Loss of Tuberous Sclerosis Complex 2 confers inflammation via dysregulation of Nuclear factor kappa-light-chain-enhancer of activated B cells.

Background: Aberrant activation of mTORC1 is clearly defined in TSC, causing uncontrolled cell growth. While mTORC1 inhibitors show efficacy to stabilise tumour growth in TSC, they are not fully curative. Disease facets of TSC that are not restored with mTOR inhibitors might involve NF-κB. The study aimed to characterise NF-κB in the context of TSC. Results: Enrichment of NF-κB-regulated genes was observed in TSC patient tumours, SEN/SEGAs, cortical tubers and a TSC tumour-derived cell line (621 - 101). Highlighting an inflammatory component of TSC, TSC cell models showed an elevated level of NF-κB and STAT3 activation. Herein, we report a dysregulated inflammatory phenotype of TSC2-deficient cells where NF-κB promotes autocrine signalling involving IL-6. Of importance, mTORC1 inhibition does not block this inflammatory signal to promote STAT3, while NF-κB inhibition was much more effective. Combined mTORC1 and NF-κB inhibition was potent at preventing anchorage-independent growth of TSC2-deficient cells, and unlike mTORC1 inhibition alone was sufficient to prevent colony regrowth after cessation of treatment. Conclusion: This study reveals autocrine signalling crosstalk between NF-κB and STAT3 in TSC cell models. Furthermore, the data presented indicate that NF-κB pathway inhibitors could be a viable adjunct therapy with the current mTOR inhibitors to treat TSC.

Granzyme B-activated IL18 potentiates αß and γδ CAR T cell immunotherapy in a tumor-dependent manner.

Interleukin (IL)18 is a potent pro-inflammatory cytokine that is activated upon caspase 1 cleavage of the latent precursor, pro-IL18. Therapeutic T cell armoring with IL18 promotes autocrine stimulation and positive modulation of the tumor microenvironment (TME). However, existing strategies are imperfect since they involve constitutive/poorly regulated activity or fail to modify the TME. Here, we have substituted the caspase 1 cleavage site within pro-IL18 with that preferred by granzyme B, yielding GzB-IL18. We demonstrate that GzB-IL18 is constitutively released but remains functionally latent unless chimeric antigen receptor (CAR) T cells are activated, owing to concomitant granzyme B release. Armoring with GzB-IL18 enhances cytolytic activity, proliferation, interferon (IFN)-γ release, and anti-tumor efficacy by a similar magnitude to constitutively active IL18. We also demonstrate that GzB-IL18 provides a highly effective armoring strategy for γδ CAR T cells, leading to enhanced metabolic fitness and significant potentiation of therapeutic activity. Finally, we show that constitutively active IL18 can unmask CAR T cell-mediated cytokine release syndrome in immunocompetent mice. By contrast, GzB-IL18 promotes anti-tumor activity and myeloid cell re-programming without inducing such toxicity. Using this stringent system, we have tightly coupled the biological activity of IL18 to the activation state of the host CAR T cell, favoring safer clinical implementation of this technology.

Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy.

γδ T-cells provide immune surveillance against cancer, straddling both innate and adaptive immunity. G115 is a clonal γδ T-cell receptor (TCR) of the Vγ9Vδ2 subtype which can confer responsiveness to phosphoantigens (PAgs) when genetically introduced into conventional αß T-cells. Cancer immunotherapy using γδ TCR-engineered T-cells is currently under clinical evaluation. In this study, we sought to broaden the cancer specificity of the G115 γδ TCR by insertion of a tumour-binding peptide into the complementarity-determining region (CDR) three regions of the TCR δ2 chain. Peptides were selected from the foot and mouth disease virus A20 peptide which binds with high affinity and selectivity to αvß6, an epithelial-selective integrin that is expressed by a range of solid tumours. Insertion of an A20-derived 12mer peptide achieved the best results, enabling the resulting G115 + A12 T-cells to kill both PAg and αvß6-expressing tumour cells. Cytolytic activity of G115 + A12 T-cells against PAg-presenting K562 target cells was enhanced compared to G115 control cells, in keeping with the critical role of CDR3 δ2 length for optimal PAg recognition. Activation was accompanied by interferon (IFN)-γ release in the presence of either target antigen, providing a novel dual-specificity approach for cancer immunotherapy.

Intratumoral pan-ErbB targeted CAR-T for head and neck squamous cell carcinoma: interim analysis of the T4 immunotherapy study.

BACKGROUND: Locally advanced/recurrent head and neck squamous cell carcinoma (HNSCC) is associated with significant morbidity and mortality. To target upregulated ErbB dimer expression in this cancer, we developed an autologous CD28-based chimeric antigen receptor T-cell (CAR-T) approach named T4 immunotherapy. Patient-derived T-cells are engineered by retroviral transduction to coexpress a panErbB-specific CAR called T1E28ζ and an IL-4-responsive chimeric cytokine receptor, 4αß, which allows IL-4-mediated enrichment of transduced cells during manufacture. These cells elicit preclinical antitumor activity against HNSCC and other carcinomas. In this trial, we used intratumoral delivery to mitigate significant clinical risk of on-target off-tumor toxicity owing to low-level ErbB expression in healthy tissues. METHODS: We undertook a phase 1 dose-escalation 3+3 trial of intratumoral T4 immunotherapy in HNSCC (NCT01818323). CAR T-cell batches were manufactured from 40 to 130 mL of whole blood using a 2-week semiclosed process. A single CAR T-cell treatment, formulated as a fresh product in 1-4 mL of medium, was injected into one or more target lesions. Dose of CAR T-cells was escalated in 5 cohorts from 1×107-1×109 T4+ T-cells, administered without prior lymphodepletion. RESULTS: Despite baseline lymphopenia in most enrolled subjects, the target cell dose was successfully manufactured in all cases, yielding up to 7.5 billion T-cells (67.5±11.8% transduced), without any batch failures. Treatment-related adverse events were all grade 2 or less, with no dose-limiting toxicities (Common Terminology Criteria for Adverse Events V.4.0). Frequent treatment-related adverse events were tumor swelling, pain, pyrexias, chills, and fatigue. There was no evidence of leakage of T4+ T-cells into the circulation following intratumoral delivery, and injection of radiolabeled cells demonstrated intratumoral persistence. Despite rapid progression at trial entry, stabilization of disease (Response Evaluation Criteria in Solid Tumors V.1.1) was observed in 9 of 15 subjects (60%) at 6 weeks post-CAR T-cell administration. Subsequent treatment with pembrolizumab and T-VEC oncolytic virus achieved a rapid complete clinical response in one subject, which was durable for over 3 years. Median overall survival was greater than for historical controls. Disease stabilization was associated with the administration of an immunophenotypically fitter, less exhausted, T4 CAR T-cell product. CONCLUSIONS: These data demonstrate the safe intratumoral administration of T4 immunotherapy in advanced HNSCC.

CAR Based Immunotherapy of Solid Tumours-A Clinically Based Review of Target Antigens.

Immunotherapy with CAR-engineered immune cells has transformed the management of selected haematological cancers. However, solid tumours have proven much more difficult to control using this emerging therapeutic modality. In this review, we survey the clinical impact of solid tumour CAR-based immunotherapy, focusing on specific targets across a range of disease indications Among the many candidates which have been the subject of non-clinical CAR T-cell research, clinical data are available for studies involving 30 of these targets. Here, we map out this clinical experience, highlighting challenges such as immunogenicity and on-target off-tumour toxicity, an issue that has been both unexpected and devastating in some cases. We also summarise how regional delivery and repeated dosing have been used in an effort to enhance impact and safety. Finally, we consider how emerging armouring systems and multi-targeted CAR approaches might be used to enhance tumour access and better enable discrimination between healthy and transformed cell types.

CAR-Based Immunotherapy of Solid Tumours-A Survey of the Emerging Targets.

Immunotherapy with CAR T-cells has revolutionised the treatment of B-cell and plasma cell-derived cancers. However, solid tumours present a much greater challenge for treatment using CAR-engineered immune cells. In a partner review, we have surveyed data generated in clinical trials in which patients with solid tumours that expressed any of 30 discrete targets were treated with CAR-based immunotherapy. That exercise confirms that efficacy of this approach falls well behind that seen in haematological malignancies, while significant toxic events have also been reported. Here, we consider approximately 60 additional candidates for which such clinical data are not available yet, but where pre-clinical data have provided support for their advancement to clinical evaluation as CAR target antigens.

Generation and application of TGFß-educated human Vγ9Vδ2 T cells.

Clinical trials that tested the antitumor activity of γδ T cells have been mostly unsuccessful. To address this, we expanded human Vγ9Vδ2 T cells in TGFß1, a cytokine which enhances their viability, trafficking properties, and intrinsic antitumor activity. This protocol summarizes the production and in vitro functional characterization of TGFß1 educated human Vγ9Vδ2 cells and highlights their compatibility with chimeric antigen receptor (CAR) engineering. We also describe in vivo testing of the antitumor activity of these CAR T cells in mice. For complete details on the use and execution of this protocol, please refer to Beatson et al. (2021).

Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation.

Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously, we demonstrated that dual co-stimulation can be effectively harnessed by a parallel (p)CAR architecture in which a CD28-containing second generation CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR). When compared to linear CARs, pCAR-engineered T-cells elicit superior anti-tumor activity in a range of pre-clinical models. Since CD19 is the best validated clinical target for cellular immunotherapy, we evaluated a panel of CD19-specific CAR and pCAR T-cells in this study. First, we generated a panel of single chain antibody fragments (scFvs) by alanine scanning mutagenesis of the CD19-specific FMC63 scFv (VH domain) and these were incorporated into second generation CD28+CD3ζ CARs. The resulting panel of CAR T-cells demonstrated a broad range of CD19 binding ability and avidity for CD19-expressing tumor cells. Each scFv-modified CAR was then converted into a pCAR by co-expression of an FMC63 scFv-targeted CCR with a 4-1BB endodomain. When compared to second generation CARs that contained an unmodified or mutated FMC63 scFv, each pCAR demonstrated a significant enhancement of tumor re-stimulation potential and IL-2 release, reduced exhaustion marker expression and enhanced therapeutic efficacy in mice with established Nalm-6 leukemic xenografts. These data reinforce the evidence that the pCAR platform delivers enhanced anti-tumor activity through effective provision of dual co-stimulation. Greatest anti-tumor activity was noted for intermediate avidity CAR T-cells and derived pCARs, raising the possibility that effector to target cell avidity is an important determinant of efficacy.

Hypoxia-sensing CAR T cells provide safety and efficacy in treating solid tumors.

Utilizing T cells expressing chimeric antigen receptors (CARs) to identify and attack solid tumors has proven challenging, in large part because of the lack of tumor-specific targets to direct CAR binding. Tumor selectivity is crucial because on-target, off-tumor activation of CAR T cells can result in potentially lethal toxicities. This study presents a stringent hypoxia-sensing CAR T cell system that achieves selective expression of a pan-ErbB-targeted CAR within a solid tumor, a microenvironment characterized by inadequate oxygen supply. Using murine xenograft models, we demonstrate that, despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumor efficacy without off-tumor toxicity. This dynamic on/off oxygen-sensing safety switch has the potential to facilitate unlimited expansion of the CAR T cell target repertoire for treating solid malignancies.

Crosstown Traffic: Lymphodepleting Chemotherapy Drives CAR T Cells.

CAR-engineered T cell immunotherapy has proven transformative in selected hematological malignancies. However, solid tumors largely remain impervious to these approaches. In addressing this challenge, Srivastava et al. in this issue demonstrate that oxaliplatin-based lymphodepleting chemotherapy promotes enhanced CAR T cell recruitment to lung tumors, boosting therapeutic impact in combination with anti-PD-L1.

Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors.

Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3ζ) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling, and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity, and specificity, pCAR T cells consistently elicit superior anti-tumor activity compared with T cells that express traditional linear CARs.

TGF-ß1 potentiates Vγ9Vδ2 T cell adoptive immunotherapy of cancer.

Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-ß1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-ß1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-ß are described, including prostaglandin E2 receptor downmodulation, TGF-ß insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.

CAR T-cell immunotherapy of MET-expressing malignant mesothelioma.

Mesothelioma is an incurable cancer for which effective therapies are required. Aberrant MET expression is prevalent in mesothelioma, although targeting using small molecule-based therapeutics has proven disappointing. Chimeric antigen receptors (CARs) couple the HLA-independent binding of a cell surface target to the delivery of a tailored T-cell activating signal. Here, we evaluated the anti-tumor activity of MET re-targeted CAR T-cells against mesothelioma. Using immunohistochemistry, MET was detected in 67% of malignant pleural mesotheliomas, most frequently of epithelioid or biphasic subtype. The presence of MET did not influence patient survival. Candidate MET-specific CARs were engineered in which a CD28+CD3ζ endodomain was fused to one of 3 peptides derived from the N and K1 domains of hepatocyte growth factor (HGF), which represents the minimum MET binding element present in this growth factor. Using an NIH3T3-based artificial antigen-presenting cell system, we found that all 3 candidate CARs demonstrated high specificity for MET. By contrast, these CARs did not mediate T-cell activation upon engagement of other HGF binding partners, namely CD44v6 or heparan sulfate proteoglycans, including Syndecan-1. NK1-targeted CARs demonstrated broadly similar in vitro potency, indicated by destruction of MET-expressing mesothelioma cell lines, accompanied by cytokine release. In vivo anti-tumor activity was demonstrated following intraperitoneal delivery to mice with an established mesothelioma xenograft. Progressive tumor regression occurred without weight loss or other clinical indicators of toxicity. These data confirm the frequent expression of MET in malignant pleural mesothelioma and demonstrate that this can be targeted effectively and safely using a CAR T-cell immunotherapeutic strategy.

The Transcription Factor ZNF683/HOBIT Regulates Human NK-Cell Development.

We identified ZNF683/HOBIT as the most highly upregulated transcription factor gene during ex vivo differentiation of human CD34+ cord blood progenitor cells to CD56+ natural killer (NK) cells. ZNF683/HOBIT mRNA was preferentially expressed in NK cells compared to other human peripheral blood lymphocytes and monocytes. During ex vivo differentiation, ZNF683/HOBIT mRNA started to increase shortly after addition of IL-15 and further accumulated in parallel to the generation of CD56+ NK cells. shRNA-mediated knockdown of ZNF683/HOBIT resulted in a substantial reduction of CD56-CD14- NK-cell progenitors and the following generation of CD56+ NK cells was largely abrogated. The few CD56+ NK cells, which escaped the developmental inhibition in the ZNF683/HOBIT knockdown cultures, displayed normal levels of NKG2A and KIR receptors. Functional analyses of these cells showed no differences in degranulation capacity from control cultures. However, the proportion of IFN-γ-producing cells appeared to be increased upon ZNF683/HOBIT knockdown. These results indicate a key role of ZNF683/HOBIT for the differentiation of the human NK-cell lineage and further suggest a potential negative control on IFN-γ production in more mature human NK cells.

Intracavitary 'T4 immunotherapy' of malignant mesothelioma using pan-ErbB re-targeted CAR T-cells.

Malignant mesothelioma remains an incurable cancer. We demonstrated that mesotheliomas expressed EGFR (79.2%), ErbB4 (49.0%) and HER2 (6.3%), but lacked ErbB3. At least one ErbB family member was expressed in 88% of tumors. To exploit ErbB dysregulation in this disease, patient T-cells were engineered by retroviral transduction to express a panErbB-targeted chimeric antigen receptor (CAR), co-expressed with a chimeric cytokine receptor that allows interleukin (IL)-4 mediated CAR T-cell proliferation. This combination is referred to as T4 immunotherapy. T-cells from mesothelioma patients were uniformly amenable to T4 genetic modification and expansion/enrichment thereafter using IL-4. Patient-derived T4+ T-cells were activated upon contact with a panel of four mesothelioma cell lines, leading to cytotoxicity and cytokine release in all cases. Adoptive transfer of T4 immunotherapy to SCID Beige mice with an established bioluminescent LO68 mesothelioma xenograft was followed by regression or eradication of disease in all animals. Despite the established ability of T4 immunotherapy to elicit cytokine release syndrome in SCID Beige mice, therapy was very well tolerated. These findings provide a strong rationale for the clinical evaluation of intracavitary T4 immunotherapy to treat mesothelioma.

Targeting of Aberrant αvß6 Integrin Expression in Solid Tumors Using Chimeric Antigen Receptor-Engineered T Cells.

Expression of the αvß6 integrin is upregulated in several solid tumors. In contrast, physiologic expression of this epithelial-specific integrin is restricted to development and epithelial re-modeling. Here, we describe, for the first time, the development of a chimeric antigen receptor (CAR) that couples the recognition of this integrin to the delivery of potent therapeutic activity in a diverse repertoire of solid tumor models. Highly selective targeting αvß6 was achieved using a foot and mouth disease virus-derived A20 peptide, coupled to a fused CD28+CD3 endodomain. To achieve selective expansion of CAR T cells ex vivo, an IL-4-responsive fusion gene (4αß) was co-expressed, which delivers a selective mitogenic signal to engineered T cells only. In vivo efficacy was demonstrated in mice with established ovarian, breast, and pancreatic tumor xenografts, all of which express αvß6 at intermediate to high levels. SCID beige mice were used for these studies because they are susceptible to cytokine release syndrome, unlike more immune-compromised strains. Nonetheless, although the CAR also engages mouse αvß6, mild and reversible toxicity was only observed when supra-therapeutic doses of CAR T cells were administered parenterally. These data support the clinical evaluation of αvß6 re-targeted CAR T cell immunotherapy in solid tumors that express this integrin.

Targeting of Tumor-Associated Glycoforms of MUC1 with CAR T Cells.

We read with interest the manuscript by June and colleagues published recently in Immunity in which they describe targeting of aberrantly glycosylated tumor-associated cell membrane mucin MUC1 using chimeric antigen receptor-engineered human T cells (Posey et al., 2016). In that study, the authors used a second generation 4-1BB costimulatory-molecule-based chimeric antigen receptor (CAR) (Imai et al., 2004) in which targeting was achieved using a single-chain variable fragment (scFv) derived from the 5E5 antibody. This CAR selectively binds MUC1 that carries the Tn or sialyl (S)Tn glycan. Both of these truncated glycans are aberrantly expressed on the MUC1 glycoprotein in a spectrum of malignancies and consequently represent attractive targets for immunotherapeutic exploitation.

Preclinical in vivo modeling of cytokine release syndrome induced by ErbB-retargeted human T cells: identifying a window of therapeutic opportunity?

The ErbB network is dysregulated in many solid tumors. To exploit this, we have developed a chimeric Ag receptor (CAR) named T1E28z that targets several pathogenetically relevant ErbB dimers. T1E28z is coexpressed with a chimeric cytokine receptor named 4αß (combination termed T4), enabling the selective expansion of engineered T cells using IL-4. Human T4(+) T cells exhibit antitumor activity against several ErbB(+) cancer types. However, ErbB receptors are also expressed in several healthy tissues, raising concerns about toxic potential. In this study, we have evaluated safety of T4 immunotherapy in vivo using a SCID beige mouse model. We show that the human T1E28z CAR efficiently recognizes mouse ErbB(+) cells, rendering this species suitable to evaluate preclinical toxicity. Administration of T4(+) T cells using the i.v. or intratumoral routes achieves partial tumor regression without clinical or histopathologic toxicity. In contrast, when delivered i.p., tumor reduction is accompanied by dose-dependent side effects. Toxicity mediated by T4(+) T cells results from target recognition in both tumor and healthy tissues, leading to release of both human (IL-2/IFN-γ) and murine (IL-6) cytokines. In extreme cases, outcome is lethal. Both toxicity and IL-6 release can be ameliorated by prior macrophage depletion, consistent with clinical data that implicate IL-6 in this pathogenic event. These data demonstrate that CAR-induced cytokine release syndrome can be modeled in mice that express target Ag in an appropriate distribution. Furthermore, our findings argue that ErbB-retargeted T cells can achieve therapeutic benefit in the absence of unacceptable toxicity, providing that route of administration and dose are carefully optimized.

Synergistic Chemoimmunotherapy of Epithelial Ovarian Cancer Using ErbB-Retargeted T Cells Combined with Carboplatin.

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy, underscoring the need for better therapies. Adoptive immunotherapy using genetically targeted T cells represents a promising new treatment for hematologic malignancies. However, solid tumors impose additional obstacles, including the lack of suitable targets for safe systemic therapy and the need to achieve effective T cell homing to sites of disease. Because EOC undergoes transcœlomic metastasis, both of these challenges may be circumvented by T cell administration to the peritoneal cavity. In this study, we describe such an immunotherapeutic approach for EOC, in which human T cells were targeted against the extended ErbB family, using a chimeric Ag receptor named T1E28z. T1E28z was coexpressed with a chimeric cytokine receptor named 4αß (combination termed T4), enabling the selective ex vivo expansion of engineered T cells using IL-4. Unlike control T cells, T4(+) T cells from healthy donors and patients with EOC were activated by and destroyed ErbB(+) EOC tumor cell lines and autologous tumor cultures. In vivo antitumor activity was demonstrated in mice bearing established luciferase-expressing SKOV-3 EOC xenografts. Tumor regression was accompanied by mild toxicity, manifested by weight loss. Although efficacy was transient, therapeutic response could be prolonged by repeated T cell administration. Furthermore, prior treatment with noncytotoxic doses of carboplatin sensitized SKOV-3 tumors to T4 immunotherapy, promoting enhanced disease regression using lower doses of T4(+) T cells. By combining these approaches, we demonstrate that repeated administration of carboplatin followed by T4(+) T cells achieved optimum therapeutic benefit in the absence of significant toxicity, even in mice with advanced tumor burdens.