Celyad (Belgium)

Chimeric antigen receptor (CAR)-T cell therapy revolutionized treatment of hematological malignancies, but translation to solid tumors remains difficult. Therefore, different avenues are explored to develop novel CAR-T cell products. Nanobodies have advantageous features for CAR design, such as ease of engineering and low immunogenicity, but predictive values on nanobody characteristics for optimal CAR-T cell function remain unclear. We performed a side-by-side evaluation of 12 nanobody-based (nano)CARs against human epidermal growth factor receptor 2 (HER2). All nanoCARs showed the ability to activate a reporter T cell line upon recognition of HER2pos cells. When evaluated in primary T cells, 6/12 nanoCARs showed the strongest reactivity and cytotoxicity against HER2pos glioblastoma and breast cancer cells. Moreover, only 1/12 was able to confer potent cytotoxicity against HER2pos melanoma cells. These data add to the current view that nanobody selection for CAR design remains subject to extensive side-by-side screening. Still, superior cytotoxicity across multiple solid tumor cell lines—determined using in vitro assays evaluating activation, cytokine secretion, and target cell-specific killing—led to selection and further characterization of a lead nanoCAR 1R59b, showing tumor control in an in vivo xenograft model, providing a promising HER2 nanoCAR for further (pre)clinical investigation.

BackgroundChimeric antigen receptor (CAR) T-cells have achieved unprecedented success treating various cancers, with complete responses exceeding 90% in several hematological malignancies. However, these remarkable outcomes have not been replicated in other types of cancers, particularly solid tumors. In fact, solid tumors present additional layers of complexity that significantly hamper the effectiveness of immune cell therapies. Consequently, further advancements are necessary in CAR T-cells to enhance their persistence, overcome exhaustion and enable them to cope with the immunosuppressive tumor microenvironment. Gene-editing technologies, especially CRISPR/Cas9, have proven effective in introducing a limited number of edits in T-cells. Nevertheless, the success of CAR T-cell therapies for solid tumors require the simultaneous engineering of multiple cellular features. Achieving this level of multiplexing with gene-editing technologies has proven challenging in terms of scalability and manufacturability. Moreover, the inherent mechanism of action of gene-editing technologies carries the risk of introducing unintended, irreversible genomic alterations. Preclinical data convincingly demonstrate that undesired mutations and chromosomal aberrations are not merely hypothetical concerns. Non-gene editing technologies are a viable alternative capable of circumventing many of these issues. In this context, we describe an effective, safe and scalable non-gene edited approach with a high degree of multiplexing capability for the engineering of CAR T-cells.Methods miRNA scaffolds highly expressed in T-cells were redirected against genes of interest using custom-designed, shRNA-based guide sequences and were tested for their knock-down efficiency. The best performing scaffolds were then combined into a chimeric miRNA cluster able to target up to five genes of interest. The cluster knock-down efficiency and stability with a wide variety of guide sequences were validated against functionally relevant target genes in primary T-cells.ResultsPreviously, we introduced a miRNA-based shRNA platform capable of targeting up to four genes simultaneously. In this study, we further advanced this technology by expanding the platform to a 5-plex system. The novel chimeric cluster demonstrated high efficiency in knocking down five highly relevant genes in CAR T-cells simultaneously. Notably, our non-gene editing technology enabled independent modulation of each target gene to achieve the desired expression levels, thus fine-tuning the functional outcomes based on the specific biology of each target.ConclusionsOur data validate the efficacy of our technology for introducing up to five functionally relevant edits in CAR T-cells simultaneously. The 5-plex miRNA-based shRNA platform stands out as an easy, safe, and effective strategy for multiplex engineering of CAR T-cells.

Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.

To design slow resorption patient-specific bone graft whose properties of bone regeneration are increased by its geometry and composition and to assess it in in-vitro and in-vivo models.A graft composed by hydroxyapatite (HA) and β-TCP was designed as a cylinder with 3D gyroid porosities and 7 mm medullary space based on swine's anatomy. It was produced using a stereolithography 3D-printing machine (V6000, Prodways).Sterile bone grafts impregnated with or without a 10µg/mL porcine BMP-2 (pBMP-2) solution were implanted into porcine femurs in a bone loss model. Bone defect was bi-weekly evaluated by X-ray during 3 months. After sacrifice, microscanner and non-decalcified histology analysis were conducted on biopsies.Finally, osteoblasts were cultured inside the bone graft or in monolayer underneath the bone graft. Cell viability, proliferation, and gene expression were assessed after 7 and 14 days of cell culture (n=3 patients).3D scaffolds were successfully manufactured with a composition of 80% HA and 20% β-TCP ±5% with indentation compressive strength of 4.14 MPa and bending strength of 11.8MPa.In vivo study showed that bone regeneration was highly improved in presence of pBMP-2. Micro-CT shows a filling of the gyroid sinuses of the implant (Figure 1).In vitro, the presence of BMP2 did not influence the viability of the osteoblasts and the mortality remained below 3%. After 7 days, the presence of BMP2 in the scaffold significantly increased by 85 and 65% the COL1A1 expression and by 8 and 33-fold the TNAP expression by osteoblasts in the monolayer or in the scaffold, respectively. This BMP2 effect was transient in monolayer and did not modify gene expression at day 14.BMP2-impregnated bone graft is a promising patient-personalized 3D-printed solution for bone defect regeneration, by promoting neighboring host cells recruitment and solid new bone formation.For any figures and tables, please contact the authors directly.

This last decade, chimeric antigen receptor (CAR) T-cell therapy has become a real treatment option for patients with B-cell malignancies, while multiple efforts are being made to extend this therapy to other malignancies and broader patient populations. However, several limitations remain, including those associated with the time-consuming and highly personalized manufacturing of autologous CAR-Ts. Technologies to establish “off-the-shelf” allogeneic CAR-Ts with low alloreactivity are currently being developed, with a strong focus on gene editing technologies. Although these technologies have many advantages, they have also strong limitations including double-strand breaks in the DNA with associated multiple safety risks as well as the lack of modulation. As an alternative, non-gene editing technologies provide an interesting approach to support the development of allogeneic CAR-Ts in the future, with possibilities of fine-tuning gene expression and easy development. Here we will review the different ways allogeneic CAR-Ts can be manufactured and discuss which technologies are currently used. The biggest hurdles for successful therapy of allogeneic CAR-Ts will be summarized and finally an overview of the current clinical evidence for allogeneic CAR-Ts in comparison to its autologous counterpart will be given.

BackgroundCo-inhibitory receptors, such as PD-1 and LAG-3, have a crucial role in regulating T-cell activity, as their expression on the cell surface upon chronic T-cell activation is associated with T-cell exhaustion. Immunotherapies directed against co-inhibitory receptors exhibited unprecedented efficacy in several cancer indications. However, many patients do not respond to these therapies and some tumor types remain largely refractory. A key determinant of CAR T-cell failure against solid tumors is T-cell exhaustion induced by checkpoint inhibitors on cancer cells. The coordinated targeting of different co-inhibitory receptors may help overcome these hurdles. Here we show that the simultaneous downregulation of co-inhibitory receptors PD-1, LAG-3, TIM-3 and TIGIT endows CAR T-cells with superior efficacy.MethodsA microRNA (miRNA)-based short hairpin RNA (shRNA) platform was developed, to allow for the tunable modulation of multiple target genes simultaneously. The platform was equipped with shRNA-derived guide sequences (shGuides) targeting PD-1, LAG-3, TIM-3 and TIGIT, and combined with an anti-CD19 CAR. The shRNA platform-equipped CAR T-cells were challenged with target cancer cells expressing the co-inhibitory receptors’ ligands. The impact of the simultaneous downregulation of the four co-inhibitory receptors was assessed in vitro by monitoring T-cell activation (via surface activation markers and cytokine secretion), killing activity, and persistence (via repeated challenges with the target cells).ResultsThe miRNA-derived shRNA platform targeting PD-1, LAG-3, TIM-3 and TIGIT led to a significant downregulation of the four co-inhibitory receptors in the CAR T-cells. Upon activation by the target cancer cells, the simultaneous knock-down of the four genes enhanced cytokine secretion compared to CAR T-cells not engineered with the shRNA platform. Moreover, the CAR T-cells carrying the quadruple knock-down showed superior killing ability, increased persistence and prolonged activity when repeatedly challenged in sequential cycles with the target cancer cells.ConclusionsThese data validate our technology for the effective introduction of multiple functionally relevant edits in CAR T-cells. Co-inhibitory receptors concomitantly contribute at regulating T-cell responses, making it difficult to inhibit or knock-out multiple receptors together. With our approach we proved the feasibility of the simultaneous knock-down of PD-1, LAG-3, TIM-3 and TIGIT. Moreover, we show increased performance of the engineered CAR T-cells, suggesting that the simultaneous downregulation of the four co-inhibitory receptors may improve the antitumor activity of adoptive cell therapy. This strategy may empower the CAR T-cells to successfully target tumors reliant on high expression of immune checkpoint molecules and therefore often refractory to immunotherapy, such as solid tumors.

BackgroundChimeric antigen receptor (CAR) T-cells have revolutionized the treatment of hematological malignancies. However, the current autologous approach to CAR T-cell production suffers several drawbacks (difficulty to obtain high quality starting material from patients, limited manufacturing capacity, complex logistics, long vein-to-vein time, high costs), to the point that to date only approximately 10% of eligible patients can receive the treatment. Allogeneic ‘off-the-shelf’ CAR T-cells could help circumvent these issues, but their successful clinical application is hampered by the difficulty to manage graft-versus-host disease (GvHD) and host-versus-graft (HvG). A variety of genetic engineering strategies have been proposed to address these issues, all burdened by the complexity of multiple, potentially risky genome edits. Here we describe a non-gene edited one-step approach for the effective generation of allogeneic CAR T-cells. To this aim, we silenced CD3ζ to target T-cell receptor (TCR) expression, and we downregulated β2M and CIITA to target HLA class I and HLA class II expression, respectively. We aimed at preventing apoptosis induced by CD95L, often expressed by cancer cells, by knocking down CD95.MethodsA microRNA (miRNA)-based short hairpin RNA (shRNA) platform was developed, to allow for the tunable modulation of multiple target genes simultaneously. The platform was equipped with shRNA-derived guide sequences (shGuides) targeting CD3ζ, β2M, CIITA and CD95 simultaneously, and combined with an anti-CD19 CAR. Each shRNA was screened and assessed functionally in vitro.ResultsThe miRNA-derived shRNA platform targeted against CD3ζ, β2M, CIITA and CD95 in the CAR construct led to a substantial downregulation of TCR, HLA class I, HLA class II and CD95. This, in turn, granted the engineered CAR T-cells protection in in vitro assays mimicking GvHD, HvG and CD95L-induced autophagy, ultimately leading to significantly improved CAR T-cell survival. Notably, thanks to the tunability of gene expression allowed by our platform, we were able to achieve functionally relevant downregulation of HLA class I to avoid recognition by donor T-cells, ensuring at the same time sufficient expression to let CAR T-cells escape NK recognition, without the need of further editing (e.g., HLA E overexpression).ConclusionsThese data validate our technology for the effective introduction of multiple functionally relevant edits in CAR T-cells. Moreover, we show in vitro proof-of-concept that our approach can improve allogeneic CAR T-cell viability by allowing evasion from GvHD, HvG and CD95L-induced autophagy. Our technology stands out as an easy, safe, and effective strategy for the generation of allogeneic CAR T-cells.

BackgroundManipulating protein expression to generate cells with a specific desired phenotype is one of the central goals of engineered cell therapy. Short Hairpin RNA (shRNA) is a well-established approach to reduce protein expression through the targeted degradation of messenger RNA transcripts. However, the use of shRNA in the Chimeric Antigen Receptor (CAR) T cell therapy has been limited. We have recently shown that single shRNA incorporated into a CAR expression vector can knockdown expression of the target antigen when expressed on the CAR T itself to avoid fratricide or expression of some elements of the T cell receptor (TCR) to generate allogeneic CAR T cells. An attraction of the shRNA approach is to express multiple shRNA from the same vector that can regulate protein expression thereby optimzing CAR T cell phenotype.MethodsRetroviral vectors encoding a CAR targeting a well-studied antigen (generally BCMA) co-expressing a tag for cell enrichment and identification along with shRNA multiplexed were generated. The shRNA multiplexed were inserted within a microRNA (miR) framework to enable expression from a single PolII promoter (the retroviral LTR promoter). Functional assessment of target knockdown target in T cells along with retroviral titers was determinedResultsOur products in ongoing clinical development have employed a miR196a2 scaffold enabling the expression of the desired shRNA driven by the same promoter as that used for the CAR and other transgenes. Multiplexing the miR196a2 scaffold to express multiple shRNA (targeting CD247, beta 2 Microglobulin and CD95) was successful in terms of target knockdown but an obvious reduction in retroviral titer was observed. These titer reductions were variable between the duplex and triplex shRNA constructs examined but were uniformly low when considering clinical development. A proprietary scaffold was developed that coupled expression of duplexed and triplexed shRNA while also elevating vector titer by at least 2-3x.ConclusionsMultiplexing shRNA within a single vector format with scaffolds that ensure co-linked expression of the shRNA with therapeutic transgenes is a highly attractive approach to generate CAR T cells with bespoke, desired phenotypes. However, simply multiplexing shRNA using a currently clinical-used scaffold (miR196a2) resulted in reductions in vector titer. Engineering further proprietary scaffolds were produced that maintained shRNA expression but elevated retroviral titer to a level which does not preclude clinical development. These developments now provide the opportunity to develop second generation clinical candidates using shRNA multiplexed technology.