CAR-512

A review.Endogenous and engrafted T cells specifically recognize and eliminate tumor cells in model organisms and in humans.However, not all resident T cells are created equal in their therapeutic potential.This has galvanized investigators in the academic community to fashion T cells ex vivo for improved in vivo human applications.This has led to a series of remarkable clin. observations describing reduction and possibly elimination of tumor burdens based on administration of T cells that are genetically modified to enforce expression of chimeric antigen receptors (CARs).The prototypical CAR derives the specificity for tumor-associated antigen (TAA) by grafting the single-chain variable fragment region (for example, from a monoclonal antibody) onto an extracellular framework that is fused to an endodomain containing one or more signaling mols. (for example, co-opted from the T-cell receptor signaling complex).CARs directly bind to TAA without the need for antigen presentation by human leukocyte antigen resulting in the fully competent activation of genetically modified T cells (defined at a min. as antigen-dependent proliferation, cytokine production, serial killing and protection from activation-induced cell death).The field of applied immunotherapy administering CAR⁺ T cell has progressed from immunol. to immunotherapy based on three parallel tracts (Figure 1).The first tract encompasses the preclin. (i) discoveries of CARs and attendant signaling, (ii) understanding of T-cell subpopulations that contribute to the lysis of tumor cells and their contribution to persistence (sometimes referred to as memory), (iii) the investigation of gene transfer using viral and non-viral approaches to stably and reliably integrate transgenes and (iv) approaches to the physiol. activation and propagation of T cells that maintain their ability to recycle the effector functions in the tumor microenvironment.The second tract is the ability to safely manipulate the candidate research participant to both receive and tolerate one or more infusions of CAR⁺ T cells.The lymphopenic recipient appears to be the preferred patient for the sustained engraftment of T cells, including genetically modified T cells.Following adoptive immunotherapy, standard operating procedures are in place to manage tumor lysis syndrome, cytokine release syndrome and macrophage activation syndrome.The third tract is the bioprocessing to scale up and validate the production of CAR⁺ T cells in compliance with current good manufacturing practice (cGMP) for Phase I and II trials and is the basis of the manuscripts that this editorial is introducing.The series of reviews that accompany this editorial chiefly focus on the manufacture and release of clin.-grade T cells and the genetic modification to express introduced immunoreceptors.The articles describe the translation from discovery in the laboratory, to pressure-testing technologies in compliance with current good laboratory practice (cGLP), to production under cGMP.The experience of investigators and their teams seeking to administer CAR⁺ T cells is primarily driven by academic practices as this is currently the nexus for innovation ('tinkering') and the test bed for bioprocessing.Currently, most CAR⁺ T cells are both manufactured and infused at multiple points of care that are widely geog. distributed (Figure 2).This has both benefits and limitations.The upside of multiple groups undertaking the production of T cells in parallel is that this introduces desired heterogeneity between the academic facilities with inbuilt competition to obtain first-in-human clin. data.The phys. placement of facilities operating in compliance with cGMP in close proximity with recipients shortens the feedback loop between the infusing and manufacturing teams, which spurs innovation and real-time problem solving.The downside of academics producing and releasing CAR⁺ T cells is that most communities operate with scarce resources that stymie the implementation of new technologies, such as concerning automation and unbreached systems.Furthermore, the diversified approaches to manufacturing can complicate the assessment of one group's data compared with another.Biopharmaceutical companies will attempt to reduce variation in manufacturing as they seek registration of CAR⁺ T cells by introducing harmonization across their manufacturing facilities.Their general approach is to achieve this using centralized manufacturing in which CAR⁺ T cells are produced and released at a geog. distance from the candidate patient.However, the field of adoptive immunotherapy has a dearth of data as to whether centralized manufacturing of patient- and 3^rd party-derived CAR⁺ T cells is broadly feasible and does not result in ruinous costs being passed on to the recipients and their families.The administration of clin.-grade CAR⁺ T cells into thousands rather than tens to hundreds of recipients a year is a welcome goal to broaden the number of patients that can clin. benefit from this immunotherapy.Increasingly, specialized vendors are emerging to assist with this culturing and processing of genetically modified T cells.However, as the industry gears up to undertake this scale-up, there is a heightened tension between the cultures of innovation and application.Patients, oncologists, investigators and investors desire (immediate) access to the existing CAR⁺ T-cell therapies.This places a premium on the execution of manufacturing plans and the logistics therein.However, there is recognition that the current approaches to the fabrication of CAR species along with their insertion into propagated T cells and subsets needs to be refined and in some cases redesigned to meet the challenge of overcoming inter- and intra-tumor diversity of TAA expression.This is manifested by the drive to personalize T-cell therapies.A challenge is to maintain emphasis on new approaches to bioprocessing while at the same time codifying the supply chain of CAR⁺ T cells.This is where academia and industry may coexist, with the former developing and testing new ideas regarding genetic modification of T cells and the latter scaling to achieve global impact on health care.Such academic and industrial partnerships have recently emerged (for example, University of Pennsylvania with Novartis; Memorial Sloan Kettering Cancer Center and Fred Hutchinson Cancer Research Center with Juno Therapeutics; National Cancer Institute with Kite Pharma; Baylor College of Medicine with Bluebird Bio and Celgene) with other relationships in the works.It remains to be seen whether this coupling will keep the flame of innovation alive given the pressures around commercialization and its attendant needs such as protecting the intellectual property, which can limit timely communications and collaborations.For example, will market forces be sufficient to manufacture T cells for oncol. (as well as non-cancerous) diagnosis for which there are few candidate recipients.Will CAR⁺ T cells be manufactured in centralized facilities operating as stand-alone GMP facilities or will their production be undertaken at diverse locoregional facilities operating with a mandate to individualize the genetically modified product.The implementation of CAR⁺ T cells is now embraced by both nonprofit academic and for profit biopharmaceutical communities.However, achieving the widespread distribution of CAR⁺ T cells remains a critical step to the successful deployment of this gene therapy, which has led to the recent commercialization of T-cell therapies.For academic investigators, bioprocessing in compliance with cGLP and cGMP has largely remained an unfunded mandate that is necessary to overcome the 'valley of death' as biologics, such as T cells, move from the bench to the bedside.The clin. successes of CAR⁺ T cells in early-phase clin. trials has spurred renewed interest in bioprocessing in order to overcome the critical hurdles associated with delivering T cells to multiple bedsides.Indeed, the human application of CAR⁺ T cells is attempting to move from the boutique to the mainstream.This will require overcoming multiple problems, but perhaps chief among them is bioprocessing and the associated issues of distribution.The will to deliver multiple T-cell products to multiple recipients has moved from academia to industry as the for-profit entities have the resources and inclination to build an 'assembly line' to manufacture CAR⁺ T cells.'Amateurs talk tactics, professionals study logistics' (attributed to General Omar Bradley) is an apt quote for the current state of affairs regarding CAR⁺ T cells, as this era of applied immunol. appears to be about 'connecting the dots'.However, the dots should be connected in a cost-effective manner in order to bring the cost of goods under control.Else, the premise of CAR⁺ T cells will not achieve its promise.

Sarcomas are rare, mesenchymal tumors, representing about 10-15% of all childhood cancers. GD2 is a suitable target for chimeric antigen receptor (CAR) T-cell therapy due to its overexpression in several solid tumors. In this preclinical study, we investigated the potential use of iCasp9.2A.GD2.CAR-CD28.4-1BBζ (CAR.GD2) T-cells as a treatment option for patients who have GD2-positive sarcomas and we sought to identify factors shaping hostile tumor microenvironment in this setting. GD2 expression was evaluated by flow-cytometry on primary tumor biopsies of pediatric sarcoma patients. GD2 expression in sarcoma cells was also evaluated in response to an enhancer of zeste homolog 2 (EZH2) inhibitor (Tazemetostat). The antitumor activity of CAR.GD2 T-cells was evaluated both in vitro and in vivo preclinical models of orthotopic and/or metastatic soft-tissue and bone sarcomas. GD2 expression was detected in 55% of the primary tumors. Notably, the Osteosarcoma and Alveolar Rhabdomyosarcomas subtypes exhibited the highest GD2 expression levels, while Ewing sarcoma showed the lowest. CAR.GD2 T-cells show a significant tumor control both in vitro and in vivo models of GD2-expressing tumors. Pretreatment with an EZH2 inhibitor (Tazemetostat) upregulating GD2 expression, sensitizes GD2^dim sarcoma cells to CAR.GD2 T-cells cytotoxic activity. Moreover, in mouse models of disseminated Rhabdomyosarcomas and orthotopic Osteosarcoma, CAR.GD2 T-cells showed both a vigorous anti-tumor activity and long-term persistence as compared to un-transduced T-cells. The presence of immunosuppressive murine myeloid-derived suppressor (MDSC) cells significantly reduces long-term anti-tumour activity of infused CAR.GD2 T-cells. Tumor-derived G-CSF was found to be one of the key factors driving expansion of immunosuppressive murine and human MDSC, thus indirectly limiting the efficacy of CAR.GD2 T-cells. Our preclinical data strongly suggest that CAR.GD2 T-cells hold promise as a potential therapeutic option for the treatment of patients with GD2-positive sarcomas. Strategies to tackle hostile immunosuppressive MDSC are desirable to optimize CAR.GD2 T-cell activity.