Gabrielle Romain

Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia.

Individual Motile CD4+ T Cells Can Participate in Efficient Multikilling through Conjugation to Multiple Tumor Cells

Liadi, Singh, and colleagues used Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to show that CD4+ CD19-chimeric antigen receptor (CAR+) T cells participate in multikilling of tumor cells with slower kinetics of killing than CD8+CAR+ T cells, but high motility subgroups of both T-cell subsets have similar kinetics. T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR+ T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. Cancer Immunol Res; 3(5); 473–82. ©2015 AACR. See related commentary by June, p. 470

IgG Fc domains that bind C1q but not effector Fc[gamma] receptors delineate the importance of complement-mediated effector functions

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Automated profiling of individual cell–cell interactions from high-throughput time-lapse imaging microscopy in nanowell grids (TIMING)

MOTIVATION There is a need for effective automated methods for profiling dynamic cell-cell interactions with single-cell resolution from high-throughput time-lapse imaging data, especially, the interactions between immune effector cells and tumor cells in adoptive immunotherapy. RESULTS Fluorescently labeled human T cells, natural killer cells (NK), and various target cells (NALM6, K562, EL4) were co-incubated on polydimethylsiloxane arrays of sub-nanoliter wells (nanowells), and imaged using multi-channel time-lapse microscopy. The proposed cell segmentation and tracking algorithms account for cell variability and exploit the nanowell confinement property to increase the yield of correctly analyzed nanowells from 45% (existing algorithms) to 98% for wells containing one effector and a single target, enabling automated quantification of cell locations, morphologies, movements, interactions, and deaths without the need for manual proofreading. Automated analysis of recordings from 12 different experiments demonstrated automated nanowell delineation accuracy >99%, automated cell segmentation accuracy >95%, and automated cell tracking accuracy of 90%, with default parameters, despite variations in illumination, staining, imaging noise, cell morphology, and cell clustering. An example analysis revealed that NK cells efficiently discriminate between live and dead targets by altering the duration of conjugation. The data also demonstrated that cytotoxic cells display higher motility than non-killers, both before and during contact. CONTACT broysam@central.uh.edu or nvaradar@central.uh.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Detection and isolation of auto-reactive human antibodies from primary B cells

The isolation of human monoclonal antibodies (hmAb) has emerged as a versatile platform in a wide variety of contexts ranging from vaccinology to therapeutics. In particular, the presence of high titers of circulating auto-antibodies is implicated in the pathology and outcome of autoimmune diseases. Therefore, the molecular characterization of these hmAb provides an avenue to understanding the pathogenesis of autoimmune diseases. Additionally, the phenotype of the auto-reactive B cells may have direct relevance for therapeutic intervention. In this report, we describe a high-throughput single-cell assay, microengraving, for the screening, characterization and isolation of anti-citrullinated protein antibodies (ACPA) from peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis (RA) patients. Stimulated B cells are profiled at the single-cell level in a large array of sub-nanoliter nanowells (∼10(5)), assessing both the phenotype of the cells and their ability to secrete cyclic-citrullinated peptide (CCP)-specific antibodies. Single B cells secreting ACPA are retrieved by automated micromanipulation, and amplification of the immunoglobulin (Ig) heavy and light chains is performed prior to recombinant expression. The methodology offers a simple, rapid and low-cost platform for isolation of auto-reactive antibodies from low numbers of input cells and can be easily adapted for isolation and characterization of auto-reactive antibodies in other autoimmune diseases.

Quantitative high-throughput single-cell cytotoxicity assay for T cells.

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials(1-4). There are several advantages to using CAR(+) T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR(+) T cells in the event of host toxicity(5). Delineating the optimal functions of CAR(+) T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo(6,7). While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets(8). Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes(9). In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

Single-cell profiling of dynamic cytokine secretion and the phenotype of immune cells

Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.

Beyond Autoantibodies: Biological Roles Of Human Autoreactive B Cells In Rheumatoid Arthritis Revealed By Whole Transcriptome Profiling

Although the contribution of B-cell derived autoreactive antibodies to rheumatoid arthritis (RA) has been studied extensively, the autoantibody-independent roles of B cells in the progression of the disease is not well-defined. Here we present the first comprehensive transcriptome profile of human autoreactive B cells in an autoimmune disease by performing RNA-sequencing of citrulline-specific B cells from RA patients. In order to facilitate a comprehensive understanding of the profile of these citrulline-specific (RA-CCPPOS) B cells, we performed comparative analyses to both citrulline-negative (RA-CCPNEG) B cells from the same donors, and identified 431 differentially expressed genes (DEGs); and hemagglutinin-specific (HA) B cells from healthy individuals and identified 1658 DEGs. Three-way comparisons of these B cell populations demonstrated that RA-CCPPOS B cells, in comparison to the RA-CCPNEG B cells, demonstrate a potential role in protein citrullination and inflammation; RA-CCPPOS B cells in comparison to HA-specific B cells demonstrate RA-specific signatures like the expression of pro-inflammatory cytokines, chemokines, costimulatory molecules and B-cell activation cascades; and all B cells from RA patients demonstrated a significant impact of the multitude of TNF signaling pathways. Furthermore, transcription factor profiling suggested that cyclic AMP (cAMP) related pathways and downstream signaling molecules are selectively enriched in RA-CCPPOS cells in comparison to the other two B cell subsets. We advanced the understanding of the citrulline reactive B cells in RA pathophysiology by documenting and validating two novel observations in independent cohorts of patients: (1) the expression of IL15Rα is restricted to citrulline-specific cells within RA patients and the concentration of soluble IL15Rα is elevated in the sera of RA patients, (2) B cells from RA patients are capable of producing epidermal growth factor ligand, amphiregulin (AREG) which in turn has a direct impact on the mechanistic effectors of RA, osteoclasts and fibroblastlike synoviocytes (FLS). Overall, our comprehensive dataset identifies several existing FDA-approved drugs that can potentially be repurposed for RA and can serve as a foundation for studying the multi-faceted roles of B cells in other autoimmune diseases.

278. Next-Generation Non-Viral Gene Transfer to Redirect T-Cell Specificity

Non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system has been successfully tested in humans to express a chimeric antigen receptor (CAR) to redirect T-cell specificity to CD19. This system has been modified to (i) improve the design of the CD19-specific CAR and (ii) reduce the time in culture to 14 days. Our previous clinical trials infused T cells expressing a 2nd generation CAR (designated CD19RCD28) with an IgG4-Fc stalk that activated via chimeric CD28 and CD3ζ. To evaluate the length of extracellular domain on function, we tested four CD19-specific CARs with two long [IgG4-Fc (CD19RCD28) and EQ (L235E and N297Q) mutant IgG4-Fc (CD19R*CD28)], medium (CD8α hinge, CD19RCD8CD28) and short (12aa IgG1 hinge, CD19R12aaCD28) stalks which all signaled through chimeric CD28 and CD3ζ endodomains. Generation of our T cells is based on electro-transfer of CARs coded by the SB system and antigen-specific stimulation through activating and K562-derived propagating cells (AaPC) in the presence of exogenous cytokines. After electro-transfer of SB-derived DNA plasmids, T cells were selectively propagated with either a new two-weekly (2x) or standard four-weekly (4x) additions of AaPC. All genetically modified T cells were capable of specific lysis of CD19+ tumor targets and producing IFN-γ in response to CD19+ stimulator cells. Serial killing was tested using massively parallel microscopy to observe single T cells and we observed that CDl9RCD8CD28+ T cells exhibited superior ability to partake in multiple killing events. CAR+ T cells were further tested in vivo for their ability to control CD19+ leukemia in a mouse model of minimal residual disease as well as established disease (Figure A and BFigure A and B). We found that T cells expressing modified CARs (CD19R*CD28, CD19RCD8CD28, CD19R12aaCD28) with reduced ability to bind to Fc gamma receptors (FcγR) were able to control leukemia more efficiently in mice compared to T cells expressing CD19RCD28. The CD19RCD8CD28 CAR was superior in controlling disease in the model of minimal residual disease compared with the CAR design evaluated in our prior clinical trials. T cells expressing CD19R*CD28 and CD19RCD8CD28 were then evaluated in 2x stimulation cycle. Both the 4x CAR+ T cells had similar CAR expression (>70%) whereas the 2x CAR+ T cells exhibited reduced CAR expression (~40%). The 2x CAR+ T cells expressed markers associated with less differentiated state of naive-like and memory T cells when compared to 4x CAR+ T cells, which was supported by measurement of mRNA species using bar-coded probes. The efficacy of the CAR+ T cells was tested in mice bearing established CD19+ leukemia and we observed superior survival in mice receiving the 2x CAR+ T cells compared with the 4x CAR+ T cells (Figure CFigure C). These data depict that length of extracellular domain and its associated binding to FcγR improves T-cell effector functions and that decreasing the time in culture can improve control of leukemia in vivo. These data support the use of cDl9RCD8CD28 testing in a next-generation clinical trial (IND# 16474).View Large Image | Download PowerPoint Slide

Abstract B082: Migratory T cells demonstrate superior persistence and enhanced tumor control

Adoptive cell therapy (ACT) based on the transfer of chimeric antigen receptor (CAR) T cells rendered specific for CD19 has demonstrated significant anti-tumor effects in patients with refractory CD19+ B-cell malignancies. There are however no biomarkers that can predict the potency of T-cell infusion products and thus the ability to enrich functionally superior cells is lacking. Existing clinical data supports at least two important functions of the T cells in mediating tumor regression: (a) immediate cytotoxicity to enable tumor cell killing, and (b) long-term persistence to ensure lasting and durable responses. We demonstrate here that migratory capacity of T cells can be used as a biomarker to segregate functionally superior cells that display enhanced polyfunctionality in vitro and superior tumor control in vivo. The simplicity of sorting cells based on motility and the scalability of the approach imply that these can be routinely adapted in standard GMP settings to enrich for functional T cells among any heterogeneous population. Populations of CD19-specific chimeric antigen receptor (CAR) T cells were biomanufactured using our standard clinical protocol. Timelapse Imaging In Nanowell Grids (TIMING) was used to enable the simultaneous quantification of the interaction between thousands of individual tumor-specific CD8+ T cells and multiple tumor cells. These results demonstrated that IFN-gamma secretion was the most common function elicited upon tumor cell conjugation. We found that CD8+ T cells with killing ability, especially serial killing ability, required shorter durations of target cell conjugation in comparison to IFN-gamma secreting mono-functional cells, indicating rapid synapse termination by T cells capable of killing versus cytokine secretion. Tracking the velocities of these cells by time-lapse imaging revealed that these serial killer T cells (with or without IFN-gamma secretion) had a high out-of-contact basal motility. In order to gain a better understanding of the phenotypic characteristics of the migratory T cells, we performed molecular profiling of T cells identified only by their basal motility (in the absence of tumor cells) using single-cell multiplexed transcriptional profiling, microscopy and flow cytometry, and found that the costimulatory molecules CD2 and CD244, and the chemokine receptor CXCR3 were expressed at higher levels in highly motile T cells compared to the non-motile T cells. In order to test the hypothesis that migratory capacity can serve as a biomarker T cell polyfunctionality, we next sorted motile cells using a modified migration assay, and confirmed that the migrating cells were enriched in highly motile cells. We characterized these cells phenotypically by flow-cytometry and demonstrated that T-cell differentiation markers including CCR7, CD95 and CD45RA were not differentially expressed between the motile and non-motile cells. Consistently however, the migrated cells displayed superior killing and serial killing in comparison to the non-migrated T cells. This superior in vitro polyfunctionality of migratory T cells was confirmed in a mouse xenograft model of leukemia in which motile CAR+ T cells were superior in controlling the disease in comparison to either their non-motile counterparts or the parent unsorted population. In aggregate, these results demonstrate the utility of our TIMING single cell methodology in uncovering not only the dynamic profile of T-cell behavior but also the ability to identify subpopulations of T-cell with enhanced polyfunctionality. Our studies support the use of motility as a surrogate and selective marker of higher CAR+ T cell bioactivity. These results also open up avenues to molecularly engineer T cells for an increased motility that could translate to better in vivo outcomes. Note: This abstract was not presented at the conference. Citation Format: Gabrielle Romain, Harjeet Singh, Ivan Liadi, Jay R Adolacion, Laurence J.N. Cooper, Navin Varadarajan. Migratory T cells demonstrate superior persistence and enhanced tumor control [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B082.