Stefan Evers

Bio: Stefan Evers is an academic researcher from Hoffmann-La Roche. The author has contributed to research in topics: Proteome & Fibroblast activation protein, alpha. The author has an hindex of 16, co-authored 41 publications receiving 2129 citations.

The Where, the When, and the How of Immune Monitoring for Cancer Immunotherapies in the Era of Checkpoint Inhibition.

Abstract: Clinical trials with immune checkpoint inhibitors have provided important insights into the mode of action of anticancer immune therapies and potential mechanisms of immune escape. Development of the next wave of rational clinical combination strategies will require a deep understanding of the mechanisms by which combination partners influence the battle between the immune system's capabilities to fight cancer and the immune-suppressive processes that promote tumor growth. This review focuses on our current understanding of tumor and circulating pharmacodynamic correlates of immune modulation and elaborates on lessons learned from human translational research with checkpoint inhibitors. Actionable tumor markers of immune activation including CD8(+)T cells, PD-L1 IHC as a pharmacodynamic marker of T-cell function, T-cell clonality, and challenges with conduct of trials that ask scientific questions from serial biopsies are addressed. Proposals for clinical trial design, as well as future applications of peripheral pharmacodynamic endpoints as potential surrogates of early clinical activity, are discussed. On the basis of emerging mechanisms of response and immune escape, we propose the concept of the tumor immunity continuum as a framework for developing rational combination strategies.

Mannose receptor-mediated regulation of serum glycoprotein homeostasis.

Abstract: Carbohydrates are thought to function as tags that mark circulatory glycoproteins for rapid clearance. To examine the role of the mannose receptor (MR) in glycoprotein clearance, we generated mice genetically deficient in MR. MR −/− mice were defective in clearing proteins bearing accessible mannose and N -acetylglucosamine residues and had elevated levels of eight different lysosomal hydrolases. Proteomic analysis of MR −/− and control mouse sera showed that an additional 4 out of 52 proteins identified were elevated in MR −/− serum. Each of these is up-regulated during inflammation and wound healing. Thus, MR appears to operate as an essential regulator of serum glycoprotein homeostasis.

Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines.

Abstract: We developed cergutuzumab amunaleukin (CEA-IL2v, RG7813), a novel monomeric CEA-targeted immunocytokine, that comprises a single IL-2 variant (IL2v) moiety with abolished CD25 binding, fused to the C-terminus of a high affinity, bivalent carcinoembryonic antigen (CEA)-specific antibody devoid of Fc-mediated effector functions. Its molecular design aims to (i) avoid preferential activation of regulatory T-cells vs. immune effector cells by removing CD25 binding; (ii) increase the therapeutic index of IL-2 therapy by (a) preferential retention at the tumor by having a lower dissociation rate from CEA-expressing cancer cells vs. IL-2R-expressing cells, (b) avoiding any FcγR-binding and Fc effector functions and (c) reduced binding to endothelial cells expressing CD25; and (iii) improve the pharmacokinetics, and thus convenience of administration, of IL-2. The crystal structure of the IL2v-IL-2Rβγ complex was determined and CEA-IL2v activity was assessed using human immune effector cells. Tumor targeting was investigated in tumor-bearing mice using 89Zr-labeled CEA-IL2v. Efficacy studies were performed in (a) syngeneic mouse models as monotherapy and combined with anti-PD-L1, and in (b) xenograft mouse models in combination with ADCC-mediating antibodies. CEA-IL2v binds to CEA with pM avidity but not to CD25, and consequently did not preferentially activate Tregs. In vivo, CEA-IL2v demonstrated superior pharmacokinetics and tumor targeting compared with a wild-type IL-2-based CEA immunocytokine (CEA-IL2wt). CEA-IL2v strongly expanded NK and CD8+ T cells, skewing the CD8+:CD4+ ratio toward CD8+ T cells both in the periphery and in the tumor, and mediated single agent efficacy in syngeneic MC38-CEA and PancO2-CEA models. Combination with trastuzumab, cetuximab and imgatuzumab, all of human IgG1 isotype, resulted in superior efficacy compared with the monotherapies alone. Combined with anti-PD-L1, CEA-IL2v mediated superior efficacy over the respective monotherapies, and over the combination with an untargeted control immunocytokine. These preclinical data support the ongoing clinical investigation of the cergutuzumab amunaleukin immunocytokine with abolished CD25 binding for the treatment of CEA-positive solid tumors in combination with PD-L1 checkpoint blockade and ADCC competent antibodies.

Gene Expression Changes Triggered by Exposure of Haemophilus influenzae to Novobiocin or Ciprofloxacin: Combined Transcription and Translation Analysis

Abstract: The responses of Haemophilus influenzae to DNA gyrase inhibitors were analyzed at the transcriptional and the translational level. High-density microarrays based on the genomic sequence were used to monitor the expression levels of >80% of the genes in this bacterium. In parallel the proteins were analyzed by two-dimensional electrophoresis. DNA gyrase inhibitors of two different functional classes were used. Novobiocin, as a representative of one class, inhibits the ATPase activity of the enzyme, thereby indirectly changing the degree of DNA supercoiling. Ciprofloxacin, a representative of the second class, obstructs supercoiling by inhibiting the DNA cleavage-resealing reaction. Our results clearly show that different responses can be observed. Treatment with the ATPase inhibitor Novobiocin changed the expression rates of many genes, reflecting the fact that the initiation of transcription for many genes is sensitive to DNA supercoiling. Ciprofloxacin mainly stimulated the expression of DNA repair systems as a response to the DNA damage caused by the stable ternary complexes. In addition, changed expression levels were also observed for some genes coding for proteins either annotated as “unknown function” or “hypothetical” or for proteins not directly involved in DNA topology or repair. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. {"type":"entrez-nucleotide","attrs":{"text":"AJ297131","term_id":"10880347","term_text":"AJ297131"}}AJ297131 and {"type":"entrez-nucleotide","attrs":{"text":"AL135960","term_id":"6635875","term_text":"AL135960"}}AL135960.]

Two-dimensional map of the proteome of Haemophilus influenzae.

Abstract: We have constructed a two-dimensional database of the proteome of Haemophilus influenzae, a bacterium of medical interest of which the complete genome, comprising about 1742 open reading frames, has been sequenced. The soluble protein fraction of the microorganism was analyzed by two-dimensional electrophoresis, using immobilized pH gradient strips of various pH regions, gels with different acrylamide concentrations and buffers with different trailing ions. In order to visualize low-copy-number gene products, we employed a series of protein extraction and sample application approaches and several chromatographic steps, including heparin chromatography, chromatofocusing and hydrophobic interaction chromatography. We have also analyzed the cell envelope-bound protein fraction using either immobilized pH gradient strips or a two-detergent system with a cationic detergent in the first and an anionic detergent in the second-dimensional separation. Different proteins (502) were identified by matrix-assisted laser desorption/ionization mass spectrometry and amino acid composition analysis. This is at present one of the largest two-dimensional proteome databases.

Alternative activation of macrophages

Abstract: The classical pathway of interferon-gamma-dependent activation of macrophages by T helper 1 (T(H)1)-type responses is a well-established feature of cellular immunity to infection with intracellular pathogens, such as Mycobacterium tuberculosis and HIV. The concept of an alternative pathway of macrophage activation by the T(H)2-type cytokines interleukin-4 (IL-4) and IL-13 has gained credence in the past decade, to account for a distinctive macrophage phenotype that is consistent with a different role in humoral immunity and repair. In this review, I assess the evidence in favour of alternative macrophage activation in the light of macrophage heterogeneity, and define its limits and relevance to a range of immune and inflammatory conditions.

Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

Abstract: We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

Elements of cancer immunity and the cancer–immune set point

Abstract: Immunotherapy is proving to be an effective therapeutic approach in a variety of cancers. But despite the clinical success of antibodies against the immune regulators CTLA4 and PD-L1/PD-1, only a subset of people exhibit durable responses, suggesting that a broader view of cancer immunity is required. Immunity is influenced by a complex set of tumour, host and environmental factors that govern the strength and timing of the anticancer response. Clinical studies are beginning to define these factors as immune profiles that can predict responses to immunotherapy. In the context of the cancer-immunity cycle, such factors combine to represent the inherent immunological status - or 'cancer-immune set point' - of an individual.

Tolerogenic dendritic cells.

Abstract: Dendritic cells (DCs) have several functions in innate and adaptive immunity. In addition, there is increasing evidence that DCs in situ induce antigen-specific unresponsiveness or tolerance in central lymphoid organs and in the periphery. In the thymus DCs generate tolerance by deleting self-reactive T cells. In peripheral lymphoid organs DCs also induce tolerance to antigens captured by receptors that mediate efficient uptake of proteins and dying cells. Uptake by these receptors leads to the constitutive presentation of antigens on major histocompatibility complex (MHC) class I and II products. In the steady state the targeting of DC antigen capture receptors with low doses of antigens leads to deletion of the corresponding T cells and unresponsiveness to antigenic rechallenge with strong adjuvants. In contrast, if a stimulus for DC maturation is coadministered with the antigen, the mice develop immunity, including interferon-gamma-secreting effector T cells and memory T cells. There is also new evidence that DCs can contribute to the expansion and differentiation of T cells that regulate or suppress other immune T cells. One possibility is that distinct developmental stages and subsets of DCs and T cells can account for the different pathways to peripheral tolerance, such as deletion or suppression. We suggest that several clinical situations, including autoimmunity and certain infectious diseases, can be influenced by the antigen-specific tolerogenic role of DCs.

TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells.

Abstract: Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor β (TGFβ) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFβ-blocking and anti-PD-L1 antibodies reduced TGFβ signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFβ shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.