Novel strategies exploiting interleukin-12 in cancer immunotherapy.

The structural integrity of vaccine antigens is critical to the generation of protective antibody responses, but the impact of protease activity on vaccination in vivo is poorly understood. We characterized protease activity in lymph nodes and found that antigens were rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions, whereas low protease activity and antigen degradation rates were detected in the vicinity of follicular dendritic cells (FDCs). Correlated with these findings, immunization regimens designed to target antigen to FDCs led to germinal centers dominantly targeting intact antigen, whereas traditional immunizations led to much weaker responses that equally targeted the intact immunogen and antigen breakdown products. Thus, spatially compartmentalized antigen proteolysis affects humoral immunity and can be exploited. Description Location, location, location To generate protective immune responses, vaccine antigens are designed to mimic native protein structures on foreign pathogens. After vaccination, antigens are transported from the injection site by lymphatic vessels to the draining lymph nodes, tissues where immune responses are initiated. Aung et al. found that in order to mount a successful antibody response, incoming antigens need to be localized to specific sites within the lymph nodes called follicles (see the Perspective by Kalia and Hickman). Those antigens that were not rapidly directed to follicles were destroyed by proteases. Vaccines that resulted in speedy delivery of antigens to follicles preserved intact antigen and generated better antibody responses. These findings may have implications for the design of vaccines against difficult to neutralize or genetically variable pathogens. —PNK and SMH Spatially heterogeneous protease activity within lymph nodes modulates humoral responses to vaccination. INTRODUCTION Antigens in vaccines are designed to induce antibodies that bind to key epitopes on the surfaces of pathogens, promoting microbe elimination. Despite advances in the engineering of immunogens that closely mimic native pathogen protein structures, not all vaccines elicit protective humoral immunity. We hypothesized that the integrity of vaccine antigens in vivo could be an important factor. Extracellular antigen degradation in lymph nodes (LNs) could limit the generation of protective antibody responses and also create competing irrelevant responses against antigen breakdown products. However, vaccine antigen stability in vivo is poorly understood. RATIONALE Extracellular protease activity within the LN has not been studied, and how such potential proteolytic activity could affect immunogen structure and subsequent immune responses is unknown. We used HIV immunogens as a model system and measured antigen stability in the LN, identified degradative proteases, and explored vaccination strategies to maximize the delivery of intact antigens to B cells. RESULTS We investigated antigen stability within the LN by conjugating antigens with dyes that undergo a loss in fluorescence resonance energy transfer (FRET) upon structural breakdown. After vaccination, antigen was rapidly degraded in the subcapsular sinus (SCS) and extrafollicular regions of the LN within 48 hours, with approximately half of the antigen breakdown occurring extracellularly. By contrast, antigen localized to B cell follicles remained intact. To determine the cause of antigen degradation, we examined protease expression and activity in LNs. RNA sequencing, histology, and imaging zymography revealed that extracellular proteases and protease activity were present at high levels in the SCS and extrafollicular regions, but were present at low levels within follicles. The identified metalloproteases degraded antigen in vitro, and their inhibition in vivo increased intact antigen levels within the LN. Although antigen captured by follicular dendritic cells (FDCs) has been reported to be retained in an intact state, the mechanisms for this preservation have remained unclear. We tested the importance of low follicular protease activity by adoptively transferring protease-expressing polyclonal B cells into the LNs of immunized mice. We observed a significant decrease in FDC-captured antigen stability, indicating that low follicular protease activity is important for intact antigen retention. Motivated by these findings, we tested vaccination strategies to maximize the humoral response to intact antigen. Immunization by “extended dosing” regimens or nanoparticle formulations that rapidly targeted antigen to FDCs were compared with traditional bolus-soluble antigen vaccination (in which little follicular uptake of antigen is observed). FDC-targeting vaccinations led to large germinal centers (GCs), with high numbers of B cells recognizing intact antigen. By contrast, traditional bolus immunization resulted in small GCs with B cells that equally recognized intact antigen and breakdown products. Similar patterns of response were observed in serum antibody titers. CONCLUSION Here, we have shown that rapid antigen degradation can occur in LNs after vaccination except within follicles. This degradation is mediated by spatially compartmentalized proteolysis within the tissue. Vaccine strategies promoting rapid follicular antigen localization strongly promoted antibody generation against intact antigen without amplifying responses to irrelevant antigen breakdown products. Such approaches may enable more effective vaccines against difficult pathogens such as HIV. Stability of FDC-captured antigen after immunization. Left and middle: HIV antigens (magenta) localized to FDCs (cyan) that reside among cells in the follicle (green) 3 days after immunization. Right: FRET analysis of antigen structural integrity within the outlined region shows that antigen localized to FDCs remains intact. Scale bar, 10 μm.

Low protease activity in B cell follicles promotes retention of intact antigens after immunization

Clinical immunotherapy of solid tumors elicits durable responses only in a minority of patients, largely due to the highly immunosuppressive tumor microenvironment (TME). Although rational combinations of vaccine adjuvants with inflammatory cytokines or immune agonists that relieve immunosuppression represent an appealing therapeutic strategy against solid tumors, there are unavoidable nonspecific toxicities due to the pleiotropy of cytokines and undesired activation of off‐target cells. Herein, a Zn2+ doped layered double hydroxide (Zn‐LDH) based immunomodulating adjuvant, which not only relieves immunosuppression but also elicits robust antitumor immunity, is reported. Peritumorally injected Zn‐LDH sustainably neutralizes acidic TME and releases abundant Zn2+, promoting a pro‐inflammatory network composed of M1‐tumor‐associated macrophages, cytotoxic T cells, and natural‐killer cells. Moreover, the Zn‐LDH internalized by tumor cells effectively disrupts endo‐/lysosomes to block autophagy and induces mitochondrial damage, and the released Zn2+ activates the cGas‐STING signaling pathway to induce immunogenic cell death, which further promotes the release of tumor‐associated antigens to induce antigen‐specific cytotoxic T lymphocytes. Unprecedentedly, merely injection of Zn‐LDH adjuvant, without using any cytotoxic inflammatory cytokines or immune agonists, significantly inhibits the growth, recurrence, and metastasis of solid tumors in mice. This study provides a rational bottom‐up design of potent adjuvant for cancer metalloimmunotherapy against solid tumors.

A Peritumorally Injected Immunomodulating Adjuvant Elicits Robust and Safe Metalloimmunotherapy against Solid Tumors

Engineering Self-Assembling Protein Nanoparticles for Therapeutic Delivery

Cancer immunotherapy efficacy is largely limited by the suppressive tumor immune microenvironment (TIME) where antitumor immune cells are inhibited and tumor antigens continue to mutate or be lost. To remodel the TIME, we here applied weakly alkaline layered double hydroxide nanoparticles (LDH NPs) to neutralize the excess acid and block autophagy of tumor cells for neoadjuvant cancer immunotherapy. Peritumoral injection of LDH NPs provided a long-term and efficient acid-neutralization in the TIME, blocked the lysosome-mediated autophagy pathway in tumor cells, and increased the levels of antitumor tumor-associated macrophages and T cells. These LDH NPs captured tumor antigens released in the tumor tissues and effectively inhibited the growth of both melanoma and colon tumors in vivo. These findings indicate that LDH NPs, as an immunomodulator and adjuvant, successfully "awaken" and promote the host innate and adaptive immune systems, showing promising potential for solid tumor immunotherapy.

Efficient Immunotherapy of Drug-Free Layered Double Hydroxide Nanoparticles via Neutralizing Excess Acid and Blocking Tumor Cell Autophagy.

Anti-tumour inflammatory cytokines are highly toxic when administered systemically. Here, in multiple syngeneic mouse models, we show that the intratumoural injection of recombinantly expressed cytokines bound tightly to the common vaccine adjuvant aluminium hydroxide (alum) (via ligand exchange between hydroxyls on the surface of alum and phosphoserine residues tagged to the cytokine by an alum-binding peptide) leads to weeks-long retention of the cytokines in the tumours, with minimal side effects. Specifically, a single dose of alum-tethered interleukin-12 induced substantial interferon-γ-mediated T-cell and natural-killer-cell activities in murine melanoma tumours, increased tumour antigen accumulation in draining lymph nodes and elicited robust tumour-specific T-cell priming. Moreover, intratumoural injection of alum-anchored cytokines enhanced responses to checkpoint blockade, promoting cures in distinct poorly immunogenic syngeneic tumour models and eliciting control over metastases and distant untreated lesions. Intratumoural treatment with alum-anchored cytokines represents a safer and tumour-agnostic strategy to improving local and systemic anticancer immunity. 

/pdf/intratumourally-injected-alum-tethered-cytokines-elicit-2nu50w57.pdf

Intratumourally injected alum-tethered cytokines elicit potent and safer local and systemic anticancer immunity

http://www.cell.com/article/S2211124721017216/pdf

Franz Kronthaler

Papers

Intratumourally injected alum-tethered cytokines elicit potent and safer local and systemic anticancer immunity

Mannose-binding lectin and complement mediate follicular localization and enhanced immunogenicity of diverse protein nanoparticle immunogens