Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons.

Antigen processing is critical for therapeutic vaccines to generate epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often limits the quantity of epitopes released. We address this challenge using machine learning to ascribe a proteasomal degradation score to epitope sequences. Epitopes with varying scores were translocated into cells using nontoxic anthrax proteins. Epitopes with a low score show pronounced immunogenicity due to antigen processing, but epitopes with a high score show limited immunogenicity. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by these vaccines in clinical settings.

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells.

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR FMC63 as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion of both murine CD19 CAR-T cells in a syngeneic model and human CAR-T cells in a humanized mouse model of B cell acute lymphoblastic leukemia (B-ALL), and enhanced control of leukemia progression. This approach thus enables vaccine boosting to be applied to any clinically-relevant CAR-T cell product.

Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons.

Antigen processing is critical for producing epitope peptides that are presented by HLA molecules for T cell recognition. Therapeutic vaccines aim to harness these epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often reduces vaccine efficacy through limiting the quantity of epitopes released. Here, we set out to improve antigen processing by harnessing protein degradation signals called degrons from the ubiquitin-proteasome system. We used machine learning to generate a computational model that ascribes a proteasomal degradation score between 0 and 100. Epitope peptides with varying degron activities were synthesized and translocated into cells using nontoxic anthrax proteins: protective antigen (PA) and the N-terminus of lethal factor (LFN). Immunogenicity studies revealed epitope sequences with a low score (<25) show pronounced T-cell activation but epitope sequences with a higher score (>75) provide limited activation. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity, through conserving the epitope region but varying the flanking sequence. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by vaccine therapeutics in clinical settings.

Optimization of an alum-anchored clinical HIV vaccine candidate.

In the ongoing effort to develop a vaccine against HIV, vaccine approaches that promote strong germinal center (GC) responses may be critical to enable the selection and affinity maturation of rare B cell clones capable of evolving to produce broadly neutralizing antibodies. We previously demonstrated an approach for enhancing GC responses and overall humoral immunity elicited by alum-adjuvanted protein immunization via the use of phosphoserine (pSer) peptide-tagged immunogens that stably anchor to alum particles via ligand exchange with the alum particle surface. Here, using a clinically relevant stabilized HIV Env trimer termed MD39, we systematically evaluated the impact of several parameters relevant to pSer tag composition and trimer immunogen design to optimize this approach, including phosphate valency, amino acid sequence of the trimer C-terminus used for pSer tag conjugation, and structure of the pSer tag. We also tested the impact of co-administering a potent saponin/monophosphoryl lipid A (MPLA) nanoparticle co-adjuvant with alum-bound trimers. We identified MD39 trimer sequences bearing an optimized positively-charged C-terminal amino acid sequence, which, when conjugated to a pSer tag with four phosphates and a polypeptide spacer, bound very tightly to alum particles while retaining a native Env-like antigenicity profile. This optimized pSer-trimer design elicited robust antigen-specific GC B cell and serum IgG responses in mice. Through this optimization, we present a favorable MD39-pSer immunogen construct for clinical translation.

Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity.

Chimeric antigen receptor (CAR) T cell therapy effectively treats human cancer, but the loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T cells triggers the engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited the priming of endogenous anti-tumor T cells. This process was accompanied by shifts in CAR T metabolism toward oxidative phosphorylation (OXPHOS) and was critically dependent on CAR-T-derived IFN-γ. Antigen spreading (AS) induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR antigen negative, and heterogeneous tumor control was further enhanced by the genetic amplification of CAR T IFN-γ expression. Thus, CAR-T-cell-derived IFN-γ plays a critical role in promoting AS, and vaccine boosting provides a clinically translatable strategy to drive such responses against solid tumors.

Universal redirection of CAR T cells against solid tumours via membrane-inserted ligands for the CAR.

The effectiveness of chimaeric antigen receptor (CAR) T cell therapies for solid tumours is hindered by difficulties in the selection of an effective target antigen, owing to the heterogeneous expression of tumour antigens and to target antigen expression in healthy tissues. Here we show that T cells with a CAR specific for fluorescein isothiocyanate (FITC) can be directed against solid tumours via the intratumoural administration of a FITC-conjugated lipid-poly(ethylene)-glycol amphiphile that inserts itself into cell membranes. In syngeneic and human tumour xenografts in mice, 'amphiphile tagging' of tumour cells drove tumour regression via the proliferation and accumulation of FITC-specific CAR T cells in the tumours. In syngeneic tumours, the therapy induced the infiltration of host T cells, elicited endogenous tumour-specific T cell priming and led to activity against distal untreated tumours and to protection against tumour rechallenge. Membrane-inserting ligands for specific CARs may facilitate the development of adoptive cell therapies that work independently of antigen expression and of tissue of origin.

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

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.

Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccines.

The recent clinical success of multiple mRNA-based SARS-CoV-2 vaccines has proven the potential of RNA formulated in lipid nanoparticles (LNPs) in humans, and products based on base-modified RNA, sequence-optimized RNA, and self-replicating RNAs formulated in LNPs are all in various stages of clinical development. However, much remains to be learned about critical parameters governing the manufacturing and use of LNP-RNA formulations. One important issue that has received limited attention in the literature to date is the identification of optimal storage conditions for LNP-RNA that preserve long-term activity of the formulations. Here, we analyzed the physical structure, in vivo expression characteristics, and functional activity of alphavirus-derived self-replicating RNA (repRNA)-loaded LNPs encoding HIV vaccine antigens following storage in varying temperatures, buffers, and in the presence or absence of cryoprotectants. We found that for lipid nanoparticles with compositions similar to clinically-used LNPs, storage in RNAse-free PBS containing 10% (w/v) sucrose at -20 °C was able to maintain vaccine stability and in vivo potency at a level equivalent to freshly prepared vaccines following 30 days of storage. LNPs loaded with repRNA could also be lyophilized with retention of bioactivity.

Phosphate-mediated coanchoring of RBD immunogens and molecular adjuvants to alum potentiates humoral immunity against SARS-CoV-2.

There is a need for additional rapidly scalable, low-cost vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to achieve global vaccination. Aluminum hydroxide (alum) adjuvant is the most widely available vaccine adjuvant but elicits modest humoral responses. We hypothesized that phosphate-mediated coanchoring of the receptor binding domain (RBD) of SARS-CoV-2 together with molecular adjuvants on alum particles could potentiate humoral immunity by promoting extended vaccine kinetics and codelivery of vaccine components to lymph nodes. Modification of RBD immunogens with phosphoserine (pSer) peptides enabled efficient alum binding and slowed antigen clearance, leading to notable increases in germinal center responses and neutralizing antibody titers in mice. Adding phosphate-containing CpG or saponin adjuvants to pSer-RBD:alum immunizations synergistically enhanced vaccine immunogenicity in mice and rhesus macaques, inducing neutralizing responses against SARS-CoV-2 variants. Thus, phosphate-mediated coanchoring of RBD and molecular adjuvants to alum is an effective strategy to enhance the efficacy of SARS-CoV-2 subunit vaccines.

A particulate saponin/TLR agonist vaccine adjuvant alters lymph flow and modulates adaptive immunity.

Saponins are potent and safe vaccine adjuvants, but their mechanisms of action remain incompletely understood. Here, we explored the properties of several saponin formulations, including immune-stimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA). We found that MPLA self-assembles with saponins to form particles physically resembling ISCOMs, which we termed saponin/MPLA nanoparticles (SMNP). Saponin-containing adjuvants exhibited distinctive mechanisms of action, altering lymph flow in a mast cell­dependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally related adjuvant AS01B in mice, and primed robust germinal center B cell, TFH, and HIV tier 2 neutralizing antibodies in nonhuman primates. Together, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest that SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.

The injury response to DNA damage in live tumor cells promotes antitumor immunity.

Although immune checkpoint blockade (ICB) has strong clinical benefit for treating some tumor types, it fails in others, indicating a need for additional modalities to enhance the ICB effect. Here, we identified one such modality by using DNA damage to create a live, injured tumor cell adjuvant. Using an optimized ex vivo coculture system, we found that treating tumor cells with specific concentrations of etoposide, mitoxantrone, or doxorubicin markedly enhanced dendritic cell­mediated T cell activation. These immune-enhancing effects of DNA damage did not correlate with immunogenic cell death markers or with the extent of apoptosis or necroptosis; instead, these effects were mediated by live injured cells with activation of the DNA-PK, ATR, NF-κB, p38 MAPK, and RIPK1 signaling pathways. In mice, intratumoral injection of ex vivo etoposide­treated tumor cells in combination with systemic ICB (by anti-PD-1 and anti-CTLA4 antibodies) increased the number of intratumoral CD103+ dendritic cells and circulating tumor-antigen­specific CD8+ T cells, decreased tumor growth, and improved survival. These effects were absent in Batf3−/− mice and in mice in which the DNA-damaging drug was injected directly into the tumor, due to DNA damage in the immune cells. The combination treatment induced complete tumor regression in a subset of mice that were then able to reject tumor rechallenge, indicating that the injured cell adjuvant treatment induced durable antitumor immunological memory. These results provide a strategy for enhancing the efficacy of immune checkpoint inhibition in tumor types that do not respond to this treatment modality by itself.

Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization.

Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2, and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely, viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non-neutralizing or weakly neutralizing ("off-target") antibodies. Using our recently developed electron microscopy polyclonal epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.

Exploiting albumin as a mucosal vaccine chaperone for robust generation of lung-resident memory T cells.

Tissue-resident memory T cells (TRMs) can profoundly enhance mucosal immunity, but parameters governing TRM induction by vaccination remain poorly understood. Here, we describe an approach exploiting natural albumin transport across the airway epithelium to enhance mucosal TRM generation by vaccination. Pulmonary immunization with albumin-binding amphiphile conjugates of peptide antigens and CpG adjuvant (amph-vaccines) increased vaccine accumulation in the lung and mediastinal lymph nodes (MLNs). Amph-vaccines prolonged antigen presentation in MLNs over 2 weeks, leading to 25-fold increased lung-resident T cell responses over traditional immunization and enhanced protection from viral or tumor challenge. Mimicking such prolonged exposure through repeated administration of soluble vaccine revealed that persistence of both antigen and adjuvant was critical for optimal TRM induction, mediated through T cell priming in MLNs after prime, and directly in the lung tissue after boost. Thus, vaccine persistence strongly promotes TRM induction, and amph-conjugates may provide a practical approach to achieve such kinetics in mucosal vaccines.

Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization.

Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2 and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non or weakly neutralizing ("off-target") antibodies. Using our recently developed electron microscopy epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.

Author Correction: A Engineered immunogen binding to alum adjuvant enhances humoral immunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Engineered immunogen binding to alum adjuvant enhances humoral immunity.

Adjuvants are central to the efficacy of subunit vaccines. Aluminum hydroxide (alum) is the most commonly used vaccine adjuvant, yet its adjuvanticity is often weak and mechanisms of triggering antibody responses remain poorly understood. We demonstrate that site-specific modification of immunogens with short peptides composed of repeating phosphoserine (pSer) residues enhances binding to alum and prolongs immunogen bioavailability. The pSer-modified immunogens formulated in alum elicited greatly increased germinal center, antibody, neutralizing antibody, memory and long-lived plasma cell responses compared to conventional alum-adsorbed immunogens. Mechanistically, pSer-immunogen:alum complexes form nanoparticles that traffic to lymph nodes and trigger B cell activation through multivalent and oriented antigen display. Direct uptake of antigen-decorated alum particles by B cells upregulated antigen processing and presentation pathways, further enhancing B cell activation. These data provide insights into mechanisms of action of alum and introduce a readily translatable approach to significantly improve humoral immunity to subunit vaccines using a clinical adjuvant.

Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor.

Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment. Amph-ligand boosting triggered massive CAR-T expansion, increased donor cell polyfunctionality, and enhanced antitumor efficacy in multiple immunocompetent mouse tumor models. We demonstrate two approaches to generalizing this strategy to any chimeric antigen receptor, enabling this simple non-human leukocyte antigen-restricted approach to enhanced CAR-T functionality to be applied to existing CAR-T designs.

Enhancement of Peptide Vaccine Immunogenicity by Increasing Lymphatic Drainage and Boosting Serum Stability.

Antitumor T-cell responses have the potential to be curative in cancer patients, but the induction of potent T-cell immunity through vaccination remains a largely unmet goal of immunotherapy. We previously reported that the immunogenicity of peptide vaccines could be increased by maximizing delivery to lymph nodes (LNs), where T-cell responses are generated. This was achieved by conjugating the peptide to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG (DSPE-PEG) to promote albumin binding, which resulted in enhanced lymphatic drainage and improved T-cell responses. Here, we expanded upon these findings and mechanistically dissected the properties that contribute to the potency of this amphiphile-vaccine (amph-vaccine). We found that multiple linkage chemistries could be used to link peptides with DSPE-PEG, and further, that multiple albumin-binding moieties conjugated to peptide antigens enhanced LN accumulation and subsequent T-cell priming. In addition to enhancing lymphatic trafficking, DSPE-PEG conjugation increased the stability of peptides in serum. DSPE-PEG peptides trafficked beyond immediate draining LNs to reach distal nodes, with antigen presented for at least a week in vivo, whereas soluble peptide presentation quickly decayed. Responses to amph-vaccines were not altered in mice deficient in the albumin-binding neonatal Fc receptor (FcRn), but required Batf3-dependent dendritic cells (DCs). Amph-peptides were processed by human DCs equivalently to unmodified peptides. These data define design criteria for enhancing the immunogenicity of molecular vaccines to guide the design of next-generation peptide vaccines. Cancer Immunol Res; 6(9); 1025-38. ©2018 AACR.

Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses.

Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte-associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8+ T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.

Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells.

Tumor cells disseminate into compartments that are poorly accessible from circulation, which necessitates high doses of systemic chemotherapy. However, the effectiveness of many drugs, such as the potent topoisomerase I poison SN-38, is hampered by poor pharmacokinetics. To deliver SN-38 to lymphoma tumors in vivo, we took advantage of the fact that healthy lymphocytes can be programmed to phenocopy the biodistribution of the tumor cells. In a murine model of disseminated lymphoma, we expanded autologous polyclonal T cells ex vivo under conditions that retained homing receptors mirroring lymphoma cells, and functionalized these T cells to carry SN-38-loaded nanocapsules on their surfaces. Nanocapsule-functionalized T cells were resistant to SN-38 but mediated efficient killing of lymphoma cells in vitro. Upon adoptive transfer into tumor-bearing mice, these T cells served as active vectors to deliver the chemotherapeutic into tumor-bearing lymphoid organs. Cell-mediated delivery concentrated SN-38 in lymph nodes at levels 90-fold greater than free drug systemically administered at 10-fold higher doses. The live T cell delivery approach reduced tumor burden significantly after 2 weeks of treatment and enhanced survival under conditions where free SN-38 and SN-38-loaded nanocapsules alone were ineffective. These results suggest that tissue-homing lymphocytes can serve as specific targeting agents to deliver nanoparticles into sites difficult to access from the circulation, and thus improve the therapeutic index of chemotherapeutic drugs with unfavorable pharmacokinetics.