Liposomal phytohemagglutinin: In vivo T‐cell activator as a novel pan‐cancer immunotherapy

Abstract Immunotherapy is an attractive approach for treating cancer. T‐cell engagers (TCEs) are a type of immunotherapy that are highly efficacious; however, they are challenged by weak T‐cell activation and short persistence. Therefore, alternative solutions to induce greater activation and persistence of T cells during TCE immunotherapy is needed. Methods to activate T cells include the use of lectins, such as phytohemagglutinin (PHA). PHA has not been used to activate T cells in vivo, for immunotherapy, due to its biological instability and toxicity. An approach to overcome the limitations of PHA while also preserving its function is needed. In this study, we report a liposomal PHA which increased PHA stability, reduced toxicity and performed as an immunotherapeutic that is able to activate T cells for the use in future cancer immunotherapies to circumvent current obstacles in immunosuppression and T‐cell exhaustion.

Commonly used compounds to improve activation of T cells ex vivo include antibody-conjugated beads, as well as small molecules and lectins, such as phorbol 12-myristate 13-acetate (PMA), ionomycin, concanavalin A and phytohemagglutinin (PHA). 3,4 Beads cannot be used in vivo, since they will be mainly accumulating in filtrating organs, such as the liver, and missing the target tumour tissue. PMA and ionomycin stimulate T cells by activating protein kinase C 5 ; however, their use is limited by their carcinogenic potential. 6 Concanavalin A and PHA, both lectins, stimulate T cells by binding to glycoproteins on the T-cell receptor. 5,7,8 PHA is more potent compared to concanavalin A, yet PHA has not been used to activate T cells in vivo due to its biological instability, short bioavailability profile and toxicity (ie agglutination of red and white blood cells) leading to death. [8][9][10] Herein, we propose an alternative solution for the lower activation of TCE immunotherapy. In order to take full advantage of PHA as an immune activator, while also preserving its function, we encapsulated PHA in a liposome to increase its stability, reduce toxicity and create an immunotherapeutic circumventing current obstacles in immunosuppression and T-cell exhaustion.

| RE SULTS AND D ISCUSS I ON
Liposomal formulations of PHA were prepared using the thin-film hydration method 11,12 in which PHA is encapsulated in the core of the liposome particles ( Figure 1A). Next, we investigated the ability of the PHA-loaded liposomes to activate T cells compared to free PHA. We found that the T-cell activation marker CD25 increased in expression correlating with PHA concentration, regardless of its formulation, and at 1 mg/ml, around 90% of T cells had an increase in CD25 expression ( Figure 1B). We then examined the effect of PHA on T-cell survival, as a marker for toxicity. No change in T-cell survival was observed at 24 h for either formulations of PHA ( Figure 1C). These results demonstrate that the liposomal formulation maintained the desired effect of T-cell activation without changing the toxicity profile in vitro.
The main limitation of PHA is its low bioavailability and toxicity in vivo. Therefore, we investigated the effect of the liposomal formulation on PHA's pharmacokinetic profile and toxicity in vivo. As In brief, 25 mg of PHA was dissolved in 500 µl of 0.1 M sodium carbonate, excess of AF647 was added, stirred for one hour at room temperature, and unbound AF647 was removed using dialysis. Immunocompetent C57BL/6 mice (Charles River Laboratories, strain 027, female, 52 days old) were injected intravenously with free or liposomal AF647-PHA (10 mg/kg; n = 3 per group), and blood serum was analysed at 0.25, 2, 4, 8 and 24 h using a fluorescent plate reader (Ex/Em = 644/665). Statistical significance between two formulation calculated by 2-way anova is indicated by * (p < 0.05). (E) Mice survival at increasing concentrations of free or liposomal PHA. C57BL/6 mice (n = 4 per concentration, per condition) were injected with increasing doses (0, 10, 25 and 50 mg/kg) of free or liposomal PHA and closely monitored for survival for three days. (F) Activation of T cells at increasing concentrations of free or liposomal PHA in vivo. C57BL/6 mice (n = 4 per concentration, per condition) were injected intravenously with 10, 25 or 50 mg/kg of free or liposomal PHA. Three days following injection, blood was extracted, and T-cell activation was measured as the downregulation of CD62L expression on CD3+ T cells by flow cytometry. Activated T cells were presented as % CD3 T cells minus the T cells with high CD62L expression. Statistical significance between free and liposomal PHA calculated by 2-way anova is indicated by * (p < 0.05). (G) Multiple myeloma (MM) survival at increasing concentrations of free or liposomal PHA. Fluorescently labelled (DiO) MM cell line OPM2 was cultured with or without PBMC isolated T cells and was treated with 1 mg/ml of free or liposomal PHA for 24 h, and the survival of MM cells was analysed using flow cytometry as count of OPM2 cells normalized against counting beads. Statistical significance between MM versus MM + T cells groups is indicated by * (p < 0.05); in MM + T cells condition, statistical significance of free or liposomal PHA compared to control is indicated by # (p < 0.05). (H) Tumour progression in MM-bearing mice upon free of liposomal PHA treatment. Luciferase-expressing mice myeloma 5TGM1 cells (1 × 10 6 cells/mouse) were injected intravenously into 20 C57BL/KaLwRij mice. One week post-inoculation, mice were randomly divided into 4 groups (n = 5) and treated intravenously with  Figure 1H). The control cohort died at day 22, 10 mg/kg free and liposomal PHA had 60% of mice survived past day 31; whereas 50 mg/kg liposomal PHA-treated mice had 100% survival up to day 35 ( Figure 1I).
Moreover, typical cancer immunotherapies such as CAR-T cells and TCEs target a specific marker on the tumour cell for elimination; however, this leads to antigen-less clones and relapse. 13,14 BCMA and CS1 are both popular MM targets for immunotherapy, and previous therapies targeting each of these antigens were shown to induce a decrease in the expression and induce antigenless tumour escape in MM. 15 We therefore tested the effect of the liposomal PHA on the expression of these antigens in MM cells.
We observed that liposomal PHA did not reduce the expression of BCMA or CS1 expression on MM cells ( Figure 1J). This is because the liposomal PHA does not depend on a specific antigen.
Finally, and due to the fact that the liposomal PHA activates T cells generically regardless of the tumour target, we exploited the use of liposomal PHA for the treatment of a variety of cancers such as leukaemia, breast cancer, lung cancer, and glioma.