Andrew Kaiser

Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

Sunitinib pretreatment improves tumor-infiltrating lymphocyte expansion by reduction in intratumoral content of myeloid-derived suppressor cells in human renal cell carcinoma.

Targeted therapy with sunitinib, pazopanib or everolimus has improved treatment outcome for patients with metastatic renal cell carcinoma patients (RCC). However, despite considerable efforts in sequential or combined modalities, durable remissions are rare. Immunotherapy like cytokine therapy with interleukin-2, T cell checkpoint blockade or adoptive T cell therapies can achieve long-term benefit and even cure. This raises the question of whether combining targeted therapy with immunotherapy could also be an effective treatment option for RCC patients. Sunitinib, one of the most frequently administered therapeutics in RCC patients has been implicated in impairing T cell activation and proliferation in vitro. In this work, we addressed whether this notion holds true for expansion of tumor-infiltrating lymphocytes (TILs) in sunitinib-treated patients. We compared resected primary RCC tumor material of patients pretreated with sunitinib with resection specimen from sunitinib-naïve patients. We found improved TIL expansion from sunitinib-pretreated tumor digests. These TIL products contained more PD-1 expressing TIL, while the regulatory T cell infiltration was not altered. The improved TIL expansion was associated with reduced intratumoral myeloid-derived suppressor cell (MDSC) content. Depletion of MDSCs from sunitinib-naïve RCC tissue-digest improved TIL expansion, proving the functional relevance of the MDSC alteration by sunitinib. Our in vivo results do not support previous in vitro observations of sunitinib inhibiting T cell function, but do provide a possible rationale for the combination of sunitinib with immunotherapy.

A tandem CD19/CD20 CAR lentiviral vector drives on-target and off-target antigen modulation in leukemia cell lines

BackgroundClinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse. To target hematologic malignancies with a chimeric antigen receptor (CAR) that targets two antigens with a single vector, and thus potentially lessen the chance of leukemic escape mutations, a tandem-CAR approach was investigated.MethodsAntigen binding domains from the FMC63 (anti-CD19) and Leu16 (anti-CD20) antibodies were linked in differing configurations to transmembrane and T cell signaling domains to create tandem-CARs. Expression on the surface of primary human T cells was induced by transduction with a single lentiviral vector (LV) encoding the tandem-CAR. Tandem-CARs were compared to single antigen targeting CARs in vitro and in vivo, and to an admixture of transduced cells expressing each CAR in vivo in immunodeficient (NSG) disease-bearing mice.ResultsTandem constructs efficient killed the Raji leukemia cell line both in vitro and in vivo. Tandem CARs generated less cytokine than the CD20 CAR, but similar to CD19 CARs, on their own. In co-culture experiments at low effector to target ratios with both single- and tandem- CAR-T cells, a rapid down-modulation of full-length CD19 expression was seen on leukemia targets. There also was a partial down-modulation of CD22, and to a lesser degree, of CD20. Our data also highlight the extreme sensitivity of the NALM-6 cell line to general lymphocyte-mediated cytotoxicity. While single and tandem constructs were effective in vivo in a standard setting, in a high-disease burden setting, the tandem CAR proved both effective and less toxic than an admixture of transduced T cell populations expressing single CARs.ConclusionTandem CARs are equally effective in standard disease models to single antigen specificity CARs, and may be both more effective and less toxic in a higher disease burden setting. This may be due to optimized cell killing with more moderate cytokine production. The rapid co-modulation of CD19, CD20, and CD22 may account for the ability to rapidly evolve escape mutants by selecting for leukemic clones that not require these target antigens for continued expansion.

Minimizing leukemia escape: implementing a dual anti-CD20- and CD19-scFv-based chimeric antigen receptor (CAR)

Adoptive immunotherapy with chimeric antigen receptor (CAR) transduced T lymphocytes has shown promising results in both pediatric and adult B cell malignancies. Nevertheless, both CAR-based and antibody-based anti-CD19 therapies, e.g. blinatumomab, have seen treatment failures attributed to the loss of CD19 or an epitope of CD19 on the surface of the malignant B cell. It may be possible to overcome antigen escape by targeting two tumor antigens simultaneously, i.e. CD19 and CD20 using a tandem construct with two scFv-based CAR binding domains. Lentiviral vectors encoding chimeric antigen receptors comprised of anti-CD19 and anti-CD20 targeting domains expressed alone or in tandem were transduced into T cells from healthy donors to generate the corresponding CAR19, CAR20, CAR19_20 (CD19 scFv more distal to the T cell plasma membrane) and CAR20_19 T cells (CD20 scFv distal). The transduced T cells were 50-70% CAR positive as determined by protein L flow cytometric analysis. Expression of CAR proteins of the expected molecular weight was confirmed by Western blot analysis of transduced T cells. When CAR-transduced T cells were combined with CD19+CD20+ Raji target cells, but not CD19-CD20- K562 cells, all four CAR T cell types demonstrated comparable efficient killing of leukemia targets (E:T ratio >2), and target-dependent induction of IFN-γ, as measured in co-culture supernatants by ELISA. We then began a series of in vitro co-culture experiments where we used very low E:T ratios to examine the potential for CAR-induced antigen loss on surviving leukemia cells. All CAR-T cells expressing an anti-CD19 scFv induced rapid loss of CD19 surface expression. In contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Upon flow cytometric analysis of surviving leukemia cells on day 5, CD19 expression was reduced to 3%, 48%, 73%, 90% and 93% of control when co-cultured with CAR T cells expressing CAR19_20, CAR20_19, CAR19, CAR20, and control T cells, respectively. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration. In conclusion, tandem CAR T cells are as effective as single CAR19 or CAR20 T cells in leukemia cell killing. Importantly, dual scFv-expressing CARs are more potent in preventing tumor antigen escape via target antigen down-regulation.

CD137+CD154− Expression As a Regulatory T Cell (Treg)-Specific Activation Signature for Identification and Sorting of Stable Human Tregs from In Vitro Expansion Cultures

Regulatory T cells (Tregs) are an attractive therapeutic tool for several different immune pathologies. Therapeutic Treg application often requires prolonged in vitro culture to generate sufficient Treg numbers or to optimize their functionality, e.g., via genetic engineering of their antigen receptors. However, purity of clinical Treg expansion cultures is highly variable, and currently, it is impossible to identify and separate stable Tregs from contaminating effector T cells, either ex vivo or after prior expansion. This represents a major obstacle for quality assurance of expanded Tregs and raises significant safety concerns. Here, we describe a Treg activation signature that allows identification and sorting of epigenetically imprinted Tregs even after prolonged in vitro culture. We show that short-term reactivation resulted in expression of CD137 but not CD154 on stable FoxP3+ Tregs that displayed a demethylated Treg-specific demethylated region, high suppressive potential, and lack of inflammatory cytokine expression. We also applied this Treg activation signature for rapid testing of chimeric antigen receptor functionality in human Tregs and identified major differences in the signaling requirements regarding CD137 versus CD28 costimulation. Taken together, CD137+CD154− expression emerges as a universal Treg activation signature ex vivo and upon in vitro expansion allowing the identification and isolation of epigenetically stable antigen-activated Tregs and providing a means for their rapid functional testing in vitro.

455. Automated Lentiviral Transduction of T Cells with CARS Using the CliniMACS Prodigy

T lymphocytes are exhibiting enormous potential in early phase clinical trials in patients with haematological malignancies. However, the complex procedures involved in the ex-vivo modification of T cells is labour intensive and currently limited to a small number of centres with the required infrastructure and expertise. To simplify procedures and widen applicability for clinical therapies, we have adopted the CliniMACS Prodigy platform to automate these multifaceted cell manufacturing processes. We found efficient lentiviral transduction of human T cells in a GMP compliant manner and demonstrate the feasibility of implementing this device in the manufacture of chimeric antigen receptor (CAR) based T cell immunotherapies. Eight automated T cell Transduction (TCT) processes have been performed using a self-inactivating third generation lentiviral vector encoding a CD19 specific CAR (CAR19), three of which using a clinical grade vector for final stage validation studies. Either fresh or cryopreserved peripheral blood mononuclear cells from non-mobilised leukapheresis from healthy donors were loaded onto the CliniMACS Prodigy using single use closed tubing sets. All cells were cultured in TexMACS media and activated with TransAct™. Transduction occurred 24-28hours post activation and cells were expanded for up to 8 days in the CentriCult-Unit enabling stable cell culture conditions and automated cell feeding. Finally, cells were harvested and cryopreserved to assess the functional capabilities of CAR19 T cells. Small scale comparison transductions were run in parallel to assess the efficiency of the automated T-cell modification process. The mean T cell expansion during automated cell cultivation was 16.2x (range 5.4-28.4x) with an average yield over 8 days of 14.5×108 total lymphocytes from a starting lymphocyte count of 1×108. This was comparable to cell expansion achieved in manual small scale experiments under the same activation conditions, 18.1x (range 11.5-27.5x). Successful transduction was also observed in the automated system with a mean transduction efficiency of 49.1% (range 23.9-64.9% CAR19+ T cells) which was again similar to transduction efficiencies achieved in manual small scale controls (mean of 51.8%). Flow cytometry analysis of the final product showed a high purity of CD45+CD3+ T cells (mean = 94%) with a relatively high frequency of CD8+ T cells (mean 48.9%). Further immunophenotyping revealed the bulk of the T cell product to be a mix of stem cell memory and central memory based on CD45RA, CD62L and CD95 expression with minimal expression of the T cell exhaustion marker PD-1. Additionally, CAR19 T cells generated using the automated procedure, were functional in cytotoxic activity both in vitro and in an in vivo mouse model. Importantly, these data are comparable to data generated from previous GMP manufacture of CAR19 T cells using the WAVE bioreactor with X-Vivo15 media and magnetic beads conjugated with anti-CD3/CD28 antibodies with the added advantage of increased simplicity in manufacture. In summary, we have demonstrated the feasibility of the CliniMACS Prodigy platform for the generation of CAR+ T cells for adoptive immunotherapy. Automated activation, transduction and expansion resulted in clinically relevant doses of CAR19 T cells with greatly reduced ‘hands-on’ operator time. Given the closed-system nature of the device, and automated features, the CliniMACS Prodigy should widen applicability of T-cell engineering beyond centres with highly specialised infrastructures.

653. Sialyl Glycolipid Stage-Specific Embryonic Antigen 4 (SSEA4) - A Novel Target for CAR T Cell Therapy of Solid Cancers

Owing to their capacity to eradicate tumors, T cells represent an attractive means for immunomodulation in cancer immunotherapy. In this context, chimeric antigen receptor (CAR) - based therapies are receiving increasing attention. The combination of antibody-derived specificity with T cell effector function renders the immune cells MHC-independent and even enables targeting of antigens for which there is immunological tolerance. T cells, genetically modified with CAR, have shown impressive success in the treatment of leukemia. However, the application of CAR T cells to the treatment of solid tumors remains challenging due to the lack of truly cancer-specific targets and an immunosuppressive tumor microenvironment hostile to T cells.We have identified the sialyl glycolipid stage-specific embryonic antigen 4 (SSEA4) as an epitope whose expression strongly correlates with metastasis and chemoresistence in triple negative breast cancer cells (TNBC). Single chain antibody fragments (scFv) were derived from an antibody that specifically recognizes this sialyl-glycolipid and were cloned into a lentiviral expression vector encoding a CAR containing an IgG1 spacer domain with CD137 and CD3z signaling domains. Healthy donor T cells were enriched by magnetic cell sorting and activated with TransActTM Reagent, a colloidal nanomatrix-based activation reagent, before lentiviral transduction of the anti-SSEA4 CAR expression construct. Engagement of SSEA4 by CAR expressing T cells induced T cell degranulation, secretion of inflammatory cytokines and resulted in an effective killing of SSEA4 expressing target cells.As TNBC patients are exposed to multiple rounds of chemotherapy and SSEA4 expression is found enriched in residual tumor cells surviving chemotherapy, a combinatorial approach using chemotherapy followed with CAR T cell therapy holds great promise to improve treatment outcome and overall survival of TNBC patients. Having assessed the performance of different anti-SSEA4 CAR constructs in vitro, current studies are focusing on in vivo functionality using mouse models.

Towards automated manufacturing of clinical scale gene-modified T cells

Adoptive immunotherapy using gene-modified T cells redirected against cancer has proven clinical efficacy and tremendous potential in several medical fields. However, such personalized medicine faces several challenges in the complexity associated with the current clinical manufacturing methods, which hampers dissemination. Conventionally, the preparation of autologous gene-modified T cells comprises many (open) handling steps, is labor intensive and is not adapted to treat large numbers of patients or for commercial manufacturing. Moreover, the cell-manufacturing process requires extensive training of personnel as well as a dedicated infrastructure, which restricts these clinical procedures to very few institutions worldwide. In order to face these challenges, Miltenyi Biotec has dedicated large efforts to further enable automation of cell manufacturing by developing a unique cell processing platform, the CliniMACS® Prodigy, which enables the automated manufacturing of clinical grade gene-modified T cells in a closed single-use tubing set. Starting from leukapheresis or whole blood products, the automated process enables magnetic labeling and enrichment of T cells, their subsequent stimulation, gene-modification with lentiviral vectors, expansion and final formulation with minimal user interaction. Within the process a novel stimulatory reagent has been implemented: MACS GMP TransAct™ in combination with TexMACS GMP Medium. TransAct is a colloidal reagent developed for polyclonal T cell stimulation that is soluble and can be removed by washing. The reagent is biodegradable, sterile filtered, and suitable for potent T cell activation, gene-modification, and expansion. Clinically relevant numbers of functional gene-modified T cells (>109) have been generated within 10-14 days using the automated manufacturing process. The flexibility and ease-of-use associated with this device and the developed process for clinical scale production of engineered T cells creates a solution for the treatment of large patient groups and facilitates economic commercial-scale manufacturing.