchored protein without and with a serine-glycine linker connect-ing it to the GPI anchor (Figure F3.11 below).
These constructs allowed testing of the L19 CAR in three different formats: CD8 stalk, IgG1 hinge or an Fc domain spacer. CARs typically have to have the optimal spacer domain selected empirical-ly. The L19-CD8 spacer CAR was taken forward for further testing.
3.2.2 In vivo work
We started with an immunocompromised model: CD19 CAR versus Raji tumour xenografts in NSG mice. This showed no benefit of either the CAR or Darleukin (not shown).
We next moved to an Immunocompetent model: GD2 transgenic CT26 tumours in Balb/c. The aim was to demonstrate additive benefit of Darleukin support to CAR therapy in a physiological context. For immunocompetent modelling, we employed the CT26 model challenged by GD2-CAR therapy. CT26 is a Balb/c derived colon carcinoma for which Eva has generated a single cell clone transgenic for GD2.
3.2.3 Conclusion:
There appears to be synergy between GD2 CAR and Darleukin but not with the L19 CAR. We plan to repeat the in vivo experiment to increase the power of the combined animal work. In addition, detailed microenvironmental analysis is ongoing.
3.3 Work-package 3
3.3.1 Introduction
The aims of WP3 involved several approaches to enhance adoptive immunotherapy. These ap-proaches included engineering CAR T-cells to secrete cytokines and antibody fragments as well as combining genome engineering strategies with CAR T-cells to enhance potency.
The former approaches are still being written up and cannot be disclosed in this document, but what is summarized below is the first experience with PD1 genome editing in an adoptive transfer model which has been published by Menger et al3.
3.3.2 Genome editing to enhance adoptive immunotherapy
Despite the promising efficacy of adoptive cell therapies including TILs and CAR T-cell therapies, complete response rates remain relatively low and outcomes in other cancers are less impressive. The immunosuppressive nature of the tumor microenvironment and the expression of immune-inhibitory ligands, such as PD-L1/CD274 by the tumor and stroma are considered key factors limit-ing efficacy. The addition of checkpoint inhibitors (CPI) to ACT protocols bypasses some mecha-nisms of immunosuppression, but associated toxicities remain a significant concern. To overcome PD-L1 mediated immunosuppression and reduce CPI-associated toxicities, we used TALEN tech-nology to render tumor-reactive T cells resistant to PD-1 signaling. We set out to demonstrate that inactivation of the PD-1 gene in melanoma-reactive CD8 þ T cells and in fibrosarcoma-reactive polyclonal T cells enhanced the persistence of PD-1 gene-modified T cells at the tumor site and in-creased tumor control.
Only the pair targeting the exon 2 sequence (Fig. F3.15A) caused detectable mismatch-identified mutagenesis (up to 27%) in the PD-1 gene, correlating with loss of PD-1 expression in up to 15% of electroporated EL4 cells. To investigate whether PD-1 inactivation of primary tumor-reactive T cells provided superior antitumor activity against the poorly immunogenic mouse melanoma B16 model, we used CD8 T-cell receptor transgenic (TCR Tg) cells specific to the melanoma differentiation an-tigen gp-100 (pmel-1).
In vitro activated pmel-1 T cells were electroporated with control GFP mRNA (CD8 wt ) or PD-1–targeting TALEN mRNA (CD8 PD-1Ex2 ), delivering a high transfection efficiency of >85% as confirmed by GFP expression. Western blot analysis confirmed transient TALEN expression one day (d1) after transfection (Fig. F3.15B) and Miseq analysis of the targeted sequence showed a high frequency of mutations (53% non-homologous end joining, NHEJ; Fig. F3.15C). Three days after transfection, PD-1–negative cells were enriched using magnetic beads before ACT; 1x10^6 CD8 wt or CD8 PD-1Ex2 pmel-1 cells and rhIL-2 were administered to B16 tumor-bearing mice.
Significantly, enhanced tumor control was consistently observed in mice receiving CD8 PD-1Ex2 cells compared with those treated with CD8 wt cells (Fig. F3.15D). Six days after transfer in vivo, a significant enrichment of tumor-infiltrating PD-1–negative pmel-1 cells was observed in mice treat-ed with CD8 PD-1Ex2 T cells (48.2%; Fig. 1E, top),which contributed to a 2-fold increase in the total num-ber of tumor-infiltrating pmel-1 cells (Fig. 1E, bottom).
To define the therapeutic potential and functional impact of PD-1 gene inactivation in a more phys-iologically relevant T cell population, we performed PD-1 gene editing in polyclonal tumor-reactive lymphocytes (TRL) from C57BL/6 SJL mice challenged with MCA205 cells. CD4+and CD8+ T cells were isolated from the tumor (TILs) and tumor-draining lymph node (TDLN) via positive selection. After electroporation of the donor TRL,we observed a transfection efficiency>65% and we identi-fied TALEN-induced PD-1 gene mutations by Mi seq analys is in 26%of CD4+ Tcells and almost 40% of CD8+ Tcells.
In parallel to the generation of the donor mock-transfected (TRL wt ) and PD-1–edited TRL cells (TRL PD-1Ex2 ), we challenged a separate group of C57BL/6 mice with MCA205 tumors. Four days later, tumor-bearing mice were treated with TBI followed by ACT with TRL wt or TRL PD-1Ex2 cells and rhIL-2. Six days after transfer, analysis of PD-1 expression on TILs from mice re-ceiving TRL wt or TRL PD-1EX2 revealed a 15% to 20% average increase in PD-1T cells (Fig. 13.6A and B). Consistent with our findings in the TCR Tg model, a significant accumulation of PD-1- cells was observed only
PD-1 gene inactivation significantly increased the activity of our ACT protocol, and anti-tumor ac-tivity was dependent on both CD8+ and CD4+ T cells as administration of depleting anti-CD8 or MHC-II–blocking antibodies ablated antitumor responses (Fig. 3.17). No evidence of tumor recur-rence was observed after MCA205 re-challenge on day 50 in mice that had previously rejected tu-mors (data no shown).
Our data demonstrate the feasibility and potential clinical relevance of gene-editing approaches, conferring superior in vivo activity in the context of adoptive cell therapy protocols. Of relevance, we observed no evidence of toxicity (weight and physiologic alterations) after ACT. To the best of our knowledge, this was the first proof-of-concept study illustrating enhancement and persistence of antitumor responses using targeted genome editing of primary tumor reactive Tcells.
Further, our data indicate that the primary mechanism by which PD-1 gene inactivation affects tu-mor-reactive Tcells is by regulating their ability to survive rather than increasing their activity on a per cell basis, which aligns with the original description of the role of PD-1 signaling in T-cell apoptosis.
Although we investigated PD-1as the initial proof-of-concept target, the technology and approach described potentially allow the permanent disruption of other inhibitory check-points (one or more), considerably advancing the design of the next generation of cancer immunotherapies.
Potential Impact:
4.1 Impact of ATECT research output
ATECT has been at the forefront of engineered T-cell therapy and has considerable impact in the field. Clinical and research output from the ATECT consortium has the potential to develop new CAR T-cell approaches which are easier and cheaper to manufacture.
CAR T-cell therapy has shown activity in lymphoid malignancy, however sold cancer are considered more challenging due to poorer T-cell engraftment and survival within the tumour bed. Demonstration that genetic editing of checkpoint blockade receptors in tumour-specific T-cells can enhance their engraftment and survival within the tumour leading to increased activity.
4.1.1 Off-the-shelf allogeneic / gene-edited CAR T-cells
ATECT delivered the first-in-man genome editing CAR T-cells2. The UCART approach initially de-scribed in ATECT has been adopted by Servier, Pfizer and more recently a large bio-pharma start-up AlloGene. More generally, the clinical data generated set a considerable regulatory and techno-logical precedent and opened the field to similar approaches including approaches using different genome editing platforms such as CrispR/CAS9 and Zinc Finger Nucleases. The initial clinical data generated by ATECT opens the way for “off-the-shelf” CAR T-cells which potentially increases the availability and reduces the cost of this treatment approach.
4.1.2 Automated production of CAR T-cells
In addition, the CARD was to our knowledge the first CAR T-cell study to use an automated manufacturing platform. Although the products for this study will be manufactured individually for each patient, again use of an automated platform reduces costs and potentially increases availability of CAR T-cells for more patients so this was an important proof of concept and heralds wide-spread adoption of the Miltenyi Prodigy platform by CAR T-cell companies globally.
4.1.3 Allogeneic 2nd party CAR T-cells
Allogeneic haematopoietic stem cell transplantation (HSCT) is still a mainstay of treating relapsed or high-risk ALL. Enhancement or consolidation of HSCT with CAR donor leukocyte infusion is an attractive strategy and has been considered in the past but concerns over causing GvHD have lim-ited clinical exploration. In ATECT, 2nd party (HSCT donor) derived CD19 CAR T-cells were tested in the setting of relapse post HSCT. Low incidence of GvHD sets the stage for earlier intervention, likely prophylactically, as CAR19 DLI. This will change the face of HSCT for B-cell malignancies.
4.1.4 Genome editing for enhanced T-cell activity
Genome editing can be deployed to modify other T-cell attributes in addition to the propensity for T-cells to cause GvHD. In ATECT, we explored the disruption of the PD1 gene by editing in adop-tively transferred tumour-specific T-cells. This work has led to similar approaches using other ge-nome editing platforms as well as translation by others into clinical studies.
4.1.5 Other scientific impacts
CAR T-cells face inhibitory signals in the tumour microenvironment, and prevention of this local immune suppressive effect will likely be critical in permitting effective tumour control. As yet un-published data regarding perturbation of the microenvironment may impact on the field particularly when translated to clinical studies.
4.2 Dissemination
4.2.1 Section A (public) published manuscripts
1. Title: Enhanced CAR T cell expansion and pro-longed persistence in pediatric ALL patients treated with a low affinity CD19CAR, Authors: Ghorashian et al, Reference: Nature Medicine (2019, in press), Open Access: No
2. Title: Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells., Authors: Qasim et al, Reference: Sci Transl Med. 2017 Jan 25;9(374), Open Access: No
3. Title: TALEN-Mediated Inactivation of PD-1 in Tumor-Reactive Lymphocytes Promotes Intratumoral T-cell Persistence and Rejection of Established Tumors, Authors: Menger et al. Reference: Cancer Res. 2016 Apr 15;76(8):2087-93,, Open Access: YES
4. Title: The end of omics? High dimensional single cell analysis in precision medicine., Authors: Galli E et al., Reference: Eur J Immunol. 2019 Feb;49(2):212-220, Open Access: No
5. Title: Graft-versus-host disease, but not graft-versus-leukemia immunity, is mediated by GM-CSF-licensed myeloid cells. Tugues S et al., Reference: Sci Transl Med. 2018 Nov 28;10(469), Open Access: No
4.2.2 Scientific or technical presentations
1. Title: Advanced T-cell applications for Cancer, Authors: Martin Pule, Meeting: Tandem BMT Meeting ASBMT 2016, Open Access: Yes
2. Title: CAR T-cell therapy for Lymphoma, Authors: Martin Pule, Meeting: British Society for Haematology 2016, Open Access: No
3. Title: CAR T-cell therapy for Leukemia, Authors: Martin Pule, Meeting: European Haematology Association 2016, Open Access: No
4. Title: Lentiviral purification and concentration using genetically encoded packaging cell factor, Authors: Leila Mekkaoui and Martin Pule, Meeting: European Society for Cell and Gene Therapy 2017, Open Access: Yes
5. Title: Construction and Pre-clinical Evaluation of a new anti-CD19 Chimeric Antigen Receptor, Authors: Ann Kramer and Martin Pule, Meeting: European Society for Cell and Gene Therapy 2016, Open Access: Yes
6. Title: T-cell engineering for Cancer Applications, Authors: Martin Pule, Meeting: International Society for Cell Therapy 2017, Open Access: No
7. Title: T-cell engineering for Cancer Applications, Authors: Martin Pule, Meeting: EORTC meeting 2017, Open Access: No
8. Title: Advanced T-cell engineering for Cancer Applications, Authors: Martin Pule, Meeting: SITC meeting 2017, Open Access: No
9. Title: Advanced T-cell engineering for Cancer Applications, Authors: Martin Pule, Meeting: Keystone meeting 2018, Open Access: No
10. Title: First in man gene-edited CAR T-cell thera-py, Authors: Waseem Qasim, Meeting: American Society for Haematology 2016, Open Access: No
11. Title: Automated CAR T-cell manufacture with the Miltenyi Prodigy, Authors: Claire Roddie, Meeting: American Society for Haematology 2018, Open Access: No
4.3.2 Public Engagement
“War in the Blood” is a 90-minute documentary aired on the 7th of July 2019 on BBC2 channel. It was produced by Minnow Films and directed by Arthur Carey. It features two investigators of ATECT – Martin Pule and Claire Roddie and follows two patients enrolled onto experimental phase I CAR T-cell clinical studies. One of the patients is recruited to the CARD study. The documentary shows the complex interplay between science teams, clinical teams and with the clinical trial sub-jects – the patients at the heart of the matter.
http://www.minnowfilms.co.uk/in-production/War_in_the_Blood.html
https://www.bbc.co.uk/programmes/m0006nzt
List of Websites:
Project website: https://atect-fp7.org/
Scientific Coordinator Contact:
Dr Martin Pule
Senior Lecturer, consultant Haematologist
Department of Haematology
Cancer Institute, University College London
72 Huntley Street – London – WC1E 6BT – UK
E-mail:m.pule@ucl.ac.uk
Administrative Coordinator Contact:
Mr Panos Papoutsis
European Project Manager
European Research and Innovation Office, University College London
Maple House, 7th floor, 149 Tottenham Court Road, London W1T 7NF
E-mail:p.papoutsis@ucl.ac.uk
The ATECT consortium aimed to improve Chimeric Antigen Receptor (CAR) T-cell cancer therapy.
Work-page 1: The main aim of work-package was to address the cost and complexity of CAR T-cell manufacturing processes. One aspect of this was to explore the use of allogeneic CD19 CAR T-cells in two clinical studies. One clinical study (“CARD”) used matched allogeneic CAR T-cells. In addition, this study explored the use of automated manufacture of CD19 CAR T-cells. Manufactur-ing has been successful. Clinical data from this study will be presented late 2019 or early 2020.
A second more complex allogeneic approach was explored in the “UCART19” study. This ap-proach uses fully mismatched donor CD19 CAR T-cells. To overcome graft-versus-host disease (GvHD), concomitant TALEN mediated disruption of the TCR gene was performed; to overcome rejection, the CD52 gene was similarly disrupted allowing use of the powerful immunosuppressant CD52 targeting therapeutic antibody Alemtuzumab as conditioning. Two patients with infant B-ALL (in whom autologous CAR T-cell production) received UCART19 and outcome from these patients was reported as the first-in-man experience with genome-edited CAR T-cells. A key partner for this work, Cellectis, left the consortium and UCART19 studies continued in a phase I study in children and adults outside the consortium.
A third approach was developed in work-package 1: targeting of AML with an allogeneic approach. Based on what we learned from clinical exploration of UCART19, a protein-based allogeneic ap-proach was developed which simplified manufacture. Since AML stem-cells are heterogeneous in terms of antigen expression between and even within patients, a multi-targeting strategy was de-vised. The consortium has taken this project close to the point of clinical translation.
Work-package 2 focused on exploiting neovascularization which is an intrinsic part of the biology of most cancers. L19 is an antibody which recognizes the extracellular B domain of fibronectin. L19-IL2 (Darleukin) is an immunocytokine which is directed to neovascularization. We explored whether co-administration of L19-IL2 would enhance CAR T-cell engraftment at the tumour site. In addition, we asked whether co-expression of an L19 CAR along with the cancer antigen CAR would enhance CAR T-cell activity by disrupting the microenvironment. We found that combina-tion of L19-IL2 with CAR enhanced activity, but the L19-CAR did not.
Work-package 3 focused on a number of different ways to overcome the hostile microenviron-ment. One approach was to use genome editing to disrupt inhibitory receptors on adoptively trans-ferred T-cells. Targeting PD1 as an example inhibitory receptor, we showed increased immune ac-tivity. Other approaches included engineering T-cells to release immune blockade inhibitory anti-bodies or immune-activatory cytokines.
Overall, the ATECT consortium achieved several landmarks: the first-in-man genome edited al-logeneic CAR T-cells, the first CAR T-cells manufactured by automated production and the first demonstration in vivo of enhancing T-cell activity by disruption of the PD1 gene by genome edit-ing. In addition, ATECT explored several ways of enhancing activity of adoptively transferred T-cells.
Project Context and Objectives:
2.1 Introduction:
Chimeric Antigen Receptors (CARs), generated by fusing the antigen-binding region of a monoclo-nal antibody (mAb) to intracellular T-cell signalling domains1, hold the promise to revolutionize cancer treatment. Introduction of genes coding for CARs into T-cells using integrating vectors en-dow antigen recognition independent of MHC restriction. Recent clinical data leave no doubt that this new form of cancer therapy can be remarkably effective, engendering long-lived remissions in patients with refractory disease.
There are, however, considerable barriers to be overcome to take this new form of therapy. Some of these barriers are practical – for instance can we develop ways of making such products cheaper and easier. Other barriers relate to engraftment of engineered T-cells in the face of hostile microen-vironment. To allow CAR T-cell therapy to be more widely applicable, advanced cellular engineer-ing approaches need to be applied to overcome these barriers.
2.2 Technological Background:
The ATECT consortium proposed to address these limitations through advanced cellular engineer-ing. The central technological theme of this consortium is the application of gene editing strategies alongside advanced standard methods of genetic modification with insertional vectors, which we describe as a combination of advanced “positive” and “negative” engineering (figure F2.1).
T-cell engineering strategies for Cancer therapy, either Chimeric Antigen Receptors (CARs) or TCR transfer holds promise to revolutionize cancer treatment. There are, however, considerable barriers to be overcome to take this form of therapy to a format that can benefit all EU citizens with a wide range of common cancers. The aim of this consortium is to exploit advances in T-cell engineering to allow the full potential of CAR therapy to be unleashed.
At present, CAR therapy requires a bespoke autologous therapeutic product for each patient. This greatly limits practicality, scalability and commercialisation. The development of a strategy for creation of universal engineered T-cells is the first key aim of this consortium. There is an in-creased appreciation of the immunological hostilities (CAR) T-cells face in the tumour microenvi-ronment, and prevention of this local immune suppressive effect will likely be critical in permitting effective tumour control.
The second main aim of this proposal is therefore to engineer CAR T-cells to be resistant to the hostile microenvironment. CAR T-cells can only be effective if they can access the tumour site. Exploiting the fact that neo-angiogenesis is a hallmark of neoplastic progression, the third aim of the consortium is to utilise endothelial cues of neo-angiogenesis to direct CAR T-cell migration and activity.
We planned to develop strategies to engineer T-cells to be highly effective anti-cancer agents. A key part of this is to engineer them to be resistant to the hostile environment that tumour cells gen-erate around them. A key part of this is to engineer the T-cell to secrete cytokines into the tumour bed.
2.3 Partner Organizations:
Partner 1: UCL | University College London : UCL is Europe’s leading health research multidis-ciplinary university with the one of the world's major concentrations of biomedical researchers grouping 8,000 staff and 22,000 studentst.. UCL has a particular expertise in cell and Gene Thera-py. Much of the work of the ATECT consortium will be executed at the UCL Cancer Institute.
Partner 2: CTX | Cellectis Therapeutics: Founded in France in 1999, the Cellectis Group is based on highly specific DNA engineering technologies. Cellectis is today one of the world leading com-panies in the field of genome engineering. The Group has a workforce of 230 employees working on 5 sites worldwide: Paris & Evry in France. Cellectis Therapeutics is a subsidiary of Cellectis. Its aim is innovative strategies for the development of wide-reaching therapeutic treatments based on core cellectis technology.
Partner 3: PHL | Philogen: Philogen was founded in 1996 with the mission to develop new bio-pharmaceuticals for the treatment of angiogenesis-related disorders, such as cancer and rheumatoid arthritis. The company has been a pioneer in the isolation, engineering and clinical development of lead products capable of targeting angiogenesis in vivo and has been the first in the world to demonstrate that human monoclonal antibodies can efficiently and selectively target the tumor neo- vasculatur.
Partner 4: NKI | Netherlands Cancer Institute: The Netherlands Cancer Institute was established in 1913. The Institute accommodates approximately 550 scientists and scientific support personnel, 53 medical specialists, 180 beds, an out-patient clinic that receives 183,000 patients each year, 5 operating theatres and 9 irradiation units. It is the only dedicated cancer centre in The Netherlands and maintains an important role as a national and international centre of scientific and clinical ex-pertise, development and training.
Partner 5: UZH | University of Zurich: Founded in 1833, the University of Zurich is Switzer-land’s largest university, with a current enrolment of over 26,000 students. UZH is made up of sev-en faculties covering approximately 100 different subject areas. As a member of the “League of European Research Universities” (LERU), UZH is one of Europe’s most prestigious research insti-tutions.. Most of the ATECT will be carried out at the Institute of experimental Immunology at the University of Zurich. Read more about the the [UZH], or the [Institute of experimental Immunology].
Project Results:
3. DESCRIPTION OF FINDINGS
3.1 Findings from work-package 1
3.1.1 Introduction for WP1
WP1 is the clinical study work-package for ATECT. In this WP, we aimed to explore the use of al-logeneic CAR T-cell therapy in two clinical studies in B-cell malignancies and to prepare for a fur-ther clinical study in acute myeloid leukaemia (AML).
The two allogeneic CAR T-cell studies tested two different approaches to allogeneic CAR T-cells. With the first study (CARD), matched donors were used as the source of CAR T-cells in the setting of allogeneic haematopoietic stem cell transplant (allo HSCT). In the second study (UCART19), completely mismatched donors were used, and allogeneic effects abrogated by genome editing.
The third aim of this work-package was to develop an allogeneic CAR T-cell strategy for AML.
3.1.2 Automated manufacture and allogeneic 2nd party CAR T-cells for B-cell malignancies
The CARD study aimed to test 2nd party CD19-specific CAR T-cells in the setting of patients with with B-cell malignancies who had relapsed after allo-HSCT. The CAR T-cells would be derived from the HSCT donors. The main clinical question to be asked was the incidence of graft-versus host disease (GvHD). In addition, CARD was the first study to open which used an automated manufacturing process since we could be guaranteed of excellent starting material from the allo-HSCT donors.
The transgene used for the CARD study is shown in figure F3.1 below and incorporated a suicide gene called RQR8 along with a novel CD19 CAR based on the 4G7 binder (figure F3.1).
An automated manufacturing process was developed for this clinical study. This was based on the Miltenyi Prodigy device. At the time this study was initiated, all CAR T-cell products were generat-ed using an open process. This requires very specialized clean-rooms and skilled operators. The Miltenyi Prodigy device is an automated closed fluid handling system with integrated culture sys-tem. Much lower stringency of environment is required for its operation and far less technician time.
The CARD study obtained regulatory approval from the UK regulator in 2017. At the time of writ-ing of this report, we have manufactured donor-derived CD19 CAR T-cell products for 12 patients. This demonstrates feasibility of the concept of approaching, consenting and harvesting matched donors for the manufacture of CAR T-cell products where autologous collections are not possible.
The details of the products are outlined in the table 4 below. All met release criteria (one QP release is pending). Using the CliniMACS Prodigy, the median transduction efficiency of the products at a MOI of 5 was 41.5% (range 13.0- 56.1%) and the CD3 viability was uniformly excellent at >90% for all products. Using this device there was enrichment for naïve and central memory phenotypes within the products. PD-1 expression demonstrated that the final products did not exhibit and ex-hausted phenotype.
CARD clinical data has not yet been presented or published so cannot be included in this final re-port. While we have recruited the number we planned to on original writing of the ATECT proposal (9 patients), given the data are interesting and we have altered conditioning on the study, CARD has now been extended to 14 patients and will proceed beyond the end of ATECT.
A clinical update for CARD is scheduled for the American Society for Haematology meeting in 2019. Any updates or clinical publications will be posted to the ATECT website.
3.1.2 Allogeneic 3rd party CAR T-cells for B-cell malignancies
The UCART19 study aimed to test fully allogeneic “off-the-shelf” CAR T-cells. At time of writing this proposal, CAR T-cell products were autologous. There are considerable limitations to autolo-gous CAR T-cell production. The main limitation is the time taken to produce a CAR T-cell prod-uct. A patient with a rapidly advancing malignancy may not have 3-4 weeks a typical production / release. Further, a patient who has had treatment with multiple rounds of combination chemothera-py, or other treatments such as radiotherapy and allo-HSCT may be T-cell lymphopenic or may have low quality T-cells. Finally, autologous manufacture is “bespoke” and hence cannot make use of economies of scale for manufacture.
Allogeneic approaches are complicated by two immunologic phenomena. Firstly, CAR T-cells ex-pressing a native TCR can cause graft-versus-host disease (GvHD) if the native TCR reacts against the recipient. GvHD is very well understood in allogeneic HSCT, particularly with significant mis-match such as in haploidentical HSCT. Further, transfusion associated GvHD (taGvHD) gives per-haps the best indication of the possibility of GvHD in a completely mismatched setting. In the transfusion era before leukodepletion, packed red cells occasionally caused ta GvHD due to pas-senger lymphocytes often in recipients who were immunosuppressed. As few as 10,000 lympho-cytes were considered sufficient to cause fatal taGvHD.
The other immunological consideration is that of immune rejection of CAR T-cells by the recipient. It was known that a reasonable period of engraftment is required for activity, and in B-ALL a long period of engraftment appeared needed for long-term disease control. From experience with alloge-neic HSCT, the usual conditioning with Fludarabine and Cyclophosphamide seemed unlikely to prevent rejection. Strategies to disrupt the HLA locus would result in the now “null” CAR T-cells being vulnerable to NK cell depletion.
The strategy we devised used TALEN directed genome editing of two loci: the TCR alpha locus and the CD52 locus. Disruption of the TCR alpha locus would lead to disruption of TCR and hence CD3 expression and thus prevent GvHD. CD52 is a densely expressed small GPI anchored protein whose precise function is unknown. CD52 is a pan-lymphoid marker. Alemtuzumab (Campath 1H) is a highly lytic therapeutic antibody currently marketed by Sanofi which targets CD52. Disruption of CD52 expression would allow addition of Alemtuzumab to the standard Fludarabine / Cyclo-phosphamide conditioning which would deplete the recipient’s T-cells but would not target the CAR T-cells. The manufacturing strategy is detailed in the figure below.
Given that this was a highly experimental approach, a phase 0 approach was taken. A considerable unmet need for therapeutic options is infant ALL. CAR T-cells cannot be currently generated from infants. Universal CAR19 (UCART19) T cells by lentiviral transduction of non–human leukocyte antigen–matched donor cells and simultaneous transcription activator-like effector nuclease (TALEN)–mediated gene editing of T cell receptor a chain and CD52 gene loci from two health do-nors. Two infants with relapsed refractory CD19 + B cell acute lymphoblastic leukemia received lymphodepleting chemotherapy and anti-CD52 serotherapy, followed by a single-dose infusion of UCART19 cells. These findings were reported by Qasim et al2 and key findings are summarized in figures F3.4 and F3.5 below.
Molecular remissions were achieved within 28 days in both infants, and UCART19 cells persisted until conditioning ahead of successful allogeneic stem cell transplantation. Both children remain in remission now over 2 years following this procedure. Some aplasia and some signs of possible GvHD were noted but resolved following transplant. This phase 0 experiment demonstrated the therapeutic potential of gene-editing technology and the UCART19 approach.
Cellectis left the ATECT consortium however, and consequently the UCART19 as planned was not continued within the consortium. However, two UCART19 studies did start outside the consortium in adult and paediatric B-ALL respectively (NCT02746952 and NCT02808442). This has recently been reported on at the American Society of Haematology
American Society for Haematology, 2019, #896
Preliminary Data on Safety, Cellular Kinetics and Anti-Leukemic Activity of UCART19, an Al-logeneic Anti-CD19 CAR T-Cell Product, in a Pool of Adult and Pediatric Patients with High-Risk CD19+ Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia.
Presented by: Reuben Benjamin
UCART19 is an allogeneic, genetically modified CAR T-cell product (anti-CD19 scFv- 41BB-CD3ζ) manufactured from healthy donor T cells, in which TRAC and CD52 genes have been knocked out to allow its administration in non-HLA matched patients (pts).
Aims: Preliminary safety/anti leukemic and cellular kinetics data of UCART19 administered to pediatric and adult patients with R/R B-ALL are reported.
Methods: Data of patients included in the ongoing CALM study (adult) and PALL study (pediat-ric) have been pooled. CALM is a dose-escalation study (approximate dose level [DL] 1: 1x105 cells/kg, DL2: 1x106 cells/kg, DL3: 3x106 cells/kg) and PALL is testing a unique dose of 1.1 to 2.3x106 cells/kg. Eligible patients presented with a morphological disease (>5%) or MRD load ≥1x10-3. A lymphodepleting regimen (LD) combining cyclophosphamide (1500 mg/m² in CALM, 120 mg/kg in PALL) and fludarabine (90 mg/m2 in CALM, 150 mg/m2 in PALL) without (FC) or with alemtuzumab (FCA) (1 mg/kg) was administered one week before UCART19 infu-sion on Day 0 (D0). Cellular kinetics of UCART19 was assessed by flow cytometry (flow) in CALM and by vector copy number in PALL.
Results: As of 15 July 2018, 20 pts had received at least one UCART19 infusion, including 13 pts in CALM (6 at DL1; 6 at DL2 and 1 at DL3) and 7 pts in PALL. Seventeen pts had completed D28 evaluation (1 pt died at D15, 2 pts have not reached D28 yet). Prior to LD, blasts % in the bone marrow (median [range]) was 6% [0-68%] in PALL and 25% [0-96%] in CALM. Seventeen pts received LD with FCA and 3 pts received LD with FC.
Safety was evaluable in 18/20 pts. Cytokine release syndrome (CRS) was reported in 17/18 pts and was mild and reversible in the majority of cases (2 G1, 12 G2, 2 G3, 1 G4). One pt died in context of CRS G4 and neutropenic sepsis. According to data available to date, the severity of CRS does not seem to follow the levels of serum cytokines (IL-6, IL-10 and IFNγ). Mild self-limited neurotoxicity events were reported in 6 pts (5 G1 and 1 G2). Two pts (1 infant and 1 adult) developed G1 acute skin GvHD (non-biopsy proven for 1 pt) that was reversible with steroids.
Grade 1-4 viral infections were reported in 8/18 pts. The majority of events recovered, except in 2 pts who died after allo-SCT in context of prolonged cytopenia (defined as persistent G4 beyond D42 post UCART19 infusion): 1 child with BK virus infection and 1 adult with adenovirus infection.
A further 4 pts developed prolonged cytopenia and all recovered after allo-SCT.
Cellular kinetic data was available for 18/20 pts. UCART19 was detectable in blood from D7. Peak expansion was observed in 72% (13/18) pts between D10 and D17, mostly at D14, with a median persistence duration of 28 days. Expansion occurred at each dose tested; without consistent relationship between administered dose and magnitude of expansion. One patient treated at DL2 presented a high expansion and a long persistence (very low detection by flow at D120). UCART19 persistence could not be assessed beyond D42-D56 in 3 pts because the remaining CARs were ablated by the transplant conditioning regimen. No expansion was observed in 5/18 pts in whom early lymphocyte recovery was detected from D14. Of these 5 pts, 3 did not receive alemtuzumab. The role of clinical status, tumor burden and lymphodepleting regimen on UCART19 expansion is under investigation.
Anti-leukemic activity was evaluable in 16/20 pts (1 pt died at D15, 1 pt was not assessed at D28, 2 pts have not yet reached D28). After UCART19 infusion, 88% of evaluable pts (14/16) achieved CR or CRi by D28 or D42 and 86% (12/14) of these pts were MRD negative (MRD- stands for < 1x10-4 copies) by flow or qPCR. Two out of 16 pts had no expansion and showed refractory disease. Among 12 pts achieving MRD-, 5 pts remain in molecular remission 4.5 to 16.4 months post UCART19. In total, 11 pts underwent allo-SCT (5 in PALL and 6 in CALM). Preliminary data suggests that in the majority of pts, anti-leukemic activity is linked with CAR expansion.
Conclusion: Pooled data of 20 pts show an acceptable and manageable safety profile of UCART19. Severe CRS was reported in 15%. Only 2 G1 cutaneous acute GvHD were observed and no severe neurotoxicity was reported. In this heavily pre-treated population, 88% pts of evaluable pts (14/16) achieved CR or CRi of which 86% (12/14) achieved MRD-. All pts who achieved MRD- had evidence of UCART19 expansion. Updated data, including data for the highest dose level in CALM study, will be presented (NCT 02746952, NCT02808442).
3.1.3 Allogeneic CAR T-cells for AML using a sekdel platform
WP1.1 in ATECT concerned itself with an allogeneic platform that relied on genome-editing of the TCR receptor locus. During this work, which included some early clinical experience, we deter-mined several limitations of that approach: (1) Genome editing and CAR T-cell expression was un-coupled which made sorting difficult; (2) Genome editing resulted in translocations and also, (3) a very low level of contaminating TCR positive CAR T-cells was needed to prevent graft-versus-host disease (GvHD).
We have developed an alternative approach termed “TCR-KDEL”. The TCR/CD3 complex assem-bles together in the endoplasmic reticulum aligning several complementary trans-membrane polar residues together. If one of these protein components is missing, the entire TCR/CD3 complex fails to assemble and unassembled components are ubiquitinated. We propose to direct one of these components to the Golgi apparatus using a scFv-KDEL.
To test this, we have incorporated CD3-kdel into a tri-cistronic cassette with a CD19 CAR. We used a CD19 CAR since this is the gold-standard targeting approach and allows us to compare activity with control (CD19 CAR alone).
The CD19 CAR/KDEL was tested in vitro in comparison with CD19 CAR alone and no difference in function was observed (not shown). Next CD19 CAR was compared with CD19 CAR/KDEL in a stress model of efficacy and also a long-term model of xeno-GvHD.
We have validated the TCR/CD3 KDEL approach with CD19. In addition, we have developed a targeting array for AML. This is not yet published, so cannot be disclosed in this report but once published will be posted on the ATECT website.
3.2 Work-package 2
3.2.1 Introduction
Work-package 2 focused on the application of a remarkable molecule L19-IL2 (Darleukin). devel-oped by partner Philogen, to CAR T-cell therapy. Darleukin targets IL2 to the extracellular domain B of fibronectin (EDF-B). The fully human version of this immunocytokine (Darleukin®) has been tested in a randomized Phase IIb study in patients with metastatic melanoma, in combination with dacarbazine versus dacarbazine monotherapy. A registrational Phase II clinical trial for the intra-lesional administration in melanoma skin lesions is also ongoing.
Since L19-IL2 localizes precisely at sites of disseminated lymphoma we wished to explore whether combining CAR T-cells with Darleukin would allow superior CAR T-cell expansion and persistence given that cytokine would be delivered to the tumour bed
In addition, we considered the possibility of exploiting L19 for direct CAR targeting – could direct destruction of the tumour microenvironment enhance CAR T-cell activity?
3.2.2 Combining neovascularization targeting and CAR T-cell therapy
Neovascularization is a hallmark of many cancers. Partner Philogen has developed an antibody “L19” which targets the ED-B domain which is a selective marked of neovascularization. Notably, L19 crosses the species barrier to mice which allows for immunocompetent animal models.
We tested two approaches for combining the targeting of neovascularization with CAR tested in a GD2 CAR model: (a) CAR in combination with an L19-IL2 fusion (immunocytokine, Darleukin) and (b) Co-expressing L19 as a CAR with the GD2 CAR. Early on we identified potential synergy between GD2 CAR and immunocytokine but not between CAR and L19 (figure F3.10 below).
3.2.1 Validating the L19 CAR
To test the L19 CAR, we cloned the ED-B domain as a membrane anchored fragment to allow functional L19 CAR function without having to recapitulate a basement membrane in vitro. The ED-B domain was cloned as a trans-membrane protein by cloning in frame with the CD8 stalk; the ED-B was also cloned as a GPI anchored protein without and with a serine-glycine linker connect-ing it to the GPI anchor (Figure F3.11 below).
These constructs allowed testing of the L19 CAR in three different formats: CD8 stalk, IgG1 hinge or an Fc domain spacer. CARs typically have to have the optimal spacer domain selected empirical-ly. The L19-CD8 spacer CAR was taken forward for further testing.
3.2.2 In vivo work
We started with an immunocompromised model: CD19 CAR versus Raji tumour xenografts in NSG mice. This showed no benefit of either the CAR or Darleukin (not shown).
We next moved to an Immunocompetent model: GD2 transgenic CT26 tumours in Balb/c. The aim was to demonstrate additive benefit of Darleukin support to CAR therapy in a physiological context. For immunocompetent modelling, we employed the CT26 model challenged by GD2-CAR therapy. CT26 is a Balb/c derived colon carcinoma for which Eva has generated a single cell clone transgenic for GD2.
3.2.3 Conclusion:
There appears to be synergy between GD2 CAR and Darleukin but not with the L19 CAR. We plan to repeat the in vivo experiment to increase the power of the combined animal work. In addition, detailed microenvironmental analysis is ongoing.
3.3 Work-package 3
3.3.1 Introduction
The aims of WP3 involved several approaches to enhance adoptive immunotherapy. These ap-proaches included engineering CAR T-cells to secrete cytokines and antibody fragments as well as combining genome engineering strategies with CAR T-cells to enhance potency.
The former approaches are still being written up and cannot be disclosed in this document, but what is summarized below is the first experience with PD1 genome editing in an adoptive transfer model which has been published by Menger et al3.
3.3.2 Genome editing to enhance adoptive immunotherapy
Despite the promising efficacy of adoptive cell therapies including TILs and CAR T-cell therapies, complete response rates remain relatively low and outcomes in other cancers are less impressive. The immunosuppressive nature of the tumor microenvironment and the expression of immune-inhibitory ligands, such as PD-L1/CD274 by the tumor and stroma are considered key factors limit-ing efficacy. The addition of checkpoint inhibitors (CPI) to ACT protocols bypasses some mecha-nisms of immunosuppression, but associated toxicities remain a significant concern. To overcome PD-L1 mediated immunosuppression and reduce CPI-associated toxicities, we used TALEN tech-nology to render tumor-reactive T cells resistant to PD-1 signaling. We set out to demonstrate that inactivation of the PD-1 gene in melanoma-reactive CD8 þ T cells and in fibrosarcoma-reactive polyclonal T cells enhanced the persistence of PD-1 gene-modified T cells at the tumor site and in-creased tumor control.
Only the pair targeting the exon 2 sequence (Fig. F3.15A) caused detectable mismatch-identified mutagenesis (up to 27%) in the PD-1 gene, correlating with loss of PD-1 expression in up to 15% of electroporated EL4 cells. To investigate whether PD-1 inactivation of primary tumor-reactive T cells provided superior antitumor activity against the poorly immunogenic mouse melanoma B16 model, we used CD8 T-cell receptor transgenic (TCR Tg) cells specific to the melanoma differentiation an-tigen gp-100 (pmel-1).
In vitro activated pmel-1 T cells were electroporated with control GFP mRNA (CD8 wt ) or PD-1–targeting TALEN mRNA (CD8 PD-1Ex2 ), delivering a high transfection efficiency of >85% as confirmed by GFP expression. Western blot analysis confirmed transient TALEN expression one day (d1) after transfection (Fig. F3.15B) and Miseq analysis of the targeted sequence showed a high frequency of mutations (53% non-homologous end joining, NHEJ; Fig. F3.15C). Three days after transfection, PD-1–negative cells were enriched using magnetic beads before ACT; 1x10^6 CD8 wt or CD8 PD-1Ex2 pmel-1 cells and rhIL-2 were administered to B16 tumor-bearing mice.
Significantly, enhanced tumor control was consistently observed in mice receiving CD8 PD-1Ex2 cells compared with those treated with CD8 wt cells (Fig. F3.15D). Six days after transfer in vivo, a significant enrichment of tumor-infiltrating PD-1–negative pmel-1 cells was observed in mice treat-ed with CD8 PD-1Ex2 T cells (48.2%; Fig. 1E, top),which contributed to a 2-fold increase in the total num-ber of tumor-infiltrating pmel-1 cells (Fig. 1E, bottom).
To define the therapeutic potential and functional impact of PD-1 gene inactivation in a more phys-iologically relevant T cell population, we performed PD-1 gene editing in polyclonal tumor-reactive lymphocytes (TRL) from C57BL/6 SJL mice challenged with MCA205 cells. CD4+and CD8+ T cells were isolated from the tumor (TILs) and tumor-draining lymph node (TDLN) via positive selection. After electroporation of the donor TRL,we observed a transfection efficiency>65% and we identi-fied TALEN-induced PD-1 gene mutations by Mi seq analys is in 26%of CD4+ Tcells and almost 40% of CD8+ Tcells.
In parallel to the generation of the donor mock-transfected (TRL wt ) and PD-1–edited TRL cells (TRL PD-1Ex2 ), we challenged a separate group of C57BL/6 mice with MCA205 tumors. Four days later, tumor-bearing mice were treated with TBI followed by ACT with TRL wt or TRL PD-1Ex2 cells and rhIL-2. Six days after transfer, analysis of PD-1 expression on TILs from mice re-ceiving TRL wt or TRL PD-1EX2 revealed a 15% to 20% average increase in PD-1T cells (Fig. 13.6A and B). Consistent with our findings in the TCR Tg model, a significant accumulation of PD-1- cells was observed only
PD-1 gene inactivation significantly increased the activity of our ACT protocol, and anti-tumor ac-tivity was dependent on both CD8+ and CD4+ T cells as administration of depleting anti-CD8 or MHC-II–blocking antibodies ablated antitumor responses (Fig. 3.17). No evidence of tumor recur-rence was observed after MCA205 re-challenge on day 50 in mice that had previously rejected tu-mors (data no shown).
Our data demonstrate the feasibility and potential clinical relevance of gene-editing approaches, conferring superior in vivo activity in the context of adoptive cell therapy protocols. Of relevance, we observed no evidence of toxicity (weight and physiologic alterations) after ACT. To the best of our knowledge, this was the first proof-of-concept study illustrating enhancement and persistence of antitumor responses using targeted genome editing of primary tumor reactive Tcells.
Further, our data indicate that the primary mechanism by which PD-1 gene inactivation affects tu-mor-reactive Tcells is by regulating their ability to survive rather than increasing their activity on a per cell basis, which aligns with the original description of the role of PD-1 signaling in T-cell apoptosis.
Although we investigated PD-1as the initial proof-of-concept target, the technology and approach described potentially allow the permanent disruption of other inhibitory check-points (one or more), considerably advancing the design of the next generation of cancer immunotherapies.
Potential Impact:
4.1 Impact of ATECT research output
ATECT has been at the forefront of engineered T-cell therapy and has considerable impact in the field. Clinical and research output from the ATECT consortium has the potential to develop new CAR T-cell approaches which are easier and cheaper to manufacture.
CAR T-cell therapy has shown activity in lymphoid malignancy, however sold cancer are considered more challenging due to poorer T-cell engraftment and survival within the tumour bed. Demonstration that genetic editing of checkpoint blockade receptors in tumour-specific T-cells can enhance their engraftment and survival within the tumour leading to increased activity.
4.1.1 Off-the-shelf allogeneic / gene-edited CAR T-cells
ATECT delivered the first-in-man genome editing CAR T-cells2. The UCART approach initially de-scribed in ATECT has been adopted by Servier, Pfizer and more recently a large bio-pharma start-up AlloGene. More generally, the clinical data generated set a considerable regulatory and techno-logical precedent and opened the field to similar approaches including approaches using different genome editing platforms such as CrispR/CAS9 and Zinc Finger Nucleases. The initial clinical data generated by ATECT opens the way for “off-the-shelf” CAR T-cells which potentially increases the availability and reduces the cost of this treatment approach.
4.1.2 Automated production of CAR T-cells
In addition, the CARD was to our knowledge the first CAR T-cell study to use an automated manufacturing platform. Although the products for this study will be manufactured individually for each patient, again use of an automated platform reduces costs and potentially increases availability of CAR T-cells for more patients so this was an important proof of concept and heralds wide-spread adoption of the Miltenyi Prodigy platform by CAR T-cell companies globally.
4.1.3 Allogeneic 2nd party CAR T-cells
Allogeneic haematopoietic stem cell transplantation (HSCT) is still a mainstay of treating relapsed or high-risk ALL. Enhancement or consolidation of HSCT with CAR donor leukocyte infusion is an attractive strategy and has been considered in the past but concerns over causing GvHD have lim-ited clinical exploration. In ATECT, 2nd party (HSCT donor) derived CD19 CAR T-cells were tested in the setting of relapse post HSCT. Low incidence of GvHD sets the stage for earlier intervention, likely prophylactically, as CAR19 DLI. This will change the face of HSCT for B-cell malignancies.
4.1.4 Genome editing for enhanced T-cell activity
Genome editing can be deployed to modify other T-cell attributes in addition to the propensity for T-cells to cause GvHD. In ATECT, we explored the disruption of the PD1 gene by editing in adop-tively transferred tumour-specific T-cells. This work has led to similar approaches using other ge-nome editing platforms as well as translation by others into clinical studies.
4.1.5 Other scientific impacts
CAR T-cells face inhibitory signals in the tumour microenvironment, and prevention of this local immune suppressive effect will likely be critical in permitting effective tumour control. As yet un-published data regarding perturbation of the microenvironment may impact on the field particularly when translated to clinical studies.
4.2 Dissemination
4.2.1 Section A (public) published manuscripts
1. Title: Enhanced CAR T cell expansion and pro-longed persistence in pediatric ALL patients treated with a low affinity CD19CAR, Authors: Ghorashian et al, Reference: Nature Medicine (2019, in press), Open Access: No
2. Title: Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells., Authors: Qasim et al, Reference: Sci Transl Med. 2017 Jan 25;9(374), Open Access: No
3. Title: TALEN-Mediated Inactivation of PD-1 in Tumor-Reactive Lymphocytes Promotes Intratumoral T-cell Persistence and Rejection of Established Tumors, Authors: Menger et al. Reference: Cancer Res. 2016 Apr 15;76(8):2087-93,, Open Access: YES
4. Title: The end of omics? High dimensional single cell analysis in precision medicine., Authors: Galli E et al., Reference: Eur J Immunol. 2019 Feb;49(2):212-220, Open Access: No
5. Title: Graft-versus-host disease, but not graft-versus-leukemia immunity, is mediated by GM-CSF-licensed myeloid cells. Tugues S et al., Reference: Sci Transl Med. 2018 Nov 28;10(469), Open Access: No
4.2.2 Scientific or technical presentations
1. Title: Advanced T-cell applications for Cancer, Authors: Martin Pule, Meeting: Tandem BMT Meeting ASBMT 2016, Open Access: Yes
2. Title: CAR T-cell therapy for Lymphoma, Authors: Martin Pule, Meeting: British Society for Haematology 2016, Open Access: No
3. Title: CAR T-cell therapy for Leukemia, Authors: Martin Pule, Meeting: European Haematology Association 2016, Open Access: No
4. Title: Lentiviral purification and concentration using genetically encoded packaging cell factor, Authors: Leila Mekkaoui and Martin Pule, Meeting: European Society for Cell and Gene Therapy 2017, Open Access: Yes
5. Title: Construction and Pre-clinical Evaluation of a new anti-CD19 Chimeric Antigen Receptor, Authors: Ann Kramer and Martin Pule, Meeting: European Society for Cell and Gene Therapy 2016, Open Access: Yes
6. Title: T-cell engineering for Cancer Applications, Authors: Martin Pule, Meeting: International Society for Cell Therapy 2017, Open Access: No
7. Title: T-cell engineering for Cancer Applications, Authors: Martin Pule, Meeting: EORTC meeting 2017, Open Access: No
8. Title: Advanced T-cell engineering for Cancer Applications, Authors: Martin Pule, Meeting: SITC meeting 2017, Open Access: No
9. Title: Advanced T-cell engineering for Cancer Applications, Authors: Martin Pule, Meeting: Keystone meeting 2018, Open Access: No
10. Title: First in man gene-edited CAR T-cell thera-py, Authors: Waseem Qasim, Meeting: American Society for Haematology 2016, Open Access: No
11. Title: Automated CAR T-cell manufacture with the Miltenyi Prodigy, Authors: Claire Roddie, Meeting: American Society for Haematology 2018, Open Access: No
4.3.2 Public Engagement
“War in the Blood” is a 90-minute documentary aired on the 7th of July 2019 on BBC2 channel. It was produced by Minnow Films and directed by Arthur Carey. It features two investigators of ATECT – Martin Pule and Claire Roddie and follows two patients enrolled onto experimental phase I CAR T-cell clinical studies. One of the patients is recruited to the CARD study. The documentary shows the complex interplay between science teams, clinical teams and with the clinical trial sub-jects – the patients at the heart of the matter.
http://www.minnowfilms.co.uk/in-production/War_in_the_Blood.html
https://www.bbc.co.uk/programmes/m0006nzt
List of Websites:
Project website: https://atect-fp7.org/
Scientific Coordinator Contact:
Dr Martin Pule
Senior Lecturer, consultant Haematologist
Department of Haematology
Cancer Institute, University College London
72 Huntley Street – London – WC1E 6BT – UK
E-mail:m.pule@ucl.ac.uk
Administrative Coordinator Contact:
Mr Panos Papoutsis
European Project Manager
European Research and Innovation Office, University College London
Maple House, 7th floor, 149 Tottenham Court Road, London W1T 7NF
E-mail:p.papoutsis@ucl.ac.uk