CHIR-12.12, an Antagonist Anti-CD40 Antibody, Exhibits Greater ADCC Than Rituximab Against a Variety of Malignant B Cells: Evaluation of FcγRIIIa Polymorphism and ADCC Response.

Abstract We have generated a novel, fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, using XenoMouse® mice (Abgenix, Inc.) and have previously demonstrated that it inhibits normal human B cell proliferation and survival and has potent ADCC against primary CLL and NHL cells. CHIR-12.12 and the anti-CD20 monoclonal antibody rituximab were compared for their relative ADCC activity against a variety of malignant human B-cell lines expressing both CD40 and CD20 antigens, including two lymphoma cell lines (Daudi, Namalwa), two multiple myeloma cell lines (ARH77, IM-9), a B-ALL cell line (CCRF-SB), and a B-CLL cell line (EHEB). All cell lines expressed both CD20 and CD40 antigens, and the number of cell surface CD20 molecules per cell were 2.6- to 30.8-fold higher than CD40. For all target cell lines, despite the greater number of CD20 receptors, CHIR-12.12 showed greater maximum cell lysis and a lower ED50 than rituximab. ADCC activity of rituximab is known to correlate with the FcγRIIIa genotype of the effector cells. The homozygous valine (V/V) or heterozygous valine/phenylalanine (V/F) polymorphisms at aa158 are associated with greater cell lysis than is the homozygous F/F polymorphism. The role of the FcγRIIIa aa158 genotype as it relates to CHIR-12.12 activity was explored using Daudi lymphoma target cells and effector NK cells purified from human donors expressing the three polymorphisms. CHIR-12.12 induced potent ADCC with NK cells of all three genotypes (ED50s of 4, 2, and 0.4 pM for F/F, V/F, and V/V, respectively). The rituximab ED50s were 53, 21, and 9 pM for F/F, V/F, and V/V, respectively. Comparison of affinity of the FcγRIIIa F and V alleles for CHIR-12.12 and rituximab using Biacore® analysis showed that CHIR-12.12 bound the F allele with a 4.6-fold higher affinity than rituximab (2.8 μM versus 13 μM, respectively). These data demonstrate that CHIR-12.12 is a more potent ADCC mediator than rituximab, even with human NK cells of the aa158 F/F genotype. CHIR-12.12 is currently in Phase I clinical trials for B-cell malignancies.

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In Vitro and In Vivo Effects of CT-011, a Humanized Anti–PD-1 Monoclonal Antibody, in Combination with Rituximab against Human B-Cell Lymphomas.

Blood ◽

10.1182/blood.v114.22.724.724 ◽

2009 ◽

Vol 114 (22) ◽

pp. 724-724

Author(s):

Fuliang Chu ◽

Myriam Foglietta ◽

Hong Qin ◽

Rakesh Sharma ◽

Qing Yi ◽

...

Keyword(s):

Monoclonal Antibody ◽

T Cells ◽

B Cell ◽

Nk Cells ◽

Tumor Cells ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

In Vivo Effects ◽

Human B Cell

Abstract Abstract 724 Background: Programmed death (PD)–1 is an inhibitory receptor that impairs the function of activated T-cells and natural killer (NK) cells when engaged by its ligands PD-L1 or PD-L2. We have previously demonstrated that PD-1 is markedly up-regulated in intratumoral and peripheral blood CD4+ and CD8+ T cells in patients with follicular lymphoma (FL), a finding associated with impaired T-cell function, suggesting that PD-1 blockade may improve FL immune control. CT-011, a humanized anti PD-1 monoclonal antibody, was previously studied in a phase I clinical trial in patients with advanced hematological malignancies. CT-011 was well tolerated and induced sustained elevations of CD4+ T cells in the peripheral blood. More importantly, apparent clinical benefit was observed in six patients, including one patient with FL who had large tumor masses that achieved a durable complete remission lasting >14 months. Here, we studied the in vitro and in vivo effects of CT-011 on T-cell and/or NK-cell immune responses against human B-cell lymphoma and the hypothesis that CT-011 may improve tumor control when combined with rituximab, a chimeric anti-CD20 monoclonal antibody for the treatment of human FL. Materials and Methods: To determine the effects of CT-011 on antitumor T cells, intratumoral T cells were isolated from primary FL tumor samples, and cultured with or without autologous tumor cells in the presence or absence of CT-011 or isotype control antibody (50 μg/ml each) for 5 days, and tested for proliferation by 3H thymidine incorporation assay. To determine the effects of CT-011 on NK cells, peripheral blood mononuclear cells (PBMCs) derived from normal donors or patients with FL were cultured in the presence or absence of CT-011 (50 μg/ml) with or without IL-2 for 96 hours and analyzed for expression of various activating receptors including CD16, CD32, CD64, Fas ligand, NKG2D, NKp30, NKp44, and NKp46. The in vivo effects of CT-011 were tested in two B-cell lymphoma xenograft models. Ramos and RL lymphoma tumor cells were injected subcutaneously into nude and SCID mice, respectively, and CT-011 (10 μg/mouse) was injected weekly with or without rituximab starting approximately 7–10 days after tumor inoculation. Results: We observed that CT-011 significantly increased the proliferation of intratumoral T cells in response to autologous tumor cells compared with isotype control antibody. Treatment with CT-011 enhanced the expression of Fas ligand, CD32, CD64, and NKp30 on human NK cells in the presence of IL-2 as compared with PBMCs treated with IL-2 alone or media control. In the RL lymphoma xenograft model in SCID mice, treatment with CT-011 significantly delayed tumor growth (P≤0.05) and improved survival (P≤0.01) compared with control mice injected with saline. In a Ramos lymphoma xenograft model in nude mice, treatment with CT-011 and rituximab eradicated established tumors in a significant proportion of mice (P≤0.05) and markedly improved survival compared with rituximab alone or saline. Conclusions: Taken together, these studies suggest that blockade of PD-1 with CT-011 enhances the function of anti-tumor T-cells and augments the expression of activating receptors on NK cells. Treatment with CT-011 led to improved tumor control against human B-cell lymphoma in xenograft models and the combined use of CT-011 and rituximab was more effective that rituximab alone. These results provide the rationale to test the combination of CT-011 with rituximab in patients with B-cell lymphoma, given that the combination is likely to be complementary and may even be synergistic, leading to enhanced clinical efficacy without increasing toxicity. The development of such approaches that activate both the innate (NK-cells) and adaptive (T-cells) immune systems is likely to minimize the emergence of immune escape variants and improve clinical outcome in patients with lymphoma. A clinical trial evaluating CT-011 in combination with rituximab is planned in patients with relapsed FL. Disclosures: Rodionov: Cure Tech Ltd.: Employment. Rotem-Yehudar:Cure Tech Ltd.: Employment.

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Decitabine Or 5-Azacitidine Pre-Treatment Does Not Compromise The Novel Fc-Engineered Antibody Mab 33.1-Mediated ADCC Against Primary AML Blasts - A Rationale For Combination Therapy

Blood ◽

10.1182/blood.v122.21.3959.3959 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3959-3959

Author(s):

Shun He ◽

Carolyn Cheney ◽

Susan P. Whitman ◽

Jianhua Yu ◽

Sumithira Vasu ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Cell Lines ◽

Nk Cell ◽

Hl60 Cell ◽

Adcc Activity ◽

Term Survival ◽

Effector Cells ◽

Blast Cells ◽

Pre Treatment

Abstract Introduction Acute Myeloid leukemia (AML) in patients older than 60 years is a devastating diagnosis with long-term survival rates of 10%. Elderly patients have poor survival both due to chemoresistance and presence of concomitant comorbidities rendering them ineligible for induction chemotherapy. Hence novel treatment options are warranted in this patient population. Promising activity of monoclonal antibodies such as alemtuzumab and rituximab for chronic lymphocytic leukemia (CLL) and rituximab for lymphomas has raised the potential use of antibody therapies in AML. CD33 is expressed on greater than 90% of AML blast cells while absent from all non-hematopoietic tissues. Hence CD33 is a viable target for antibody-based therapeutics in AML. Here, we tested the ex vivo efficacy of the mAb 33.1, a fully human anti-CD33 antibody Fc-engineered for increased binding to Fcγ receptors on AML cell lines and primary AML blasts. The goals of this study are to evaluate 1) the efficacy of mAb33.1 on purified allogeneic and autologous natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against primary AML Blasts; 2) to evaluate efficacy of mAb 33.1 in combination with azanucleosides (i.e. decitabine, 5-azacitidine) that are currently used in AML therapy on NK cell-mediated ADCC against primary AML blasts; and 3) to correlate the levels of surface expression of CD33 on AML blasts to the mAb 33.1 mediated ADCC. Methods mAb 33.1 mediated NK cell activation was determined by NK degranulation as determined by CD107a induction, and ADCC was determined by standard 4-hour 51Cr-release assay. An AML cell line HL60 and a total of 15 AML blast samples were used as targets in this study. NK cells enriched from normal donor PBMC (for allogeneic assays) or sorted from AML blast samples (for autologous assays) were used as effector cells. Results The mAb 33.1 induced potent ADCC activity (>40%) compared to control non-Fc engineered antibody at the concentration of 10 μg/ml in the HL60 cell line. For the AML blasts, mAb 33.1 mediated significantly higher ADCC activity when compared to the control antibody (p<0.05). The relative cytotoxicity mediated by mAb 33.1 varied among different patients, ranging from 4.4% to 65.8%. Subsequent quantification of CD33 showed that there is a positive correlation between ADCC activity and the number of surface CD33 molecules on the AML blasts. Induction of CD107a expression was also observed in both allogeneic and autologous NK cells when the blasts were labeled with mAb 33.1. Pre-treatment of the NK cells and/or target blasts with decitabine or 5-azacitidine for 48hrs, did not alter the mAb 33.1 mediated ADCC activity or CD107 induction. Conclusion mAb33.1 mediated potent ADCC activity and NK activation against AML cell lines and primary AML blasts. Both autologous and allogeneic NK cell-mediated ADCC against primary blast cells from AML patients was observed. The level of NK cell-mediated ADCC was positively associated with the levels of the surface CD33 expression on target AML blasts. Pre-treatment of either AML blasts and/or NK effector cells with Decitabine or 5-azacitidine did not compromise mAb 33.1-mediated ADCC. These pre-clinical studies support further clinical development of mAb 33.1 in combination with relevant anti-AML therapies such as decitabine or 5-azacitidine in patients with CD33 expression. Disclosures: Heider: boehringer-ingelheim: Employment.

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Natural Killer Cells with NKDG2D Cytotoxicity but NKp30-Dull Phenotype Predominate in Myelodysplastic Syndrome (MDS).

Blood ◽

10.1182/blood.v106.11.3436.3436 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3436-3436

Author(s):

Fanqi Bai ◽

Jeffrey S. Painter ◽

Cantor Alan ◽

Zou JianXiang ◽

Sheng Wei ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Line ◽

Cell Lines ◽

Nk Cell ◽

B Cell Lymphoma ◽

Target Cells ◽

Nkg2d Ligands ◽

Human Stress ◽

Normal Controls

Abstract Natural Killer (NK) function in patients with MDS as measured by non-MHC-restricted cytotoxicity and activation-dependent cell cytotoxicity (ADCC) are reduced in patients with MDS, however, the mechanisms of the functional impairment are not known. Tumor cytolysis occurs through orchestrated control by inhibitory NK receptors (NKRs) and activating NKRs, which control signaling events that lead to polarized movement of perforin-containing granules toward the NK-tumor contact area. We found that NK cells from 23 out of 35 patients with MDS (66%) displayed reduced lysis of K562 tumor cells compared to age-matched normal controls (p<0.01). To better characterize this defect, we evaluated patient NK function against differential tumor targets including the MDS1 cell line established from an MDS patients. We found that MDS1 incited non-MHC-restricted lysis. Unactivated PBMCs, unactivated NK cells, NK cell lines (NK92 and NKL) but not purified unactivated T cells from normal donors killed MDS1 in 4-hr 51Cr-release assays. Normal NK cells and NK cell lines were also found to rapidly redistrubute perforin granules after exposure to MDS1suggesting that a perforin-dependent lytic pathway was activated. We then performed simultaneous cytolytic assays with K562, MDS1, and the 721.221 B cell lymphoma cell line as target cells. We found that NK cells from MDS patients had greater lytic activity against MDS1 (average 24% vs. average 8% at 50:1 Effector:Target ratio, respectively, p<0.01) Antibody-blocking experiments demonstrated that the NKL cell line and PBMCs from 8 out of 10 MDS patients predominantly used the NKG2D activating receptor to kill MDS1. Consistent with this finding, we showed that MDS1 cells express the major human stress-inducible endogenous proteins MICA and MICB, which are NKG2D ligands. In contast, lysis by NK92 cells and normal PBMCs was not appreciably reduced by NKG2D blocking antibodies suggesting that other unidentified NKR(s) also mediate lysis. To identify the NKRs expressed in MDS patients, we performed immunophenotyping for both the activating NKRs and inhibitory NKRs compared to age-matched normal controls. We found that two activating receptors, NKp30 and CD244 (2B4), were significantly reduced on NK cells from all MDS patients regardless of their ability to lyse NK targets. Inhibitory NKR expression and function were normal. Interestingly, NKG2D expression correlated with reduced cytolytic function. Similar to studies on normal NK cells with low NKp30 and NKp46 (NCRdull) phenotypes, these results suggest that low NKp30 expression leads to predominant NKG2D utilization for tumor cell lysis, which is reduced in MDS patients with defective NK function. Our findings provide critical information about potential importance for immunosurviellance through NKG2D-NKG2D ligands.

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The Anti-CD20 Monoclonal Antibody GA101 Displays More Robust Anti-Tumor Activity Versus Rituximab In Waldenstrom's Macroglobulinemia (WM)

Blood ◽

10.1182/blood.v116.21.4904.4904 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4904-4904 ◽

Cited By ~ 2

Author(s):

Guang Yang ◽

Christina Hanzis ◽

Sigitas Verselis ◽

Lian Xu ◽

Zachary Hunter ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Death ◽

B Cell ◽

Nk Cells ◽

Healthy Donor ◽

B Cell Depletion ◽

Adcc Activity ◽

Direct Cell ◽

Anti Cd20

Abstract Abstract 4904 Background: Rituximab is an IgG class CD20-directed monoclonal antibody used in the treatment of B-cell malignancies, including WM. We and others have previously demonstrated dependence for IgG class therapeutic antibodies on polymorphisms at FcγRIIIA-158. Approximately half of WM patients express V/V or V/F, and the remainder half express F/F at this polymorphic locus. Patients with WM expressing FcγRIIIA-158 V/V or V/F show improved rituximab single agent activity, as well as attainment of deeper responses (VGPR or CR) with combination Rituximab therapy. GA101 is a novel humanized anti-CD20 antibody with a glyco-engineered Fc domain that exhibits increased Fcg receptor binding and ADCC activity. Methods: In this study, we examined the in vitro activity of GA101 and Rituximab against WM cells, and also examined the activity of these antibodies in context of FcγRIIIA-158 polymorphisms. ADCC activity for GA101 and Rituximab was assessed using genotyped healthy donor derived NK cells against BCWM.1 WM cells, as well autologous NK cells against the patient's own lymphoplasmacytic cells. In vitro B-cell depletion and direct cell death induction assays were also performed. Results: We observed significantly greater ADCC activity against WM cells for GA101 versus Rituximab in both healthy donor, as well as autologous NK cell assays. GA101 mediated ADCC activity was particularly more robust versus Rituximab in patients expressing FcγRIIIA-158 F/F versus V/V or V/F (Figure 1). In addition, GA101 induced significant direct cell death against WM lymphoplasmacytic cells, as well as in vitro B-cell depletion assays in comparison to Rituximab, which exhibited little direct cell death induction activity. Nuclear translocation of apoptosis inducing factor (AIF) was observed following GA101 by immunofluorescence microscopy. Conclusions: GA101 is associated with enhanced ADCC activity relative to Rituximab by NK cells, particularly for those subjects expressing FcγRIIIA-158 F/F. In addition, GA101 demonstrated direct cell death in WM lymphoplasmacytic cells through an AIF mediated caspase-independent pathway. These studies provide the framework for the investigation of GA101 in WM, and suggest particular benefit for those patients who express FcγRIIIA-158 F/F. Disclosures: No relevant conflicts of interest to declare.

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Targeting AML Using an Fc-Engineered BST1/CD157 Monoclonal Antibody

Blood ◽

10.1182/blood.v124.21.987.987 ◽

2014 ◽

Vol 124 (21) ◽

pp. 987-987 ◽

Cited By ~ 1

Author(s):

Christina Krupka ◽

Anna Jansen ◽

Irène Lassmann ◽

Jon Terrett ◽

Dee Aud ◽

...

Keyword(s):

Nk Cells ◽

Cell Lines ◽

Target Cell ◽

Research Funding ◽

Functional Characterization ◽

Cell Lysis ◽

Primary Diagnosis ◽

Target Antigen ◽

Adcc Activity ◽

Raji Cells

Abstract Antibody-based immunotherapy represents a promising strategy to target and eliminate acute myeloid leukemia (AML) tumor cells. We evaluated BST1/CD157, a novel, slow internalizing surface antigen, for its suitability as immunotherapeutic target in AML. An Fc-optimized antibody against BST1/CD157 was developed (MEN1112/OBT357NF), which binds to the Fc-receptor on natural killer (NK) cells with enhanced affinity. The target antigen expression profile of BST1/CD157 on AML bulk cells as well as on leukemic stem cells (LSC) was analyzed with flow cytometry using a commercially available antibody. BST1/CD157 expression was detected in > 97% of AML patients at primary diagnosis (n=81) and at time of relapse (n=20) using specific fluorescence intensity (SFI, positivity: SFI > 1. 5). Importantly, BST1/CD157 was also expressed on CD34+/CD38-cells (n=20), the compartment with a high percentage of LSCs. Functional titration experiments of MEN1112/OBT357NF showed antibody-mediated lysis of U937 cells in the low picomolar range (EC50 = 30–140 pM) and demonstrated superior ADCC activity compared to its non Fc-engineered parental analogue. To evaluate the additive effect of the complement cascade, healthy donor (HD) monocytes were co-incubated with autologous NK cells and MEN1112/OBT357NF in the presence or absence of 5% donor serum. After 20 hours of incubation, a significantly enhanced lysis efficacy by MEN1112/OBT357NF through the addition of serum compared to control cultures without serum (p=0.02) was demonstrated. For further functional characterization of MEN1112/OBT357NF standard chromium release assays using AML cell lines and NK cells from HDs were performed. In this setting, efficient target cell lysis (30–50%) at various effector cell:target (E:T) ratios (50:1–6:1) was observed. Compared to Rituximab-mediated lysis of RAJI cells, MEN1112/OBT357NF triggered more efficient target cell lysis at much lower effector cell concentrations (33% lysis of OCI-AML3 cells vs. 11% lysis of RAJI cells at E:T 6:1). Furthermore, the cytotoxic potential of NK cells from AML patients after intense double induction chemotherapy on AML cell lines was tested. The results were highly variable with efficient lysis in about one third of samples tested (n=10). As a positive control NK cells from HDs were used which showed superior lysis as compared to patient-derived NK cells. In an autologous set-up using NK cells from either primary diagnosis or at time of remission, we could show efficient lysis of primary AML cells in one of two patient samples (patient in complete remission) in the presence of MEN1112/OBT357NF. In summary, our analysis demonstrates that BST1/CD157 is a promising target antigen for antibody-based therapy in AML. The effective in-vitro ADCC activity supports further development of MEN1112/OBT35NF as an immunotherapy for patients with AML. Disclosures Krupka: Oxford BioTherapeutics Ltd: Research Funding. Jansen:Oxford BioTherapeutics Ltd: Research Funding. Aud:Oxford BioTherapeutics Ltd: Employment. Dusek:Oxford BioTherapeutics Ltd: Employment. Bisht:Oxford BioTherapeutics Ltd: Employment. Pombo-Villar:Oxford BioTherapeutics Ltd: Employment, Equity Ownership. Rohlff:Oxford BioTherapeutics Ltd: Employment. Subklewe:Oxford BioTherapeutics Ltd: Research Funding.

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Interferon-gamma gene expression in human B-cell lines: induction by interleukin-2, protein kinase C activators, and possible effect of hypomethylation on gene regulation

Blood ◽

10.1182/blood.v80.3.724.bloodjournal803724 ◽

1992 ◽

Vol 80 (3) ◽

pp. 724-732 ◽

Cited By ~ 47

Author(s):

Y Pang ◽

Y Norihisa ◽

D Benjamin ◽

RR Kantor ◽

HA Young

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Interleukin 2 ◽

Cell Types ◽

Chain Gene ◽

Ifn Gamma ◽

Kinase C ◽

Human B Cell

Human interferon-gamma (IFN-gamma) is an important immunomodulatory protein produced predominantly by T cells and large granular lymphocytes (LGLs). Whereas large amounts of data have been accumulated regarding IFN gamma gene expression in these two cell types, little information about IFN gamma expression in other cell types exists. In this study, we have analyzed the production of IFN gamma by the Epstein- Barr virus (EBV)-positive B-cell line, JLP(c), derived from a patient with Burkitt's lymphoma, and another human B-cell line, PA682BM-1, which was derived from an acquired immunodeficiency syndrome patient. Southern blot analysis indicates the presence of an Ig heavy chain gene rearrangement, but no rearrangement of the T-cell receptor beta chain gene or IFN gamma gene in these B-cell lines. Both cell lines were found to express surface IgD and other B-cell surface markers, thus confirming their B-cell lineage. Analysis for surface Ig, cytoplasmic Ig, and secreted Ig indicates that the two cell lines are in relatively early stages of the B-cell differentiation pathway. We now report that PA682BM-1 can be triggered by the protein kinase C (PKC) activators, phorbol 12-myristate 13-acetate (PMA) and (-)Indolactam-v, to secrete IFN gamma, whereas JLP(c) cells spontaneously produce low levels of IFN gamma that can be enhanced by PKC activators and interleukin-2 (IL-2). After activation of the cell lines with IL-2, (-)Indolactam-v, and PMA, increases in cytoplasmic messenger RNAs (mRNAs) of IFN gamma and the IL- 2 receptor chains were also observed. The induction of IFN gamma mRNA and protein by IL-2 was completely blocked by a monoclonal antibody to IL-2 receptor p75 (beta chain), but not by the monoclonal antibody to p55 (alpha chain). Analysis of IFN gamma genomic DNA indicates that the gene is not amplified, but that hypomethylation in the 5′ noncoding region of the IFN gamma gene has occurred in the B-cell line from the Burkitt's lymphoma patient that spontaneously produces IFN gamma. This finding suggests that the methylation state of the promoter region may play an important role in the control of IFN gamma gene expression in B cells.

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Enhanced Cytotoxicity of Monoclonal Antibody SGN-40 and Immunomodulatory Drug IMiD3 Against Human Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.1498.1498 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1498-1498

Author(s):

Yu-Tzu Tai ◽

Laurence Catley ◽

Xian-Feng Li ◽

Makoto Hamasaki ◽

Klaus Podar ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Growth Inhibition ◽

Nk Cells ◽

Cell Lines ◽

Target Cells ◽

Specific Lysis ◽

Effector Cells ◽

Human Multiple Myeloma ◽

Apoptotic Effects

Abstract SGN-40, a humanized immoglobulin G1 (IgG1) anti-CD40 monoclonal antibody, mediates cytotoxicity against human multiple myeloma (MM) cells via several mechanisms in vitro. These include induction of cytotoxic ligands of TNFR family and suppression of IL-6-induced proliferative and antiapoptotic effects, as well as antibody-dependent cell-mediated cytotoxicity (ADCC). Since > 80% of primary patient MM cells express CD40, targeting CD40 using SGN-40 presents a potential novel treament strategy, and a phase I clinical study of SGN-40 in patients with refractory or recurrent MM is ongoing. We recently reported that Thalidomide and immunomodulatory drugs (IMiDs) target both MM cells and the bone marrow (BM) microenvironment, and activate NK cells via induction of IL-2 production. In the present study, we therefore evaluated the effects of IMiD3 on the direct antiproliferative and apoptotic effects of SGN-40, as well as on ADCC against both MM cell lines and patient MM cells (CD40+CD138++). SGN-40 and IMiD3 induced synergistic growth inhibition, assayed by [3H] thymidine uptake, in dexamethasone (Dex)-sensitive MM.1S and Dex-resistant MM.1R lines, 2 other CD40-positive MM cell lines, as well as 2 patient MM cells. The temporal sequence of SGN-40 and IMiD3 treatment did not alter growth inhibition. The combination of SGN-40 and IMiD3 significantly increased MM apoptosis, evidenced by enhanced cleavage of caspase 3/8/PARP and increased subG0 cells compared with either single agent. The addition of IMiD3 to target cells and effector cells moderately increased specific lysis in any MM cell line, whereas pretreatment of target cells with IMiD3 significantly augmented sensitivity of all MM lines to ADCC and pretreatment of effector cells also improved specific MM cell lysis. In addition, preincubation of both effector and tumor cells with IMiD3 greatly enhanced specific lysis of MM cell lines and 2 patient MM cells in ADCC assay, associated with a significant increase 38+3% in natural killer cells (CD56+CD16+ and CD14-CD3-) following IMiD3 treatment. IMiD3 not only improved natural cytotoxicity of NK cells, but also significantly induced the CD56dimCD16+CD3- NK subset, which is a more potent mediator of ADCC against MM than the CD56bright NK subset. Moreover, IMiD3 treatment upregulates CD40L expression on CD56+CD3- NK effectors: IMiD3 (2 μM) induces CD40L upregulation equivalent to IL-2 (1000 unit/ml). Finally, combined SGN-40 and IMiD3 augments NK cell proliferation, which is associated with enhanced AKT/NF-kB and ERK activation. Taken together, our studies show that the addition of IMiD3 to SGN-40 results in synergistic cytotoxicity mediated via direct antiproliferative and apoptotic effects therefore increased sensitivity of MM cells to ADCC by inducing the cytotoxic NK subset. These studies establish the framework for the development of SGN-40 and IMiD3 in a new treament paradigm to both target MM cells directly and to induce immune effectors against MM.

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Transfection of NK Cells with mRNA or Lentivirus Expressing Chimeric Antigen Receptors Results in Highly Efficient Killing of Lymphoid Malignancies and Compares Favorably with Monoclonal Antibody-Directed ADCC.

Blood ◽

10.1182/blood.v114.22.1696.1696 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1696-1696 ◽

Cited By ~ 1

Author(s):

Laurent Boissel ◽

Monica Betancur ◽

Richard A. Van Etten ◽

Hans-Georg Klingemann

Keyword(s):

Monoclonal Antibody ◽

Nk Cells ◽

Cell Line ◽

Cell Lines ◽

Receptor Expression ◽

Antigen Receptors ◽

Chimeric Antigen Receptors ◽

Highly Efficient ◽

Lentivirus Transduction ◽

Anti Cd20

Abstract Abstract 1696 Poster Board I-722 NK cells frequently do not kill malignant lymphoid target cells except through antibody dependent cellular cytotoxicity (ADCC) when combined with monoclonal antibodies (mAb). Here we compared the ability of the human NK cell line NK-92 to lyse lymphoid cell lines and primary chronic lymphoid leukemia (CLL) cells after transfection with either mRNA or lentivirus coding for Chimeric Antigen Receptors (CAR) against CD19 or CD20. Electroporation (Genepulser, Biorad) of 10 μg mRNA (coding for GFP, CD19-CAR or CD20-CAR) into NK-92 cells was performed at 300V and 150 μF (Leuk. Res 2009;33:1255). For lentivirus transduction, CD19-CAR and CD20-CAR were cloned into a pCL20c IRES-GFP vector and lentivirus stocks for GFP, CD19-CAR and CD20-CAR were obtained from 293T packaging cells. NK-92 cells were transduced by two successive rounds of spinfection in the presence of 8 μg/ml protamine sulfate. Mean transfection efficiencies were 57.2% ± 6.6 for electroporation with mRNA, and 32.6% ± 4.1 for lentivirus transduction. The cytotoxity of the transfected/transduced NK-92 cells against the reference Raji cell line was not affected. The NK-92 resistant cell lines SUP-B15 (CD19+/CD20-) and TMD5 (CD19+/CD20+) became highly sensitive to lysis by transfected NK-92 expressing CD19-CAR, while only TMD5 became sensitive to transfected NK-92 expressing CD20-CAR. Importantly, mRNA-transfected NK-92 showed two-fold higher killing compared to lentivirus-transduced NK-92 despite similar receptor expression. As expected, expression of CAR following mRNA transfection was temporary (48 hours), whereas lentivirus transduced NK-92 maintained CAR expression and cytolytic function for indefinite periods in culture. In order to obtain higher target cell killing by lentivirus transduced cells, NK-92 cells could be highly enriched by cell sorting. Subsequently, we compared CAR-directed killing with ADCC on primary B-CLL patient samples, using NK-92 cells genetically modified by lentivirus expressing anti-CD20 CAR versus high affinity Fc receptor-expressing (FcRγIIIA) NK-92 cells combined with the anti-CD20 monoclonal antibody Rituximab. Initially all CLL cells were resistant to killing by NK-92. Anti-CD20 CAR transfected NK-92 cells showed significantly higher cytotoxicity than ADCC against patients' cells, with less patient-to-patient variability. In conclusions, transfection of NK cells with mRNA coding for CAR against lymphoid surface antigens is a highly efficient method that can easily be scaled up to produce clinical grade material. Lentivirus transduced NK-92 cells show stable expression of CAR, but efficient killing requires sorting or selection for transfected cells. CAR-directed killing by NK-92 compares favorably with anti-CD20 mAb mediated ADCC against primary CLL targets and either approach, alone or in combination, could have clinical relevance. Disclosures Klingemann: ZelleRx Corp.: Co-founder and shareholder.

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CD38 Protects Tumor Cells from NK Lysis

Blood ◽

10.1182/blood.v126.23.5389.5389 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5389-5389

Author(s):

Anlai Wang ◽

Guang Yang ◽

Zhili L Song ◽

Francisco J Adrian

Keyword(s):

Nk Cells ◽

Tumor Cells ◽

Cell Lines ◽

B Cell Lymphoma ◽

Cell Lysis ◽

Protective Role ◽

Target Cells ◽

Median Percentage ◽

Protective Function ◽

Cd38 Expression

Abstract Expression of the type II glycoprotein CD38 in hematological malignancies is abnormally high compared to that in normal cells of hematopoietic origin. Across all malignancies, the highest CD38 expression levels are detected on the surface of multiple myeloma (MM) cells. The high CD38 level has prompted the development of anti-CD38 antibody-based therapies, like daratumumab and isatuximab (SAR650984), that are promising therapeutics for the treatment of patients with MM. Cellular functions associated with CD38 include co-receptor mediated signaling, cell adhesion and ecto-enzymatic conversion of NAD to modulate calcium signaling and adenosine synthesis; but other than allowing antibody-mediated engagement of innate immunity, the role of CD38 in MM is not fully understood. In order to investigate a potential immune-protective role for CD38, we evaluated a panel of 15MM and 6 diffuse large B-cell lymphoma (DLBCL) cell lines with varying levels of CD38 for their susceptibility to NK-mediated lysis. Briefly, calcein AM pulsed target cells were incubated with NK-92.CD16 NK cells (E:T=3:1) for 1 hr and cell lysis measured as a function of the fluorescent calcein released into the supernatant. In an initial experiment, we observed that the percentage of cell lysis for all lines with >100,000 CD38s per cell was low, below 10%. The cell lines with <100,000 CD38s per cell were more susceptible to NKs, with a median percentage cell lysis of around 25%. The observed levels of cell lysis were not correlated with the levels of released IFNγ or TNFα. The differential lytic activity against the target cells led to the hypothesis that CD38 might have a role in protecting the tumor cells from NK-mediated lysis. To investigate this, we first knocked down CD38 in SUDHL-8 cells using shRNA, decreasing the levels of CD38 from 246,000 to 51,000 per cell. This resulted in an increase in the percentage of lysed cells from 25% to more than 50%, likely reflecting a loss of protection against the NK cells. We next overexpressed CD38 in U266 cells, increasing the CD38 levels from 9,000 to 327,000 per cell. As a result, the percentage of lysis decreased from more than 60% to around 30%, indicating that U266 cells are now more resistant to the lysis by NK cells. The protection that CD38 seems to confer to CD38-overexpressing U266 cells is completely lost by addition of isatuximab which triggers CD16-mediated lysis (antibody dependent cellular toxicity, ADCC). Whether the protective function of CD38 described here is a direct or an indirect protection mechanism is currently under investigation. Disclosures Off Label Use: isatuximab (SAR650984, anti-CD38 antibody). Song:Sanofi: Employment. Adrian:Sanofi: Employment.

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G protein subunit G alpha 16 expression is restricted to progenitor B cells during human B-cell differentiation

Blood ◽

10.1182/blood.v85.7.1836.bloodjournal8571836 ◽

1995 ◽

Vol 85 (7) ◽

pp. 1836-1842 ◽

Cited By ~ 21

Author(s):

MY Mapara ◽

K Bommert ◽

RC Bargou ◽

C Leng ◽

C Beck ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

B Cell Lymphoma ◽

Cell Phenotype ◽

Low Grade ◽

B Cell Differentiation ◽

Human B Cell

Recently G alpha 16, a new guanosine triphosphate (GTP) binding protein alpha subunit has been described to be specifically expressed in human hematopoietic cells. Expression of G alpha 16 was observed in human cell lines of myelomonocytic and T-lymphocytic origin, but not in human B-cell lines Raji and IM9. We studied the expression of G alpha 16 in human B cells corresponding to different stages of B-cell differentiation by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The human Burkitt's lymphoma cell lines Raji, Ramos, BJAB, the lymphoblastoid cell line SKW6.4, and the plasmocytoma cell line U266 were devoid of G alpha 16. In contrast, G alpha 16 was detected in the human progenitor B cell lines Reh and Nalm-6. Using the mu+, k-cell line BLIN-1 (pre-B cell phenotype) and its derived subclone 1E8 (surface mu+, k+; B-cell phenotype) G alpha 16 expression was found to disappear on transition from pre-B to B-cell differentiation stage. The analysis of a broad panel of human neoplastic B lymphocytes ranging from progenitor B-acute lymphatic leukemia (pre-pre-B-ALL), common acute leukemias (cALL), pre-B-ALL, mature B-ALL to low grade B-cell lymphoma (chronic lymphocytic leukemia of B-cell type, leukemic centrocytic non-Hodgkins lymphoma [NHL], hairy cell leukemia) showed that G alpha 16 expression is limited to progenitor and pre-B-ALL cells. Therefore, we conclude that within B-cell differentiation, G alpha 16 is expressed solely during early B cell ontogeny and downregulated during differentiation. Thus, G alpha 16 might be an important regulator involved in signaling processes in progenitor B cells.

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