Preclinical Development of CAR T Cells with Antigen-Inducible IL18 Enforcement to Treat GD2-Positive Solid Cancers.

PURPOSE: Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. EXPERIMENTAL DESIGN: We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. RESULTS: Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. CONCLUSIONS: These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022-501725-21-00).

Magnetic CAR T cell purification using an anti-G4S linker antibody.

Chimeric antigen receptor (CAR) redirected T cells are successfully employed in the combat against several hematological malignancies, however, are often compromised by low transduction rates making refinement of the CAR T cell products necessary. Here, we report a broadly applicable enrichment protocol relying on marking CAR T cells with an anti-glycine4-serine (G4S) linker antibody followed by magnetic activated cell sorting (MACS). The protocol is broadly applicable since the G4S peptide is an integral part of the vast majority of CARs as it links the VH and VL recognition domains. We demonstrate the feasibility by using the canonical second generation CARs specific for CEA and Her2, respectively, obtaining highly purified CAR T cell products in a one-step procedure without impairing cell viability. The protocol is also applicable to a dual specific CAR (tandem CAR). Except for CD39, T cell activation/exhaustion markers were not upregulated after separation. Purified CAR T cells retained their functionality with respect to antigen-specific cytokine secretion, cytotoxicity, and the capacity to proliferate and eliminate cognate tumor cells upon repetitive stimulation. Collectively, the one-step protocol for purifying CAR T cells extends the toolbox for preclinical research and specifically for clinical CAR T cell manufacturing.

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.

Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T-cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and redirection of various immune cell types, including conventional T-cells, TCRγ/δ T-cells, regulatory T-cells, and NK-cells. In T-cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T-cells exhibited comparable leukemia control to T cell receptor alpha constant (TRAC)-replaced and lentivirus-transduced CAR-T-cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK-cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.

The effector program of human CD8 T cells supports tissue remodeling.

CD8 T lymphocytes are classically viewed as cytotoxic T cells. Whether human CD8 T cells can, in parallel, induce a tissue regeneration program is poorly understood. Here, antigen-specific assay systems revealed that human CD8 T cells not only mediated cytotoxicity but also promoted tissue remodeling. Activated CD8 T cells could produce the epidermal growth factor receptor (EGFR)-ligand amphiregulin (AREG) and sensitize epithelial cells for enhanced regeneration potential. Blocking the EGFR or the effector cytokines IFN-γ and TNF could inhibit tissue remodeling. This regenerative program enhanced tumor spheroid and stem cell-mediated organoid growth. Using single-cell gene expression analysis, we identified an AREG+, tissue-resident CD8 T cell population in skin and adipose tissue from patients undergoing abdominal wall or abdominoplasty surgery. These tissue-resident CD8 T cells showed a strong TCR clonal relation to blood PD1+TIGIT+ CD8 T cells with tissue remodeling abilities. These findings may help to understand the complex CD8 biology in tumors and could become relevant for the design of therapeutic T cell products.

Fine-tuning the antigen sensitivity of CAR T cells: emerging strategies and current challenges.

Chimeric antigen receptor (CAR) T cells are "living drugs" that specifically recognize their target antigen through an antibody-derived binding domain resulting in T cell activation, expansion, and destruction of cognate target cells. The FDA/EMA approval of CAR T cells for the treatment of B cell malignancies established CAR T cell therapy as an emerging pillar of modern immunotherapy. However, nearly every second patient undergoing CAR T cell therapy is suffering from disease relapse within the first two years which is thought to be due to downregulation or loss of the CAR target antigen on cancer cells, along with decreased functional capacities known as T cell exhaustion. Antigen downregulation below CAR activation threshold leaves the T cell silent, rendering CAR T cell therapy ineffective. With the application of CAR T cells for the treatment of a growing number of malignant diseases, particularly solid tumors, there is a need for augmenting CAR sensitivity to target antigen present at low densities on cancer cells. Here, we discuss upcoming strategies and current challenges in designing CARs for recognition of antigen low cancer cells, aiming at augmenting sensitivity and finally therapeutic efficacy while reducing the risk of tumor relapse.

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.

Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3 ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3 ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3 ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3 ζ-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant ( TRAC )-replaced and lentivirus-transduced CAR-T cells in vivo . Tuning of CD3 ζ-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC -edited CAR-T cells, integration of a Her2-CAR into CD3 ζ conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3 ζ gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3 ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes. Key points: Integration of ζ-deficient CARs into CD3 ζ gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use CD3 ζ-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions.

CD44v6 specific CAR-NK cells for targeted immunotherapy of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major challenge for current therapies. CAR-T cells have shown promising results in blood cancers, however, their effectiveness against solid tumors remains a hurdle. Recently, CD44v6-directed CAR-T cells demonstrated efficacy in controlling tumor growth in multiple myeloma and solid tumors such as HNSCC, lung and ovarian adenocarcinomas. Apart from CAR-T cells, CAR-NK cells offer a safe and allogenic alternative to autologous CAR-T cell therapy. In this paper, we investigated the capacity of CAR-NK cells redirected against CD44v6 to execute cytotoxicity against HNSCC. Anti-CD44v6 CAR-NK cells were generated from healthy donor peripheral blood-derived NK cells using gamma retroviral vectors (gRVs). The NK cell transduction was optimized by exploring virus envelope proteins derived from the baboon endogenous virus envelope (BaEV), feline leukemia virus (FeLV, termed RD114-TR) and gibbon ape leukemia virus (GaLV), respectively. BaEV pseudotyped gRVs induced the highest transduction rate compared to RD114-TR and GaLV envelopes as measured by EGFP and surface CAR expression of transduced NK cells. CAR-NK cells showed a two- to threefold increase in killing efficacy against various HNSCC cell lines compared to unmodified, cytokine-expanded primary NK cells. Anti-CD44v6 CAR-NK cells were effective in eliminating tumor cell lines with high and low CD44v6 expression levels. Overall, the improved cytotoxicity of CAR-NK cells holds promise for a therapeutic option for the treatment of HNSCC. However, further preclinical trials are necessary to test in vivo efficacy and safety, as well to optimize the treatment regimen of anti-CD44v6 CAR-NK cells against solid tumors.

IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities.

Chimeric antigen receptor (CAR) modified T cells can induce complete remissions in patients with advanced hematological malignancies. Nevertheless, the efficacy is mostly transient and remains so far poor in the treatment of solid tumors. Crucial barriers to long-term CAR T cell success encompass loss of functional capacities known as "exhaustion", among others. To extend CAR T cell functionality, we reduced interferon regulatory factor 4 (IRF4) levels in CAR T cells using a one-vector system encoding a specific short-hairpin (sh) RNA along with constitutive CAR expression. At baseline, CAR T cells with downregulated IRF4 showed equal cytotoxicity and cytokine release compared to conventional CAR T cells. However, under conditions of repetitive antigen encounter, IRF4low CAR T cells displayed enhanced functionality with superior cancer cell control in the long-term compared with conventional CAR T cells. Mechanistically, the downregulation of IRF4 in CAR T cells resulted in prolonged functional capacities and upregulation of CD27. Moreover, IRF4low CAR T cells were more sensitive to cancer cells with low levels of target antigen. Overall, IRF4 downregulation capacitates CAR T cells to recognize and respond to target cells with improved sensitivity and endurance.

CAR T cells as micropharmacies against solid cancers: Combining effector T-cell mediated cell death with vascular targeting in a one-step engineering process.

To enhance the potency of chimeric antigen receptor (CAR) engineered T cells in solid cancers, we designed a novel cell-based combination strategy with an additional therapeutic mode of action. CAR T cells are used as micropharmacies to produce a targeted pro-coagulatory fusion protein, truncated tissue factor (tTF)-NGR, which exerts pro-coagulatory activity and hypoxia upon relocalization to the vascular endothelial cells that invade tumor tissues. Delivery by CAR T cells aimed to induce locoregional tumor vascular infarction for combined immune-mediated and hypoxic tumor cell death. Human T cells that were one-vector gene-modified to express a GD2-specific CAR along with CAR-inducible tTF-NGR exerted potent GD2-specific effector functions while secreting tTF-NGR that activates the extrinsic coagulation pathway in a strictly GD2-dependent manner. In murine models, the CAR T cells infiltrated GD2-positive tumor xenografts, secreted tTF-NGR into the tumor microenvironment and showed a trend towards superior therapeutic activity compared with control cells producing functionally inactive tTF-NGR. In vitro evidence supports a mechanism of hypoxia-mediated enhancement of T cell cytolytic activity. We conclude that combined CAR T cell targeting with an additional mechanism of antitumor action in a one-vector engineering strategy is a promising approach to be further developed for targeted treatment of solid cancers.

CAR and TCR form individual signaling synapses and do not cross-activate, however, can co-operate in T cell activation.

In engineered T cells the CAR is co-expressed along with the physiological TCR/CD3 complex, both utilizing the same downstream signaling machinery for T cell activation. It is unresolved whether CAR-mediated T cell activation depends on the presence of the TCR and whether CAR and TCR mutually cross-activate upon engaging their respective antigen. Here we demonstrate that the CD3ζ CAR level was independent of the TCR associated CD3ζ and could not replace CD3ζ to rescue the TCR complex in CD3ζ KO T cells. Upon activation, the CAR did not induce phosphorylation of TCR associated CD3ζ and, vice versa, TCR activation did not induce CAR CD3ζ phosphorylation. Consequently, CAR and TCR did not cross-signal to trigger T cell effector functions. On the membrane level, TCR and CAR formed separate synapses upon antigen engagement as revealed by total internal reflection fluorescence (TIRF) and fast AiryScan microscopy. Upon engaging their respective antigen, however, CAR and TCR could co-operate in triggering effector functions through combinatorial signaling allowing logic "AND" gating in target recognition. Data also imply that tonic TCR signaling can support CAR-mediated T cell activation emphasizing the potential relevance of the endogenous TCR for maintaining T cell capacities in the long-term.

A CD19-Anti-ErbB2 scFv Engager Protein Enables CD19-Specific CAR T Cells to Eradicate ErbB2+ Solid Cancer.

The efficacy of CD19-specific CAR T cells in the treatment of leukemia/lymphoma relies, at least in part, on the unique properties of the particular CAR and the presence of healthy B cells that enhance the target cell lysis and cytokine secretion through repetitive stimulation. Here, we report to apply the same CAR to target solid tumors, such as ErbB2+ carcinoma. CD19 CAR T cells are redirected towards the ErbB2+ cells by a fusion protein that is composed of the herceptin-derived anti-ErbB2 scFv 4D5 linked to the CD19 exodomain. The CD19-4D5scFv engager enabled CD19 CAR T cells to recognize the ErbB2+ cancer cells and to suppress the ErbB2+ tumor growth. The primary killing capacity by the ErbB2-redirected CD19 CAR T cells was as efficient as by the ErbB2 CAR T cells, however, adding CD19+ B cells furthermore reinforced the activation of the CD19 CAR T cells, thereby improving the anti-tumor activities. The ErbB2-redirected CD19 CAR T cells, moreover, showed a 100-fold superior selectivity in targeting cancer cells versus healthy fibroblasts, which was not the case for the ErbB2 CAR T cells. The data demonstrate that the CD19 CAR T cells can be high-jacked by a CD19-scFv engager protein to attack specifically solid cancer, thereby expanding their application beyond the B cell malignancies.

Do Treg Speed Up with CARs? Chimeric Antigen Receptor Treg Engineered to Induce Transplant Tolerance.

Adoptive transfer of regulatory T cells (Treg) can induce transplant tolerance in preclinical models by suppressing alloantigen-directed inflammatory responses; clinical translation was so far hampered by the low abundance of Treg with allo-specificity in the peripheral blood. In this situation, ex vivo engineering of Treg with a T-cell receptor (TCR) or chimeric antigen receptor (CAR) provides a cell population with predefined specificity that can be amplified and administered to the patient. In contrast to TCR-engineered Treg, CAR Treg can be redirected toward a broad panel of targets in an HLA-unrestricted fashion' making these cells attractive to provide antigen-specific tolerance toward the transplanted organ. In preclinical models, CAR Treg accumulate and amplify at the targeted transplant, maintain their differentiated phenotype, and execute immune repression more vigorously than polyclonal Treg. With that, CAR Treg are providing hope in establishing allospecific, localized immune tolerance in the long term' and the first clinical trials administering CAR Treg for the treatment of transplant rejection are initiated. Here, we review the current platforms for developing and manufacturing alloantigen-specific CAR Treg and discuss the therapeutic potential and current hurdles in translating CAR Treg into clinical exploration.

Targeting colorectal cancer cells using AND-gated adaptor RevCAR T-cells.

Despite the success of chimeric antigen receptor (CAR) T-cells especially for treating hematological malignancies, critical drawbacks, such as "on-target, off-tumor" toxicities, need to be addressed to improve safety in translating to clinical application. This is especially true, when targeting tumor-associated antigens (TAAs) that are not exclusively expressed by solid tumors but also on hea9lthy tissues. To improve the safety profile, we developed switchable adaptor CAR systems including the RevCAR system. RevCAR T-cells are activated by cross-linking of bifunctional adaptor molecules termed target modules (RevTM). In a further development, we established a Dual-RevCAR system for an AND-gated combinatorial targeting by splitting the stimulatory and co-stimulatory signals of the RevCAR T-cells on two individual CARs. Examples of common markers for colorectal cancer (CRC) are the carcinoembryonic antigen (CEA) and the epithelial cell adhesion molecule (EpCAM), while these antigens are also expressed by healthy cells. Here we describe four novel structurally different RevTMs for targeting of CEA and EpCAM. All anti-CEA and anti-EpCAM RevTMs were validated and the simultaneous targeting of CEA+ and EpCAM+ cancer cells redirected specific in vitro and in vivo killing by Dual-RevCAR T-cells. In summary, we describe the development of CEA and EpCAM specific adaptor RevTMs for monospecific and AND-gated targeting of CRC cells via the RevCAR platform as an improved approach to increase tumor specificity and safety of CAR T-cell therapies.

CAR Triggered Release of Type-1 Interferon Limits CAR T-Cell Activities by an Artificial Negative Autocrine Loop.

The advent of chimeric antigen receptor (CAR) T cells expedited the field of cancer immunotherapy enabling durable remissions in patients with refractory hematological malignancies. T cells redirected for universal cytokine-mediated killing (TRUCKs), commonly referred to as "fourth generation" CAR T-cells, are designed to release engineered payloads upon CAR-induced T-cell activation. Building on the TRUCK technology, we aimed to generate CAR T-cells with a CAR-inducible artificial, self-limiting autocrine loop. To this end, we engineered CAR T-cells with CAR triggered secretion of type-1 interferons (IFNs). At baseline, IFNα and IFNß CAR T-cells showed similar capacities in cytotoxicity and cytokine secretion compared to conventional CAR T-cells. However, under "stress" conditions of repetitive rounds of antigen stimulation using BxPC-3 pancreas carcinoma cells as targets, anti-tumor activity faded in later rounds while being fully active in destructing carcinoma cells during first rounds of stimulation. Mechanistically, the decline in activity was primarily based on type-1 IFN augmented CAR T-cell apoptosis, which was far less the case for CAR T-cells without IFN release. Such autocrine self-limiting loops can be used for applications where transient CAR T-cell activity and persistence upon target recognition is desired to avoid lasting toxicities.

Decitabine-Mediated Upregulation of CSPG4 in Ovarian Carcinoma Cells Enables Targeting by CSPG4-Specific CAR-T Cells.

The addition of CAR-T cells to the armamentarium of immunotherapy revigorated the field of oncology by inducing long-lasting remissions in patients with relapsing/refractory hematological malignancies. Nevertheless, in the lion's share of patients diagnosed with solid tumors, CAR-T-cell therapy so far failed to demonstrate satisfactory anti-tumor activity. A crucial cause of resistance against the antigen-specific attack of CAR-T cells is predicated on the primary or secondary absence of suitable target antigens. Thus, the necessity to create a broad repertoire of different target antigens is vital. We aimed to evaluate the potential of the well-established melanoma antigen chondroitin sulfate proteoglycan 4 (CSPG4) as an inducible antigen in ovarian cancer cells, using CSPG4-negative SKOV-3 ovarian cancer cells as a model. Based on the hypomethylating activity of the FDA-approved drug decitabine, we refined a protocol to upregulate CSPG4 in the majority of decitabine-treated SKOV-3 cells. CSPG4-specific CAR-T cells generated by mRNA-electroporation showed CSPG4-directed cytokine secretion and cytotoxicity towards decitabine-treated SKOV-3. Another ovarian cancer cell line (Caov-3) and the neoplastic cell line 293T behaved similar. In aggregate, we generated proof-of-concept data paving the way for the further exploration of CSPG4 as an inducible antigen for CAR-T cells in ovarian cancer.

Treatment with Living Drugs: Pharmaceutical Aspects of CAR T Cells.

BACKGROUND: Adoptive therapy with genetically modified T cells achieves spectacular remissions in advanced hematologic malignancies. In contrast to conventional drugs, this kind of therapy applies viable autologous T cells that are ex vivo genetically engineered with a chimeric antigen receptor (CAR) and are classified as advanced therapy medicinal products. SUMMARY: As "living drugs," CAR T cells differ from classical pharmaceutical drugs as they provide a panel of cellular capacities upon CAR signaling, including the release of effector molecules and cytokines, redirected cytotoxicity, CAR T cell amplification, active migration, and long-term persistence and immunological memory. Here, we discuss pharmaceutical aspects, the regulatory requirements for CAR T cell manufacturing, and how CAR T cell pharmacokinetics are connected with the clinical outcome. KEY MESSAGES: From the pharmacological perspective, the development of CAR T cells with high translational potential needs to address pharmacodynamic markers to balance safety and efficacy of CAR T cells and to address pharmacokinetics with respect to trafficking, homing, infiltration, and persistence of CAR T cells.

A Low Dose of Pure Cannabidiol Is Sufficient to Stimulate the Cytotoxic Function of CIK Cells without Exerting the Downstream Mediators in Pancreatic Cancer Cells.

Despite numerous studies conducted over the past decade, the exact role of the cannabinoid system in cancer development remains unclear. Though research has focused on two cannabinoid receptors (CB1, CB2) activated by most cannabinoids, CB2 holds greater attention due to its expression in cells of the immune system. In particular, cytokine-induced killer cells (CIKs), which are pivotal cytotoxic immunological effector cells, express a high-level of CB2 receptors. Herein, we sought to investigate whether inducing CIK cells with cannabidiol can enhance their cytotoxicity and if there are any possible counter effects in its downstream cascade of phosphorylated p38 and CREB using a pancreatic ductal adenocarcinoma cell line (PANC-1). Our results showed that IL-2 modulates primarily the expression of the CB2 receptor on CIK cells used during ex vivo CIK expansion. The autophagosomal-associated scaffold protein p62 was found to co-localize with CB2 receptors in CIK cells and the PANC-1 cell line. CIK cells showed a low level of intracellular phospho-p38 and, when stimulated with cannabidiol (CBD), a donor specific variability in phospho-CREB. CBD significantly decreases the viability of PANC-1 cells presumably by increasing the cytotoxicity of CIK cells. Taken together, in our preclinical in vitro study, we propose that a low effective dose of CBD is sufficient to stimulate the cytotoxic function of CIK without exerting any associated mediator. Thus, the combinatorial approach of non-psychoactive CBD and CIK cells appears to be safe and can be considered for a clinical perspective in pancreatic cancer.

GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18.

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs ("T cells redirected for universal cytokine-mediated killing"), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD2 using the CliniMACS Prodigy® system using a recently described "all-in-one" lentiviral vector combining constitutive anti-GD2 CAR expression and inducible IL-18. Starting with 0.84 x 108 and 0.91 x 108 T cells after enrichment of CD4+ and CD8+ we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 109 and 3.6 x 109 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD2-CAR mediated activation after co-cultivation with GD2-expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD2-expressing target cells indicated by an increased expression of activation markers (e.g. CD25, CD69) on both CD4+ and CD8+ T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-2, granzyme B, IFN-γ, perforin, TNF-α). Manufactured TRUCKs showed a specific cytotoxicity towards GD2-expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD2-expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products.

Ex Vivo Generation of CAR Macrophages from Hematopoietic Stem and Progenitor Cells for Use in Cancer Therapy.

Chimeric antigen receptor (CAR) T-cell therapies have shown impressive results in patients with hematological malignancies; however, little success has been achieved in the treatment of solid tumors. Recently, macrophages (MΦs) were identified as an additional candidate for the CAR approach, and initial proof of concept studies using peripheral blood-derived monocytes showed antigen-redirected activation of CAR MΦs. However, some patients may not be suitable for monocyte-apheresis, and prior cancer treatment regimens may negatively affect immune cell number and functionality. To address this problem, we here introduce primary human hematopoietic stem and progenitor cells (HSPCs) as a cell source to generate functional CAR MΦs ex vivo. Our data showed successful CAR expression in cord blood (CB)-derived HSPCs, with considerable cell expansion during differentiation to CAR MΦs. HSPC-derived MΦs showed typical MΦ morphology, phenotype, and basic anti-bacterial functionality. CAR MΦs targeting the carcinoembryonic antigen (CEA) and containing either a DAP12- or a CD3ζ-derived signaling domain showed antigen redirected activation as they secreted pro-inflammatory cytokines specifically upon contact with CEA+ target cells. In addition, CD3ζ-expressing CAR MΦs exhibited significantly enhanced phagocytosis of CEA+ HT1080 cells. Our data establish human HSPCs as a suitable cell source to generate functional CAR MΦs and further support the use of CAR MΦs in the context of solid tumor therapy.