Live Target Cells Research Articles

A real-time antibody-dependent cellular phagocytosis assay by live cell imaging

Antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which antibody-opsonized targets (pathogens or cells) activate the Fc receptors on the surface of phagocytes to induce phagocytosis, resulting in internalization and degradation of pathogens or target cells through phagosome acidification. Besides NK cells-mediated antibody-dependent cellular cytotoxicity (ADCC), tumor-infiltrated monocytes and macrophages can directly kill tumor cells in the presence of tumor antigen-specific antibodies through ADCP, representing another attractive strategy for cancer immunotherapy. Even though several methods have been developed to measure ADCP, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study we established a new ADCP assay to identify therapeutical monoclonal antibodies (mAbs) that facilitate macrophages phagocytosis of live target cells. We used Incucyte, an imaging system for live cell analysis. By labeling the live target cells with a pH sensitive dye (pHrodo), we successfully monitored the ADCP in real time. We demonstrated that our image-based assay is robust and quantitative, suitable for screening and characterization of therapeutical mAbs that directly kill target cells through ADCP. Furthermore, we found different subtypes of macrophages have distinct ADCP activities using both mouse and human primary macrophages differentiated in vitro. By studying various mAbs with mutations in their Fc regions using our assay, we showed that the variants with increased binding to Fc gamma receptors (FcγRs) have enhanced ADCP activities.

Discovery of Live Cell Selective Fluorescent Probes and Elucidation of Their Mechanisms: Case Study of B Cell Selective Probe CDgB.

The development of small-molecule fluorescent probes for specific immune cell identification offers an economical alternative to expensive antibodies. Moreover, it enables the identification of live target cells and provides insights into the distinct properties of cells, leveraging their specific staining mechanisms. This chapter presents a comprehensive elucidation of the methodology employed for screening fluorescent compounds using flow cytometry measurements. A novel analytical approach is proposed to distinguish a fluorescent compound with a specific carbon length for B lymphocytes, involving an assessment of the staining index and the predominant ratio of immune cells. Moreover, a protocol is presented for investigating the staining mechanisms of these probes by employing cell mimicking models such as small unilamellar vesicles (SUVs).

Hyperactive Rac stimulates cannibalism of living target cells and enhances CAR-M-mediated cancer cell killing.

The 21kD GTPase Rac is an evolutionarily ancient regulator of cell shape and behavior. Rac2 is predominantly expressed in hematopoietic cells where it is essential for survival and motility. The hyperactivating mutation Rac2E62K also causes human immunodeficiency, although the mechanism remains unexplained. Here, we report that in Drosophila, hyperactivating Rac stimulates ovarian cells to cannibalize neighboring cells, destroying the tissue. We then show that hyperactive Rac2E62K stimulates human HL60-derived macrophage-like cells to engulf and kill living T cell leukemia cells. Primary mouse Rac2+/E62K bone-marrow-derived macrophages also cannibalize primary Rac2+/E62K T cells due to a combination of macrophage hyperactivity and T cell hypersensitivity to engulfment. Additionally, Rac2+/E62K macrophages non-autonomously stimulate wild-type macrophages to engulf T cells. Rac2E62K also enhances engulfment of target cancer cells by chimeric antigen receptor-expressing macrophages (CAR-M) in a CAR-dependent manner. We propose that Rac-mediated cell cannibalism may contribute to Rac2+/E62K human immunodeficiency and enhance CAR-M cancer immunotherapy.

Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction

BackgroundDirect cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses.MethodsCardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry.ResultsWe found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair.ConclusionsFibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.Graphical

Flow Cytometry Analysis of Cell-Surface Markers to Identify Human Naïve Pluripotent Stem Cells.

Cell-surface proteins provide excellent biomarkers to identify specific cell types and resolve heterogeneous cell populations. The analysis of cell-surface proteins by flow cytometry produces robust and quantitative information with single-cell resolution, and allows live target cells to be purified and characterized or re-cultured. Studies using antibody screens, proteomics, and candidate analysis have identified a comprehensive set of proteins that are expressed on the surface of naïve and primed human pluripotent stem cells. These findings have led to the development of suitable protein markers and antibodies to accurately distinguish between these two cell types. Here, a detailed protocol is provided that uses multi-color flow cytometry to analyze cell-surface protein expression in naïve and primed human pluripotent stem cells. This method enables the unambiguous identification of pluripotent cell types and the opportunity to sort target cells including during cell state transitions. The protocol can be combined to additionally investigate the expression of reporter genes and other informative features, such as DNA content.

Correlates of Neutralizing/SARS-CoV-2-S1-Binding Antibody Response With Adverse Effects and Immune Kinetics in BNT162b2-Vaccinated Individuals

Background: While mRNA vaccines against SARS-CoV-2 have been exceedingly effective in preventing symptomatic viral infection, the features of immune response remain to be clarified.Methods: In the present prospective observational study, 225 healthy individuals in Kumamoto General Hospital, Japan, who received two BNT162b2 doses in February 2021, were enrolled. Correlates of BNT162b2-elicited SARS-CoV-2-neutralizing activity (50% neutralization titer: NT50; assessed using infectious virions and live target cells) with SARS-CoV-2-S1-binding-IgG and -IgM levels, adverse effects (AEs), ages, and genders were examined. The average half-life of neutralizing activity and the average time length for the loss of detectable neutralizing activity were determined and the potency of serums against variants of concerns was also determined.Findings: Significant rise in NT50s was seen in serums on day 28 post-1st dose. A moderate inverse correlation was seen between NT50s and ages, but no correlation was seen between NT50s and AEs. NT50s and IgG levels on day 28 post-1st dose and pain scores following the 2nd shot were greater in women than in men. The average half-life of neutralizing activity in the vaccinees was approximately 67.8 days and the average time length for their serums to lose the detectable neutralizing activity was 198.3 days. While serums from elite-responders (NT50s >1,500-fold: the top 4% among all participants’ NT50s) potently to moderately blocked the infectivity of variants of concerns, some serums with moderate NT50s failed to block the infectivity of a beta strain.Interpretation: BNT162b2-elicited immune response has no significant association with AEs. BNT162b2-efficacy is likely diminished to under detection limit by 6-7 months post-1st shot. High-level neutralizing antibody-containing serums potently to moderately block the infection of SARS-CoV-2 variants; however, a few moderate-level neutralizing antibody-containing serums failed to do so. If BNT162b2-elicited immunity memory is short, an additional vaccine or other protective measures would be needed.Funding Information: This research was supported in part by a grant from the Japan Agency for Medical Research and Development to Maeda (grant number JP20fk0108260, 20fk0108502) and to Mitsuya (grant number 20fk0108502), and in part by a grant for MHLW Research on Emerging and Re-emerging Infectious Diseases and Immunization Program to Maeda (grant number JPMH20HA1006) from Ministry of Health, Labor and Welfare, and in part by a grant for COVID-19 to Mitsuya (grant number 19A3001) and Maeda (grant number 20A2003D) from the Intramural Research Program of National Center for Global Health and Medicine, and in part by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health (Mitsuya).Declaration of Interests: Matsushima, Noda, Sato, and Yoshida are employees of SysmexCorporation.Other authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.Ethics Approval Statement: The Ethics Committees from the Kumamoto General Hospital and NCGM approved this study (Kumamoto General Hospital No. 180, and NCGM-G-004176-00, respectively). Each participant provided a written informed consent, and this study abided by the Declaration of Helsinki principles.

Multifunctional Microparticles with Stimulation and Sensing Capabilities for Facile NK Cell Activity Assay.

Natural killer (NK) cells are a subset of innate lymphoid cells playing an important role in immune surveillance and early defense against infection and cancer. They recognize and directly kill infected or transformed cells. At the same time, they produce various cytokines and chemokines to regulate other immune cells. NK cell activity can be a useful marker for health screenings because impaired NK cell functions may indicate a more susceptible environment for infection or tumor development. Currently, most NK cell activity assays are focused on measuring either cytokine secretion, in particular, interferon γ (IFN-γ), or cytotoxicity against target cells such as K562, thus only providing partial information on NK cell activity. In order to develop a comprehensive test for measuring NK cell function, cytotoxicity and cytokine secretion ability should be measured simultaneously. In addition, current NK cell assays are performed by stimulating NK cells with cocktails of cytokines, antibody-coated beads, or live target cells. In this study, we developed multifunctional microparticles for NK cell activity assay (MNAs) that allow simultaneous stimulation and sensing various NK cell activities, including cytokine secretion and cytotoxicity. The surfaces of MNAs are decorated with multiple functional biomolecules, including antibodies that stimulate NK cells by engaging NK cell activating receptors, antibodies that can capture cytokines secreted by NK cells, and a peptide sensor that reacts with granzyme B, a key molecule released by NK cells for cytotoxicity. The performances of MNAs are assessed using flow cytometry and live cell imaging. NK cell activity is measured by simply mixing MNAs with NK cells and performing flow cytometry, and the results are comparable to those measured by standard NK cell activity assays.

Generation of a novel affibody molecule targeting Chlamydia trachomatis MOMP

Chlamydia trachomatis (C. trachomatis) is the leading cause of preventable blindness worldwide and the most prevalent cause of bacterial sexually transmitted diseases. At present, there is no available vaccine, and recurrences after antibiotics treatment are substantial problems. Major outer membrane protein (MOMP) accounts for 60% of the outer mass of C. trachomatis, functioning as trimeric porin, and it is highly antigenic. Therefore, MOMP is the most promising candidate for vaccine developing and target therapy of Chlamydia. Affibody, a new class of affinity ligands derived from the Z-domain in the binding region of Staphylococcus aureus protein A, has been the focus of researchers as a viable alternative to antibodies. In this study, the MOMP-targeted affibody molecule (ZMOMP:461) was screened by phage-displayed peptide library. Further, the affinity and specificity were characterized by surface plasmon resonance (SPR) and Western blot. Immunofluorescence assay (IFA) indicated that the MOMP-binding affibody could recognize native MOMP in HeLa229 cells infected C. trachomatis. Immunoprecipitation assay confirmed further that ZMOMP:461 molecule specifically recognizes the epitope on relaxed trimer MOMP. Our findings provide strong evidence that affibody molecule (ZMOMP:461) serves as substitute for MOMP antibody for biological applications and has a great potential for delivering drugs for target therapy.Key points• We screened a novel affibody molecule ZMOMP:461 targeting Chlamydia trachomatis MOMP.• ZMOMP:461 recognizes the recombinant and native MOMP with high affinity and specificity.• ZMOMP:461 could be internalized into live target cells.

Abstract 1182: New bioluminescent strategies to measure kinetics of immune cell-mediated cytotoxicity

Abstract The number of immunotherapeutic strategies for treating cancer have expanded in recent years, increasing the need for assays that can be used to assess their effectiveness. To facilitate studies of these immunotherapies, we developed bioluminescent methods that could rapidly and sensitively measure target cell cytotoxicity. Two approaches were developed. The first method utilizes a bioluminescent enzymatic activity assay for a well-accepted cytotoxicity marker, lactate dehydrogenase (LDH), which is rapidly released from dead cells. With the improved sensitivity of the bioluminescent assay, lower levels of LDH can be measured, resulting in the detection of fewer dead cells (LOD< 10 cells). Cytotoxicity in both 2D and 3D cell cultures can be monitored over time by removing samples of medium at different time points. Here we present examples in which this assay was used to test the effectiveness of immunotherapeutic treatments including antibody-drug conjugates (ADC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In ADCC models, cell killing was detectable at early time points (by 2 hours). Increases in the cytotoxicity of individual samples could be followed over time, from early time points to 24 hours. In the second approach we developed a novel bioluminescent assay based on the specific labeling of target cells. With this method, intracellular proteins in live target cells are covalently labeled. Upon target cell death, the labeled proteins are released from the cell and the label is quantitated using bioluminescence detection reagents. Using this assay, we were able to quantitate the killing of target cells in both ADCC and CAR-T applications. By adding the detection reagents directly to the medium at the start of the ADCC reaction, cell death was monitored in real-time for up to 6 hours. Neither assay requires genetic engineering and both involve no or minimal target cell preparation. They are applicable to multiple cell types and the non-lytic nature of these approaches provides kinetic capabilities and opportunities for multiplexing. The ability to rapidly quantitate cytotoxicity will facilitate the functional analysis of immunotherapies. Citation Format: Maggie L. Bach, Natasha Karassina, Peter Hofsteen, Donna Leippe, James Cali, Jolanta Vidugiriene. New bioluminescent strategies to measure kinetics of immune cell-mediated cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1182.

CD16-IL15-CLEC12A Trispecific Killer Engager (TriKE) Drives NK Cell Expansion, Activation, and Antigen Specific Killing of Cancer Stem Cells in Acute Myeloid Leukemia

Our group developed a 161533 trispecific killer engager (TriKE) molecule to target acute myeloid leukemia (AML) cells using Natural Killer (NK) cells. This molecule contains an anti-CD16 camelid nanobody to activate NK cells, an anti-CD33 single chain variable fragment (scFv) to engage cancer targets, and an IL-15 molecule that drives NK cell priming, expansion and survival. Using an earlier version of this molecule, we have shown that the CD33 TriKE is effective at activating NK cells against AML targets in vitro and in vivo. This preclinical data has lead to the establishment of a clinical trial in refractory AML patients at the University of Minnesota, set to open Q3 2018. While these previous studies have validated the use of TriKEs as an effective strategy of harnessing NK cells in cancer immunotherapy, CD33 has limitations as a target antigen.The high mortality and poor five-year survival rates (26%) for AML patients can be attributed to chemotherapy resistance and disease relapse. A majority of chemotherapy resistant leukemia stem cells (LSCs), that are hypothesized to facilitate relapse, do not express CD33. In addition, all hematopoietic stem cells and normal myeloid cells express CD33, thus targeting this antigen can lead to severe defects in hematopoiesis and on-target/off-tumor toxicity. To address these limitations, we developed a TriKE that targets CLEC12A or C-type lectin-like molecule 1 (CLL-1). CLEC12A is highly expressed on AML cells and over 70% of CD33 negative cells express CLEC12A. It has been attributed as a stem cell marker in AML, being selectively overexpressed in LSCs. CLEC12A is expressed by CD34+/CD38- LSCs but not normal CD34+/CD38- hematopoietic stem cells in regenerating bone marrow, thus minimizing off-target effects.The CLEC12A TriKE was developed in a mammalian cell system to ensure that appropriate post-translational modifications are present. We confirmed that the TriKE binds specifically to HL-60 and THP-1 target cells that express CLEC12A compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMCs) with the CLEC12A TriKE drives a significant increase in NK cell specific proliferation over 7 days as measured by CellTrace dilution compared to treatment with a CLEC12A scFv or IL-15 alone (69.7 ± 6.7% vs 11.9 ± 2.5% vs 38.4 ± 7.3%) (Figure 1A). To measure NK cell killing, we conducted an IncuCyte Zoom assay. Here, HL-60 target cells were labeled with a caspase 3/7 reagent where a color change indicates target cell death. The CLEC12A TriKE was able to induce more target cell killing than CLEC12A scFv or IL-15 as measured by number of live target cells at the end of the 48 hour assay (53.9 ± 1.9% vs 103.3 ± 3.4% vs 71.1 ± 1.4%). The CLEC12A TriKE induces an increase in NK cell degranulation, measured by CD107a expression against HL-60 AML tumor targets in a 4 hour functional assay compared to treatment with CLEC12A scFv or IL-15 alone (62.3 ± 1.1% vs 19.4 ± 3.8% vs 27.5 ± 4.9%). In this assay, there is also an increase in cytokine production, measured by IFNg and TNFa respectively (16.7 ± 4.2% vs 2.3 ± 1.5% vs 4.7 ± 1.9% and 18.0 ± 5.1% vs 2.5 ± 1.7% vs 4.6 ± 2.5%) (Figure 1B). We observe a similar enhanced functional response with THP-1 AML tumor targets. In these functional assays, treatment with the CLEC12A TriKE produced less background activation compared to the CD33 TriKE, indicating less off-target effects on PBMCs. To confirm the clinical relevance of this molecule, we tested the efficacy of the CLEC12A TriKE against primary AML targets. AML blasts were identified as SSC low, CD45 intermediate and CD34 high cells. Out of the 9 AML samples tested, 7 expressed high levels of CD33 (70.4 ± 6.3%) and CLEC12A (78.1 ± 5.2%). In functional assays with these samples, the CLEC12A TriKE was able to induce greater CD107a and IFNg expression, and enhanced killing of tumor targets as measured by a live/dead stain compared to CLEC12A scFv or IL-15 (Figure 1C). In these assays, the efficacy of the CLEC12A TriKE was comparable to the CD33 TriKE. Our data demonstrates that the CLEC12A TriKE drives NK cell specific proliferation, degranulation, cytokine secretion, and killing of tumor targets in vitro. Apart from AML, CLEC12A is expressed on cancer cells and LSCs in patients with myelodysplastic syndromes (MDS). These findings highlight the clinical potential of the CLEC12A TriKE individually or in combination with the CD33 TriKE for the treatment of MDS and AML. [Display omitted] DisclosuresVallera:GT Biopharma: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding.

Research progress of the cell microencapsulation

Cell microencapsulation aims to wrap the living target cells by one or several materials with good biological compatibility and semipermeable membrane properties. Cell microencapsulation not only can achieve immune isolation and prevent the attacks by macromolecular immunes and immune cells, but also can allow the free access of metabolites, small molecule nutrients and bioactive substances to the microcapsule. With the continuous progress of interdisciplinary technologies, cell microencapsulation shows increasing application prospects of making up a variety of limitations of organ transplantation. Moreover, with the development and maturation of cell microencapsulation, it has shown a strong advantage in regenerative medicine, which will certainly promote the rapid development of artificial cells and artificial organs. In this paper, the preparation of cell microcapsules, the effects of the outer membrane of microcapsules on immunological macromolecules and cytokines, the immunogenicity of the outer membrane, and the representative applications of cell microencapsulation were summarized. Key words: Cell microencapsulation; Immune isolation; Cytokines; Organ transplantation

A Real-Time Cytotoxicity Assay as an Alternative to the Standard Chromium-51 Release Assay for Measurement of Human NK and T Cell Cytotoxic Activity.

This unit describes the monitoring and quantification of cellular cytotoxicity using a non-radioactive and real-time cytotoxic assay. The extent of target-cell lysis is monitored over time by imaging and quantifying live fluorescent target cells using a cell-imaging multimode reader. This assay is performed in a 96 well plate in optimized culture conditions at 37°C in the presence of 5% CO2 . The basic protocol describes natural killer cell-mediated cytotoxic assay that can be adapted to include antibodies blocking inhibitory NK receptors or triggering antibody-dependent cell-mediated cytotoxicity (ADCC). The assay is also suitable for antigen specific T-cell cytotoxic assays. Until now, the standard chromium 51 (51 Cr)-release assay has remained the sole sensitive assay but its major drawbacks include cost and hazard of handling radioactivity. The real-time cytotoxic assay is therefore an effective alternative providing a robust and sensitive assay that accurately monitors lysis of target cells over time. © 2017 by John Wiley & Sons, Inc.

56 - A high-throughput image cytometry-based screening method for the detection of IL2-induced peripheral blood mononuclear cell-mediated cytotoxicity

Cell-mediated cytotoxicity assays have been an important functional test for investigating the cytotoxic effect of immune effector on target cancer cells. Cytotoxicity assays have traditionally been performed using the 51Chromium (51Cr) release assay, which involves labeling the tumor cells (target) with radioisotopes. When the target cells are lysed by the immune cells (effector), they release the entrapped radioactive 51Chromium, which is measured to determine the level cytotoxicity induced by the effector cells. The 51Chromium release assay is highly hazardous, time-consuming, and can only acquire end point readout. Furthermore, it can generate inconsistent results due to batch variation, ability of the target cell for 51Cr uptake, and low sensitivity. In this work, we demonstrate a novel high-throughput cytotoxicity screening assay using the Celigo imaging-cytometry method. First, live K562 target cells were stained with Calcein AM, and were co-cultured with PBMCs from two healthy donors in a 96-well plate in the presence or absence of IL-2. Direct cell counting of Calcein positive cells was used to calculate the changes in number of live target cells in the same well from T = 0 to T = 4 hours. Thus, accurate cell-mediated killing was determined by normalizing results based on time = 0. The donor PBMCs were added to each well with Calcein-stained target cells at different Effector-to-Target (E:T) ratios. The 96-well microplate was then scanned and analyzed using the Celigo image cytometer at different time points to measure the % lysis of target cell. Although it could be argued that release of a dye may not actually represent cytotoxicity by effector cells, nonetheless much like 51Cr release assay percent cytotoxicity was directly proportionate to the duration of the assay, E:T ratio used, and amount of activating cytokine present in the media, all as expected. Further, when PBMCs from both donors showed near equal level of cytotoxicity at 4 hr; upon calculation of the NK cell lytic unit, it was revealed that one donor possessed much higher level of effector function than the other . The proposed image cytometry method can scan and analyze the entire well area of the entire 96-well plate in 7 min, which can be utilized to perform high-throughput screening of potential antibody- or chemical-based cancer drug, which could be a more efficient method for academic, industry, and clinical research.

A real-time digital bio-imaging system to quantify cellular cytotoxicity as an alternative to the standard chromium-51 release assay

Abstract Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard chromium 51Cr-release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non-radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentive to find alternative solutions to 51Cr. We took advantage of the recent development of cell-imaging multimode readers to develop a novel non-radioactive and real-time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target-cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96-well plates using the cell-imaging multimode reader Cytation™ 5 from Biotek. We have developed classical NK cell assays in the presence or absence of blocking antibodies and antibody-dependent cell-mediated cytotoxicity (ADCC). We show that in these assays, cell killing occurs within the first two hours with a half maximum killing reached after 30 minutes. This technology has numerous applications such as NK and T cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.

The anti-tumor efficacy of 3C23K, a glyco-engineered humanized anti-MISRII antibody, in an ovarian cancer model is mainly mediated by engagement of immune effector cells.

Ovarian cancer is the leading cause of death in women with gynecological cancers and despite recent advances, new and more efficient therapies are crucially needed. Müllerian Inhibiting Substance type II Receptor (MISRII, also named AMHRII) is expressed in most ovarian cancer subtypes and is a novel potential target for ovarian cancer immunotherapy. We previously developed and tested 12G4, the first murine monoclonal antibody (MAb) against human MISRII. Here, we report the humanization, affinity maturation and glyco-engineering steps of 12G4 to generate the Fc-optimized 3C23K MAb, and the evaluation of its in vivo anti-tumor activity. The epitopes of 3C23K and 12G4 were strictly identical and 3C23K affinity for MISRII was enhanced by a factor of about 14 (KD = 5.5 × 10−11 M vs 7.9 × 10−10 M), while the use of the EMABling® platform allowed the production of a low-fucosylated 3C23K antibody with a 30-fold KD improvement of its affinity to FcγRIIIa. In COV434-MISRII tumor-bearing mice, 3C23K reduced tumor growth more efficiently than 12G4 and its combination with carboplatin was more efficient than each monotherapy with a mean tumor size of 500, 1100 and 100 mm3 at the end of treatment with 3C23K (10 mg/kg, Q3-4D12), carboplatin (60 mg/kg, Q7D4) and 3C23K+carboplatin, respectively. Conversely, 3C23K-FcKO, a 3C23K form without affinity for the FcγRIIIa receptor, did not display any anti-tumor effect in vivo. These results strongly suggested that 3C23K mechanisms of action are mainly Fc-related. In vitro, antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) were induced by 3C23K, as demonstrated with human effector cells. Using human NK cells, 50% of the maximal lysis was obtained with a 46-fold lower concentration of low-fucosylated 3C23K (2.9 ng/ml) than of 3C23K expressed in CHO cells (133.35 ng/ml). As 3C23K induced strong ADCC with human PBMC but almost none with murine PBMC, antibody-dependent cell phagocytosis (ADCP) was then investigated. 3C23K-dependent (100 ng/ml) ADCP was more active with murine than human macrophages (only 10% of living target cells vs. about 25%). These in vitro results suggest that the reduced ADCC with murine effectors could be partially balanced by ADCP activity in in vivo experiments. Taken together, these preclinical data indicate that 3C23K is a new promising therapeutic candidate for ovarian cancer immunotherapy and justify its recent introduction in a phase I clinical trial.

Abstract P3-03-06: Characterization of HER2-positive breast cancer (BC) cells selected for tolerance to trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC)

Abstract Cellular mechanisms of trastuzumab resistance include alteration(s) in cell signaling pathways (PTEN loss, activation of PI3K/Akt signaling), steric hindrance of antibody binding (by Muc-1/Muc-3), over-activation of alternate receptor kinases (HER3/c-Met/IGF-1R), and proteolysis of HER2 extracellular domain harboring target epitopes for antibody-based therapeutics. Prior studies of trastuzumab resistance have focused largely on cells selected ex vivo with the antibody in absence of human immune effector cells. We developed a selection model, wherein human HER2 positive BC cells (BT474, SKBR3) were subjected to acute ADCC (>90% cell death), trastuzumab concentration 100ug/mL, effector-target ratio 100:1, using human peripheral blood mononuclear cells (PBMCs) as effectors. Surviving cells were allowed to recover to confluence over 8-10 weeks, for 10 total rounds of ADCC selection ex vivo. Mock-treated parent, IgG1 isotype control, trastuzumab, and PBMCs alone were used as controls. ADCC assays based on calcein fluorescent labeling of live target cells, revealed significant reduction (maximum 20%, p<0.005) in cell lysis in immune-selected BT474 cell lines compared to parental controls (immune-selected SKBR3 cells exhibited a non-significant trend towards reduced ADCC). Transcriptome-wide next-generation RNA sequencing (Illumina NextSeq 500, 2 x 75 bp paired-end, median of 46 million paired-end reads/sample), coupled with pathway enrichment analysis (Reactome), followed by q-PCR validation, confirmed significant changes in expression in immune-selected cells (compared to parent control) for genes including: ALDH1, ANK1, TMPRSS3, HINT1, DNM2, TNNC1, COL4A4 in BT474; and ALDH1, ANK1, CAMP1, CPE, IDO1 in SKBR3 cells. Whole-genome sequencing (Illumina HiSeq X, 150 bp paired end, 30x coverage) elucidated 180 genes with single nucleotide variations (SNVs) in immune-selected cells compared to parent in BT474 cells, and 215 genes in SKBR3 cells. Thirty-four SNVs were shared in both cell lines. Further screening and validation confirm genes with SNVs demonstrating significant transcript up-regulation. These include: COL4A3, LEP, SOX-9 in BT474; and HLA-B, TNFRSF10B, HLA-B, PSMA6 in SKBR3. In further phenotypic analysis, ADCC-conditioned BT474 cells exhibit an elongated fibroblast-like morphology with multiple processes, in contrast to control. Immune-selected SKBR3 cells (and not BT474 cells) demonstrate significantly increased motility compared to control in transwell migration assays (p<0.001), and demonstrated increased cell proliferation (MTT assay, 10-15%, 48h; p=0.0242) as compared to parent controls. Our data indicate immune-selection by effector cells contributes to ADCC tolerance in vitro, and is associated with distinct genotypic and phenotypic alterations. Future investigation will determine whether Fc-engineered MAbs (afucosylated), antibody drug conjugates (T-DM1), or potentiation of ADCC by co-stimulatory agonist CD137 antibodies will re-sensitize ADCC-tolerance. This investigation will help to elucidate potentially targetable pathways that emerge from immune-selection with trastuzumab. Citation Format: Biswas T, Fritzemeier R, Mark A, Meißner T, Young B, Jones BL, Pegram M. Characterization of HER2-positive breast cancer (BC) cells selected for tolerance to trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-06.

A real-time digital bio-imaging system to quantify cellular cytotoxicity as an alternative to the standard chromium-51 release assay.

Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard 51 Cr-release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non-radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentives to find alternative solutions to 51 Cr. We took advantage of the recent development of cell-imaging multimode readers to develop a novel non-radioactive and real-time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target-cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96-well plates. We have developed classical natural killer cell assays in the presence or absence of blocking antibodies and antibody-dependent cell-mediated cytotoxicity. We show that in these assays, cell killing occurs within the first 2 hr with half maximum killing reached after 30 min. This technology has numerous applications such as natural killer and T-cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.

Toxin Transport by A-B Type of Toxins in Eukaryotic Target Cells and Its Inhibition by Positively Charged Heterocyclic Molecules.

A-B types of toxins are among the most potent bacterial protein toxins produced by gram-positive bacteria. Prominent examples are the tripartite anthrax toxin of Bacillus anthracis and the different A-B type clostridial toxins that are the causative agents of severe human and animal diseases and could serve as biological weapons. The components of all these toxins comprise one binding/transport (B) subunit and one or two separate, non-linked enzymatically active (A) subunits. The A and B subunits are separately produced and secreted by the pathogenic gram-positive bacteria and must assemble on the surface of eukaryotic target cells to form biologically active toxin complexes. The B components are cleaved by proteases to generate the biologically active species that binds to receptors on the surface of the target cells and form there oligomers which bind the A subunits. The AB complexes are internalized by receptor-mediated endocytosis and reach early or late endosomes that become acidified. Subsequently, the B components form channels in endosomal membranes that are indispensable for the transport of the enzymatic subunits across these membranes into the cytosol of target cells via the trans-membrane channels. In addition to the channels formed by the B components, host cell factors including chaperones and further folding helper enzymes are involved in the import of the enzymatic subunits into the cytosol of eukaryotic cells. Positively charged heterocyclic molecules, such as chloroquine and related aminoquinolinium and azolopyridinium salts have been shown in recent years to bind with high affinity to the channels formed by the B components of binary toxins. Since binding to the B components is also a prerequisite for transport of the A components across the endosomal membranes the channel blockers also prevent transport of the A subunits into the host cell cytosol. The inhibition of toxin uptake into cells by such pharmacological compounds should also be of clinically interest because the toxins are the major virulence factors causing anthrax on the one hand and severe enteric disease on the other hand. Therefore, the novel toxin inhibitors should be attractive compounds for an application in combination with antibiotics to prevent or treat the diseases associated with binary toxins. Here the different processes involved in channel block in vitro and inhibition of intoxication of living target cells are reviewed in some detail.

Abstract 2123: Antibody-dependent cell-mediated cytotoxicity (ADCC) tolerance to monoclonal antibody therapeutics in human breast and colon cancer cell lines

Abstract Resistance to monoclonal antibody (mAb) therapy limits clinical utility. ADCC is a significant mechanism of action of mAb-elicited tumor response. Postulated mechanisms of mAb resistance include, alteration in cell signaling pathways (PI3K/Akt/Ras-MAPK signaling), over activation/expression of alternate receptor kinases (c-Met/ IGF-1R), or proteolysis of extracellular domains harboring target epitopes. However, most studies on mAb resistance have utilized cancer cells conditioned with mAbs alone, in absence of immune effector cells. We developed an in vitro selection model wherein human HER2 positive breast cancer cell lines (BT474, SKBR3) and EGFR-expressing colorectal cancer cell lines (HT29, DLD1) were subjected to acute ADCC (>90% cell death) with high effector-target (100:1) ratio repeatedly with trastuzumab and cetuximab, respectively, using human peripheral blood mononuclear cells (PBMCs) as effectors. Surviving cells were allowed to recover to confluence over 8-10 weeks for 10 total rounds of ADCC selection. Internal controls included mock-treated parental, isotype-IgG1, effector cells only, and mAb alone. In vitro ADCC assays based on calcein fluorescent labeling of live target cells, demonstrated significant reduction (20%, p<0.005) in cell lysis in immune-selected cell lines compared to parental controls. ADCC-conditioned HT29 cells demonstrated 5-fold reduction in EGFR gene copy number by fluorescence in situ hybridization (FISH, p<0.0001), 2-fold reduction in mRNA level (qPCR, p< 0.01) and 1.8-fold reduction in protein level (immunoblot densitometry, p<0.01). In BT474 breast cells, ADCC-conditioned cells acquired elongated fibroblast-like morphology. Transcriptome-wide next-generation RNA sequencing (Illumina NextSeq 500, 2 × 150 bp paired-end, with over 100 million paired-end reads/sample), coupled with bioinformatic analyses (Reactome pathway database) revealed gene expression changes in immune-selected cells. These involve changes in key immune signaling pathways such as NK-cell and T-cell maturation, class I MHC antigen processing, and immune regulatory pathways, among others. Further validation through q-PCR analyses and loss/ gain of expression is ongoing. Future investigation will also determine whether mAb combination with agonist CD137 mAb (co-stimulatory NK receptor) or Fc-engineered mAbs (e.g. afucosylated) will re-sensitize resistant cells. Our data indicate immune-selection by effector cells contributes to ADCC resistance in vitro. Such investigation may help to elucidate potentially targetable pathways/ markers that emerge from immune-selection with mAbs and effector cells. Citation Format: Tanuka Biswas, Rebecca Fritzemeier, Adam Mark, Tobias Meißner, Brandon M. Young, Mark D. Pegram. Antibody-dependent cell-mediated cytotoxicity (ADCC) tolerance to monoclonal antibody therapeutics in human breast and colon cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2123.

A high-throughput image cytometry-based screening method for the detection of IL2-induced peripheral blood mononuclear cell-mediated cytotoxicity

Abstract Cell-mediated cytotoxicity assays have been an important functional test for investigating the cytotoxic effect of immune effector on target cancer cells. Cytotoxicity assays have traditionally been performed using the 51Chromium (51Cr) release assay, which involves labeling the tumor cells (target) with radioisotopes. The 51Chromium release assay is highly hazardous, time-consuming, and can only acquire end point readout. Furthermore, it can generate inconsistent results due to batch variation, ability of the target cell for 51Cr uptake, and low sensitivity. In this work, we demonstrate a novel high-throughput cytotoxicity screening assay using the Celigo imaging-cytometry method. First the live K562 target cells were stained with Calcein AM, and were co-cultured with PBMCs from two healthy donors in a flat- bottom 96-well plate in the presence or absence of IL-2. Direct cell counting of Calcein positive cells was implied to calculate the changes in number of live target cells in the same well from T = 0 to T = 4 hours. Thus, accurate cell-mediated killing was determined using the “self-referencing” calculation, which could be a more direct method for assessing cytotoxicity than 51Cr release. The donor PBMCs were added to each well with different Calcein-stained target cells at Effector-to-Target (E:T) ratios. The 96-well microplate was scanned and analyzed using the Celigo image cytometer at different time points to measure the % lysis of target cell. The proposed image cytometry method can scan and analyze in 7 min, which can be utilized to perform high-throughput screening of potential antibody- or chemical-based cancer drug, which could be a more efficient method for academic, industry, and clinical research.