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.

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 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.

Impaired natural and CD16-mediated NK cell cytotoxicity in patients with WAS and XLT: ability of IL-2 to correct NK cell functional defect

Blood ◽  
2004 ◽  
Vol 104 (2) ◽  
pp. 436-443 ◽  
Author(s):  
Angela Gismondi ◽  
Loredana Cifaldi ◽  
Cinzia Mazza ◽  
Silvia Giliani ◽  
Silvia Parolini ◽  
...  
Keyword(s):  
Nk Cells ◽  
Molecular Mechanisms ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Target Cells ◽  
Cell Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Functional Defect ◽  
Nk Cell Cytotoxicity ◽  
Syndrome Protein

Abstract In this study we show that Wiskott-Aldrich syndrome protein (WASp), a critical regulator of actin cytoskeleton that belongs to the Scar/WAVE family, plays a crucial role in the control of natural killer (NK) cell cytotoxicity. Analysis of NK cell numbers and cytotoxic activity in patients carrying different mutations in the WASP coding gene indicated that although the percentage of NK cells was normal or increased, natural cytotoxicity and antibody-mediated NK cell cytotoxicity were inhibited in all patients with the classical WAS phenotype and in most patients carrying mutations associated with the X-linked thrombocytopenia (XLT) phenotype. The inhibition of NK cell-mediated cytotoxicity was associated with the reduced ability of WAS and XLT NK cells to form conjugates with susceptible target cells and to accumulate F-actin on binding. Treatment with interleukin-2 (IL-2) corrected the functional defects of NK cells by affecting their ability to bind to sensitive target cells and to accumulate F-actin. In addition, we provide information on the molecular mechanisms that control WASp function, demonstrating that binding of NK cells to sensitive targets or triggering through CD16 by means of reverse antibody-dependent cellular cytotoxicity (ADCC) rapidly activates Cdc42. We also found that WASp undergoes tyrosine phosphorylation upon CD16 or β2-integrin engagement on NK cells. (Blood. 2004;104:436-443)

Pseudoexpression of Glucocorticoid-Induced TNF-Related (GITR) Ligand Upon Coating of Cancer Cells by Platelets Impairs NK Cell Anti-Tumor Reactivity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3193-3193 ◽  
Author(s):  
Theresa Placke ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
Hans-Georg Kopp
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Immune Surveillance ◽  
Substantial Reduction ◽  
Cell Activity ◽  
Target Cells ◽  
Tumor Immune Escape ◽  
Tumor Spread

Abstract Abstract 3193 NK cells as part of the innate immune system substantially contribute to cancer immune surveillance. They prevent tumor progression and metastasis due to their ability to mediate cellular cytotoxicity and to produce cytokines like IFN-γ, which, among others, stimulates subsequent adaptive immune responses. NK reactivity results from an integrative response emerging upon recognition of multiple ligands for activating and inhibitory NK cell receptors including various members of the TNFR family. Apart from the direct interaction with their target cells, NK cell activity is further influenced by the reciprocal interplay with various other hematopoietic cells like e.g. dendritic cells. Metastatic tumor spread in experimental animals is dramatically reduced in thrombopenic mice. Additional depletion of NK cells reverses this effect, indicating that platelets may impair NK anti-tumor reactivity. However, the underlying mechanisms have not been fully elucidated, especially in humans. Recently, we demonstrated that NK anti-tumor immunity is impaired by platelet-derived TGF-β, which is released upon interaction of platelets with tumor cells (Kopp et al., Cancer Res. 2009). Here we report that the ligand for the TNFR family member GITR (GITRL) is upregulated on megakaryocytes during maturation resulting in substantial GITRL expression by platelets. Since we recently identified GITR as inhibitory NK receptor involved in tumor immune escape (e.g., Baltz et al., Blood 2008, Baessler et al., Cancer Res. 2009) we investigated how platelet-derived GITRL influences platelet function and NK immune surveillance. Signaling via GITRL into platelets upon interaction with NK-expressed GITR or recombinant GITR-Ig fusion protein did not alter platelet activation as revealed by analysis of the activation marker CD62P and release of TGF-β. Interestingly, we found that GITRL-negative tumor cells rapidly get coated by platelets, which confers a seemingly GITRL-positive phenotype. “GITRL pseudoexpression” on tumor cells caused a substantial reduction of NK cell cytotoxicity and cytokine production. This reduced NK reactivity was not due to induction of apoptosis via GITR and could be restored by addition of a blocking GITR antibody. Thus, coating of tumor cells by platelets inhibits NK reactivity, which is in part mediated by platelet-derived GITRL. Our data provide a functional basis for the previously observed finding that platelets increase metastasis i.e. by enabling evasion of tumor cells from NK-mediated immune surveillance. Disclosures: No relevant conflicts of interest to declare.

Expression and Immunomodulatory Function of RANKL in Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 245-245
Author(s):  
Benjamin J Schmiedel ◽  
Tina Baessler ◽  
Miyuki Azuma ◽  
Lothar Kanz ◽  
Helmut R. Salih
Keyword(s):  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Immune Surveillance ◽  
Leukemia Cells ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Hematopoietic Malignancies ◽  
Immunomodulatory Function

Abstract Abstract 245 The TNF family member RANKL and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but have also been shown to influence progression of malignancies like breast cancer (Tan et al., Nature 2011), myeloma (Sordillo et al., Cancer 2003) and CLL (Secchiero et al. J Cell Physiol. 2006). NK cells are cytotoxic lymphocytes that play an important role in tumor immune surveillance especially of hematopoietic malignancies. Their reactivity is influenced by a variety of activating and inhibitory molecules expressed by their target cells including several members of the TNF family. Recently, we reported that RANK, upon interaction with RANKL which can be expressed by malignant hematopoietic cells, mediates signals that impair NK reactivity (Schmiedel et al., Blood 2010 116,21:893–893). Here we extended these analyses and comprehensively studied the expression and immunomodulatory function of RANKL in leukemia. Analysis of primary leukemia cells revealed substantial RANKL surface expression in a high proportion of the investigated cases (AML, 47 of 65 (72%); ALL, 16 of 21 (76%); CML, 6 of 10 (60%); CLL, all 54 (100%)). Signaling via surface-expressed RANKL into the malignant cells mediated the release of cytokines like TNF, IL-6, IL-8 and IL-10 which have been shown to act as autocrine and paracrine growth and survival factors in leukemia. Moreover, the factors released upon RANKL signaling upregulated RANK expression on NK cells. In line, NK cells from leukemia patients (n=75) displayed significantly (p<0.001, Mann-Whitney U-test) higher RANK expression compared to healthy controls (n=30) confirming our notion that RANK-RANKL interaction may contribute to leukemia pathophysiology. We further found that RANK-RANKL interaction, beyond directly inhibiting NK cell function via RANK, may contribute to evasion of leukemia cells from NK immunosurveillance by creating an NK inhibitory cytokine milieu. This was revealed by impaired cytotoxicity and degranulation in response to leukemia targets following exposure of the NK cells to the factors released upon RANKL signaling by leukemia cells. Notably, the RANKL-mediated cytokine release of leukemia cells could be disrupted by the clinically approved RANKL antibody Denosumab/AMG162. Thus, RANKL signaling may trigger a “vicious cycle” comprising of release of immunosuppressive cytokines and also upregulation of RANK on NK cells. The latter both directly inhibits NK reactivity and may result in augmented RANKL signaling into leukemia cells. Our data suggest that therapeutic modulation of the RANK/RANKL system e.g. with Denosumab/AMG162, which is approved for treatment of osteolysis, may be a promising strategy to reinforce NK reactivity against hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Engineering Anti-AML Antibodies for Improved NK Cell ADCC

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3629-3629
Author(s):  
Vladimir Senyukov ◽  
William Kelton ◽  
Nishant Mehta ◽  
George Georgiou ◽  
Dean Lee
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Adoptive Immunotherapy ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Cell Culture Supernatant ◽  
Current Therapy ◽  
Target Cells ◽  
Wild Type ◽  
Related Donor

Abstract Abstract 3629 Acute myeloid leukemia (AML) is an aggressive malignancy for which current therapy fails to provide durable remission in approximately half of cases. Natural killer (NK) cells, as a key component of innate immunity, have recently shown clinical potential for adoptive immunotherapy against AML, particular when the donor and recipient are KIR mismatched. In addition to patients who do not have a suitable related donor, approximately 30% of patients bear all three families of KIR ligands and therefor cannot benefit from KIR mismatch. Thus, finding a related donor with predicted KIR mismatch is a major obstacle for adoptive NK cell immunotherapy. The majority of peripheral blood NK cells express CD16a (FcγRIIIa), which is the most potent receptor among the activating receptors that NK cells posses. NK cells express CD16a in association with disulflde-linked homo- or hetero-dimers of FcRγ or CD3ζ. Clustering of CD16a initiated by binding to the Fc-portion of IgG1 or IgG3 that opsonize target cells induces signals strong enough to overcome KIR inhibition. Thus, combining NK cell adoptive immunotherapy with Abs against tumor antigens could help overcome the limitations of KIR mismatching. Indeed, many promising anticancer Abs have failed in clinical trials because of insufficient efficacy, which, at least in part, may result from low affinity CD16a binding. Indeed, it was shown that the affinity between Fc and FcγRs correlates with cytotoxicity in cell-based assays and that the Abs with optimized FcγR affinity induced strong cytotoxicity against targeted tumor cells. CD33 is expressed on the blast cells of most cases of AML and represents a suitable antigen for antibody-based therapies. Lintuzumab, an unconjugated, humanized anti-CD33 mAb (HuM195), failed to improve patient outcomes in two randomized trials when combined with conventional chemotherapy. Gemtuzumab ozogamicin, an anti-CD33 mAb conjugated to the calicheamicin, in combination with chemotherapy, improved survival in a subset of AML patients, but has been withdrawn from US market by safety concerns. We optimized the FcγR affinity of HuM195 mAb (mNuM195) by cloning into pMaz-IgH Herceptin recipient vector containing S239D, A330L, I332E mutations that, as previously shown, leads to significant improvement of IgG1 binding to CD16a. To generate control wild type variant (wHuM195) we cloned the variable domains of HuM195 into pMaz-IgH Herceptin. Plasmids were transfected into HEK293F, and Abs were purified from cell culture supernatant with protein A resin, eluted with glycine HCL, and then the samples were buffer exchanged into PBS pH 7.4 for long-term storage. This S239D-A330L-I332E triple mutation in Fc portion of IgG1 did not affect antigen-biding affinity for CD33 target protein but showed more than 14-fold higher binding to CD16a than the wild type variant. The mHuM195 Abs increased cytotoxic activity of expanded human NK cells in Calcein AM-release assay when used in concentration as low as 0.01 μg/ml to pretreat murine thymoma EL-4 cells gene-modified to express human CD33 (ADCC, Mean±SD: 38.7±2.25% vs 11.7±3.49% for optimized vs wild type HuM195, and 5±3.15% without Abs, E:T ratio 2:1). We obtained the similar results when using K562 as targets, which naturally express CD33. K562 cells pretreated with mHuM195 Abs induced degranulation in 34±5.25% of NK cells where wHuM195 did so only in 17±4.6% of NK cells. Thus, optimization of HuM195 Ab to improve CD16a affinity results in dramatic increases NK cell cytotoxic activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1456-1463 ◽  
Author(s):  
Siao-Yi Wang ◽  
Emilian Racila ◽  
Ronald P. Taylor ◽  
George J. Weiner
Keyword(s):  
Cell Activation ◽  
Complement Fixation ◽  
Nk Cell ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Antitumor Effects ◽  
Surface Enhanced ◽  
The Impact ◽  
The Relationship

Abstract Antibody-dependent cellular cytotoxicity (ADCC) and complement fixation both appear to play a role in mediating antitumor effects of monoclonal antibodies (mAbs), including rituximab. We evaluated the relationship between rituximab-induced complement fixation, natural killer (NK)–cell activation, and NK cell–mediated ADCC. Down-modulation of NK- cell CD16 and NK-cell activation induced by rituximab-coated target cells was blocked by human serum but not heat-inactivated serum. This inhibition was also observed in the absence of viable target cells. C1q and C3 in the serum were required for these inhibitory effects, while C5 was not. An antibody that stabilizes C3b on the target cell surface enhanced the inhibition of NK-cell activation induced by rituximab-coated target cells. Binding of NK cells to rituximab-coated plates through CD16 was inhibited by the fixation of complement. C5-depleted serum blocked NK cell–mediated ADCC. These data suggest that C3b deposition induced by rituximab-coated target cells inhibits the interaction between the rituximab Fc and NK-cell CD16, thereby limiting the ability of rituximab-coated target cells to induce NK activation and ADCC. Further studies are needed to define in more detail the impact of complement fixation on ADCC, and whether mAbs that fail to fix complement will be more effective at mediating ADCC.

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.

scholarly journals CD33 Delineates Two Functionally Distinct NK Cell Populations Divergent in Cytokine Production and Antibody-Mediated Cellular Cytotoxicity

2022 ◽  
Vol 12 ◽  
Author(s):  
Maryam Hejazi ◽  
Congcong Zhang ◽  
Sabrina B. Bennstein ◽  
Vera Balz ◽  
Sarah B. Reusing ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cytokine Production ◽  
Nk Cell ◽  
Functional Divergence ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Transcriptional Signature ◽  
Promising Target

The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56dim NK cells that do generally not express CD33 in vivo. RNAseq analysis revealed that upregulation of CD33+ NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56bright (CD117high, CD16low) and CD56dim NK cells (high expression of granzyme B and perforin). CD33+ NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33− subset. Moreover, CD33+ NK cells showed superior production of IFNγ and TNFα, whereas CD33− NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33+ NK cells combining efficient target cell killing and cytokine production, or alternatively CD33− NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.

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