scholarly journals Bispecific antibody-mediated redirection of NKG2D-CAR natural killer cells facilitates dual targeting and enhances antitumor activity

2021 ◽  
Vol 9 (10) ◽  
pp. e002980
Author(s):  
Congcong Zhang ◽  
Jasmin Röder ◽  
Anne Scherer ◽  
Malena Bodden ◽  
Jordi Pfeifer Serrahima ◽  
...  
Keyword(s):  
T Cells ◽  
Antitumor Activity ◽  
Nk Cells ◽  
Cancer Cells ◽  
Natural Killer ◽  
Chimeric Antigen Receptor ◽  
Peripheral Blood ◽  
Antigen Receptor ◽  
Scfv Antibody ◽  
Effector Cells

BackgroundNatural killer group 2D (NKG2D) is an activating receptor of natural killer (NK) cells and other lymphocytes that mediates lysis of malignant cells through recognition of stress-induced ligands such as MICA and MICB. Such ligands are broadly expressed by cancer cells of various origins and serve as targets for adoptive immunotherapy with effector cells endogenously expressing NKG2D or carrying an NKG2D-based chimeric antigen receptor (CAR). However, shedding or downregulation of NKG2D ligands (NKG2DL) can prevent NKG2D activation, resulting in escape of cancer cells from NKG2D-dependent immune surveillance.MethodsTo enable tumor-specific targeting of NKG2D-expressing effector cells independent of membrane-anchored NKG2DLs, we generated a homodimeric recombinant antibody which harbors an N-terminal single-chain fragment variable (scFv) antibody domain for binding to NKG2D, linked via a human IgG4 Fc region to a second C-terminal scFv antibody domain for recognition of the tumor-associated antigen ErbB2 (HER2). The ability of this molecule, termed NKAB-ErbB2, to redirect NKG2D-expressing effector cells to ErbB2-positive tumor cells of different origins was investigated using peripheral blood mononuclear cells, ex vivo expanded NK cells, and NK and T cells engineered with an NKG2D-based chimeric receptor.ResultsOn its own, bispecific NKAB-ErbB2 increased lysis of ErbB2-positive breast carcinoma cells by peripheral blood-derived NK cells endogenously expressing NKG2D more effectively than an ErbB2-specific IgG1 mini-antibody able to induce antibody-dependent cell-mediated cytotoxicity via activation of CD16. Furthermore, NKAB-ErbB2 synergized with NK-92 cells or primary T cells engineered to express an NKG2D-CD3ζ chimeric antigen receptor (NKAR), leading to targeted cell killing and greatly enhanced antitumor activity, which remained unaffected by soluble MICA known as an inhibitor of NKG2D-mediated natural cytotoxicity. In an immunocompetent mouse glioblastoma model mimicking low or absent NKG2DL expression, the combination of NKAR-NK-92 cells and NKAB-ErbB2 effectively suppressed outgrowth of ErbB2-positive tumors, resulting in treatment-induced endogenous antitumor immunity and cures in the majority of animals.ConclusionsOur results demonstrate that combining an NKAB antibody with effector cells expressing an activating NKAR receptor represents a powerful and versatile approach to simultaneously enhance tumor antigen-specific as well as NKG2D-CAR and natural NKG2D-mediated cytotoxicity, which may be particularly useful to target tumors with heterogeneous target antigen expression.

A Novel NKp44-Based Chimeric Antigen Receptor That Targets Multiple Types of Cancer

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3517-3517 ◽  
Author(s):  
Yasushi Kasahara ◽  
Changsu Shin ◽  
Sakiko Yoshida ◽  
Takayuki Takachi ◽  
Nobuhiro Kubo ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cancer Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Surface Expression ◽  
Binding Domain ◽  
Human T Cells ◽  
Immunoglobulin Domain ◽  
Tm Domain

Abstract Genetic modification of T cells with an artificial tumor-targeting chimeric antigen receptor (CAR) is a new approach for adoptive cell therapy for cancer. Defining cell surface molecules that are both selectively expressed on cancer cells and can be safely targeted with T cells or NK cells is a significant challenge in this research field. NKp44 is a member of the natural cytotoxicity receptor (NCR) families and also known as NCR2. Expression of NKp44 is limited to activating NK cells, which leads to a marked increase in cytotoxicity against tumors. The receptor contains one extracellular immunoglobulin domain, type I transmembrane (TM) domain, and intracellular (IC) domain, and its surface expression seems to require binding of the TM domain to adaptor molecules of DAP12 accessory protein that contains ITAMs. The ligand for NKp44 is considered damage-associated molecular pattern molecules, which have been reported to be expressed by various types of cancer cells but not by healthy cells. Therefore, a wide range of cancer cells may be safely targeted if the ligand-binding domain of this receptor is used in a construction of a chimeric antigen receptor (CAR) as an antigen recognition site, instead of using single chain variable domains derived from monoclonal antibody. We created several NKp44-based CAR constructs, which shares the extracellular NKp44 IG domain as a ligand-binding domain. Surface expression levels and subsequent functional properties can differ among T cells or NK cells transduced with novel CARs with different structural characteristics. We thus tested whether swapping the domains other than the antigen-binding domain affected expression and function. The CAR genes were retrovirally transduced into human primary T cells according to a standard method. We also transduced human primary NK cells with NKp44-based CARs, by a previously reported method (Imai C, et al. Blood 2005), to compare the expression pattern of the CAR in NK cells with that in human T cells. Retroviral transfer of wild type NKp44 gene and a construct harboring IC(p44) both did not induce NKp44 surface expression (Fig 1A,B). By sharp contrast, primary NK cells were able to express the CAR protein on the cell surface after transfer of these two genes. Removal of the IC(p44) [EH(p44)-TM(p44)-IC(CD3z)] allowed slight surface expression in T cells (Fig1C). The replacement of TM(p44) with TM(CD8a) resulted in higher surface expression in T cells (Fig 1D). These observations indicated the presence of IC(p44) as well as TM(p44) in the CAR constructs hampered surface expression in T cells most likely due to the lack of DAP12 expression. In addition to TM replacement, replacement of EH(p44) with EH(CD8a) markedly increased surface expression of the CAR (Fig 1E). Similarly, we tested use of CD28 domains instead of CD8a. Surprisingly, as different from the case of CD8a, the construct EH(p44)-TM(CD28)-IC(CD3z) yielded highest surface expression among the all CAR constructs created in this study in T cells as well as in NK cells (Fig1F), while the replacement of EH(p44) of the abovementioned CAR with EH(CD28) resulted in marked reduction of the CAR expression (Fig 1G). We confirmed surface expression of NKp44 ligand with flow cytometric analysis using recombinant human NKp44 Fc chimera protein (R&D Systems, McKinley Place, Minneapolis, USA) on various tumor cell lines including myeloid leukemia (K562, THP-1, U937, KY821, HL60), T-cell leukemia (PEER, MOLT4, HSB2), Burkitt lymphoma (Raji), BCR-ABL-positive B-ALL (OP-1), osteosarcoma (MG63, NOS1, NOS2, NOS10, U2OS, SaOS2), rhabdomyosarcoma (Rh28, RMS-YM), neuroblastoma (SK-N-SH, NB1, NB16, IMR32), and cervical carcinoma (Hela). Function of the best construct [EH(p44)-TM(CD28)-IC(CD3z)] was further evaluated. Primary T cells transduced with this NKp44-based CAR exerted powerful cytotoxicity against tumor cell lines tested and produced interferon-g and granzyme B, while GFP-transduced T cells and control T cells transduced with truncated NKp44-based CAR did not. In conclusion, we have created a novel CAR based on the antigen-binding property derived from NKp44 receptor immunoglobulin domain. This CAR should be effective to redirect T cells as well as NK cells against various types of cancer including hematological malignancies. Figure 1 Schematic representation of gene constructs and their surface expression of NKp44-based CARs in human T cells and NK cells. Figure 1. Schematic representation of gene constructs and their surface expression of NKp44-based CARs in human T cells and NK cells. Disclosures Imai: Juno Therapeutics: Patents & Royalties.

scholarly journals Chimeric Antigen Receptor T Cells with Dissociated Signaling Domains Exhibit Focused Antitumor Activity with Reduced Potential for Toxicity In Vivo

2013 ◽  
Vol 1 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Evripidis Lanitis ◽  
Mathilde Poussin ◽  
Alex W. Klattenhoff ◽  
Degang Song ◽  
Raphael Sandaltzopoulos ◽  
...  
Keyword(s):  
T Cells ◽  
Antitumor Activity ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Signaling Domains

scholarly journals Manufacture of Chimeric Antigen Receptor T Cells from Mobilized Cyropreserved Peripheral Blood Stem Cell Units Depends on Monocyte Depletion

2019 ◽  
Vol 25 (2) ◽  
pp. 223-232 ◽  
Author(s):  
Annette Künkele ◽  
Christopher Brown ◽  
Adam Beebe ◽  
Stephanie Mgebroff ◽  
Adam J. Johnson ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Chimeric Antigen Receptor ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Antigen Receptor ◽  
Blood Stem Cell

scholarly journals Mesothelin-Specific Chimeric Antigen Receptor mRNA-Engineered T Cells Induce Antitumor Activity in Solid Malignancies

2013 ◽  
Vol 2 (2) ◽  
pp. 112-120 ◽  
Author(s):  
Gregory L. Beatty ◽  
Andrew R. Haas ◽  
Marcela V. Maus ◽  
Drew A. Torigian ◽  
Michael C. Soulen ◽  
...  
Keyword(s):  
T Cells ◽  
Antitumor Activity ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Receptor Mrna ◽  
Solid Malignancies ◽  
Engineered T Cells

scholarly journals NKG2C+CD57+ Natural Killer Cells May Play an Important Role in Maintaining Virus-Specific Immune Reconstitution Post-Allogeneic HSCT

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3327-3327
Author(s):  
Debora Queiros ◽  
Susanne Luther-Wolf ◽  
Eva M Weissinger ◽  
Arnold Ganser
Keyword(s):  
T Cells ◽  
Healthy Volunteers ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Immune Reconstitution ◽  
Blood Samples ◽  
Allogeneic Hsct ◽  
Cell Counts ◽  
Cmv Reactivation

Abstract Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for malignant hematological diseases in adults. Due to the delayed immune reconstitution after HSCT, human cytomegalovirus (CMV) can reactivate, leading to prolonged hospitalization and increased morbidity and even mortality. Natural Killer (NK) cells have recently been described to undergo persistent reconfiguration in response to CMV-reactivation. Here we analyzed the presence and expansion of CMV-specific NK cells in patients after allogeneic HSCT. Methods: A multicolor flow cytometry panel for monitoring the CMV-specific NK cell (NKG2C+CD57+) reconstitution and expression of activating receptors was established. Reconstitution of CMV-specific NK cells was assessed in peripheral blood samples from 67 CMV-seropositive patients. The samples were collected and analyzed between day 0 and 100 post-HSCT at intervals of 7-10 days. Monitoring of CMV-reactivation by CMV-pp65 expression and reconstitution of CMV-specific T cells (CMV-CTLs) was done routinely in our laboratory, using 7 commercially available, certified CMV-tetramers, allowing for comparison of CMV-CTL and NKG2C+CD57+ NK cells. For further immunological tests, PBMCs from CMV-seropositive healthy volunteers were isolated by density gradient centrifugation. NK cells were negatively selected by magnetic bead separation. Additional purification of NKG2C+CD57+ NK cellswas achieved by cell sorting. Selected NK cells were expanded by co-culture with irradiated allogeneic PBMCs as feeder cells and the medium was supplemented with PHA and IL-2. Expanded CD57+NKG2C+ NK cells were KIR-typed. Results: Our patient cohort consisted of 67 patients after allogeneic HSCT with a median age of 59 years (range: 20-75). Forty-two patients (62.7%) were transplanted for acute leukemia, 54 (80.6%) received reduced intensity conditioning (RIC) and 62 (92.5%) received anti-thymocyte-antibodies globulin (ATG). GvHD-prophylaxis was cyclosporine A (CsA) in combination with mycophenolate motefil (MMF) for 82.1% of the patients and 77.6% were transplanted from matched donors. Thirty-three (49.2%) patients reactivated CMV (median age: 59.5 years, range 28-75; median day of reactivation: 38 days post-HSCT, range: 19-54). A significant increase in the absolute cell counts of NKG2C+CD57+ NK cells was observed after CMV reactivation, when compared to patients who did not reactivate CMV (p<0.0001). Interestingly, we observed a decreased expression of the CD8-molecule on NK cells during CMV-reactivation. CD8-expression on NK cells was previously described to be associated with a more cytotoxic phenotype of NK cells, this decrease may be a consequence of apoptosis following lytic activity. Monitoring for an additional activation marker, NKG2D, showed a significant increased expression after CMV reactivation (p=0.006), demonstrating not only the activating regulation of NK cells, but also, the co-stimulatory effects on T cell proliferation and cytokine production. Remarkably, when comparing NKG2C+CD57+ NK cells with CMV-specific T cells (Figure 1), both cell populations show similar kinetics of expansion, with an increase in the absolute cell counts during and after CMV-reactivation. NKG2C+CD57+ preliminary expansion-studies were performed using peripheral blood samples from CMV-seropositive healthy volunteers. After two weeks in culture, an expansion of up to 3100-fold was achieved. Further studies to assess the proliferative capacity of NKG2C+CD57+ subpopulation and its functional properties post-HSCT, are ongoing. In addition, an extensive panel of cytokines and chemokines excreted by the NKG2C+CD57+cells will be studied in order to evaluate their recruitment ability of other cell-types. Conclusion: Taken together, our results indicate that NK cells undergo a dynamic modulation and expansion of this population occurs in response to CMV-reactivation. Additionally, NKG2C+CD57+ NK cells may substitute for missing CMV-specific T cells shortly after HSCT and may play an important role in sustaining the immune reconstitution after CMV-reactivation. This study shows that NKG2C+CD57+ NK cells can be selected and expanded in vitro, holding promise for adoptive transfer in patients with recurrent CMV-reactivations. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Granulocyte Colony-Stimulating Factor Effectively Mobilizes TCR γδ and NK Cells Providing an Allograft Potentially Enhanced for the Graft-Versus-Leukemia Effect for Allogeneic Stem Cell Transplantation

2021 ◽  
Vol 12 ◽  
Author(s):  
Lia Minculescu ◽  
Henrik Sengelov ◽  
Hanne Vibeke Marquart ◽  
Lars Peter Ryder ◽  
Anne Fischer-Nielsen ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Cell Compartment ◽  
Effector Cells ◽  
Graft Versus Leukemia ◽  
Hla Dr

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potential cure for patients with hematological malignancies but substantial risks of recurrence of the malignant disease remain. TCR γδ and NK cells are perceived as potent innate effector cells in HSCT and have been associated with post-transplant protection from relapse in clinical studies. Immunocompetent cells from the donor are crucial for patient outcomes and peripheral blood stem cells (PBSC) are being increasingly applied as graft source. G-CSF is the preferential mobilizing agent in healthy donors for PBSC grafts, yet effects of G-CSF on TCR γδ and NK cells are scarcely uncovered and could influence the graft composition and potency of these cells. Therefore, we analyzed T and NK cell subsets and activation markers in peripheral blood samples of 49 donors before and after G-CSF mobilization and—for a subset of donors—also in the corresponding graft samples using multicolor flowcytometry with staining for CD3, CD4, CD8, TCRαβ, TCRγδ, Vδ1, Vδ2, HLA-DR, CD45RA, CD197, CD45RO, HLA-DR, CD16, CD56, and CD314. We found that TCR γδ cells were mobilized and harvested with an efficiency corresponding that of TCR αβ cells. For TCR γδ as well as for TCR αβ cells, G-CSF preferentially mobilized naïve and terminally differentiated effector (TEMRA) cells over memory cells. In the TCR γδ cell compartment, G-CSF preferentially mobilized cells of the nonVδ2 types and increased the fraction of HLA-DR positive TCR γδ cells. For NK cells, mobilization by G-CSF was increased compared to that of T cells, yet NK cells appeared to be less efficiently harvested than T cells. In the NK cell compartment, G-CSF-stimulation preserved the proportion of CD56dim NK effector cells which have been associated with relapse protection. The expression of the activating receptor NKG2D implied in anti-leukemic responses, was significantly increased in both CD56dim and CD56bright NK cells after G-CSF stimulation. These results indicate differentiated mobilization and altering properties of G-CSF which could improve the effects of donor TCR γδ and NK cells in the processes of graft-versus-leukemia for relapse prevention after HSCT.

scholarly journals IKZF3 deficiency potentiates chimeric antigen receptor T cells targeting solid tumors

2021 ◽  
Author(s):  
tian chi ◽  
yan zou
Keyword(s):  
T Cells ◽  
Cancer Cells ◽  
Solid Tumors ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Xenograft Model ◽  
Cytokine Signaling ◽  
General Strategy ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptor (CAR) T cell therapy has been successful in treating hematological malignancy, but solid tumors remain refractory. Here, we demonstrated that knocking out transcription factor IKZF3 in HER2-specific CAR T cells targeting breast cancer cells did not affect proliferation or differentiation of the CAR T cells in the absence of tumors, but markedly enhanced killing of the cancer cells in vitro and in a xenograft model. Furthermore, IKZF3 KO had similar effects on the CD133-specific CAR T cells targeting glioblastoma cells. AlphaLISA and RNA-seq analyses indicate that IKZF3 KO increased the expression of genes involved in cytokine signaling, chemotaxis and cytotoxicity. Our results suggest a general strategy for enhancing CAR T efficacy on solid tumors.

scholarly journals B and T Immunoregulation: A New Insight of B Regulatory Lymphocytes in Autism Spectrum Disorder

2021 ◽  
Vol 15 ◽  
Author(s):  
Andrea De Giacomo ◽  
Concetta Domenica Gargano ◽  
Marta Simone ◽  
Maria Giuseppina Petruzzelli ◽  
Chiara Pedaci ◽  
...  
Keyword(s):  
Autism Spectrum Disorder ◽  
Immune Response ◽  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Autism Spectrum ◽  
Spectrum Disorder ◽  
B Cell Memory

Introduction: Autism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized by a complex pathogenesis, by impairment social communication and interaction, and may also manifest repetitive patterns of behavior. Many studies have recognized an alteration of the immune response as a major etiological component in ASDs. Despite this, it is still unclear the variation of the function of the immune response.Aim: Our aim is to investigate the levels of immunological markers in peripheral blood of children with ASD such as: regulatory B and T cells, memory B and natural killer (NK) cells.Materials and Methods: We assessed various subsets of immune cells in peripheral blood (regulatory B and T cells, B-cell memory and natural killer cells) by multi-parametric flow cytometric analysis in 26 ASD children compared to 16 healthy controls (HCs) who matched age and gender.Results: No significant difference was observed between B-cell memory and NK cells in ASDs and HCs. Instead, regulatory B cells and T cells were decreased (p &lt; 0.05) in ASD subjects when compared to HCs.Discussion: Regulatory B and T cells have a strategic role in maintaining the immune homeostasis. Their functions have been associated with the development of multiple pathologies especially in autoimmune diseases. According to our study, the immunological imbalance of regulatory B and T cells may play a pivotal role in the evolution of the disease, as immune deficiencies could be related to the severity of the ongoing disorder.

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