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.

IL12 integrated into the CAR exodomain converts CD8+ T cells to poly-functional NK-like cells with superior killing of antigen-loss tumors.

Chimeric antigen receptor (CAR)-redirected T cell therapy often fails to control tumors in the long term due to selecting cancer cells that downregulated or lost CAR targeted antigen. To reprogram the functional capacities specifically of engineered CAR T cells, we inserted IL12 into the extracellular moiety of a CD28-ζ CAR; both the CAR endodomain and IL12 were functionally active, as indicated by antigen-redirected effector functions and STAT4 phosphorylation, respectively. The IL12-CAR reprogrammed CD8+ T cells toward a so far not recognized natural killer (NK) cell-like signature and a CD94+CD56+CD62Lhigh phenotype closely similar, but not identical, to NK and cytokine induced killer (CIK) cells. In contrast to conventional CAR T cells, IL12-CAR T cells acquired antigen-independent, human leukocyte antigen E (HLA-E) restricted cytotoxic capacities eliminating antigen-negative cancer cells in addition to eliminating cancer cells with CAR cognate antigen. Simultaneous signaling through both the CAR endodomain and IL12 were required for inducing maximal NK-like cytotoxicity; adding IL12 to conventional CAR T cells was not sufficient. Antigen-negative tumors were attacked by IL12-CAR T cells, but not by conventional CAR T cells. Overall, we present a prototype of a new family of CARs that augments tumor recognition and elimination through expanded functional capacities by an appropriate cytokine integrated into the CAR exodomain.

Anti-CD19 CAR T cells potently redirected to kill solid tumor cells.

Successful CAR T cell therapy for the treatment of solid tumors requires exemplary CAR T cell expansion, persistence and fitness, and the ability to target tumor antigens safely. Here we address this constellation of critical attributes for successful cellular therapy by using integrated technologies that simplify development and derisk clinical translation. We have developed a CAR-CD19 T cell that secretes a CD19-anti-Her2 bridging protein. This cell therapy strategy exploits the ability of CD19-targeting CAR T cells to interact with CD19 on normal B cells to drive expansion, persistence and fitness. The secreted bridging protein potently binds to Her2-positive tumor cells, mediating CAR-CD19 T cell cytotoxicity in vitro and in vivo. Because of its short half-life, the secreted bridging protein will selectively accumulate at the site of highest antigen expression, ie. at the tumor. Bridging proteins that bind to multiple different tumor antigens have been created. Therefore, antigen-bridging CAR-CD19 T cells incorporate critical attributes for successful solid tumor cell therapy. This platform can be exploited to attack tumor antigens on any cancer.

Designed Ankyrin Repeat Protein (DARPin) to target chimeric antigen receptor (CAR)-redirected T cells towards CD4+ T cells to reduce the latent HIV+ cell reservoir.

Chimeric Antigen Receptor (CAR)-redirected T cells show great efficacy in the patient-specific therapy of hematologic malignancies. Here, we demonstrate that a DARPin with specificity for CD4 specifically redirects and triggers the activation of CAR engineered T cells resulting in the depletion of CD4+ target cells aiming for elimination of the human immunodeficiency virus (HIV) reservoir.

IL7-IL12 Engineered Mesenchymal Stem Cells (MSCs) Improve A CAR T Cell Attack Against Colorectal Cancer Cells.

Chimeric antigen receptor (CAR) redirected T cells are efficacious in the treatment of leukemia/lymphoma, however, showed less capacities in eliminating solid tumors which is thought to be partly due to the lack of cytokine support in the tumor lesion. In order to deliver supportive cytokines, we took advantage of the inherent ability of mesenchymal stem cells (MSCs) to actively migrate to tumor sites and engineered MSCs to release both IL7 and IL12 to promote homeostatic expansion and Th1 polarization. There is a mutual interaction between engineered MSCs and CAR T cells; in presence of CAR T cell released IFN-γ and TNF-α, chronic inflammatory Th2 MSCs shifted towards a Th17/Th1 pattern with IL2 and IL15 release that mutually activated CAR T cells with extended persistence, amplification, killing and protection from activation induced cell death. MSCs releasing IL7 and IL12 were superior over non-modified MSCs in supporting the CAR T cell response and improved the anti-tumor attack in a transplant tumor model. Data demonstrate the first use of genetically modified MSCs as vehicles to deliver immuno-modulatory proteins to the tumor tissue in order to improve the efficacy of CAR T cells in the treatment of solid malignancies.

Blocking CD30 on T Cells by a Dual Specific CAR for CD30 and Colon Cancer Antigens Improves the CAR T Cell Response against CD30- Tumors.

Chimeric antigen receptor (CAR)-engineered T cells are efficacious in controlling advanced leukemia and lymphoma, however, they fail in the treatment of solid cancer, which is thought to be due to insufficient T cell activation. We revealed that the immune response of CAR T cells with specificity for carcinoembryonic antigen (CEA) was more efficacious against CEA+ cancer cells when simultaneously incubated with an anti-CD30 immunotoxin or anti-CD30 CAR T cells, although the targeted cancer cells lack CD30. The same effect was achieved when the anti-CD30 single-chain variable fragment (scFv) was integrated into the extracellular domain of the anti-CEA CAR. Improvement in T cell activation was due to interfering with the T cell CD30-CD30L interaction by the antagonistic anti-CD30 scFv HRS3; an agonistic anti-CD30 scFv or targeting the high-affinity interleukin-2 (IL-2) receptor was not effective. T cells with the anti-CD30/CEA CAR showed superior immunity against established CEA+ CD30- tumors in a mouse model. The concept is broadly applicable since anti-CD30/TAG72 CAR T cells also showed improved elimination of TAG72+ CD30- cancer cells. Taken together, targeting CD30 on CAR T cells by the HRS3 scFv within the anti-tumor CAR improves the redirected immune response against solid tumors.

Functionality and Cell Senescence of CD4/ CD8-Selected CD20 CAR T Cells Manufactured Using the Automated CliniMACS Prodigy® Platform.

Clinical studies using autologous CAR T cells have achieved spectacular remissions in refractory CD19+ B cell leukaemia, however some of the patient treatments with CAR T cells failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes for CD20 CAR T cells from healthy donor leukapheresis using the automated CliniMACS Prodigy® platform. Starting with a CD4/CD8-positive selection, a high purity of a median of 97% T cells with a median 65-fold cell expansion was achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached in a median of 23%. CD20 CAR T cells showed a good specific IFN-γ secretion after cocultivation with CD20+ target cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase in telomere length during the manufacturing process, while telomere length remained consistent in one and decreased in another process. In conclusion, this shows for the first time that beside heterogeneity among healthy donors, CAR T cell products also differ regarding cell senescence, even for cells manufactured in a standardised automated process.

CD28-ζ CAR T Cells Resist TGF-ß Repression through IL-2 Signaling, Which Can Be Mimicked by an Engineered IL-7 Autocrine Loop.

Adoptive cell therapy with chimeric antigen receptor (CAR)-redirected T cells induced spectacular regressions of leukemia and lymphoma, however, failed so far in the treatment of solid tumors. A cause is thought to be T cell repression through TGF-ß, which is massively accumulating in the tumor tissue. Here, we show that T cells with a CD28-ζ CAR, but not with a 4-1BB-ζ CAR, resist TGF-ß-mediated repression. Mechanistically, LCK activation and consequently IL-2 release and autocrine IL-2 receptor signaling mediated TGF-ß resistance; deleting the LCK-binding motif in the CD28 CAR abolished both IL-2 secretion and TGF-ß resistance, while IL-2 add-back restored TGF-ß resistance. Other γ-cytokines like IL-7 and IL-15 could replace IL-2 in this context. This is demonstrated by engineering IL-2 deficient CD28ΔLCK-ζ CAR T cells with a hybrid IL-7 receptor to provide IL-2R ß chain signaling upon IL-7 binding. Such modified T cells showed improved CAR T cell activity against TGF-ß+ tumors. Data draw the concept that an autocrine loop resulting in IL-2R signaling can make CAR T cells more potent in staying active against TGF-ß+ solid tumors.

CD20-CD19 Bispecific CAR T Cells for the Treatment of B-Cell Malignancies.

The treatment of leukemia/lymphoma by chimeric antigen receptor (CAR) redirected T cells with specificity for CD19 induced complete remissions in the majority of patients, with a realistic hope for cure. However, recent follow-up data revealed a substantial risk of relapse through leukemic cells that lack the CAR targeted antigen. In this situation, a bispecific CAR with binding domains for CD19 and CD20 is aimed at recognizing leukemic cells with only one cognate antigen. The anti-CD20-CD19 bispecific CAR induced a full T-cell response upon engagement of CD19 or CD20 on target cells showing a true "OR" gate recognition in redirecting T-cell activation. T cells with the anti-CD20-CD19 CAR efficiently killed patients' chronic lymphocytic leukemia cells in vitro. The bispecific CAR T cells cleared pediatric acute lymphocytic leukemia with a mixed CD19+CD20+/CD20- phenotype from the blood and bone marrow of transplanted mice, while anti-CD20 CAR T cells left CD20- leukemic cells behind without curing the disease. Data indicate the superior anti-leukemic activity in the control of leukemia, implying that the anti-CD20-CD19 bispecific CAR T cells may reduce the risk of relapse through antigen-loss leukemic cells in the long term.

Most Do, but Some Do Not: CD4⁺CD25- T Cells, but Not CD4⁺CD25⁺ Treg Cells, Are Cytolytic When Redirected by a Chimeric Antigen Receptor (CAR).

Evidences are accumulating that CD4⁺ T cells can physiologically mediate antigen specific target cell lysis. By circumventing major histocompatibility complex (MHC)-restrictions through an engineered chimeric antigen receptor (CAR), CD4⁺ T cells lyse defined target cells as efficiently as do CD8⁺ T cells. However, the cytolytic capacity of redirected CD4⁺CD25- T cells, in comparison with CD4⁺CD25⁺ regulatory T (Treg) cells was so far not thoroughly defined. Treg cells require a strong CD28 signal together with CD3ζ for activation. We consequently used a CAR with combined CD28-CD3ζ signalling for redirecting CD4⁺CD25- T cells and CD4⁺CD25⁺ Treg cells from the same donor. CAR redirected activation of these T cell subsets and induced a distinct cytokine pattern with high IL-10 and a lack of IL-2 release by Treg cells. Despite strong antigen-specific activation, CAR Treg cells produced only weak target cell lysis, whereas CD4⁺CD25- CAR T cells were potent killers. Cytolysis did not correlate with the target cell sensitivity to Fas/FasL mediated killing; CD4⁺CD25- T cells upregulated perforin and granzyme B upon CAR activation, whereas Treg cells did less. The different cytolytic capacities of CAR redirected conventional CD4⁺ cells and Treg cells imply their use for different purposes in cell therapy.

Shared target antigens on cancer cells and tissue stem cells: go or no-go for CAR T cells?

INTRODUCTION: Adoptive therapy with chimeric antigen receptor (CAR) T cells redirected towards CD19 produces remissions of B cell malignancies, however, it also eradicates healthy B cells sharing the target antigen. Such 'on-target off-tumor' toxicity raises serious safety concerns when the target antigen is also expressed by tissue stem cells, with the risk of lasting tissue destruction. Areas covered: We discuss CAR T cell targeting of activation antigens versus lineage associated antigens on the basis of recent experimental and animal data and the literature in the field. Expert commentary: Targeting an activation associated antigen which is transiently expressed by stem cells seems to be safe, like CAR T cells targeting CD30 spare CD30+ hematopoietic stem and progenitor cells while eliminating CD30+ lymphoma cells, whereas targeting lineage associated antigens which increase in expression during cell maturation, like folate receptor-ß and CD123, is of risk to destruct tissue stem cells.

Chimeric antigen receptor T cells targeting Fc µ receptor selectively eliminate CLL cells while sparing healthy B cells.

Adoptive cell therapy of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR)-modified T cells targeting CD19 induced lasting remission of this refractory disease in a number of patients. However, the treatment is associated with prolonged "on-target off-tumor" toxicities due to the targeted elimination of healthy B cells demanding more selectivity in targeting CLL cells. We identified the immunoglobulin M Fc receptor (FcµR), also known as the Fas apoptotic inhibitory molecule-3 or TOSO, as a target for a more selective treatment of CLL by CAR T cells. FcµR is highly and consistently expressed by CLL cells; only minor levels are detected on healthy B cells or other hematopoietic cells. T cells with a CAR specific for FcµR efficiently responded toward CLL cells, released a panel of proinflammatory cytokines and lytic factors, like soluble FasL and granzyme B, and eliminated the leukemic cells. In contrast to CD19 CAR T cells, anti-FcµR CAR T cells did not attack healthy B cells. T cells with anti-FcµR CAR delayed outgrowth of Mec-1-induced leukemia in a xenograft mouse model. T cells from CLL patients in various stages of the disease, modified by the anti-FcµR CAR, purged their autologous CLL cells in vitro without reducing the number of healthy B cells, which is the case with anti-CD19 CAR T cells. Compared with the currently used therapies, the data strongly imply a superior therapeutic index of anti-FcµR CAR T cells for the treatment of CLL.

Superior Therapeutic Index in Lymphoma Therapy: CD30(+) CD34(+) Hematopoietic Stem Cells Resist a Chimeric Antigen Receptor T-cell Attack.

Recent clinical trials with chimeric antigen receptor (CAR) redirected T cells targeting CD19 revealed particular efficacy in the treatment of leukemia/lymphoma, however, were accompanied by a lasting depletion of healthy B cells. We here explored CD30 as an alternative target, which is validated in lymphoma therapy and expressed by a broad variety of Hodgkin's and non-Hodgkin's lymphomas. As a safty concern, however, CD30 is also expressed by lymphocytes and hematopoietic stem and progenitor cells (HSPCs) during activation. We revealed that HRS3scFv-derived CAR T cells are superior since they were not blocked by soluble CD30 and did not attack CD30(+) HSPCs while eliminating CD30(+) lymphoma cells. Consequently, normal hemato- and lymphopoiesis was not affected in the long-term in the humanized mouse; the number of blood B and T cells remained unchanged. We provide evidence that the CD30(+) HSPCs are protected against a CAR T-cell attack by substantially lower CD30 levels than lymphoma cells and higher levels of the granzyme B inactivating SP6/PI9 serine protease, which furthermore increased upon activation. Taken together, adoptive cell therapy with anti-CD30 CAR T cells displays a superior therapeutic index in the treatment of CD30(+) malignancies leaving healthy activated lymphocytes and HSPCs unaffected.

Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone.

Chimeric antigen receptor (CAR)-modified T cells emerged as effective tools in the immunotherapy of cancer but can produce severe on-target off-tissue toxicities. This risk can conceivably be overcome, at least partially, by transient transfection. The design of CARs, however, has so far not been optimized for use in non-permanent T cell modification. Here we compared the performance of T cells modified with three different first- and second-generation CARs, each specific for MCSP (HMW-MAA) which is commonly expressed by melanoma cells. Upon RNA transfer, the expression of all receptors was limited in time. The second-generation CARs, which combined CD28-CD3ζ signaling, were expressed at higher levels and more prolonged than first-generation CARs with CD3ζ only. The CD28 domain increased the cytokine production, but had only an indirect effect on the lytic capacity, by prolonging the CAR expression. Especially for the second-generation CARs, the scFv clearly impacted the level and duration of CAR expression and the T cell performance. Thus, we identified a CAR high in both expression and anti-tumor cell reactivity. T cells transfected with this CAR increased the mean survival time of mice after challenge with melanoma cells. To facilitate clinical application, this CAR was used to redirect T cells from late-stage melanoma patients by RNA transfection. These T cells mediated effective antigen-specific tumor cell lysis and release of pro-inflammatory cytokines, even after cryoconservation of the transfected T cells. Taken together, the analysis identified a CAR with superior anti-melanoma performance after RNA transfer which is a promising candidate for clinical exploration.

Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma.

Adoptive T-cell therapy recently achieved impressive efficacy in early phase trials, in particular in hematologic malignancies, strongly supporting the notion that the immune system can control cancer. A current strategy of favor is based on ex vivo-engineered patient T cells, which are redirected by a chimeric antigen receptor (CAR) and recognize a predefined target by an antibody-derived binding domain. Such CAR T cells can substantially reduce the tumor burden as long as the targeted antigen is present on the cancer cells. However, given the tremendous phenotypic diversity in solid tumor lesions, a reasonable number of cancer cells are not recognized by a given CAR, considerably reducing the therapeutic success. This article reviews a recently described strategy for overcoming this shortcoming of the CAR T-cell therapy by modulating the tumor stroma by a CAR T-cell-secreted transgenic cytokine like interleukin-12 (IL-12). The basic process is that CAR T cells, when activated by their CAR, deposit IL-12 in the targeted tumor lesion, which in turn attracts an innate immune cell response toward those cancer cells that are invisible to CAR T cells. Such TRUCKs, T cells redirected for universal cytokine-mediated killing, exhibited remarkable efficacy against solid tumors with diverse cancer cell phenotypes, suggesting their evaluation in clinical trials.

Second mitochondria-derived activator of caspase (SMAC) mimetic potentiates tumor susceptibility toward natural killer cell-mediated killing.

Resistance to apoptosis is a hallmark of cancer, and represents an important mechanism of how tumor cells resist immune cell destruction. Mitochondria are the central regulators of the apoptotic machinery by releasing pro-apoptotic factors including cytochrome c and second mitochondria-derived activator of caspase (SMAC) upon mitochondrial outer membrane permeabilization (MOMP). Small molecules activating MOMP such as BH3 mimetics or antagonizers of the inhibitor of apoptosis proteins (IAPs) such as SMAC mimetics have recently engendered new optimism for a more individualized and effective cancer therapy. Here we show that a SMAC mimetic potentiates cancer cell killing by natural killer (NK) cells through reactivation of tumor cell apoptosis. Specifically, the SMAC mimetic enhances the susceptibility of tumor cells toward NK cell-mediated effector mechanisms involving death receptors and cytolytic granules containing perforin and granzymes by relieving caspase activity. Our data highlight for the first time the specific use of SMAC mimetics for boosting immune cell-mediated immunotherapy, representing a novel and promising approach in the treatment of cancer.

Antigen-Specific T-Cell Activation Independently of the MHC: Chimeric Antigen Receptor-Redirected T Cells.

Adoptive T-cell therapy has recently shown promise in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T-cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC) expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR) which consists in the extracellular part of an antibody-derived domain for binding with a "tumor-associated antigen" and in the intracellular part of a T-cell receptor (TCR)-derived signaling moiety for T-cell activation. The specificity of CAR-mediated T-cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T-cell targeting by an engineered CAR in comparison to TCR modified T cells and the impact of the CAR activation threshold on redirected T-cell activation. Finally we review most significant progress recently made in early stage clinical trials to treat cancer.

Arming cytokine-induced killer cells with chimeric antigen receptors: CD28 outperforms combined CD28-OX40 "super-stimulation".

Cytokine-induced killer (CIK) cells raised interest for use in cellular antitumor therapy due to their capability to recognize and destroy autologous tumor cells in a HLA-independent fashion. The antitumor attack of CIK cells, predominantly consisting of terminally differentiated CD8(+)CD56(+) cells, can be improved by redirecting by a chimeric antigen receptor (CAR) that recognizes the tumor cell and triggers CIK cell activation. The requirements for CIK cell activation were, however, so far less explored and are likely to be different from those of "younger" T cells. We revealed that CD28 and OX40 CARs produced higher interferon- secretion as compared with the first-generation ζ-CAR; CD28-ζ and the third-generation CD28-ζ-OX40 CAR, however, performed similar in modulating most CIK cell effector functions. Compared with the CD28-ζ CAR, however, the CD28-ζ-OX40 CAR accelerated terminal maturation of CD56(+) CIK cells producing high frequencies in activation-induced cell death (AICD) and reduced antitumor efficiency in vivo. Consequently, CD28-ζ CAR CIK cells of CD56(-) phenotype were superior in redirected tumor cell elimination. CAR-mediated CIK cell activation also increased antigen-independent target cell lysis; the CD28-ζ CAR was more efficient than the CD28-ζ-OX40 CAR. Translated into therapeutic strategies, CAR-redirected CIK cells benefit from CD28 costimulation; "super-costimulation" by the CD28-ζ-OX40 CAR, however, performed less in antitumor efficacy due to increased AICD.