Amy K. Erbe

NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy

Natural killer (NK) cells play a major role in cancer immunotherapies that involve tumor-antigen targeting by monoclonal antibodies (mAbs). NK cells express a variety of activating and inhibitory receptors that serve to regulate the function and activity of the cells. In the context of targeting cells, NK cells can be “specifically activated” through certain Fc receptors that are expressed on their cell surface. NK cells can express FcγRIIIA and/or FcγRIIC, which can bind to the Fc portion of immunoglobulins, transmitting activating signals within NK cells. Once activated through Fc receptors by antibodies bound to target cells, NK cells are able to lyse target cells without priming, and secrete cytokines like interferon gamma to recruit adaptive immune cells. This antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells is utilized in the treatment of various cancers overexpressing unique antigens, such as neuroblastoma, breast cancer, B cell lymphoma, and others. NK cells also express a family of receptors called killer immunoglobulin-like receptors (KIRs), which regulate the function and response of NK cells toward target cells through their interaction with their cognate ligands that are expressed on tumor cells. Genetic polymorphisms in KIR and KIR-ligands, as well as FcγRs may influence NK cell responsiveness in conjunction with mAb immunotherapies. This review focuses on current therapeutic mAbs, different strategies to augment the anti-tumor efficacy of ADCC, and genotypic factors that may influence patient responses to antibody-dependent immunotherapies.

Increasing the Clinical Efficacy of NK and Antibody-Mediated Cancer Immunotherapy: Potential Predictors of Successful Clinical Outcome Based on Observations in High-Risk Neuroblastoma

Disease recurrence is frequent in high-risk neuroblastoma (NBL) patients even after multi-modality aggressive treatment [a combination of chemotherapy, surgical resection, local radiation therapy, autologous stem cell transplantation, and cis-retinoic acid (CRA)]. Recent clinical studies have explored the use of monoclonal antibodies (mAbs) that bind to disialoganglioside (GD2), highly expressed in NBL, as a means to enable immune effector cells to destroy NBL cells via antibody-dependent cell-mediated cytotoxicity (ADCC). Preclinical data indicate that ADCC can be more effective when appropriate effector cells are activated by cytokines. Clinical studies have pursued this by administering anti-GD2 mAb in combination with ADCC-enhancing cytokines (IL2 and GM-CSF), a regimen that has demonstrated improved cancer-free survival. More recently, early clinical studies have used a fusion protein that consists of the anti-GD2 mAb directly linked to IL2, and anti-tumor responses were seen in the Phase II setting. Analyses of genes that code for receptors that influence ADCC activity and natural killer (NK) cell function [Fc receptor (FcR), killer immunoglublin-like receptor (KIR), and KIR-ligand (KIR-L)] suggest patients with anti-tumor activity are more likely to have certain genotype profiles. Further analyses will need to be conducted to determine whether these genotypes can be used as predictive markers for favorable therapeutic outcome. In this review, we discuss factors that affect response to mAb-based tumor therapies such as hu14.18-IL2. Many of our observations have been made in the context of NBL; however, we will also include some observations made with mAbs targeting other tumor types that are consistent with results in NBL. Therefore, we hypothesize that the NBL observations discussed here may also be relevant to mAb therapy for other cancers, in which ADCC is known to play a role.

Human NK cells maintain licensing status and are subject to killer immunoglobulin-like receptor (KIR) and KIR-ligand inhibition following ex vivo expansion

Infusion of allogeneic NK cells is a potential immunotherapy for both hematopoietic malignancies and solid tumors. Interactions between killer immunoglobulin-like receptors (KIR) on human NK cells and KIR-ligands on tumor cells influence the magnitude of NK function. To obtain sufficient numbers of activated NK cells for infusion, one potent method uses cells from the K562 human erythroleukemia line that have been transfected to express activating 41BB ligand (41BBL) and membrane-bound interleukin 15 (mbIL15). The functional importance of KIRs on ex vivo expanded NK cells has not been studied in detail. We found that after a 12-day co-culture with K562-mbIL15-41BBL cells, expanded NK cells maintained inhibition specificity and prior in vivo licensing status determined by KIR/KIR-ligand interactions. Addition of an anti-CD20 antibody (rituximab) induced NK-mediated antibody-dependent cellular cytotoxicity and augmented killing of CD20+ target cells. However, partial inhibition induced by KIR/KIR-ligand interactions persisted. Finally, we found that extended co-cultures of NK cells with stimulatory cells transduced to express various KIR-ligands modified both the inhibitory and activating KIR repertoires of the expanded NK cell product. These studies demonstrate that the licensing interactions known to occur during NK ontogeny also influence NK cell function following NK expansion ex vivo with HLA-null stimulatory cells.

Neuroblastoma Patients' KIR and KIR-Ligand Genotypes Influence Clinical Outcome for Dinutuximab-based Immunotherapy: A Report from the Children's Oncology Group

Purpose: In 2010, a Childrens Oncology Group (COG) phase III randomized trial for patients with high-risk neuroblastoma (ANBL0032) demonstrated improved event-free survival (EFS) and overall survival (OS) following treatment with an immunotherapy regimen of dinutuximab, GM-CSF, IL2, and isotretinoin compared with treatment with isotretinoin alone. Dinutuximab, a chimeric anti-GD2 monoclonal antibody, acts in part via natural killer (NK) cells. Killer immunoglobulin-like receptors (KIR) on NK cells and their interactions with KIR-ligands can influence NK cell function. We investigated whether KIR/KIR-ligand genotypes were associated with EFS or OS in this trial. Experimental Design: We genotyped patients from COG study ANBL0032 and evaluated the effect of KIR/KIR-ligand genotypes on clinical outcomes. Cox regression models and log-rank tests were used to evaluate associations of EFS and OS with KIR/KIR-ligand genotypes. Results: In this trial, patients with the “all KIR-ligands present” genotype as well as patients with inhibitory KIR2DL2 with its ligand (HLA-C1) together with inhibitory KIR3DL1 with its ligand (HLA-Bw4) were associated with improved outcome if they received immunotherapy. In contrast, for patients with the complementary KIR/KIR-ligand genotypes, clinical outcome was not significantly different for patients who received immunotherapy versus those receiving isotretinoin alone. Conclusions: These data show that administration of immunotherapy is associated with improved outcome for neuroblastoma patients with certain KIR/KIR-ligand genotypes, although this was not seen for patients with other KIR/KIR-ligand genotypes. Further investigation of KIR/KIR-ligand genotypes may clarify their role in cancer immunotherapy and may enable KIR/KIR-ligand genotyping to be used prospectively for identifying patients likely to benefit from certain cancer immunotherapy regimens. Clin Cancer Res; 24(1); 189–96. ©2017 AACR. See related commentary by Cheung and Hsu, p. 3

HLA-Bw4-I-80 isoform differentially influences clinical outcome as compared to HLA-Bw4-T-80 and HLA-A-Bw4 isoforms in rituximab or dinutuximab-based cancer immunotherapy

Killer-cell immunoglobulin-like receptors (KIRs) are a family of glycoproteins expressed primarily on natural killer cells that can regulate their function. Inhibitory KIRs recognize MHC class I molecules (KIR-ligands) as ligands. We have reported associations of KIRs and KIR-ligands for patients in two monoclonal antibody (mAb)-based trials: (1) A Children’s Oncology Group (COG) trial for children with high-risk neuroblastoma randomized to immunotherapy treatment with dinutuximab (anti-GD2 mAb) + GM-CSF + IL-2 + isotretinion or to treatment with isotretinoin alone and (2) An Eastern Cooperative Oncology Group (ECOG) trial for adults with low-tumor burden follicular lymphoma responding to an induction course of rituximab (anti-CD20 mAb) and randomized to treatment with maintenance rituximab or no-maintenance rituximab. In each trial, certain KIR/KIR-ligand genotypes were associated with clinical benefit for patients randomized to immunotherapy treatment (immunotherapy in COG; maintenance rituximab in ECOG) as compared to patients that did not receive the immunotherapy [isotretinoin alone (COG); no-maintenance (ECOG)]. Namely, patients with both KIR3DL1 and its HLA-Bw4 ligand (KIR3DL1+/HLA-Bw4+ genotype) had improved clinical outcomes if randomized to immunotherapy regimens, as compared to patients with the KIR3DL1+/HLA-Bw4+ genotype randomized to the non-immunotherapy regimen. Conversely, patients that did not have the KIR3DL1+/HLA-Bw4+ genotype showed no evidence of a difference in outcome if receiving the immunotherapy vs. no-immunotherapy. For each trial, HLA-Bw4 status was determined by assessing the genotypes of three separate isoforms of HLA-Bw4: (1) HLA-B-Bw4 with threonine at amino acid 80 (B-Bw4-T80); (2) HLA-B-Bw4 with isoleucine at amino acid 80 (HLA-B-Bw4-I80); and (3) HLA-A with a Bw4 epitope (HLA-A-Bw4). Here, we report on associations with clinical outcome for patients with KIR3DL1 and these separate isoforms of HLA-Bw4. Patients randomized to immunotherapy with KIR3DL1+/A-Bw4+ or with KIR3DL1+/B-Bw4-T80+ had better outcome vs. those randomized to no-immunotherapy, whereas for those with KIR3DL1+/B-Bw4-I80+ there was no evidence of a difference based on immunotherapy vs. no-immunotherapy. Additionally, we observed differences within treatment types (either within immunotherapy or no-immunotherapy) that were associated with the genotype status for the different KIR3DL1/HLA-Bw4-isoforms. These studies suggest that specific HLA-Bw4 isoforms may differentially influence response to these mAb-based immunotherapy, further confirming the involvement of KIR-bearing cells in tumor-reactive mAb-based cancer immunotherapy.

Donor selection for ex vivo-expanded natural killer cells as adoptive cancer immunotherapy

Department of Human Oncology, University of Wisconsin, Madison, WI, USA Department of Pediatrics, University of Wisconsin, Madison, WI, USA *Author for correspondence: Tel.: +1 608 263 9069; Fax: +1 608 263 4226; pmsondel@humonc.wisc.edu

FCGR Polymorphisms Influence Response to IL-2 in Metastatic Renal Cell Carcinoma

Purpose: Fc-gamma receptors (FCGRs) are expressed on immune cells, bind to antibodies, and trigger antibody-induced cell-mediated antitumor responses when tumor-reactive antibodies are present. The affinity of the FCGR/antibody interaction is variable and dependent upon FCGR polymorphisms. Prior studies of patients with cancer treated with immunotherapy indicate that FCGR polymorphisms can influence antitumor response for certain immunotherapies that act via therapeutically administered mAbs or via endogenous tumor-reactive antibodies induced from tumor antigen vaccines. The previously published “SELECT” trial of high-dose aldesleukin (HD-IL2) for metastatic renal cell carcinoma resulted in an objective response rate of 25%. We evaluated the patients in this SELECT trial to determine whether higher-affinity FCGR polymorphisms are associated with outcome. Experimental Design: SNPs in FCGR2A, FCGR3A, and FCGR2C were analyzed, individually and in combination, for associations between genotype and clinical outcome. Results: When higher-affinity genotypes for FCGR2A, FCGR3A, and FCGR2C were considered together, they were associated with significantly increased tumor shrinkage and prolonged survival in response to HD-IL2. Conclusions: Although associations of higher-affinity FCGR genotype with clinical outcome have been demonstrated with mAb therapy and with idiotype vaccines, to our knowledge, this is the first study to show associations of FCGR genotypes with outcome following HD-IL2 treatment. We hypothesize that endogenous antitumor antibodies may engage immune cells through their FCGRs, and HD-IL2 may enhance antibody-induced tumor destruction, or antibody-enhanced tumor antigen presentation, via augmented activation of innate or adaptive immune responses; this FCGR-mediated immune activity would be augmented through immunologically favorable FCGRs. Clin Cancer Res; 23(9); 2159–68. ©2016 AACR.

Suppression of the mouse double minute 4 gene causes changes in cell cycle control in a human mesothelial cell line responsive to ultraviolet radiation exposure.

The TP53 tumor suppressor gene is the most frequently inactivated gene in human cancer identified to date. However, TP53 mutations are rare in human mesotheliomas, as well as in many other types of cancer, suggesting that aberrant TP53 function may be due to alterations in its regulatory pathways. Mouse double minute 4 (MDM4) has been shown to be a key regulator of TP53 activity, both independently as well as in concert with its structural homolog, Mouse Double Minute 2 (MDM2). The purpose of this study was to characterize the effects of MDM4 suppression on TP53 and other proteins involved in cell cycle control before and after ultraviolet (UV) exposure in MeT5a cells, a nonmalignant human mesothelial line. Short hairpin RNA (shRNA) was used to investigate the impact of MDM4 on TP53 function and cellular transcription. Suppression of MDM4 was confirmed by Western blot. MDM4 suppressed cells were analyzed for cell cycle changes with and without exposure to UV. Changes in cell growth as well as differences in the regulation of direct transcriptional targets of TP53, CDKN1A (cyclin‐dependent kinase 1α, p21) and BAX, suggest a shift from cell cycle arrest to apoptosis upon increasing UV exposure. These results demonstrate the importance of MDM4in cell cycle regulation as well as a possible role inthe pathogenesis of mesothelioma‐type cancers. Environ. Mol. Mutagen. 2009.

Tumor-Specific Inhibition of In Situ Vaccination by Distant Untreated Tumor Sites

Untreated tumor sites antagonize the systemic and local antitumor immune response to an in situ vaccination regimen. This effect is radiation sensitive and may be mediated by tumor-specific regulatory T cells harbored in the untreated tumor sites. In situ vaccination is an emerging cancer treatment strategy that uses local therapies to stimulate a systemic antitumor immune response. We previously reported an in situ vaccination effect when combining radiation (RT) with intratumor (IT) injection of tumor-specific immunocytokine (IC), a fusion of tumor-specific antibody and IL2 cytokine. In mice bearing two tumors, we initially hypothesized that delivering RT plus IT-IC to the “primary” tumor would induce a systemic antitumor response causing regression of the “secondary” tumor. To test this, mice bearing one or two syngeneic murine tumors of B78 melanoma and/or Panc02 pancreatic cancer were treated with combined external beam RT and IT-IC to the designated “primary” tumor only. Primary and secondary tumor response as well as animal survival were monitored. Immunohistochemistry and quantitative real-time PCR were used to quantify tumor infiltration with regulatory T cells (Treg). Transgenic “DEREG” mice or IgG2a anti–CTLA-4 were used to transiently deplete tumor Tregs. Contrary to our initial hypothesis, we observed that the presence of an untreated secondary tumor antagonized the therapeutic effect of RT + IT-IC delivered to the primary tumor. We observed reciprocal tumor specificity for this effect, which was circumvented if all tumors received RT or by transient depletion of Tregs. Primary tumor treatment with RT + IT-IC together with systemic administration of Treg-depleting anti–CTLA-4 resulted in a renewed in situ vaccination effect. Our findings show that untreated tumors can exert a tumor-specific, Treg-dependent, suppressive effect on the efficacy of in situ vaccination and demonstrate clinically viable approaches to overcome this effect. Untreated tumor sites antagonize the systemic and local antitumor immune response to an in situ vaccination regimen. This effect is radiation sensitive and may be mediated by tumor-specific regulatory T cells harbored in the untreated tumor sites. Cancer Immunol Res; 6(7); 825–34. ©2018 AACR.

Genotyping Single Nucleotide Polymorphisms and Copy Number Variability of the FCGRs Expressed on NK Cells

Natural killer (NK) cells are one of the main effector immune cells involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Upon recognition of cell-bound IgG antibodies, which occurs through Fc gamma receptors (FCGRs) expressed on the cell surface of NK cells, NK cells become activated and lyse target tumor or infected cells. The FCGRs, FCGR3A and FCGR2C, expressed on the surface of NK cells have single nucleotide polymorphisms (SNPs) that result in differential activity of NK cells. In addition to SNP genetic variation within each of these genes, the FCGRs are subject to copy number variation (CNV), which leads to variable protein expression levels on the cell surface. Studies have found that FCGR genotype for FCGR3A and FCGR2C is associated with variation in the response to immunotherapy.Due to high sequence homology within FCGR3 and FCGR2 families, there are difficulties associated with genotyping these specific receptors related to cross-amplification of non-targeted FCGRs. To improve specificity for both FCGR3A and FCGR2C, Rnase-H (RH) primers were designed to amplify specifically FCGR3A (while not co-amplifying FCGR3B) and FCGR2C (while not co-amplifying FCGR2B). In addition, fluorescently labeled locked nucleic acid (LNA) probes provide additional precision for determination of the SNPs within both FCGR3A and FCGR2C. For CNV determination, separate fluorescently labeled probes for FCGR3A, and for FCGR2C, can be used with the same RH primers for each gene. These probes can be combined in the same well with control primers/probe for a known diploid gene and used to calculate the copy number of both FCGR3A and FCGR2C. Here we provide new detailed methodology that allows for the specific amplification of these FCGRs in a single PCR reaction, allowing for genotyping of both the SNPs and CNVs using real-time PCR.