CAR'TCR-T cells co-expressing CD33-CAR and dNPM1-TCR as superior dual-targeting approach for AML treatment.

Acute myeloid leukemia (AML), a fast-progressing hematological malignancy affecting myeloid cells, is typically treated with chemotherapy or hematopoietic stem cell transplantation. However, approximately half of the patients face relapses and 5-year survival rates are poor. With the goal to facilitate dual-specificity, boosting anti-tumor activity, and minimizing the risk for antigen escape, this study focused on combining chimeric antigen receptor (CAR) and T cell receptor (TCR) technologies. CAR'TCR-T cells, co-expressing a CD33-CAR and a transgenic dNPM1-TCR, revealed increased and prolonged anti-tumor activity in vitro, particularly in case of low target antigen expression. The distinct transcriptomic profile suggested enhanced formation of immunological synapses, activation, and signaling. Complete elimination of AML xenografts in vivo was only achieved with a cell product containing CAR'TCR-T, CAR-T, and TCR-T cells, representing the outcome of co-transduction with two lentiviral vectors encoding either CAR or TCR. A mixture of CAR-T and TCR-T cells, without CAR'TCR-T cells, did not prevent progressive tumor outgrowth and was comparable to treatment with CAR-T and TCR-T cells individually. Overall, our data underscore the efficacy of co-expressing CAR and transgenic TCR in one T cell, and might open a novel therapeutic avenue not only for AML but also other malignancies.

Automated manufacture of ΔNPM1 TCR-engineered T cells for AML therapy.

Acute myeloid leukemia (AML) is a heterogeneous malignancy that requires further therapeutic improvement, especially for the elderly and for subgroups with poor prognosis. A recently discovered T cell receptor (TCR) targeting mutant nucleophosmin 1 (ΔNPM1) presents an attractive option for the development of a cancer antigen-targeted cellular therapy. Manufacturing of TCR-modified T cells, however, is still limited by a complex, time-consuming, and laborious procedure. Therefore, this study specifically addressed the requirements for a scaled manufacture of ΔNPM1-specific T cells in an automated, closed, and good manufacturing practice-compliant process. Starting from cryopreserved leukapheresis, 2E8 CD8-positive T cells were enriched, activated, lentivirally transduced, expanded, and finally formulated. By adjusting and optimizing culture conditions, we additionally reduced the manufacturing time from 12 to 8 days while still achieving a clinically relevant yield of up to 5.5E9 ΔNPM1 TCR-engineered T cells. The cellular product mainly consisted of highly viable CD8-positive T cells with an early memory phenotype. ΔNPM1 TCR CD8 T cells manufactured with the optimized process showed specific killing of AML in vitro and in vivo. The process has been implemented in an upcoming phase 1/2 clinical trial for the treatment of NPM1-mutated AML.

Combining CSPG4-CAR and CD20-CCR for treatment of metastatic melanoma.

The prognosis for patients with metastatic melanoma is poor and treatment options are limited. Genetically-engineered T cell therapy targeting chondroitin sulfate proteoglycan 4 (CSPG4), however, represents a promising treatment option, especially as both primary melanoma cells as well as metastases uniformly express CSPG4. Aiming to prevent off-tumor toxicity while maintaining a high cytolytic potential, we combined a chimeric co-stimulatory receptor (CCR) and a CSPG4-directed second-generation chimeric antigen receptor (CAR) with moderate potency. CCRs are artificial receptors similar to CARs, but lacking the CD3ζ activation element. Thus, T cells expressing solely a CCR, do not induce any cytolytic activity upon target cell binding, but are capable of boosting the CAR T cell response when both CAR and CCR engage their target antigens simultaneously. Here we demonstrate that co-expression of a CCR can significantly enhance the anti-tumor response of CSPG4-CAR T cells in vitro as well as in vivo. Importantly, this boosting effect was not dependent on co-expression of both CCR- and CAR-target on the very same tumor cell, but was also achieved upon trans activation. Finally, our data support the idea of using a CCR as a powerful tool to enhance the cytolytic potential of CAR T cells, which might open a novel therapeutic window for the treatment of metastatic melanoma.

CD62L as target receptor for specific gene delivery into less differentiated human T lymphocytes.

Chimeric antigen receptor (CAR)-expressing T cells are a complex and heterogeneous gene therapy product with variable phenotype compositions. A higher proportion of less differentiated CAR T cells is usually associated with improved antitumoral function and persistence. We describe in this study a novel receptor-targeted lentiviral vector (LV) named 62L-LV that preferentially transduces less differentiated T cells marked by the L-selectin receptor CD62L, with transduction rates of up to 70% of CD4+ and 50% of CD8+ primary T cells. Remarkably, higher amounts of less differentiated T cells are transduced and preserved upon long-term cultivation using 62L-LV compared to VSV-LV. Interestingly, shed CD62L neither altered the binding of 62L-LV particles to T cells nor impacted their transduction. The incubation of 2 days of activated T lymphocytes with 62L-LV or VSV-LV for only 24 hours was sufficient to generate CAR T cells that controlled tumor growth in a leukemia tumor mouse model. The data proved that potent CAR T cells can be generated by short-term ex vivo exposure of primary cells to LVs. As a first vector type that preferentially transduces less differentiated T lymphocytes, 62L-LV has the potential to circumvent cumbersome selections of T cell subtypes and offers substantial shortening of the CAR T cell manufacturing process.

Combined targeting of soluble latent TGF-ß and a solid tumor-associated antigen with adapter CAR T cells.

Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble factors such as TGF-ß. TGF-ß plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-ß within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-ß, both in vitro and in vivo. Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-ß and tumor cell killing by targeting CD66c as TAA in vivo. In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-ß.

Adapter-Mediated Transduction with Lentiviral Vectors: A Novel Tool for Cell-Type-Specific Gene Transfer.

Selective gene delivery to a cell type of interest utilizing targeted lentiviral vectors (LVs) is an efficient and safe strategy for cell and gene therapy applications, including chimeric antigen receptor (CAR)-T cell therapy. LVs pseudotyped with measles virus envelope proteins (MV-LVs) have been retargeted by ablating binding to natural receptors while fusing to a single-chain antibody specific for the antigen of choice. However, the broad application of MV-LVs is hampered by the laborious LV engineering required for every new target. Here, we report the first versatile targeting system for MV-LVs that solely requires mixing with biotinylated adapter molecules to enable selective gene transfer. The analysis of the selectivity in mixed cell populations revealed transduction efficiencies below the detection limit in the absence of an adapter and up to 5000-fold on-to-off-target ratios. Flexibility was confirmed by transducing cell lines and primary cells applying seven different adapter specificities in total. Furthermore, adapter mixtures were applied to generate CAR-T cells with varying CD4/CD8-ratios in a single transduction step. In summary, a selective and flexible targeting system was established that may serve to improve the safety and efficacy of cellular therapies. Compatibility with a wide range of readily available biotinylated molecules provides an ideal technology for a variety of applications.

Automated, scaled, transposon-based production of CAR T cells.

BACKGROUND: There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition. METHODS: We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR. RESULTS: We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1-3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns. CONCLUSIONS: We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.

Anti-CD19 CARs displayed at the surface of lentiviral vector particles promote transduction of target-expressing cells.

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future.

Automated generation of gene-edited CAR T cells at clinical scale.

The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality of the starting material and the different manufacturing steps are associated with production failure and product contamination. Here, we present an automated T cell engineering process to produce off-the-shelf chimeric antigen receptor (CAR) T cells on an extended CliniMACS Prodigy platform containing an in-line electroporation unit. This setup was used to combine lentiviral delivery of a CD19-targeting CAR with transfer of mRNA encoding a TRAC locus-targeting transcription activator-like effector nuclease (TALEN). In three runs at clinical scale, the T cell receptor (TCR) alpha chain encoding TRAC locus was disrupted in >35% of cells with high cell viability (>90%) and no detectable off-target activity. A final negative selection step allowed the generation of TCRα/ß-free CAR T cells with >99.5% purity. These CAR T cells proliferated well, maintained a T cell memory phenotype, eliminated CD19-positive tumor cells, and released the expected cytokines when exposed to B cell leukemia cells. In conclusion, we established an automated, good manufacturing practice (GMP)-compliant process that integrates lentiviral transduction with electroporation of TALEN mRNA to produce functional TCRα/ß-free CAR19 T cells at clinical scale.

Vectofusin-1 Improves Transduction of Primary Human Cells with Diverse Retroviral and Lentiviral Pseudotypes, Enabling Robust, Automated Closed-System Manufacturing.

Cell and gene therapies are finally becoming viable patient treatment options, with both T cell- and hematopoietic stem cell (HSC)-based therapies being approved to market in Europe. However, these therapies, which involve the use of viral vector to modify the target cells, are expensive and there is an urgent need to reduce manufacturing costs. One major cost factor is the viral vector production itself, therefore improving the gene modification efficiency could significantly reduce the amount of vector required per patient. This study describes the use of a transduction enhancing peptide, Vectofusin-1®, to improve the transduction efficiency of primary target cells using lentiviral and gammaretroviral vectors (LV and RV) pseudotyped with a variety of envelope proteins. Using Vectofusin-1 in combination with LV pseudotyped with viral glycoproteins derived from baboon endogenous retrovirus, feline endogenous virus (RD114), and measles virus (MV), a strongly improved transduction of HSCs, B cells and T cells, even when cultivated under low stimulation conditions, could be observed. The formation of Vectofusin-1 complexes with MV-LV retargeted to CD20 did not alter the selectivity in mixed cell culture populations, emphasizing the precision of this targeting technology. Functional, ErbB2-specific chimeric antigen receptor-expressing T cells could be generated using a gibbon ape leukemia virus (GALV)-pseudotyped RV. Using a variety of viral vectors and target cells, Vectofusin-1 performed in a comparable manner to the traditionally used surface-bound recombinant fibronectin. As Vectofusin-1 is a soluble peptide, it was possible to easily transfer the T cell transduction method to an automated closed manufacturing platform, where proof of concept studies demonstrated efficient genetic modification of T cells with GALV-RV and RD114-RV and the subsequent expansion of mainly central memory T cells to a clinically relevant dose.

Highly Efficient and Selective CAR-Gene Transfer Using CD4- and CD8-Targeted Lentiviral Vectors.

Chimeric antigen receptor (CAR)-modified T cells have revealed promising results in the treatment of cancer, but they still need to overcome various hurdles, including a complicated manufacturing process. Receptor-targeted lentiviral vectors (LVs) delivering genes selectively to T cell subtypes may facilitate and improve CAR T cell generation, but so far they have resulted in lower gene delivery rates than conventional LVs (vesicular stomatitis virus [VSV]-LV). To overcome this limitation, we studied the effect of the transduction enhancer Vectofusin-1 on gene delivery to human T cells with CD4- and CD8-targeted LVs, respectively, encoding a second-generation CD19-CAR in conjunction with a truncated version of the low-affinity nerve growth factor receptor (ΔLNGFR) as reporter. Vectofusin-1 significantly enhanced the gene delivery of CD4- and CD8-LVs without a loss in target cell selectivity and killing capability of the generated CAR T cells. Notably, delivery rates mediated by VSV-LV were substantially reduced by Vectofusin-1. Interestingly, a transient off-target signal in samples treated with Vectofusin-1 was observed early after transduction. However, this effect was not caused by uptake and expression of the transgene in off-target cells, but rather it resulted from cell-bound LV particles having ΔLNGFR incorporated into their surface. The data demonstrate that gene transfer rates in the range of those mediated by VSV-LVs can be achieved with receptor-targeted LVs.

Automated Manufacturing of Potent CD20-Directed Chimeric Antigen Receptor T Cells for Clinical Use.

The clinical success of gene-engineered T cells expressing a chimeric antigen receptor (CAR), as manifested in several clinical trials for the treatment of B cell malignancies, warrants the development of a simple and robust manufacturing procedure capable of reducing to a minimum the challenges associated with its complexity. Conventional protocols comprise many open handling steps, are labor intensive, and are difficult to upscale for large numbers of patients. Furthermore, extensive training of personnel is required to avoid operator variations. An automated current Good Manufacturing Practice-compliant process has therefore been developed for the generation of gene-engineered T cells. Upon installation of the closed, single-use tubing set on the CliniMACS Prodigy™, sterile welding of the starting cell product, and sterile connection of the required reagents, T cells are magnetically enriched, stimulated, transduced using lentiviral vectors, expanded, and formulated. Starting from healthy donor (HD) or lymphoma or melanoma patient material (PM), the robustness and reproducibility of the manufacturing of anti-CD20 specific CAR T cells were verified. Independent of the starting material, operator, or device, the process consistently yielded a therapeutic dose of highly viable CAR T cells. Interestingly, the formulated product obtained with PM was comparable to that of HD with respect to cell composition, phenotype, and function, even though the starting material differed significantly. Potent antitumor reactivity of the produced anti-CD20 CAR T cells was shown in vitro as well as in vivo. In summary, the automated T cell transduction process meets the requirements for clinical manufacturing that the authors intend to use in two separate clinical trials for the treatment of melanoma and B cell lymphoma.

DARPins: an efficient targeting domain for lentiviral vectors.

We have recently developed a retargeting system for lentiviral vectors (LVs) that relies on the pseudotyping of LVs with engineered measles virus (MV) glycoproteins (hemagglutinin (H) and fusion protein (F)). Specificity is provided through display of a single-chain antibody (scFv) as targeting domain by fusion to the MV-H protein. As an alternative to scFv, designed ankyrin repeat proteins (DARPins) can be selected to become high-affinity binders to any kind of target molecule. In this study six HER2/neu-specific DARPins exhibiting different affinities and binding to different HER2/neu epitopes were applied as targeting domains. All H-DARPin fusion proteins were efficiently expressed on the cell surface. Upon coexpression with F, syncytia formation was observed in HER2/neu positive cells only and correlated directly with the HER2/neu receptor density. All H-DARPin proteins incorporated into LVs, albeit at different levels. The vectors only transduced HER2/neu-positive cells, while HER2/neu-negative cells remained untransduced. Highest titers were observed with one particular DARPin binding to the membrane distal domain of HER2/neu with medium affinity. When applied in vivo systemically, HER2/neu-targeted LVs showed exclusive gene expression in HER2/neu positive tumor tissue, while vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors mainly transduced cells in spleen and liver. Thus, DARPins are a promising alternative to scFvs for retargeting of LVs.

Liver cancer protease activity profiles support therapeutic options with matrix metalloproteinase-activatable oncolytic measles virus.

Primary and secondary cancers of the liver are a significant health problem with limited treatment options. We sought here to develop an oncolytic measles virus (MV) preferentially activated in liver tumor tissue, thus reducing infection and destruction of healthy tissue. We documented that in primary tumor tissue, urokinase-type plasminogen activator and especially matrix metalloproteinase-2 (MMP-2) are significantly more active than in adjacent nontumorous tissue. We then generated variants of the MV fusion protein by inserting different MMP substrate motifs at the protease cleavage site and identified the motif PQGLYA as the most efficient cleavage site as determined by syncytia formation on protease-positive tumor cells. The corresponding MMP-activatable oncolytic MV-MMPA1 virus was rescued and shown to be strongly restricted on primary human hepatocytes and healthy human liver tissue, while remaining as effective as the parental MV in the tumor tissue sections. Our findings underline the clinical potency of the MMP activation concept as a strategy to generate safer oncolytic viruses for the treatment of primary and secondary cancers of the liver.