Active immunotherapy for C5a-mediated inflammation using adjuvant-free self-assembled peptide nanofibers.

The terminal protein in the complement cascade C5a is a potent inflammatory molecule and chemoattractant that is involved in the pathology of multiple inflammatory diseases including sepsis and arthritis, making it a promising protein to target with immunotherapies. Active immunotherapies, in which patients are immunized against problematic self-molecules and generate therapeutic antibodies as a result, have received increasing interest as an alternative to traditional monoclonal antibody treatments. In previous work, we have designed supramolecular self-assembling peptide nanofibers as active immunotherapies with defined combinations of B- and T-cell epitopes. Herein, the self-assembling peptide Q11 platform was employed to generate a C5a-targeting active immunotherapy. Two of three predicted B-cell epitope peptides from C5a were found to be immunogenic when displayed within Q11 nanofibers, and the nanofibers were capable of reducing C5a serum concentrations following immunization. Contrastingly, C5a's precursor protein C5 maintained its original concentration, promising to minimize side effects heretofore associated with C5-targeted therapies. Immunization protected mice against an LPS-challenge model of sepsis, and it reduced clinical severity in a model of collagen-antibody induced arthritis. Together, this work indicates the potential for targeting terminal complement proteins with active immunotherapies by leveraging the immunogenicity of self-assembled peptide nanomaterials. STATEMENT OF SIGNIFICANCE: Chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease are currently treated primarily with monoclonal antibodies against key inflammatory mediators. While helpful for many patients, they have high non-response rates, are costly, and commonly fail as anti-drug antibodies are raised by the patient. The approach we describe here explores a fundamentally different treatment paradigm: raising therapeutic antibody responses with an active immunotherapy. We employ innovative supramolecular peptide nanomaterials to elicit neutralizing antibody responses against complement component C5a and demonstrate therapeutic efficacy in preclinical mouse models of sepsis and rheumatoid arthritis. The strategy reported may represent a potential alternative to monoclonal antibody therapies.

Characterization and root cause analysis of immunogenicity to pasotuxizumab (AMG 212), a prostate-specific membrane antigen-targeting bispecific T-cell engager therapy.

Introduction: In oncology, anti-drug antibody (ADA) development that significantly curtails response durability has not historically risen to a level of concern. The relevance and attention ascribed to ADAs in oncology clinical studies have therefore been limited, and the extant literature on this subject scarce. In recent years, T cell engagers have gained preeminence within the prolific field of cancer immunotherapy. These drugs whose mode of action is expected to potently stimulate anti-tumor immunity, may potentially induce ADAs as an unintended corollary due to an overall augmentation of the immune response. ADA formation is therefore emerging as an important determinant in the successful clinical development of such biologics. Methods: Here we describe the immunogenicity and its impact observed to pasotuxizumab (AMG 212), a prostate-specific membrane antigen (PSMA)-targeting bispecific T cell engager (BiTE®) molecule in NCT01723475, a first-in-human (FIH), multicenter, dose-escalation study in patients with metastatic castration-resistant prostate cancer (mCRPC). To explain the disparity in ADA incidence observed between the SC and CIV arms of the study, we interrogated other patient and product-specific factors that may have explained the difference beyond the route of administration. Results: Treatment-emergent ADAs (TE-ADA) developed in all subjects treated with at least 1 cycle of AMG 212 in the subcutaneous (SC) arm. These ADAs were neutralizing and resulted in profound exposure loss that was associated with contemporaneous reversal of initial Prostate Surface Antigen (PSA) responses, curtailing durability of PSA response in patients. Pivoting from SC to a continuous intravenous (CIV) administration route remarkably yielded no subjects developing ADA to AMG 212. Through a series of stepwise functional assays, our investigation revealed that alongside a more historically immunogenic route of administration, non-tolerant T cell epitopes within the AMG 212 amino acid sequence were likely driving the high-titer, sustained ADA response observed in the SC arm. Discussion: These mechanistic insights into the AMG 212 ADA response underscore the importance of performing preclinical immunogenicity risk evaluation as well as advocate for continuous iteration to better our biologics.

BPIFA1 is a secreted biomarker of differentiating human airway epithelium.

In vitro culture and differentiation of human-derived airway basal cells under air-liquid interface (ALI) into a pseudostratified mucociliated mucosal barrier has proven to be a powerful preclinical tool to study pathophysiology of respiratory epithelium. As such, identifying differentiation stage-specific biomarkers can help investigators better characterize, standardize, and validate populations of regenerating epithelial cells prior to experimentation. Here, we applied longitudinal transcriptomic analysis and observed that the pattern and the magnitude of OMG, KRT14, STC1, BPIFA1, PLA2G7, TXNIP, S100A7 expression create a unique biosignature that robustly indicates the stage of epithelial cell differentiation. We then validated our findings by quantitative hemi-nested real-time PCR from in vitro cultures sourced from multiple donors. In addition, we demonstrated that at protein-level secretion of BPIFA1 accurately reflects the gene expression profile, with very low quantities present at the time of ALI induction but escalating levels were detectable as the epithelial cells terminally differentiated. Moreover, we observed that increase in BPIFA1 secretion closely correlates with emergence of secretory cells and an anti-inflammatory phenotype as airway epithelial cells undergo mucociliary differentiation under air-liquid interface in vitro.

Multiepitope supramolecular peptide nanofibers eliciting coordinated humoral and cellular antitumor immune responses.

Subunit vaccines inducing antibodies against tumor-specific antigens have yet to be clinically successful. Here, we use a supramolecular α-helical peptide nanofiber approach to design epitope-specific vaccines raising simultaneous B cell, CD8+ T cell, and CD4+ T cell responses against combinations of selected epitopes and show that the concurrent induction of these responses generates strong antitumor effects in mice, with significant improvements over antibody or CD8+ T cell-based vaccines alone, in both prophylactic and therapeutic subcutaneous melanoma models. Nanofiber vaccine-induced antibodies mediated in vitro tumoricidal antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The addition of immune checkpoint and phagocytosis checkpoint blockade antibodies further improved the therapeutic effect of the nanofiber vaccines against murine melanoma. These findings highlight the potential clinical benefit of vaccine-induced antibody responses for tumor treatments, provided that they are accompanied by simultaneous CD8+ and CD4+ responses, and they illustrate a multiepitope cancer vaccine design approach using supramolecular nanomaterials.

Immunogenicity assessment of bispecific antibody-based immunotherapy in oncology.

With increasing numbers of bispecific antibodies (BsAbs) and multispecific products entering the clinic, recent data highlight immunogenicity as an emerging challenge in the development of such novel biologics. This review focuses on the immunogenicity risk assessment (IgRA) of BsAb-based immunotherapies for cancer, highlighting several risk factors that need to be considered. These include the novel scaffolds consisting of bioengineered sequences, the potentially synergistic immunomodulating mechanisms of action (MOAs) from different domains of the BsAb, as well as several other product-related and patient-related factors. In addition, the clinical relevance of anti-drug antibodies (ADAs) against selected BsAbs developed as anticancer agents is reviewed and the advances in our knowledge of tools and strategies for immunogenicity prediction, monitoring, and mitigation are discussed. It is critical to implement a drug-specific IgRA during the early development stage to guide ADA monitoring and risk management strategies. This IgRA may include a combination of several assessment tools to identify drug-specific risks as well as a proactive risk mitigation approach for candidate or format selection during the preclinical stage. The IgRA is an on-going process throughout clinical development. IgRA during the clinical stage may bridge the gap between preclinical immunogenicity prediction and clinical immunogenicity, and retrospectively guide optimization efforts for next-generation BsAbs. This iterative process throughout development may improve the reliability of the IgRA and enable the implementation of effective risk mitigation strategies, laying the foundation for improved clinical success.

HIV envelope antigen valency on peptide nanofibers modulates antibody magnitude and binding breadth.

A major challenge in developing an effective vaccine against HIV-1 is the genetic diversity of its viral envelope. Because of the broad range of sequences exhibited by HIV-1 strains, protective antibodies must be able to bind and neutralize a widely mutated viral envelope protein. No vaccine has yet been designed which induces broadly neutralizing or protective immune responses against HIV in humans. Nanomaterial-based vaccines have shown the ability to generate antibody and cellular immune responses of increased breadth and neutralization potency. Thus, we have developed supramolecular nanofiber-based immunogens bearing the HIV gp120 envelope glycoprotein. These immunogens generated antibody responses that had increased magnitude and binding breadth compared to soluble gp120. By varying gp120 density on nanofibers, we determined that increased antigen valency was associated with increased antibody magnitude and germinal center responses. This study presents a proof-of-concept for a nanofiber vaccine platform generating broad, high binding antibody responses against the HIV-1 envelope glycoprotein.

Modular complement assemblies for mitigating inflammatory conditions.

Complement protein C3dg, a key linkage between innate and adaptive immunity, is capable of stimulating both humoral and cell-mediated immune responses, leading to considerable interest in its use as a molecular adjuvant. However, the potential of C3dg as an adjuvant is limited without ways of controllably assembling multiple copies of it into vaccine platforms. Here, we report a strategy to assemble C3dg into supramolecular nanofibers with excellent compositional control, using ß-tail fusion tags. These assemblies were investigated as therapeutic active immunotherapies, which may offer advantages over existing biologics, particularly toward chronic inflammatory diseases. Supramolecular assemblies based on the Q11 peptide system containing ß-tail-tagged C3dg, B cell epitopes from TNF, and the universal T cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only ß-tail-C3dg and lacking the TNF B cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with ß-tail-C3dg caused the expansion of an autoreactive C3dg-specific T cell population, which may act to dampen the immune response, preventing excessive inflammation. These findings indicate that molecular assemblies displaying C3dg warrant further development as active immunotherapies.

Multifactorial Design of a Supramolecular Peptide Anti-IL-17 Vaccine Toward the Treatment of Psoriasis.

Current treatments for chronic immune-mediated diseases such as psoriasis, rheumatoid arthritis, or Crohn's disease commonly rely on cytokine neutralization using monoclonal antibodies; however, such approaches have drawbacks. Frequent repeated dosing can lead to the formation of anti-drug antibodies and patient compliance issues, and it is difficult to identify a single antibody that is broadly efficacious across diverse patient populations. As an alternative to monoclonal antibody therapy, anti-cytokine immunization is a potential means for long-term therapeutic control of chronic inflammatory diseases. Here we report a supramolecular peptide-based approach for raising antibodies against IL-17 and demonstrate its efficacy in a murine model of psoriasis. B-cell epitopes from IL-17 were co-assembled with the universal T-cell epitope PADRE using the Q11 self-assembling peptide nanofiber system. These materials, with or without adjuvants, raised antibody responses against IL-17. Exploiting the modularity of the system, multifactorial experimental designs were used to select formulations maximizing titer and avidity. In a mouse model of psoriasis induced by imiquimod, unadjuvanted nanofibers had therapeutic efficacy, which could be enhanced with alum adjuvant but reversed with CpG adjuvant. Measurements of antibody subclass induced by adjuvanted and unadjuvanted formulations revealed strong correlations between therapeutic efficacy and titers of IgG1 (improved efficacy) or IgG2b (worsened efficacy). These findings have important implications for the development of anti-cytokine active immunotherapies and suggest that immune phenotype is an important metric for eliciting therapeutic anti-cytokine antibody responses.

Self-Assembling Peptide Gels for 3D Prostate Cancer Spheroid Culture.

Progress in prostate cancer research is presently limited by a shortage of reliable in vitro model systems. The authors describe a novel self-assembling peptide, bQ13, which forms nanofibers and gels useful for the 3D culture of prostate cancer spheroids, with improved cytocompatibility compared to related fibrillizing peptides. The mechanical properties of bQ13 gels can be controlled by adjusting peptide concentration, with storage moduli ranging between 1 and 10 kPa. bQ13's ability to remain soluble at mildly basic pH considerably improved the viability of encapsulated cells compared to other self-assembling nanofiber-forming peptides. LNCaP cells formed spheroids in bQ13 gels with similar morphologies and sizes to those formed in Matrigel or RADA16-I. Moreover, prostate-specific antigen (PSA) is produced by LNCaP cells in all matrices, and PSA production is more responsive to enzalutamide treatment in bQ13 gels than in other fibrillized peptide gels. bQ13 represents an attractive platform for further tailoring within 3D cell culture systems.

Progress Toward the Clinical Translation of Bioinspired Peptide and Protein Assemblies.

Supramolecular materials composed of proteins and peptides have been receiving considerable attention toward a range of diseases and conditions from vaccines to drug delivery. Owing to the relative newness of this class of materials, the bulk of work to date has been preclinical. However, examples of approved treatments particularly in vaccines, dentistry, and hemostasis demonstrate the translational potential of supramolecular polypeptides. Critical milestones in the clinical development of this class of materials and currently approved supramolecular polypeptide therapies are described in this study. Additional examples of not-yet-approved materials that are steadily advancing toward clinical use are also featured. Spherical assemblies such as virus-like particles, designed protein nanoparticles, and spherical peptide amphiphiles are highlighted, followed by fiber-forming systems such as fibrillizing peptides, fiber-forming peptide-amphiphiles, and filamentous bacteriophages.