Preclinical immunogenicity risk assessment of biotherapeutics using CD4 T cell assays.

T-cell dependent antibody responses to biotherapeutics remain a challenge to the optimal clinical application of biotherapeutics because of their capacity to impair drug efficacy and their potential to cause safety issues. To minimize this clinical immunogenicity risk, preclinical assays measuring the capacity of biotherapeutics to elicit CD4 T cell response in vitro are commonly used. However, there is considerable variability in assay formats and a general poor understanding of their respective predictive value. In this study, we evaluated the performance of three different CD4 T cell proliferation assays in their capacity to predict clinical immunogenicity: a CD8 T cell depleted peripheral blood mononuclear cells (PBMC) assay and two co-culture-based assays between dendritic cells (DCs) and autologous CD4 T cells with or without restimulation with monocytes. A panel of 10 antibodies with a wide range of clinical immunogenicity was selected. The CD8 T cell depleted PBMC assay predicted the clinical immunogenicity in four of the eight highly immunogenic antibodies included in the panel. Similarly, five antibodies with high clinical immunogenicity triggered a response in the DC: CD4 T cell assay but the responses were of lower magnitude than the ones observed in the PBMC assay. Remarkably, three antibodies with high clinical immunogenicity did not trigger any response in either platform. The addition of a monocyte restimulation step to the DC: CD4 T cell assay did not further improve its predictive value. Overall, these results indicate that there are no CD4 T cell assay formats that can predict the clinical immunogenicity of all biotherapeutics and reinforce the need to combine results from various preclinical assays assessing antigen uptake and presentation to fully mitigate the immunogenicity risk of biotherapeutics.

Bispecific Antibody Designed for Targeted NK Cell Activation and Functional Assessment for Biomedical Applications.

Natural killer (NK) cells serve as key innate effectors and their activity has been considered a prognostic biomarker in diverse human diseases. Currently, NK cell functional assays have several problems primarily related to adequate preparation, labeling, or treatment of target cells, which are cumbersome and often hamper consistent sensitivity for NK cells. Here, bispecific antibodies (BsAb's) targeting NKG2D and 2B4 receptors, whose combination mounts selective cytotoxicity and IFN-γ production of NK cells, are developed as acellular, consistent, and easy-to-use strategies for assessing NK cell functions. These NK cell activator BsAb's (NKABs) are constructed in symmetric dual bivalent formats with different interdomain spacings [immunoglobulin G (IgG)-single-chain variable fragment (scFv) and dual-variable domain (DVD)-Ig] and kappa constant (Cκ)-scFv format linking two scFv's with a Cκ domain. These NKABs are specific and superior to a combination of monospecific antibodies for NK cell activation. NKAB elicits both direct cytotoxicity and IFN-γ production via integration of NKG2D and 2B4 signals. Moreover, stimulation with NKAB IgG-scFv and Cκ-scFv reveals defective NK cell functions in X-linked lymphoproliferative disease involving 2B4 dysfunction in NK cells and multiple myeloma in peripheral blood mononuclear cells and whole blood, respectively. Hence, this work provides a proof of concept that NKAB facilitates the reliable and comprehensive measurement of NK cell function in clinical settings for diagnostic and prognostic purposes.

Assessment of cellular response to mitogens in long-term allogeneic hematopoietic stem cell transplantation survivors.

Infection is one of the major causes of death in hematopoietic stem cell transplantation (HSCT) survivors. Precise assessments of immune function after HSCT will be critical in establishing appropriate treatment and prophylaxis, such as re-vaccination. Although several surrogate markers for prediction of clinical outcomes after HSCT have been proposed, definitive markers of immune reconstitution and data on those markers in long-term survivors are lacking. In this study, cellular response to mitogens was assessed and clinical features associated with a poor response to mitogens were investigated in long-term allogeneic HSCT survivors. Age at transplantation and age at the time of mitogen stimulation test were each identified as significant risk factors for poor response to phytohemagglutinin and concanavalin A, respectively (P < 0.001 each). However, time elapsed since transplantation was not found to be correlated with responsiveness to mitogens in this study. Prospective, in-depth studies on immune reconstitution are needed to establish appropriate prophylaxis against infections after HSCT and a schedule for re-vaccination.

Improved prediction of HLA antigen presentation hotspots: Applications for immunogenicity risk assessment of therapeutic proteins.

Immunogenicity risk assessment is a critical element in protein drug development. Currently, the risk assessment is most often performed using MHC-associated peptide proteomics (MAPPs) and/or T-cell activation assays. However, this is a highly costly procedure that encompasses limited sensitivity imposed by sample sizes, the MHC repertoire of the tested donor cohort and the experimental procedures applied. Recent work has suggested that these techniques could be complemented by accurate, high-throughput and cost-effective prediction of in silico models. However, this work covered a very limited set of therapeutic proteins and eluted ligand (EL) data. Here, we resolved these limitations by showcasing, in a broader setting, the versatility of in silico models for assessment of protein drug immunogenicity. A method for prediction of MHC class II antigen presentation was developed on the hereto largest available mass spectrometry (MS) HLA-DR EL data set. Using independent test sets, the performance of the method for prediction of HLA-DR antigen presentation hotspots was benchmarked. In particular, the method was showcased on a set of protein sequences including four therapeutic proteins and demonstrated to accurately predict the experimental MS hotspot regions at a significantly lower false-positive rate compared with other methods. This gain in performance was particularly pronounced when compared to the NetMHCIIpan-3.2 method trained on binding affinity data. These results suggest that in silico methods trained on MS HLA EL data can effectively and accurately be used to complement MAPPs assays for the risk assessment of protein drugs.

Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms.

Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro. All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.

A highly polarized TH2 bladder response to infection promotes epithelial repair at the expense of preventing new infections.

Urinary tract infections (UTIs) typically evoke prompt and vigorous innate bladder immune responses, including extensive exfoliation of the epithelium. To explain the basis for the extraordinarily high recurrence rates of UTIs, we examined adaptive immune responses in mouse bladders. We found that, following each bladder infection, a highly T helper type 2 (TH2)-skewed immune response directed at bladder re-epithelialization is observed, with limited capacity to clear infection. This response is initiated by a distinct subset of CD301b+OX40L+ dendritic cells, which migrate into the bladder epithelium after infection before trafficking to lymph nodes to preferentially activate TH2 cells. The bladder epithelial repair response is cumulative and aberrant as, after multiple infections, the epithelium was markedly thickened and bladder capacity was reduced relative to controls. Thus, recurrence of UTIs and associated bladder dysfunction are the outcome of the preferential focus of the adaptive immune response on epithelial repair at the expense of bacterial clearance.

Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice.

Interleukin-3 (IL-3) is an important hematopoietic growth factor and immunregulatory cytokine. Although activated T helper cells represent a main source of IL-3, other cell types have been reported to express this cytokine. However, precise identification and quantification of the cells that produce IL-3 in vivo have not been performed. Therefore, we used a CRISPR/Cas approach to engineer mice containing a bicistronic mRNA linking a readily identifiable reporter, enhanced green fluorescent protein (ZsGreen1), to IL-3 expression. To characterize these novel reporter mice, we first examined ZsGreen1 expression by CD4 T cells subsets primed and activated in vitro. We found that activated Th1 cells expressed ∼4-fold higher levels of ZsGreen1 as compared to Th0 and Th2 cells. Endogenous IL-3 expression remained intact although reporter Th1 cells secreted ∼33 % less IL-3 than similarly activated wild-type cells. To characterize the ability of reporter mice to accurately mark IL-3-producing cells in vivo, we infected mice with Nippostrongylus brasiliensis. Low but significant numbers of ZsGreen1+ CD4 T cells were detected in the mesenteric lymph nodes and lung following both primary and secondary infection. No difference in basophil and intestinal mast cell numbers were observed between infected reporter and wild-type mice indicating that reporter mice secreted IL-3 levels in vivo that results in IL-3-driven biological activities which are indistinguishable from those observed in corresponding wild-type mice. These IL-3 reporter mice will be a valuable resource to investigate IL-3-dependent immune responses in vivo.

Assessment of IFNγ responsiveness in patient-derived xenografts.

Patient-derived xenografts are a useful tool in cancer immunology, as they allow researchers to study human cancers in vivo when starting with a relatively small amount of human tumor tissue. These models make it possible to study tumor cell-intrinsic changes that occur in response to external stimuli including cytokines like interferon gamma (IFNγ) that are important for effective anti-tumor immune responses. IFNγ responsiveness can be measured by assessing surface expression of MHC class I on tumor cells, the molecule on which tumor antigens are presented to cytotoxic T cells in the tumor microenvironment. Low levels of MHC class I and lack of responsiveness have been associated with resistance to T-cell directed therapies like immune checkpoint inhibitors. In this chapter, we present a protocol for an assay to screen patient-derived xenografts for their responsiveness to IFNγ. The results of this assay can be used as a starting point for uncovering cancer cell-intrinsic mechanisms of resistance to immunotherapies in patient tumors.

Assessment of the genes and molecular mechanisms of B cells activation through systems biology approaches.

Two classes of T helper lymphocytes, Th1 and Th2, have different roles in B cell activation based on specific cytokines. To understand the difference of molecular mechanisms of B cell activation, the microarray dataset of B cells co-cultured with type 1 and 2 T helper, Be1 and Be2, were investigated. After quality assessment, using the GEO2R tool, the GSE84948 dataset was re-analyzed. Genes with adjusted p-value ⩽ 0.05 were assumed as differentially expressed (DE). The protein-protein interaction (PPI) networks were constructed using CluePedia plugin of Cytoscape, and analyzed by NetworkAnalyzer tool and MCODE plugin. Using ClueGO plugin of Cytoscape software, gene ontology (GO) analysis was performed. The comparison of Be1 and Be2 cells with naive B cells revealed 8742 and 8748 DE genes, respectively. The topology analysis of PPI networks predicted central genes. Among these, Jak3, Actrt3, and Pik3cb genes were determined as crucial genes in Be1 network. Prkx, Smarca4, and Jak2 genes were defined as critical genes in Be2 PPI network. GO analysis with PPI networks genes resulted in the promotion of immune system activation. In conclusion, we explored holistic methods for molecular assay of the difference between B cell activation mechanisms with Th1 and Th2.

Assessment of TCR signal strength of antigen-specific memory CD8+ T cells in human blood.

Assessment of the quality and the breadth of antigen (Ag)-specific memory T cells in human samples is of paramount importance to elucidate the pathogenesis and to develop new treatments in various diseases. T-cell receptor (TCR) signal strength, primarily controlled by TCR affinity, affects many fundamental aspects of T-cell biology; however, no current assays for detection of Ag-specific CD8+ T cells can assess their TCR signal strength in human samples. Here, we provide evidence that interferon regulatory factor 4 (IRF4), a transcription factor rapidly upregulated in correlation with TCR signal strength, permits the assessment of the TCR signal strength of Ag-specific CD8+ T cells in human peripheral blood mononuclear cells (PBMCs). Coexpression of IRF4 and CD137 sensitively detected peptide-specific CD8+ T cells with extremely low background in PBMCs stimulated for 18 hours with MHC class I peptides. Our assay revealed that human memory CD8+ T cells with high-affinity TCRs have an intrinsic ability to highly express CD25. Furthermore, HIV-specific CD8+ T cells in chronic HIV+ subjects were found to display primarily low-affinity TCRs with low CD25 expression capacity. Impairment in the functions of HIV-specific CD8+ T cells might be associated with their suboptimal TCR signals, as well as impaired responsiveness to interleukin-2.

Autoreactive, Low-Affinity T Cells Preferentially Drive Differentiation of Short-Lived Memory B Cells at the Expense of Germinal Center Maintenance.

B cell fate decisions within a germinal center (GC) are critical to determining the outcome of the immune response to a given antigen. Here, we characterize GC kinetics and B cell fate choices in a response to the autoantigen myelin oligodendrocyte glycoprotein (MOG) and compare the response with a standard model foreign antigen. Both antigens generate productive primary responses, as evidenced by GC development, circulating antigen-specific antibodies, and differentiation of memory B cells. However, in the MOG response, the status of the cognate T cell partner drives preferential B cell differentiation to a memory phenotype at the expense of GC maintenance, resulting in a truncated GC. Reduced plasma cell differentiation is largely independent of T cell influence. Interestingly, memory-phenotype B cells formed in the MOG GC are not long lived, resulting in a failure of the B cell response to secondary challenge.

The Functional Assessment of T cells.

It is important to know what kind of T cell populations is involved in various disease states and to know the state of T cell functions involved in the disease. When T cell antigen receptors (TCR) recognize a specific antigen, the cell transmits a signal by a transduction mechanism within the T cell's cytoplasm. This signal initiates gene transcription essential for differentiation and activation of T cells. In this chapter, we will describe the methods of analyzing the transcribed mRNA and detecting the translated product in order to know the activation state of T cells.

Toward precision manufacturing of immunogene T-cell therapies.

Cancer can be effectively targeted using a patient's own T cells equipped with synthetic receptors, including chimeric antigen receptors (CARs) that redirect and reprogram these lymphocytes to mediate tumor rejection. Over the past two decades, several strategies to manufacture genetically engineered T cells have been proposed, with the goal of generating optimally functional cellular products for adoptive transfer. Based on this work, protocols for manufacturing clinical-grade CAR T cells have been established, but these complex methods have been used to treat only a few hundred individuals. As CAR T-cell therapy progresses into later-phase clinical trials and becomes an option for more patients, a major consideration for academic institutions and industry is developing robust manufacturing processes that will permit scaling-out production of immunogene T-cell therapies in a reproducible and efficient manner. In this review, we will discuss the steps involved in cell processing, the major obstacles surrounding T-cell manufacturing platforms and the approaches for improving cellular product potency. Finally, we will address the challenges of expanding CAR T-cell therapy to a global patient population.

Sensitivity and specificity of the lymphocyte transformation test in drug reaction with eosinophilia and systemic symptoms causality assessment.

BACKGROUND: Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe delayed hypersensitivity reaction. The determination of drug causality is complex. The lymphocyte transformation test (LTT) has been reported positive in more than 50% of DRESS cases. Nevertheless, the sensitivity and specificity of LTT specifically in DRESS have not yet been established. Rechallenge with the culprit drug is contraindicated and cannot be used as gold standard for sensitivity and specificity determination. OBJECTIVE: To estimate the sensitivity and specificity of LTT in a clinically defined series of patients with DRESS. METHODS: Some 41 patients diagnosed with DRESS were included in the study. The results of the algorithm of the Spanish Pharmacovigilance System were used as the standard for a correct diagnosis of drug causality. A standard LTT was performed with involved drugs in acute or recovery samples. A stimulation index (SI) ≥2 in at least one concentration except for beta-lactams (SI ≥3) and contrast media (SI ≥4) was considered positive. Contingency tables and ROC curves were used for analysis. RESULTS: Sensitivity and specificity of LTT in the recovery phase of DRESS were 73% and 82%, respectively, whereas in the acute phase, they were only 40% and 30%, respectively. Comparison of skin tests and LTT confirmed a higher sensitivity and specificity of LTT in DRESS. LTT showed high sensitivity (S) and specificity (Sp) for anticonvulsants (S 100%, Sp 100%; P = .008), anti-TB drugs (S 87.5%, Sp 100%; P = .004), and beta-lactams (S 73%, Sp 100%; P = .001). ROC curves revealed that the best criteria for LTT positivity for all drugs are SI ≥2 in at least one concentration, increasing overall sensitivity to 80%, and for beta-lactams from 73% to 92%. CONCLUSIONS AND CLINICAL RELEVANCE: LTT is a good diagnostic tool for drug causality in DRESS, mainly when performed in the recovery phase.

An optimized protocol for the in vitro generation and functional analysis of human PD1/PD-L1 signal.

Programmed cell death-1 (PD1) is an inhibitory receptor expressed on the activated T and B cells. Binding of PD1 to its ligands, PD-L1 and PD-L2 has led to deliver an inhibitory signal into the activated T cells. Recently, blocking PD1/PD-L1 pathway has emerged as a new treatment paradigm across a broad spectrum of malignancies. Remarkable clinical responses of monoclonal antibodies specific for PD-1 or its ligands in patients with many different types of cancer, attracted several pharmaceutical companies and researchers to investigate the agents that block PD1/PD-L1 signal. The safety and efficacy of the agents are needed to examine in the preclinical studies. In this study, we optimized a facile and cost-effective protocol for in vitro generation and functional analysis of human PD1/PD-L1 pathway. Activation of CD8 + CD279 + T cell was performed by anti-CD3 and D28 antibodies and the recombinant PD-L1 was used for inactivation of T cells through PD1/PD-L1 pathway. In this protocol, T-cell cytokine production (IL-2 and IFN-γ) and proliferation assay confirmed that a measurable PD1/PD-L1 signal was generated. We expected that in vitro PD1/PD-L1 signal that has been optimized in this study will serve as a valuable protocol for preclinical studies involving PD1/PD-L1 pathway.

T-cell Receptors for Clinical Therapy: In Vitro Assessment of Toxicity Risk.

Adoptive therapy with T-cell receptor (TCR)-engineered T cells has shown promising results in the treatment of patients with tumors, and the number of TCRs amenable for clinical testing is expanding rapidly. Notably, adoptive therapy with T cells is challenged by treatment-related side effects, which calls for cautious selection of target antigens and TCRs that goes beyond their mere ability to induce high T-cell reactivity. Here, we propose a sequence of in vitro assays to improve selection of TCRs and exemplify risk assessments of on-target as well as off-target toxicities using TCRs directed against cancer germline antigens. The proposed panel of assays covers parameters considered key to safety, such as expression of target antigen in healthy tissues, determination of a TCR's recognition motif toward its cognate peptide, and a TCR's cross-reactivity toward noncognate peptides. Clin Cancer Res; 23(20); 6012-20. ©2017 AACR.

Heterogeneity assessment of functional T cell avidity.

The potency of cellular immune responses strongly depends on T cell avidity to antigen. Yet, functional avidity measurements are rarely performed in patients, mainly due to the technical challenges of characterizing heterogeneous T cells. The mean functional T cell avidity can be determined by the IFN-γ Elispot assay, with titrated amounts of peptide. Using this assay, we developed a method revealing the heterogeneity of functional avidity, represented by the steepness/hillslope of the peptide titration curve, documented by proof of principle experiments and mathematical modeling. Our data show that not only natural polyclonal CD8 T cell populations from cancer patients, but also monoclonal T cells differ strongly in their heterogeneity of functional avidity. Interestingly, clones and polyclonal cells displayed comparable ranges of heterogeneity. We conclude that besides the mean functional avidity, it is feasible and useful to determine its heterogeneity (hillslope) for characterizing T cell responses in basic research and patient investigation.

Comparative assessment of lipid based nano-carrier systems for dendritic cell based targeting of tumor re-initiating cells in gynecological cancers.

We aimed to identify an optimum nano-carrier system to deliver tumor antigen to dendritic cells (DCs) for efficient targeting of tumor reinitiating cells (TRICs) in gynecological malignancies. Different lipid based nano-carrier systems i.e. liposomes, ethosomes and solid lipid nanoparticles (SLNPs) were examined for their ability to activate DCs in allogeneic settings. Out of these three, the most optimized formulation was subjected for cationic and mannosylated surface modification and pulsed with DCs for specific targeting of tumor cells. In both allogeneic and autologous trials, SLNPs showed a strong ability to activate DCs and orchestrate specific immune responses for targeting TRICs in gynecological malignancies. Our findings suggest that the mannosylated form of SLNPs is a suitable molecular vector for DC based therapeutics. DCs pulsed with mannosylated SLNPs may be utilized as adjuvant therapy for specific removal of TRICs to benefit patients from tumor recurrence.

Self-reactivity as the necessary cost of maintaining a diverse memory T-cell repertoire.

The adaptive immune system is expected to protect the host from infectious agents and malignancies, while avoiding robust activation against self-peptides. However, T cells are notoriously inept at protection whenever the pathogen or tumor is persistent in the body for longer periods of time. While this has been thought of as an adaptation to limit the immunopathology from continued effector T-cell responses, it is also likely an extension of the T cell's intrinsic mechanisms which evolved to tolerate self-peptides. Here we deliberate on how the need to tolerate self-peptides might stem from a paradoxical requirement-the utility of such molecules in maintaining a diverse repertoire of pathogen-specific memory T cells in the body. Understanding the mechanisms underlying this intriguing nexus, therefore, has the potential to reveal therapeutic strategies not only for improving immune responses to chronic infections and tumors but also the long-term efficacy of vaccines aimed at cellular immune responses.

Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets.

CD8(+) T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with <20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore, we observed superior cytotoxic effector function in TEM compared with TCM, and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase, and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM Collectively, this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity, and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications.