NK Cell Antibody-dependent Cellular Cytotoxicity Research Articles

T-cell help in the tumor microenvironment enhances rituximab-mediated NK-cell ADCC

AbstractRituximab (RTX) and other monoclonal antibodies (mAbs) that bind directly to malignant cells are of great clinical value but are not effective for all patients. A major mechanism of action of RTX is antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Prior in vitro studies in our laboratory demonstrated that T cells contribute to maintaining the viability and cytotoxic potential of NK cells activated by anti-CD20–coated target B cells. Here, we conducted studies using a novel mouse model and clinical correlative analysis to assess whether T-cell help contribute to RTX-mediated NK-cell ADCC in the tumor microenvironment (TME) in vivo. A humanized mouse model was developed using Raji lymphoma cells and normal donor peripheral blood mononuclear cells that allows for control of T-cell numbers in the lymphoma TME. In this model, NK-cell viability and CD16 and CD25 expression dropped after RTX in the absence of T cells but increased in the presence of T cells. RTX therapy was more effective when T cells were present and was ineffective when NK cells were depleted. In patients with indolent lymphoma, fine needle aspirates were obtained before and ∼1 week after treatment with a RTX-containing regimen. There was a strong correlation between CD4+ T cells as well as total T cells in the pretherapy TME and an increase in NK-cell CD16 and CD25 expression after RTX. We conclude that T-cell help in the TME enhances RTX-mediated NK-cell viability and ADCC.

Generation and optimization of off-the-shelf immunotherapeutics targeting TCR-Vβ2+ T cell malignancy

Current treatments for T cell malignancies encounter issues of disease relapse and off-target toxicity. Using T cell receptor (TCR)Vβ2 as a model, here we demonstrate the rapid generation of an off-the-shelf allogeneic chimeric antigen receptor (CAR)-T platform targeting the clone-specific TCR Vβ chain for malignant T cell killing while limiting normal cell destruction. Healthy donor T cells undergo CRISPR-induced TRAC, B2M and CIITA knockout to eliminate T cell-dependent graft-versus-host and host-versus-graft reactivity. Second generation 4-1BB/CD3zeta CAR containing high affinity humanized anti-Vβ scFv is expressed efficiently on donor T cells via both lentivirus and adeno-associated virus transduction with limited detectable pre-existing immunoreactivity. Our optimized CAR-T cells demonstrate specific and persistent killing of Vβ2+ Jurkat cells and Vβ2+ patient derived malignant T cells, in vitro and in vivo, without affecting normal T cells. In parallel, we generate humanized anti-Vβ2 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) by Fc-engineering for NK cell ADCC therapy.

Impact of HIV-1 Vpu-mediated downregulation of CD48 on NK-cell-mediated antibody-dependent cellular cytotoxicity.

HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC.

The human cytomegalovirus-specific and UL40-mediated imprint in the natural killer cell repertoire is associated with antibody-mediated rejection in lung transplant recipients.

CD16+ natural killer (NK-) cells play, together with donor-specific antibodies (DSA) and via antibody-dependent cellular cytotoxicity (ADCC), an important role in the pathogenesis of antibody-mediated rejection (ABMR) in lung-transplant recipients (LTRs). Cytotoxic CD16+NKG2C+ NK cells proliferate in response to human Cytomegalovirus (HCMV) infections via the presentation of HCMV-encoded and highly polymorphic UL40 peptides. In our study, we aimed to clarify whether infections with HCMV-strains carrying different UL40 peptide variants are associated with the shift of the NK cell repertoire and the development of ABMR in LTRs. We included 30 DSA+ABMR+, 30 DSA+ABMR- and 90 DSA-ABMR- LTRs. In all patients, 1 episode of high-level HCMV-replication occurred. In all DSA+ABMR+ LTRs, HCMV-replication occurred prior to ABMR diagnosis. The association of HCMV UL40 variants with the expansion of CD16+ NK cell subsets and ABMR was assessed in NK cell proliferation and ADCC assays. Our study revealed that the VMAPRTLIL and VMTPRTLVL UL40 variants were significantly overrepresented in DSA+ABMR+ LTRs. Both peptides were associated with a pronounced proliferation of cytotoxic and proinflammatory CD16+NKG2C+ NK cells. The stimulation with both peptides led to a shift of the NK cell repertoire towards CD16+NKG2C+ NK cells, which was associated with strong ADCC responses after stimulation with endothelial cells and plasma from DSA+ABMR+ LTRs. Distinct UL40 peptide variants of the infecting HCMV-strain are associated with the development of ABMR after lung transplantation, due to a shift towards a highly cytotoxic CD16+NKG2C+ NK cell population. These peptides are thus potential prognostic markers for ABMR.

Comprehensive genetic and functional analyses of Fc gamma receptors influence on response to rituximab therapy for autoimmunity.

Rituximab is widely used to treat autoimmunity but clinical response varies. Efficacy is determined by the efficiency of B-cell depletion, which may depend on various Fc gamma receptor (FcγR)-dependent mechanisms. Study of FcγR is challenging due to the complexity of the FCGR genetic locus. We sought to assess the effect of FCGR variants on clinical response, B-cell depletion and NK-cell-mediated killing in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). A longitudinal cohort study was conducted in 835 patients [RA=573; SLE=262]. Clinical outcome measures were two-component disease activity score in 28-joints (2C-DAS28CRP) for RA and British Isles Lupus Assessment Group (BILAG)-2004 major clinical response (MCR) for SLE at 6 months. B-cells were evaluated by highly-sensitive flow cytometry. Single nucleotide polymorphism and copy number variation for genes encoding five FcγRs were measured using multiplex ligation-dependent probe amplification. Exvivo studies assessed NK-cell antibody-dependent cellular cytotoxicity (ADCC) and FcγR expression. In RA, carriage of FCGR3A-158V and increased FCGR3A-158V copies were associated with greater 2C-DAS28CRP response (adjusted for baseline 2C-DAS28CRP). In SLE, MCR was associated with increased FCGR3A-158V, OR 1.64 (95% CI 1.12-2.41) and FCGR2C-ORF OR 1.93 (95% CI 1.09-3.40) copies. 236/413 (57%) patients with B-cell data achieved complete depletion. Homozygosity for FCGR3A-158V and increased FCGR3A-158V copies were associated with complete depletion in combined analyses. FCGR3A genotype was associated with rituximab-induced ADCC, and increased NK-cell FcγRIIIa expression was associated with improved clinical response and depletion invivo. Furthermore, disease status and concomitant therapies impacted both NK-cell FcγRIIIa expression and ADCC. FcγRIIIa is the major low affinity FcγR associated with rituximab response. Increased copies of the FCGR3A-158V allele (higher affinity for IgG1), influences clinical and biological responses to rituximab in autoimmunity. Enhancing FcγR-effector functions could improve the next generation of CD20-depleting therapies and genotyping may stratify patients for optimal treatment protocols. Medical Research Council, National Institute for Health and Care Research, Versus Arthritis.

Abstract 4216: Tipifarnib inhibits the secretion of tumor-derived small extracellular vesicles and enhances the immunotherapeutic efficacy of dinutuximab in neuroblastoma

Abstract Anti-GD2 monoclonal antibody (Dinutuximab) immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. However, 40% of patients fail to respond or develop resistance to the treatment, and the molecular mechanisms by which this occurs remain poorly understood. Here we utilize the syngeneic 9464D-GD2 mouse model to investigate the role of neuroblastoma tumor-derived small extracellular vesicles (sEVs) in developing resistance to the anti-GD2 monoclonal antibody Dinutuximab. Neuroblastoma-derived sEVs significantly attenuated the efficacy of Dinutuximab treatment in vivo. Mechanistically, both RNA-sequencing and flow cytometry analysis of whole tumors demonstrated that neuroblastoma-derived sEVs modulate immune cells tumor infiltration upon Dinutuximab treatment to create an immunosuppressive tumor microenvironment, which contains more tumor-associated macrophages (TAMs) and fewer tumor-infiltrating NK cells, in addition to suppression of splenic NK cell maturation in vivo. Moreover, neuroblastoma-derived sEVs suppressed Dinutuximab-mediated NK cell antibody-dependent cellular cytotoxicity in vitro. Importantly, Tipifarnib, a farnesyltransferase inhibitor that inhibits sEV secretion, drastically enhanced the efficacy of Dinutuximab in vivo and reversed the immunosuppressive effects of neuroblastoma-derived sEVs. Notably, Tipifarnib modulated immature myeloid cells in the bone marrow to disfavor the formation of CD11b+Ly6ChighLy6Glow subpopulations, which are the precursors for TAMs. Taken together, these preclinical findings uncover a novel mechanism by which neuroblastoma-derived sEVs modulate the immune system to promote resistance to Dinutuximab and suggest that Tipifarnib-mediated inhibition of sEV secretion may serve as a viable treatment strategy to improve immunotherapy in high-risk patients. Citation Format: Xiaoming Liu, Carson A. Wills, Longgui Chen, Jiawen Zhang, Yuanjun Zhao, Mi Zhou, Jeffrey M. Sundstrom, Todd D. Schell, Vladimir S. Spiegelman, Megan M. Young, Hong-Gang Wang. Tipifarnib inhibits the secretion of tumor-derived small extracellular vesicles and enhances the immunotherapeutic efficacy of dinutuximab in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4216.

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

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

Paralysis of the cytotoxic granule machinery is a new cancer immune evasion mechanism mediated by chitinase 3-like-1

BackgroundNatural killer (NK) cells require a functional lytic granule machinery to mediate effective antitumor responses. Evading the lytic cargo deployed at the immune synapse (IS) could be a critical step...

Balancing the CD38 Expression on Effector and Target Cells in Daratumumab-Mediated NK Cell ADCC against Multiple Myeloma.

Simple SummaryWe tracked the cytotoxic potential of NK cells towards multiple myeloma cells in daratumumab-mediated antibody-dependent cellular cytotoxicity assays. These cytotoxicity levels could be directly correlated to the expression of the target antigen (CD38) and to the percentage of fratricide between effector cells. Increasing the expression of CD38 on target cells or neutralizing CD38 on effector cells changed the equilibrium between target and effector cell lysis and promoted multiple myeloma cell death. This study highlights the importance of a balanced CD38 expression on target and effector cells and attempts to alter this balance will affect the susceptibility of MM cells towards daratumumab-mediated cellular toxicity. Multiple myeloma (MM) is an incurable cancer characterized by the proliferation and accumulation of monoclonal plasma cells in the bone marrow. The monoclonal anti-CD38 daratumumab has taken a central place in the different treatment regimens for newly diagnosed and relapsed, refractory myeloma. In this study, we correlated the NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and potential fratricide induced by daratumumab with CD38-expression levels on both effector and target cells. We show that CD38 expression can be modulated by adding all-trans retinoic acid (ATRA) or interferon-α to MM cells to further fine-tune these effects. In addition, we observed that ADCC becomes inefficient when fratricide occurs and both ADCC and fratricide depend on the balance between CD38 expression on effector and target cells. However, the addition of adjuvants (retinoic acid or interferon-α) to myeloma cells or the inhibition of fratricide using a CD38-blocking nanobody on NK-cells can reverse this balance towards ADCC and thus promote lysis of target cells by ADCC. ATRA and interferon-α increased the CD38 expression at the surface of MM cells about three-fold and two-fold, respectively. This increase was of interest for MM cells with low CD38 expression, that became susceptible to daratumumab-mediated ADCC after preincubation. A CD38-blocking nanobody prevented the binding of daratumumab to these NK-cells and blunted the fratricidal effect on effector NK cells. In conclusion, our study highlights the importance of a balanced CD38 expression on target and effector cells and attempts to alter this balance will affect the susceptibility of MM cells towards daratumumab-mediated ADCC.

T cells, particularly activated CD4+ cells, maintain anti-CD20-mediated NK cell viability and antibody dependent cellular cytotoxicity

Anti-CD20 monoclonal antibody (mAb) therapy is a mainstay of therapy for B cell malignancies, however many patients fail to respond or eventually develop resistance. The current understanding of mechanisms responsible for this resistance is limited. When peripheral blood mononuclear cells of healthy donors were cultured with Raji cells for 7 days, rituximab (RTX) induced NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), enhanced NK cell viability and increased or maintained NK expression of CD56, CD16, CD57 and KIR. T cells, mainly CD4+, mediated these changes in a contact-dependent manner, with local T cell production of IL2 playing a central role. Similar findings were found when autologous B cells were used as target cells demonstrating the need for T cell help was not due to allogenic reaction. Results with other anti-CD20 and anti-EGFR antibodies were consistent. Small numbers of T cells activated by anti-CD3/CD28 beads or bispecific antibody enhanced RTX-mediated NK cell ADCC, viability and phenotypical changes. Pathway analysis of bulk NK cell mRNA sequencing after activation by RTX with and without T cells was consistent with T cells maintaining the viability of the activated NK cells. These findings suggest T cell help, mediated in large part by local production of IL2, contributes to NK cell ADCC and viability, and that activating T cells in the tumor microenvironment, such as through the use of anti-CD3 based bispecific antibodies, could enhance the efficacy of anti-CD20 and other mAb therapies where NK-mediated ADCC is a primary mechanism of action.

Abstract 1630: FceR1g negative NK-cells (g-NK) enhance antibody-dependent cellular cytotoxicity and in vivo efficacy of therapeutic monoclonal antibodies against hematologic malignanices

Abstract Monoclonal antibodies (mAbs) are a central component of hematologic malignancy therapy; however, not all patients initially respond to these agents and resistance is a major clinical issue. A major mechanism of action for these mAbs in vivo is NK cell antibody-dependent cellular cytotoxicity (ADCC). Recent studies led to the discovery of a novel subset of human NK cells that lack expression of FceR1g (“g-NK cells”) and have a multi-fold increase in ADCC activity after CD16 crosslinking. Here, we validate the potency of g-NKs as an off-the shelf cellular immunotherapy to enhance the efficacy of therapeutic mAbs. g-NK cells were enriched and expanded using a proprietary method from CMV-positive blood donors selected for high levels of circulating g-NKs. For ADCC cytotoxicity assays, expanded NK cells were sorted by flow cytometry into populations of “conventional” NK cells (cNK), adaptive (NKG2C+) NK cells, and g-NK cells. After sorting, NK-cells were co-cultured 4 hr at 37° C with Raji or MM.1S target cells (1e4 cells) at varying NK-cell:target cell ratios. For in vivo persistence studies a single dose of 1e7 g-NK or cNK cells was injected into NOD scid gamma (NSG) mice. For in vivo efficacy studies with rituximab (RIT), NSG mice were inoculated with 5e5 luciferase-labeled Raji lymphoma cells and dosed weekly with 15e6 g-NK cells +/-200 ug RIT or vehicle. For in vivo efficacy studies with daratumumab (DARA) and elotuzumab (ELO), NSG mice were inoculated with 1e6 luciferase-labeled MM.1S cells and dosed every 6d with expanded g-NKs (20e6), resting cNKs (3e5, equivalent to physiological levels of NK cells/kg in humans, and +/- 10 ug DARA or 10 ug ELO. In all studies, mice received IL-15 intraperitoneally every 3 days. Our expansion method was able to expand g-NKs from selected CMV-seropositive donors ~800-fold (n=8). By flow cytometry we found an increase in the percentage of g-NK from 28% of NK cells at input to 82% post-expansion. In vitro ADCC assays demonstrated that g-NKs enhanced cytotoxicity of RIT to Raji cells (70% at 1:1 E:T) when compared to both NKG2C+ NKs (29%) and cNKs (27%) (p<0.001). Notably, we saw similar ADCC of g-NK cells whether used fresh or thawed after viable freezing (70% vs. 63%, p=0.71). In addition, g-NK cells enhanced cytotoxicity of DARA and ELO to MM.1S cells (58% and 54% at 1:1 E:T) when compared to cNK (14% and 12% at 1:1 E:T) (p<0.001). We confirmed in NSG mice that g-NK persistence was markedly improved (>90%) over cNKs in blood at multiple time points (p<0.001), and spleen at Day 22 (p<0.001). Efficacy studies using Raji cells showed the combination of g-NK and RIT led to a >90% reduction in tumor burden versus RIT alone (p<0.001). Evaluation of expanded g-NKs in a disseminated orthotopic xenograft model of multiple myeloma found a dramatic reduction (>70%) in tumor burden with DARA+g-NK vs. DARA alone or DARA+cNK (p<0.001). Similar results were found with ELO. Here we demonstrate that adoptive transfer of expanded g-NK cells markedly enhances anti-tumor ADCC of therapeutic mAbs in several preclinical models. Importantly, because adoptive transfer of NK-cells in humans does not result in severe graft-versus-host disease, we propose that this long-lived, highly potent NK-cell therapy could be administered in an “off-the-shelf” manner to supercharge mAb efficacy. Citation Format: Austin Basil Bigley, Nadia H. Agha, Shanae Spade, Gaetano Dipierro, Ronald Martell, Byron C. Hann, Nina Shah, Arun P. Wiita. FceR1g negative NK-cells (g-NK) enhance antibody-dependent cellular cytotoxicity and in vivo efficacy of therapeutic monoclonal antibodies against hematologic malignanices [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1630.

Induction of NK Cell Reactivity against B-Cell Acute Lymphoblastic Leukemia by an Fc-Optimized FLT3 Antibody.

Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism by which antitumor antibodies mediate therapeutic efficacy. At present, we evaluate an Fc-optimized (amino acid substitutions S239D/I332E) FLT3 antibody termed 4G8-SDIEM (FLYSYN) in patients with acute myeloid leukemia (NCT02789254). Here we studied the possibility to induce NK cell ADCC against B-cell acute lymphoblastic leukemia (B-ALL) by Fc-optimized FLT3 antibody treatment. Flow cytometric analysis confirmed that FLT3 is widely expressed on B-ALL cell lines and leukemic cells of B-ALL patients. FLT3 expression did not correlate with that of CD20, which is targeted by Rituximab, a therapeutic monoclonal antibody (mAb) employed in B-ALL treatment regimens. Our FLT3 mAb with enhanced affinity to the Fc receptor CD16a termed 4G8-SDIE potently induced NK cell reactivity against FLT3-transfectants, the B-ALL cell line SEM and primary leukemic cells of adult B-ALL patients in a target-antigen dependent manner as revealed by analyses of NK cell activation and degranulation. This was mirrored by potent 4G8-SDIE mediated NK cell ADCC in experiments with FLT3-transfectants, the cell line SEM and primary cells as target cells. Taken together, the findings presented in this study provide evidence that 4G8-SDIE may be a promising agent for the treatment of B-ALL, particularly in CD20-negative cases.

Tissue driven influences on human NK cell development, function and residence

Abstract Natural killer (NK) cells are innate immune cells with the inherent ability to kill tumor and virus infected cells without any prior priming. Recent studies have demonstrated the ability of NK cells to develop adaptive memory and tissue residence. However, our understanding of human NK cells in tissues and their site-specific properties is limited. Here we use our unique tissue resource established in collaboration with LiveOnNY to elucidate the diversity of human NK cell phenotypic subsets, functional profile and transcriptional signatures in non-mucosal and mucosal tissue sites. We identified tissue-specific patterns of NK cell maturation and show that NK cell subset (immature and mature) and memory NK cell distribution is a function of tissue site which is not affected by age. Our results also show that while NK cells from different tissue sites express similar levels of granzyme B, NK cell antibody-dependent cellular cytotoxicity response and cytokine driven activation may be influenced by tissue site. Whole transcriptome profiling of NK cell subsets revealed that mature NK cells from all the sites (blood, bone marrow, spleen, lung and LN) have very similar transcriptional profiles. Interestingly, immature NK cells from all tissue sites have very different transcriptional profiles compared to their blood counterpart and are enriched for tissue resident memory T cell transcriptional signature. Furthermore, we show that immature NK cells show site-specific expression patterns of tissue residence surface markers. Overall, our data demonstrates the effects of tissue microenvironment on the developmental and function potential of tissue NK cells and provides insight into the development of future NK cell mediated immunotherapies.

Determination of optimum vitamin D3 levels for NK cell-mediated rituximab- and obinutuzumab-dependent cellular cytotoxicity.

Vitamin D3 (25-OH-D3) deficiency impairs rituximab-dependent cellular cytotoxicity and the outcome of patients with diffuse large B-cell and follicular lymphomas (DLBCL). Since the optimum 25-OH-D3 serum levels for NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) are unknown, we determined the 25-OH-D3 serum levels associated with maximum NK cell-mediated ADCC. CD20 antibody-loaded CD20+ B-cell lymphoma cell lines were cultured with NK cells and ADCC activity was determined by lactate dehydrogenase release assays. Using a newly developed formula, pre-defined 25-OH-D3 serum levels were achieved with high individual precision over a wide range. NK cells from 20 healthy individuals killed antibody-treated CD20+ lymphoma cells in a concentration- and E:T ratio-dependent manner with obinutuzumab displaying a stronger ADCC activity than rituximab. Maximum NK-cell activity and ADCC were observed at 65ng/ml 25-OH-D3, the middle of the normal range (30-100ng/ml). 25-OH-D3 serum levels around this range should be the target in interventional trials aiming at improving NK cell-mediated ADCC by 25-OH-D3 substitution. Lower levels do not provide significant ADCC improvements in individuals with 25-OH-D3 deficiency or insufficiency and might result in the failure of interventions with 25-OH-D3.

Differential regulation of human monocytes and NK cells by antibody-opsonized tumors.

The monocyte network is important for therapeutic efficacy of antibody therapies against cancer. One mechanism which monocytes/macrophages use to kill cancer cells is phagocytosis. Using trastuzumab and human breast cancer cell lines as a model, we used flow cytometry to evaluate the importance of avidity, antigen density, Fcγ receptor (FcγR) expression, and FcγR polymorphisms in human monocyte phagocytosis. By increasing avidity for the tumor through the addition of pertuzumab to trastuzumab, there was a two-to-threefold increase in phagocytosis potency against the HCC1419 cell line compared to antibodies alone, while NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) failed to increase tumor cell death. Consistent with increasing the avidity through multiple antibodies, antigen density significantly enhanced phagocytosis with breast cancer cell lines that were HER2 gene-amplified compared to non-amplified tumor cells. Confirmation that high antigen density enhanced phagocytosis was obtained when HER2 was overexpressed in HER2 non-amplified cell lines. In contrast, NK cell ADCC failed to distinguish differences in tumor cell death when comparing gene-amplified and non-amplified breast cancer cell lines. The level of phagocytosis was influenced by FcγRIIa and FcγRIIIa expression. Most monocytes are FcγRIIIa-, and the induction of the receptor significantly enhances antibody-dependent phagocytosis. Although both receptors are involved, when blocked FcγRIIIa had a greater influence on phagocytosis. Furthermore, the polymorphism FcγRIIIa 158V significantly enhanced phagocytosis; whereas FcγRIIa 131H polymorphism appeared to improve phagocytosis but was not statistically significant. Targeting of monocytes for enhanced phagocytosis may improve the effectiveness of therapeutic antibodies to improve clinical outcomes.

Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain.

Antibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and nonstoichiometric and may affect an antibody's binding to its target. Moreover, an emerging field for therapeutics is antibody-drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation are crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human descent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full-length IgG.

The IL-15 Superagonist ALT-803 Enhances NK Cell ADCC and in Vivo Clearance of B Cell Lymphomas Directed By an Anti-CD20 Monoclonal Antibody

Anti-CD20 monoclonal antibodies (mAbs) have provided an important therapeutic passive immunotherapy approach for B cell malignancies. Typically anti-CD20 mAbs are combined with traditional chemotherapy and/or radiation therapy that have the potential for serious long term adverse complications. Novel approaches are needed to improve anti-CD20 mAb anti-lymphoma efficacy with agents that do not result in long term toxicity. ALT-803 is a super agonist IL-15 variant bound to an IL-15Ralpha – Fc fusion and represents a novel immunostimulatory agent for NK and T cells. Notably, ALT-803 exhibits extended in vivo pharmacokinetics and altered biodistribution compared to recombinant human IL-15. We hypothesized that ALT-803 would enhance anti-CD20-mAb-(rituximab) directed NK cell antibody dependent cellular cytotoxicity (ADCC) against B cell lymphomas. ALT-803 at concentrations of 0.35-35 ng/mL potentiated rituximab-triggered NK cell ADCC against the Raji (27% vs. 74%, E:T 25:1, P<0.01) and Daudi (39% vs. 84%, E:T 2:1, P<0.05) B cell lymphoma lines in vitro. Moreover, the activation of NK cells with ALT-803 significantly increased ADCC against primary human follicular lymphoma cells in vitro (11% vs. 33% at a 2.5:1 E:T ratio, P<0.001, N=5 primary follicular lymphomas, N=15 NK cell donors). One mechanism whereby ALT-803 may be modulating human NK cells is via the induction of cytotoxic effector molecules. After 24-48 hour in vitro activation with ALT-803 (0.35-350 ng/mL) an increased expression of granzyme B and perforin were observed in primary human NK cells (P<0.05). The effectiveness of ALT-803 enhancement of NK cell ADCC against B cell lymphoma cell lines was assessed with two in vivo mouse models. First, Daudi cells were engrafted into SCID mice (that have an intact NK cell compartment), and groups were treated with vehicle, rituximab (10 mg/kg), ALT-803 (0.2 mg/kg), or ALT-803+rituximab at day 15 and 18, and assessed for lymphoma percentages in the bone marrow at day 22. Mice treated with ALT-803+rituximab had significantly reduced Daudi B cell burden, compared to rituximab, ALT-803, or vehicle treatment (vehicle versus ALT-803+rituximab, 38% vs. 5%, P<0.01). At doses of 0.2-0.02 mg/kg the effect of ALT-803 on rituximab mediated lymphoma clearance was demonstrated (P<0.02 compared to vehicle treatment). Further, the survival of mice treated with ALT-803+rituximab was significantly longer compared vehicle control, rituximab alone, and ALT-803 alone groups (see KM survival curves, Figure, P<0.05). In our second in vivo model, malignant Raji B cells expressing luciferase were injected into immunodeficient NOD-SCID-gamma-c-/- (NSG) mice (100,000/mouse, day 0) followed by primary human NK cells (4 million / mouse, day 3), and treated (initiated on day 3) with vehicle, ALT-803 (0.05 mg/kg q3-4 days), rituximab (10 mg/kg day 3), or ALT-803+rituximab. At day 16, ALT-803+rituximab exhibited a significant reduction in Raji signal compared to the control groups (P<0.05). These results were confirmed in a second approach where an increased Raji cell numbers were engrafted (1 million / mouse, day 0), primary human NK cells (4 million / mouse) were infused on day 3, and groups of mice were treated with rituximab (5 mg/kg) or ALT-803 (0.2 mg/kg q3-4 days)+rituximab (mean photons per second at day 34, 3.8x109 versus 3.1x108, respectively, P<0.05). ALT-803 was well tolerated at all of the administered dose levels in combination with rituximab. Thus, ALT-803 represents an effective immunostimulatory agent that augments NK cell cytotoxic potential and ADCC against malignant follicular lymphoma and B cell lines in vitro, and significantly increases rituximab-directed clearance of B cell lymphoma by NK cells in two in vivo models. Based on these findings, a phase 1/2 clinical trial of ALT-803 plus rituximab is planned for patients with relapsed/refractory indolent non-Hodgkin lymphomas. [Display omitted] DisclosuresLiu:Altor BioScience Corporation: Employment. Kong:Altor BioScience Corporation: Employment. Jeng:Altor BioScience Corporation: Employment. Rhode:Altor BioScience Corporation: Employment. Wong:Altor BioScience Corporation: Employment.

An Fc-Engineered CD133 Antibody for Induction of NK Cell Reactivity Against Cancer Cells

CD133 is expressed in various types of solid tumors where it predicts an unfavourable clinical outcome (e.g. Linuma et al., J Clin Oncol. 2011). Moreover, CD133 has been shown to be preferentially expressed by cancer stem cells (CSC), as e.g. demonstrated by its usability to enrich CSCs containing populations within cancer cell preparations (e.g. Zeppernick et al., Nature 2008). As CSC display marked resistance to chemo- and radiation therapy, great efforts are presently made to develop immunotherapeutic approaches to specifically target CSC without harming healthy tissue. We here report on the preclinical characterization of a monoclonal antibody (mAb) directed towards CD133 for induction of NK cell reactivity against cancer cells. Two different anti-CD133 antibody clones (W6B3 and 293C3) were used to study expression levels of CD133 in different cell lines derived from solid tumors (A172 and U87MG, glioblastoma; CaCo-2, Colo-205, HCT-8, HT-29 and SW48, colorectal cancer; MDA-MB-231, MDA-MB-468 and BT474, breast cancer; WERI-Rb1, retinoblastoma). CD133 was recognized by W6B3 and 293C3 in 4 and 5 out of the 11 cell lines, respectively. The latter mAb was then chosen for the development of chimeric mAbs with either a wildtype human Fc part (293C3-WT) or a variant containing amino acid exchanges (S239D/I332E) to enhance its affinity to the activating Fc receptor CD16 (293C3-SDIE) and thereby its capacity to mediate antibody dependent cellular cytotoxicity (ADCC), an important antibody property that largely contributes to the clinical success of antitumor mAbs. After confirming the binding specificity of both 293C3-WT and 293C3-SDIE we employed cytotoxicity assays to comparatively analyze their ability to induce NK cell ADCC against different solid tumor cell lines. In all cases, profound induction of tumor cell lysis upon application of 293C3-SDIE was observed, and the therapeutic effect of the Fc-optimized mAb clearly exceeded that of its counterpart containing a wild type Fc part. Notably, colony forming unit assays did not reveal any toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells. Thus, treatment with 293C3-SDIE constitutes a promising novel strategy for immunotherapy of cancer, in particular considering its ability to preferentially target CSC which is presently under study. DisclosuresNo relevant conflicts of interest to declare.

Development and Preclinical Characterization of an Fc-Optimized CD133 Antibody for Induction of NK Cell Reactivity Against Myeloid Leukemia

NK cells are cytotoxic lymphocytes that play a major role in anti-tumor immunity and largely contribute to the efficacy of allogenic stem cell transplantation (SCT) in leukemia. Another clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of monoclonal antibodies (mAb) like Rituximab, a feature considered critical for the therapeutic success of antibody treament. Modifications of the human IgG1 Fc-part in anti-tumor antibodies lead to markedly improved capability to recruit Fc-receptor bearing effector cells as highlighted by the improved clinical efficacy of the Fc-engineered CD20 antibody Ofatumumab as compared to its unmodified counterpart Rituximab in CLL. So far, no immunotherapeutic antibodies are available for the treatment of myeloid leukemias. Here we report on the development and preclinical characterization of an Fc-optimized mAb directed towards CD133, which is expressed on a wide variety of malignant cell types. As a first step we evaluated binding of three different mouse anti-human CD133 mAbs (clones AC133, W6B3 and 293C3) to 20 primary AML and 6 primary CML samples in order to identify a clone with optimal binding characteristics. AC133 and W6B3 comparably bound to the leukemic cells in 11/20 AML and 5/6 CML samples. In contrast, binding of 293C3 was observed in 18/20 AML cases and 5/6 CML cases. Thus, 293C3 recognizes a different epitope than the other two antibody clones, which is expressed in a high proportion of myeloid leukemia cases. Accordingly, 293C3 was selected for generating chimeric mAbs with either a wildtype Fc part (293C3-WT) or a variant containing distinct modifications (S239D/I332E) to enhance its affinity to the activating Fc receptor CD16 (293C3-SDIE). The binding specificity of 293C3-WT and 293C3-SDIE was validated by FACS in analyses with CD133 transfectants and mock controls. When comparing 293C3-WT and 293C3-SDIE with regard to their immunostimulatory properties, we found that already 293C3-WT induced NK cell ADCC against primary leukemia cells as revealed by analyses of degranulation and target cell lysis. These effects were by far exceeded by treatment with 293C3-SDIE, confirming the functional relevance of the SDIE modification in its Fc part. Notably, treatment with 293C3-SDIE also enhanced the reactivity of NK cells against CD133-positive AML cells in an autologous setting. Considering the expression of CD133, among others, on healthy hematopoietic progenitor cells, we further performed colony forming unit assays with healthy bone marrow cells, which did not reveal any toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells. Thus, 293C3-SDIE constitutes an attractive immunotherapeutic compound which we envisage in particular for the elimination of minimal residual disease in CD133 bearing leukemia, especially in the context of allogenic SCT. DisclosuresNo relevant conflicts of interest to declare.

Fc-optimized NKG2D-Fc constructs induce NK cell antibody-dependent cellular cytotoxicity against breast cancer cells independently of HER2/neu expression status.

The ability of NK cells to mediate Ab-dependent cellular cytotoxicity (ADCC) largely contributes to the clinical success of antitumor Abs, including trastuzumab, which is approved for the treatment of breast cancer with HER2/neu overexpression. Notably, only ∼25% of breast cancer patients overexpress HER2/neu. Moreover, HER2/neu is expressed on healthy cells, and trastuzumab application is associated with side effects. In contrast, the ligands of the activating immunoreceptor NKG2D (NKG2DL) are selectively expressed on malignant cells. In this study, we took advantage of the tumor-associated expression of NKG2DL by using them as target Ags for NKG2D-IgG1 fusion proteins optimized by amino acid exchange S239D/I332E in their Fc part. Compared to constructs with wild-type Fc parts, fusion proteins carrying the S239D/I332E modification (NKG2D-Fc-ADCC) mediated highly enhanced degranulation, ADCC, and IFN-γ production of NK cells in response to breast cancer cells. NKG2D-Fc-ADCC substantially enhanced NK reactivity also against HER2/neu-low targets that were unaffected by trastuzumab, as both compounds mediated their immunostimulatory effects in strict dependence of target Ag expression levels. Thus, in line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to its highly increased affinity to CD16, NKG2D-Fc-ADCC potently enhances NK cell reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, NKG2D-Fc-ADCC may constitute an attractive means for immunotherapy especially of HER2/neu-low or -negative breast cancer.