Pseudotyped Lentiviral Vectors: One Vector, Many Guises

Viruses have evolved specialized molecular mechanisms to transfer their genome efficiently into host cells. Viruses can be repurposed into viral vectors to achieve controlled gene transfer to desired cells. One of the most popular class of vectors, lentiviral vectors, transduce mammalian cells efficiently. Lentiviral vectors are pseudotyped with various heterologous viral envelopes to alter their tropism. While the most common example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), many other viral proteins have also been used. Pseudotyping lentiviral vectors with a diverse set of naturally occurring or engineered viral envelopes has allowed targeted infection of specific cell types. Many exciting studies are further uncovering new specificities and shortcomings of pseudotyped lentiviral vectors. These studies will expand the toolbox to make lentiviral vectors that cater to the specific requirements of transduction. In this review, we provide a comprehensive overview of various viral envelope pseudotypes used with LVs, their specificity, advantages, and drawbacks

Personalized T cell-mediated cancer immunotherapy: progress and challenges

Immunotherapies are yielding effective treatments for several previously untreatable cancers. Until recently, vaccines and adoptive cell therapies have been designed to target public tumor antigens common to multiple patients rather than private antigens specific to a single patient. Due to the difficulty of identifying public antigens that are expressed exclusively on tumor cells, these studies have yielded both clinical successes and serious immune-related adverse events. Multiple avenues of research now underscore the centrality of tumor-specific mutated private antigens to endogenous anti-tumor immunity. Immunotherapies that target these neoantigens may enable safer and more durable tumor regression, but personalized targeting presents a number of challenges. Foremost among these is to develop processes that accelerate advancement from neoantigen discovery to use of these neoantigens as vaccines or as targets for adoptive cell therapies. Exome sequencing has facilitated discovery of neoantigens for melanoma and other highly mutated cancers. New technologies – possibly proceeding from T cell receptor repertoire sequencing – are needed to identify antigens for cancers with low mutational burden and few neoantigens. In this review, we discuss progress toward personalizing T cell-mediated immunotherapy for cancer as well as challenges going forward

Pseudotyped Lentiviral Vectors: One Vector, Many Guises

Viruses have evolved specialized molecular mechanisms to transfer their genome efficiently into host cells. Viruses can be repurposed into viral vectors to achieve controlled gene transfer to desired cells. One of the most popular class of vectors, lentiviral vectors, transduce mammalian cells efficiently. Lentiviral vectors are pseudotyped with various heterologous viral envelopes to alter their tropism. While the most common example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), many other viral proteins have also been used. Pseudotyping lentiviral vectors with a diverse set of naturally occurring or engineered viral envelopes has allowed targeted infection of specific cell types. Many exciting studies are further uncovering new specificities and shortcomings of pseudotyped lentiviral vectors. These studies will expand the toolbox to make lentiviral vectors that cater to the specific requirements of transduction. In this review, we provide a comprehensive overview of various viral envelope pseudotypes used with LVs, their specificity, advantages, and drawbacks

T Cell Receptor Immunotherapy Drives Human Immunodeficiency Virus Evolution in Humanized Mice

Effective CD8+ T cell responses targeted to the KK10 epitope of HIV presented by HLA-B*27:05, a protective HLA allele, correlate with the ability to control infection without antiretroviral therapy (ART). Here, we report an immunotherapy approach using two B*27:05-KK10-specific T Cell Receptors (TCRs) isolated from HIV controllers. Immunocompromised mice engrafted with human Hematopoietic Stem/Progenitor Cells (HSPCs) encoding for the TCRs showed differentiation into functionally active engineered T cells. Following infection with HIV, both TCRs showed sustained, albeit modest, viral suppression over 32 weeks, accompanied by a concomitant increase in CD4+ T cells. Sequencing of viral quasi-species from the plasma of infected mice demonstrated clear evidence for viral evolution under selection pressure from the TCRs. The most commonly observed mutation in the KK10 epitope was L6M, which preserved viral fitness but showed attenuated recognition by the TCRs. These studies show that TCR-immunotherapy was able to suppress HIV infection long-term while driving HIV evolution in humanized mice

T Cell Receptor Immunotherapy Drives Human Immunodeficiency Virus Evolution in Humanized Mice

Effective CD8+ T cell responses targeted to the KK10 epitope of HIV presented by HLA-B*27:05, a protective HLA allele, correlate with the ability to control infection without antiretroviral therapy (ART). Here, we report an immunotherapy approach using two B*27:05-KK10-specific T Cell Receptors (TCRs) isolated from HIV controllers. Immunocompromised mice engrafted with human Hematopoietic Stem/Progenitor Cells (HSPCs) encoding for the TCRs showed differentiation into functionally active engineered T cells. Following infection with HIV, both TCRs showed sustained, albeit modest, viral suppression over 32 weeks, accompanied by a concomitant increase in CD4+ T cells. Sequencing of viral quasi-species from the plasma of infected mice demonstrated clear evidence for viral evolution under selection pressure from the TCRs. The most commonly observed mutation in the KK10 epitope was L6M, which preserved viral fitness but showed attenuated recognition by the TCRs. These studies show that TCR-immunotherapy was able to suppress HIV infection long-term while driving HIV evolution in humanized mice

T cell antigen discovery via signaling and antigen-presenting bifunctional receptors

CD8^+ T cells recognize and eliminate tumors in an antigen-specific manner. Despite progress in characterizing the antitumor T cell repertoire and function, the identification of target antigens remains a challenge. Here we describe the use of chimeric receptors called signaling and antigen-presenting bifunctional receptors (SABRs) in a cell-based platform for T cell receptor (TCR) antigen discovery. SABRs present an extracellular complex comprising a peptide and major histocompatibility complex (MHC), and induce intracellular signaling via a TCR-like signal after binding with a cognate TCR. We devised a strategy for antigen discovery using SABR libraries to screen thousands of antigenic epitopes. We validated this platform by identifying the targets recognized by public TCRs of known specificities. Moreover, we extended this approach for personalized neoantigen discovery

T cell receptors for the HIV KK10 epitope from patients with differential immunologic control are functionally indistinguishable

HIV controllers (HCs) are individuals who can naturally control HIV infection, partially due to potent HIV-specific CD8+ T cell responses. Here, we examined the hypothesis that superior function of CD8+ T cells from HCs is encoded by their T cell receptors (TCRs). We compared the functional properties of immunodominant HIV-specific TCRs obtained from HLA-B*2705 HCs and chronic progressors (CPs) following expression in primary T cells. T cells transduced with TCRs from HCs and CPs showed equivalent induction of epitope-specific cytotoxicity, cytokine secretion, and antigen-binding properties. Transduced T cells comparably, albeit modestly, also suppressed HIV infection in vitro and in humanized mice. We also performed extensive molecular dynamics simulations that provided a structural basis for similarities in cytotoxicity and epitope cross-reactivity. These results demonstrate that the differential abilities of HIV-specific CD8+ T cells from HCs and CPs are not genetically encoded in the TCRs alone and must depend on additional factors

T cell antigen discovery via trogocytosis

T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide–major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide–MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy

T cell receptors for the HIV KK10 epitope from patients with differential immunologic control are functionally indistinguishable

HIV controllers (HCs) are individuals who can naturally control HIV infection, partially due to potent HIV-specific CD8+ T cell responses. Here, we examined the hypothesis that superior function of CD8+ T cells from HCs is encoded by their T cell receptors (TCRs). We compared the functional properties of immunodominant HIV-specific TCRs obtained from HLA-B*2705 HCs and chronic progressors (CPs) following expression in primary T cells. T cells transduced with TCRs from HCs and CPs showed equivalent induction of epitope-specific cytotoxicity, cytokine secretion, and antigen-binding properties. Transduced T cells comparably, albeit modestly, also suppressed HIV infection in vitro and in humanized mice. We also performed extensive molecular dynamics simulations that provided a structural basis for similarities in cytotoxicity and epitope cross-reactivity. These results demonstrate that the differential abilities of HIV-specific CD8+ T cells from HCs and CPs are not genetically encoded in the TCRs alone and must depend on additional factors

T cell antigen discovery via trogocytosis

T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide–major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide–MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy