scholarly journals 606 SAR444245 (THOR-707), an engineered non-alpha IL-2, enhances NK mediated antibody-dependent cellular cytotoxicity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A636-A636
Author(s):  
Julie-Ann Gavigan ◽  
Nicole Acuff ◽  
Christen Buetz ◽  
Michael Lampa ◽  
Chaomei Shi ◽  
...  
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
Nk Cells ◽  
Cell Lines ◽  
Cell Biology ◽  
Nk Cell ◽  
Cellular Cytotoxicity ◽  
Natural Amino Acid ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Dose Dependent

BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-natural amino acid serving as a bioconjugation site for a single PEG. The non-natural amino acid is positioned to enable the PEG bioconjugation to obscure block binding to the IL-2 alpha receptor, while retaining near-native affinity with the intermediate affinity βγ IL-2 receptor. The non-alpha features of SAR444245 minimize activation of immune suppressive regulatory CD4+ T cells, while retaining activity on CD8+ T cells and NK cells expressing the IL-2 βγ receptors. NK cells exert anti-tumor activity through antibody dependent cellular cytotoxicity (ADCC) of IgG antibodies as well as antibody independent mechanisms.MethodsHere, we utilized a panel of human primary PBMC based immunoassays and transcriptomic analysis to evaluate whether SAR444245 may improve ADCC function of IgG1 anti-tumor target antibodies.ResultsWe characterized the ability of SAR444245 to enhance the cytolytic function of NK cells towards the prototypic NK target cell K562 as well as to modulate NK cell ADCC in combination with EGFR or CD20-targeting antibodies. In vitro assays demonstrated that SAR444245 can activate NK cells, promote NK cell proliferation and improve cytotoxicity of NK cells against K562 cells and across a panel of human EGFR and CD20 positive cell lines. In PBMC based ADCC assays with 1ug/ml of antibody, SAR444245 improved ADCC function maximally by 9-fold for an anti-EGFR antibody and at 5-fold for an anti-CD20 antibody. SAR444245 exhibited dose-dependent enhancement of NK cell ADCC function. Notably, this activity was observed in cell lines expressing varying levels of EGFR and CD20. SAR444245 treatment was associated with dose dependent increases in NK cell degranulation and IFN-γ production. Transcriptomic profiling revealed that SAR444245 had broad effects on NK cell biology leading to changes in inhibitory and activating receptors.ConclusionsIn summary, these results indicate that SAR444245 can enhance the cytolytic activity of NK cells and enhance the ADCC effect of tumor-directed antibodies by activating NK cells.

scholarly journals Anti-tumor effects of NK cells and anti-PD-L1 antibody with antibody-dependent cellular cytotoxicity in PD-L1-positive cancer cell lines

2020 ◽  
Vol 8 (2) ◽  
pp. e000873 ◽  
Author(s):  
Ji-Eun Park ◽  
Seong-Eun Kim ◽  
Bhumsuk Keam ◽  
Ha-Ram Park ◽  
Soyeon Kim ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Nk Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Nk Cell ◽  
Cancer Cell Lines ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Healthy Donors ◽  
Nk Cell Cytotoxicity

BackgroundAlthough programmed cell death-1/programmed death-ligand 1 (PD-L1) inhibitors show remarkable antitumor activity, a large portion of patients with cancer, even those with high PD-L1-expressing tumors, do not respond to their effects. Most PD-L1 inhibitors contain modified fragment crystallizable region (Fc) receptor binding sites to prevent antibody-dependent cellular cytotoxicity (ADCC) against PD-L1-expressing non-tumor cells. However, natural killer (NK) cells have specific antitumor activity in the presence of tumor-targeting antibody through ADCC, which could enhance NK cell-induced cytotoxicity. We evaluated the antitumor efficacy of ADCC via anti-PD-L1 monoclonal antibodies (mAbs) and NK cells against several PD-L1-positive cancer cell lines.MethodsVarious cancer cell lines were used as target cell lines. Surface PD-L1 expression was analyzed by flow cytometry. IMC-001 and anti-hPD-L1-hIgG1 were tested as anti-PD-L1 mAbs with ADCC and atezolizumab as an anti-PD-L1 mAb without ADCC. NK cell cytotoxicity was measured by 51Cr-release assay and CD107a degranulation assay. Also, live cell imaging was performed to evaluate cytotoxicity in a single-cell level. NK-92-CD16 (CD16-transduced NK-92 cell line) and peripheral blood mononuclear cells from healthy donors, respectively, were used as an effector cell. FcγRIIIa (CD16a)-V158F genotyping was performed for healthy donors.ResultsWe demonstrated that the cytotoxicity of NK-92-CD16 cells toward PD-L1-positive cancer cell lines was significantly enhanced in the presence of anti-PD-L1 mAb with ADCC. We also noted a significant increase in primary human NK cell cytotoxicity against PD-L1-positive human cancer cells when cocultured with anti-PD-L1 mAb with ADCC. Moreover, NK cells expressing a FCGR3A high-affinity genotype displayed higher anti-PD-L1 mAb-mediated ADCC lysis of tumor cells than donors with a low-affinity genotype.ConclusionThese results suggest that NK cells induce an ADCC response in combination with anti-PD-L1 mAbs, which helps promote ADCC antitumor activity against PD-L1-positive tumors. This study provides support for NK cell immunotherapy against high PD-L1-expressing tumors in combination with ADCC through anti-PD-L1 mAbs.

Quantification and Characterization of Peripheral Blood Natural Killer (NK) Cells Mediating Rituximab and Alemtuzumab Dependent Cytotoxicity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2808-2808
Author(s):  
Olaf Penack ◽  
Lars Fischer ◽  
Chiara Gentilini ◽  
Anne M. Asemissen ◽  
Carmen Scheibenbogen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Cell Activation ◽  
Nk Cell ◽  
Surface Expression ◽  
Leukemia Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Specific Lysis ◽  
Flow Cytometric

Abstract Background: Recent data suggest that NK cell mediated antibody dependent cellular cytotoxicity (ADCC) is a major mechanism of action of the anti-CD20 monoclonal antibody (mAb) rituximab and the anti-CD52 mAb alemtuzumab, which are frequently applied in patients with non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. However, the exact mechanisms leading to NK cell activation are not completely understood and the cytotoxic subpopulation of peripheral blood NK cells mediating ADCC remains to be defined. In order to quantify and characterize the NK cells mediating ADCC, we used a novel flow cytometric assay, which detects the lytic granule membrane protein CD107a as a marker for NK cell degranulation. Methods: PBMCs from healthy individuals were coincubated at 37°C for 3 h with different human leukemia and lymphoma cell lines. In each tube, containing 200μl effector/target suspension (4x105 cells), 15μl of PE-Cy5 conjugated anti-CD107a monoclonal antibody was added prior to incubation. To assess antibody dependent cellular cytotoxicity (ADCC) saturating concentrations (10μg/ml) of rituximab or alemtuzumab were used. After the first 1 h 5μl of the secretion inhibitor 2 mM monensin was added. At the end of coincubation cells were stained with mAbs (CD56, CD3, NKG2D, CD69, CD94, NKp30, NK46) for flow cytometry. NK cell-mediated cytotoxicity (specific lysis) was analyzed by flow cytometric detection of propidium iodide uptake. Results: After coincubation with NK sensitive K562 cells up to 6% of CD56+ cells expressed CD107a, indicating that a subpopulation of NK cells releases cytotoxic granules after contact with these target cells. In contrast, coincubation with NK-resistant leukemia cells (ML2, EHEB, DAUDI, RAJI, AM0-1, YT-1) was not followed by an increased surface expression of CD107a. However, when rituximab was added to CD20+ lymphoma or leukemia cells (EHEB, DAUDI, RAJI) we observed that up to 15% of NK cells expressed CD107a after coincubation. In contrast no increased CD107a surface expression was observed when rituximab was added to the CD20− cell lines AMO-0 and YT-1, which excludes unspecific NK cell activation. When alemtuzumab was added to the CD52+ cell lines AMO-1, DAUDI, EHEB, RAJI and YT-1, surface expression of CD107a on NK cells was increased considerably. The majority of degranulating NK cells had the phenotype: CD56dim, CD69+, NKG2D+, NKp30−, NKp46− and CD94−. Furthermore we found that the CD107a assay can also visualize ADCC under clinical conditions as we observed increased numbers of NK cells degranulating in response to CD20+ lymphoma cell lines in patients with non-Hodgkin’s lymphoma treated with rituximab. The number of degranulating NK cells was closely related to the concentration of rituximab and the effector:target ratio, showing a maximum at a ratio of 1:1 and concentrations above 5μg/ml. CD107a surface expression and specific lysis demonstrated a strong positive correlation (r2 = 0.99), confirming that NK cell cytotoxicity can be assessed by this method. Conclusion: The CD107a assay represents a promising new method not only for assessment of natural cytotoxicity on a single cell level but also for determination of ADCC in vitro and in patients treated with mAb. In clinical practice, it may help to find optimal doses and time schedules for the treatment with different mAbs.

Lenalidomide Strongly Enhances Natural Killer (NK) Cell Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) of Rituximab Treated Non-Hodkin’s Lymphoma Cell Lines In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3714-3714 ◽  
Author(s):  
Lei Wu ◽  
Peter Schafer ◽  
George Muller ◽  
David Stirling ◽  
J. Blake Bartlett
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Dependent Manner ◽  
Activation Markers ◽  
Early Results ◽  
Ifn Γ ◽  
Dose Dependent

Abstract Lenalidomide (Revlimid® is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone is for the treatment of multiple myeloma patients who have received at least one prior therapy. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL). Potential mechanisms of action include anti-angiogenic, anti-proliferative and immunomodulatory activities. Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Furthermore, lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo. We have utilized an in vitro ADCC system to assess the ability of lenalidomide to directly enhance human NK cell function in response to therapeutic antibodies, such as rituximab (chimeric anti-CD20 mAb). Isolated NK cells produced little or no IFN-γ in response to IgG and/or IL-2 or IL-12. However, pre-treatment of NK cells with lenalidomide greatly enhanced IFN-γ production by NK cells in a dose-dependent manner. In a functional ADCC assay, NHL cell lines (Namalwa, Farage & Raji) were pre-coated with rituximab and exposed to NK cells pre-treated with lenalidomide in the presence of either exogenous IL-2 or IL-12. After 4 hours in culture the viability of the tumor cells was assessed. Lenalidomide consistently and synergistically increased the killing of tumor cells in a dose-dependent manner and up to >4-fold compared to rituximab alone. Rituximab alone had only a small effect in this model and there was no killing of cells in the absence of rituximab. The presence of either exogenous IL-2 or IL-12 was required to see enhanced killing by lenalidomide. In cancer patients lenalidomide has been shown to increase serum IL-12 levels and is also known to induce IL-2 production by T cells in vitro. Potential mechanisms for enhanced ADCC include increased signaling through NK FCγ receptors and/or IL-2 or IL-12 receptors. However, we found that these receptors are unaffected by lenalidomide, although downstream effects on NK signaling pathways are likely and are being actively investigated. In conclusion, we have shown that lenalidomide strongly enhances the ability of rituximab to induce ADCC mediated killing of NHL cells in vitro. This provides a strong rationale for combination of these drugs in patients with NHL and CLL.

Fc-Engineered NKG2D-IgG1 Fusion Proteins Target Leukemia Cells for Antibody-Dependent Cellular Cytotoxicity (ADCC) of NK Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1537-1537 ◽  
Author(s):  
Julia Hilpert ◽  
Katrin Baltz-Ghahremanpour ◽  
Benjamin J Schmiedel ◽  
Lothar Kanz ◽  
Gundram Jung ◽  
...  
Keyword(s):  
Nk Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity

Abstract Abstract 1537 The capability of anti-tumor antibodies to recruit Fc-receptor (FcR) bearing effector cells like NK cells, a feature considered critical for therapeutic success, can be markedly improved by modifications of the human IgG1 part. At present, Fc-engineered antibodies targeting leukemia cells are yet not available. The various ligands of the NK cell-activating immunoreceptor NKG2D (NKG2DL) are generally absent on healthy cells but upregulated on malignant cells of various origins including leukemia. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as target-antigens for Fc-optimized NKG2D-IgG1 fusion proteins targeting leukemia cells for antibody-dependent cellular cytotoxicity (ADCC) and IFN-g production of NK cells. NKG2D-IgG1 fusion proteins with distinct modifications in their Fc portion were generated as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT), the mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcgRIIIa receptor but comparable binding to NKG2DL-expressing target cells. Functional analyses with allogenic NK cells and leukemia cell lines as well as primary leukemic cells of AML and CLL patients revealed that NKG2D-Fc-KO significantly (p<0.05, Mann-Whitney U test) reduced NK cytotoxicity and IFN-g production (about 20% and 30% reduction, respectively), which can be attributed to blockade of NKG2DL-mediated activating signals. Treatment with NKG2D-Fc-WT significantly (p<0.05, Mann-Whitney U test) enhanced NK reactivity (about 20% and 100% increase in cytotoxicity and cytokine production, respectively). The effects observed upon treatment with NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT resulting in at least doubled NK ADCC and IFN-g production compared to NKG2D-Fc-WT. When applied in combination with Rituximab in analyses with CLL cells, a clear additive effect resulting in a more than four-fold increase of ADCC and FcgRIIIa-induced IFN-g production was observed. The NKG2D-Fc fusion proteins did not induce NK reactivity against healthy blood cells, which is in line with the tumor-restricted expression of NKG2DL. Of note, treatment with NKG2D-Fc-ADCC also significantly (p<0.05, Mann-Whitney U test) enhanced reactivity (up to 70% increase) of NK cells against NKG2DL-positive AML and CLL cells among patient PBMC in an autologous setting. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing leukemia cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to their highly increased affinity to the FcgRIIIa receptor, NKG2D-Fc-ADCC potently enhances NK anti-leukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, Fc-modified NKG2D-Ig may thus constitute an attractive means for immunotherapy of leukemia. Disclosures: No relevant conflicts of interest to declare.

Different NK cell–activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity

Blood ◽  
2009 ◽  
Vol 114 (19) ◽  
pp. 4117-4127 ◽  
Author(s):  
Stephanie M. Wood ◽  
Marie Meeths ◽  
Samuel C. C. Chiang ◽  
Anne Grete Bechensteen ◽  
Jaap J. Boelens ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Reciprocal Relationship ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Loss Of Function ◽  
Lytic Granules ◽  
Molecular Events ◽  
Cell Release

Abstract The autosomal recessive immunodeficiencies Griscelli syndrome type 2 (GS2) and familial hemophagocytic lymphohistiocytosis type 3 (FHL3) are associated with loss-of-function mutations in RAB27A (encoding Rab27a) and UNC13D (encoding Munc13-4). Munc13-4 deficiency abrogates NK-cell release of perforin-containing lytic granules induced by signals for natural and antibody-dependent cellular cytotoxicity. We demonstrate here that these signals fail to induce degranulation in resting NK cells from Rab27a-deficient patients. In resting NK cells from healthy subjects, endogenous Rab27a and Munc13-4 do not colocalize extensively with perforin. However, phorbol 12-myristate 13-acetate and ionomycin stimulation or conjugation to susceptible target cells induced myosin-dependent colocalization of Rab27a and Munc13-4 with perforin. Unexpectedly, individual engagement of receptors leukocyte functional antigen-1, NKG2D, or 2B4 induced colocalization of Rab27a, but not Munc13-4, with perforin. Conversely, engagement of antibody-dependent cellular cytotoxicity receptor CD16 induced colocalization of Munc13-4, but not Rab27a, with perforin. Furthermore, colocalization of Munc13-4 with perforin was Rab27a-dependent. In conclusion, Rab27a or Munc13-4 recruitment to lytic granules is preferentially regulated by different receptor signals, demonstrating that individual target cell ligands regulate discrete molecular events for lytic granule maturation. The data suggest Rab27a facilitates degranulation at an early step yet highlight a reciprocal relationship between Munc13-4 and Rab27a for degranulation.

scholarly journals Pre-Clinical and Clinical Immunomodulatory Effects of Pomalidomide or CC-92480 in Combination with Bortezomib in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1613-1613
Author(s):  
Chad C Bjorklund ◽  
Michael Amatangelo ◽  
Hsiling Chiu ◽  
Jian Kang ◽  
Tiziana Civardi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Granzyme B ◽  
Target Cells ◽  
Publicly Traded ◽  
Dose Dependent ◽  
Privately Held

Abstract Background: Pomalidomide (POM) is an established agent in relapsed/refractory (R/R) multiple myeloma (MM) with direct cytotoxicity against MM cells and immunostimulatory activities in multiple cell types including T cells and NK cells. CC-92480 is a novel Aiolos/Ikaros degrading cereblon E3 ligase modulator (CELMoD ®) agent is currently being investigated in combination with the proteasome inhibitor (PI) bortezomib (BTZ) and corticosteroid dexamethasone (DEX), or with DEX only in R/R MM (CC-92480-MM-002 and CC-92480-MM-001). Previous results indicate that triplet combination of POM/BTZ/DEX may enhance some T, B and NK cell subpopulations, overcoming immunosuppression when compared to BTZ/DEX-only treated patients (Rao et al, 2019). Mechanisms of action (MOA) of CC-92480- and POM-mediated substrate depletion occurs via ubiquitination and proteasome degradation, where BTZ has been speculated as potentially antagonistic as a PI. Here, we report pre-clinical and clinical observations of an immune MOA of CC-92480 or POM in combination with BTZ. Results: To mimic the clinical pharmacokinetics, BTZ was utilized as a high-dose pulse method alone and in combination with POM or CC-92480, followed by flow cytometric measurements of Aiolos and Ikaros protein abundance in healthy donor (HD) T cells. The addition of BTZ modestly delayed CRBN-dependent substrate depletion compared to single agent POM or CC-92480; however, this effect was only apparent at early time points (1-6 hr) where the effect was negligible by 24 hr. To understand the functional implications of BTZ combination, we conducted CD3-stimulated PBMC-mediated cytotoxicity assay against H929 MM target cells in a co-culture model. The efficiency of POM or CC-92480 induced PBMC-mediated killing in a dose dependent manner (~65% increase compared to DMSO) were similar at a 100-fold lower dose range of CC-92480 compared to POM, with the effect not being altered by co-treatment with BTZ. These data were replicated with a POM or CC-92480 treated supernatant stimulation of purified NK cells co-culture, which induced an 80% reduction in target cell viability with the BTZ combination having no negative effects on CELMoD-mediated activity. Cytokine analysis on PBMC supernatants treated with either POM or CC-92480 in the absence or presence of BTZ-pulse showed a dose-dependent increase in IL-2 (&gt;2.4-fold) and Granzyme B (&gt;3.1-fold), which were not impacted by BTZ co-treatment. As a secondary readout on activation status, we measured multiple signaling molecules and activation markers on the cell surface of T and NK cell subsets in CD3 stimulated HD PBMCs treated with dose-dependent POM or CC-92480 with or without co-treatment of BTZ. Compared to DMSO controls, elevated expression levels of CD25 (IL2RA), CD278 (ICOS), Granzyme B, CD134 (OX40R) and HLA-DR were observed with both POM and CC-92480 on CD4, CD8 and NK cells demonstrating a CELMoD-mediated increase in immune activation. These effects were not impacted by the co-treatment of BTZ. Examination of peripheral blood samples from MM patients enrolled in the CC-92480-MM-001/002 (NCT03374085/NCT03989414) clinical trials revealed that CC-92480 promoted potent immunomodulation when administered in combination with DEX and with BTZ/DEX. These data included increased numbers of activated and central memory T cells, as well as increased Ki67+ proliferating T and NK cell populations compared to samples collected during the screening period before any drugs had been administered, consistent with earlier observation of POM in combination with BTZ/DEX treated patients. Conclusions: Taken together, these data demonstrate that POM and CC-92480 are potent immunomodulatory agents with enhanced induction of PBMC and NK mediated cell killing of MM tumor cells and activation of T and NK cells, at 100-fold lower concentrations of CC-92480 compared to POM. Additionally, we showed that combination with BTZ in preclinical assays and in the clinical setting did not antagonistically affect the immunostimulatory ability of POM or CC-92480. Disclosures Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kang: BMS: Current equity holder in publicly-traded company. Civardi: Bristol Myers Squibb: Current Employment. Katz: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Maciag: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hagner: BMS: Current Employment, Current equity holder in publicly-traded company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Bahlis: Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Richardson: Oncopeptides: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Protocol Intelligence: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Regeneron: Consultancy; AstraZeneca: Consultancy; AbbVie: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Fc-Engineered RANK-Fc Fusion Proteins for Neutralization of Soluble RANKL and Induction of Antibody-Dependent Cellular Cytotoxicity (ADCC) Against Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3039-3039
Author(s):  
Benjamin J Schmiedel ◽  
Carolin Scheible ◽  
Tina Baessler ◽  
Constantin M Wende ◽  
Stefan Wirths ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Nk Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Soluble Form ◽  
Target Cells ◽  
Published Data ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity

Abstract Abstract 3039 Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NF-κB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin as key regulators of bone remodelling. Multiple myeloma (MM) disrupts the balance within this molecule system towards osteoclastogenesis and bone destruction. Neutralization of RANKL by the monoclonal antibody Denosumab (AMG162) is presently being evaluated for treatment of both non-malignant and malignant osteolysis. We found, in line with previously published data, that primary MM cells (9 of 10) express substantial levels of RANKL at the cell surface and that MM cells directly release RANKL in soluble form (sRANKL). Next we evaluated the possibility to combine neutralization of sRANKL with targeting of MM cells for antibody-dependent cellular cytotoxicity (ADCC) of NK cells utilizing RANK-Ig fusion proteins with modified Fc portions. Compared to wildtype RANK-Fc, our mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (RANK-Fc-ADCC) and abrogated (RANK-Fc-KO) affinity, respectively, to the NK cell FcγRIIIa, but comparable capacity to neutralize RANKL in binding competition and osteoclast formation assays. Analyses with RANKL transfectants and RANKL-negative controls confirmed the high and lacking potential of the RANK-Fc-ADCC and the RANK-Fc-KO to induce NK ADCC, respectively, and ascertained that the RANK-Fc-ADCC specifically induced NK cell lysis of RANKL-expressing but not RANKL-negative target cells. Most notably, in cultures of NK cells with RANKL-expressing primary MM cells RANK-Fc-ADCC potently enhanced NK cell degranulation, cytokine release and MM cells lysis due to enhanced NK reactivity. Thus, our Fc-engineered RANK-Fc-ADCC fusion protein may both neutralize detrimental effects of sRANKL and enhance NK anti-tumor reactivity by targeting RANKL-expressing malignant cells thereby constituting an attractive immunotherapeutic means for treatment of MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD38-Deficient, CD16-Engineered NK Cells Exhibit Enhanced Antibody-Dependent Cellular Cytotoxicity without NK Cell Fratricide to Augment Anti-Myeloma Immunity in Combination with Daratumumab

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3224-3224
Author(s):  
Karrune Woan ◽  
Ryan Bjordahl ◽  
Frank Cichocki ◽  
Svetlana Gaidarova ◽  
Cameron Pride ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Nk Cell ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Myeloma Cells ◽  
Cell Immunotherapy

Abstract Daratumumab targets the cell surface protein CD38 and is the only FDA approved monoclonal antibody that has demonstrated single agent efficacy in relapsed refractory myeloma. CD38 is broadly expressed in the immune system, and its high expression on multiple myeloma cells allows for effective targeting by daratumumab. Daratumumab induces myeloma cell death through multiple mechanisms, including complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and perhaps most importantly, antibody-dependent cellular cytotoxicity (ADCC). ADCC is mediated by binding of the antibody Fc region to the CD16 Fc receptor expressed on natural killer (NK) cells. Engagement of CD16 induces NK cell activation and target cell cytolysis. However, because CD38 is also expressed on the surface of NK cells, daratumumab treatment can induce NK cell fratricide, which likely impairs the effectiveness of ADCC-mediated targeting and elimination of myeloma. In addition, NK cell function is often suppressed or absent in patients with myeloma, as a result of the tumor itself or from its therapy, further reducing the effectiveness of daratumumab. Collectively, preclinical and clinical observations suggest a potential therapeutic benefit of maintaining NK cell numbers and function in patients to support daratumumab-mediated ADCC and augment the treatment of multiple myeloma. We have developed an off-the-shelf NK cell immunotherapy derived from genetically engineered, induced pluripotent stem cells (iPSC) for enhanced ADCC in combination with daratumumab. iPSCs were engineered to express a high-affinity, non-cleavable CD16 construct (hnCD16) in combination with complete bi-allelic disruption of the CD38 gene (hnCD16 CD38-/-), and the engineered iPSCs were subsequently differentiated into NK (iNK) cells. We hypothesized that CD38-deficient iNK cells would exhibit improved survival by avoiding daratumumab-induced NK cell fratricide, while expression of the hnCD16 transgene would enhance ADCC against myeloma cells in combination with daratumumab. Genetic modification was confirmed in hnCD16 CD38-/- iNK cells by flow cytometry, demonstrating abrogation of CD38 expression (Fig. 1A) and constitutive high expression of CD16 (Fig. 1B). Additionally, hnCD16 iNK cells and hnCD16 CD38-/- iNK cells expressed similar levels of SLAMF7/CD319 (the target of elotuzumab) and NKG2A (Fig. 1C and D). No significant difference in iNK cell differentiation, expansion, maturation, activation, or ability to mediate natural cytotoxicity was observed. In contrast to previous reports, we observed no effect of CD38-deficiency on CD16-mediated calcium flux between hnCD16 iNK cells and hnCD16 CD38-/- iNK cells (Figure 1E). In vitro culture of NK cells in the presence of daratumumab led to NK cell fratricide for both peripheral blood-derived NK cells and hnCD16 iNK cells (Fig. 1F). Daratumumab-induced NK cell fratricide was dependent upon expression of both CD16 and CD38, as unmodified iNK with low CD16 levels (~20% of cells) showed reduced cell death in the presence of daratumumab, which was entirely absent in hnCD16 CD38-/- iNK cells (Fig. 1F). This data was confirmed by extended culture of NK cells with RPMI-8226 tumor spheroids in the presence or absence of daratumumab. The number of hnCD16 iNK cells and peripheral blood NK cells were significantly reduced compared to hnCD16 CD38-/- iNK cells (p>0.005, Fig. 1 G). Importantly, hnCD16 CD38-/- iNK cells were better able to mediate ADCC towards MM1.S multiple myeloma cells compared to hnCD16 iNK cells (Fig. 1H). Taken together, these data support our hypothesis that targeted knock out of CD38 on NK cells alleviates daratumumab-induced NK cell fratricide that occurs through the crosslinking of CD16 and CD38 on neighboring NK cells, leading to augmented anti-myeloma immunity. These data provide a translatable, proof of concept study demonstrating precision genetic engineering of iPSC to generate off-the-shelf NK cell immunotherapy to enhance daratumumab mediated ADCC in multiple myeloma. We propose a strategy of off-the-shelf hnCD16 CD38-/- iNK infusion in combination with daratumumab to overcome NK cell depletion effects of CD38 targeted agents and to improve myeloma patient outcomes. Figure 1. Figure 1. Disclosures Bjordahl: Fate Therapeutics Inc.: Employment. Cichocki:Fate Therapeutics Inc.: Consultancy, Research Funding. Gaidarova:Fate Therapeutics Inc: Employment. Pride:Fate Therapeutics Inc.: Employment. Kaufman:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

Comparative Analysis of LFA-1 Activation State and Functional Involvement in Enhanced Cytotoxicity of NK Cell Lines KHYG-1 and NK-92

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4909-4909
Author(s):  
Garnet Suck ◽  
Suet-Mien Tan ◽  
Sixian Chu ◽  
Madelaine Niam ◽  
Tsyr Jong Lim ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Nk Cell ◽  
Divalent Cations ◽  
Affinity Ligand ◽  
Activated State ◽  
Functional Involvement ◽  
Nk Cytotoxicity

Abstract Background: KHYG-1 and NK-92 are highly cytotoxic IL-2 dependent NK cell lines. NK cytotoxicity is regulated through an array of receptors with activating and inhibitory functions for spontaneous elimination of pathogen infected or tumor cells. Important roles have been described for the leukocyte function-associated antigen (LFA)- 1 adhesion receptor (αLβ2) in NK cytotoxicity. In T cells, the integrin LFA-1 occurs in a non-functional, bent form and requires activation, through divalent cations e.g., for ligand binding. In marked contrast, IL-2 stimulated NK cells could directly engage the LFA-1 ligand intercellular adhesion molecules (ICAM)-1, thereby inducing conjugate formation, granule polarization, degranulation, and tumor cell lysis (Barber DF et al., 2004. JI 173:3653–9). Strikingly, in the NK cell line KHYG-1 granules were constitutively polarized (clustered around the MTOC; Suck G et al., 2006. Int Immunol 18:1347–54) and LFA-1 downstream signaling molecules, the spleen tyrosine kinase (Syk) and extracellular signal-regulated MAP kinase (ERK) constitutively phosphorylated (Suck G et al., 2005. Exp Hem. 33:1160–71). These previous findings prompted us to investigate LFA-1 activation state and functional involvement in cytotoxicity in KHYG-1 and NK- 92. Methods: Adhesion assays were performed on immobilized ICAMs and % adherence determined in a fluorescence plate reader (Tang XY et al., 2008 JI, 180:4793–804). LFA-1 was activated with Mg/EGTA (5 mM MgCl2 and 1.5 mM EGTA), monoclonal antibody (mAb) KIM185 (10 mg/mL) or both; LFA-1 mAb, clone MHM24, was used for blocking studies, 1–10 mg/ml and mAb KIM127 for immunoprecipitation; cytotoxicity, conjugate formation, and degranulation (CD107a) were measured by Flow cytometry and cell morphology imaged by phase contrast (Olympus, 1X70) or confocal microscopy (Zeiss, LSM510 Meta/Nikon A1). Excel software was employed for statistical analyses. Results: In cell binding assays KHYG-1 and NK-92 showed constitutive adhesion to the LFA1-1 ligand ICAM-1, 61% +/− 5.8% SD and 55% +/− 7.5% SD, respectively. However, only 22% NK-92 and 10.5% KHYG-1 cells adhered to the lower affinity ligand ICAM-3 and activation with Mg/EGTA or KIM185 was required to increase binding to 73% in KHYG- 1 and to 62% in NK-92. Immunoprecipitation experiments with the activation reporter mAb KIM127 revealed an activated extended LFA-1 conformation in both cell lines. Together these results suggested an intermediate affinity activation state for the LFA- 1 population in KHYG-1 and NK-92. Despite such similarities, ICAM-1 engagement triggered pronounced cell spreading in KHYG-1, similar to phorbol ester stimulated T cells, but not in NK-92 or primary NK cells. It is conceivable that the capacity to undergo cytoskeletal remodeling in KHYG-1 may be impaired as potentially also reflected in the constitutively polarized granule state. In addition, LFA-1 blocking studies with LFA- 1 antibody MHM24 did not inhibit target conjugate formation in KHYG-1, compared to partial inhibition in NK-92, and cytotoxicity against K562 was only about 40% diminished in KHYG-1, compared to almost complete abrogation (maximum 98%) in NK-92. Nevertheless, overall cytolytic potential against K562 was comparable among the two cell lines, which implicated important functions for other receptors at least in KHYG-1. Conclusion: Results suggested a constitutively activated intermediate affinity state for LFA-1 in KHYG-1 and NK-92. It is conceivable that a similar activated state occurs in primary NK cells. Interestingly, although cytoskeletal dysfunction was indicated in KHYG-1, cytotoxicity of the cell line was unaffected, rendering it a valuable model for the study of alternative pathways.

scholarly journals IL-15 enhanced antibody-dependent cellular cytotoxicity mediated by NK cells and macrophages

2018 ◽  
Vol 115 (46) ◽  
pp. E10915-E10924 ◽  
Author(s):  
Meili Zhang ◽  
Bernard Wen ◽  
Olga M. Anton ◽  
Zhengsheng Yao ◽  
Sigrid Dubois ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Xenograft Model ◽  
Fold Increase ◽  
Antitumor Action ◽  
Host Immune System ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Syngeneic Mouse Model

The goal of cancer immunotherapy is to stimulate the host immune system to attack malignant cells. Antibody-dependent cellular cytotoxicity (ADCC) is a pivotal mechanism of antitumor action of clinically employed antitumor antibodies. IL-15 administered to patients with metastatic malignancy by continuous i.v. infusion at 2 μg/kg/d for 10 days was associated with a 38-fold increase in the number and activation status of circulating natural killer (NK) cells and activation of macrophages which together are ADCC effectors. We investigated combination therapy of IL-15 with rituximab in a syngeneic mouse model of lymphoma transfected with human CD20 and with alemtuzumab (Campath-1H) in a xenograft model of human adult T cell leukemia (ATL). IL-15 greatly enhanced the therapeutic efficacy of both rituximab and alemtuzumab in tumor models. The additivity/synergy was shown to be associated with augmented ADCC. Both NK cells and macrophages were critical elements in the chain of interacting effectors involved in optimal therapeutic responses mediated by rituximab with IL-15. We provide evidence supporting the hypothesis that NK cells interact with macrophages to augment the NK-cell activation and expression of FcγRIV and the capacity of these cells to become effectors of ADCC. The present study supports clinical trials of IL-15 combined with tumor-directed monoclonal antibodies.

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