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1.
Plant Biotechnol J ; 21(6): 1254-1269, 2023 06.
Article in English | MEDLINE | ID: mdl-36811226

ABSTRACT

Immune checkpoint inhibitors (ICIs) have achieved huge clinical success. However, many still have limited response rates, and are prohibitively costly. There is a need for effective and affordable ICIs, as well as local manufacturing capacity to improve accessibility, especially to low-to-middle income countries (LMICs). Here, we have successfully expressed three key ICIs (anti-PD-1 Nivolumab, anti-NKG2A Monalizumab, and anti-LAG-3 Relatimab) transiently in Nicotiana benthamiana and Nicotiana tabacum plants. The ICIs were expressed with a combination of different Fc regions and glycosylation profiles. They were characterized in terms of protein accumulation levels, target cell binding, binding to human neonatal Fc receptors (hFcRn), human complement component C1q (hC1q) and various Fcγ receptors, as well as protein recovery during purification at 100 mg- and kg-scale. It was found that all ICIs bound to the expected target cells. Furthermore, the recovery during purification, as well as Fcγ receptor binding, can be altered depending on the Fc region used and the glycosylation profiles. This opens the possibility of using these two parameters to fine-tune the ICIs for desired effector functions. A scenario-based production cost model was also generated based on two production scenarios in hypothetical high- and low-income countries. We have shown that the product accumulation and recovery of plant production platforms were as competitive as mammalian cell-based platforms. This highlights the potential of plants to deliver ICIs that are more affordable and accessible to a widespread market, including LMICs.


Subject(s)
Neoplasms , Nicotiana , Animals , Humans , Nicotiana/genetics , Immune Checkpoint Inhibitors , Receptors, IgG , Mammals
2.
Front Immunol ; 13: 1065495, 2022.
Article in English | MEDLINE | ID: mdl-36713444

ABSTRACT

Objectives: Vγ9Vδ2 T-cells are a subset of T-cells with a crucial role in immunosurveillance which can be activated and expanded by multiple means to stimulate effector responses. Little is known about the expression of checkpoint molecules on this cell population and whether the ligation of these molecules can regulate their activity. The aim of this study was to assess the expression of both activatory and inhibitory receptors on Vγ9Vδ2 T-cells to assess potential avenues of regulation to target with immunotherapy. Methods: Expression of various activatory and inhibitory receptors was assessed on Vγ9Vδ2 T-cells by flow cytometry following activation and expansion using zoledronic acid (ZA) and Bacillus Calmette-Guérin (BCG). Expression of these markers and production of effector molecules was also examined following co-culture with various tumour cell targets. The effect of immune checkpoint blockade on Vγ9Vδ2 T-cells was also explored. Results: Vγ9Vδ2 T-cells expressed high levels of activatory markers both at baseline and following stimulation. Vγ9Vδ2 T-cells expressed variable levels of inhibitory checkpoint receptors with many being upregulated following stimulation. Expression of these markers is further modulated upon co-culture with tumour cells with changes reflecting activation and effector functions. Despite their high expression of inhibitory receptors when cultured with tumour cells expressing cognate ligands there was no effect on Vδ2+ T-cell cytotoxic capacity or cytokine production with immune checkpoint blockade. Conclusions: Our work suggests the expression of checkpoint receptors present on Vγ9Vδ2 T-cells which may provide a mechanism with the potential to be utilised by tumour cells to subvert Vγ9Vδ2 T-cell cytotoxicity. This work suggests important candidates for blockade by ICI therapy in order to increase the successful use of Vγ9Vδ2 T-cells in immunotherapy.


Subject(s)
Neoplasms , T-Lymphocytes , Humans , Immune Checkpoint Inhibitors/metabolism , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Lymphocyte Activation , Immunotherapy , Neoplasms/therapy , Neoplasms/metabolism
3.
Front Immunol ; 10: 1535, 2019.
Article in English | MEDLINE | ID: mdl-31333666

ABSTRACT

Objective: We have previously shown that increased circulating interleukin-6 (IL-6) results in enhanced CD4+ T cell signaling via signal transduction and activator of transcription-3 (STAT3) in early rheumatoid arthritis (RA). We tested the hypothesis that transcriptional "imprinting" of T-cells by this mechanism skews downstream effector responses, reinforcing immune dysregulation at a critical, but targetable, disease phase. Methods: We modeled naïve CD4+ T cell exposure to pathophysiological concentrations of IL-6 in vitro, assessing the dynamic transcriptional and functional consequences for downstream effector cells utilizing microarray and flow cytometry. Fresh blood from treatment-naïve early arthritis patients was phenotyped in parallel for comparison. Results: T cell sensitivity to IL-6 was most marked in the naïve subset, and related to gp130 rather than IL-6R expression. Exposure of healthy naïve CD4+ T cells to IL-6 induced the same STAT3 target genes as previously seen to discriminate RA patients from disease controls. After TCR stimulation IL-6 pre-exposed cells exhibited enhanced proliferative capacity, activation, and a propensity toward Th1 differentiation, compared to non-exposed cells. An entirely analogous phenotype was observed in early RA compared to control CD4+ T cells. Conclusions: Sustained IL-6 exposure at a critical point in the natural history of RA "primes" the adaptive immune system to respond aberrantly to TCR stimulation, potentiating disease induction with implications for the optimal timing of targeted therapy.


Subject(s)
Arthritis, Rheumatoid/immunology , CD4-Positive T-Lymphocytes/immunology , Interleukin-6/immunology , Models, Immunological , Signal Transduction/immunology , Transcription, Genetic/immunology , Adult , Aged , Aged, 80 and over , Arthritis, Rheumatoid/pathology , Female , Humans , Male , Middle Aged , Receptors, Antigen, T-Cell/immunology , STAT3 Transcription Factor/immunology
4.
Curr Opin Rheumatol ; 30(2): 207-214, 2018 03.
Article in English | MEDLINE | ID: mdl-29206659

ABSTRACT

PURPOSE OF REVIEW: Rheumatoid arthritis is a systemic disease of evolving immune dysregulation that culminates in joint destruction and disability. The principle by which pro-inflammatory cytokines may be therapeutically targeted to abrogate disease is well established, but has yet to translate into reliable cures for patients. Emerging insights into cytokine-mediated pathobiology during rheumatoid arthritis development are reviewed, and their implications for future treatment strategies considered. RECENT FINDINGS: Accumulating data highlight cytokine perturbations before the clinical onset of rheumatoid arthritis. Some of these have now been linked to the arthritogenic activation of autoantibodies and associated pain and bone destruction in affected joints. These observations suggest cytokines may trigger the transition from systemic immunity to arthritis. Cytokine exposure could furthermore 'prime' synovial stromal cells to perpetuate a dominant pro-inflammatory environment. By facilitating cross-talk between infiltrating immune cells and even sustaining ectopic lymphoid structure development in some cases, cytokine interplay ultimately underpins the failure of arthritis to resolve. SUMMARY: Successful therapeutic stratification will depend upon an increasingly sophisticated appreciation of how dominant players amongst cytokine networks vary across time and anatomical space during incipient rheumatoid arthritis. The prize of sustained remission for all patients justifies the considerable effort required to achieve this understanding.


Subject(s)
Arthritis, Rheumatoid/immunology , Cytokines/immunology , Arthritis, Rheumatoid/diagnosis , Arthritis, Rheumatoid/drug therapy , Autoantibodies/immunology , Chronic Disease , Disease Progression , Humans
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