Preparation and Characterization of Cell Membranes for Cancer Immunotherapy

Cancer immunotherapy has advanced rapidly over the past decade leading to clinical approval of immune checkpoint blockade and adoptive cell transfer therapies. Further efforts into development of therapeutic vaccines had generated promising results in pre-clinical and clinical studies. Here, we demonstrate novel methodology for preparation of cell membranes into nano-sized vesicles and the development of characterization methods via nanoparticle flow cytometry. Cancer cell membranes from murine melanoma cell line expressing model antigen, ovalbumin, were used for generation of PEGylated vehicles (PEG-NPs), which efficiently delivered endogenous membrane-associated cancer antigens to the draining lymph nodes after subcutaneous administration. PEG-NPs were efficiently taken up by dendritic cells and, when dosed with a potent adjuvant, led to antigen-specific T cell activation and proliferation approximately 4-fold greater than treatment with traditional freeze-thaw lysates. In combination with immune checkpoint blockade (anti-PD-1 treatment), our vaccination approach led to therapeutic cure of 63% of mice and persistent memory responses rejecting additional tumor rechallenge. We further utilized our nanoparticle platform by using adjuvant-matured dendritic cells (DCs) generating MPLA-activated dendritic cell membrane vesicles ((MPLA)DC-MVs). This preparation led to nanoparticles carrying T cell activation ligands (CD80 and CD86) and promoted their proliferation activation in vitro compared to antigen peptide alone, as demonstrated by 2-fold increase in proliferation and 5- to 8-fold increase in live cell numbers and expression of CD25 activation marker. In addition, (MPLA)DC-MVs, but not unstimulated DC-MVs, resulted in activation of immature dendritic cells in vitro, indicated by 2- and 1.3-fold greater expression of CD40 and CD80, respectively. Administration of this formulation in vivo together with OVA peptide epitope led to 2-fold enhanced expansion and maintenance of antigen-specific T cells compared to peptide alone in mice that received adoptive cell transfer or had established OVA-expressing tumors. These studies had demonstrated the use for cell membranes in immunotherapy as vaccine vehicles, but further characterization and optimization could allow for improved efficacy, prompting us to adopt flow cytometry methods aimed at nanoparticle analysis. The technique was established by analysis of lipid-based synthetic formulations focused on demonstrating effective fluorescence detection and separation of individual particle populations. Proof of concept studies were used to confirm presence of ovalbumin on membrane-derived vesicles with antibody staining. Finally, we had utilized this technique to examine antigen display on hepatitis virus C vaccine formulation in order to determine if broadly neutralizing antibodies can bind efficiently and thus if they can be raised in mice immunized with these formulations. Our studies demonstrate that similar levels of broadly neutralizing antibody binding to nanoparticles translate to similar level of protection against cross-strain viral challenge. Taken together, this work has generated a foundation for further research into the use of cell membranes as nanoparticles for immunotherapeutic approaches and techniques necessary for their characterization.PHDPharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/145958/1/ochyl_1.pd

Immunogenic Cell Death Amplified by Co-localized Adjuvant Delivery for Cancer Immunotherapy

Despite their potential, conventional whole-cell cancer vaccines prepared by freeze-thawing or irradiation have shown limited therapeutic efficacy in clinical trials. Recent studies have indicated that cancer cells treated with certain chemotherapeutics, such as mitoxantrone, can undergo immunogenic cell death (ICD) and initiate antitumor immune responses. However, it remains unclear how to exploit ICD for cancer immunotherapy. Here, we present a new material-based strategy for converting immunogenically dying tumor cells into a powerful platform for cancer vaccination and demonstrate their therapeutic potential in murine models of melanoma and colon carcinoma. We have generated immunogenically dying tumor cells surface-modified with adjuvant-loaded nanoparticles. Dying tumor cells laden with adjuvant nanodepots efficiently promote activation and antigen cross-presentation by dendritic cells in vitro and elicit robust antigen-specific CD8α+ T-cells in vivo. Furthermore, whole tumor-cell vaccination combined with immune checkpoint blockade leads to complete tumor regression in 78% of CT26 tumor-bearing mice and establishes long-term immunity against tumor recurrence. Our strategy presented here may open new doors to "personalized" cancer immunotherapy tailored to individual patient's tumor cells. Keywords: cancer immunotherapy; cancer vaccine; Cell engineering; innunogenic cell death; nanoparticl

Self‐healing encapsulation and controlled release of vaccine antigens from PLGA microparticles delivered by microneedle patches

There is an urgent need to reduce reliance on hypodermic injections for many vaccines to increase vaccination safety and coverage. Alternative approaches include controlled release formulations, which reduce dosing frequencies, and utilizing alternative delivery devices such as microneedle patches (MNPs). This work explores development of controlled release microparticles made of poly (lactic‐co‐glycolic acid) (PLGA) that stably encapsulate various antigens though aqueous active self‐healing encapsulation (ASE). These microparticles are incorporated into rapid‐dissolving MNPs for intradermal vaccination.PLGA microparticles containing Alhydrogel are loaded with antigens separate from microparticle fabrication using ASE. This avoids antigen expsoure to many stressors. The microparticles demonstrate bi‐phasic release, with initial burst of soluble antigen, followed by delayed release of Alhydrogel‐complexed antigen over approximately 2 months in vitro. For delivery, the microparticles are incorporated into MNPs designed with pedestals to extend functional microneedle length. These microneedles readily penetrate skin and rapidly dissolve to deposit microparticles intradermally. Microparticles remain in the tissue for extended residence, with MNP‐induced micropores resealing readily. In animal models, these patches generate robust immune responses that are comparable to conventional administration techniques. This lays the framework for a versatile vaccine delivery system that could be self‐applied with important logistical advantages over hypodermic injections.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147859/1/btm210103-sup-0001-supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147859/2/btm210103_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147859/3/btm210103.pd

Engineered Ovalbumin Nanoparticles for Cancer Immunotherapy

Ovalbumin (OVA) is a protein antigen that is widely used for eliciting cellular and humoral immune responses in cancer immunotherapy. As an alternative to solute OVA, engineering approach is developed herein towards protein nanoparticles (pNPs) based on reactive electrospraying. The resulting pNPs are comprised of polymerized OVA, where individual OVA molecules are chemically linked via poly(ethylene glycol) (PEG) units. Controlling the PEG/OVA ratio allows for fine‐tuning of critical physical properties, such as particle size, elasticity, and, at the molecular level, mesh size. As the PEG/OVA ratio decreased, OVA pNPs are more effectively processed by dendritic cells, resulting in higher OT‐I CD8+ cells proliferation in vitro. Moreover, pNPs with lower PEG/OVA ratios elicit enhanced lymphatic drainage in vivo and increased uptake by lymph node macrophages, dendritic cells, and B cells, while 500 nm OVA pNPs show poor draining lymph nodes delivery. In addition, pNPs with lower PEG/OVA ratios result in higher anti‐OVA antibody titers in vivo, suggesting improved humoral immune responses. Importantly, OVA pNPs result in significantly increased median survival relative to solute OVA antigen in a mouse model of B16F10‐OVA melanoma. This work demonstrates that precisely engineered OVA pNPs can improve the overall anti‐tumor response compared to solute antigen.As an alternative to solute antigens for cancer immunotherapy, protein nanoparticles (pNPs) comprised of polymerized antigen linked by poly(ethylene glycol) units are developed based on reactive electrospraying. This engineering approach allows fine tuning the physico‐chemical properties of pNPs such as particle size, elasticity, and mesh size. These properties are related to pNPs enhanced antigen‐specific immune responses and improved anti‐tumor efficacy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163384/3/adtp202000100-sup-0001-SuppMat.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163384/2/adtp202000100.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163384/1/adtp202000100_am.pd

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Immunogenic Cell Death Amplified by Co-localized Adjuvant Delivery for Cancer Immunotherapy

Despite their potential, conventional whole-cell cancer vaccines prepared by freeze–thawing or irradiation have shown limited therapeutic efficacy in clinical trials. Recent studies have indicated that cancer cells treated with certain chemotherapeutics, such as mitoxantrone, can undergo immunogenic cell death (ICD) and initiate antitumor immune responses. However, it remains unclear how to exploit ICD for cancer immunotherapy. Here, we present a new material-based strategy for converting immunogenically dying tumor cells into a powerful platform for cancer vaccination and demonstrate their therapeutic potential in murine models of melanoma and colon carcinoma. We have generated immunogenically dying tumor cells surface-modified with adjuvant-loaded nanoparticles. Dying tumor cells laden with adjuvant nanodepots efficiently promote activation and antigen cross-presentation by dendritic cells in vitro and elicit robust antigen-specific CD8α⁺ T-cells in vivo. Furthermore, whole tumor-cell vaccination combined with immune checkpoint blockade leads to complete tumor regression in ∼78% of CT26 tumor-bearing mice and establishes long-term immunity against tumor recurrence. Our strategy presented here may open new doors to “personalized” cancer immunotherapy tailored to individual patient’s tumor cells

Self-encapsulating Poly(lactic-<i>co</i>-glycolic acid) (PLGA) Microspheres for Intranasal Vaccine Delivery

Herein we describe a formulation of self-encapsulating poly­(lactic-<i>co</i>-glycolic acid) (PLGA) microspheres for vaccine delivery. Self-healing encapsulation is a novel encapsulation method developed by our group that enables the aqueous loading of large molecules into premade PLGA microspheres. Calcium phosphate (CaHPO₄) adjuvant gel was incorporated into the microspheres as a protein-trapping agent for improved encapsulation of antigen. Microspheres were found to have a median size of 7.05 ± 0.31 μm, with a w/w loading of 0.60 ± 0.05% of ovalbumin (OVA) model antigen. The formulation demonstrated continuous release of OVA over a 49-day period. Released OVA maintained its antigenicity over the measured period of >21 days of release. C57BL/6 mice were immunized via the intranasal route with prime and booster doses of OVA (10 μg) loaded into microspheres or coadministered with cholera toxin B (CTB), the gold standard of mucosal adjuvants. Microspheres generated a Th2-type response in both serum and local mucosa, with IgG antibody responses approaching those generated by CTB. The results suggest that this formulation of self-encapsulating microspheres shows promise for further study as a vaccine delivery system