**6. Proinflammatory IA1: enhancing the immune response to cancer**

T cells plays a critical role in the anticancer inflammatory responses. An effective anticancer proinflammatory T cell response is dependent upon the activation of Teff cells. Normally, T cells are activated upon ligation of their antigen receptors with specific cognate antigens [68]. However, because of the low frequency of cancer antigen-specific lymphocytes, the immune response to cancers can be initially, and all too often remains, weak. While previous studies have attempted to enhance the anticancer T cell response using pan T cell mitogens (e.g., phytohemagglutinin; PHA), cytokines (e.g., IL-2), or monoclonal antibodies (e.g., anti-CD3 and anti-CD28) the overly robust T cell response arising from these approaches often induced significant systemic toxicity leading to the suspension or abrogation of multiple clinical trials [69–74]. In contrast, in an allorecognition response only 1–10% of T cells are alloreactive [75]. Hence, the IA1 therapeutic, derived from a bioreactor allorecognition response (MLR), is expected to activate endogenous T cells in a more controlled manner, with less toxicity.

To assess IA1's ability to enhance the anticancer activity of resting PBMC, cells were treated for 24 hours with IA1 and overlaid on HeLa and SH-4 cancers cells. Cancer cell proliferation was then followed for 168 hours. Importantly, IA1 exerted no toxicity to resting PBMC but, as shown in **Figure 4**, induced significant activation (e.g., proliferation) of resting CD3+ (CD4+ and CD8+ ) skewed towards proinflammatory subsets thus decreasing the Teff:Treg ratio. However, as predicted by the biology of the alloresponse, IA1-mediated T cell proliferation was much more restrained than that induced by the anti-CD3/anti-CD28 or PHA stimulation [43]. This finding suggests that the systemic toxicity, relative to pan T cell activators, should be greatly reduced. Crucially, IA1-activated PBMC demonstrated a potent inhibition of cancer cell (HeLa and SH-4 melanoma) proliferation relative to the resting PBMC (**Figure 6**). The anti-proliferation effect of IA1-activated PBMC was noted within ~12 hours vs. 4–5 days for resting cells. These findings demonstrate that miRNA-enriched therapeutics can be biomanufactured from the secretome and can induce a potent proinflammatory, anticancer, effect on resting lymphocytes.

The potential utility and use of IA1 in Adoptive Cell Therapy (ACT) is diagrammatically shown in **Figure 6**. The bioproduction of IA1 is both inexpensive and rapid (5 days) and the IA1 can be stored for long periods (several months frozen in the laboratory; data not shown). Moreover, neither IA1 or TA1 production actually requires donor specific tissues (PBMC) making these secretome-based therapeutics an "offthe-shelf" immune adjuvant. Most importantly for patient care, *ex vivo* activation of lymphocytes is rapid (24 hours). The rapidity of this approach is in stark contrast to the weeks to months necessary for production and expansion of CAR-T cells. Hence, IA1 activation of autologous PBMC could be employed as a first line therapy or, potentially, be used as an immunotherapeutic bridge while CAR-T cells are produced. Due to the simplicity and low cost of the approach, multiple rounds could be used as necessary with large numbers of autologous PBMC employed. Indeed, due to the ability to infuse large numbers of IA1 treated autologous cells, enhanced recognition

#### **Figure 6.**

*Schematic presentation of use and efficacy of the IA1 secretome therapeutic. Left panels: the enhanced efficacy of treated PBMC is supported by photomicrographs of allogenic PBMC responding to HeLa cells. As shown, after 72 hours incubation, resting (weak responders; left) PBMC show limited interaction when overlaid on HeLa cells. In contrast, the same PBMC, when treated for 24 hours with IA1, show a robust enhanced interaction (right) with the HeLa cell monolayer. Moreover, when IA1-treated PBMC are overlaid on SH-4 melanoma cells a greatly enhanced anti-cancer effect is noted relative to untreated PBMC. Shown are the growth profiles (as measured by electrical impedance) of SH-4 treated with either the SYN (derived from the secretome of resting PBMC) or IA1therapeutics. PBMC:SH-4 ratios included 50:1, 25:1 and 10:1. Right panels: bioreactor production of IA1 secretome is readily accomplished using an allogeneic MLR. Potential source materials include PBMC donors (A and B), autologous cells (dotted arrow), lymphocytic cell lines, or leukoreduction filters from blood collection bags. The secretome is collected at day 5 for processing into IA1 (Figure 4). IA1 is stable for months when aliquoted and frozen. Weak to absent immune response to both the primary tumor and metastatic sites allows for cancer progression. PBMC (D) from the patient can be treated* ex vivo *for 24 hours with IA1 and then reinfused into the individual where they show enhanced recognition and killing of the primary tumor and, potentially, improved immune surveillance at metastatic sites. Derived from Ref. [43].*

of not only the primary tumor but metastatic sites as well could be achieved thus improving long-term survival. Of note, similarly to our use of the tolerogenic TA1 in NOD mice (**Figures 4** and **5**), IA1 could be directly injected into the recipient yielding a systemic proinflammatory reset of the immune system [40].

#### **7. Conclusions**

The immunomodulation of the endogenous immune system has become a major focus in treating a broad range of clinical conditions ranging from tissue/ organ engraftment, autoimmune disease and cancer therapy. While significant clinical advancements have been made in immunotherapy, substantial challenges remain. One target of interest is the biologic/clinical desire to induce a persistent systemic immunological reset that could reduce both the need for chronic therapy and reduce the potential toxicities associated with current immunomodulatory approaches. Recent studies have demonstrated that miRNA are key regulators of cellular processes involved in both tolerogenic and proinflammatory immune responses and mediate immune cell proliferation and differentiation. Using an alloresponse bioreactor secretome system we have demonstrated that miRNA-based therapeutics can be reproducibly manufactured that can systemically reorient the immune system to either a tolerogenic or proinflammatory state by simultaneously

**83**

*Modulating the T Lymphocyte Immune Response via Secretome Produced…*

modulating both regulatory and effector T cell subsets thus skewing the Treg:Teff cell ratio to favor tolerance or inflammation. The tolerogenic TA1 therapeutic is derived from polymer-mediated immunocamouflage of the alloresponse reaction while the inflammatory IA1 preparation is derived from the alloresponse itself. The secretomes from these reactions are processed to maintain the miRNA within the secretome. In contrast to most miRNA therapeutic tactics, our approach has been to mimic the "complex pattern of miRNA expression" seen in protolerogenic or proinflammatory states. This "complex" approach was predicated by the inherent nature of miRNA bioregulation in that there is a low probability that altered expression of a single, or even a few, miRNA would exert a potent and definitive biological response. As shown, this approach successfully results in significant and, in mice, systemic and persistent changes to the immune system. The tolerogenic TA1 proved useful in reducing the onset and incidence of autoimmune diabetes in the NOD mouse while the proinflammatory IA1 therapeutic greatly enhanced the efficacy of human T cells to recognize and kill cancer cells without inducing the systemic inflammatory response seen with mitogens or monoclonal antibody (e.g., anti-CD3/CD28) therapies. Moreover, this approach can simultaneously modulate both regulatory and effect T cell subtype. The successful development of this miRNAimmunomodulatory approach may prove useful in facilitating organ engraftment, treating autoimmune disease and enhancing the endogenous anticancer response.

This work was supported by grants from the Canadian Institutes of Health Research (Grant no. 123317; MDS), Canadian Blood Services (MDS) and Health Canada (MDS). The views expressed herein do not necessarily represent the view of the federal government of Canada. We thank the Canada Foundation for Innovation and the Michael Smith Foundation for Health Research for infrastructure funding at the University of British Columbia Centre for Blood Research. The funders had no role in study design, data collection and analysis, decision to publish, or prepara-

Canadian Blood Services is pursuing patents related to the production and utilization of the described acellular immunomodulatory agents. Canadian Blood Services, a not-for-profit organization responsible for collecting, manufacturing and distributing blood and blood products to all Canadians (except Quebec), is the assignee for relevant patents. MDS, DW and WMT are inventors on these patents. XY has no conflicts of interest beyond being paid by Canadian Blood Services.

*DOI: http://dx.doi.org/10.5772/intechopen.86598*

**Acknowledgements**

tion of the manuscript.

**Conflict of interest**

*Modulating the T Lymphocyte Immune Response via Secretome Produced… DOI: http://dx.doi.org/10.5772/intechopen.86598*

modulating both regulatory and effector T cell subsets thus skewing the Treg:Teff cell ratio to favor tolerance or inflammation. The tolerogenic TA1 therapeutic is derived from polymer-mediated immunocamouflage of the alloresponse reaction while the inflammatory IA1 preparation is derived from the alloresponse itself. The secretomes from these reactions are processed to maintain the miRNA within the secretome. In contrast to most miRNA therapeutic tactics, our approach has been to mimic the "complex pattern of miRNA expression" seen in protolerogenic or proinflammatory states. This "complex" approach was predicated by the inherent nature of miRNA bioregulation in that there is a low probability that altered expression of a single, or even a few, miRNA would exert a potent and definitive biological response. As shown, this approach successfully results in significant and, in mice, systemic and persistent changes to the immune system. The tolerogenic TA1 proved useful in reducing the onset and incidence of autoimmune diabetes in the NOD mouse while the proinflammatory IA1 therapeutic greatly enhanced the efficacy of human T cells to recognize and kill cancer cells without inducing the systemic inflammatory response seen with mitogens or monoclonal antibody (e.g., anti-CD3/CD28) therapies. Moreover, this approach can simultaneously modulate both regulatory and effect T cell subtype. The successful development of this miRNAimmunomodulatory approach may prove useful in facilitating organ engraftment, treating autoimmune disease and enhancing the endogenous anticancer response.
