Cells For Cellular Immunotherapy Research Articles

Abstract 2851: Moving on: Embracing γδ T-cell biology to create truly next generation immunotherapy concepts

Abstract Chimeric antigen receptor (CAR) modification of αβ T cells has revolutionized the field of oncology, driving the focus of attention to the immune system to target and fight malignancies. Currently available αβ T-cell therapies come with many challenges, including alloreactivity, off-target toxicities, cytokine release syndrome, limited survival of infused cells and the necessity to gene edit cells to overcome graft vs. host disease. Whilst some of these limitations can be overcome with complex and costly gene-engineering approaches, utilizing innate immune cells which are inherently non-HMC restricted and able to orchestrate wider immune responses might provide an alternative strategy avoiding many of these obstacles. Many groups developed protocols to grow and modify natural killer cells and invariant natural killer T-cells whilst others, including ours, focused on the development of Vδ1 γδ T-cell-based immunotherapies. Currently, these cell types are evaluated in the clinic in both non-engineered and engineered versions. Most of these engineering principles have been directly translated from the αβ CAR-T field without taking into consideration the biology of innate immune cells: they utilize αβ T-cell specific co-stimulatory molecules and signaling cassettes as well as armoring strategies developed for αβ T-cells. Vδ1 T-cells are tissue resident lymphocytes, which for most of their lifetime remain in epithelia rich tissues. They consistently survey tissues and monitor malignant transformation using a variety of natural cytotoxicity receptors, NK like receptors and the γδ TCR. In contrast to αβ T-cells, Vδ1 T-cells do not follow the ‘two-signal theory’: they must integrate multiple signals from an array of receptors in order to discriminate between healthy and malignant cells. We thus propose that CAR strategies exclusively utilizing CD3ζ activation domains are not using the full potential of innate immune cells for cellular immunotherapy. Another distinguishing feature of Vδ1 T-cells is the absence of traditional autocrine cytokine feedback loops seen in αβ T-cells. Our results demonstrate that traditional cytokine armoring strategies utilizing forced secretion of soluble or membrane-tethered IL-15 are detrimental to the biology of Vδ1 T-cells. By dissecting the IL-15 receptor biology we are now able to create cells that not only survive and grow in the absence of exogenous IL-15 but become more sensitive to endogenous levels. Applying γδ T-cell biology and an adapted understanding of the IL-15 pathway, we have now created novel engineering strategies to tailor specificity, potency, and proliferation of Vδ1 T-cells even in the presence of CAR whilst maintaining the cells’ inherent ability to discriminate between healthy and malignant cells. Preclinical evaluation of these concepts is ongoing with the aim to develop next generation tailored Vδ1T-cell immunotherapies. Citation Format: Jyothi Kumaran, Rajeev Karattil, André Simoes, Rebecca Alade, Mihil Patel, Liz Wood, Gonzalo Mercado Vico, Amy Lane, Sara Tamagno, Sarah Edwards, Andrea Venuso, Sam Illingworth, Alice Brown, Michael Koslowski, Istvan Kovacs, Oliver Nussbaumer. Moving on: Embracing γδ T-cell biology to create truly next generation immunotherapy concepts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2851.

Abstract B64: Coexpression of a CD19-OX40-CD3ζ CAR with membrane-bound IL-15 enhances natural killer cell function

Abstract Introduction: Chimeric antigen receptors (CARs) have been used successfully to retarget T cells in patients with hematologic malignancies. Natural killer (NK) cells offer an alternative to T cells for cellular immunotherapy and are suitable for allogeneic use as they are not HLA-restricted and patients who receive NK cell treatment do not develop graft-versus-host disease (GVHD). Therefore, NK cells can provide an attractive alternative for “off-the-shelf” cellular therapy. Here, we investigated multiple approaches to modify and enhance CD19 CAR expression on NK cells. It has been previously reported that costimulatory domains play an important role in proliferation, efficacy, and persistence of CAR-T cells both in vitro and in vivo. To understand how CAR structure effects the functional behavior of NK cells, we assessed the capability of various costimulatory signaling domains, including CD28, OX40, CD28-41BB, etc., all coupled to CD3ζ, to enhance CD19 CAR signaling and cytotoxicity in NK cells. We demonstrated that NK activity and persistence can be elevated by simultaneous expression of chimeric constructs directing the expression of a CD19 CAR and a membrane-bound form of IL-15 (mbIL-15). Methods: NK cells were generated by coculture of peripheral blood mononuclear cells (PBMC) with genetically modified irradiated K562 feeder cells. NK cells were transduced at a MOI of 1-2 with a gamma-retrovirus encoding a CD19 CAR and mbIL-15. NK expansion and CAR expression were evaluated by flow cytometry. In vitro cytotoxicity of transduced NK cells was measured using both flow cytometry and the IncuCyte S3 live cell analysis system. The in vivo activity of engineered NK cells was further assessed in a xenograft tumor model in NSG mice, using a Nalm6 leukemia cell line. Results: CD19 CAR constructs containing various costimulatory domains were all expressed in expanded and transduced NK cells. CAR expression in multiple donor NK cells was typically between 60-90%. In cytotoxicity assays, CD19 CAR constructs containing the costimulatory domains of OX40, CD28, or CD27 generally exhibited the greatest cytotoxic activity against Nalm6 and Raji tumor cell lines in vitro. High expression of these three CD19 CAR constructs was also maintained over the course of 4 weeks in addition to sustained NK proliferation and cell numbers, indicating an increase in NK cell persistence. In comparing these constructs in an in vivo Nalm6 tumor model, all three constructs demonstrated greater antitumor efficacy relative to unmodified NK cells. The CD19 CAR construct containing the OX40 costimulatory domain showed moderately enhanced efficacy than the CD28 or CD27 variants in vivo. Conclusion: Transduction of NK cells with CD19-OX40-CD3ζ CAR and mbIL15 increases their cytotoxic activity and persistence. Based on these data, further development of NK CD19 CAR for clinical use will be pursued. Citation Format: Luxuan G. Buren, Chao Guo, Yanying Fan, Alexander Aronov, Xiumin Wu, Daofeng Liu, Ming-Hong Xie, Sasha Lazetic, Ivan H. Chan, James B. Trager. Coexpression of a CD19-OX40-CD3ζ CAR with membrane-bound IL-15 enhances natural killer cell function [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B64.

A simple method for in vitro preparation of natural killer cells from cord blood

BackgroundCord Blood (CB) has been considered a promising source of natural killer (NK) cells for cellular immunotherapy. However, it is difficult to expand the large numbers of highly pure NK cells from CB without cell sorting and feeder cells/multiple cytokines. In this study, we try to develop a simple, safe and economical method for ex vivo expansion and purification of NK cells from CB without cell sorting and feeder cells/multiple cytokines.ResultsThe large numbers (mean: 1.59 × 1010) of highly pure (≥90%) NK cells from CB could be obtained through interleukin-2, group A streptococcus and zoledronate stimulation of mononuclear cells using the 21-day culture approach. When compared to resting NK cells, expanded NK cells were a higher expression of activating receptors CD16, NKG2D, NKp30, NKp44, NKp46 and activating markers CD62L and CD69, while the inhibitory receptors, CD158a and CD158b remained largely unchanged. In addition, these cells showed a higher concentration of IFN-γ, TNF-α and GM-CSF secretion and cytotoxicity to K562 cells and acute myeloid leukemia targets than resting NK cells.ConclusionWe develop a simple, safe and economical method to obtain high yield, purity, and functionality NK cells from CB without cell sorting and feeder cells/multiple cytokines.

Inducible Gene Switches with Memory in Human T Cells for Cellular Immunotherapy.

Cell-based therapies that employ engineered T cells—including those modified to express chimeric antigen receptors (CARs)—to target cancer cells have demonstrated promising responses in clinical trials. However, engineered T cell responses must be regulated to prevent severe side effects such as cytokine storms and off-target responses. Here we present a class of recombinase-based gene circuits that will enable inducible, one-time state switching in adoptive T cell therapy using an FDA-approved drug, creating a generalizable platform that can be used to control when and how strongly a gene is expressed. These circuits exhibit memory such that induced T cells will maintain any changes made even when the drug inducer is removed. This memory feature avoids prolonged drug inducer exposure, thus reducing the complexity and potential side effect associated with the drug inducer. We have utilized these circuits to control the expression of an anti-Her2-CAR, demonstrating the ability of these circuits to regulate CAR expression and T cell activity. We envision this platform can be extended to regulate other genes involved in T cell behavior for applications in various adoptive T cell therapies.

In Vitro and in Vivo Imaging of Initial T-B Cell Interactions in the Setting of B Cell-Based Cancer Immunotherapy

In recent years, there has been a growing interest in the use of B cells for cellular immunotherapy, since B cell-based cancer vaccines have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated (CD40) B cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo.The dynamic interactions of human antigen-presenting cells and antigen-specific T cells were observed by time-lapse videomicroscopy. The migratory and chemoattractant potential of CD40B cells was analyzed by flow cytometry and standard transwell migration assays. GFP+ CD40B cells or CD40B cells isolated from Luciferase+mice were used for subsequent in vivo studies.Murine CD40B cells show similar migratory and chemotactic characteristics compared to human CD40B cells. Upon CD40-activation, B cells upregulate the important molecules involved in lymh node homing (CD62L, CCR7/CDCR4), which are functional and induce chemotaxis of T cells in vitro. Striking differences were observed for interactions of human CD40B cells or DCs with T cells. Antigen-loaded CD40B cells differ from immature and mature DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived (7.5 min) and sequential interactions with cognate T cells. In vivo, CD40B cells migrate to the spleen and the lymph nodes, where they enrich in the B cell zone before traveling to B cell/ T cell boundary close to the T cell zone.CD40B cell interactions with T cells are dynamic and short-lived and thereby differ from DCs. Taken together, the migration behavior of CD40B cells and their interaction with T cells underline their potential as cellular adjuvant for cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

T Cell Receptor Gene Transfer to Virus-Specific T Cells for Cellular Anti-Tumor Immunotherapy.

Patients with relapsed hematological malignancies after allogeneic stem cell transplantation (alloSCT) can be successfully treated by donor lymphocyte infusions (DLI). Since DLI consists of a variety of T cells with different specificities, the benificial anti-leukemic effect of DLI is often accompanied by Graft-versus-Host Disease (GvHD). Genetic modification of T cells to express T cell receptors (TCR) with defined anti-tumor specificity would be an attractive strategy to specifically eradicate the malignant cells without induction of GvHD. We previously demonstrated that transfer of the minor histocompatibility antigen HA-2 specific TCR to CMV specific T cells led to the generation of T cells with dual specificity for CMV as well as HA-2. CMV and EBV specific T cells are ideal target cells for TCR gene transfer, since the majority of human individuals have high frequencies of these T cells due to latent persistence of CMV and EBV. In addition, based on their virus specificity, these T cells do not induce GvHD in an alloSCT setting, and we hypothesize that due to frequent encounter with viral antigens, TCR transferred virus specific T cells will survive for a prolonged period of time in vivo. The aim of this study is to develop a clinical grade method for the generation of TCR transduced virus specific T cells for cellular immunotherapy. CMV and EBV specific T cells were isolated from healthy individuals using pentamers in combination with clinical grade available anti-biotin magnetic beads. Isolation by pentamer-coated beads induced stimulation, expansion and efficient transduction of virus specific T cells, leading to the generation of cell lines with high frequencies of virus specific (>80%) and transduced (20–40%) T cells. T cells were transduced with multi-cistronic retroviral vectors encoding the α and β chains of the HA-2 TCR linked by an IRES or 2A-like sequence. No differences in transduction efficiency and TCR surface expression were observed between the IRES and 2A-like vectors. The transduced virus specific T cells were shown to exhibit dual specificity and tetramer staining of the introduced TCR correlated with specific lysis of target cells endogenously-expressing HA-2. Furthermore, variation in surface expression of the introduced TCR was observed between T cells with different virus specificities. T cells directed against the HLA-A1 epitope of CMV-pp50, for example, efficiently expressed the HA-2 TCR, whereas T cells specific for the HLA-B8 epitope of EBV-EBNA-3A did not express the introduced TCR. Functional analyses demonstrated that TCR-transduced pp50 specific T cells were dual specific, recognizing HA-2 as well as pp50 positive target cells, whereas TCR-engineered EBNA-3A specific T cells were primarily EBNA-3A specific. The efficiency of surface expression of the transferred TCR was shown to be determined by intrinsic properties of the TCRs, illustrating that for TCR gene transfer purposes TCRs need to be selected that exhibit high competition potential, whereas recipient T cells need to express endogenous TCRs with low competition potential. For clinical application, TCRs will be transferred to virus specific T cells selected for their capacity to efficiently express the introduced TCR without loss of virus specificity. The safety, clinical and immunological efficacy of TCR gene transfer to virus specific T cells as cellular anti-tumor immunotherapy after alloSCT will be investigated in a clinical phase I/II study.

Interferon-gamma expressing EBV LMP2A-specific T cells for cellular immunotherapy

To generate therapeutic T cells for adoptive immunotherapy, T cells specific to Epstein-Barr virus LMP2A were enriched on the basis of antigen-specific production of interferon-gamma (IFNγ). The enriched T cells, contained over 60% LMP2A-specific effectors, were polyclonal and targeted multiple LMP2A epitopes. A high proportion of the enriched T cells produced the Th1 cytokines interleukin (IL)-2 and granulocyte monocyte colony stimulating factor, while few cells expressed the Th2 cytokines IL4 and IL10. The enriched T cells specifically lysed LMP2A-expressing target cells, with concomitant production of IFNγ and surface expression of CD107, suggesting the involvement of the granule exocytosis-mediated cytolytic pathway. In addition, the enriched T cells expressed CD45RO, CD28 and CD27, but not CD45RA, consistent with a differentiation stage capable of self-renewal for long-term persistence. LMP2A-specific T cells enriched based on IFNγ-production may provide improved efficacy for the treatment of Epstein-Barr virus related malignancy.

“Designer” Cells for Cellular Immunotherapy: Zinc Finger Nuclease (ZFN) Stimulated Targeted Recombination for the Simultaneous Disruption of the Endogenous Glucocorticoid Receptor and Site-Specific Addition of the IL13-Zetakine.

Immunotherapy utilizing genetically-modified cytolytic T-lymphocytes (CTL) provides a promising therapeutic approach for treating a variety of diseases, including cancer.We have demonstrated that CTLs encoding a chimeric T-cell receptor (IL13-zetakine), consisting of an extracellular IL-13 domain and a cytoplasmic CD3 domain, can be re-directed to target malignant glioma both in vitro and in animal models. This chimera re-targets the antigen-specific effector functions of modified CTLs to recognize glioblastomas due to the high expression of IL13R in these tumors. However, practical application of this approach is limited by the fact that patients undergoing surgical resection of the tumor often require treatment with glucocorticoids to control the resulting inflammation. Such treatment blocks the activity of the re-directed CTL clones and thus inhibits their therapeutic action.To overcome this limitation and render these tumor-specific CTLs resistant to glucocorticoids we have chosen to employ engineered ZFNs to specifically disrupt the endogenous glucocorticoid receptor (GR) gene. Heterodimeric ZFNs, consisting of the cleavage domain of the restriction enzyme FokI linked to engineered zinc finger DNA-binding domains, can be designed to specifically cleave a predetermined site in the genome. We have shown that these ZFN-induced double strand breaks can promote homologous recombination with high efficiency. In the present study we have investigated the use of ZFNs to simultaneously effect functional inactivation of human GR via specifically targeting the integration of the IL13-zetakine expression cassette into the GR locus itself. We can show that GR-specific ZFNs cleave their intended target sequences with high specificity and efficiency - resulting in the disruption of GR and the creation of glucocorticoid resistant cells. Moreover, we can demonstrate that these ZFNs coupled with an appropriate IL13-zetakine containing donor-DNA molecule can stimulate the integration of this chimeric T-cell receptor directly into the GR locus. Thus, this procedure results in the simultaneous knockout of GR and addition of the IL13-zetakine in a genetically defined manner.These data support the notion that ZFN-modified cells can be engineered to express chimeric antigen receptors from a predetermined genomic locus and may provide a general approach to generating effective cellular immunotherapy strategies.

Highly Efficient Expansion of Human CD4+CD25+ Regulatory T Cells for Cellular Immunotherapy in Patients with Graft-Versus-Host Disease

CD4+CD25+ regulatory T cells (T(REG)) are engaged in the regulation of murine and human immune responses as well as graft-versus-host disease (GvHD) after allogeneic stem-cell transplantation. Despite their suppression of GvHD they do not impair graft-versus-tumor activity in the mouse, which makes T(REG) especially attractive candidates for cellular immunotherapy. T(REG) comprise only 5% to 10% of CD4+ T cells in peripheral blood and are naturally anergic, which prevented their use as therapeutic suppressor cells in the context of autoimmune or alloimmune reactions so far. We therefore developed an in vitro expansion protocol for human T(REG), breaking their anergy with anti-CD3/anti-CD28-coupled paramagnetic beads and a combination of interleukin (IL)-2 and IL-15. Highly purified human T(REG) can be expanded 285-fold to 1000-fold within 20 days and keep their phenotype as well as all their suppressor functions even in the context of stimulation with mature allogeneic dendritic cells. However, we demonstrate that FoxP3 is not a reliable marker for human T(REG) as it is transiently inducible in CD4+CD25- cells upon activation with cytokines or via their T cell receptor. In addition, we successfully expanded CD4+CD25+ cells from patients after allogeneic stem-cell transplantation with or without GvHD and show that different suppressor functions might be lost independently, demonstrating that human T(REG) biology is likely more complicated than previously thought.

Human dendritic Langerhans cells generated in vitro from CD34+ progenitors can prime naive CD4+ T cells and process soluble antigen.

Earlier studies have concluded that fresh Langerhans cells (LC) are able to capture and process native Ags, whereas cultured LC have lost these functions while acquiring the capacity to prime naive T cells. Herein we studied the functions of human dendritic/Langerhans cells (d-Lc) generated in vitro by culturing CD34+ hemopoietic progenitor cells in the presence of granulocyte-macrophage CSF (GM-CSF) + TNF-alpha. Less than 50 d-Lc were found to strongly stimulate the proliferation of 2.5 x 10(4) allogeneic naive CD4+ T cells. Furthermore, six to 50 d-Lc induced half-maximal proliferation of naive syngeneic CD4+ cord blood T cells, in the presence of picomolar concentrations of superantigens. During the alloreaction, the CD4+ T cells were expanded up to 100-fold within two successive stimulation cycles with the same d-Lc, and the recovered T cells were specific for the d-Lc alloantigen. HLA-matched tetanus toxoid (TT)-specific T cell clones were found to proliferate in response to TT presented by CD1a+ d-Lc. Finally, electron microscopy demonstrated that CD1a+ d-Lc were able to capture an Ag (gold-labeled Igs) through receptor-mediated endocytosis. Thus, in vitro generated d-Lc can prime naive T cells and process native Ags, a property that might eventually prove useful for priming Ag-specific naive T cells for cellular immunotherapy.