Search

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in the treatment of hematologic malignancies. Unfortunately, it has limited efficacy against solid tumors, even when the targeted antigens are well expressed. A better understanding of the underlying mechanisms of CAR T cell therapy resistance in solid tumors is necessary to develop strategies to improve efficacy. Here we report that solid tumors release small extracellular vesicles (sEVs) that carry both targeted tumor antigens and the immune checkpoint protein PD-L1. These sEVs acted as cell-free functional units to preferentially interact with cognate CAR T cells and efficiently inhibited their proliferation, migration, and function. In syngeneic mouse tumor models, blocking tumor sEV secretion not only boosted the infiltration and anti-tumor activity of CAR T cells but also improved endogenous anti-tumor immunity. These results suggest that solid tumors use sEVs as an active defense mechanism to resist CAR T cells and implicate tumor sEVs as a potential therapeutic target to optimize CAR T cell therapy against solid tumors.

A fundamental limitation of T cell therapies in solid tumors is loss of inflammatory effector functions, such as cytokine production and proliferation. Here, we target a regulatory axis of T cell inflammatory responses, Regnase-1 and Roquin-1, to enhance antitumor responses in human T cells engineered with two clinical-stage immune receptors. Building on previous observations of Regnase-1 or Roquin-1 knockout in murine T cells or in human T cells for hematological malignancy models, we found that knockout of either Regnase-1 or Roquin-1 alone enhances antitumor function in solid tumor models, but that knockout of both Regnase-1 and Roquin-1 increases function further than knockout of either regulator alone. Double knockout of Regnase-1 and Roquin-1 increased resting T cell inflammatory activity and led to at least an order of magnitude greater T cell expansion and accumulation in xenograft mouse models, increased cytokine activity, and persistence. However double knockout of Regnase-1 and Roguin-1 also led to a lymphoproliferative syndrome and toxicity in some mice. These results suggest that regulators of immune inflammatory functions may be interesting targets to modulate to improve antitumor responses.

Viral engineered chimeric antigen receptor (CAR) T cell therapies are potent, targeted cancer immunotherapies, but their permanent CAR expression can lead to severe adverse effects. Nonviral messenger RNA (mRNA) CAR T cells are being explored to overcome these drawbacks, but electroporation, the most common T cell transfection method, is limited by cytotoxicity. As a potentially safer nonviral delivery strategy, here, sequential libraries of ionizable lipid nanoparticle (LNP) formulations with varied excipient compositions were screened in comparison to a standard formulation for improved mRNA delivery to T cells with low cytotoxicity, revealing B10 as the top formulation with a 3-fold increase in mRNA delivery. When compared to electroporation in primary human T cells, B10 LNPs induced comparable CAR expression with reduced cytotoxicity while demonstrating potent cancer cell killing. These results demonstrate the impact of excipient optimization on LNP performance and support B10 LNPs as a potent mRNA delivery platform for T cell engineering.

Cancer is becoming increasingly understood not only as a disease of pathological cells but also as one of immune hypofunction. The heterogenous and patient-specific nature of cancer further underscores the need for personalized cellular therapies, which are currently produced ex vivo. Gene-modulating approaches, such as therapeutic RNAs and improved viral vectors, now bring us closer toward strategies for mitigating disease, particularly for diseases that benefit from altering gene or transgene expression profiles in pathological or therapeutic immune cells. An advancing toolbox of technologies and trends toward simplifying personalized therapies foreshadow opportunities for direct, in vivo precision medicine against cancer.

Genetically engineered T cell immunotherapies have provided remarkable clinical success to treat B cell acute lymphoblastic leukaemia by harnessing a patient's own T cells to kill cancer, and these approaches have the potential to provide therapeutic benefit for numerous other cancers, infectious diseases and autoimmunity. By introduction of either a transgenic T cell receptor or a chimeric antigen receptor, T cells can be programmed to target cancer cells. However, initial studies have made it clear that the field will need to implement more complex levels of genetic regulation of engineered T cells to ensure both safety and efficacy. Here, we review the principles by which our knowledge of genetics and genome engineering will drive the next generation of adoptive T cell therapies.

T-cell-based immunotherapies hold promise for the treatment of many types of cancer, with three approved products for B-cell malignancies and a large pipeline of treatments in clinical trials. However, there are several challenges to their broad implementation. These include insufficient expansion of adoptively transferred T cells, inefficient trafficking of T cells into solid tumours, decreased T-cell activity due to a hostile tumour microenvironment and the loss of target antigen expression. Together, these factors restrict the number of therapeutically active T cells engaging with tumours. Nanomaterials are uniquely suited to overcome these challenges, as they can be rationally designed to enhance T-cell expansion, navigate complex physical barriers and modulate tumour microenvironments. Here, we present an overview of nanomaterials that have been used to overcome clinical barriers to T-cell-based immunotherapies and provide our outlook of this emerging field at the interface of cancer immunotherapy and nanomaterial design. This Review discusses the progress and the remaining challenges for the clinical application of nanomaterial-based T-cell immunotherapies.

T-cell-based immunotherapies hold promise for the treatment of many types of cancer, with three approved products for B-cell malignancies and a large pipeline of treatments in clinical trials. However, there are several challenges to their broad implementation. These include insufficient expansion of adoptively transferred T cells, inefficient trafficking of T cells into solid tumours, decreased T-cell activity due to a hostile tumour microenvironment and the loss of target antigen expression. Together, these factors restrict the number of therapeutically active T cells engaging with tumours. Nanomaterials are uniquely suited to overcome these challenges, as they can be rationally designed to enhance T-cell expansion, navigate complex physical barriers and modulate tumour microenvironments. Here, we present an overview of nanomaterials that have been used to overcome clinical barriers to T-cell-based immunotherapies and provide our outlook of this emerging field at the interface of cancer immunotherapy and nanomaterial design.

Engineered immune cells as therapies is a rapidly growing field that can successfully address complex and refractory disease in ways traditional molecular therapeutics cannot. Currently, engineering these cells entails viral-mediated genomic integration of desired genes or electroporation-based delivery of gene-editing machinery, notably the Clustered regularly interspaced short palindromic repeats - CRISPR-associated (CRISPR-Cas9) system. Recent advancements in nonviral delivery methods and improved Cas-based gene editors are providing new tools that may enable the production of consistent and precisely defined therapeutic immune cells. Combined with ideas borrowed from synthetic biology, these engineering advances are poised to help create the next generation of immune cell therapies with even greater precision and functionality to address complex diseases.

Protection against mucosally transmitted infections probably requires immunity at the site of pathogen entry(1), yet there are no mucosal adjuvant formulations licensed for human use. Polyethyleneimine (PEI) represents a family of organic polycations used as nucleic acid transfection reagents in vitro and DNA vaccine delivery vehicles in vivo(2,3). Here we show that diverse PEI forms have potent mucosal adjuvant activity for viral subunit glycoprotein antigens. A single intranasal administration of influenza hemagglutinin or herpes simplex virus type-2 (HSV-2) glycoprotein D with PEI elicited robust antibody-mediated protection from an otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen, which were taken up by antigen-presenting cells in vitro and in vivo, promoted dendritic cell trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host double-stranded DNA that triggered Irf3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use.

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-197CN54), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-197CN54Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.

Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly related Env surface (gp120) antigens produced in different systems: (a) mammalian (293 FreeStyle™ cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), which confer mainly paucimannosidic glycans; (c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic™), which impart high-mannose, hybrid and complex glycans without sialic acid; and (d) 293F cells, which impart high-mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4 binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic-acid-containing gp120 was significantly more immunogenic than the sialylated version when administered in two different adjuvants, and induced higher titers of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic-acid-imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations that should be considered when selecting expression systems for glycosylated antigens to be used for structure-function studies and for vaccine use. © 2010 Elsevier Ltd.

The new intradermal DNA delivery technique, termed DNA tattooing might overcome the discrepancy between the encouraging immunogenicity results obtained with DNA vaccines in murine studies and the poor results obtained in non-human primates and humans, the so called "simian barrier". Here, we demonstrate a 10- to 100-fold increase in the magnitude of vaccine specific T-cell responses in peripheral blood from DNA tattooed rhesus macaques, as compared to T-cell responses in animals immunized via intramuscular (IM) route. A marked increase in the magnitude of the antigen specific T-cell responses as well as an increase in the number of animals responding to the immunogens was observed. These findings in non-human primates suggest that similar results may be observed in humans. Clinical trials are planned to validate tattooing as an optimal method of DNA vaccine delivery in humans. © 2008 Elsevier Ltd. All rights reserved.

We evaluated 52 different E. coli expressed pneumococcal proteins as immunogens in a BALB/c mouse model of S. pneumoniae lung infection. Proteins were selected based on genetic conservation across disease-causing serotypes and bioinformatic prediction of antibody binding to the target antigen. Seven proteins induced protective responses, in terms of reduced lung burdens of the serotype 3 pneumococci. Three of the protective proteins were histidine triad protein family members (PhtB, PhtD and PhtE). Four other proteins, all bearing LPXTG linkage domains, also had activity in this model (PrtA, NanA, PavB and Eng). PrtA, NanA and Eng were also protective in a CBA/N mouse model of lethal pneumococcal infection. Despite data inferring widespread genomic conservation, flow-cytometer based antisera binding studies confirmed variable levels of antigen expression across a panel of pneumococcal serotypes. Finally, BALB/c mice were immunized and intranasally challenged with a viulent serotype 8 strain, to help understand the breadth of protection. Those mouse studies reaffirmed the effectiveness of the histidine triad protein grouping and a single LPXTG protein, PrtA.

The genetic diversity of HIV-1 represents a major challenge in vaccine development. In this study, we establish a rationale for eliminating HIV-1–infected cells by targeting cellular immune responses against stable human endogenous retroviral (HERV) antigens. HERV DNA sequences in the human genome represent the remnants of ancient infectious retroviruses. We show that the infection of CD4+ T cells with HIV-1 resulted in transcription of the HML-2 lineage of HERV type K [HERV-K(HML-2)] and the expression of Gag and Env proteins. HERV-K(HML-2)–specific CD8+ T cells obtained from HIV-1–infected human subjects responded to HIV-1–infected cells in a Vif-dependent manner in vitro. Consistent with the proposed mode of action, a HERV-K(HML-2)–specific CD8+ T cell clone exhibited comprehensive elimination of cells infected with a panel of globally diverse HIV-1, HIV-2, and SIV isolates in vitro. We identified a second T cell response that exhibited cross-reactivity between homologous HIV-1-Pol and HERV-K(HML-2)-Pol determinants, raising the possibility that homology between HIV-1 and HERVs plays a role in shaping, and perhaps enhancing, the T cell response to HIV-1. This justifies the consideration of HERV-K(HML-2)–specific and cross-reactive T cell responses in the natural control of HIV-1 infection and for exploring HERV-K(HML-2)–targeted HIV-1 vaccines and immunotherapeutics.

The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.

T-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific responses were detected. At these high peptide concentrations, we detected false-positive responses, three of which were mapped to an HIV-1 Gag peptide contaminant. Thus, HERV-K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected by 15mer peptide mapping.

Env-specific IgG and IgM were detected in 25 and 60%, respectively, of volunteers immunized with NYVAC expressing clade C gpl20. The serum sample with the highest IgM titre but undetectable IgG neutralized the homologous isolate with a reciprocal IC 90 titre of 7.8 in the absence of complement, and 24.4 in the presence of complement (P= 0.0003). These results suggest that vaccine-induced, Env-specific IgM may have antiviral activity and should be subjected to further investigation.

Polyethyleneimine (PEI) is an organic polycation used extensively as a gene and DNA vaccine delivery reagent. Although the DNA targeting activity of PEI is well documented, its immune activating activity is not. We recently reported that PEI has robust mucosal adjuvanticity when administered intranasally with glycoprotein antigens. Here, we show that PEI has strong immune activating activity after systemic delivery. PEI administered subcutaneously with viral glycoprotein (HIV-1 gp140) enhanced antigen-specific serum IgG production in the context of mixed Th1/Th2-type immunity. PEI elicited higher titers of both antigen binding and neutralizing antibodies than alum in mice and rabbits and induced an increased proportion of antibodies reactive with native antigen. In an intraperitoneal model, PEI recruited neutrophils followed by monocytes to the site of administration and enhanced antigen uptake by antigen-presenting cells. The Th bias was modulated by PEI activation of the Nlrp3 inflammasome; however its global adjuvanticity was unchanged in Nlrp3-deficient mice. When coformulated with CpG oligodeoxynucleotides, PEI adjuvant potency was synergistically increased and biased toward a Th1-type immune profile. Taken together, these data support the use of PEI as a versatile systemic adjuvant platform with particular utility for induction of secondary structure-reactive antibodies against glycoprotein antigens.

The enormous sequence diversity of HIV remains a major roadblock to the development of a prophylactic vaccine and new approaches to induce protective immunity are needed. Endogenous retrotransposable elements (ERE) such as endogenous retrovirus K (ERV)-K and long interspersed nuclear element-1 (LINE-1) are activated during HIV-1-infection and could represent stable, surrogate targets to eliminate HIV-1-infected cells. Here, we explored the hypothesis that vaccination against ERE would protect macaques from acquisition and replication of simian immunodeficiency virus (SIV). Following vaccination with antigens derived from LINE-1 and ERV-K consensus sequences, animals mounted immune responses that failed to delay acquisition of SIVsmE660. We observed no differences in acute or set point viral loads between ERE-vaccinated and control animals suggesting that ERE-specific responses were not protective. Indeed, ERE-specific T cells failed to expand anamnestically in vivo following infection with SIVsmE660 and did not recognize SIV-infected targets in vitro, in agreement with no significant induction of targeted ERE mRNA by SIV in macaque CD4+ T cells. Instead, lower infection rates and viral loads correlated significantly to protective TRIM5α alleles. Cumulatively, these data demonstrate that vaccination against the selected ERE consensus sequences in macaques did not lead to immune-mediated recognition and killing of SIV-infected cells, as has been shown for HIV-infected human cells using patient-derived HERV-K-specific T cells. Thus, further research is required to identify the specific nonhuman primate EREs and retroviruses that recapitulate the activity of HIV-1 in human cells. These results also highlight the complexity in translating observations of the interplay between HIV-1 and human EREs to animal models.

Rhesus macaques ( Macaca mulatta ) have played a valuable role in the development of human immunodeficiency virus (HIV) vaccine candidates prior to human clinical trials. However, changes and/or improvements in immunogen quality in the good manufacturing practice (GMP) process or changes in adjuvants, schedule, route, dose, or readouts have compromised the direct comparison of T-cell responses between species. Here we report a comparative study in which T-cell responses from humans and macaques to HIV type 1 antigens (Gag, Pol, Nef, and Env) were induced by the same vaccine batches prepared under GMP and administered according to the same schedules in the absence and presence of priming. Priming with DNA (humans and macaques) or alphavirus (macaques) and boosting with NYVAC induced robust and broad antigen-specific responses, with highly similar Env-specific gamma interferon (IFN-γ) enzyme-linked immunospot assay responses in rhesus monkeys and human volunteers. Persistent cytokine responses of antigen-specific CD4 + and CD8 + T cells of the central memory as well as the effector memory phenotype, capable of simultaneously eliciting multiple cytokines (IFN-γ, interleukin 2, and tumor necrosis factor alpha), were induced. Responses were highly similar in humans and primates, confirming earlier data indicating that priming is essential for inducing robust NYVAC-boosted IFN-γ T-cell responses. While significant similarities were observed in Env-specific responses in both species, differences were also observed with respect to responses to other HIV antigens. Future studies with other vaccines using identical lots, immunization schedules, and readouts will establish a broader data set of species similarities and differences with which increased confidence in predicting human responses may be achieved.

© 2008 2008 Elsevier Ltd. All rights reserved. The best hope for curbing the HIV-1 pandemic lies in the development of a prophylactic vaccine, a challenge that has eluded vaccinologists since the discovery of HIV-1 in 1983. HIV-1 is genetically highly diverse both within infected individuals and globally. Strains vary substantially, especially in the envelope glycoproteins (Env), the only targets for virus-neutralizing antibodies. Antigenic variation, combined with multiple other immune evasion strategies, enable the virus to evade neutralizing antibodies in naturally infected hosts or in recipients of the vaccine candidates tested thus far. In vivo control of viremia appears to rely on CD8 cytotoxic T-lymphocyte (CTL) responses and such responses are frequently, but not universally, detected in highly exposed seronegative persons. Novel vaccine strategies that induce CTL responses in macaque models challenged with simian immunodeficiency virus (SIV) or SIV recombinant for HIV-1 Env (termed simian-human immunodeficiency virus) do not prevent infection but allow the control of viremia and prevention of disease progression. Conversely, infusion of broadly neutralizing monoclonal antibodies does prevent infection in macaques. An ideal vaccine strategy would probably induce both robust CTL responses and broadly neutralizing antibodies.

NYVAC-C (vP2010), a recombinant vector expressing HIV subtype C gag, pol, env and nef antigens, was tested in a phase I study in healthy, HIV negative volunteers in London and Lausanne. Twenty-four participants were randomised to receive NYVAC-C (20) or matching placebo (4) at weeks 0 and 4, and assessed for safety and immunogenicity over 48 weeks. There were no serious adverse events, and no clinical or laboratory abnormalities or other events that led to withdrawal, interruption or dose reduction of the NYVAC-C/placebo. Half of the 10 assessed responded in the ELISpot assay under stringent criteria, which informed the sample size for a DNA-NYVAC-C comparison to NYVAC-C alone.

More than 20 years have passed since the human immunodeficiency virus (HIV)-1 was identified as the cause of AIDS (acquired immune deficiency syndrome). With rapid early progress, the development of a vaccine was predicted within 2–10 years. However, over two decades later, we have seen only a single vaccine candidate complete Phase III clinical efficacy trials. These trials showed the vaccine was not able to protect the trial volunteers from HIV infection or subsequently modify the early clinical progression. It is now accepted that the initial optimism in the field was misguided, as the complexity of the problem stretched beyond the known horizons of vaccinology, immunology and retrovirology. In the intervening 20 years, unprecedented research efforts have pushed the cutting edge of these fields forward to such a degree that we are better able to put the problem of developing a vaccine for HIV-1 into context. Now it is time to examine the prospects for HIV-1 vaccines and ascertain whether real progress is being made.