The Limitations of Current T Cell-Driven Anticancer Immunotherapies Can Be Overcome with an Original Extracellular-Vesicle-Based Vaccine Strategy.

The emergence of tumors associated with defects in immune surveillance often involve the impairment of key functions of T lymphocytes. Therefore, several anticancer immunotherapies have focused on the induction/strengthening of the tumor-specific activity of T cells. In particular, strategies based on immune checkpoint inhibitors, CAR-T cells, and mRNA vaccines share a common goal of inducing/recovering an effective antitumor cytotoxic activity, often resulting in either exhausted or absent in patients' lymphocytes. In many instances, these approaches have been met with success, becoming part of current clinic protocols. However, the most practiced strategies sometimes also pay significant tolls in terms of adverse events, a lack of target specificity, tumor escape, and unsustainable costs. Hence, new antitumor immunotherapies facing at least some of these issues need to be explored. In this perspective article, the characteristics of a novel CD8+ T cell-specific anticancer vaccine strategy based on in vivo-engineered extracellular vesicles are described. How this approach can be exploited to overcome at least some of the limitations of current antitumor immunotherapies is also discussed.

SARS-CoV-2-Specific CD8+ T-Cells in Blood but Not in the Lungs of Vaccinated K18-hACE2 Mice after Infection.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 enters the host by infecting nasal ciliated cells. Then, the virus can spread towards the oropharyngeal cavity and the pulmonary tissues. The antiviral adaptive immunity is promptly induced in response to the virus's detection, with virus-specific T-lymphocytes appearing before antiviral antibodies. Both the breadth and potency of antiviral CD8+ T-cell immunity have a key role in containing viral spread and disease severity. Current anti-SARS-CoV-2 vaccines do not impede the virus's replication in the upper respiratory tract, and there is consensus on the fact that the best potency of the antiviral immune response in both blood and the upper respiratory tract can be reached upon infection in vaccinees (i.e., breakthrough infection). However, whether the antiviral CD8+ T-cells developing in response to the breakthrough infection in the upper respiratory tract diffuse to the lungs is also still largely unknown. To fill the gap, we checked the CD8+ T-cell immunity elicited after infection of K18-hACE2 transgenic mice both at 3 weeks and 3 months after anti-spike vaccination. Virus-specific CD8+ T-cell immunity was monitored in both blood and the lungs before and after infection. By investigating the de novo generation of the CD8+ T-cells specific for SARS-CoV-2 viral proteins, we found that both membrane (M) and/or nucleocapsid (N)-specific CD8+ T-cells were induced at comparable levels in the blood of both unvaccinated and vaccinated mice. Conversely, N-specific CD8+ T-cells were readily found in the lungs of the control mice but were either rare or absent in those of vaccinated mice. These results support the idea that the hybrid cell immunity developing after asymptomatic/mild breakthrough infection strengthens the antiviral cell immunity in the lungs only marginally, implying that the direct exposition of viral antigens is required for the induction of an efficient antiviral cell immunity in the lungs.

Antiviral effect of SARS-CoV-2 N-specific CD8+ T cells induced in lungs by engineered extracellular vesicles.

Induction of effective immunity in the lungs should be a requisite for any vaccine designed to control the severe pathogenic effects generated by respiratory infectious agents. We recently provided evidence that the generation of endogenous extracellular vesicles (EVs) engineered for the incorporation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 Nucleocapsid (N) protein induced immunity in the lungs of K18-hACE2 transgenic mice, which then can survive the lethal virus infection. However, nothing is known about the ability of the N-specific CD8+ T cell immunity in controlling viral replication in the lungs, a major pathogenic signature of severe disease in humans. To fill the gap, we investigated the immunity generated in the lungs by N-engineered EVs in terms of induction of N-specific effectors and resident memory CD8+ T lymphocytes before and after virus challenge carried out three weeks and three months after boosting. At the same time points, viral replication extents in the lungs were evaluated. Three weeks after the second immunization, virus replication was reduced in mice best responding to vaccination by more than 3-logs compared to the control group. The impaired viral replication matched with a reduced induction of Spike-specific CD8+ T lymphocytes. The antiviral effect appeared similarly strong when the viral challenge was carried out 3 months after boosting, and associated with the persistence of N-specific CD8+ T-resident memory lymphocytes. In view of the quite low mutation rate of the N protein, the present vaccine strategy has the potential to control the replication of all emerging variants.

How Do Anti-SARS-CoV-2 mRNA Vaccines Protect from Severe Disease?

COVID-19 pathogenesis develops in two phases. First, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 spreads within the epithelial cells of the mucosa of upper and, possibly, lower respiratory tracts. While the virus dissemination can be controlled by an emerging adaptive host immune response, if the virus diffuses to the pulmonary alveoli, a potentially lethal mechanism can arise in the second phase. It consists of an uncontrolled burst of cytokines/inflammatory factors (i.e., cytokine storm), leading to the insurgence of respiratory symptoms and, consequently, multi-organ failures. Messenger (m)RNA-based vaccines represent the most innovative approach in terms of prophylaxis against SARS-CoV-2-induced disease. The cumulating data indicate that the response to mRNA vaccines is basically ineffective to counteract the viral replication in the upper respiratory tracts, while showing efficacy in containing the development of severe disease. Considering that the antiviral immunity elicited by intramuscularly delivered mRNA vaccines is expected to show similar quantitative and qualitative features in upper and lower respiratory tracts, the different outcomes appear surprising and deserve accurate consideration. In this review, a still unexplored mechanism accounting for the mRNA vaccine effect against severe disease is proposed. Based on well-established experimental evidence, a possible inhibitory effect on alveolar macrophages as a consequence of the diffusion of the extracellular and/or cell-associated Spike protein can be envisioned as a key event counteracting the cytokine storm. This benefit, however, may be associated with defects in the immune functions of macrophages in other tissues whose possible consequences deserve careful evaluation.

Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein.

We propose an innovative anti-SARS-CoV-2 immune strategy based on extracellular vesicles (EVs) inducing an anti-SARS-CoV-2 N CD8+ T cytotoxic lymphocyte (CTL) immune response. We previously reported that the SARS-CoV-2 N protein can be uploaded at high levels in EVs upon fusion with Nefmut, i.e., a biologically inactive HIV-1 Nef mutant incorporating into EVs at quite high levels. Here, we analyze the immunogenic properties in human cells of EVs engineered with SARS-CoV-2 N fused at the C-terminus of either Nefmut or a deletion mutant of Nefmut referred to as NefmutPL. The analysis of in vitro-produced EVs has supported the uploading of N protein when fused with truncated Nefmut. Mice injected with DNA vectors expressed each fusion protein developed robust SARS-CoV-2 N-specific CD8+ T cell immune responses. When ex vivo human dendritic cells were challenged with EVs engineered with either fusion products, the induction of a robust N-specific CTL activity, as evaluated by both CD107a and trogocytosis assays, was observed. Through these data we achieved the proof-of-principle that engineered EVs can be instrumental to elicit anti-SARS-CoV-2 CTL immune response in human cells. This achievement represents a mandatory step towards the upcoming experimentations in pre-clinical models focused on intranasal administration of N-engineered EVs.

Biological and Immune Responses to Current Anti-SARS-CoV-2 mRNA Vaccines beyond Anti-Spike Antibody Production.

Several vaccine strategies are now available to fight the current SARS-CoV-2 pandemic. Those based on the administration of lipid-complexed messenger(m)RNA molecules represent the last frontiers in terms of technology innovation. mRNA molecules coding for the SARS-CoV-2 Spike protein are intramuscularly injected, thereby entering cells by virtue of their encapsulation into synthetic lipid nanovesicles. mRNA-targeted cells express the Spike protein on their plasma membrane in a way that it can be sensed by the immune system, which reacts generating anti-Spike antibodies. Although this class of vaccines appears as the most effective against SARS-CoV-2 infection and disease, their safety and efficiency are challenged by several factors included, but not limited to the following: emergence of viral variants, lack of adequate pharmacokinetics/pharmacodynamics studies, inability to protect oral mucosa from infection, and antibody waning. Emergence of viral variants can be a consequence of mass vaccination carried out in a pandemic time using suboptimal vaccines against an RNA virus. On the other hand, understanding the remainder flaws could be of some help in designing next generation anti-SARS-CoV-2 vaccines. In this commentary, issues regarding the fate of injected mRNA, the tissue distribution of the induced antiviral antibodies, and the generation of memory B cells are discussed. Careful evaluation of both experimental and clinical observations on these key aspects should be taken into account before planning booster administration, vaccination to non-at-risk population, and social restrictions.

Extracellular Vesicles and Their Use as Vehicles of Immunogens.

Healthy cells constitutively release lipid bilayered vesicles of different sizes and recognizing different biogenesis, collectively referred to as extracellular vesicles (EVs). EVs can be distinguished in exosomes and microvesicles. Biological and biomedical research on EVs is an emerging field that is rapidly growing. Many EV features including biogenesis, cell uptake, and functions still require unambiguous elucidation. Nevertheless, it has been well established that EVs are involved in communication among cells, tissues, and organs under both healthy and disease conditions by virtue of their ability to deliver macromolecules to target cells. Here, we summarize most recent findings regarding biogenesis, structure, and functions of both exosomes and microvesicles. In addition, the use of EVs as delivery tools to induce CD8+ T cell immunity is addressed compared to current designs exploiting enveloped viral vectors and virus-like particles. Finally, we describe a both safe and original approach conceived for the induction of strong CTL immunity against antigens uploaded in EVs constitutively released by muscle cells.

Strong SARS-CoV-2 N-Specific CD8+ T Immunity Induced by Engineered Extracellular Vesicles Associates with Protection from Lethal Infection in Mice.

SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with the Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses of 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut-based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy.

Virus-Induced CD8+ T-Cell Immunity and Its Exploitation to Contain the SARS-CoV-2 Pandemic.

The current battle against Severe Acute Respiratory Syndrome (SARS)-Coronavirus-2 benefits from the worldwide distribution of different vaccine formulations. All anti-SARS-CoV-2 vaccines in use are conceived to induce anti-Spike neutralizing antibodies. However, this strategy still has unresolved issues, the most relevant of which are: (i) the resistance to neutralizing antibodies of emerging SARS-CoV-2 variants and (ii) the waning of neutralizing antibodies. On the other hand, both pre-clinical evidence and clinical evidence support the idea that the immunity sustained by antigen-specific CD8+ T lymphocytes can complement and also surrogate the antiviral humoral immunity. As a distinctive feature, anti-SARS-CoV-2 CD8+ T-driven immunity maintains its efficacy even in the presence of viral protein mutations. In addition, on the basis of data obtained in survivors of the SARS-CoV epidemic, this immunity is expected to last for several years. In this review, both the mechanisms and role of CD8+ T-cell immunity in viral infections, particularly those induced by SARS-CoV and SARS-CoV-2, are analyzed. Moreover, a CD8+ T-cell-based vaccine platform relying on in vivo engineered extracellular vesicles is described. When applied to SARS-CoV-2, this strategy was proven to induce a strong immunogenicity, holding great promise for its translation into the clinic.

Long-Term Antitumor CD8+ T Cell Immunity Induced by Endogenously Engineered Extracellular Vesicles.

We developed an innovative method to induce antigen-specific CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This approach employs a DNA vector expressing a mutated HIV-1 Nef protein (Nefmut) deprived of the anti-cellular effects typical of the wild-type isoform, meanwhile showing an unusual efficiency of incorporation into EVs. This function persists even when foreign antigens are fused to its C-terminus. In this way, Nefmut traffics large amounts of antigens fused to it into EVs spontaneously released by the recipient cells. We previously provided evidence that mice injected with a DNA vector expressing the Nefmut/HPV16-E7 fusion protein developed an E7-specific CTL immune response as detected 2 weeks after the second immunization. Here, we extended and optimized the anti-HPV16 CD8+ T cell immune response induced by the endogenously engineered EVs, and evaluated the therapeutic antitumor efficacy over time. We found that the co-injection of DNA vectors expressing Nefmut fused with E6 and E7 generated a stronger anti-HPV16 immune response compared to that observed in mice injected with the single vectors. When HPV16-E6 and -E7 co-expressing tumor cells were implanted before immunization, all mice survived at day 44, whereas no mice injected with either void or Nefmut-expressing vectors survived until day 32 after tumor implantation. A substantial part of immunized mice (7 out of 12) cleared the tumor. When the cured mice were re-challenged with a second tumor cell implantation, none of them developed tumors. Both E6- and E7-specific CD8+ T immunities were still detectable at the end of the observation time. We concluded that the immunity elicited by engineered EVs, besides counteracting and curing already developed tumors, was strong enough to guarantee the resistance to additional tumor attacks. These results can be of relevance for the therapy of both metastatic and relapsing tumors.

The C-Terminal Domain of Nefmut Is Dispensable for the CD8+ T Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles.

Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nefmut fused at its C-terminus to viral and tumor antigens elicit a potent, effective, and anti-tolerogenic CD8+ T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nefmut-derivatives released by muscle cells. In the perspective of a possible translation into the clinic of the Nefmut-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nefmut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nefmut to associate with EVs. The EV-anchoring function was also preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. Most important, the Nefmut C-terminal deletion did not affect levels, quality, and diffusion at distal sites of the antigen-specific CD8+ T immunity. We concluded that the C-terminal Nefmut truncation does not influence stability, EV-anchoring, and CD8+ T cell immunogenicity of the fused antigen. Hence, the C-terminal deleted Nefmut may represent a safer alternative to the full-length isoform for vaccines in humans.

Simultaneous CD8+ T-Cell Immune Response against SARS-Cov-2 S, M, and N Induced by Endogenously Engineered Extracellular Vesicles in Both Spleen and Lungs.

Most advanced vaccines against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 are designed to induce antibodies against spike (S) protein. Differently, we developed an original strategy to induce CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This is a new vaccination approach based on intramuscular injection of DNA expression vectors coding for a biologically inactive HIV-1 Nef protein (Nefmut) with an unusually high efficiency of incorporation into EVs, even when foreign polypeptides are fused to its C-terminus. Nanovesicles containing Nefmut-fused antigens released by muscle cells can freely circulate into the body and are internalized by antigen-presenting cells. Therefore, EV-associated antigens can be cross-presented to prime antigen-specific CD8+ T-cells. To apply this technology to a strategy of anti-SARS-CoV-2 vaccine, we designed DNA vectors expressing the products of fusion between Nefmut and different viral antigens, namely N- and C-terminal moieties of S (referred to as S1 and S2), M, and N. We provided evidence that all fusion products are efficiently uploaded in EVs. When the respective DNA vectors were injected in mice, a strong antigen-specific CD8+ T cell immunity became detectable in spleens and, most important, in lung airways. Co-injection of DNA vectors expressing the diverse SARS-CoV-2 antigens resulted in additive immune responses in both spleen and lungs. Hence, DNA vectors expressing Nefmut-based fusion proteins can be proposed for new anti-SARS-CoV-2 vaccine strategies.

The conundrum of current anti-SARS-CoV-2 vaccines.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has given rise to the urgent need for vaccines and therapeutic interventions to address the spread of the SARS-CoV-2 virus. SARS-CoV-2 vaccines in development, and those being distributed currently, have been designed to induce neutralizing antibodies using the spike protein of the virus as an immunogen. However, the immunological correlates of protection against the virus remain unknown. This raises questions about the efficacy of current vaccination strategies. In addition, safety profiles of several vaccines seem inadequate or have not yet been evaluated under controlled experimentation. Here, evidence from the literature regarding the efforts already made to identify the immunological correlates of protection against SARS-CoV-2 infection are summarized. Furthermore, key biological features of most of the advanced vaccines and considerations regarding their safety and expected efficacy are highlighted.

Exploiting Manipulated Small Extracellular Vesicles to Subvert Immunosuppression at the Tumor Microenvironment through Mannose Receptor/CD206 Targeting.

Immunosuppression at tumor microenvironment (TME) is one of the major obstacles to be overcome for an effective therapeutic intervention against solid tumors. Tumor-associated macrophages (TAMs) comprise a sub-population that plays multiple pro-tumoral roles in tumor development including general immunosuppression, which can be identified in terms of high expression of mannose receptor (MR or CD206). Immunosuppressive TAMs, like other macrophage sub-populations, display functional plasticity that allows them to be re-programmed to inflammatory macrophages. In order to mitigate immunosuppression at the TME, several efforts are ongoing to effectively re-educate pro-tumoral TAMs. Extracellular vesicles (EVs), released by both normal and tumor cells types, are emerging as key mediators of the cell to cell communication and have been shown to have a role in the modulation of immune responses in the TME. Recent studies demonstrated the enrichment of high mannose glycans on the surface of small EVs (sEVs), a subtype of EVs of endosomal origin of 30-150 nm in diameter. This characteristic renders sEVs an ideal tool for the delivery of therapeutic molecules into MR/CD206-expressing TAMs. In this review, we report the most recent literature data highlighting the critical role of TAMs in tumor development, as well as the experimental evidences that has emerged from the biochemical characterization of sEV membranes. In addition, we propose an original way to target immunosuppressive TAMs at the TME by endogenously engineered sEVs for a new therapeutic approach against solid tumors.

Engineered Extracellular Vesicles/Exosomes as a New Tool against Neurodegenerative Diseases.

Neurodegenerative diseases are commonly generated by intracellular accumulation of misfolded/aggregated mutated proteins. These abnormal protein aggregates impair the functions of mitochondria and induce oxidative stress, thereby resulting in neuronal cell death. In turn, neuronal damage induces chronic inflammation and neurodegeneration. Thus, reducing/eliminating these abnormal protein aggregates is a priority for any anti-neurodegenerative therapeutic approach. Although several antibodies against mutated neuronal proteins have been already developed, how to efficiently deliver them inside the target cells remains an unmet issue. Extracellular vesicles/exosomes incorporating intrabodies against the pathogenic products would be a tool for innovative therapeutic approaches. In this review/perspective article, we identify and describe the major molecular targets associated with neurodegenerative diseases, as well as the antibodies already developed against them. Finally, we propose a novel targeting strategy based on the endogenous engineering of extracellular vesicles/exosomes constitutively released by cells of the central nervous system.

N-Terminal Fatty Acids of NEFMUT Are Required for the CD8+ T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles.

We recently described a cytotoxic CD8+ T lymphocyte (CTL) vaccine platform based on the intramuscular (i.m.) injection of DNA eukaryotic vectors expressing antigens of interest fused at the C-terminus of HIV-1 Nefmut, i.e., a functionally defective mutant that is incorporated at quite high levels into exosomes/extracellular vesicles (EVs). This system has been proven to elicit strong CTL immunity against a plethora of both viral and tumor antigens, as well as inhibit both transplantable and orthotopic tumors in mice. However, a number of open issues remain regarding the underlying mechanism. Here we provide evidence that hindering the uploading into EVs of Nefmut-derived products by removing the Nefmut N-terminal fatty acids leads to a dramatic reduction of the downstream antigen-specific CD8+ T-cell activation after i.m. injection of DNA vectors in mice. This result formally demonstrates that the generation of engineered EVs is part of the mechanism underlying the in vivo induced CD8+ T-cell immunogenicity. Gaining new insights on the EV-based vaccine platform can be relevant in view of its possible translation into the clinic to counteract both chronic and acute infections as well as tumors.

Extracellular vesicle-mediated intercellular communication in HIV-1 infection and its role in the reservoir maintenance.

HIV-1 infection is efficiently controlled by combination anti-retroviral therapy (cART). However, despite preventing disease progression, cART does not eradicate virus infection which persists in a latent form for an individual's lifetime. The latent reservoir comprises memory CD4+ T lymphocytes, macrophages, and dendritic cells; however, for the most part, the reservoir is generated by virus entry into activated CD4+ T lymphocytes committed to return to a resting state, even though resting CD4+ T lymphocytes can be latently infected as well. The HIV-1 reservoir is not recognized by the immune system, is quite stable, and has the potential to re-seed systemic viremia upon cART interruption. Viral rebound can occur even after a long period of cART interruption. This event is most likely a consequence of the extended half-life of the HIV-1 reservoir, the maintenance of which is not clearly understood. Several recent studies have identified extracellular vesicles (EVs) as a driving force contributing to HIV-1 reservoir preservation. In this review, we discuss recent findings in the field of EV/HIV-1 interplay, and then propose a mechanism through which EVs may contribute to HIV-1 persistence despite cART. Understanding the basis of the HIV-1 reservoir maintenance continues to be a matter of great relevance in view of the limitations of current strategies aimed at HIV-1 eradication.

The Intracellular Delivery Of Anti-HPV16 E7 scFvs Through Engineered Extracellular Vesicles Inhibits The Proliferation Of HPV-Infected Cells.

PURPOSE: Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nefmut. It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation. METHODS: The Nefmut gene was fused in frame at its 3'-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nefmut-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nefmut/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nefmut/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay. RESULTS: The Nefmut/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nefmut/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nefmut/anti-HPV16-E7 scFv. CONCLUSION: Our data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.

Development of a novel human phage display-derived anti-LAG3 scFv antibody targeting CD8+ T lymphocyte exhaustion.

BACKGROUND: Lymphocyte-activation gene (LAG)3 is a 498 aa transmembrane type I protein acting as an immune inhibitory receptor. It is expressed on activated lymphocytes, natural killer cells and plasmacytoid dendritic cells. In activated lymphocytes, LAG3 expression is involved in negative control of cell activation/proliferation to ensure modulation and control of immune responses. In view of its deregulated expression in tumor-infiltrating lymphocytes, LAG3, together with the additional immune checkpoint inhibitors CTLA4 and PD1, is considered a major target in order to reverse the immunosuppression typically mounting in oncologic diseases. Since many patients still fail to respond to current immune checkpoints-based therapies, the identification of new effective immune inhibitors is a priority in the ongoing fight against cancer. RESULTS: We identified a novel human single-chain variable fragment (scFv) Ab against a conformational epitope of LAG3 by in vitro phage display technology using the recombinant antigen as a bait. This scFv (referred to as F7) was characterized in terms of binding specificity to both recombinant antigen and human LAG3-expressing cells. It was then rebuilt into an IgG format pre-optimized for clinical usage, and the resulting bivalent construct was shown to preserve its ability to bind LAG3 on human cells. Next, we analyzed the activity of the anti-LAG3 scFvF7 using two different antigen-specific CD8+ T lymphocyte clones as target cells. We proved that the reconstituted anti-LAG3 F7 Ab efficiently binds the cell membrane of both cell clones after peptide-activation. Still more significantly, we observed a striking increase in the peptide-dependent cell activation upon Ab treatment as measured in terms of IFN-γ release by both ELISA and ELISPOT assays. CONCLUSIONS: Overall, the biotechnological strategy described herein represents a guiding development model for the search of novel useful immune checkpoint inhibitors. In addition, our functional data propose a novel candidate reagent for consideration as a cancer treatment.

Tumor cells endowed with professional antigen-presenting cell functions prime PBLs to generate antitumor CTLs.

Intrinsic genetic instability of tumor cells leads to continuous production of mutated proteins referred to as tumor-specific neoantigens. Generally, they are recognized as nonself products by the host immune system. However, an effective adaptive response clearing neoantigen-expressing cells is lost in tumor diseases. Most advanced therapeutic strategies aim at inducing neoantigen-specific immune activation through personalized approaches. They include tumor cell exome sequencing, human leukocyte antigen (HLA) typing, synthesis, and injection of peptides/RNA with adjuvants. Here, we propose an innovative method to induce a CD8+ T cytotoxic lymphocyte (CTL) immune response against tumor neoantigens bypassing the steps needed in current therapeutic strategies of personalized vaccination. We assumed that tumor cells can be the most efficient and precise factory of major histocompatibility complex (MHC) class I-associated, tumor neoantigen-derived peptides. Hence, endowing tumor cells with professional antigen-presenting functions would prime CD8+ T lymphocytes towards a response against nonself tumor antigens. To explore this possibility, both adenocarcinoma and melanoma human cells were engineered to express both CD80 and CD86 costimulatory molecules. HLA-matched lymphocytes were then primed through cocultivation with the engineered tumor cells. The generation of tumor-specific CD8+ T lymphocytes was tested through the combined analysis of cell activation markers, formation of immunologic synapses, generation of tumor antigen-specific CD8+ T lymphocytes, and cytotoxic activity. Our data consistently indicate that tumor cells endowed with professional antigen-presenting functions can generate an effective tumor-specific CTL immune response. This finding may open avenues towards the development of innovative antitumor immunotherapies. KEY MESSAGES: We established a novel method to induce antitumor CTLs without a need to identify TAAs and/or tumor neoantigens. This strategy relies on transducing tumor cells with a retroviral vector expressing both CD80 and CD86. In this way, tumor cells prime naïve CD8+ T lymphocytes in a way that CTLs killing the same tumor cells are generated. These findings open the way towards preclinical assays in the perspective to introduce this antitumor immunotherapy strategy in clinic.