*1.3. B Cell Based Therapy*

It is known that T1D is affiliated with the loss of tolerance by autoreactive (isletreactive) B cells [90,91]. B lymphocytes (cells), in addition to T lymphocytes (cells), work directly with the adaptive immune system to produce cellular and humoral defense mechanisms for protection against infections or tumors [90,92]. Therefore, the depletion of B cells makes an individual highly vulnerable to opportunistic infections. However, to prevent autoimmunity from occurring, the B cells must be suppressed, rehabilitated, or culled [92]. There is also a correlation between the number of CD20<sup>+</sup> B cells and a decreasing pancreatic beta-cell count [90]. In the current realm of diabetes treatment, there exists a need to discover effective immunotherapy methods, as current treatment by daily insulin injection(s) is not a sustainable way to treat diabetes and is not a cure. The role of B cells in non-obese mice models has shown to arrest the disease development around the preinsulitis stage [93]. Therefore, B cell immunotherapy is proposed as an effective treatment for T1D patients [61].

To target B cells, monoclonal antibodies are being used to identify surface antigen markers on the surface of the cells. The biomarkers targeted on the B cell surface are those involved in the processes of maturation, differentiation, and survival [61,90,93]. Through mechanisms including, but not limited to, complement-dependent cytotoxicity, antibodydependent cellular cytotoxicity, and induction of apoptosis, the antibodies are able to induce cell death [61]. Current approaches to antibody B cell therapy include the use of rituximab (RTX), which has proven to show low efficacy in treatment results due to the following factors: detrimental side effects caused by B cell depletion, rapid reemergence of autoreactive B cells post RTX treatment, and the depletion of regulatory cells as collateral of RTX treatment [90]. Pancreas-localized B cells may also be resistant to RTX-mediated deletion, as indicated by a down-regulation of the CD20<sup>+</sup> surface marker [93]. Due to confounding effects, it is difficult to manipulate the dose and duration of RTX treatment, hindering the effectiveness and clear outcome of the treatment [61]. Combination therapy

is another approach used to deplete B cell levels with aims to control glycemia and reverse T1D. Through combination therapy, antigens and antibodies are administered together to achieve a therapeutic outcome that offers better protection than administering the agents alone [62,94,95]. Oral administration of insulin in combination with anti-CD20 antibodies shows low efficacy in reversal when compared to administering proinsulin DNA with an anti-CD20 antibody [94,95]. However, the aforementioned method is moderately effective in the prevention of T1D. Thus, the combination therapy approach to an immunological treatment of diabetes shows (limited) efficacy in the prevention of T1D, and little to no efficacy in the reversal of T1D.

A treatment is needed that can deplete B cells before the onset of hyperglycemia—a point where it is too late to arrest disease progression [62,93]. B cell depletion has been shown to arrest diabetes progression at the pre-insulitis stage, prior to T cell islet infiltration and insulitis development [96]. Further, specificity for autoreactive B cells is needed, so that pan-B cell-depletion is limited, and global immunosuppression does not occur [96–98]. The use of a site-specific receptor-mediated drug delivery system, in conjunction with small interfering RNA (siRNA), to target antiapoptotic factor B cell lymphoma/leukemia 2 (BCl2), is an effective method of gene silencing in B cell immunotherapy [99]. This can be achieved through nanomedicine, by using synthetic polymer-based nanoparticles conjugated with siRNA to specifically target autoreactive B cells [100]. Nanomedicine uses nanoscale substances and materials to monitor and treat human biological systems. Nanoparticles (NP's), a major proponent of nanomedicine, are known to couple various properties with reduced toxicity, in comparison to other bio-interfaces, all at the scale of nanometers [30].

Varying polymers such as chitosan, calcium pectinate zinc oxide, alginate, casein, and other polyester or polycationic acrylic polymers have proven to be effective oral administrative carriers for immunomodulators, insulin, and other engineered vaccines to treat T1D [98]. These polymers are characterized by nanoporous structures, in which various therapeutic agents such as insulin, proinsulin DNA, and siRNAs can be conjugated, and released upon conformational change when glucose levels fluctuate [95,98]. An increase in glucose levels, for example, would induce a conformational change in the nanoparticles, allowing for conjugated materials in the nanopores to be released and directed to the immune cells through a mechanism of biodegradation.

T1D is known to be characterized by the presence of autoantibodies (AABs) and the spreading of islet autoantigens (AAGs) during a prolonged, subclinical period, prior to the detection/diagnosis of T1D, during which it's expected that seroconversion will occur early in age, with epitope spreading indicative of disease progression [101,102]. The mechanisms leading to the generation of AABs and AAGs, and thus the onset of T1D, are considered genetic, though environmental factors can play a part. Through genetic mechanisms, T cells generate blueprints for the construction of autoreactive B cells, which in turn present betacell antigens to autoreactive T cells, creating a vicious loop which aids in the development and progression of T1D [102,103]. Thus, it is essential that an immunotherapeutic treatment for B cell intervention is fabricated to break the detrimental loop which leads to the overall reduction of beta cells, which can be approached by nanomedicine. To induce (therapeutic) genetic expression within the B cell environment, NPs may be enveloped with CRISPR-cas9 (a gene editing system that allows for DNA repair, deletion, or modification) that expresses various cofactors to decrease expression and induce deletion of the autoreactive B cells [103]. In turn, the reduction of autoreactive B cells would result in increased B cell tolerance, and a reduction in the decrease of beta cells. Limited studies have produced results on immunotherapeutic approaches to treating T1D with NPs by targeting B cells, however there are studies that show a successful depletion of B cells in varying autoimmune diseases, leading to the prevention, and, in some cases, a reversal of the disease [101,102,104]. An immunotherapeutic approach to treating T1D through depleting autoreactive B cells poses an effective method for the actual reversal of T1D, which is largely due to individual genetic predispositions. These genetic predispositions may be combated by using CRISPR-cas9

or other immunotherapeutic agents conjugated with the prospective cofactors to induce the cellular and humoral responses, which aims to deplete the autoreactive B cells. It is important to consider the various stages of T1D during immunotherapy, as genetic and environmental conditions will impact the efficacy of the treatment.

Thus, it is indicative that a dynamic nano-delivery system must be constructed that can bind immunotherapeutic agents and release them upon environmental and physiological change. Furthermore, the ability to have controlled release of the agents in the system allows for more tunability in response to varying conditions, environments, and stimuli. Thus, nanoparticles are highly desirable in their role as a vaccine candidate, aiming to break immune tolerance and monitor glycemic activity. In comparison to methods such as surface antigen targeting through monoclonal antibodies, nanoparticle administration to specific sites on autoreactive B cells is gaining more and more attention and relevance in present-day studies, as summarized in Table 2.

**Table 2.** Summary of strategies for targeting and regulating B cell population and function towards T1D.

