Antibody-dependent Cellular Cytotoxicity Assays Research Articles

Broad Impairment of Natural Killer Cells from Operationally Tolerant Kidney Transplanted Patients.

The role of natural killer (NK) cells in organ transplantation is controversial. This study aims to decipher their role in kidney transplant tolerance in humans. Previous studies highlighted several modulated genes involved in NK cell biology in blood from spontaneously operationally tolerant patients (TOLs; drug-free kidney-transplanted recipients with stable graft function). We performed a phenotypic, functional, and genetic characterization of NK cells from these patients compared to kidney-transplanted patients with stable graft function under immunosuppression and healthy volunteers (HVs). Both operationally TOLs and stable patients harbored defective expression of the NKp46 activator receptor and lytic molecules perforin and granzyme compared to HVs. Surprisingly, NK cells from operationally TOLs also displayed decreased expression of the CD16 activating marker (in the CD56Dim NK cell subset). This decrease was associated with impairment of their functional capacities upon stimulation, as shown by lower interferon gamma (IFNγ) production and CD107a membranous expression in a reverse antibody-dependent cellular cytotoxicity (ADCC) assay, spontaneous lysis assays, and lower target cell lysis in the 51Cr release assay compared to HVs. Conversely, despite impaired K562 cell lysis in the 51Cr release assay, patients with stable graft function harbored a normal reverse ADCC and even increased amounts of IFNγ+ NK cells in the spontaneous lysis assay. Altogether, the strong impairment of the phenotype and functional cytotoxic capacities of NK cells in operationally TOLs may accord with the establishment of a pro-tolerogenic environment, despite remaining highly activated after transplantation in patients with stable graft function.

Antibody-Dependent Cellular Cytotoxicity Responses to Seasonal Influenza Vaccination in Older Adults.

Older adults are at high risk of influenza disease, but generally respond poorly to vaccination. Antibody-dependent cellular cytotoxicity (ADCC) may be an important component of protection against influenza infection. An improved understanding of the ADCC response to influenza vaccination in older adults is required. We studied sera samples from 3 groups of subjects aged ≥65 years (n = 16-17/group) receiving the 2008/2009 seasonal trivalent influenza vaccine (TIV). Subjects had minimal pre-existing hemagglutination inhibiting (HAI) antibodies and TIV induced either no, low, or high HAI responses. Serum ADCC activity was analyzed using Fc receptor cross-linking, NK cell activation, and influenza-infected cell killing. Most subjects from TIV nonresponder, low responder, and high responder groups had detectable ADCC antibodies prevaccination, but baseline ADCC was not predictive of HAI vaccine responsiveness. Interestingly, ADCC and HAI responses tracked closely across all groups, against all 3 TIV hemagglutinins, and in all ADCC assays tested. Older adults commonly have pre-existing ADCC antibodies in the absence of high HAI titers to circulating influenza strains. In older vaccinees, ADCC response mirrored HAI antibodies and was readily detectable despite high postvaccination HAI titers. Alternate measures of vaccine responsiveness and improved vaccinations in this at-risk group are needed.

A mathematical model of antibody-dependent cellular cytotoxicity (ADCC)

Immunotherapies exploit the immune system to target and kill cancer cells, while sparing healthy tissue. Antibody therapies, an important class of immunotherapies, involve the binding to specific antigens on the surface of the tumour cells of antibodies that activate natural killer (NK) cells to kill the tumour cells. Preclinical assessment of molecules that may cause antibody-dependent cellular cytotoxicity (ADCC) involves co-culturing cancer cells, NK cells and antibody in vitro for several hours and measuring subsequent levels of tumour cell lysis. Here we develop a mathematical model of such an in vitro ADCC assay, formulated as a system of time-dependent ordinary differential equations and in which NK cells kill cancer cells at a rate which depends on the amount of antibody bound to each cancer cell. Numerical simulations generated using experimentally-based parameter estimates reveal that the system evolves on two timescales: a fast timescale on which antibodies bind to receptors on the surface of the tumour cells, and NK cells form complexes with the cancer cells, and a longer time-scale on which the NK cells kill the cancer cells. We construct approximate model solutions on each timescale, and show that they are in good agreement with numerical simulations of the full system. Our results show how the processes involved in ADCC change as the initial concentration of antibody and NK-cancer cell ratio are varied. We use these results to explain what information about the tumour cell kill rate can be extracted from the cytotoxicity assays.

Functional, Biophysical, and Structural Characterization of Human IgG1 and IgG4 Fc Variants with Ablated Immune Functionality.

Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2σ with barely detectable activity in antibody-dependent cellular cytotoxicity, phagocytosis, complement activity, and Fcγ receptor binding assays. Here, we extend that work to IgG1 and IgG4 antibodies, alternative subtypes which may offer advantages over IgG2 antibodies. In several in vitro and in vivo assays, the IgG1σ and IgG4σ variants showed equal or even lower Fc-related activities than the corresponding IgG2σ variant. In particular, IgG1σ and IgG4σ variants demonstrate complete lack of effector function as measured by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and in vivo T-cell activation. The IgG1σ and IgG4σ variants showed acceptable solubility and stability, and typical human IgG1 pharmacokinetic profiles in human FcRn-transgenic mice and cynomolgus monkeys. In silico T-cell epitope analyses predict a lack of immunogenicity in humans. Finally, crystal structures and simulations of the IgG1σ and IgG4σ Fc domains can explain the lack of Fc-mediated immune functions. These variants show promise for use in those therapeutic antibodies and Fc fusions for which the Fc domain should be immunologically “silent”.

Natural Killer (NK) Cell Education Differentially Influences HIV Antibody-Dependent NK Cell Activation and Antibody-Dependent Cellular Cytotoxicity.

Immunotherapy using broadly neutralizing antibodies (bNAbs) endowed with Fc-mediated effector functions has been shown to be critical for protecting or controlling viral replication in animal models. In human, the RV144 Thai trial was the first trial to demonstrate a significant protection against HIV infection following vaccination. Analysis of the correlates of immune protection in this trial identified an association between the presence of antibody-dependent cellular cytotoxicity (ADCC) mediated by immunoglobulin G (IgG) antibodies (Abs) to HIV envelope (Env) V1/V2 loop structures and protection from infection, provided IgA Abs with competing specificity were not present. Systems serology analyses implicated a broader range of Ab-dependent functions in protection from HIV infection, including but not limited to ADCC and Ab-dependent NK cell activation (ADNKA) for secretion of IFN-γ and CCL4 and expression of the degranulation marker CD107a. The existence of such correlations in the absence of bNAbs in the RV144 trial suggest that NK cells could be instrumental in protecting against HIV infection by limiting viral spread through Fc-mediated functions such as ADCC and the production of antiviral cytokines/chemokines. Beside the engagement of FcγRIIIa or CD16 by the Fc portion of anti-Env IgG1 and IgG3 Abs, natural killer (NK) cells are also able to directly kill infected cells and produce cytokines/chemokines in an Ab-independent manner. Responsiveness of NK cells depends on the integration of activating and inhibitory signals through NK receptors, which is determined by a process during their development known as education. NK cell education requires the engagement of inhibitory NK receptors by their human leukocyte antigen ligands to establish tolerance to self while allowing NK cells to respond to self cells altered by virus infection, transformation, stress, and to allogeneic cells. Here, we review recent findings regarding the impact of inter-individual differences in NK cell education on Ab-dependent functions such as ADCC and ADNKA, including what is known about the HIV Env epitope specificity of ADCC competent Abs and the conformation of HIV Env on target cells used for ADCC assays.

Abstract 644: CA125/MUC16 suppresses antibody-dependent cellular cytotoxicity of IgG1-based therapies via perturbation of antibody Fc-Receptor engagement on immune effector cells

Abstract Background: Human cancers employ a number of mechanisms to evade host immune responses. Here we report the effects of the tumor-shed antigen CA125 (MUC16) on suppressing IgG1-mediated antibody-dependent cellular cytotoxicity (ADCC). This evidence stems from prespecified subgroup analysis of an 1100 patient Phase 3 clinical trial testing the investigational agent farletuzumab, a monoclonal antibody (mAb) to folate receptor alpha, plus standard-of-care in patients with recurrent ovarian cancer. In this study, patients with low levels of CA125 [no greater than 3X the upper limit of normal (< 3X ULN)] treated with farletuzumab compared to placebo demonstrated improvements in both progression free survival (HR 0.49, p = 0.0028) and overall survival (HR 0.44, p = 0.0108). Farletuzumab’s pharmacologic activity is mediated in part through ADCC. Here we show that CA125 inhibits farletuzumab’s ADCC activity by suppressing antibody interaction with the CD16a Fc-Receptor on effector cells. Methods: Functional assays employing Jurkat-hCD16 reporter cells as well as primary NK cells were used to measure ADCC activity. CA125 isolates from various patients and cell lines were used in ADCC assays to monitor CA125 effects on immune-effector function. Multiple isogenic tumor cells expressing endogenous CA125 and their CA125-null counterparts were also used to monitor CD16a-mediated ADCC activity. CD16a+ cell binding assays were used to measure the effects of CA125 inhibition on CD16a Fc-Receptor interaction and activation. Recombinant CD16a and humanized mAbs were used in ELISA format to measure molecular binding. Results: Functional assays showed that exogenously added tumor-shed CA125 was able to suppress ADCC mediated by IgG1-type mAbs against several target cell lines. This CA125-mediated ADCC suppression was also observed in cells naturally expressing membrane-bound CA125 but not in isogenic CA125-null cells. Molecular studies suggest that CA125-mediated immunosuppression is caused by the perturbation of antibody interaction with the CD16a Fc-Receptor and that this effect is specific to IgG-type mAbs. Conclusions: Here we demonstrate that the tumor-shed antigen CA125 can elicit immunosuppression by blocking the interaction of tumor-targeting mAbs and CD16a Fc-Receptor on immune effector cells. This effect appears to be through a direct interaction of CA125 with antibody-CD16a proteins. These findings support a biological mechanism for the clinical observation that relapsed ovarian cancer patients with low CA125 blood levels have improved clinical responses to the experimental agent farletuzumab. These findings also provide new insights on the potential biological mechanisms for which tumors produce tumor-shed antigens such as CA125 to suppress host anti-tumor immune responses. Citation Format: J. Bradford Kline, Rina Kennedy, Shawn Fernando, Jennifer McDonough, Earl Albone, Elizabeth B. Somers, Wenquan Wang, Charles Schweizer, Luigi Grasso, Nicholas C. Nicolaides. CA125/MUC16 suppresses antibody-dependent cellular cytotoxicity of IgG1-based therapies via perturbation of antibody Fc-Receptor engagement on immune effector cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 644. doi:10.1158/1538-7445.AM2017-644

Abstract SY09-01: Next-generation anti-CTLA-4 antibodies

Abstract The activity of ipilimumab as a single agent and in combination with nivolumab (anti-PD-1) in melanoma, as well as the use of ipilimumab/nivolumab combinations in other malignancies, has confirmed the importance of CTLA-4 blockade in immunotherapy. The antitumor effect of this treatment also results in significant immune-related adverse events that limit dosing and result in patient discontinuation. We have taken two approaches to alter the activity of ipilimumab so as to improve its potency and its safety profile. One approach is to enhance the antibody-dependent cellular cytotoxicity (ADCC) activity of ipilimumab in order to increase the potential for Treg depletion at the tumor site; this would be expected to increase the activity of the antibody. The second approach is to produce a prodrug form of ipilimumab (an anti-CTLA-4 Probody therapeutic) that will have reduced activity systemically, but will become proteolytically cleaved at the tumor site to produce the fully functional antibody; the goal of this approach is to reduce the adverse event profile while retaining the antitumor activity of ipilimumab. It has previously been shown that antitumor activity of anti-CTLA-4 antibodies in mouse models of cancer is dependent on the ability of the antibody to bind activating FcγRs and mediate ADCC against Tregs at the tumor site (1, 2). Although human IgG1 Abs have been shown to be effective mediators of ADCC in patients with hematologic malignancies, it is still unclear whether ipilimumab mediates Treg depletion in solid tumors. Using in vitro ADCC assays, we have found that a nonfucosylated (NF) version of ipilimumab (ipilimumab-NF) has increased activity compared to ipilimumab. Ipilimumab-NF also demonstrates increased IL-2 secretion in peripheral mononuclear cells treated with the superantigen SEB as compared to ipilimumab. Transgenic mice that express human FcγRs in the place of mouse FcγRs were used to investigate the activity of anti-mouse surrogate CTLA-4 antibody engineered with either a human IgG1 or human IgG1-NF Fc region in a mouse tumor model. In these mice, the IgG1-NF version of anti-mouse CTLA-4 was found to significantly increase antitumor activity and Treg depletion at the tumor site compared to the IgG1. These data suggest that the clinical activity of ipilimumab could be enhanced by use of the nonfucosylated version of the Ab. In addition, ipilimumab-NF was tested for its ability to enhance a vaccine response in Mauritian cynomolgus macaques. Ipilimumab-NF was shown to result in increased vaccine-induced T-cell responses compared to ipilimumab using two replication-incompetent adenovirus serotype 5 viral vectors encoding SIV antigens as assessed by MHC-I tetramers and IFN-gamma ELISPOT in Mauritian cynomolgus macaques expressing the common allele, Mafa-A1*063. In a second approach, using Probody platform technology developed by CytomX, we have developed an anti-CTLA-4 Probody therapeutic (Probody Tx) based on ipilimumab. Probody Txs utilize a masking peptide that binds to the antigen-binding site of the Ab to reduce target binding. The mask extends from the light chain of the Ab via a linker sequence that contains cleavage sites for proteases preferentially active at the tumor site relative to healthy tissue. The ipilimumab Probody Tx binds to CTLA-4 with significantly lower affinity than the parental antibody and has reduced activity in in vitro assays. When tested in a mouse tumor model using human CTLA-4 KI mice, the ipilimumab-Probody Tx has comparable antitumor activity and Treg depletion at the tumor compared to ipilimumab. In contrast, ipilimumab-Probody Tx-treated mice show reduced levels of activated peripheral Tregs compared to ipilimumab-treated mice, even at doses 8-fold higher than are required for antitumor efficacy, consistent with reduced activity of the Probody Tx outside the tumor microenvironment. The development of next-generation anti-CTLA-4 antibodies holds promise for improving the utility of ipilimumab for single-agent or combination therapy. The two improvements to ipilimumab outlined above could each lead to a superior therapeutic outcome and merit further investigation.

Surrogate CD16-expressing effector cell lines for determining the bioactivity of therapeutic monoclonal antibodies

Traditional antibody dependent cellular cytotoxicity (ADCC) assays use donor derived natural killer (NK) or peripheral blood mononuclear cells, but donor genetic variability and the technically challenging nature of the assay means that alternative in vitro assay formats are required. We explored the utility of two reporter gene cell lines, the J2 and J9, as surrogate effector cells for ADCC assays. Both express the ADCC relevant Fcγ receptor CD16, crosslinking of which leads to firefly luciferase expression. For anti-CD20 rituximab and anti-HER2 trastuzumab (both IgG1 monoclonal antibodies, mAbs) a dose dependent firefly luciferase response was observed exclusively in the presence of their respective targets, representing the molecular interaction which potentiates ADCC activity. Importantly, both surrogate effector and NK cell based assays gave statistically similar values for rituximab ADCC activity. Increased engagement with target cell bound mAbs was determined to be cytotoxic for the J2 and J9 cell lines at the assay end point (at which luciferase expression is measured). However, use of the J9 cells containing the constitutively expressed renilla luciferase gene enabled data normalisation and corrected for fluctuations in both cell number and viability providing an advantage over currently available surrogate effector cell-lines. Abrogated ADCC activity with IgG4 mAbs, but enhanced activity with an IgG1 non-fucosylated mAb, was seen with the J9 cell line, as expected. Additionally, two rituximab products (biosimilars in development) with similar binding by flow cytometry, N-glycan profiles using HPLC and CD16 binding by surface plasmon resonance showed comparable ADCC activity to Mabthera. The ADCC activity of another anti-CD20 mAb, ofatumumab, reported only with primary cell based assays to date was also measured. This is the first report of a dual reporter gene based ADCC assay.

T0001, a variant of TNFR2-Fc fusion protein, exhibits improved Fc effector functions through increased binding to membrane-bound TNFα.

T0001 is a recombinant human TNFR-Fc fusion protein mutant; it exhibits higher affinity to TNFα than etanercept and is now being tested in a Phase 1 study in China (ClinicalTrials.gov Identifier: NCT02481180). T0001 can inhibit the binding of soluble TNFα (sTNFα) or membrane-bound TNFα (mTNFα) to TNF receptors. When bound to mTNFα, the Fc-bearing TNFα antagonists have the potential to induce Fc-mediated effects, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) as well as outside-to-inside signals (apoptosis mainly). Recent studies have shown that ADCC may also play an important role in Crohn's disease (CD) and ulcerative colitis (UC). In this study, T0001 presented a higher binding activity on mTNFα than etanercept and similar binding activity with adalimumab and infliximab. Upon the addition of sTNFα, adalimumab and infliximab showed significantly increased binding to FcγRIIIa and C1q than T0001 and etanercept. T0001 exhibited significantly higher ADCC and CDC activity than etanercept, and the potency and the reporter response of T0001 were very close to adalimumab and infliximab in ADCC reporter gene assays. And the similar potency of T0001 was also corroborated by PMBC-based ADCC assay. T0001, but not etanercept could induce apoptosis, while adalimumab and infliximab were more effective. These results suggest that T0001 may not only exert improved efficacy in treating rheumatoid arthritis (RA) because of its high affinity to sTNFα but also has a therapeutic potential in CD and UC due to its increased binding to mTNFα with resultant Fc-associated functions (ADCC, in particular) and improved apoptosis.

Antibody-dependent-cellular-cytotoxicity-inducing antibodies significantly affect the post-exposure treatment of Ebola virus infection

Passive immunotherapy with monoclonal antibodies (mAbs) is an efficacious treatment for Ebola virus (EBOV) infections in animal models and humans. Understanding what constitutes a protective response is critical for the development of novel therapeutic strategies. We generated an EBOV-glycoprotein-pseudotyped Human immunodeficiency virus to develop sensitive neutralizing and antibody-dependent cellular cytotoxicity (ADCC) assays as well as a bioluminescent-imaging-based mouse infection model that does not require biosafety level 4 containment. The in vivo treatment efficiencies of three novel anti-EBOV mAbs at 12 h post-infection correlated with their in vitro anti-EBOV ADCC activities, without neutralizing activity. When they were treated with these mAbs, natural killer cell (NK)-deficient mice had lower viral clearance than WT mice, indicating that the anti-EBOV mechanism of the ADCC activity of these mAbs is predominantly mediated by NK cells. One potent anti-EBOV mAb (M318) displayed unprecedented neutralizing and ADCC activities (neutralization IC50, 0.018 μg/ml; ADCC EC50, 0.095 μg/ml). These results have important implications for the efficacy of antiviral drugs and vaccines as well as for pathogenicity studies of EBOV.

Modulating IgG effector function by Fc glycan engineering

IgG antibodies contain a conserved N-glycosylation site on the Fc domain to which a complex, biantennary glycan is attached. The fine structures of this glycan modulate antibody effector functions by affecting the binding affinity of the Fc to diverse Fc receptor family members. For example, core fucosylation significantly decreases antibody-dependent cellular cytotoxicity (ADCC), whereas terminal α2,6-sialylation plays a critical role in the anti-inflammatory activity of human i.v. immunoglobulin therapy. The effect of specific combinations of sugars in the glycan on ADCC remains to be further addressed, however. Therefore, we synthesized structurally well-defined homogeneous glycoforms of antibodies with different combinations of fucosylation and sialylation and performed side-by-side in vitro FcγR-binding analyses, cell-based ADCC assays, and in vivo IgG-mediated cellular depletion studies. We found that core fucosylation exerted a significant adverse effect on FcγRIIIA binding, in vitro ADCC, and in vivo IgG-mediated cellular depletion, regardless of sialylation status. In contrast, the effect of sialylation on ADCC was dependent on the status of core fucosylation. Sialylation in the context of core fucosylation significantly decreased ADCC in a cell-based assay and suppressed antibody-mediated cell killing in vivo. In contrast, in the absence of fucosylation, sialylation did not adversely impact ADCC.

Tocilizumab (Anti-IL-6R) Suppressed TNFα Production by Human Monocytes in an In Vitro Model of Anti-HLA Antibody-Induced Antibody-Dependent Cellular Cytotoxicity.

BackgroundWe previously demonstrated that natural killer (NK) cells activated via FcγRIIIa (CD16) interactions with anti-HLA antibodies binding to peripheral blood mononuclear cells (PBMCs) in the in vitro antibody-dependent cellular cytotoxicity (ADCC) assay produced IFNγ. Here we investigate if other CD16 bearing cells are responsive to alloantigen via alloantibody in the in vitro ADCC and if the ADCC-induced cytokine reactions and cytotoxicity can be modified by the anti-interleukin 6 receptor (IL-6R) monoclonal antibody, Tocilizumab (TCZ).MethodsWhole blood from a normal individual was incubated overnight with irradiated allo-PBMCs pretreated with anti-HLA antibody positive (in vitro ADCC) or negative sera (mixed lymphocyte reaction [MLR]), with or without TCZ or control IgG. IFNγ+, TNFα+ or IL-6+ cell% in NK cells, monocytes and CD8+ T cells were enumerated by cytokine flow cytometry. ADCC using PBMCs (effector) and Farage B cells (FB, target) with anti-HLA antibody positive sera, with or without TCZ, was measured by flow cytometry.ResultsIFNγ+ and/or TNFα+ cell% in NK cells, monocytes and CD8+ T cells were elevated in the ADCC compared to the MLR condition. IL-6+ cells were significantly increased in ADCC versus MLR (10.2 ± 4.8% vs 2.7 ± 1.5%, P = 0.0003), but only in monocytes. TCZ treatment significantly reduced TNFα+ cell% in monocytes in ADCC, but had no effect on other cytokine+ cells. TCZ showed no effect on cytotoxicity in ADCC.ConclusionsIFNγ, TNFα, and IL-6 production induced by HLA antibody-mediated CD16 bearing cell activation in NK cells, monocytes, and CD8+ T cells suggests a potential role for ADCC and these inflammatory cytokines in mediation of antibody-mediated rejection. TCZ suppressed TNFα production in monocytes in the ADCC condition, suggesting a role of IL-6/IL-6R pathway in monocytes activation. Inhibition of this pathway could reduce the inflammatory cascade induced by alloantibody, although the inhibitory effect on cytotoxicity is minimal.

Abstract B66: An integrated immuno-oncology platform using high-throughput cell based assays, gene editing and genomic screens in immune cells

Abstract Horizon is establishing a powerful in vitro immuno-oncology platform with the capability to identify combinations of agents that will synergise with checkpoint inhibitors, determine methods for making safer and more effective cell therapies and find novel targets for immuno-therapy. Horizon previously reported the establishment of a platform consisting of a suite of high-throughput cell-based assays modelling a variety of oncology relevant immune reactions. The platform includes primary human immune-cell based assays for T cell activation, Mixed lymphocyte reaction (MLR), Tumor cell lysis, Antibody-dependent cellular cytotoxicity (ADCC), Complement-dependent cytotoxicity (CDC) and Natural killer cell cytotoxicity assays that have been validated with appropriate clinically approved antibodies (e.g. nivolumab, blinotumamab, & rituximab). Miniaturized to a 384-well format and supported by automation at each experimental step the platform is highly customizable in terms of testing agents that either enhance or inhibit immune cell functions. We will present ongoing efforts to identify synergistic activities with other relevant therapeutics likely to bring benefits to patients. In addition to the continued development of our high throughput immuno-oncology cell based assays, we have expanded our cell engineering know how combined with CRISPR-Cas9 gene editing technology to build new models to understand better the immune response and its subversion in tumorigenesis. By modifying specific genes of interest in immune cells we aim to robustly identify and validate new targets for the clinic. For example, we have used high efficiency gene editing with CRISPR-Cas9 in human primary T cells to knock out and knock in genes using Neon transfection systems. Using this methodology we can rapidly generate primary T cell models lacking specific checkpoint proteins (such as PD-1) to better understand how T-cell signalling pathways interact and to nominate novel targets suitable for ex vivo gene editing. We have also employed pooled CRISPR-Cas9 screens to investigate the effects of metabolic changes on T cell biology. CD3+ T cells have been infected with a one vector lentiviral system that delivers both Cas9 and sgRNAs targeting genes involved in cellular metabolism. Results from these screens should identify potential targets involved in T cell metabolism that affect their capacity to respond to proliferative stimuli in the form of anti-CD3 and anti-CD28 antibodies. We anticipate that these and other data generated using libraries targeting essential genes will be invaluable for the design of more complex immuno-oncology screens. Overall, Horizon's integrated immuno-oncology platform will enable large scale interrogation of prospective immunotherapies, either alone or in combination, and could be useful for the discovery of novel checkpoint components, for deciphering the underlying mechanisms promoting an immunosuppressive tumor microenvironment and for the understanding of how pathways crucial for an anti-tumor immune response interact. Thus, this platform could contribute substantially to the discovery and development of future immunotherapies. Citation Format: An Frank, Sujatha Kumar, Christina Ghirelli, Kim Hoenderdos, Tabasum Huseni, Lauren Thibault, Lydia Kifle, Nava Almog, Felicia Zhao, Simon Scrace, Anatoly Myaskovsky, Chris Lowe, Janine Steiger, Nicola McCarthy, Jonathan Moore. An integrated immuno-oncology platform using high-throughput cell based assays, gene editing and genomic screens in immune cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B66.

Development of a novel anti-human aspartyl-(asparaginyl) β-hydroxylase monoclonal antibody with diagnostic and therapeutic potential.

Human aspartyl-(asparaginyl)-β-hydroxylase (HAAH) has recently been the subject of several studies, as it was previously observed to be overexpressed in numerous types of carcinoma cells and tissues in patient tumor samples. HAAH has been implicated in tumor invasion and metastasis, indicating that it may be an important target and biomarker for tumor diagnosis and treatment. However, the immunological tools currently available for the study of this protein, including monoclonal antibodies, are limited, as is the present knowledge regarding the role of HAAH in tumor therapy and diagnosis. In the present study, a recombinant C-terminal domain of HAAH was expressed in Pichia pastoris and a novel monoclonal antibody (mAb) targeting HAAH (HAAH-C) was constructed. Immunofluorescence and antibody-dependent cellular cytotoxicity (ADCC) assays were used to demonstrate the specificity and ADCC activity of this antibody. The results demonstrated that this anti-C-terminal HAAH mAB, in combination with an existing anti-N terminal HAAH mAb, exhibited a high response to native HAAH from carcinoma cell culture supernatant, as measured with a double antibody sandwich enzyme-linked immunosorbent assay. This validated novel mAB-HAAH-C may prompt further studies into the underlying mechanisms of HAAH, and the exploration of its potential in tumor diagnosis and therapy.

Humans from Wuchereria bancrofti endemic area elicit substantial immune response to proteins of the filarial parasite Brugia malayi and its endosymbiont Wolbachia

BackgroundIn the past, immune responses to several Brugia malayi immunodominant antigens have been characterized in filaria-infected populations; however, little is known regarding Wolbachia proteins. We earlier cloned and characterized few B. malayi (trehalose-6-phosphate phosphatase, Bm-TPP and heavy chain myosin, BmAF-Myo) and Wolbachia (translation initiation factor-1, Wol Tl IF-1 and NAD+-dependent DNA ligase, wBm-LigA) proteins and investigated the immune responses, which they triggered in animal models. The current study emphasizes on immunological characteristics of these proteins in three major categories of filarial endemic zones: endemic normal (EN, asymptomatic, amicrofilaraemic; putatively immune), microfilariae carriers (MF, asymptomatic but microfilaraemic), and chronic filarial patients (CP, symptomatic and mostly amicrofilaraemic).MethodsImmunoblotting and ELISA were carried out to measure IgG and isotype antibodies against these recombinant proteins in various clinical categories. Involvement of serum antibodies in infective larvae killing was assessed by antibody-dependent cellular adhesion and cytotoxicity assay. Cellular immune response was investigated by in vitro proliferation of peripheral blood mononuclear cells (PBMCs) and reactive oxygen species (ROS) generation in these cells after stimulation.ResultsImmune responses of EN and CP displayed almost similar level of IgG to Wol Tl IF-1 while other three proteins had higher serum IgG in EN individuals only. Specific IgA, IgG1, IgG3 and IgM to Bm-TPP were high in EN subjects, while BmAF-Myo additionally showed elevated IgG2. Enhanced IgA and IgG3 were detected in both EN and CP individuals in response to Wol Tl IF-1 antigen, but IgG1 and IgM were high only in EN individuals. wBm-LigA and BmAF-Myo exhibited almost similar pattern of antibody responses. PBMC isolated from EN subjects exhibited higher proliferation and ROS generation when stimulated with all three proteins except for Wol Tl IF-1.ConclusionsOverall, these findings display high immunogenicity of all four proteins in human subjects and revealed that the EN population was exposed to both B. malayi and Wolbachia proteins simultaneously. In addition, immune responses to Wol Tl IF-1 suggest possible role of this factor in Wolbachia-induced pathological responses while immune responses to other three proteins suggest that these can be explored further as vaccine candidates.

CD33-Directed Chimeric Antigen Receptor (CAR) T Cells for the Treatment of Acute Myeloid Leukemia (AML)

Therapies for acute myeloid leukemia (AML) have not improved patient long-term survival for decades. Novel treatment options, such as chimeric antigen receptor (CAR) T cells, are needed for patients with this disease. However, AML cells lack ideal targeting antigens that are safe to target with CAR T cells. CD33, a commonly targeted antigen, is expressed in about 85-90% of AML cases but is also present on normal myeloid progenitors and myelocytes. Our aim was to engineer and validate CD33-directed CAR T cells, with the intention to open a phase I clinical trial in patients with relapsed AML. Given the potential toxicity associated with targeting CD33 in patients, an elimination gene was included in the construct design to allow CAR T cell clearance after disease eradication.We constructed a CAR specific for CD33 by utilizing the variable heavy and light chains of the humanized M-195 antibody (HuM-195), CD28 and zeta signaling domains, and the IL-12 gene. To facilitate CAR T cell elimination if necessary, a truncated epidermal growth factor receptor (EGFRt) gene was also inserted into the retroviral vector to create the EGFRt/HuM195-28z/IL-12 CAR construct. Retroviral transduction with this tri-cistronic construct resulted in high transduction efficiency of human T cells, as determined by detection of EGFRt with fluorescently-labeled cetuximab or CAR with fluorescently-labeled CD33 molecule by flow cytometry. T cells transduced with the EGFRt/HuM195-28z/IL-12 CAR secreted functional IL-12, as assessed by culturing CAR cell supernatant with peripheral blood mononuclear cells and detecting IFN-g produced in response to IL-12. When stimulated through the CAR by co-culturing with the CD33+ AML cell line Molm-13, EGFRt/HuM195-28z/IL-12 CAR T cells proliferated and produced significant levels of the pro-inflammatory cytokines IFN-g and IL-2. In addition, EGFRt/HuM195-28z/IL-12 CAR T cells mediated significantly cytotoxicity against Molm-13 at a range of effector-to-target ratios in standard 51Cr-release assays, as compared to control CAR T cells.The in vivo anti-tumor efficacy of EGFRt/HuM195-28z/IL-12 CAR T cells was tested in two preclinical mouse models of AML. First, SCID/Beige mice were xenografted with Molm-13 cells. Mice subsequently treated with EGFRt/HuM195-28z/IL-12 CAR T cells exhibited significant long-term survival, as compared to untreated or control CAR T cell-treated mice (p = <0.0001). In addition, NSG mice were engrafted with patient-derived CD33+ AML (PDX) cells. PDX mice treated with EGFRt/HuM195-28z/IL-12 CAR T cells exhibited reduction of peripheral CD33+ disease, as compared to untreated or control CAR T cell-treated mice. These studies demonstrate the capacity of the EGFRt/HuM195-28z/IL-12 CAR to redirect the specific anti-tumor function of T cells to CD33+ AML tumor cells. Ongoing studies aim to validate elimination of CAR T cells via EGFRt in vitro in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays, as well as in mice are currently underway. These data support the utilization of CD33-specific CAR T cells in the clinic for patients with relapsed AML as a means to decrease disease burden prior to consolidative therapies such as allogeneic transplantation. DisclosuresBrentjens:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Influenza virus-specific antibody dependent cellular cytoxicity induced by vaccination or natural infection

Influenza viruses are responsible for substantial morbidity and mortality during seasonal epidemics. Vaccination is the most effective method to prevent infection, however due to antigenic drift of the viral surface protein hemagglutinin (HA), annual influenza virus vaccination is required. In addition to seasonal viruses, certain (avian) influenza A viruses of other subtypes, like H5N1 or H7N9, cause sporadic zoonotic infections. Therefore, the availability of game-changing novel vaccines that induce “universal” immune responses to a wide variety of influenza A virus subtypes is highly desirable. The quest for universal influenza vaccines has fueled the interest in broadly-reactive antibodies specific for the stalk of hemagglutinin (HA) and biological activities of antibodies other than direct virus neutralization, like antibody-dependent cellular cytotoxicity (ADCC). In the present study, we investigated the ADCC response upon influenza virus vaccination and infection in humans using a robust ADCC assay that is based on the use of recombinant HA and a continuous NK cell line that expresses FcγRIII (CD16). This assay offers advantages over existing methods, like ease to perform and possibilities to standardize. We showed that HA-specific ADCC mediating antibodies are induced by vaccination with adjuvanted trivalent seasonal and monovalent H1N1pdm09 inactivated vaccines, and by infection with H1N1pdm09 virus. In addition, the use of chimeric influenza HA with a H1 stem but antigenically irrelevant head domain derived from an avian virus allowed detection of H1-stalk-specific ADCC mediating antibodies. This assay will facilitate the assessment of ADCC mediating serum antibodies after (universal) influenza vaccination or infection and may define ADCC activity as a correlate of (cross-) protection in the future.

Short Communication: Small-Molecule CD4 Mimetics Sensitize HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity by Antibodies Elicited by Multiple Envelope Glycoprotein Immunogens in Nonhuman Primates.

Recent studies have linked antibody Fc-mediated effector functions with control of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus infections. Interestingly, the presence of antibodies with potent antibody-dependent cellular cytotoxicity (ADCC) activity in RV144 vaccine trial participants correlated inversely with HIV-1 acquisition risk. These antibodies were recently found to recognize epitopes on the HIV-1 envelope (Env) glycoprotein exposed upon Env-CD4 binding. Accordingly, small-molecule CD4 mimetics (CD4mc) that induce Env to sample the CD4-bound conformation were shown to sensitize HIV-1-infected cells to ADCC mediated by sera from HIV-1-infected individuals. However, it remains unknown whether antibodies elicited through immunization can also mediate CD4mc-induced ADCC. In this study, we tested the capacity of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from Env-vaccinated nonhuman primates using a FACS-based ADCC assay. In parallel, we evaluated the ability of CD4mc to sensitize HIV-1 viral particles to neutralization by sera from these immunized animals. We found that the vaccine-induced antibodies were able to mediate ADCC and viral neutralization in the presence, but not the absence, of CD4mc. Thus, CD4mc are capable of sensitizing HIV-1-infected cells to ADCC and infectious viral particles to neutralization by easy-to-elicit antibodies that are otherwise unable to mediate these activities.

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.

SYD985, a Novel Duocarmycin-Based HER2-Targeting Antibody-Drug Conjugate, Shows Antitumor Activity in Uterine Serous Carcinoma with HER2/Neu Expression.

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer. Up to 35% of USC may overexpress the HER2/neu oncogene at strong (i.e., 3+) levels by IHC while an additional 40% to 50% express HER2/neu at moderate (2+) or low (1+) levels. We investigated the efficacy of SYD985, (Synthon Biopharmaceuticals), a novel HER2-targeting antibody-drug conjugate (ADC) composed of the mAb trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin) in USC. We also compared the antitumor activity of SYD985 in head-to-head experiments to trastuzumab emtansine (T-DM1), a FDA-approved ADC, against multiple primary USC cell lines expressing different levels of HER2/neu in in vitro and in vivo experiments. Using antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability, and bystander killing assays as well as propidium iodide-based flow cytometry assays and multiple in vivo USC mouse xenograft models, we demonstrate for the first time that SYD985 is a novel ADC with activity against USC with strong (3+) as well as low to moderate (i.e., 1+/2+) HER2/neu expression. SYD985 is 10- to 70-fold more potent than T-DM1 in comparative experiments and, unlike T-DM1, it is active against USC demonstrating moderate/low or heterogeneous HER2/neu expression. Clinical studies with SYD985 in patients harboring chemotherapy-resistant USC with low, moderate, and high HER2 expression are warranted. Mol Cancer Ther; 15(8); 1900-9. ©2016 AACR.