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.

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.

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.

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.

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.

Targeting two co-operating cytokines efficiently shapes immune responses.

For simultaneously mobilizing the adaptive and innate immune system against cancer, we fused interleukin (IL)-2 and IL-12 to generate a dual cytokine moiety that is targeted to neoplastic lesions by an antibody-binding domain. This approach elicits a broader attack of the immune system against cancer than the use of each cytokine alone.

Young T Cells Age During a Redirected Anti-Tumor Attack: Chimeric Antigen Receptor-Provided Dual Costimulation is Half the Battle.

Adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells showed spectacular efficacy in the treatment of leukemia in recent early phase trials. Patient's T cells were ex vivo genetically engineered with a CAR, amplified and re-administered to the patient. While T cells mediating the primary response were predominantly of young effector and central memory phenotype, repetitive antigen engagement irreversible triggers T cell maturation leaving late memory cells with the KLRG1(+) CD57(+) CD7(-) CCR7(-) phenotype in the long-term. These cells preferentially accumulate in the periphery, are hypo-responsive upon TCR engagement and prone to activation-induced cell death. A recent report indicates that those T cells can be rescued by CAR provided CD28 and OX40 (CD134) stimulation. We discuss the strategy with respect to prolong the anti-tumor response and to improve the over-all efficacy of adoptive cell therapy.

Survivin blockade sensitizes rhabdomyosarcoma cells for lysis by fetal acetylcholine receptor-redirected T cells.

Cellular immunotherapy may provide a strategy to overcome the poor prognosis of metastatic and recurrent rhabdomyosarcoma (RMS) under the current regimen of polychemotherapy. Because little is known about resistance mechanisms of RMS to cytotoxic T cells, we investigated RMS cell lines and biopsy specimens for expression and function of immune costimulatory receptors and anti-apoptotic molecules by RT-PCR, Western blot analysis, IHC, and cytotoxicity assays using siRNA or transfection-modified RMS cell lines, together with engineered RMS-directed cytotoxic T cells specific for the fetal acetylcholine receptor. We found that costimulatory CD80 and CD86 were consistently absent from all RMSs tested, whereas inducible T-cell co-stimulator ligand (ICOS-L; alias B7H2) was expressed by a subset of RMSs and was inducible by tumor necrosis factor α in two of five RMS cell lines. Anti-apoptotic survivin, along with other inhibitor of apoptosis (IAP) family members (cIAP1, cIAP2, and X-linked inhibitor of apoptosis protein), was overexpressed by RMS cell lines and biopsy specimens. Down-regulation of survivin by siRNA or pharmacologically in RMS cells increased their susceptibility toward a T-cell attack, whereas induction of ICOS-L did not. Treatment of RMS-bearing Rag(-/-) mice with fetal acetylcholine receptor-specific chimeric T cells delayed xenograft growth; however, this happened without definitive tumor eradication. Combined blockade of survivin and application of chimeric T cells in vivo suppressed tumor proliferation during survivin inhibition. In conclusion, survivin blockade provides a strategy to sensitize RMS cells for T-cell-based therapy.

Adoptive immunotherapy with redirected T cells produces CCR7- cells that are trapped in the periphery and benefit from combined CD28-OX40 costimulation.

Adoptive therapy of cancer with genetically redirected T cells showed spectacular efficacy in recent trials. A body of preclinical and clinical data indicate that young effector and central memory T cells perform superior in a primary antitumor response; repetitive antigen engagement, however, drives T-cell maturation to terminally differentiated cells associated with the loss of CCR7, which enables T cells to persist in peripheral tissues. In this work, we explored the antitumor efficacy of CCR7(-) T cells when redirected in an antigen-dependent fashion by a chimeric antigen receptor (CAR) toward tumors in the periphery. CAR-engineered CCR7(-) T cells more efficiently accumulated at the tumor site, secreted more IFN-γ, expressed higher amounts of cytotoxic molecules, and showed superior tumor cell lysis compared to the younger CCR7(+) cells. CCR7(-) T cells, however, were more prone to spontaneous and activation-induced cell death, which could be counteracted by simultaneous CD28 and OX40 (CD134) costimulation. Consequently, the combined CD28-ζ-OX40 signaling CAR rescued CCR7(-) T cells from apoptosis, which then produced more efficient antitumor efficacy than CCR7(+) T cells redirected by the same CAR. Data suggest that T-cell therapy will benefit from combined CD28-ζ-OX40 stimulation in the long-term by rescuing continuously generated CCR7(-) T cells for an antitumor attack.

An IL12-IL2-antibody fusion protein targeting Hodgkin's lymphoma cells potentiates activation of NK and T cells for an anti-tumor attack.

Successful immunotherapy of Hodgkin's disease is so far hampered by the striking unresponsiveness of lymphoma infiltrating immune cells. To mobilize both adoptive and innate immune cells for an anti-tumor attack we fused the pro-inflammatory cytokines IL2 and IL12 to an anti-CD30 scFv antibody in a dual cytokine fusion protein to accumulate both cytokines at the malignant CD30(+) Hodgkin/Reed-Sternberg cells in the lymphoma lesion. The tumor-targeted IL12-IL2 fusion protein was superior in activating resting T cells to amplify and secrete pro-inflammatory cytokines compared to targeted IL2 or IL12 alone. NK cells were also activated by the dual cytokine protein to secrete IFN-γ and to lyse target cells. The tumor-targeted IL12-IL2, when applied by i.v. injection to immune-competent mice with established antigen-positive tumors, accumulated at the tumor site and induced tumor regression. Data demonstrate that simultaneous targeting of two cytokines in a spatial and temporal simultaneous fashion to pre-defined tissues is feasible by a dual-cytokine antibody fusion protein. In the case of IL12 and IL2, this produced superior anti-tumor efficacy implying the strategy to muster a broader immune cell response in the combat against cancer.

Antibody-IL2 fusion proteins for tumor targeting.

Increasing insight into the misbalance and poor activity of tumor infiltrating immune cells raised interest to activate and improve an antitumor immune response by accumulating IL2 in the tumor tissue. IL2 can be targeted as part of an antibody-cytokine fusion protein to the tumor tissue by a single chain fragment of variable regions (scFv) antibody recognizing a tumor-associated antigen. IL2 can moreover be combined with IL12 in a dual cytokine fusion protein, which simultaneously targets both cooperating cytokines to the tumor in order to improve the activation of both T cells and innate immune cells. We here describe in detail the construction, expression, and functional testing of antibody-IL2 fusion proteins and provide a protocol to determine the biodistribution of such proteins in animal models.