Immunomagnetic separation in LAK generation in patients with breast cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15220-e15220
Author(s):  
Tatiana V. Shamova ◽  
Oleg I. Kit ◽  
Anastasia O. Sitkovskaya ◽  
Elena Yu. Zlatnik ◽  
Elena S. Bondarenko ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Nk Cells ◽  
Immunomagnetic Separation ◽  
Target Cells ◽  
Double Positive ◽  
Immune Effector ◽  
Hla Dr ◽  
Ifn Γ

e15220 Background: The purpose of the study was to evaluate the effect of IL-2 and INF-γ on the proliferation and phenotype of lymphocytes in patients with breast cancer (BC) after T-regs removal from the pool in vitro. Methods: Lymphocytes were isolated from the blood of 11 patients with stage II BC, T-regs were removed by immunomagnetic separation, and samples were cultured for 6 days with cytokines: IL-2 - 0.1/1 μg, INF-γ - 10 IU and their combinations. Results: The level of NK cells in the samples with IL-2 was maintained throughout the experiment, with IFN-γ - gradually decreased, and in the control (samples without stimulation) it decreased sharply. In experimental samples (IL-2, IL-2+IFN-γ), the amount of NKT remained at the initial level by day 6, and in the control it decreased by 2 times (p < 0.05). The number of CD335+ NK cells with high cytotoxicity against target cells increased after 3 days in the test samples with IL-2 by 5.2 times, and after 6 days - by 11.6 times in comparison with the control (p < 0.05). On day 6, the level of double-positive T cells increased by 1.8 times in co-culturing with IL-2+INF-γ (1 μg/10 IU), (p < 0.05). The percentage of double-negative T cells increased in the dynamics of cultivation from days 1 to 6, in some cases statistically significantly (IL-2 - 1 μg: by 2 times; IL-2+INF-γ - 0.1 μg/10 IU: by 1.8 times; p < 0.05). The percentage of CD4+CD38+ cells on day 6 of incubation with IL-2 at both doses increased by 1.8-2 times compared with the control, and CD8+CD38+ by 4-6 times. The percentage of CD4+HLA-DR+ cells after 6 days of cultivation with IL-2 increased by 6 times, and CD8+HLA-DR+ by 8-9 times. Cultivation with INF-γ reduced the stimulating effect, and in some cases it did not appear. The combined effect of IL-2 and INF-γ on day 6 led to an increase in the proportion of CD4+CD25+CD127dim cells by 1.8 times in comparison with the control (p < 0.05), and when exposed to INF-γ only, the number of these cells remained at the control levels. Conclusions: INF-γ is not an effective inducer of activation of immune effector cells, while the effect of IL-2, both alone and in combination with INF-γ, leads to a significant increase in the proportion of cytotoxic cells, as well as to activation of the T cell unit, including CD4 + CD25 + CD127dim.

scholarly journals Engineered IL-7 Receptor Enhances the Therapeutic Effect of AXL-CAR-T Cells on Triple-Negative Breast Cancer

2020 ◽  
Vol 2020 ◽  
pp. 1-13 ◽  
Author(s):  
Zhenhui Zhao ◽  
Yan Li ◽  
Wei Liu ◽  
Xun Li
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Xenograft Model ◽  
Target Cells ◽  
Car T Cells ◽  
Car T

Triple-negative breast cancer (TNBC) is a very aggressive malignant type of tumor that currently lacks effective targeted therapies. In hematological malignancies, chimeric antigen receptor T (CAR-T) cells have shown very significant antitumor ability; however, in solid tumors, the efficacy is poor. In order to apply CAR-T cells in the treatment of TNBC, in this study, constitutively activated IL-7 receptor (C7R) that has been reported is used to enhance the antitumor function of constructed CAR-T cells by ourselves. Using in vitro coincubation experiments with target cells and in vivo antitumor experiments in mice, we found that the coexpressed C7R can significantly improve the activation, cell proliferation, and cytotoxicity of CAR-T cells. In addition, the in vivo experiments suggested that the enhanced CAR-T cells displayed significant antitumor activity in a TNBC subcutaneous xenograft model, in which in vivo, the survival time of CAR-T cells was prolonged. Together, these results indicated that CAR-T cells that coexpress C7R may be a novel therapeutic strategy for TNBC.

scholarly journals Effector Mechanisms of CD8+ HLA-DR+ T Cells in Breast Cancer Patients Who Respond to Neoadjuvant Chemotherapy

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6167
Author(s):  
Rubén Osuna-Gómez ◽  
Cristina Arqueros ◽  
Carla Galano ◽  
Maria Mulet ◽  
Carlos Zamora ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
T Cell ◽  
Neoadjuvant Chemotherapy ◽  
Hormone Receptors ◽  
Cytotoxic T Lymphocyte ◽  
Breast Cancer Patients ◽  
Effective Response ◽  
Hla Dr ◽  
Ifn Γ

Cytotoxic T lymphocyte (CTLs) activation is an independent predictor of response to neoadjuvant chemotherapy (NACT) in breast cancer (BC) patients. Here, we go deeper into the function of CD8+ HLA-DR+T cells from NACT treated HER2 negative BC patients. Flow cytometry analysis revealed that CD8+ HLA-DR+ T cell percentage was increased in NACT responder (R) compared to non-responder (NR) patients. R patients with ER-/PR- hormone receptors had the highest CD8+HLA-DR+T cell frequencies, while no differences were found when patients were classified according to cancer stage or menopause status. Interestingly, the cytotoxicity and production of anti-tumor cytokines were enhanced when CD8+ HLA-DR+ T cells from healthy donors were cultured with plasma from R, but not from NR patients. The induced anti-tumor profile of CD8+ HLA-DR+ T cells was associated with plasmatic IL-12 and IFN-γ levels, increased cytokines in R patients. IL-12 or IFN-γ neutralization decreased cytotoxic activity and TNF-α production by cultured CD8+ HLA-DR+ T cells in R plasma presence. All these data suggest that an effective response to NACT in BC patients is associated with increased IL-12 or IFN-γ levels involved in the induction of cytotoxic and pro-inflammatory mechanisms in CD8+ HLA-DR+ T cells.

scholarly journals Multi-Antigen Primed T Cells Promote Apoptosis of Acute Myeloid Leukemia (AML)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4794-4794
Author(s):  
Ebtesam Nafie ◽  
Mathias Oelke ◽  
Melissa Valerio ◽  
Sojung Kim ◽  
Ivan Rodriguez ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Time Dependent ◽  
Target Cells ◽  
Ifn Γ ◽  
Acute Myeloid

Abstract Introduction Acute myeloid leukemia (AML), the most common acute leukemia in adults, is characterized by uncontrolled proliferation of immuature myeloid cells. Despite newly approved drugs, AML remains largely incurable due to the persistence of the leukemia stem cell (LSC) population which lie quiescent in the bone marrow niche. Immunotherapy has potential to eradicate LSCs, however, no unique LSC immunophenotype has been identified. Moreover, it is necessary to simultaneously target multiple antigens (Ags) to prevent immune escape and to overcome refractory disease. We present in vitro studies in support of a therapeutic platform capable of targeting multiple intracellular Ags which could meet this challenge. The adoptive transfer of activated T cells primed to engage diverse AML associated epitopes by ex vivo exposure to artificial Ag presenting cells (aAPC) has the potential to eliminate both primary leukemia blasts and LSCs. Hypothesis Ex vivo enrichment and expansion (E+E) of antigen-educated CD8+ T cells recognizing 5 peptides derived from 3 proteins, Cyclin A1, PRAME and WT1, can selectively identify, engage, and kill AML cell lines or patient-derived (PD) AML blasts in a HLA A*02:01 restricted manner in vitro. Materials and Methods T cells from the peripheral blood mononuclear cell fraction of a healthy HLA A*02:01 donor were enriched for antigen-educated CD8+/CD4 -T cells. These cells were cultured with nanoparticles decorated with the 5 peptides and a costimulatory protein, resulting in the activation and expansion of those T cells expressing the cognate T cell receptors. These cells are composed of ~97% abT cells, 3% gdT cells and ~13% T scm, 41.5% T cm, 39.5%T em, 6%T emra and 1% T n. Results Ex vivo expanded educated T cells exhibit target-specific anti-AML activity. T cell mediated cell apoptosis of HLA-matched THP1 cells is dose and time-dependent. At 10:1 effector to target (E:T) ratio, ~28% apoptosis occurred at 24 hrs, while apoptosis was at basal levels when antigen non-educated T cells were used (data not shown). Studies were extended to PD AML cells (Fig. 1A: 012; Fig. 1B: 415) where antigen educated T cells elicited rapid (&lt;16 hrs) and extensive (~90%) apoptosis of target PD AML cells at all E:T ratios examined. Time lapse photography of T cell/PD AML incubations revealed antigen-educated T cells clustered around AML cells (Fig. 2A), a fraction of the latter disappearing over the course of 12 hrs while PD AML cells incubated with non-educated T cells (Fig. 2B) remained viable over 12 hrs. Furthermore, there is little or no T cell movement or clustering in the wells with unprimed, non-active T cells. Release of IFN-γ by educated T cells. T cells (Fig.3A: antigen-educated through E+E) were incubated at E:T::5:1 for 24 to 48 hrs and IFN-γ in supernatants measured. The fold difference over non-educated T cells incubated with AML cells for the same time is shown and can reach over 5-fold. IFN-γ accumulation was time-dependent where antigen-educated T cells were combined with HLA-A2 matched THP1 or PD AML cells (012, 415, 470). Educated T cells were not active against target cells lacking HLA-A2 (K562) demonstrating HLA restricted killing (Fig. 3B). Additionally, antigen-educated T cells incubated without any target released slightly more IFN-γ than non-educated T cells under similar conditions but AML cells fail to stimulate IFN-γ release from non-educated T cells (data not shown). Conclusions We demonstrate HLA restricted cytotoxic activity of antigen-educated T cells against THP1 AML cells and PD AML blasts as shown by flow cytometry and microscopy. Consistent with target cell death, the supernatants from assays with antigen-educated T cells and HLA A*02:01 AML target cells exhibited over 5-fold more IFN-γ than media from assays of non-educated cells under identical conditions. Under these in vitro conditions, PD AML blasts were more readily killed than THP1 cells perhaps due to higher target antigen density (data not shown). These results support the use of multi-antigen-educated T cells for adoptive transfer to treat AML. To investigate the safety and establish the recommended phase II dose, a multi-center Phase I clinical study is underway in relapsed AML post-allo-HCT (NCT 04284228). Future studies will incorporate new antigens to enable broader targeting of a heterogeneous population of AML within and across patients Figure 1 Figure 1. Disclosures Oelke: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy.

scholarly journals Efficacy of Targeting SARS-CoV-2 by CAR-NK Cells

2020 ◽  
Author(s):  
Minh Ma ◽  
Saiaditya Badeti ◽  
Ke Geng ◽  
Dongfang Liu
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Clinical Success ◽  
Neutralizing Antibody ◽  
Third Party ◽  
Target Cells ◽  
Novel Approach ◽  
Cell Immunotherapy

ABSTRACTSARS-CoV-2, which causes COVID-19 disease, is one of greatest global pandemics in history. No effective treatment is currently available for severe COVID-19 disease. One strategy for implementing cell-based immunity involves the use of chimeric antigen receptor (CAR) technology. Unlike CAR T cells, which need to be developed using primary T cells derived from COVID-19 patients with lymphopenia, clinical success of CAR NK cell immunotherapy is possible through the development of allogeneic, universal, and ‘off-the-shelf’ CAR-NK cells from a third party, which will significantly broaden the application and reduce costs. Here, we develop a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2. CAR-NK cells were generated using the scFv domain of CR3022 (henceforth, CR3022-CAR-NK), a broadly neutralizing antibody for SARS-CoV-1 and SARS-CoV-2. CR3022-CAR-NK cells can specifically bind to RBD of SARS-CoV-2 and pseudotyped SARS-CoV-2 S protein, and can be activated by pseudotyped SARS-CoV-2-S viral particles in vitro. Further, CR3022-CAR-NK cells can specifically kill pseudo-SARS-CoV-2 infected target cells. Thus, ‘off-the-shelf’ CR3022-CAR-NK cells may have the potential to treat patients with severe COVID-19 disease.

in vitro generation of IFN-γ-producing Listeria-specific T cells is dependent on IFN-γ production by non-NK cells

1995 ◽  
Vol 160 (2) ◽  
pp. 211-216 ◽  
Author(s):  
Fei Song ◽  
Goro Matsuzaki ◽  
Masao Mitsuyama ◽  
Kikuo Nomoto
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Ifn Γ

MicroRNA-Deficient Murine NK Cells Exhibit Impaired Development and Survival but Enhanced IFN-γ Production In Vitro and In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 357-357 ◽  
Author(s):  
Ryan P Sullivan ◽  
Jeffrey W Leong ◽  
Stephanie E Schneider ◽  
Catherine R Keppel ◽  
Elizabeth Germino ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Biology ◽  
Nk Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
Target Cells ◽  
Ifn Γ ◽  
Nk Cell Receptor

Abstract Abstract 357 NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small regulatory RNAs that control a wide variety of cellular processes by specific targeting of mRNA 3'UTRs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. While miRNA expression and regulation of adaptive T and B lymphocytes are well established, their role in the regulation of NK cell biology remains unclear. We postulated that miRNAs serve an essential role in orchestrating NK cell development and activation. To test this hypothesis, we combined lymphocyte-restricted hCD2-Cre transgenic, Rosa26-YFP-Cre-reporter, and Dicer1 ‘floxed' mice. In this model, 25–50% of Dicer1 wt/wt NK cells are YFP+ marking expression of Cre. As expected, YFP+ NK cells from Dicer1 fl/fl and fl/wt mice were confirmed to excise Dicer1, and exhibit decreased total miRNA content based on Nanostring profiling and real-time qPCR (Dicer1 fl/fl: P<0.001, fl/wt: P<0.01). MiRNA-deficient Dicer1 fl/fl mice exhibited reduced YFP+ NK percentages (spleen Dicer1 fl/fl: 14±4%, fl/wt: 35±7%, wt/wt 36±7%, P<0.001) as well as reduced absolute numbers of YFP+ NK cells [spleen Dicer1 fl/fl: 3.4±0.6×10E5, fl/wt: 6.3±1.7×10E5, wt/wt 6.1±.99×10E5, P<0.01]. In addition, Dicer1 fl/fl mice had reductions NK cell precursors in the BM (stage 2–3 NK precursors mean decrease 70±14% in Dicer1 fl/fl compared to wt/wt, P <0.01). Further, Dicer1 fl/fl NK cells exhibited reduced survival ex vivo when cultured in medium (P<0.01), low dose- (P<0.01), or high dose-IL-15 (P<0.01). These data collectively indicate that Dicer1-dependent miRNAs regulate NK cell development and homeostasis, and the net effect of miRNA loss is impaired NK development and/or survival. However, in our model Dicer1-deficient mature NK cells exhibited enhanced functionality; a finding that contrasts to less NK selective miRNA-deficient NK cell models (Bezman et al. J Immunol 185:3835, 2010). Degranulation (CD107a+, a surrogate for cytotoxicity) was enhanced in vitro in response to YAC-1 tumor target cells (P<0.05) and activating NK cell receptor ligation (P<0.001). This was unlikely due to alteration in activating NK cell receptor expression since the surface density of NKG2D and NKp46 were not affected by miRNA deficiency. Moreover, interferon-gamma (IFN-γ) production was enhanced in vitro in miRNA-deficient NK cells in response to IL-12+IL-15 (P<0.01), YAC-1 tumor target cells (P<0.01), and activating NK cell receptor ligation (P<0.001). Further, evaluation of NK cells 36 hours after infection with MCMV resulted in significantly increased IFN-γ production (% NK YFP+IFN-γ+) in Dicer1 fl/fl (64± 4.9%) vs. fl/wt (52±11%, p <0.01) or vs. wt/wt (41±6%, p <0.001) in vivo. MiRs-15/16 were identified as abundant miRNAs in NK cells that had reduced expression in Dicer1 fl/fl NK cells, and are predicted to target the murine IFN-γ 3'UTR. This targeting was validated in vitro, by transfecting 293T cells with miRNA-15/16 or control over-expression vectors and a sensor plasmid that places luciferase under the control of the murine IFN-γ 3'UTR (34% decrease, P<0.01). Moreover, the targeting was direct, since miR-15/16 targeting of IFN-γ was abrogated after mutation of two predicted binding sites in the IFN-γ 3'UTR. These data indicated that miR-15/16 may regulate IFN-γ translation by resting NK cells. Thus, our study suggests that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival and/or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell development/survival and effector function. Disclosures: No relevant conflicts of interest to declare.

Transfer of Immunity Using Vaccinate and Boost Strategy with Bispecific Antibody Armed T Cells (BATs) in Metastatic Breast Cancer Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3459-3459
Author(s):  
Archana Thakur ◽  
Joseph P. Uberti ◽  
Voravit Ratanatharathorn ◽  
Lawrence G. Lum
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Breast Cancer Cells ◽  
Bispecific Antibody ◽  
Metastatic Breast ◽  
Gm Csf ◽  
Cancer Immunity ◽  
Ifn Γ ◽  
Breast Cancer Immunity

Abstract Background. In our recent phase I immunotherapy (IT) trial in 23 women with metastatic breast cancer (MBC), 8 infusions of activated T cells (ATC) armed with anti-CD3 x anti-HER2 bispecific antibody (HER2Bi) given in combination with interleukin-2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) induced specific anti-breast cancer immunity. This study investigated whether specific cellular and humoral anti-breast cancer immunity induced by infusions of HER2 bispecific antibody armed T cells (BATs) could be transferred after high dose chemotherapy (HDC) and stem cell transplant (SCT) in MBC patients. Methods. T cell were activated with OKT3 and expanded in IL-2. ATC were harvested, armed with HER2Bi, and cryopreserved in 8 doses for twice weekly infusions for 4 weeks along with IL-2 and GM-CSF. Seven to 14 days after the last infusion of BATs, the patient was leukapheresed again to obtain and expand immune T cells. Multiple infusions of immune ATC after the HDC and SCT were given to boost the transferred cellular and humoral immune responses which were monitored up to 24 months. Results. Six of 8 MBC patients enrolled were evaluable in the protocol, no dose-limiting toxicity, delays in engraftment, or life-threatening infections were observed. Five of 6 evaluable patients exhibited increased anti- breast cancer cytotoxicity and IFN-γ Elispots after vaccination with BATs and up to 18 months post SCT. Serum and culture supernatants from in vitro antibody synthesis assay showed gradual increases in anti-SK-BR-3 IgG levels after SCT. Serum cytokine profile showed increases in IL-12 and Th1 cytokines. One out of 6 evaluable patients who rapidly progressed showed poor immune response, had high serum levels of Th2 cytokines and no evidence of transfer of immunity. Flow cytometry analysis of Vβ repertoire pattern in PBMC collected post IT and post SCT indicate transfer of the major Vβ clones post SCT that produced IFN-γ upon stimulation with breast cancer cells (Fig. 1). A significant correlation (r=1.0; p<0.002) between immune ATC cytotoxicity directed at breast cancer cells and time to progression (TTP) suggests that more robust vaccinations with a Th1 shift in cytokine profiles can lead to clinical benefit (Fig. 2). Conclusions. Our pilot study suggests that cellular and humoral immunity was transferred and boosted using immune T cells after SCT. There was robust reconstitution of T and B cell functions early after SCT as evidenced by CTL and NK activity, IFN-γ EliSpots, in vivo/in vitro antibody synthesis, and Th1 cytokine responses.BATs induced endogenous anti- breast cancer cellular, humoral and innate immunity that could be detected after SCT and may have provided clinically meaningful anti-tumor immunity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lymphocyte Co-Expression of CD38/HLA-DR Is a Marker for Anti-Tumor Activity in Hodgkin Lymphoma in Vitro: Evidence for a PD1-Independent Mechanism

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4129-4129
Author(s):  
Meret Henry ◽  
Steven Buck ◽  
Batool Al-Qanber ◽  
Manisha Gadgeel ◽  
Sureyya Savasan
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Nk Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Incubation Time ◽  
Hla Dr ◽  
Anti Tumor Activity

Abstract Introduction The immunosuppressive tumor microenvironment has become of increasing interest in Hodgkin lymphoma (HL), in particular due to the recent success of checkpoint inhibitors (CI) as part of therapeutic strategies. The mechanism of these agents action in HL, however, remains elusive. Some studies have shown that cytotoxic T-lymphocytes may not be responsible for clinical efficacy, and that tumor-associated macrophages may also be targeted by these agents. We recently described a positive association between increasing proportion of CD38/HLA-DR co-positive lymphocyte in affected nodal tissue and clinical outcome in children with HL. We developed an in vitro model to further evaluate our findings, in this study. Methods Peripheral blood mononuclear cells collected from healthy volunteers were used to generate effector cytoxic T lymphocytes (CTL), natural killer (NK) and CD4 positive T (CD4+T) cells by incubating with IL-15 and IL-2. Both a short-term (4-day) incubation and a longer incubation was used to generate lymphocytes with low-level and higher CD38/HLA-DR co-positive cells, respectively. CD3/CD8 co-positive CTL, NK (CD56-positive/CD3-negative) and CD4-positive T cells were isolated using MACS system. In addition to CD38/HLA-DR expression, isolated cells were evaluated for expression of CD279 (PD-1) and CD274 (PDL-1) by flow cytometry. Two HL cell lines, HDLM-2 (nodular sclerosis HL) and KMH-2 (mixed cellular HL) were used as targets in effector cell-mediated cytotoxicity experiments. Cells were incubated at various effector:target ratios, and HL cell death was measured with a flow cytometric cell-mediated cytotoxicity assay. The resuts were corrected for alloreactive cell elimination. Blocking antibodies against PD-1 and PDL-1 were used for cytoxicity experiments, using CTL and CD4-positive T cells as effector cells, as well. Results Higher CD38/HLA-DR co-expression was seen in CTL after longer incubation (day 11) with IL-2 and IL-15, while peak expression was reached earlier in NK cells (day 4). Both CTL and NK cells demonstrate cytotoxicity against HDLM-2 and KMH-2 cell lines. Cytotoxicity was increased, as evidenced by lower (lethal unit 20%) LU20 effector:target ratio levels, at day 11 of incubation (compared to day 5) for CTL for both cell lines: 0.5 (day 5) vs 0.29 (day 11) for KMH-2 (p=0.02) and 1.1 (day 5) vs. 0.4 (day 11) for HDLM-2 (p=0.15) cells. There was no difference between cytotoxicity with CTL compared to NK cells for either cell line at day 11. CTL, NK cells, and CD4-positive T cells all expressed both PD-1 and PDL-1, with no difference between cell types in percent positivity or mean channel intensity after cytokine incubation. PD-1 expression increased with incubation time in CTL, peaking at 50.5% on day 10, as opposed to NK cells, where it peaked at day 5-7. The co-expression of CD38/HLA-DR was higher in CTL compared to CD4-positive T cells (79% vs. 37% at day 7). PD-1 blockade did not inhibit CTL or CD4-positive T cell-mediated cytotoxicity in either cell line. This was also the case after PDL-1 blockade on tumor cells, indicating PD-1/PDL-1 pathway-independent HL cell elimination by CD38/HLA-DR co-positive CTL and CD4-positive T cells. Discussion Our results indicate that higher CD38/HLA-DR co-expression in CTL was associated with superior elimination of HL cells in vitro supporting our recent in vivo findings. Induced co-expression of CD38/HLA-DR was higher in CTL compared with NK cells and reached a peak level earlier in NK cells. Increasing expression of PD-1 and PDL-1 was observed for all three effectors cells with longer incubation time. Interestingly, there was no change in CTL or CD4-positive T cell-mediated cytoxicity of HL cells following PD-1 and PDL-1 blockade in vitro. In conclusion, both CTL and NK cells are effective against HL cells. The anti-tumor activity of CTL correlated with increasing levels of CD38/HLA-DR expression in our experimental model. Cytotoxicity was enhanced despite increased expression of PD-1, and, therefore, appears to be independent of the PD-1/PDL-1 pathway, suggesting involvement of other operational mechanisms. This model could be useful in further elucidating the interactions between immune effectors and HL cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional Characterization of Gamma Delta T Cells and Allogeneic Activated NK Cells As Effector Cells for Tafasitamab (MOR208)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3801-3801
Author(s):  
Jung Hyun Her ◽  
Dominik Pretscher ◽  
Sungyoo Cho ◽  
Yu-Kyeong Hwang ◽  
Timm Hoeres ◽  
...  
Keyword(s):  
T Cells ◽  
Combination Therapy ◽  
B Cell ◽  
Nk Cells ◽  
Effector Cell ◽  
Γδ T Cells ◽  
Target Cells ◽  
Effector Cells

Introduction Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal antibody that binds to the human B cell surface antigen CD19. CD19 is broadly and homogeneously expressed across different B cell malignancies, including diffuse large B cell lymphoma (DLBCL), and amplifies B cell receptor signaling and induces tumor cell proliferation. Tafasitamab is currently in clinical development in patients with relapsed or refractory DLBCL in combination with the immunomodulatory drug lenalidomide (L-MIND study) and the chemotherapeutic agent bendamustine (B-MIND study). The modes of action of tafasitamab comprise antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity (apoptosis). Tafasitamab carries two amino acid exchanges in the Fc region to increase its affinity to Fcγ receptors, including FcγRIIIa. FcγRIIIa plays a key role in mediating ADCC and is expressed on the surface of natural killer (NK) cells, as well as the majority of γδ T cells. MG4101 (a novel therapeutic agent consisting of cryopreserved, ex vivo-expanded, highly activated NK cells) has demonstrated potent anticancer activity in preclinical in vitro and in vivo studies. Currently, MG4101 is in clinical development in patients with malignant lymphoma and advanced solid tumors. Here, we have characterized two FcγRIIIa receptor-expressing cell types, γδ T cells and NK cells (MG4101), as effector cells for tafasitamab in vitro and explored the concept of supplementing MG4101 during tafasitamab therapy using disseminated in vivo models of non-Hodgkin's lymphoma. Methods γδ T cells (CD3+/γδ T cell receptor+) were derived from different donors by stimulation of peripheral blood mononuclear cells with zoledronate/IL-2 for 9-10 days. These were applied as effector cells in in vitro ADCC assays with tafasitamab in Mino and Jeko-1 mantle cell lymphoma (MCL) cell lines, as well as primary patient-derived chronic lymphocytic lymphoma (CLL) and MCL cells. Further, effector cell activity of MG4101 was assessed using tafasitamab-mediated ADCC assays in Raji and Ramos Burkitt's lymphoma cells. The concept of combining tafasitamab with allogeneic effector cell therapy in vivo was studied in two therapeutic survival models of disseminated lymphoma in SCID mice. In the Raji model, tafasitamab (0.05 µg/mouse) was given on Day 1 after intravenous (IV) tumor inoculation, while MG4101 (2x107 cells/mouse) was given on Days 1, 3, 6, 8 and 10. In the Ramos model, tafasitamab (10 mg/kg) and MG4101 (2x107 cells/mouse) were applied twice weekly for 3 weeks starting on Days 3 and 4, respectively, after IV tumor inoculation. Results Tafasitamab in combination with γδ T cells showed distinctly increased ADCC in Mino and Jeko-1 target cells in vitro, compared with a negative control IgG1 antibody. ADCC assays with patient-derived CLL and MCL target cells confirmed tafasitamab-mediated cytotoxic activity and demonstrated a clear enhancement in activity compared with the non-Fc-enhanced version of tafasitamab that was unable to induce substantial cytotoxicity. In vitro ADCC assays with tafasitamab and MG4101 on Raji and Ramos cell lines confirmed potent effector cell activity of the ex vivo-expanded, cryopreserved, allogeneic NK cells. In the disseminated Raji survival model, combination therapy with a single low dose of tafasitamab (0.05 µg) and MG4101 resulted in a distinct increase in survival of the mice with an increased life span (ILS) of 100% compared with monotherapy (ILS of 57% for tafasitamab and 50% for MG4101). Of note, the combination demonstrated a substantial and more than additive enhancement in survival in the more therapeutic Ramos survival model (Figure 1). Combination therapy with tafasitamab (10 mg/kg) and MG4101 NK cells resulted in superior antitumor activity (ILS of 103%) compared with either tafasitamab monotherapy (ILS of 49%) or MG4101 alone (ILS of 25%). Conclusions FcγRIIIa-expressing immune cell types, including NK cells and γδ T cells, are potent effector cells for tafasitamab-mediated ADCC. Combination therapy with tafasitamab and allogeneic MG4101 NK cells in vivo demonstrated a more than additive survival benefit compared with tafasitamab or MG4101 monotherapy in a disseminated therapeutic lymphoma model. Combination of tafasitamab supplemented with immune effector cells could represent a promising new approach for lymphoma therapy. Disclosures Her: GC LabCell: Employment, Patents & Royalties. Cho:GC LabCell: Employment, Patents & Royalties. Hwang:GC LabCell: Employment, Equity Ownership, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties. Steidl:MorphoSys AG: Employment. Patra:MorphoSys AG: Employment. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties.

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