Joanne A. Hammill

T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice

Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution.

Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors

BackgroundAdoptive cell transfer of tumor-specific T lymphocytes (T cells) is proving to be an effective strategy for treating established tumors in cancer patients. One method of generating these cells is accomplished through engineering bulk T cell populations to express chimeric antigen receptors (CARs), which are specific for tumor antigens. Traditionally, these CARs are targeted against tumor antigens using single-chain antibodies (scFv). Here we describe the use of a designed ankyrin repeat protein (DARPin) as the tumor-antigen targeting domain.MethodsWe prepared second generation anti-HER2 CARs that were targeted to the tumor antigen by either a DARPin or scFv. The CARs were engineered into human and murine T cells. We then compared the ability of CARs to trigger cytokine production, degranulation and cytotoxicity.ResultsThe DARPin CARs displayed reduced surface expression relative to scFv CARs in murine cells but both CARs were expressed equally well on human T cells, suggesting that there may be a processing issue with the murine variants. In both the murine and human systems, the DARPin CARs were found to be highly functional, triggering cytokine and cytotoxic responses that were similar to those triggered by the scFv CARs.ConclusionsThese findings demonstrate the utility of DARPins as CAR-targeting agents and open up an avenue for the generation of CARs with novel antigen binding attributes.

Chimeric antigen receptor–engineered T cells as oncolytic virus carriers

The use of engineered T cells in adoptive transfer therapies has shown significant promise in treating hematological cancers. However, successes treating solid tumors are much less prevalent. Oncolytic viruses (OVs) have the capacity to induce specific lysis of tumor cells and indirectly impact tumor growth via vascular shutdown. These viruses bear natural abilities to associate with lymphocytes upon systemic administration, but therapeutic doses must be very high in order to evade antibodies and other components of the immune system. As T cells readily circulate through the body, using these cells to deliver OVs directly to tumors may provide an ideal combination. Our studies demonstrate that loading chimeric antigen receptor–engineered T cells with low doses of virus does not impact receptor expression or function in either murine or human T cells. Engineered T cells can deposit virus onto a variety of tumor targets, which can enhance the tumoricidal activity of the combination treatment. This concept appears to be broadly applicable, as we observed similar results using murine or human T cells, loaded with either RNA or DNA viruses. Overall, loading of engineered T cells with OVs represents a novel combination therapy that may increase the efficacy of both treatments.

Combined mTOR inhibition and OX40 agonism enhances CD8(+) T cell memory and protective immunity produced by recombinant adenovirus vaccines.

The memory CD8+ T cell population elicited by immunization with recombinant human adenovirus serotype 5 (rHuAd5) vaccines is composed primarily of effector and effector memory cells (TEM) with limited polyfunctionality. In this study, we investigated whether treatment with immunomodulators could enhance and/or redistribute the CD8+ memory population elicited by rHuAd5. Vaccination in combination with both rapamycin (to modulate differentiation) and an OX40 agonist (to enhance costimulation) increased both the quantity and polyfunctionality of the CD8+ memory T cell population, with expansion of the TEM and memory precursor populations. Furthermore, this intervention enhanced protection against multiple virus challenges. Attenuation of adenovirus transgene expression was required to enable the combination rapamycin + OX40 agonist immunomodulatory treatment to further enhance skewing towards central memory formation, indicating that persistence of antigen expression ultimately limits development of this memory population following rHuAd5 immunization. These results demonstrate that during the expansion phase following adenovirus immunization, the level of mammalian target of rapamycin (mTOR) activity, the amount of costimulation and the duration of antigen availability act together to define the magnitude, phenotype, and functionality of memory CD8+ T cells. Modulation of these factors can be used to selectively manipulate memory formation.The memory CD8(+) T cell population elicited by immunization with recombinant human adenovirus serotype 5 (rHuAd5) vaccines is composed primarily of effector and effector memory cells (T(EM)) with limited polyfunctionality. In this study, we investigated whether treatment with immunomodulators could enhance and/or redistribute the CD8(+) memory population elicited by rHuAd5. Vaccination in combination with both rapamycin (to modulate differentiation) and an OX40 agonist (to enhance costimulation) increased both the quantity and polyfunctionality of the CD8(+) memory T cell population, with expansion of the T(EM) and memory precursor populations. Furthermore, this intervention enhanced protection against multiple virus challenges. Attenuation of adenovirus transgene expression was required to enable the combination rapamycin + OX40 agonist immunomodulatory treatment to further enhance skewing towards central memory formation, indicating that persistence of antigen expression ultimately limits development of this memory population following rHuAd5 immunization. These results demonstrate that during the expansion phase following adenovirus immunization, the level of mammalian target of rapamycin (mTOR) activity, the amount of costimulation and the duration of antigen availability act together to define the magnitude, phenotype, and functionality of memory CD8(+) T cells. Modulation of these factors can be used to selectively manipulate memory formation.

CD11b+ Dendritic Cell–Mediated Anti–Mycobacterium tuberculosis Th1 Activation Is Counterregulated by CD103+ Dendritic Cells via IL-10

Mycobacterium tuberculosis, the pathogen causing pulmonary tuberculosis (TB) in humans, has evolved to delay Th1 immunity in the lung. Although conventional dendritic cells (cDCs) are known to be critical to the initiation of T cell immunity, the differential roles and molecular mechanisms of migratory CD11b+ and CD103+ cDC subsets in anti–M. tuberculosis Th1 activation remain unclear. Using a murine model of pulmonary M. tuberculosis infection, we found that slow arrival of M. tuberculosis–bearing migratory CD11b+ and CD103+ cDCs at the draining lymph nodes preceded the much-delayed Th1 immunity and protection in the lung. Contrary to their previously described general roles in Th polarization, CD11b+ cDCs, but not CD103+ cDCs, were critically required for Th1 activation in draining lymph nodes following M. tuberculosis infection. CD103+ cDCs counterregulated CD11b+ cDC–mediated Th1 activation directly by producing the immune-suppressive cytokine IL-10. Thus, our study provides new mechanistic insights into differential Th immune regulation by migratory cDC subsets and helps to develop novel vaccines and therapies.

AMPK β1 reduces tumor progression and improves survival in p53 null mice

The AMP‐activated protein kinase (AMPK) is a heterotrimeric protein complex that is an important sensor of cellular energy status. Reduced expression of the AMPK β1 isoform has been linked to reduced survival in different cancers, but whether this accelerates tumor progression and the potential mechanism mediating these effects are not known. Furthermore, it is unknown whether AMPK β1 is implicated in tumorigenesis, and if so, what tissues may be most sensitive. In the current study, we find that in the absence of the tumor suppressor p53, germline genetic deletion of AMPK β1 accelerates the appearance of a T‐cell lymphoma that reduces lifespan compared to p53 deficiency alone. This increased tumorigenesis is linked to increases in interleukin‐1β (IL1β), reductions in acetyl‐CoA carboxylase (ACC) phosphorylation, and elevated lipogenesis. Collectively, these data indicate that reductions in the AMPK β1 subunit accelerate the development of T‐cell lymphoma, suggesting that therapies targeting this AMPK subunit or inhibiting lipogenesis may be effective for limiting the proliferation of p53‐mutant tumors.

Viral Engineering of Chimeric Antigen Receptor Expression on Murine and Human T Lymphocytes.

The adoptive transfer of a bolus of tumor-specific T lymphocytes into cancer patients is a promising therapeutic strategy. In one approach, tumor specificity is conferred upon T cells via engineering expression of exogenous receptors, such as chimeric antigen receptors (CARs). Here, we describe the generation and production of both murine and human CAR-engineered T lymphocytes using retroviruses.

The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity

Engineering T cells with chimeric antigen receptors (CARs) is an effective method for directing T cells to attack tumors, but may cause adverse side effects such as the potentially lethal cytokine release syndrome. Here the authors show that the T cell antigen coupler (TAC), a chimeric receptor that co-opts the endogenous TCR, induces more efficient anti-tumor responses and reduced toxicity when compared with past-generation CARs. TAC-engineered T cells induce robust and antigen-specific cytokine production and cytotoxicity in vitro, and strong anti-tumor activity in a variety of xenograft models including solid and liquid tumors. In a solid tumor model, TAC-T cells outperform CD28-based CAR-T cells with increased anti-tumor efficacy, reduced toxicity, and faster tumor infiltration. Intratumoral TAC-T cells are enriched for Ki-67+ CD8+ T cells, demonstrating local expansion. These results indicate that TAC-T cells may have a superior therapeutic index relative to CAR-T cells.Chimeric antigen receptors (CARs) are effective tools for directing T cell killing of tumors, but may cause adverse side effects. Here the authors show that coupling of antigen-recognition and CD3-binding in a modular format induces more efficient anti-tumour responses but reduced toxicity when compared with current CARs.

On-target off-tumor toxicity; when enhancing an NKG2D-based CAR in vitro led to severe toxicities in vivo

Engineering T cells with chimeric antigen receptors (CARs) has emerged as a promising approach to adoptive T cell therapy for cancer. We have been studying CARs that employ the ligand-binding domain of the NKG2D receptor to target tumors. NKG2D ligand expression is increased on the surface of stressed cells, like tumor cells, making this family of ligands a target of interest for cancer immunotherapies. Two CARs were constructed: 1) a fusion of the full-length NKG2D receptor and CD3z (NKG2Dζ) and 2) the extracellular domain of NKG2D fused to a second-generation CAR scaffold composed of transmembrane and intracellular domains from CD28 and the signaling domain of CD3z (NKG2D28ζ). Since surface expression of NKG2D is limited by the presence of DAP10, we also created a vector where DAP10 was co-expressed with NKG2Dζ (NKG2Dζ10). Indeed, NKG2Dζ10-CAR-T cells displayed a greater than 10-fold increase in CAR expression compared to NKG2Dζ; NKG2D28ζ showed an intermediate level of expression. T cells expressing any of the NKG2D CARs produced IFNg and TNFa in response to NKG2D ligand stimulation and efficiently killed tumor targets in vitro. However, following infusion into syngeneic hosts, we observed significant toxicity in vivo with these CAR constructs. Signs of toxicity, including poor body condition, hunched posture, labored breathing, and decreased core body temperature were observed in tumor-bearing and tumor-free mice treated with NKG2D-based CAR-T cells as compared to control mice. The severity of NKG2D CAR-T cell toxicity varied, with NKG2Dζ10 being severely toxic, NKG2D28ζ showing intermediate toxicity, and NKG2Dζ being tolerable. Clinical symptoms of toxicity and mortality rates were exacerbated when mice received chemotherapy prior to adoptive transfer of T cells expressing any of the NKG2D CARs. These observations were consistent between BALB/c and C57BL/6 hosts. Further characterization revealed that the toxicity coincided with a systemic cytokine storm and lethal levels of inflammation within the lungs. These data warn that extreme caution should be taken when using NKG2D ligands for targeted immunotherapy and demonstrate that enhancing T cell expression of strongly activating CARs can be detrimental in vivo when the CAR target is not uniquely expressed on the tumor. This research was supported by the CIHR and the TFRI.