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CFSE proliferation assay to measure the immune response of allogeneic T cells towards the modified NK cells. (A) Representative flow plots showing the frequency of CFSElow CD8⁺ (upper panel) and CD4⁺ (lower panel) T cells after 6 days of co-incubation with NK cells carrying the modifications depicted above the plots. (B) Quantification of activated CFSElow CD8⁺ (left) and CD4⁺ (right) T cells after co-incubation with the modified NK cells. Statistical analysis was performed using one-way ANOVA with Holm-Sidak testing for multiple comparisons. The higher frequency of CFSElow CD8⁺ T cells was statistically significant compared to sc-HLA-E and sc-HLA-E/B2M-KO NK cells (Box plots including median, quartiles and all data points, n = 7).
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Cellular immunotherapy using chimeric antigen receptors (CARs) so far has almost exclusively used autologous peripheral blood-derived T cells as immune effector cells. However, harvesting sufficient numbers of T cells is often challenging in heavily pre-treated patients with malignancies and perturbed hematopoiesis and perturbed hematopoiesis. Also...
Citations
... 33,40 In addition, allogeneic NK cells will be subject to recognition and rejection by the host T cells, thereby limiting their lifespan in vivo and resulting in reduced clinical activity. 41,42 Recently, many strategies have been used to prevent rejection of the allogeneic NK by the host immune cells. For example, knocking out expression of HLA class I molecules on NK cells via gene editing has been shown to prevent immune recognition by mismatch T cells. ...
... For example, knocking out expression of HLA class I molecules on NK cells via gene editing has been shown to prevent immune recognition by mismatch T cells. 42 In contrast, high dose and repeated infusion of CAR NK cells might be necessary to enhance the antitumor immune response. Our study showed that 2 doses of CD70-CAR NK cells successfully eradicated CD19 − and CD19 + mixed B-cell tumors, and induced a long-term survival, consistent with results from 2 recent studies that used a second-generation CD33-CAR bearing 4-1BB, or CD123-CAR with 2B4 costimulatory domain construct, respectively. ...
Chimeric antigen receptor (CAR)-NK cells can eliminate tumors not only through the ability of the CAR molecule to recognize antigen expressed cancer cells but also through NK cell receptors themselves. This overcomes some of the limitations of CAR-T cells, paving CAR-NK cells for safer and more effective off-the-shelf cellular therapy. In this study, CD70, a pan-target of lymphoma, specific fourth-generation CAR with 4-1BB co-stimulatory domain and IL-15 was constructed and transduced into cord blood-derived NK cells by Baboon envelope pseudotyped lenti-vector. CD70-CAR NK cells displayed superior cytotoxic activity in vitro and in vivo against CD19 negative B-cell lymphoma when compared to non-transduced NK cells and CD19-specific CAR-NK cells. Importantly, mice received two doses of CD70-CAR NK cells showed effective eradication of tumors, accompanied by increased concentration of plasma IL-15 and enhanced CAR-NK cell proliferation and persistence. Our study suggests that repetitive administration-based CAR NK-cell therapy has clinical advantage compared to single dose of CAR-NK cells for the treatment of B-cell lymphoma.
... To counteract this "host-versus-graft effect", the surface expression of HLA class I molecules on donor NK cells was abolished by beta 2-microglobulin KO to prevent killing by recipient alloreactive CD8 + T cells. Because loss of HLA class I molecules induces killing by NK cells ('missing self'-induced lysis), a single-chain HLA-E molecule was also introduced to inhibit receiver NK cells expressing CD94/NKG2A or CD94/NKG2B [158]. ...
Natural killer (NK) cell-based immunotherapies are attracting increasing interest in the field of cancer treatment. Early clinical trials have shown promising outcomes, alongside satisfactory product efficacy and safety. Recent developments have greatly increased the therapeutic potential of NK cells by endowing them with enhanced recognition and cytotoxic capacities. This review focuses on surface receptor engineering in NK cell therapy and discusses its impact, challenges, and future directions.
Most approaches are based on engineering with chimeric antigen receptors to allow NK cells to target specific tumor antigens independent of human leukocyte antigen restriction. This approach has increased the precision and potency of NK-mediated recognition and elimination of cancer cells. In addition, engineering NK cells with T-cell receptors also mediates the recognition of intracellular epitopes, which broadens the range of target peptides. Indirect tumor peptide recognition by NK cells has also been improved by optimizing immunoglobulin constant fragment receptor expression and signaling. Indeed, engineered NK cells have an improved ability to recognize and destroy target cells coated with specific antibodies, thereby increasing their antibody-dependent cellular cytotoxicity. The ability of NK cell receptor engineering to promote the expansion, persistence, and infiltration of transferred cells in the tumor microenvironment has also been explored. Receptor-based strategies for sustained NK cell functionality within the tumor environment have also been discussed, and these strategies providing perspectives to counteract tumor-induced immunosuppression.
Overall, receptor engineering has led to significant advances in NK cell-based cancer immunotherapies. As technical challenges are addressed, these innovative treatments will likely reshape cancer immunotherapy.
... Further, though in vitro functional assays yielded only modest improvements in GBM killing, intracranial in vivo tumor growth was dramatically reduced, likely due to TME specific factors, not present in in vitro systems. Additional modifications to generate fully allogeneic and patient-compatible, hypoimmunogenic iPSCs 45 are possible through HLA editing 46,47 . ...
Severe heterogeneity within glioblastoma has spurred the notion that disrupting the interplay between multiple elements on immunosuppression is at the core of meaningful anti-tumor responses. T cell immunoreceptor with Ig and ITIM domains (TIGIT) and its glioblastoma-associated antigen, CD155, form a highly immunosuppressive axis in glioblastoma and other solid tumors, yet targeting of TIGIT, a functionally heterogeneous receptor on tumor-infiltrating immune cells, has largely been ineffective as monotherapy, suggesting that disruption of its inhibitory network might be necessary for measurable responses. It is within this context that we show that the usurpation of the TIGIT − CD155 axis via engineered synNotch-mediated activation of induced pluripotent stem cell-derived natural killer (NK) cells promotes transcription factor-mediated activation of a downstream signaling cascade that results in the controlled, localized blockade of CD73 to disrupt purinergic activity otherwise resulting in the production and accumulation of immunosuppressive extracellular adenosine. Such “decoy” receptor engages CD155 binding to TIGIT, but tilts inhibitory TIGIT/CD155 interactions toward activation via downstream synNotch signaling. Usurping activities of TIGIT and CD73 promotes the function of adoptively transferred NK cells into intracranial patient-derived models of glioblastoma and enhances their natural cytolytic functions against this tumor to result in complete tumor eradication. In addition, targeting both receptors, in turn, reprograms the glioblastoma microenvironment via the recruitment of T cells and the downregulation of M2 macrophages. This study demonstrates that TIGIT/CD155 and CD73 are targetable receptor partners in glioblastoma. Our data show that synNotch-engineered pluripotent stem cell-derived NK cells are not only effective mediators of anti-glioblastoma responses within the setting of CD73 and TIGIT/CD155 co-targeting, but represent a powerful allogeneic treatment option for this tumor.
... Complete loss of HLA class I surface expression leaves the engineered cells vulnerable to lysis by natural killer (NK) cells, calling for more sophisticated engineering approaches, such as overexpression of HLA E to compensate for HLA class I loss. [71][72][73][74][75] To circumvent this issue, we selected a shGuide sequence against b2M, which can efficiently downregulate the target but still allows for $30% residual HLA class I expression (in orange in Figure 7A, right panel). This strategy allowed us to limit NK activation, measured as percentage of CD107a + NK, in the presence of CAR T cells carrying the shGuide against b2M to levels similar to those measured in presence of CAR T cells with no shRNA, and thus normally expressing HLA class I. On the contrary, b2M KO in CAR T cells by CRISPR-Cas9 led to substantial activation of NK cells (Figure 7D, left panel). ...
Genome engineering technologies are powerful tools in cell-based immunotherapy to optimize or fine-tune cell functionalities. However, their use for multiple gene edits poses relevant biological and technical challenges. Short hairpin RNA (shRNA)-based cell engineering bypasses these criticalities and represents a valid alternative to CRISPR-based gene editing. Here, we describe a microRNA (miRNA)-based multiplex shRNA platform obtained by combining highly efficient miRNA scaffolds into a chimeric cluster, to deliver up to four shRNA-like sequences. Thanks to its limited size, our cassette could be deployed in a one-step process along with all the CAR components, streamlining the generation of engineered CAR T cells. The plug-and-play design of the shRNA platform allowed us to swap each shRNA-derived guide sequence without affecting the system performance. Appropriately choosing the target sequences, we were able to either achieve a functional KO, or fine-tune the expression levels of the target genes, all without the need for gene editing. Through our strategy we achieved easy, safe, efficient, and tunable modulation of multiple target genes simultaneously. This approach allows for the effective introduction of multiple functionally relevant tweaks in the transcriptome of the engineered cells, which may lead to increased performance in challenging environments, e.g., solid tumors.
... However, these strategies are not completely effective and often toxic. 26 Though TCR T-cell therapy may be difficult to adapt across multiple patient populations due to its MHC restriction, rapidly expanding identification of epitopes for many of the common HLA types is broadening the scope of accessible targets. This will require advancements in computational and empiric screening strategies. ...
... II transactivator (CIITA) may shield donor cells; alternatively adding genes for HLA-E, alloimmune defense receptor (ADR), and immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS) have been attempted to improve the persistence and functionality of the infused allogeneic cells.[26][27][28][29]35,36 Deleting the genes of endogenous TCRα/β chains also can significantly reduce the chances of mispairing with exogenous TCR, potential off-target reactivity, and rejection.15,35 ...
... While deleting the B2M gene leads to the downregulation of all HLA class I molecules on the cell surface, it also puts these cells at risk of host NK cell-killing. Therefore, to escape NK cells' attack, non-polymorphic exogenous HLA class E and G genes can be inserted in these cells.[26][27][28] Expression of ADR on the cell surface has increased evasion of host T-cell cytotoxicity.29 ...
Recent development of methods to discover and engineer therapeutic T‐cell receptors (TCRs) or antibody mimics of TCRs, and to understand their immunology and pharmacology, lag two decades behind therapeutic antibodies. Yet we have every expectation that TCR‐based agents will be similarly important contributors to the treatment of a variety of medical conditions, especially cancers. TCR engineered cells, soluble TCRs and their derivatives, TCR‐mimic antibodies, and TCR‐based CAR T cells promise the possibility of highly specific drugs that can expand the scope of immunologic agents to recognize intracellular targets, including mutated proteins and undruggable transcription factors, not accessible by traditional antibodies. Hurdles exist regarding discovery, specificity, pharmacokinetics, and best modality of use that will need to be overcome before the full potential of TCR‐based agents is achieved. HLA restriction may limit each agent to patient subpopulations and off‐target reactivities remain important barriers to widespread development and use of these new agents. In this review we discuss the unique opportunities for these new classes of drugs, describe their unique antigenic targets, compare them to traditional antibody therapeutics and CAR T cells, and review the various obstacles that must be overcome before full application of these drugs can be realized.
... In cancer immunotherapy, the patient's immune system recognizes and rejects the infused NK cells, limits their life span in vivo, and eliminates the prospect of multiple infusions. Hoerster et al. used a genome-editing strategy and improved the lentiviral transduction procedure in primary human NK cells to render them resistant to the CD8 + T cell responses of the recipients [42]. They coexpressed a single-chain HLA-E molecule after knocking down the surface expression of the HLA class I molecules via the B2M gene targeting [42]. ...
... Hoerster et al. used a genome-editing strategy and improved the lentiviral transduction procedure in primary human NK cells to render them resistant to the CD8 + T cell responses of the recipients [42]. They coexpressed a single-chain HLA-E molecule after knocking down the surface expression of the HLA class I molecules via the B2M gene targeting [42]. They used CRISPR/Cas9 to inhibit the NK cell fratricide of B2M-knockout (KO) cells via "missing self"-induced lysis [42]. ...
... They coexpressed a single-chain HLA-E molecule after knocking down the surface expression of the HLA class I molecules via the B2M gene targeting [42]. They used CRISPR/Cas9 to inhibit the NK cell fratricide of B2M-knockout (KO) cells via "missing self"-induced lysis [42]. Importantly, in terms of phenotypic and natural cytotoxicity against several AML cell lines, these genetically edited NK cells were functionally identical to their unmodified counterparts [42]. ...
Chimeric antigen receptor (CAR) T cells and natural killer (NK) cells are genetically engineered immune cells that can detect target antigens on the surface of target cells and eliminate them following adoptive transfer. Recent progress in CAR-based therapies has led to outstanding clinical success in certain patients with leukemias and lymphomas and offered therapeutic benefits to those resistant to conventional therapies. The universal approach to stable CAR transgene delivery into the T/NK cells is the use of viral particles. Such approaches mediate semi-random transgene insertions spanning the entire genome with a high preference for integration into sites surrounding highly-expressed genes and active loci. Regardless of the variable CAR expression level based on the integration site of the CAR transgene, foreign integrated DNA fragments may affect the neighboring endogenous genes and chromatin structure and potentially change a transduced T/NK cell behavior and function or even favor cellular transformation. In contrast, site-specific integration of CAR constructs using recent genome-editing technologies could overcome the limitations and disadvantages of universal random gene integration. Herein, we explain random and site-specific integration of CAR transgenes in CAR-T/NK cell therapies. Also, we tend to summarize the methods for site-specific integration as well as the clinical outcomes of certain gene disruptions or enhancements due to CAR transgene integration. Also, the advantages and limitations of using site-specific integration methods are discussed in this review. Ultimately, we will introduce the genomic safe harbor (GSH) standards and suggest some appropriate safety prospects for CAR integration in CAR-T/NK cell therapies.
... Another factor leading to the short persistence is T-cell and host NK cell allorejection. To overcome this, knockout of HLA class I molecule and overexpression of inhibitory NK receptors have been proposed (130). ...
While cord blood (CB) is primarily utilized in allogeneic hematopoietic cell transplantation (HCT), the development of novel cell therapy products from CB is a growing and developing field. Compared to adult blood, CB is characterized by a higher percentage of hematopoietic stem cells (HSCs) and progenitor cells, less mature immune cells that retain a high capacity of proliferation, and stronger immune tolerance that requires less stringent HLA-matching when used in the allogenic setting. Given that CB is an FDA regulated product and along with its unique cellular composition, CB lends itself as a readily available and safe starting material for the development of off-the-shelf cell therapies. Moreover, non-hematologic cells such as mesenchymal stem cell (MSCs) residing in CB or CB tissue also have potential in regenerative medicine and inflammatory and autoimmune conditions. In this review, we will focus on recent clinical development on CB-derived cellular therapies in the field of oncology, including T-cell therapies such as chimeric antigen receptor (CAR) T-cells, regulatory T-cells, and virus-specific T-cells; NK-cell therapies, such as NK cell engagers and CAR NK-cells; CB-HCT and various modifications; as well as applications of MSCs in HCT.
... However, other studies have suggested an increase of natural killer (NK) cell-mediated cell death due to a lack of HLA-E/G expression required for normal immune surveillance by NK cells [72]. To overcome this challenge, lentiviral overexpression of HLA-E within B2M KO cells was able to suppress allogeneic T cell proliferation and activation without inducing NK cell activation [73]. As an alternative to maintaining HLA-E/G, another approach to reduce NK cell activity is the overexpression of CD47, which is a ubiquitously expressed immunomodulatory suppressive gene [74,75]. ...
Patients suffering from diabetes rely on the exogenous supply of insulin. Cell replacement therapy employing cadaveric islets cells has demonstrated a proof of principle for a practical cure, rendering patients insulin independent for prolonged periods of time. However, challenges remain before this innovative therapy can be widely accessed by patients in need. Donor islet material is limited, requiring the generation of an abundant source of insulin-producing pancreatic beta cells. Immunological allogeneic rejection and recurring autoreactivity contribute to eventual graft failure in all transplant recipients. Here we summarize past and current efforts to generate functional beta cells from pluripotent stem cells and highlight current knowledge on graft immune interactions. We further discuss remaining challenges of current cell replacement efforts and highlight potentially innovative approaches to aid current strategies.
... These iPSCs-NK cells could be engineered via knock-in/out of genes resulting in the generation of allogenic iPSCs-NK cells capable of suppressing MHC1 leading to avoidance of host T cell recognition. These engineered cells also over-expressed HLA-E, resulting in the inactivation of host NK cells [91]. The reprogramming of NK cells to iPSCs to express chimeric antigen receptor-NK cells (NK-CAR-iPSC-NK cells) has been shown to mediate a strong response in inhibiting tumor growth as well as enhanced in vitro and in vivo cell survival in an ovarian cancer study model when compared to that of peripheral blood-NK cells [92]. ...
Cancer recurrence and drug resistance following treatment, as well as metastatic forms of cancer, are trends that are commonly encountered in cancer management. Amidst the growing popularity of personalized medicine and targeted therapy as effective cancer treatment, studies involving the use of stem cells in cancer therapy are gaining ground as promising translational treatment options that are actively pursued by researchers due to their unique tumor-homing activities and anti-cancer properties. Therefore, this review will highlight cancer interactions with commonly studied stem cell types, namely, mesenchymal stroma/stem cells (MSC), induced pluripotent stem cells (iPSC), iPSC-derived MSC (iMSC), and cancer stem cells (CSC). A particular focus will be on the effects of paracrine signaling activities and exosomal miRNA interaction released by MSC and iMSCs within the tumor microenvironment (TME) along with their therapeutic potential as anti-cancer delivery agents. Similarly, the role of exosomal miRNA released by CSCs will be further discussed in the context of its role in cancer recurrence and metastatic spread, which leads to a better understanding of how such exosomal miRNA could be used as potential forms of non-cell-based cancer therapy.
... In allogeneic NK cell therapy, there is a risk of immune rejection by host cells, especially in cases where systemic IL-15 supplementation is used to promote NK cell expansion [86]. Studies are currently investigating strategies similar to those employed for T cells, such as modifications to MHC class I or II molecules, to navigate the issue of immune rejection [87]. Another major issue in NK cell-based ACT is the source of NK cells [88]. ...
Simple Summary
Patients with relapsed and refractory T-cell malignancies have poor outlook and limited treatment options. Recently, adoptive cell therapy has emerged as a promising therapy for patients with T-cell malignancies. In this review, we examine the current progress on adoptive cell therapy for T-cell malignancies and discuss the potential future directions.
Abstract
T-cell malignancies are often aggressive and associated with poor prognoses. Adoptive cell therapy has recently shown promise as a new line of therapy for patients with hematological malignancies. However, there are currently challenges in applying adoptive cell therapy to T-cell malignancies. Various approaches have been examined in preclinical and clinical studies to overcome these obstacles. This review aims to provide an overview of the recent progress on adoptive cell therapy for T-cell malignancies. The benefits and drawbacks of different types of adoptive cell therapy are discussed. The potential advantages and current applications of innate immune cell-based adoptive cell therapy for T cell malignancies are emphasized.
