Coengineering specificity, safety, and function into T cells for cancer immunotherapy.

Adoptive T-cell transfer (ACT) therapies, including of tumor infiltrating lymphocytes (TILs) and T cells gene-modified to express either a T cell receptor (TCR) or a chimeric antigen receptor (CAR), have demonstrated clinical efficacy for a proportion of patients and cancer-types. The field of ACT has been driven forward by the clinical success of CD19-CAR therapy against various advanced B-cell malignancies, including curative responses for some leukemia patients. However, relapse remains problematic, in particular for lymphoma. Moreover, for a variety of reasons, relative limited efficacy has been demonstrated for ACT of non-hematological solid tumors. Indeed, in addition to pre-infusion challenges including lymphocyte collection and manufacturing, ACT failure can be attributed to several biological processes post-transfer including, (i) inefficient tumor trafficking, infiltration, expansion and retention, (ii) chronic antigen exposure coupled with insufficient costimulation resulting in T-cell exhaustion, (iii) a range of barriers in the tumor microenvironment (TME) mediated by both tumor cells and suppressive immune infiltrate, (iv) tumor antigen heterogeneity and loss, or down-regulation of antigen presentation machinery, (v) gain of tumor intrinsic mechanisms of resistance such as to apoptosis, and (vi) various forms of toxicity and other adverse events in patients. Affinity-optimized TCRs can improve T-cell function and innovative CAR designs as well as gene-modification strategies can be used to coengineer specificity, safety, and function into T cells. Coengineering strategies can be designed not only to directly support the transferred T cells, but also to block suppressive barriers in the TME and harness endogenous innate and adaptive immunity. Here, we review a selection of the remarkable T-cell coengineering strategies, including of tools, receptors, and gene-cargo, that have been developed in recent years to augment tumor control by ACT, more and more of which are advancing to the clinic.

A lentiviral vector for the production of T cells with an inducible transgene and a constitutively expressed tumour-targeting receptor.

Vectors that facilitate the engineering of T cells that can better harness endogenous immunity and overcome suppressive barriers in the tumour microenvironment would help improve the safety and efficacy of T-cell therapies for more patients. Here we report the design, production and applicability, in T-cell engineering, of a lentiviral vector leveraging an antisense configuration and comprising a promoter driving the constitutive expression of a tumour-directed receptor and a second promoter enabling the efficient activation-inducible expression of a genetic payload. The vector allows for the delivery of a variety of genes to human T cells, as we show for interleukin-2 and a microRNA-based short hairpin RNA for the knockdown of the gene coding for haematopoietic progenitor kinase 1, a negative regulator of T-cell-receptor signalling. We also show that a gene encoded under an activation-inducible promoter is specifically expressed by tumour-redirected T cells on encountering a target antigen in the tumour microenvironment. The single two-gene-encoding vector can be produced at high titres under an optimized protocol adaptable to good manufacturing practices.

A bioluminescent-based probe for in vivo non-invasive monitoring of nicotinamide riboside uptake reveals a link between metastasis and NAD+ metabolism.

Nicotinamide riboside (NR) is a form of vitamin B3 and is one of the most studied compounds for the restoration of cellular NAD+ levels demonstrating clinical potential in many metabolic and age-related disorders. Despite its wide commercial availability as a powerful nutraceutical, our understanding of NR uptake by different cells and tissues is greatly limited by the lack of noninvasive in vivo imaging tools limiting its clinical translation. Here, we report the development and validation of a bioluminescent NR uptake probe (BiNR) for non-invasive longitudinal imaging of NR uptake both in vitro and in vivo. In addition, we optimized an assay that allows monitoring of NR flux without the need to transfect cells with the luciferase gene, enabling the use of the BiNR probe in clinical samples, as demonstrated with human T cells. Lastly, we used BiNR to investigate the role of NR uptake in cancer prevalence and metastases formation in triple negative breast cancer (TNBC) animal model. Our results demonstrate that NR supplementation results in a significant increase in cancer prevalence and metastases of TNBC to the brain. These results outline the important role of powerful nutraceuticals like NR in cancer metabolism and the need to personalize their use in certain patient populations.

Enforcing GLUT3 expression in CD8+ T cells improves fitness and tumor control by promoting glucose uptake and energy storage.

Despite the tremendous success of adoptive T-cell therapies (ACT) in fighting certain hematologic malignancies, not all patients respond, a proportion experience relapse, and effective ACT of most solid tumors remains elusive. In order to improve responses to ACT suppressive barriers in the solid tumor microenvironment (TME) including insufficient nutrient availability must be overcome. Here we explored how enforced expression of the high-affinity glucose transporter GLUT3 impacted tumor-directed T cells. Overexpression of GLUT3 in primary murine CD8+ T cells enhanced glucose uptake and increased glycogen and fatty acid storage, and was associated with increased mitochondrial fitness, reduced ROS levels, higher abundance of the anti-apoptotic protein Mcl-1, and better resistance to stress. Importantly, GLUT3-OT1 T cells conferred superior control of B16-OVA melanoma tumors and, in this same model, significantly improved survival. Moreover, a proportion of treated mice were cured and protected from re-challenge, indicative of long-term T cell persistence and memory formation. Enforcing expression of GLUT3 is thus a promising strategy to improve metabolic fitness and sustaining CD8+ T cell effector function in the context of ACT.

CAR T cells targeting the ganglioside NGcGM3 control ovarian tumors in the absence of toxicity against healthy tissues.

Chimeric antigen receptor (CAR) T cells have emerged as a powerful immunotherapeutic tool against certain hematological malignancies but a significant proportion of patients either do not respond or they relapse, sometimes as a result of target antigen loss. Moreover, limited clinical benefit has been reported for CAR therapy against epithelial derived solid tumors. A major reason for this is the paucity of solid tumor antigens identified to date that are broadly, homogeneously and stably expressed but not found on healthy tissues. To address this, here we describe the development and evaluation of CAR T cells directed against N-glycoslylated ganglioside monosialic 3 (NGcGM3). NGcGM3 derives from the enzymatic hydroxylation of N-acetylneuraminic acid (NAc) GM3 (NAcGM3) and it is present on the surface of a range of cancers including ovarian, breast, melanoma and lymphoma. However, while NAcGM3 is found on healthy human cells, NGcGM3 is not due to the 7deletion of an exon in the gene encoding for the enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Indeed, unlike for most mammals, in humans NGcGM3 is considered a neoantigen as its presence on tumors is the result of metabolic incorporation from dietary sources. Here, we have generated 3 CARs comprising different single chain variable fragments (scFvs) originating from the well-characterized monoclonal antibody (mAb) 14F7. We show reactivity of the CAR T cells against a range of patient tumor fragments and we demonstrate control of NGcGM3+ SKOV3 ovarian tumors in the absence of toxicity despite the expression of CMAH and presence of NGcGM3+ on healthy tissues in NSG mice. Taken together, our data indicate clinical potential for 14F7-based CAR T cells against a range of cancers, both in terms of efficacy and of patient safety.

Lenalidomide enhances CD23.CAR T cell therapy in chronic lymphocytic leukemia.

Chimeric antigen receptors (CAR)-modified T cells are an emerging therapeutic tool for chronic lymphocytic leukemia (CLL). However, in patients with CLL, well-known T-cell defects and the inhibitory properties of the tumor microenvironment (TME) hinder the efficacy of CAR T cells. We explored a novel approach combining CARs with lenalidomide, an immunomodulatory drug that tempers the immunosuppressive activity of the CLL TME. T cells from patients with CLL were engineered to express a CAR specific for CD23, a promising target antigen. Lenalidomide maintained the in vitro effector functions of CD23.CAR+ T cells effector functions in terms of antigen-specific cytotoxicity, cytokine release and proliferation. Overall, lenalidomide preserved functional CAR T-CLL cell immune synapses. In a Rag2-/-γc-/--based xenograft model of CLL, we demonstrated that, when combined with low-dose lenalidomide, CD23.CAR+ T cells efficiently migrated to leukemic sites and delayed disease progression when compared to CD23.CAR+ T cells given with rhIL-2. These observations underline the therapeutic potential of this novel CAR-based combination strategy in CLL.

Magnesium sensing via LFA-1 regulates CD8+ T cell effector function.

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T cells, enhanced effectiveness of bi-specific T cell engaging antibodies, and improved CAR T cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.

Hemicentin 1 influences podocyte dynamic changes in glomerular diseases.

Different complex mechanisms control the morphology of podocyte foot processes and their interactions with the underlying basement membrane. Injuries to this system often cause glomerular dysfunction and albuminuria. The present study aimed at identifying early markers of glomerular damage in diabetic nephropathy. For this purpose, we performed a microarray analysis on kidneys of 3-wk-old peroxisome proliferator-activated receptor-γ (PPARγ)-null and AZIP/F1 mice, which are two models of diabetic nephropathy due to lipodystrophy. This was followed by functional annotation of the enriched clusters of genes. One of the significant changes in the early stages of glomerular damage was the increase of hemicentin 1 (HMCN1). Its expression and distribution were then studied by real-time PCR and immunofluorescence in various models of glomerular damage and on podocyte cell cultures. HMCN1 progressively increased in the glomeruli of diabetic mice, according to disease severity, as well as in puromycin aminonucleoside (PA)-treated rats. Studies on murine and human podocytes showed an increased HMCN1 deposition upon different pathological stimuli, such as hyperglycemia, transforming growth factor-ß (TGF-ß), and PA. In vitro silencing studies showed that HMCN1 mediated the rearrangements of podocyte cytoskeleton induced by TGF-ß. Finally, we demonstrated an increased expression of HMCN1 in the kidneys of patients with proteinuric nephropathies. In summary, our studies identified HMCN1 as a new molecule involved in the dynamic changes of podocyte foot processes. Its increased expression associated with podocyte dysfunction points to HMCN1 as a possible marker for the early glomerular damage occurring in different proteinuric nephropathies.

Delayed Hair Follicle Morphogenesis and Hair Follicle Dystrophy in a Lipoatrophy Mouse Model of Pparg Total Deletion.

PPARγ regulates multiple aspects of skin physiology, including sebocyte differentiation, keratinocyte proliferation, epithelial stem cell survival, adipocyte biology, and inflammatory skin responses. However, the effects of its global deletion, namely of nonredundant key functions of PPARγ signaling in mammalian skin, are yet unknown because of embryonic lethality. Here, we describe the skin and hair phenotype of a whole-body PPARγ-null mouse (PpargΔ/Δ), obtained by preserving PPARγ expression in the placenta. PpargΔ/Δ mice exhibited total lipoatrophy and complete absence of sebaceous glands. Right after birth, hair follicle (HF) morphogenesis was transiently delayed, along with reduced expression of HF differentiation markers and of transcriptional regulators necessary for HF development. Later, adult PpargΔ/Δ mice developed scarring alopecia and severe perifollicular inflammation. Skin analyses in other models of lipodystrophy, AZIPtg/+ and Adipoq-Cretg/+Ppargfl/fl mice, coupled with skin graft experiments, showed that the early defects observed in hair morphogenesis were caused by the absence of adipose tissue. In contrast, the late alteration of HF cycle and appearance of inflammation were observed only in PpargΔ/Δ mice and likely were due to the lack sebaceous glands. Our findings underscore the increasing appreciation for the importance of adipose tissue-mediated signals in HF development and function.

In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric antigen receptor.

Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19(+)CD5(+)CD20(dim) B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR(+) T cells showed specific cytotoxic activity against CD23(+) tumor cell lines (average lysis 42%) and primary CD23(+) CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR(+) T cells released inflammatory cytokines (1445-fold more TNF-ß, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR(+) T cells. Redirected T cells were also effective in vivo in a CLL Rag2(-/-)γ(c)(-/-) xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR(+) T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR(+) T cells represent a selective immunotherapy for the elimination of CD23(+) leukemic cells in patients with CLL.

Chimeric T-cell receptors: new challenges for targeted immunotherapy in hematologic malignancies.

Chimeric T-cell receptors (ChTCR), are a fascinating technological step in the field of immunotherapy for orienting the activity of immune cells towards specific molecular targets expressed on the cell surface of various tumors, including hematologic malignancies. The main characteristics of ChTCR are their ability to redirect T-cell specificity and their killing/effector activity toward a selected target in a non MHC-restricted manner, exploiting the antigen binding properties of monoclonal antibodies. ChTCR are, in fact, artificial T-cell receptors constituted by an antigen-recognizing antibody molecule linked to a T-cell triggering domain. Various hematologic malignancies represent optimal targets for the exploitation of ChTCR, because of the bright expression of specific antigens on the surface of tumor cells. Thus, CD19 and CD20 have been targeted for B-cell lymphoid tumors (acute lymphoblastic leukemia-ALL, lymphomas and chronic lymphocytic leukemia-CLL), CD33 for myeloid leukemia, and CD30 for lymphomas. Even though technical and safety progresses are still needed to improve the profile of gene transfer and protein expression of ChTCR, phase 1 trials will be carried out in the near future to demonstrate the feasibility of their clinical translation and, it is be hoped, give preliminary indications about their anti-tumor efficacy.