Multifunctional NK Cell-Engaging Antibodies Targeting EGFR and NKp30 Elicit Efficient Tumor Cell Killing and Proinflammatory Cytokine Release.

In this work, we have generated novel Fc-comprising NK cell engagers (NKCEs) that bridge human NKp30 on NK cells to human epidermal growth factor receptor (EGFR) on tumor cells. Camelid-derived VHH single-domain Abs specific for human NKp30 and a humanized Fab derived from the EGFR-specific therapeutic Ab cetuximab were used as binding arms. By combining camelid immunization with yeast surface display, we were able to isolate a diverse panel of NKp30-specific VHHs against different epitopes on NKp30. Intriguingly, NKCEs built with VHHs that compete for binding to NKp30 with B7-H6, the natural ligand of NKp30, were significantly more potent in eliciting tumor cell lysis of EGFR-positive tumor cells than NKCEs harboring VHHs that target different epitopes on NKp30 from B7-H6. We demonstrate that the NKCEs can be further improved with respect to killing capabilities by concomitant engagement of FcγRIIIa and that soluble B7-H6 does not impede cytolytic capacities of all scrutinized NKCEs at significantly higher B7-H6 concentrations than observed in cancer patients. Moreover, we show that physiological processes requiring interactions between membrane-bound B7-H6 and NKp30 on NK cells are unaffected by noncompeting NKCEs still eliciting tumor cell killing at low picomolar concentrations. Ultimately, the NKCEs generated in this study were significantly more potent in eliciting NK cell-mediated tumor cell lysis than cetuximab and elicited a robust release of proinflammatory cytokines, both features which might be beneficial for antitumor therapy.

The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity.

T cell activation is subject to tight regulation to avoid inappropriate responses to self antigens. Here we show that genetic deficiency in the ubiquitin ligase Peli1 caused hyperactivation of T cells and rendered T cells refractory to suppression by regulatory T cells and transforming growth factor-ß (TGF-ß). As a result, Peli1-deficient mice spontaneously developed autoimmunity characterized by multiorgan inflammation and autoantibody production. Peli1 deficiency resulted in the nuclear accumulation of c-Rel, a member of the NF-κB family of transcription factors with pivotal roles in T cell activation. Peli1 negatively regulated c-Rel by mediating its Lys48 (K48) ubiquitination. Our results identify Peli1 as a critical factor in the maintenance of peripheral T cell tolerance and demonstrate a previously unknown mechanism of c-Rel regulation.

Affinity Maturation of B7-H6 Translates into Enhanced NK Cell-Mediated Tumor Cell Lysis and Improved Proinflammatory Cytokine Release of Bispecific Immunoligands via NKp30 Engagement.

Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format based on affinity-optimized versions of B7-H6 and the Fab arm derived from cetuximab. To enhance NKp30 binding, the solitary N-terminal IgV domain of B7-H6 (ΔB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel ΔB7-H6-derived NK cell engagers revealed an up to 45-fold-enhanced affinity for NKp30 and significantly improved NK cell-mediated, EGFR-dependent killing of tumor cells compared with the NK cell engager based on the wild-type ΔB7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-γ and TNF-α was significantly increased. Importantly, equipment of the ΔB7-H6-based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared with molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared with molecules solely triggering FcγRIIIa, including the clinically approved Ab cetuximab. Thus, incorporating affinity-matured ligands for NK cell-activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy.

Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells.

Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.

ELF5 Drives Lung Metastasis in Luminal Breast Cancer through Recruitment of Gr1+ CD11b+ Myeloid-Derived Suppressor Cells.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection.

Clinical-grade T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) to redirect their specificity to target tumor-associated antigens in vivo. We now have developed this molecular strategy to render cytotoxic T cells specific for fungi. We adapted the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-ζ (designated "D-CAR") upon binding with carbohydrate in the cell wall of Aspergillus germlings. T cells genetically modified with the Sleeping Beauty system to express D-CAR stably were propagated selectively on artificial activating and propagating cells using an approach similar to that approved by the Food and Drug Administration for manufacturing CD19-specific CAR(+) T cells for clinical trials. The D-CAR(+) T cells exhibited specificity for ß-glucan which led to damage and inhibition of hyphal growth of Aspergillus in vitro and in vivo. Treatment of D-CAR(+) T cells with steroids did not compromise antifungal activity significantly. These data support the targeting of carbohydrate antigens by CAR(+) T cells and provide a clinically appealing strategy to enhance immunity for opportunistic fungal infections using T-cell gene therapy.

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.

A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR.

Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR(+) T cells to eliminate expression of the endogenous αß T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or ß TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR(+)TCR(neg) T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.

Using protein geometry to optimize cytotoxicity and the cytokine window of a ROR1 specific T cell engager.

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity. Using ROR1 as our model TAA and highly modular camelid nanobodies, we describe the engineering of a next generation decoupled TCE that incorporates a "cytokine window" defined as a dose range in which maximal killing is reached but cytokine release may be modulated from very low for safety to nearly that induced by first generation TCEs. This latter attribute supports pro-inflammatory anti-tumor activity including bystander killing and can potentially be used by clinicians to safely titrate patient dose to that which mediates maximum efficacy that is postulated as greater than that possible using standard second generation approaches. We used a combined method of optimizing TCE mediated synaptic distance and apparent affinity tuning of the TAA binding arms to generate a relatively long but persistent synapse that supports a wide cytokine window, potent killing and a reduced propensity towards immune exhaustion. Importantly, this next generation TCE induced significant tumor growth inhibition in vivo but unlike a first-generation non-decoupled benchmark TCE that induced lethal CRS, no signs of adverse events were observed.

M9657 Is a Bispecific Tumor-Targeted Anti-CD137 Agonist That Induces MSLN-Dependent Antitumor Immunity without Liver Inflammation.

The costimulatory receptor CD137 (also known as TNFRSF9 or 4-1BB) sustains effective cytotoxic T-cell responses. Agonistic anti-CD137 cancer immunotherapies are being investigated in clinical trials. Development of the first-generation CD137-agonist monotherapies utomilumab and urelumab was unsuccessful due to low antitumor efficacy mediated by the epitope recognized on CD137 or hepatotoxicity mediated by Fcγ receptors (FcγR) ligand-dependent CD137 activation, respectively. M9657 was engineered as a tetravalent bispecific antibody (mAb2) in a human IgG1 backbone with LALA mutations to reduce binding to FCγRs. Here, we report that M9657 selectively binds to mesothelin (MSLN) and CD137 with similar affinity in humans and cynomolgus monkeys. In a cellular functional assay, M9657 enhanced CD8+ T cell-mediated cytotoxicity and cytokine release in the presence of tumor cells, which was dependent on both MSLN expression and T-cell receptor/CD3 activation. Both FS122m, a murine surrogate with the same protein structure as M9657, and chimeric M9657, a modified M9657 antibody with the Fab portion replaced with an anti-murine MSLN motif, demonstrated in vivo antitumor efficacy against various tumors in wild-type and human CD137 knock-in mice, and this was accompanied by activated CD8+ T-cell infiltration in the tumor microenvironment. The antitumor immunity of M9657 and FS122m depended on MSLN expression density and the mAb2 structure. Compared with 3H3, a murine surrogate of urelumab, FS122m and chimeric M9657 displayed significantly lower on-target/off-tumor toxicity. Taken together, M9657 exhibits a promising profile for development as a tumor-targeting immune agonist with potent anticancer activity without systemic immune activation and associated hepatotoxicity.

Impact of antibody architecture and paratope valency on effector functions of bispecific NKp30 x EGFR natural killer cell engagers.

Natural killer (NK) cells emerged as a promising effector population that can be harnessed for anti-tumor therapy. In this work, we constructed NK cell engagers (NKCEs) based on NKp30-targeting single domain antibodies (sdAbs) that redirect the cytotoxic potential of NK cells toward epidermal growth factor receptor (EGFR)-expressing tumor cells. We investigated the impact of crucial parameters such as sdAb location, binding valencies, the targeted epitope on NKp30, and the overall antibody architecture on the redirection capacity. Our study exploited two NKp30-specific sdAbs, one of which binds a similar epitope on NKp30 as its natural ligand B7-H6, while the other sdAb addresses a non-competing epitope. For EGFR-positive tumor targeting, humanized antigen-binding domains of therapeutic antibody cetuximab were used. We demonstrate that NKCEs bivalently targeting EGFR and bivalently engaging NKp30 are superior to monovalent NKCEs in promoting NK cell-mediated tumor cell lysis and that the architecture of the NKCE can substantially influence killing capacities depending on the NKp30-targeting sdAb utilized. While having a pronounced impact on NK cell killing efficacy, the capabilities of triggering antibody-dependent cellular phagocytosis or complement-dependent cytotoxicity were not significantly affected comparing the bivalent IgG-like NKCEs with cetuximab. However, the fusion of sdAbs can have a slight impact on the NK cell release of immunomodulatory cytokines, as well as on the pharmacokinetic profile of the NKCE due to unfavorable spatial orientation within the molecule architecture. Ultimately, our findings reveal novel insights for the engineering of potent NKCEs triggering the NKp30 axis.

Human CD34+ cells in experimental myocardial infarction: long-term survival, sustained functional improvement, and mechanism of action.

RATIONALE: Human CD34(+) cells have been used in clinical trials for treatment of myocardial infarction (MI). However, it is unknown how long the CD34(+) cells persist in hearts, whether the improvement in cardiac function is sustained, or what are the underlying mechanisms. OBJECTIVE: We sought to track the fate of injected human CD34(+) cells in the hearts of severe combined immune deficiency (SCID) mice after experimental MI and to determine the mechanisms of action. METHODS AND RESULTS: We used multimodality molecular imaging to track the fate of injected human CD34(+) cells in the hearts of SCID mice after experimental MI, and used selective antibody blocking to determine the mechanisms of action. Bioluminescence imaging showed that injected CD34(+) cells survived in the hearts for longer than 12 months. The PET signal from the injected cells was detected in the wall of the left ventricle. Cardiac MRI showed that left ventricular ejection fraction was significantly improved in the treated mice compared to the control mice for up to 52 weeks (P<0.05). Furthermore, treatment with anti-alpha4beta1 showed that generation of human-derived cardiomyocytes was inhibited, whereas anti-vascular endothelial growth factor (VEGF) treatment blocked the production of human-derived endothelial cells. However, the improvement in cardiac function was abolished only in the anti-VEGF, but not anti-alpha4beta1, treated group. CONCLUSIONS: Angiogenesis and/or paracrine effect, but not myogenesis, is responsible for functional improvement following CD34(+) cells therapy.

A Th1-inducing adenoviral vaccine for boosting adoptively transferred T cells.

Although the benefits of adoptive T-cell therapy can be increased by prior lymphodepletion of the recipient, this process usually requires chemotherapy or radiation. Vaccination with antigens to which the transferred T cells respond should be a less toxic means of enhancing their activity, but to date such vaccines have not been effective. We, therefore, determined which characteristics an adenoviral vaccine has to fulfill to optimally activate and expand adoptively transferred antigen-specific T cells in vivo. We evaluated (i) antigen, (ii) flagellin, a Toll-like receptor (TLR) 5 ligand, and (iii) an inhibitor of the antigen-presenting attenuator A20. Vaccination of mice before T-cell transfer with a vaccine that contained all three components dramatically enhanced the effector function of ovalbumin (OVA)-specific T cells as judged by the regression of established B16-OVA tumors compared to one- and two-component vaccines. Immunization with the three-component vaccine induced a strong Th1 environment, which was critical for the observed synergy and proved as effective as cytoxan-induced lymphodepletion in enhancing in vivo T-cell expansion. Thus, the combination of our vaccine with T-cell therapy has the potential to enhance and broaden adoptive cellular immunotherapy.

Plasmacytoid dendritic cells induce NK cell-dependent, tumor antigen-specific T cell cross-priming and tumor regression in mice.

A prerequisite for strong adaptive antiviral immunity is the robust initial activation of the innate immune system, which is frequently mediated by TLR-activated plasmacytoid DCs (pDCs). Natural antitumor immunity is often comparatively weak, potentially due to the lack of TLR-mediated activation signals within the tumor microenvironment. To assess whether pDCs are capable of directly facilitating effective antitumor immune responses, mice bearing established subcutaneous B16 melanoma tumors were administered TLR9-activated pDCs directly into the tumor. We found that TLR9-activated pDCs induced robust, spontaneous CTL cross-priming against multiple B16 tumor antigens, leading to the regression of both treated tumors and untreated tumors at distant contralateral sites. This T cell cross-priming was mediated by conventional DCs (cDCs) and was completely dependent upon the early recruitment and activation of NK cells at the tumor site. NK cell recruitment was mediated by CCR5 via chemokines secreted by pDCs, and optimal IFN-gamma production by NK cells was mediated by OX40L expressed by pDCs. Our data thus demonstrated that activated pDCs are capable of initiating effective and systemic antitumor immunity through the orchestration of an immune cascade involving the sequential activation of NK cells, cDCs, and CD8(+) T cells.

Fluorinated cannabinoid CB2 receptor ligands: synthesis and in vitro binding characteristics of 2-oxoquinoline derivatives.

Cannabinoid receptor 2 (CB2) plays an important role in human physiology and the pathophysiology of different diseases, including neuroinflammation, neurodegeneration, and cancer. Several classes of CB2 receptor ligands, including 2-oxoquinoline derivatives, have been previously reported. We report the synthesis and results of in vitro receptor binding of a focused library of new fluorinated 2-oxoquinoline CB2 ligands. Twelve compounds, 13-1618, 19, 21-24, 27, and 28 were synthesized in good yields in multiple steps. Human U87 glioma cells expressing either hCB1 (control) or hCB2 were generated via lentiviral transduction. In vitro competitive binding assay was performed using [(3)H]CP-55,940 in U87hCB1 and U87hCB2 cells. Inhibition constant (K(i)) values of compounds 13-16, 18, 19, 21-24, 27, and 28 for CB2 were >10,000, 2.8, 5.0, 2.4, 22, 0.8, 1.4, >10,000, 486, 58, 620, and 2400 nM, respectively, and those for CB1 were >10,000 nM. Preliminary in vitro results suggest that six of these compounds may be useful for therapy of neuropathic pain, neuroinflammatory diseases and immune disorders. In addition, compound 19, with its subnanomolar K(i) value, could be radiolabeled with (18)F and explored for PET imaging of CB2 expression.

Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer.

Antigen specific T cell migration to sites of infection or cancer is critical for an effective immune response. In mouse models of cancer, the number of lymphocytes reaching the tumor is typically only a few hundred, yet technology capable of imaging these cells using bioluminescence has yet to be achieved. A combination of codon optimization, removal of cryptic splice sites and retroviral modification was used to engineer an enhanced firefly luciferase (ffLuc) vector. Compared with ffLuc, T cells expressing our construct generated >100 times more light, permitting detection of as few as three cells implanted s.c. while maintaining long term coexpression of a reporter gene (Thy1.1). Expression of enhanced ffLuc in mouse T cells permitted the tracking of <3 x 10(4) adoptively transferred T cells infiltrating sites of vaccination and preestablished tumors. Penetration of light through deep tissues, including the liver and spleen, was also observed. Finally, we were able to enumerate infiltrating mouse lymphocytes constituting <0.3% of total tumor cellularity, representing a significant improvement over standard methods of quantitation including flow cytometry.

Both cultured and freshly isolated adipose tissue-derived stem cells enhance cardiac function after acute myocardial infarction.

AIMS: We assessed whether freshly isolated human adipose tissue-derived cells (fhADCs) or cultured human adipose tissue-derived stem cells (hASCs) have beneficial effects on cardiac function after myocardial infarction (MI), whether the injected cells can survive long term, and whether their effects result from direct differentiation or paracrine mechanisms. METHODS AND RESULTS: Myocardial infarction was experimentally induced in severe combined immunodeficient mice, and either fhADCs, cultured hASCs, or phosphate-buffered saline was injected into the peri-infarct region. Myocardial function improved significantly in mice treated with hASCs or fhADCs 4 weeks after MI. Immunofluorescence revealed that grafted hASCs and fhADCs underwent cardiomyogenic differentiation pathway, as indicated by expression of connexin 43 and troponin I in a fusion-independent manner. Some of the injected cells integrated with host cardiomyocytes through connexin 43, and others were incorporated into newly formed vessels. Human adipose tissue-derived stem cells survived in injured hearts up to 4 months, as detected by luciferase-based bioluminescence imaging. Vascular density was significantly increased, and fewer apoptotic cells were present in the peri-infarct region of cell-injected mice. CONCLUSION: This is the first study to systematically compare the effects of fhADCs and hASCs on myocardial regeneration. Both cell types engraft into infarcted myocardium, survive, and improve myocardial function, suggesting that fhADCs, like hASCs, are a promising alternative cell source for myocardial repair after MI.

HGAL inhibits lymphoma dissemination by interacting with multiple cytoskeletal proteins.

Human germinal center-associated lymphoma (HGAL) is an adaptor protein specifically expressed in germinal center lymphocytes. High expression of HGAL is a predictor of prolonged survival of diffuse large B-cell lymphoma (DLBCL) and classic Hodgkin lymphoma. Furthermore, HGAL expression is associated with early-stage DLBCL, thus potentially limiting lymphoma dissemination. In our previous studies, we demonstrated that HGAL regulates B-cell receptor signaling and cell motility in vitro and deciphered some molecular mechanisms underlying these effects. By using novel animal models for in vivo DLBCL dispersion, we demonstrate here that HGAL decreases lymphoma dissemination and prolongs survival. Furthermore, by using an unbiased proteomic approach, we demonstrate that HGAL may interact with multiple cytoskeletal proteins thereby implicating a multiplicity of effects in regulating lymphoma motility and spread. Specifically, we show that HGAL interacts with tubulin, and this interaction may also contribute to HGAL effects on cell motility. These findings recapitulate previous observations in humans, establish the role of HGAL in dissemination of lymphoma in vivo, and explain improved survival of patients with HGAL-expressing lymphomas.

Imaging of Sleeping Beauty-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography.

PURPOSE: We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells. PROCEDURES: We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19+ artificial antigen-presenting cells. RESULTS: After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19+ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2'-deoxy-2'-[18F]fluoro-5-ethyl-1-ß-D-arabinofuranosyl-uracil ([18F]FEAU). CONCLUSION: This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.

Genetic Engineering of T Cells to Target HERV-K, an Ancient Retrovirus on Melanoma.

PURPOSE: The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma. EXPERIMENTAL DESIGN: Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env-specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env-specific CAR(+) T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K-specific CAR(+) T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. RESULTS: We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K(+) AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env(+) tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env-specific CAR(+) T cells, we observed a significant antitumor effect. CONCLUSIONS: Adoptive cellular immunotherapy with HERV-K env-specific CAR(+) T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors.