Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human.

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4(+) Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4(+) T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4(+) Th cells upon activation, whereas the 'standard' proteasome is up-regulated in Tregs only upon activation.

BET Inhibition Enhances TNF-Mediated Antitumor Immunity.

Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8+ T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents.

A Novel Approach for Quantifying the Pharmacological Activity of T-Cell Engagers Utilizing In Vitro Time Course Experiments and Streamlined Data Analysis.

CD3-bispecific antibodies are a new class of immunotherapeutic drugs against cancer. The pharmacological activity of CD3-bispecifics is typically assessed through in vitro assays of cancer cell lines co-cultured with human peripheral blood mononuclear cells (PBMCs). Assay results depend on experimental conditions such as incubation time and the effector-to-target cell ratio, which can hinder robust quantification of pharmacological activity. In order to overcome these limitations, we developed a new, holistic approach for quantification of the in vitro dose-response relationship. Our experimental design integrates a time-independent analysis of the dose-response across different time points as an alternative to the static, "snap-shot" analysis based on a single time point commonly used in dose-response assays. We show that the potency values derived from static in vitro experiments depend on the incubation time, which leads to inconsistent results across multiple assays and compounds. We compared the potency values from the time-independent analysis with a model-based approach. We find comparably accurate potency estimates from the model-based and time-independent analyses and that the time-independent analysis provides a robust quantification of pharmacological activity. This approach may allow for an improved head-to-head comparison of different compounds and test systems and may prove useful for supporting first-in-human dose selection.

Pharmacokinetics and Pharmacodynamics of T-Cell Bispecifics in the Tumour Interstitial Fluid.

The goal of this study is to investigate the pharmacokinetics in plasma and tumour interstitial fluid of two T-cell bispecifics (TCBs) with different binding affinities to the tumour target and to assess the subsequent cytokine release in a tumour-bearing humanised mouse model. Pharmacokinetics (PK) as well as cytokine data were collected in humanised mice after iv injection of cibisatamab and CEACAM5-TCB which are binding with different binding affinities to the tumour antigen carcinoembryonic antigen (CEA). The PK data were modelled and coupled to a previously published physiologically based PK model. Corresponding cytokine release profiles were compared to in vitro data. The PK model provided a good fit to the data and precise estimation of key PK parameters. High tumour interstitial concentrations were observed for both TCBs, influenced by their respective target binding affinities. In conclusion, we developed a tailored experimental method to measure PK and cytokine release in plasma and at the site of drug action, namely in the tumour. Integrating those data into a mathematical model enabled to investigate the impact of target affinity on tumour accumulation and can have implications for the PKPD assessment of the therapeutic antibodies.

Corrigendum: Combination of T-Cell Bispecific Antibodies With PD-L1 Checkpoint Inhibition Elicits Superior Anti-Tumor Activity.

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Human immunocompetent Organ-on-Chip platforms allow safety profiling of tumor-targeted T-cell bispecific antibodies.

Traditional drug safety assessment often fails to predict complications in humans, especially when the drug targets the immune system. Here, we show the unprecedented capability of two human Organs-on-Chips to evaluate the safety profile of T-cell bispecific antibodies (TCBs) targeting tumor antigens. Although promising for cancer immunotherapy, TCBs are associated with an on-target, off-tumor risk due to low levels of expression of tumor antigens in healthy tissues. We leveraged in vivo target expression and toxicity data of TCBs targeting folate receptor 1 (FOLR1) or carcinoembryonic antigen (CEA) to design and validate human immunocompetent Organs-on-Chips safety platforms. We discovered that the Lung-Chip and Intestine-Chip could reproduce and predict target-dependent TCB safety liabilities, based on sensitivity to key determinants thereof, such as target expression and antibody affinity. These novel tools broaden the research options available for mechanistic understandings of engineered therapeutic antibodies and assessing safety in tissues susceptible to adverse events.

Combination of T-Cell Bispecific Antibodies With PD-L1 Checkpoint Inhibition Elicits Superior Anti-Tumor Activity.

T-cell Bispecific Antibodies (TCBs) elicit anti-tumor responses by cross-linking T-cells to tumor cells and mediate polyclonal T-cell expansion that is independent of T-cell receptor specificity. TCBs thus offer great promise for patients who lack antigen-specific T-cells or have non-inflamed tumors, which are parameters known to limit the response of checkpoint inhibitors. The current study deepens the understanding of TCB mode of action and elaborates on one of the adaptive resistance mechanisms following its treatment in vivo in humanized mice and syngeneic pre-clinical tumor models. Single-agent TCB treatment reduced tumor growth compared with controls and led to a 2-10-fold increase in tumor-infiltrating T-cells, regardless of the baseline tumor immune cell infiltration. TCB treatment strongly induced the secretion of CXCL10 and increased the frequency of intra-tumor CXCR3+ T-cells pointing to the potential role of the CXCL10-CXCR3 pathway as one of the mechanisms for T-cell recruitment to tumors upon TCB treatment. Tumor-infiltrating T-cells displayed a highly activated and proliferating phenotype, resulting in the generation of a highly inflamed tumor microenvironment. A molecular signature of TCB treatment was determined (CD8, PD-1, MIP-a, CXCL10, CXCL13) to identify parameters that most robustly characterize TCB activity. Parallel to T-cell activation, TCB treatment also led to a clear upregulation of PD-1 on T-cells and PD-L1 on tumor cells and T-cells. Combining TCB treatment with anti-PD-L1 blocking antibody improved anti-tumor efficacy compared to either agent given as monotherapy, increasing the frequency of intra-tumoral T-cells. Together, the data of the current study expand our knowledge of the molecular and cellular features associated with TCB activity and provide evidence that the PD-1/PD-L1 axis is one of the adaptive resistance mechanisms associated with TCB activity. This mechanism can be managed by the combination of TCB with anti-PD-L1 blocking antibody translating into more efficacious anti-tumor activity and prolonged control of the tumor outgrowth. The elucidation of additional resistance mechanisms beyond the PD-1/PD-L1 axis will constitute an important milestone for our understanding of factors determining tumor escape and deepening of TCB anti-tumor responses in both solid tumors and hematological disorders.

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

Specific components of prostanoid-signaling pathways are present in non-small cell lung cancer cells.

In the present study, we measured prostanoid synthesis and the expression of genes associated with prostanoid signaling in human non-small cell lung carcinoma (NSCLC) cell lines and in primary human tumors. Consistent with the proposed growth promoting role of PGE2, we found that NSCLC cell lines frequently co-expressed the genes encoding cyclooxygenase-2 and the prostaglandin E2 (PGE2) receptors EP1, 2 and 4 concomitant with the synthesis of PGE2. In contrast, NSCLC cells did not synthesize appreciable amounts of prostaglandin I2 (PGI2, prostacyclin), lacked PGI2 synthase (PGIS) and did not express the gene coding for the PGI2 receptor IP at detectable levels. In agreement with this finding, PGIS mRNA levels were dramatically diminished in primary human tumor samples as compared to matched normal lung tissue. Finally, thromboxane A2 (TxA2) was synthesized in NSCLC cell lines, but transcription of the gene coding for the TxA2 receptor TP was not observed in these cells. In marked contrast, lung fibroblasts synthesized all three prostanoids and their receptors at high levels. While the observed expression patterns were consistent with the existence of autocrine/paracrine PGE2 signaling loops in NSCLC cells, PGI2- and TxA2-mediated signals may play a role in tumor stroma cells.

Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment.

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3+ T cell/myeloma cell crosslinking, followed by CD4+/CD8+ T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA+ cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration.

Induction of PPARbeta and prostacyclin (PGI2) synthesis by Raf signaling: failure of PGI2 to activate PPARbeta.

A role for the nuclear receptor peroxisome proliferator-activated receptor-beta (PPARbeta) in oncogenesis has been suggested by a number of observations but its precise role remains elusive. Prostaglandin I2 (PGI2, prostacyclin), a major arachidonic acid (AA) derived cyclooxygenase (Cox) product, has been proposed as a PPARbeta agonist. Here, we show that the 4-hydroxytamoxifen (4-OHT) mediated activation of a C-Raf-estrogen receptor fusion protein leads to the induction of both the PPARbeta and Cox-2 genes, concomitant with a dramatic increase in PGI2 synthesis. Surprisingly, however, 4-OHT failed to activate PPARbeta transcriptional activity, indicating that PGI2 is insufficient for PPARbeta activation. In agreement with this conclusion, the overexpression of ectopic Cox-2 and PGI2 synthase (PGIS) resulted in massive PGI2 synthesis but did not activate the transcriptional activity of PPARbeta. Conversely, inhibition of PGIS blocked PGI2 synthesis but did not affect the AA mediated activation of PPARbeta. Our data obtained with four different cell types and different experimental strategies do not support the prevailing opinion that PGI2 plays a significant role in the regulation of PPARbeta.

Application of a MABEL Approach for a T-Cell-Bispecific Monoclonal Antibody: CEA TCB.

CEA TCB is a novel T-cell-bispecific (TCB) antibody targeting the carcinoembryonic antigen (CEA) expressed on tumor cells and the CD3 epsilon chain (CD3e) present on T cells, which is currently in Phase 1 clinical trials (NCT02324257) for the treatment of CEA-positive solid tumors. Because the human CEA (hCEA) binder of CEA TCB does not cross-react with cynomolgus monkey and CEA is absent in rodents, alternative nonclinical safety evaluation approaches were considered. These included the development of a cynomolgus monkey cross-reactive homologous (surrogate) antibody (cyCEA TCB) for its evaluation in cynomolgus monkey and the development of double-transgenic mice, expressing hCEA and human CD3e (hCEA/hCD3e Tg), as a potential alternative species for nonclinical safety studies. However, a battery of nonclinical in vitro/ex vivo experiments demonstrated that neither of the previous approaches provided a suitable and pharmacologically relevant model to assess the safety of CEA TCB. Therefore, an alternative approach, a minimum anticipated biological effect level (MABEL), based on an in vitro tumor lysis assay was used to determine the starting dose for the first-in-human study. Using the most conservative approach to the MABEL assessment, a dose of 52 µg was selected as a safe starting dose for clinical study.

In Vivo Fluorescence Imaging of the Activity of CEA TCB, a Novel T-Cell Bispecific Antibody, Reveals Highly Specific Tumor Targeting and Fast Induction of T-Cell-Mediated Tumor Killing.

PURPOSE: CEA TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody, engaging CD3ε upon binding to carcinoembryonic antigen (CEA) on tumor cells. Containing an engineered Fc region, conferring an extended blood half-life while preventing side effects due to activation of innate effector cells, CEA TCB potently induces tumor lysis in mouse tumors. Here we aimed to characterize the pharmacokinetic profile, the biodistribution, and the mode of action of CEA TCB by combining in vitro and in vivo fluorescence imaging readouts. EXPERIMENTAL DESIGN: CEA-expressing tumor cells (LS174T) and human peripheral blood mononuclear cells (PBMC) were cocultured in vitro or cografted into immunocompromised mice. Fluorescence reflectance imaging and intravital 2-photon (2P) microscopy were employed to analyze in vivo tumor targeting while in vitro confocal and intravital time-lapse imaging were used to assess the mode of action of CEA TCB. RESULTS: Fluorescence reflectance imaging revealed increased ratios of extravascular to vascular fluorescence signals in tumors after treatment with CEA TCB compared with control antibody, suggesting specific targeting, which was confirmed by intravital microscopy. Confocal and intravital 2P microscopy showed CEA TCB to accelerate T-cell-dependent tumor cell lysis by inducing a local increase of effector to tumor cell ratios and stable crosslinking of multiple T cells to individual tumor cells. CONCLUSIONS: Using optical imaging, we demonstrate specific tumor targeting and characterize the mode of CEA TCB-mediated target cell lysis in a mouse tumor model, which supports further clinical evaluation of CEA TCB. Clin Cancer Res; 22(17); 4417-27. ©2016 AACRSee related commentary by Teijeira et al., p. 4277.

A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors.

PURPOSE: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB. EXPERIMENTAL DESIGN: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells. RESULTS: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA-expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1-positive tumors. CONCLUSIONS: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286-97. ©2016 AACR.

Preclinical activity of the type II CD20 antibody GA101 (obinutuzumab) compared with rituximab and ofatumumab in vitro and in xenograft models.

We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation.

miR-155 inhibition sensitizes CD4+ Th cells for TREG mediated suppression.

BACKGROUND: In humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance. PRINCIPAL FINDINGS: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression. CONCLUSION: Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.