Objectives:Despite their anti-cancer properties, natural killer (NK) cells are discarded during CAR T cell manufacturing. NK cells display promising responses as a cell therapy, exhibiting remarkable safety at high doses. Moreover, NK cells have complementary cytokine profiles and distinct antigen-recognition pathways to supplement CAR T cell therapies. We demonstrate parallel expansion of NK cells from the normally discarded PBMC of CAR T cell production for downstream combination therapies against solid and blood-borne malignancies. Methods:CAR T cells were manufactured from buffy coats with initial isolation of T cells from PBMC. NK cells were rapidly expanded in parallel by stimulating the remaining PBMC with membrane-bound (mb)IL-21 + mbIL-15 feeder cells. Expanded NK cells were assessed through spectral flow cytometry and applied as a pretreatment prior to CAR Tcell infusion against xenograft tumor models in NOD-scid IL2Rgammanull (NSG) mice. Results:Applying feeder cells to residual PBMC stimulated 5000-fold expansions of pure NK cells. Expanded NK cells expressed various activation receptors and displayed strong in vitro cytotoxicity, which was further improved in CAR T-conditioned medium. A pretreatment of NK cells enhanced existing CAR T cell therapies, improving in vivo tumor clearance and survival against breast cancer, acute lymphoblastic leukaemia and non-Hodgkin's lymphoma. While NK cells eliminated CD19-/- variants in vitro, they were unable to suppress antigen-escape relapse in vivo. Conclusion:This study encourages the optimal use of leukapheresis product. By recycling discarded PBMC into NK cells, CAR T cell therapies can be effectively enhanced, improving tumor clearance and survival.

Chimeric antigen receptor (CAR) T cell therapies have shown remarkable efficacy in hematological malignancies, yet translation to solid tumors has been hindered by immunosuppressive tumor microenvironments, reduced T cell persistence and on-target/off-tumor toxicities. Constitutive CAR expression, typically driven by strong promoters such as EF1α, promotes tonic signaling, receptor clustering and antigen-independent activation, contributing to T cell exhaustion and adverse events. Inducible promoter systems have been proposed to improve control over CAR expression. NR4A1, a transcription factor (TF) activated during early T cell receptor (TCR) signaling, governs pathways central to T cell activation and dysfunction, making its promoter an attractive candidate for conditional CAR regulation. We compared constitutive (EF1α), synthetic inducible (6NFAT-NFκB and 2NFAT-2NurRE) and NR4A1 promoters to drive expression of a second-generation FRP5-CAR. NR4A1-driven CARs demonstrated low basal expression that was rapidly induced upon antigen encounter, reaching levels equivalent to EF1α-driven CARs while showing minimal antigen-independent signaling. Functionally, NR4A1-driven CARs mediated potent tumor lysis, preserved a less exhausted (PD-1low and TIM-3low) and more memory-like phenotype (CD62Lhigh and CD45RAhigh), and sustained robust antitumor responses in vitro and in vivo. These findings establish the NR4A1 promoter as a native, activation-inducible system to fine-tune CAR expression, while maintaining therapeutic efficacy comparable to constitutively expressed CAR T cells. This strategy provides a promising framework for advancing CAR T cell therapies against solid tumors.

[This corrects the article DOI: 10.1016/j.isci.2023.107019.].

Objectives:The anti-PD-L1 antibody avelumab has demonstrated efficacy across multiple cancer types. Avelumab primarily blocks the PD-1/L1 immune checkpoint, while inducing antibody-dependent cellular cytotoxicity (ADCC) from CD16a+ NK cells. However, subsets of patients possess lower-affinity CD16a allotypes that limit ADCC capacity of monoclonal antibodies. In this study, we modified the Fc domain of avelumab with characterised mutations to enhance ADCC across all CD16a allotypes. Methods:Comparisons between the wild-type avelumab and modified 'AveFc5M' were carried out to assess the impacts of introduced mutations on PD-L1 blockade, ADCC induction and complement-dependent cytotoxicity (CDC) induction. To assess ADCC, a range of PD-L1+ target cells were coated with mab prior to incubation with effector cells, with both high- and low-affinity CD16a allotypes represented. Human serum was employed for assessing CDC. Results:Both antibodies exhibited equivalent PD-L1 blocking activity. Notably, the modified AveFc5M displayed significantly enhanced ADCC across diverse tumor cell-effector cell combinations. Fc mutations also conferred the ability to mediate complement-dependent cytotoxicity (CDC) against a PD-L1+ lymphoma cell line. However, short term activation of NK cells or long-term expansion on artificial antigen presenting cells, led to upregulation of PD-L1 on NK cells. Addition of avelumab or AveFc5M to these NK cell populations induced a marked reduction in viable NK cells via ADCC-mediated fratricide. Conclusion:These findings demonstrate that Fc engineering of avelumab can substantially augment ADCC and CDC activity against PD-L1+ tumors. However, the enhanced effector function of AveFc5M led to NK cell fratricide, potentially limiting NK cell-dominated anti-cancer responses.

Background The development of effective therapeutic strategies for late-stage estrogen receptor-positive breast cancer (ER+) is limited by the scarcity of biologically relevant models. More recently, immunotherapies emerged as promising candidates for breast cancer treatment, however, the absence of immunocompetent models of ER+ breast cancer metastasis continues to hinder the assessment of these theraputic interventions.Methods To address this, we utilized the 129S6/SvEv mouse strain and syngeneic SSM3 cells in the assessment and development of ER+ metastasis models. As part of this study, the mammary intraductal (MIND) primary tumor model was established in the same background. In addition, a novel luciferase system was evaluated for potential use in metastasis tracking.Results Luciferase-expressing SSM3 cells enabled longitudinal in vivo imaging to track tumor growth. Histological analysis confirmed metastatic spread and tumor origin. Antares2, a novel luciferase reporter, showed high in vitro sensitivity but reduced in vivo performance. The study showed that systemic delivery of SSM3 cells with oestradiol supplementation can support metastatic tumor establishment and that MIND injections led to reliable, invasive tumor growth.Conclusions These findings highlight the potential and limitations of the 129S6/SvEv model as a syngeneic, immunocompetent system for studying ER+ breast cancer metastasis. Reporter expression may affect immunogenicity or cell fitness. Further refinement of these models will enable investigation of immune-modulatory therapies in ER+ metastatic breast cancer.

The efficacy of blocking antibodies against programmed death-1 (PD-1) and its ligand (PD-L1) is modulated by signalling through their Fc regions. The Fc region of anti-PD-1/PD-L1 antibodies, when cell-bound, represents a potential target for recognition by circulating rheumatoid factor (RF) autoantibodies. The resultant cell-associated immune complex may then provide different Fc signals to that of the PD-1/PD-L1 antibodies alone. However, little is known regarding the interaction of RF and therapeutic PD-1/PD-L1 antibodies. We report that PD-1 (pembrolizumab, nivolumab) and PD-L1 (avelumab) antibodies, when bound to their cellular targets, are recognised by both IgM-RF and IgA-RF components of RF+ patient serum. We further demonstrate that the presence of RF provides PD-1 antibodies with the ability to induce complement-dependent cytotoxicity (CDC) of a PD-1+ target cell line in the presence of human complement. Although RF provided avelumab with the ability to induce CDC in assays using rabbit complement, no CDC was observed in the presence of human complement. The presence of RF did not modulate the level of Fc receptor-triggered cellular cytotoxicity or neutrophil activation that was induced by PD-1/PD-L1 antibodies alone. This study demonstrates that RF has the potential to modulate the Fc-associated signals generated following binding of PD-1/PD-L1 antibodies. The impact of RF on their efficacy therefore merits further investigation.

Abstract Second-generation anti-CD19 chimeric antigen receptor (CAR) T-cells incorporating CD28 (1928z) are now standard of care for treatment of refractory/relapsed large B-cell and mantle cell lymphomas. However, clinical use is limited by high toxicity rates, especially cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Modifying intracellular signalling domains can influence CAR T-cell toxicity risk. Although rare, reports of CAR T-cell-derived malignancies underscore the risks of gene-modified cell therapies and the need to assess transgene insertion sites in new products. Toll-like receptor 2 (TLR2) a pattern recognition receptor, is highly expressed by activated CD8+ human T-cells, and can serve as a T-cell costimulatory signal, enhancing, memory formation, and modifying T-cell cytokine profile. In support of a phase 1 clinical trial (ENABLE-1, NCT04049513) evaluating a new third-generation anti-CD19 CAR T-cell product incorporating CD28 and TLR2 co-stimulatory domains (1928T2z), we explored the functional impact of the TLR2 domain in vitro, and characterised gene insertion sites within the 1928T2z product. Incorporating TLR2-derived Toll/IL-1 receptor resistance domain (TIR) between CD28 and CD3ζ domains in the 1928T2z CAR provided comparable CAR T-cell efficacy while producing lower levels of IFN-γ and GM-CSF, cytokines linked to CRS and ICANS pathogenesis, than 1928z. In contrast, positioning the TLR2 domain at the cytoplasmic tail (1928zT2) did not lower IFN-γ and GM-CSF production. Inhibiting TLR2 TIR function using ortho-vanillin, or through targeted mutations abrogating MYD88 recruitment and/or dimerization, impaired CAR T-cell activity, indicating the TLR2 domain contributes to 1928T2z CAR function. Gene expression identified downregulation of IL-6 signalling and upregulation of an anti-inflammatory cytokine pathway in 1928T2z cells compared to comparator CAR T-cells. A phosphoprotein array revealed that a functional TLR2 domain was associated with in higher levels of phosphorylated MEK1/2 (involved in TLR signalling), GATA3 (Th2-linked transcription factor) and CREB (a transcription factor that limits inflammatory responses). Using long-read sequencing and bioinformatics, we characterised insertion sites in a clonal line and polyclonal human CAR T-cells. Results showed mostly intronic or intergenic sites, with less than 10% mapping to exons, aligning with known lentiviral transgene integration profiles. In summary, incorporating a TLR2 co-stimulatory domain into CD28-costimulated anti-CD19 CAR T-cells modulates cytokine profile in a position-dependent manner, with TLR2 placement between CD28 and CD3ζ reducing CRS-and ICANS-linked cytokines while preserving in vitro anti-tumour activity. These findings align with our phase 1 trial data suggesting that 1928T2z CAR T-cells are associated with low incidence of CRS and ICANS, while exhibiting similar clinical efficacy to commercial comparators. Citation Format: Nathaniel Dasyam, Yasmin Nouri, Joshua Halpin, Alexander McLellan, David Eccles, Simon Fink, Felix Schaefer-ruoff, Markus Templin, Qin Le, Peng Li, Ian Hermans, Rachel Perret, Robert Weinkove. Functional, transcriptomic and genomic characterisation of CD19-directed CAR T-cells incorporating a TLR2 co-stimulatory domain [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A018.

BACKGROUND:Paediatric sarcomas, including rhabdomyosarcoma, Ewing sarcoma and osteosarcoma, represent a group of malignancies that significantly contribute to cancer-related morbidity and mortality in children and young adults. These cancers share common challenges, including high rates of metastasis, recurrence or treatment resistance, leading to a 5-year survival rate of approximately 20% for patients with advanced disease stages. Despite the critical need, therapeutic advancements have been limited over the past three decades. The advent of chimeric antigen receptor (CAR)-based immunotherapies offers a promising avenue for novel treatments. However, CAR-T cells have faced significant challenges and limited success in treating solid tumours due to issues such as poor tumour infiltration, immunosuppressive tumour microenvironments and off-target effects. In contrast, the adaptation of CAR technology for natural killer (NK) cells has demonstrated potential in both haematological and solid tumours, suggesting a new therapeutic strategy for paediatric sarcomas. METHODS:This study developed and validated a novel CAR-NK cell therapy targeting the ephrin type-A receptor-2 (EphA2) antigen, which is highly expressed in various paediatric sarcomas. RESULTS:CAR expression was successfully detected on the surface of NK cells post-electroporation, indicating successful transfection. Significantly, EphA2-specific CAR-NK cells demonstrated enhanced cytotoxic activity against several paediatric sarcoma cell lines in vitro, including those of rhabdomyosarcoma, Ewing sarcoma and osteosarcoma, compared to unmodified NK cells. Transient messenger RNA (mRNA) transfection of NK cells is a safe approach in genetic engineering, with further chemical modifications to mRNA enhancing stability of temporal EphA2-CAR expression in NK cells, thereby promoting prolonged protein expression. Additionally, in vivo EphA2-CAR-NK cells showed promising anti-cancer activity in rhabdomyosarcoma and osteosarcoma mouse models. CONCLUSIONS:The study provides a foundational basis for the clinical evaluation of EphA2-targeted CAR-NK cell therapy across a spectrum of paediatric sarcomas. The enhanced anti-tumour effects observed in vitro/vivo suggests potential for improved therapeutic outcomes in hard-to-cure paediatric sarcomas. KEY POINTS:Addressing unmet clinical needs in paediatric Sarcomas. Paediatric sarcomas, including rhabdomyosarcoma, Ewing sarcoma, and osteosarcoma, exhibit poor survival rates in advanced disease stages. The lack of significant therapeutic progress over the past three decades necessitates innovative treatment approaches. Advancing immunotherapy with CAR-NK cells. Natural killer (NK) cells modified with chimeric antigen receptors (CARs) represent a promising strategy to overcome the limitations of CAR-T cells, particularly in solid tumours. CAR-NK cells are associated with enhanced tumour targeting, reduced off-target effects, and improved safety profiles. EphA2 as a therapeutic target. EphA2, a receptor overexpressed in multiple paediatric sarcomas, is identified as a viable target for CAR-based immunotherapy due to its critical role in tumour progression and angiogenesis. Innovations in mRNA-based engineering. This study demonstrates the feasibility of transient mRNA transfection to engineer NK cells for CAR expression, offering a non-integrative and safer alternative to viral transduction. Enhancements in mRNA stability through chemical modifications, can further optimise protein expression. Preclinical efficacy of EphA2-CAR NK cells. EphA2-specific CAR-NK cells exhibit superior cytotoxicity against sarcoma cell lines in vitro and demonstrate significant anti-tumour activity in in vivo mouse models of rhabdomyosarcoma and osteosarcoma. Clinical translation potential. The findings establish a strong preclinical rationale for the clinical evaluation of EphA2-targeted CAR-NK therapy as a novel immunotherapeutic option for paediatric sarcomas. Future research directions: Combining EphA2-CAR NK cells with immune checkpoint inhibitors or other immunomodulatory agents could further enhance therapeutic outcomes and durability. Advanced preclinical models mimicking human tumour microenvironments are needed to refine and optimise this therapeutic approach.

The efficacy of antibody-targeted therapy of solid cancers is limited by the lack of consistent tumour-associated antigen expression. However, tumour-associated antigens shared with non-malignant cells may still be targeted using conditionally activated-antibodies, or by chimeric antigen receptor (CAR) T cells or CAR NK cells activated either by the tumour microenvironment or following 'unlocking' via multiple antigen-recognition. In this study, we have focused on tissue factor (TF; CD142), a type I membrane protein present on a range of solid tumours as a basis for future development of conditionally-activated BiTE or CAR T cells. TF is frequently upregulated on multiple solid tumours providing a selective advantage for growth, immune evasion and metastasis, as well as contributing to the pathology of thrombosis via the extrinsic coagulation pathway. Two well-characterised anti-TF monoclonal antibodies (mAb) were cloned into expression or transposon vectors to produce single chain (scFv) BiTE for assessment as CAR and CD28-CD3-based CAR or CD3-based BiTE. The affinities of both scFv formats for TF were determined by surface plasmon resonance. Jurkat cell line-based assays were used to confirm the activity of the BiTE or CAR constructs. The anti-TF mAb hATR-5 and TF8-5G9 mAb were shown to maintain their nanomolar affinities following conversion into a single chain (scFv) format and could be utilised as CD28-CD3-based CAR or CD3-based BiTE format. Because of the broad expression of TF on a range of solid cancers, anti-TF antibody formats provide a useful addition for the development of conditionally activated biologics for antibody and cellular-based therapy.

Background A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy.Methods We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice.Results Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells.Conclusion The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.

Artificial antigen-presenting cells (aAPCs) offer a cost effective and convenient tool for the expansion of chimeric antigen receptor (CAR)-bearing T cells and NK cells. aAPCs are particularly useful because of their ability to efficiently expand low-frequency antigen-reactive lymphocytes in bulk cultures. Commonly derived from the leukemic cell line K562, these aAPCs lack most major histocompatibility complex expression and are therefore useful for NK cell expansion without triggering allogeneic T-cell proliferation. To combat difficulties in accessing existing aAPC lines, while circumventing the iterative lentiviral gene transfers with antibody-mediated sorting required for the isolation of stable aAPC clones, we developed a single-step technique using Sleeping Beauty (SB)-based vectors with antibiotic selection options. Our SB vectors contain options of two to three genes encoding costimulatory molecules, membrane-bound cytokines as well as the presence of antibiotic-resistance genes that allow for stable transposition-based transfection of feeder cells. Transfection of K562 with SB vectors described in this study allows for the surface expression of CD86, 4-1BBL, membrane-bound (mb) interleukin (IL)-15 and mbIL-21 after simultaneous transposition and antibiotic selection using only two antibiotics. aAPCs successfully expanded NK cells to high purity (80-95%). Expanded NK cells could be further engineered by lentiviral CAR transduction. The multivector kit set is publicly available and will allow convenient and reproducible in-house production of effective aAPCs for the in vitro expansion of primary cells.

Artificial antigen-presenting cells (aAPCs) offer a cost effective and convenient tool for the expansion of chimeric antigen receptor (CAR)-bearing T cells and NK cells. aAPCs are particularly useful because of their ability to efficiently expand low-frequency antigen-reactive lymphocytes in bulk cultures. Commonly derived from the leukemic cell line K562, these aAPCs lack most major histocompatibility complex expression and are therefore useful for NK cell expansion without triggering allogeneic T-cell proliferation. To combat difficulties in accessing existing aAPC lines, while circumventing the iterative lentiviral gene transfers with antibody-mediated sorting required for the isolation of stable aAPC clones, we developed a single-step technique using Sleeping Beauty (SB)–based vectors with antibiotic selection options. Our SB vectors contain options of two to three genes encoding costimulatory molecules, membrane-bound cytokines as well as the presence of antibiotic-resistance genes that allow for stable transposition-based transfection of feeder cells. Transfection of K562 with SB vectors described in this study allows for the surface expression of CD86, 4-1BBL, membrane-bound (mb) interleukin (IL)-15 and mbIL-21 after simultaneous transposition and antibiotic selection using only two antibiotics. aAPCs successfully expanded NK cells to high purity (80–95%). Expanded NK cells could be further engineered by lentiviral CAR transduction. The multivector kit set is publicly available and will allow convenient and reproducible in-house production of effective aAPCs for the in vitro expansion of primary cells.

Equitable SARS-CoV-2 surveillance in low-resource communities lacking centralized sewers is critical as wastewater-based epidemiology (WBE) progresses. However, large-scale studies on SARS-CoV-2 detection in wastewater from low-and middle-income countries is limited because of economic and technical reasons. In this study, wastewater samples were collected twice a month from 186 urban and rural subdistricts in nine provinces of Thailand mostly having decentralized and non-sewered sanitation infrastructure and analyzed for SARS-CoV-2 RNA variants using allele-specific RT-qPCR. Wastewater SARS-CoV-2 RNA concentration was used to estimate the real-time incidence and time-varying effective reproduction number (Re). Results showed an increase in SARS-CoV-2 RNA concentrations in wastewater from urban and rural areas 14-20 days earlier than infected individuals were officially reported. It also showed that community/food markets were "hot spots" for infected people. This approach offers an opportunity for early detection of transmission surges, allowing preparedness and potentially mitigating significant outbreaks at both spatial and temporal scales.

Non-synonymous mutations in the SARS-CoV-2 spike region affect cell entry, tropism, and immune evasion, while frequent synonymous mutations may modify viral fitness. Host microRNAs, a type of non-coding RNA, play a crucial role in the viral life cycle, influencing viral replication and the host immune response directly or indirectly. Recently, we identified ten miRNAs with a high complementary capacity to target various regions of the SARS-CoV-2 genome. We filtered our candidate miRNAs to those only expressed with documented expression in SARS-CoV-2 target cells, with an additional focus on miRNAs that have been reported in other viral infections. We determined if mutations in the first SARS-CoV-2 variants of concern affected these miRNA binding sites. Out of ten miRNA binding sites, five were negatively impacted by mutations, with three recurrent synonymous mutations present in multiple SARS-CoV-2 lineages with high-frequency NSP3 : C3037U and NSP4 : G9802U/C9803U. These mutations were predicted to negatively affect the binding ability of miR-197-5p and miR-18b-5p, respectively. In these preliminary findings, using a dual-reporter assay system, we confirmed the ability of these miRNAs in binding to the predicted NSP3 and NSP4 regions and the loss/reduced miRNA bindings due to the recurrent mutations.

The monkeypox virus is excreted in the feces of infected individuals. Therefore, there is an interest in using viral load detection in wastewater for sentinel early surveillance at a community level and as a complementary approach to syndromic surveillance. We collected wastewater from 63 sewered and non-sewered locations in Bangkok city center between May and August 2022. Monkeypox viral DNA copy numbers were quantified using real-time polymerase chain reaction (PCR) and confirmed positive by Sanger sequencing. Monkeypox viral DNA was first detected in wastewater from the second week of June 2022, with a mean copy number of 16.4 copies/ml (n = 3). From the first week of July, the number of viral DNA copies increased to a mean copy number of 45.92 copies/ml. Positive samples were Sanger sequenced and confirmed the presence of the monkeypox virus. Our study is the first to detect monkeypox viral DNA in wastewater from various locations within Thailand. Results suggest that this could be a complementary source for detecting viral DNA and predicting upcoming outbreaks.

CAR T cell treatment of solid tumours is limited by poor persistence partly due to CD95 ligand (CD95L)-induced apoptosis. Both T cells and cells within the tumour microenvironment (TME) may express CD95L, triggering apoptosis in CD95-receptor-positive CAR T cells. Tonic signalling of CAR T cells may also increase CD95-dependent AICD. Because the intracellular protein c-FLIP protects T cells from AICD, we expressed c-FLIPp43 within a Her-2 targeted CAR cassette and evaluated the potential of c-FLIPp43 through in vitro functional assays and in vivo tumour-bearing xenograft model. cFLIP expression protected against CD95L-induced cell death in the Jurkat T cell lines. However, in primary human CAR T cells containing CAR-CD28 domains, c-FLIPp43 overexpression had minimal additional impact on resistance to CD95L-induded cell death. In vitro cytotoxicity against a breast cancer tumour cell line was not altered by c-FLIPp43 expression, but the expression of c-FLIPp43 in Her2-CAR T cells lowered interferon-γ secretion, without markedly affecting IL-2 levels, and c-FLIPp43-Her2-CAR T cells showed reduced anti-tumour activity in immunodeficient mice with breast cancer. The findings of this study provide a new understanding of the effects of controlling extrinsic apoptosis pathway suppression in CAR T cells, suggesting that c-FLIPp43 expression reduces anti-tumour immunity through the modulation of effector T cell pathways.

Tetracycline-inducible systems are widely used control elements for mammalian gene expression. Despite multiple iterations to improve inducibility, their use is still compromised by basal promoter activity in the absence of tetracyclines. In a mammalian system, we previously showed that the introduction of the G72V mutation in the rtTA-M2 tetracycline activator lowers the basal level expression and increases the fold-induction of multiple genetic elements in a long chimeric antigen receptor construct. In this study, we confirmed that the G72V mutation was effective in minimising background expression in the absence of an inducer, resulting in an increase in fold-expression. Loss of responsiveness due to the G72V mutation was compensated through the incorporation of four sensitivity enhancing (SE) mutations, without compromising promoter tightness. However, SE mutations alone (without G72V) led to undesirable leakiness. Although cryptic splice site removal from rtTA did not alter the inducible control of the luciferase reporter gene in this simplified vector system, this is still recommended as a precaution in more complex multi-gene elements that contain rtTA. The optimized expression construct containing G72V and SE mutations currently provides the best improvement of fold-induction mediated by the rtTA-M2 activator in a mammalian system.

Advances made in chimeric antigen receptor (CAR) T cell therapy have revolutionized the treatment and management of certain cancers. Currently, B cell malignancies have been among the few cancers to which CAR T cells have shown persistent and resilient anti-tumor responses. A growing body of evidence suggests that the persistence of CAR T cells within patients following infusion is linked to the mitochondrial fitness of the CAR T cell, which could affect clinical outcomes. Analysis of CAR T cells from patients undergoing successful treatment has shown an increase in mitochondrial mass and fusion events, and a reduction in aerobic metabolism, highlighting the importance of mitochondria in CAR T cell function. Consequently, there has been recent interest and investment in approaches that focus on mitochondrial programming. In this regard, miRNAs are promising agents in mitochondrial reprogramming for several reasons: (1) natural and artificial miRNAs are non-immunogenic, (2) one miRNA can simultaneously modulate the expression of multiple genes within a pathway, (3) the small size of a sequence required for producing mature miRNA is ideal for use in viral vectors and (4) different precursor miRNAs (pre-miRNAs) hairpins can be incorporated into a polycistronic miRNA cluster to create a miRNA cocktail. In this perspective, we describe the latest genetic engineering strategies that can be used to achieve the optimal expression of candidate miRNAs alongside a CAR construct. In addition, we include an in silico analysis of rational candidate miRNAs that could promote the mitochondrial fitness of CAR T cells.

The precision guiding of endogenous or adoptively transferred lymphocytes to the solid tumour mass is obligatory for optimal anti-tumour effects and will improve patient safety. The recognition and elimination of the tumour is best achieved when anti-tumour lymphocytes are proximal to the malignant cells. For example, the regional secretion of soluble factors, cytotoxic granules, and cell-surface molecule interactions are required for the death of tumour cells and the suppression of neovasculature formation, tumour-associated suppressor, or stromal cells. The resistance of individual tumour cell clones to cellular therapy and the hostile environment of the solid tumours is a major challenge to adoptive cell therapy. We review the strategies that could be useful to overcoming insufficient immune cell migration to the tumour cell mass. We argue that existing 'competitive' approaches should now be revisited as complementary approaches to improve CAR T and NK cell therapy.

Advances made in chimeric antigen receptor (CAR) T cell therapy have revolutionized the treatment and management of certain cancers. Currently, B cell malignancies have been among the few cancers to which CAR T cells have shown persistent and resilient anti‐tumor responses. A growing body of evidence suggests that the persistence of CAR T cells within patients following infusion is linked to the mitochondrial fitness of the CAR T cell, which could affect clinical outcomes. Analysis of CAR T cells from patients undergoing successful treatment has shown an increase in mitochondrial mass and fusion events, and a reduction in aerobic metabolism, highlighting the importance of mitochondria in CAR T cell function. Consequently, there has been recent interest and investment in approaches that focus on mitochondrial programming. In this regard, miRNAs are promising agents in mitochondrial reprogramming for several reasons: (1) natural and artificial miRNAs are non‐immunogenic, (2) one miRNA can simultaneously modulate the expression of multiple genes within a pathway, (3) the small size of a sequence required for producing mature miRNA is ideal for use in viral vectors and (4) different precursor miRNAs (pre‐miRNAs) hairpins can be incorporated into a polycistronic miRNA cluster to create a miRNA cocktail. In this perspective, we describe the latest genetic engineering strategies that can be used to achieve the optimal expression of candidate miRNAs alongside a CAR construct. In addition, we include an in silico analysis of rational candidate miRNAs that could promote the mitochondrial fitness of CAR T cells.