Ratios Of FcγRIIa and FcγRIII Binding Affinities Govern The Efficacy Of PMN-Mediated Cytotoxicity – Implications For Fc-Engineering Approaches Of Therapeutic Antibodies

Abstract Introduction Antibody-dependent cell-mediated cytotoxicity (ADCC) has been suggested to be an essential effector mechanism for the in vivo activity of tumor-targeting therapeutic monoclonal antibodies (mAbs). Thus, enhancing the affinity of human IgG1 mAbs to NK cell-expressed FcγRIIIa by glyco- or protein-engineering of their Fc moiety has been demonstrated to improve NK cell-mediated ADCC and represents a promising strategy to improve antibody therapy. However, human polymorphonuclear (PMN) cells express the homologous FcγRIIIb isoform, which does not trigger ADCC. The aim of the present study was to analyze a panel of distinct IgG1 mAbs, displaying different affinities for FcγRIIa and FcγRIII, with respect to PMN recruitment for tumor cell destruction. Methods Affinities of analyzed mAbs to distinct FcγR were determined by surface plasmon resonance technology. Induction of ADCC was assessed by 51Cr release experiments in the absence or presence of FcγRIII- or FcγRII-blocking agents to unravel the contribution of both receptors in these assays. Results Non-fucosylated or protein-engineered IgG1 variants with optimized FcγRIII binding capacities demonstrated the expected benefit in triggering NK cell- mediated ADCC but did not mediate ADCC by PMN, which could be restored by FcγRIIIb blockade. Additionally, eosinophils as well as PMN from paroxysmal nocturnal hemoglobinuria (PNH) patients – expressing no or low levels of FcgRIIIb – mediated effective ADCC with FcgRIII-optimized mAbs. Additional experiments with Fc variants displaying enhanced FcγRIIa binding or with double FcγRIIa/FcγRIII-optimized constructs demonstrated enhanced PMN-mediated ADCC compared to control mAbs. Statistical analyses revealed that FcγRIIa/FcγRIIIb affinity ratios correlated with the extent of human PMN-mediated ADCC. Conclusions To conclude, the present study represents novel findings concerning recruitment of PMN for tumor cell destruction by Fc-engineered antibodies. While PMN-mediated ADCC was completely abolished by FcγRIII-optimized antibodies through predominant FcγRIIIb binding, it was potently enhanced by optimization of FcγRIIa binding affinity. Importantly, functional analyses unraveled the ratio between FcγRIIa and FcγRIII binding affinities to control PMN-mediated ADCC activity and therefore opened new alleys in engineering of therapeutic antibodies. Disclosures: Muchhal: Xencor Inc.: Employment. Desjarlais:Xencor Inc.: Employment.

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Chromophore-enhanced in vivo tumor cell destruction using an 808-nm diode laser

Cancer Letters ◽

10.1016/0304-3835(95)03837-m ◽

1995 ◽

Vol 94 (2) ◽

pp. 125-131 ◽

Cited By ~ 88

Author(s):

Wei R. Chen ◽

Robert L. Adams ◽

Kenneth E. Bartels ◽

Robert E. Nordquist

Keyword(s):

Tumor Cell ◽

Diode Laser ◽

Cell Destruction ◽

Tumor Cell Destruction

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Venetoclax Enhances the Efficacy of Therapeutic Antibodies in B-Cell Malignancies

Blood ◽

10.1182/blood-2018-99-114677 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4177-4177

Author(s):

Fotini Vogiatzi ◽

Julia Heymann ◽

Thies Rösner ◽

Lennart Lenk ◽

Gunnar Cario ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

Caspase 3 ◽

Nk Cell ◽

Target Cells ◽

Therapeutic Antibodies ◽

B Cell Malignancies ◽

Nsg Mice ◽

Dependent Cell

Abstract The application of antibodies is a promising option in the treatment of B-cell malignancies, including acute lymphoblastic leukemia (ALL) and B-cell Non-Hodgkin lymphoma (B-NHL). Although patient outcomes have improved by applying combinations of chemotherapy and antibodies, certain patients characterized by a high expression of anti-apoptotic Bcl-2 have a poor prognosis. These include adult B-NHL patients with "double-hit lymphomas" (DHLs) and pediatric ALL patients harboring a t(17;19) translocation. Furthermore, a substantial number of Burkitt´s lymphoma (BL) patients also express Bcl-2 even though the impact of this finding on prognosis is yet unclear. Here, we examine the role of low doses of the Bcl-2 inhibitor venetoclax (VTX, 1nM) on the efficacy of the therapeutic antibodies rituximab (CD20), daratumumab (CD38) and CD19-DE (a variant of the CD19 antibody MOR208 engineered for improved effector cell binding). Natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) were evaluated. The CD20-expressing DHL cell line Carnaval and patient-derived xenograft (PDX) cells from two BL patients were used as target cells for rituximab, the CD20-negative/CD38-positive DHL cell line Will-2 was used with daratumumab and three PDX samples from t(17;19) positive ALL patients were used with CD19-DE. For the assessment of ADCP, human monocyte-derived macrophages were incubated with labelled target cells and microscopy assays were performed. NK-cell mediated ADCC was not enhanced by VTX in any of our models. However, 17-37% increases in ADCP by macrophages were detected when Carnaval cells were subjected to combinations of VTX/rituximab and when Will-2 cells were treated with VTX/daratumumab as compared to VTX or antibody alone (p=0.0318/p=0.0185 and p=0.0012/p=0.0068, respectively, Figure A). When BL PDX cells were subjected to ADCP assays with VTX/rituximab, mean phagocytosis levels were also enhanced by 26.0% and 21.0% in the combination treatment group as compared to VTX (p=0.0283) and rituximab alone (p=0.0282; Figure A). ADCP assays with t(17;19) positive ALL-PDX cells and CD19-DE confirmed these results as phagocytosis was increased to similar extents in the combination group as compared to VTX (p=0.0017) or CD19-DE alone (p=0.0323) (Figure A/B). In order to exclude that our observations were due to an enhancement of apoptosis in target cells only, we measured cleaved caspase-3 with VTX, antibodies alone or the combination of both. Cleaved caspase-3 levels were equal in all groups suggesting that the addition of VTX resulted in an apoptosis-independent activation of macrophages. In order to minimize heterogeneity in ADCP assays, phagocytosis was next examined using expanded macrophages from NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice for our assays. Regardless of target cells and antibody, the combination of antibody treatment with VTX resulted in enhanced phagocytosis by murine macrophages, confirming our results. The effects of VTX on the efficacy of rituximab were finally examined in vivo. Carnaval cells were injected intravenously into NSG mice and animals treated with VTX (100 mg/kg 5 days/week by oral gavage), rituximab alone (1 mg/kg once weekly intraperitoneally) or the combination of both (n=6/group). Mice were sacrificed when mice showed clinical lymphoma or leukemia engraftment and survival differences were assessed using Kaplan-Meier log-rank statistics. Compared to control, mice treated with VTX displayed a slight survival advantage, which was more marked in mice treated with rituximab (p=0.0020/p=0.0004, respectively, Figure C), suggesting a better efficacy of rituximab than VTX as monotherapy. Most importantly, mice treated with the combination VTX/rituximab showed significantly superior survival as compared to either VTX or rituximab alone (p=0.0023/p=0.0268, respectively, Figure C), suggesting additive effects in vivo. Altogether, we show that VTX enhances the efficacy of therapeutic antibodies in models of B-cell malignancies including PDX samples. The mechanism is most likely dependent on distinct influences of VTX on macrophage activation, e. g. by myeloid immune checkpoints. Our in vivo data suggest that this combination strategy may become a promising therapeutic option for the treatment of Bcl-2 expressing B-cell malignancies in the future. Figure. Figure. Disclosures Bourquin: Amgen: Other: Travel Support. Valerius:Affimed: Research Funding. Peipp:Affimed: Research Funding.

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562 SO-C101 induces NK cell cytotoxicity and potentiates antibody-dependent cell cytotoxicity and anti-tumor activity

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0562 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A596-A596

Author(s):

Zuzana Antosova ◽

Nada Podzimkova ◽

Marketa Jiratova ◽

Eva Nedvedova ◽

Guy de Martynoff ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Nk Cells ◽

Nk Cell ◽

Therapeutic Antibodies ◽

Cell Cytotoxicity ◽

Sequential Administration ◽

Dependent Cell ◽

Anti Tumor Activity

BackgroundSO-C101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SO-C101 specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion and activation of regulatory T cell compartment.MethodsHuman NK cell proliferation, the expression of NK cell receptors and ADCC activity of human PBMC after stimulation with SO-C101 in vitro in combination with monoclonal antibodies were detected by flow cytometry. The anti-tumor efficacy of SO-C101 in combination with Daratumumab was assessed in a multiple myeloma SCID xenograft mouse model in vivo.ResultsIn this study, we show that SO-C101 induced proliferation and expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+. Furthermore, SO-C101 induced expression of the cytotoxic receptors NKp30 and NKG2D whereas no upregulation of the inhibitory receptors CD158a, CD158b and NKG2A was detected. Both NK cell subsets activated by SO-C101 exhibited cytotoxicity towards cancer cells in vitro. Using human PBMCs, we show that SO-C101 potentiated killing of tumor cells induced by several clinically approved therapeutic monoclonal antibodies such as Cetuximab, Daratumumab and Obinutuzumab in vitro. SO-C101 and Daratumumab monotherapy treatment inhibited tumor growth in vivo, however, their combination showed the strongest anti-tumor efficacy. Specifically, sequential administration of Daratumumab, followed by SO-C101 promoted complete tumor regression, compared to partial anti-tumor responses induced by the respective monotherapies.ConclusionsSO-C101 augments the anti-tumor activity of therapeutic antibodies by increasing NK cells mediated antibody-dependent cell cytotoxicity. These results support the evaluation of SO-C101 in combination with monoclonal therapeutic antibodies in clinical studies.Ethics ApprovalThe anti-tumor efficacy studies in mice were approved by the internal ethics board of the respective contract research organization (CRO).

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Unique Toxicities and Resistance Mechanisms Associated with Monoclonal Antibody Therapy

Hematology ◽

10.1182/asheducation-2005.1.329 ◽

2005 ◽

Vol 2005 (1) ◽

pp. 329-334 ◽

Cited By ~ 34

Author(s):

Jonathan W. Friedberg

Keyword(s):

Follicular Lymphoma ◽

Single Agent ◽

Antibody Therapy ◽

Resistance Mechanisms ◽

Complement Dependent Cytotoxicity ◽

Dependent Cell ◽

Impact On Treatment ◽

Anti Cd20 ◽

In Vivo Cytotoxicity

Abstract Anti-CD20 therapy has had a truly dramatic impact on treatment and outcome of patients with follicular lymphoma. Unfortunately, the majority of responses to single-agent rituximab are incomplete, and all patients with follicular lymphoma will experience disease progression at some point following rituximab therapy. Rituximab has multiple mechanisms of inducing in vivo cytotoxicity, including antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, direct apoptotic signaling, and possible vaccinal effects. The cellular microenvironment within follicular lymphoma has a profound impact on which mechanism is dominant, and confers resistance in many situations. Both tumor-associated and host-associated factors also contribute to rituximab resistance. There are multiple potential approaches to overcoming rituximab resistance, including rational biologic combination immunotherapy, engineered antibodies, and radioimmunoconjugates. Improved ability to overcome resistance will require further elucidation of critical signaling pathways involved in rituximab induced cytotoxicity and a comprehensive understanding of interactions between its multiple mechanisms of action.

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Mechanisms of IL-21 Enhancement of Rituximab Efficacy in a Lymphoma Xenograft Model.

Blood ◽

10.1182/blood.v104.11.1404.1404 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1404-1404

Author(s):

Steve D. Hughes ◽

Ken Bannink ◽

Cecile Krejsa ◽

Mark Heipel ◽

Becky Johnson ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Antibody Therapy ◽

Xenograft Model ◽

Tumor Model ◽

Cell Types ◽

Current Evidence

Abstract Interleukin 21 (IL-21) is an IL-2 family cytokine produced by activated CD4+ T cells. Potent effects of IL-21 have been observed on the growth, survival, and functional activation of T cells, B cells, and natural killer (NK) cells. A Phase I clinical trial of IL-21 in metastatic melanoma and renal cell carcinoma is currently in progress. We recently reported that IL-21 significantly enhanced rituximab mediated clearance of CD20+ lymphoma cell lines both in vitro and in vivo, and that these effects were potentially mediated through IL-21 enhancement of NK cell capacity to effect antibody dependent cellular cytotoxicity (ADCC). Specifically, NK cells treated with IL-21 showed increased cytotoxicity, granzyme B and IFNg production. Current studies aim to further evaluate the mechanisms by which IL-21 enhances ADCC. A number of observations suggest a multi-factorial basis for IL-21 synergy with rituximab. In a xenograft tumor model, SCID mice were injected IV with HS Sultan cells on day 0. Treatment with recombinant murine IL-21 (mIL-21; starting day 1) combined with rituximab (starting day 3) resulted in significantly increased survival (70% vs. 20% on day 100), compared to rituximab alone. In separate studies, the spleens of mice treated with mIL-21 showed increased numbers of activated macrophages and granulocytes. As macrophages and granulocytes can participate in ADCC, IL-21 synergy with rituximab in vivo may be partly dependent on its activation of these cell types. We have also evaluated whether direct effects of IL-21 on lymphoma cells contribute to enhancement of rituximab efficacy. The xenogeneic B lymphoma models in which IL-21 plus rituximab exhibited enhanced survival are highly aggressive and these models were not shown to respond to treatment with mIL-21 alone. In vitro studies were performed to determine if IL-21 could potentiate the growth inhibitory and pro-apoptotic effects of rituximab. In the absence of effector cells synergistic interaction was not observed. In addition, we tested the ability of IL-21 to enhance cytotoxicity when combined with antibodies targeting non-hematopoietic tumor cells (e.g. trastuzumab). Human NK cells treated with IL-21 displayed significantly increased cytotoxicity in ADCC assays using trastuzumab to target breast cancer cells expressing varying levels of HER-2 antigen. In summary, the current evidence suggests that IL-21 can enhance antibody-mediated tumor cell lysis through activation of multiple effectors of ADCC. Thus IL-21 may prove to be broadly applicable to monoclonal antibody therapy of cancer.

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Obinutuzumab (GA101) More Potently Engages Phagocytic-Lineage Cells Resulting In Enhanced Monocyte and Macrophage Activity When Compared To Rituximab and Ofatumumab

Blood ◽

10.1182/blood.v122.21.5136.5136 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5136-5136 ◽

Cited By ~ 2

Author(s):

Sylvia Herter ◽

Christian Klein ◽

Pablo Umana ◽

Marina Bacac

Keyword(s):

Cell Death ◽

Tumor Cell ◽

Nk Cell ◽

Antibody Activity ◽

Type I ◽

Type Ii ◽

Therapeutic Antibodies ◽

Tumor Cell Death ◽

Macrophage Activity ◽

Anti Cd20

Abstract Therapeutic antibodies possess several clinically relevant mechanisms of action including cell death induction, perturbation of tumor cell signaling, activation of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and induction of adaptive immunity. Obinutuzumab (GA101) is a novel humanized, glycoengineered Type II anti-CD20 monoclonal antibody engineered for displaying enhanced FcγRIIIa (CD16) binding affinity and characterized by stronger induction of ADCC and direct tumor cell death when compared to wild-type, Type I anti-CD20 antibodies rituximab and ofatumumab. In light of the important role of phagocytic lineage cells in the mechanism of action of therapeutic antibodies, we compared GA101, rituximab and ofatumumab for their ability to trigger FcγR-dependent monocyte and macrophage effector functions. We show that, due to glycoengineering, GA101 displays superior CD16-dependent binding to monocytes, M1 and M2c macrophages in presence of nonspecific, competing, human endogenous IgGs, a situation that more closely mimics physiological conditions. Subsequently, GA101 more strongly engages monocytes and macrophages and leads to significantly higher elimination of CD20-expressing tumor cells as shown by assays detecting total antibody activity (ADCP, ADCC and direct effects). In support of the stronger GA101 activity, higher nitric-oxide (NO) levels are also detected in supernatants of tumor/macrophage co-cultures treated with antibody. Taken together, our data show that in addition to stronger NK-cell mediated ADCC and direct cell death induction due to Type II CD20 binding, GA101 more potently engages phagocytic-lineage cells resulting in enhanced monocyte and macrophage activity under conditions that more closely resemble physiological settings. Disclosures: Herter: Roche: Employment. Klein:Roche Glycart AG: Employment. Umana:Roche: Employment, Equity Ownership. Bacac:Roche: Employment.

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NK Cell Line Killing of Leukemia Cells Is Enhanced By Reverse Antibody Dependent Cell Mediated Cytotoxicity (R-ADCC) Via NKp30 and NKp44 and Target Fcγ Receptor II (CD32)

Blood ◽

10.1182/blood.v124.21.2444.2444 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2444-2444 ◽

Cited By ~ 1

Author(s):

Brent A. Williams ◽

Xinghua Wang ◽

Bertrand Routy ◽

Richard Cheng ◽

Sonam Maghera ◽

...

Keyword(s):

Esophageal Cancer ◽

Cell Line ◽

Cell Lines ◽

Nk Cell ◽

P Value ◽

Fcγ Receptor ◽

Isotype Control ◽

Nsg Mice ◽

Dependent Cell

Abstract Introduction: We are studying NK cell immunotherapy to treat acute myeloid leukemia (AML) and have focused on NK-92 and KHYG-1, CD16(-) human malignant NK cell lines. Phase I NK-92 trials show minimal toxicity; KHYG-1 has not been tested in humans. Here, we investigated modulation of cytotoxicity of NK cell lines against primary AML blasts and cell lines with monoclonal antibodies (mAb) directed against natural cytotoxicity receptors. Methods: NK cytotoxicity was assessed with a standard 4 hour Cr51 release assay at an effector to target (E:T) ratio of 10:1. NK lines were incubated with and without isotype control and mAbs against NKp30, NKp44 at various doses (0.001-10 µg/ml) for 1 hour and washed with medium prior to cytotoxicity assays. The student’s t-test was used to compare cytotoxicity data. Target cells were incubated with 100 µCi Cr51 and cell supernatants assayed on a gamma counter. NK targets (leukemic and esophageal cancer) were evaluated for Fcγ receptor expression by flow cytometry. To test the cytotoxic effect on in vivo proliferation, OCI/AML5 cells were co-incubated with irradiated KHYG-1 (iKHYG-1) +/-1 µg/ml NKp30 pretreatment for 4 hours at a 10:1 E:T ratio and injected ip into NOD/SCID gamma null (NSG) mice with survival as an endpoint analyzed with the log rank test. Results: NK-92 and KHYG-1 were both highly cytotoxic against K562 with moderate killing of OCI/AML3 and KG1 and KG1a. OCI/AML5 was highly sensitive to killing by NK-92, but resistant to KHYG-1. Pretreatment of NK-92 with mAbs against NKp30, NKp44 (10 µg/ml) yielded small increases in cytotoxicity against leukemic cell lines with NKp30 pretreatment only. Pretreatment of KHYG-1 with 10 µg/ml of anti-NKp30 or anti-NKp44 mediated fold increases in cytotoxicity above isotype control against 4 leukemia cell line targets and 4 primary AML samples (Table1). Anti-NKp30 and anti-NKp44 pretreatment of NK-92 and KHYG-1 did not enhance killing of a panel of esophageal cancer cell lines. Immunophenotyping cancer cell lines showed high expression of Fcγ receptor II (CD32), but very low expression of Fcγ receptor I (CD64) or III (CD16) on leukemia lines (K562, OCI/AML3, OCI/AML5, KG1 and KG1a), and no expression of Fcγ receptors on esophageal lines (OE-33, FLO-1, KYAE-1, SKGT-4). Regression analysis of the relationship between cytotoxic enhancement and CD32 expression of targets revealed a strong correlation for NKp30 (p<0.01; R2=0.71) and NKp44 (p<0.01; R2=0.64) pretreated KHYG-1. NSG mice injected with 2x106 OCI/AML5 cells developed progressive malignant ascites at 9 weeks requiring sacrifice, unaffected by iKHYG-1 (p=0.92). However, NKp30 pretreated iKHYG-1 improved survival versus no therapy (p<0.05) or iKHYG-1 (p<0.05) cohorts. Conclusion: We show a novel means to enhance cytotoxicity of NK cell lines many fold against primary AML cells by pretreatment with mAbs against NKp30 and NKp44. The mechanism of enhanced KHYG-1 cytotoxicity is from bound NKp30 or NKp44 becoming crosslinked when the Fc portion binds the Fcγ receptor II (CD32) on targets. This is the first demonstration of reverse antibody-dependent cell-mediated cytotoxicity (R-ADCC) with a NK cell line leading to enhanced killing of AML primary blasts in vitro and the first demonstration of R-ADCC in an in vivo model. Table 2: Effect of NKp30 or NKp44 pretreatment on KHYG-1 cytotoxicity against primary AML samples Fold lysis and p values Primary AML samples 5890 080078 0909 080179 Fold change lysis NKp30 1.7 15.7 2.7 4.9 Fold change lysis NKp44 0.9 16.3 2.8 6.2 p value NKp30 <0.05 =0.0001 <0.0001 <0.001 p value NKp44 0.9 <0.001 <0.001 <0.001 Disclosures No relevant conflicts of interest to declare.

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