Publications by authors named "Christian Klein"

346 Publications

The spectrum between substrates and inhibitors: Pinpointing the binding mode of dengue protease ligands with modulated basicity and hydrophobicity.

Bioorg Med Chem 2021 Oct 17;48:116412. Epub 2021 Sep 17.

Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology IPMB, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany. Electronic address:

Peptides can be inhibitors and substrates of proteases. The present study describes the inhibitor- vs. substrate-like properties of peptidic ligands of dengue protease which were designed to provide insight into their binding modes. Of particular interest was the localization of the cleavable peptide bond and the placement of hydrophobic elements in the binding site. The findings provide clues for the design of covalent inhibitors in which electrophilic functional groups bind to the catalytic serine, and in addition for the development of inhibitors that are less basic than the natural substrate and therefore have an improved pharmacokinetic profile. We observed a tendency of basic elements to favor a substrate-like binding mode, whereas hydrophobic elements decrease or eliminate enzymatic cleavage. This indicates a necessity to include basic elements which closely mimic the natural substrates into covalent inhibitors, posing a challenge from the chemical and pharmacokinetic perspective. However, hydrophobic elements may offer opportunities to develop non-covalent inhibitors with a favorable ADME profile and potentially improved target-binding kinetics.
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http://dx.doi.org/10.1016/j.bmc.2021.116412DOI Listing
October 2021

Early Everolimus-Facilitated Reduced Tacrolimus in Liver Transplantation: Results From the Randomized HEPHAISTOS Trial.

Liver Transpl 2021 Sep 15. Epub 2021 Sep 15.

Novartis Pharma GmbH, Nürnberg, Germany.

Everolimus-facilitated reduced-exposure tacrolimus (EVR + rTAC) at 30 days after liver transplantation (LT) has shown advantages in renal preservation. This study evaluated the effects of early initiation of EVR + rTAC in de novo LT recipients (LTRs). In HEPHAISTOS (NCT01551212, EudraCT 2011-003118-17), a 12-month, multicenter, controlled study, LTRs were randomly assigned at 7 to 21 days after LT to receive EVR + rTAC or standard-exposure tacrolimus (sTAC) with steroids. The primary objective was to demonstrate superior renal function (assessed by estimated glomerular filtration rate [eGFR]) with EVR + rTAC versus sTAC at month 12 in the full analysis set (FAS). Other assessments at month 12 included the evaluation of renal function in compliance set and on-treatment (OT) patients, efficacy (composite endpoint of graft loss, death, or treated biopsy-proven acute rejection [tBPAR] and individual components) in FAS, and safety. In total, 333 patients (EVR + rTAC, 169; sTAC, 164) were included in the FAS. A high proportion of patients was nonadherent in maintaining tacrolimus trough levels (EVR + rTAC, 36.1%; sTAC, 34.7%). At month 12, the adjusted least square mean eGFR was numerically higher with EVR + rTAC versus sTAC (76.2 versus 72.1 mL/minute/1.73 m , difference: 4.1 mL/minute/1.73 m ; P = 0.097). A significant difference of 8.3 mL/minute/1.73 m (P = 0.03) favoring EVR + rTAC was noted in the compliance set. Incidence of composite efficacy endpoint (7.7% versus 7.9%) and tBPAR (7.1% versus 5.5%) at month 12 as well as incidence of treatment-emergent adverse events (AEs) and serious AEs were comparable between groups. A lower proportion of patients discontinued EVR + rTAC than sTAC treatment (27.2% versus 34.1%). Early use of everolimus in combination with rTAC showed comparable efficacy, safety, and well-preserved renal function versus sTAC therapy at month 12. Of note, renal function was significantly enhanced in the compliance set.
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http://dx.doi.org/10.1002/lt.26298DOI Listing
September 2021

Light-Inducible Spatio-Temporal Control of TLR4 and NF-κB-Gluc Reporter in Human Pancreatic Cell Line.

Int J Mol Sci 2021 Aug 26;22(17). Epub 2021 Aug 26.

Department of Medical and Pharmaceutical Biotechnology, IMC University of Applied Sciences, 3500 Krems, Austria.

Augmented Toll-like receptor 4 (TLR4) expression was found in nearly 70% of patients with pancreatic adenocarcinoma, which is correlated with increased tumorigenesis and progression. In this study, we engineered a new light-oxygen-voltage-sensing (LOV) domain-based optogenetic cell line (opto-TLR4 PANC-1) that enables time-resolved activation of the NF-κB and extracellular-signal regulated kinases (ERK)1/2 signalling pathway upon blue light-sensitive homodimerisation of the TLR4-LOV fusion protein. Continuous stimulation with light indicated strong p65 and ERK1/2 phosphorylation even after 24 h, whereas brief light exposure peaked at 8 h and reached the ground level 24 h post-illumination. The cell line further allows a voltage-dependent TLR4 activation, which can be continuously monitored, turned on by light or off in the dark. Using this cell line, we performed different phenotypic cell-based assays with 2D and 3D cultures, with the aim of controlling cellular activity with spatial and temporal precision. Light exposure enhanced cell attachment, the formation and extension of invadopodia, and cell migration in 3D spheroid cultures, but no significant changes in proliferation or viability could be detected. We conclude that the opto-TLR4 PANC-1 cell line is an ideal tool for investigating the underlying molecular mechanisms of TLR4, thereby providing strategies for new therapeutic options.
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http://dx.doi.org/10.3390/ijms22179232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8431472PMC
August 2021

Ten years in the making: application of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins.

MAbs 2021 Jan-Dec;13(1):1967714

Roche Innovation Center Zurich, Schlieren, Switzerland.

Bispecific antibodies have recently attracted intense interest. CrossMab technology was described in 2011 as novel approach enabling correct antibody light-chain association with their respective heavy chain in bispecific antibodies, together with methods enabling correct heavy-chain association using existing pairs of antibodies. Since the original description, CrossMab technology has evolved in the past decade into one of the most mature, versatile, and broadly applied technologies in the field, and nearly 20 bispecific antibodies based on CrossMab technology developed by Roche and others have entered clinical trials. The most advanced of these are the Ang-2/VEGF bispecific antibody faricimab, currently undergoing regulatory review, and the CD20/CD3 T cell bispecific antibody glofitamab, currently in pivotal Phase 3 trials. In this review, we introduce the principles of CrossMab technology, including its application for the generation of bi-/multispecific antibodies with different geometries and mechanisms of action, and provide an overview of CrossMab-based therapeutics in clinical trials.
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http://dx.doi.org/10.1080/19420862.2021.1967714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425689PMC
October 2021

Inhibitor potency and assay conditions: A case study on SARS-CoV-2 main protease.

Proc Natl Acad Sci U S A 2021 09;118(36)

Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, 69120 Heidelberg, Germany

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http://dx.doi.org/10.1073/pnas.2106095118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8433543PMC
September 2021

Targeting of fibroblast activation protein in rheumatoid arthritis patients: imaging and ex vivo photodynamic therapy.

Rheumatology (Oxford) 2021 Aug 27. Epub 2021 Aug 27.

Department of Experimental Rheumatology, Radboudumc, Nijmegen, The Netherlands.

Objective: Activated synovial fibroblasts are key effector cells in rheumatoid arthritis (RA). Selectively depleting these based upon their expression of fibroblast activation protein (FAP) is an attractive therapeutic approach. Here we introduce FAP imaging of inflamed joints using [68Ga]Ga-FAPI-04 in an RA patient, and aim to assess feasibility of anti-FAP targeted photodynamic therapy (FAP-tPDT) ex vivo using 28H1-IRDye700DX on RA synovial explants.

Methods: Remnant synovial tissue from RA patients was processed into 6 mm biopsies and, from several patients, into primary fibroblast cell cultures. Both were treated using FAP-tPDT. Cell viability was measured in fibroblast cultures and biopsies were evaluated for histological markers of cell damage. Selectivity of the effect of FAP-tPDT was assessed using flowcytometry on primary fibroblasts and co-cultured macrophages. Additionally, one RA patient intravenously received [68Ga]Ga-FAPI-04 and was scanned using PET/CT imaging.

Results: In the RA patient,FAPI-04 PET imaging showed high accumulation of the tracer in arthritic joints with very low background signal. In vitro, FAP-tPDT induced cell death in primary RA synovial fibroblasts in a light dose dependent manner. An upregulation of cell damage markers was observed in the synovial biopsies after FAP-tPDT. No significant effects of FAP-tPDT were noted on macrophages after FAP-tPDT of neighbouring fibroblasts.

Conclusion: In this study the feasibility of selective FAP-tPDT in synovium of rheumatoid arthritis patients ex vivo is demonstrated. Furthermore, this study provides the first indication that FAP-targeted PET/CT can be used to image arthritic joints, an important step towards application of FAP-tPDT as a targeted locoregional therapy for RA.
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http://dx.doi.org/10.1093/rheumatology/keab664DOI Listing
August 2021

Collision with opponents-but not foul play-dominates injury mechanism in professional men's basketball.

BMC Sports Sci Med Rehabil 2021 Aug 19;13(1):94. Epub 2021 Aug 19.

Department of Trauma, Hand, Plastic and Reconstructive Surgery, University Medical Center Würzburg, Würzburg, Germany.

Background: To identify injury patterns and mechanisms in professional men's basketball by means of video match analysis.

Methods: In Germany, injuries are registered with the statutory accident insurance for professional athletes (VBG) by clubs or club physicians as part of occupational accident reporting. Moderate and severe injuries (absence of > 7 days) sustained during basketball competition in one of four seasons (2014-2017 and 2018-2019) in the first or second national men's league in Germany were prospectively analyzed using a newly developed standardized observation form. Season 2017-2018 was excluded because of missing video material.

Results: Video analysis included 175 (53%) of 329 moderate and severe match injuries. Contact patterns categorized according to the different body sites yielded eight groups of typical injury patterns: one each for the head, shoulders, and ankles, two for the thighs, and three for the knees. Injuries to the head (92%), ankles (76%), shoulders (70%), knees (47%), and thighs (32%) were mainly caused by direct contact. The injury proportion of foul play was 19%. Most injuries (61%) occurred in the central zone below the basket. More injuries occurred during the second (OR 1.8, p = 0.018) and fourth quarter (OR 1.8, p = 0.022) than during the first and third quarter of the match.

Conclusion: The eight identified injury patterns differed substantially in their mechanisms. Moderate and severe match injuries to the head, shoulders, knees, and ankles were mainly caused by collision with opponents and teammates. Thus, stricter rule enforcement is unlikely to facilitate safer match play.
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http://dx.doi.org/10.1186/s13102-021-00322-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8375128PMC
August 2021

Head injuries in professional football (soccer): Results of video analysis verified by an accident insurance registry.

PLoS One 2021 11;16(8):e0255695. Epub 2021 Aug 11.

Department of Trauma Surgery, University Medical Centre Regensburg, Regensburg, Germany.

Background: Video analysis is one of the most commonly applied methods for analysing football injuries.

Purpose: The objective of this study was to assess the accuracy of video analysis for recording head injuries in professional football from official matches in the four highest men's professional football leagues in Germany.

Methods: In this cohort study, head injuries detected by means of video analysis of all official matches over one season (2017-18) were compared to head injuries registered with the German statutory accident insurance.

Results: Our video analysis yielded 359 head injuries of 287 players. The comparison of head injuries found in our video analysis to those registered with the accident insurance only yielded a match in 23.1% (n = 83), which presents a rather low verification rate. The verification rates varied between the leagues (7.0-30.8%). All injuries documented in the accident insurance registry were found in the video analysis (100%). The types of head injury most often verified by the accident insurance registry (n = 83) were contusion (43.4%), bone fractures (19.3%) and skin lacerations (18.1%). Only 66 of the 359 head injuries (18.4%) resulted in absence from at least one training session and involved a mean time loss of 18.5 days (1-87 days).

Conclusion: The mismatch between the number of head injuries found in the video analysis and head injuries registered with the accident insurance is an important methodological issue in scientific research. The low verification rate seems to be due to the unclear correlation between injury severity and clinical consequences of head injuries detected by means of video analysis and the failure of football clubs to register minor head injuries with the accident insurance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0255695PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357092PMC
August 2021

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

Elife 2021 08 11;10. Epub 2021 Aug 11.

Roche Pharma Research & Early Development, Roche Innovation Center Zurich, Schlieren, Switzerland.

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.
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http://dx.doi.org/10.7554/eLife.67106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373379PMC
August 2021

Skin-Friction-Based Identification of the Critical Lines in a Transonic, High Reynolds Number Flow via Temperature-Sensitive Paint.

Sensors (Basel) 2021 Jul 28;21(15). Epub 2021 Jul 28.

National Research Council (CNR), Institute of Marine Engineering, Via di Vallerano 139, I-00128 Rome, Italy.

In this contribution, three methodologies based on temperature-sensitive paint (TSP) data were further developed and applied for the optical determination of the critical locations of flow separation and reattachment in compressible, high Reynolds number flows. The methodologies rely on skin-friction extraction approaches developed for low-speed flows, which were adapted in this work to study flow separation and reattachment in the presence of shock-wave/boundary-layer interaction. In a first approach, skin-friction topological maps were obtained from time-averaged surface temperature distributions, thus enabling the identification of the critical lines as converging and diverging skin-friction lines. In the other two approaches, the critical lines were identified from the maps of the propagation celerity of temperature perturbations, which were determined from time-resolved TSP data. The experiments were conducted at a freestream Mach number of 0.72 and a chord Reynolds number of 9.7 million in the Transonic Wind Tunnel Göttingen on a VA-2 supercritical airfoil model, which was equipped with two exchangeable TSP modules specifically designed for transonic, high Reynolds number tests. The separation and reattachment lines identified via the three different TSP-based approaches were shown to be in mutual agreement, and were also found to be in agreement with reference experimental and numerical data.
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http://dx.doi.org/10.3390/s21155106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8348098PMC
July 2021

Src/lck inhibitor dasatinib reversibly switches off cytokine release and T cell cytotoxicity following stimulation with T cell bispecific antibodies.

J Immunother Cancer 2021 07;9(7)

Roche Pharma Research and Early Development, Roche Innovation Centre Zurich, Schlieren, Switzerland.

Background: T cell engagers are bispecific antibodies recognizing, with one moiety, the CD3ε chain of the T cell receptor and, with the other moiety, specific tumor surface antigens. Crosslinking of CD3 upon simultaneous binding to tumor antigens triggers T cell activation, proliferation and cytokine release, leading to tumor cell killing. Treatment with T cell engagers can be associated with safety liabilities due to on-target on-tumor, on-target off-tumor cytotoxic activity and cytokine release syndrome (CRS). Tyrosine kinases such as SRC, LCK or ZAP70 are involved in downstream signaling pathways after engagement of the T cell receptor and blocking these kinases might serve to abrogate T cell activation when required (online supplemental material 1). Dasatinib was previously identified as a potent kinase inhibitor that switches off CAR T cell functionality.

Methods: Using an in vitro model of target cell killing by human peripheral blood mononuclear cells, we assessed the effects of dasatinib combined with 2+1 T cell bispecific antibodies (TCBs) including CEA-TCB, CD19-TCB or HLA-A2 WT1-TCB on T cell activation, proliferation and target cell killing measured by flow cytometry and cytokine release measured by Luminex. To determine the effective dose of dasatinib, the Incucyte system was used to monitor the kinetics of TCB-mediated target cell killing in the presence of escalating concentrations of dasatinib. Last, the effects of dasatinib were evaluated in vivo in humanized NSG mice co-treated with CD19-TCB. The count of CD20 blood B cells was used as a readout of efficacy of TCB-mediated killing and cytokine levels were measured in the serum.

Results: Dasatinib concentrations above 50 nM prevented cytokine release and switched off-target cell killing, which were subsequently restored on removal of dasatinib. In addition, dasatinib prevented CD19-TCB-mediated B cell depletion in humanized NSG mice. These data confirm that dasatinib can act as a rapid and reversible on/off switch for activated T cells at pharmacologically relevant doses as they are applied in patients according to the label.

Conclusion: Taken together, we provide evidence for the use of dasatinib as a pharmacological on/off switch to mitigate off-tumor toxicities or CRS by T cell bispecific antibodies.
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http://dx.doi.org/10.1136/jitc-2021-002582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8323395PMC
July 2021

Prognostic significance of FCGR2B expression for the response of DLBCL patients to rituximab or obinutuzumab treatment.

Blood Adv 2021 08;5(15):2945-2957

School of Cancer Sciences, University of Southampton, Southampton, United Kingdom.

Fc γ receptor IIB (FcγRIIB) is an inhibitory molecule capable of reducing antibody immunotherapy efficacy. We hypothesized its expression could confer resistance in patients with diffuse large B-cell lymphoma (DLBCL) treated with anti-CD20 monoclonal antibody (mAb) chemoimmunotherapy, with outcomes varying depending on mAb (rituximab [R]/obinutuzumab [G]) because of different mechanisms of action. We evaluated correlates between FCGR2B messenger RNA and/or FcγRIIB protein expression and outcomes in 3 de novo DLBCL discovery cohorts treated with R plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) reported by Arthur, Schmitz, and Reddy, and R-CHOP/G-CHOP-treated patients in the GOYA trial (NCT01287741). In the discovery cohorts, higher FCGR2B expression was associated with significantly shorter progression-free survival (PFS; Arthur: hazard ratio [HR], 1.09; 95% confidence interval [CI], 1.01-1.19; P = .0360; Schmitz: HR, 1.13; 95% CI, 1.02-1.26; P = .0243). Similar results were observed in GOYA with R-CHOP (HR, 1.26; 95% CI, 1.00-1.58; P = .0455), but not G-CHOP (HR, 0.91; 95% CI, 0.69-1.20; P = .50). A nonsignificant trend that high FCGR2B expression favored G-CHOP over R-CHOP was observed (HR, 0.67; 95% CI, 0.44-1.02; P = .0622); however, low FCGR2B expression favored R-CHOP (HR, 1.58; 95% CI, 1.00-2.50; P = .0503). In Arthur and GOYA, FCGR2B expression was associated with tumor FcγRIIB expression; correlating with shorter PFS for R-CHOP (HR, 2.17; 95% CI, 1.04-4.50; P = .0378), but not G-CHOP (HR, 1.37; 95% CI, 0.66-2.87; P = .3997). This effect was independent of established prognostic biomarkers. High FcγRIIB/FCGR2B expression has prognostic value in R-treated patients with DLBCL and may confer differential responsiveness to R-CHOP/G-CHOP.
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http://dx.doi.org/10.1182/bloodadvances.2021004770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361458PMC
August 2021

Single-nucleotide Fcγ receptor polymorphisms do not impact obinutuzumab/rituximab outcome in patients with lymphoma.

Blood Adv 2021 08;5(15):2935-2944

School of Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom.

Single-nucleotide polymorphisms (SNPs) have been shown to influence Fcγ receptor (FcγR) affinity and activity, but their effect on treatment response is unclear. We assessed their importance in the efficacy of obinutuzumab or rituximab combined with chemotherapy in untreated advanced follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) in the GALLIUM (www.clinicaltrials.gov #NCT01332968) and GOYA (#NCT01287741) trials, respectively. Genomic DNA was extracted from patients enrolled in GALLIUM (n = 1202) and GOYA (n = 1418). Key germline SNPs, FCGR2A R131H (rs1801274), FCGR3A F158V (rs396991), and FCGR2B I232T (rs1050501), were genotyped and assessed for their impact on investigator-assessed progression-free survival (PFS). In both cohorts there was no prognostic effect of FCGR2A or FCGR3A. In FL, FCGR2B was associated with favorable PFS in univariate and multivariate analyses comparing I232T with I232I, with a more modest association for rituximab-treated (univariate: hazard ratio [HR], 0.78; 95% confidence interval [CI], 0.54-1.14; P = .21) vs obinutuzumab-treated patients (HR, 0.56; 95% CI, 0.34-0.91; P = .02). Comparing T232T with I232I, an association was found for obinutuzumab (univariate: HR, 2.76; 95% CI, 1.02-7.5; P = .0459). Neither observation retained significance after multiple-test adjustment. FCGR2B was associated with poorer PFS in multivariate analyses comparing T232T with I232I in rituximab- but not obinutuzumab-treated patients with DLBCL (HR, 4.40; 95% CI, 1.71-11.32; P = .002; multiple-test-adjusted P = .03); however, this genotype was rare (n = 13). This study shows that FcγR genotype is not associated with response to rituximab/obinutuzumab plus chemotherapy in treatment-naive patients with advanced FL or DLBCL.
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http://dx.doi.org/10.1182/bloodadvances.2020003985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361457PMC
August 2021

Raman Imaging Reveals Accumulation of Hemoproteins in Plaques from Alzheimer's Diseased Tissues.

ACS Chem Neurosci 2021 08 22;12(15):2940-2945. Epub 2021 Jul 22.

Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, Chimie De La Matière Complexe, Université de Strasbourg-CNRS, 4 Rue Blaise Pascal, 67081 Strasbourg, France.

Hemes have been suggested to play a central role in Alzheimer's disease since they show high peroxidase reactivity when bound to amyloid β peptides, leading to the production of reactive oxygen species. Here we used Fourier transform infrared and Raman imaging on Alzheimer's diseased mice and human brain tissue. Our finding suggests the accumulation of hemes in the senile plaques of both murine and human samples. We compared the Raman signature of the plaques to the ones of various hemeoproteins and to the hemin-Aβ-42 complex. The detected Raman signature of the plaques does not allow identifying the type of heme accumulating in the plaques.
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http://dx.doi.org/10.1021/acschemneuro.1c00289DOI Listing
August 2021

Targeting intracellular WT1 in AML with a novel RMF-peptide-MHC specific T-cell bispecific antibody.

Blood 2021 Jul 19. Epub 2021 Jul 19.

German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.

Antibody-based immunotherapy is a promising strategy for targeting chemo-resistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell-surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated using CrossMab and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) in the context of human leukocyte antigen (HLA) A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary AML cells was mediated in ex vivo long-term co-cultures utilizing allogenic (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18) or autologous, patient-derived T cells (mean specific lysis: 54±12% after 11-14 days; ±SEM; n=8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 45.4±9.0% vs 70.8±8.3%; p=0.015; ±SEM; n=9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase I trial in patients with r/r AML (NCT04580121).
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http://dx.doi.org/10.1182/blood.2020010477DOI Listing
July 2021

Proteolysis-Targeting Chimeras Enhance T Cell Bispecific Antibody-Driven T Cell Activation and Effector Function through Increased MHC Class I Antigen Presentation in Cancer Cells.

J Immunol 2021 07 2;207(2):493-504. Epub 2021 Jul 2.

Molecular Targeted Therapy-Discovery Oncology, Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland;

The availability of Ags on the surface of tumor cells is crucial for the efficacy of cancer immunotherapeutic approaches using large molecules, such as T cell bispecific Abs (TCBs). Tumor Ags are processed through intracellular proteasomal protein degradation and are displayed as peptides on MHC class I (MHC I). Ag recognition through TCRs on the surface of CD8 T cells can elicit a tumor-selective immune response. In this article, we show that proteolysis-targeting chimeras (PROTACs) that target bromo- and extraterminal domain proteins increase the abundance of the corresponding target-derived peptide Ags on MHC I in both liquid and solid tumor-derived human cell lines. This increase depends on the engagement of the E3 ligase to bromo- and extraterminal domain protein. Similarly, targeting of a doxycycline-inducible Wilms tumor 1 (WT1)-FKBP12 fusion protein, by a mutant-selective FKBP12 degrader, increases the presentation of WT1 Ags in human breast cancer cells. T cell-mediated response directed against cancer cells was tested on treatment with a TCR-like TCB, which was able to bridge human T cells to a WT1 peptide displayed on MHC I. FKBP12 degrader treatment increased the expression of early and late activation markers (CD69, CD25) in T cells; the secretion of granzyme β, IFN-γ, and TNF-α; and cancer cell killing in a tumor-T cell coculture model. This study supports harnessing targeted protein degradation in tumor cells, for modulation of T cell effector function, by investigating for the first time, to our knowledge, the potential of combining a degrader and a TCB in a cancer immunotherapy setting.
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http://dx.doi.org/10.4049/jimmunol.2000252DOI Listing
July 2021

Co-Stimulatory versus Cell Death Aspects of Agonistic CD40 Monoclonal Antibody Selicrelumab in Chronic Lymphocytic Leukemia.

Cancers (Basel) 2021 Jun 21;13(12). Epub 2021 Jun 21.

Department of Experimental Immunology, Amsterdam University Medical Center, 1105 AZ Amsterdam, The Netherlands.

Objectives: Chronic lymphocytic leukemia (CLL) is a common form of leukemia with a heterogeneous clinical course that remains incurable due to the development of therapy resistance. In lymph node proliferation centers, signals from the microenvironment such as CD40 ligation through interaction with follicular T helper cells shield CLL cells from apoptosis. Previous observations have shown that, despite CD40-induced changes in apoptotic mediators resulting in cell survival, CD40 activation also increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. To further investigate these observations, we here studied the activity of the fully human agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile, and sensitization for cell death by aCD20 mAbs, in vitro.

Methods: CLL cells from peripheral blood were isolated by the Ficoll density method. The expression of activation markers and cytokine production following CD40 stimulation was quantified by flow cytometry and ELISA. The anti-apoptotic profile of CLL induced by stimulation was evaluated by the expression of BCL-2 proteins with Western blot, and resistance to venetoclax with flow cytometry. Cell death induced by the combination of selicrelumab and aCD20 mAbs was quantified by flow cytometry.

Results: CLL cells treated with selicrelumab upregulated co-stimulatory molecules such as CD86, TNF-α and death receptor CD95/Fas. In contrast to the CD40 ligand-transfected NIH3T3 cells, induction of resistance to venetoclax by selicrelumab was very moderate. Importantly, selicrelumab stimulation positively sensitized CLL cells to CD20-induced cell death, comparable to CD40 ligand-transfected NIH3T3 cells.

Conclusions: Taken together, these novel insights into selicrelumab-stimulatory effects in CLL may be considered for developing new therapeutic strategies, particularly in combination with obinutuzumab.
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http://dx.doi.org/10.3390/cancers13123084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234015PMC
June 2021

A Role for Xanthurenic Acid in the Control of Brain Dopaminergic Activity.

Int J Mol Sci 2021 Jun 28;22(13). Epub 2021 Jun 28.

Biopathologie de la Myéline, Neuroprotection et Stratégies Thérapeutiques, INSERM U1119, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, CRBS 1, Rue Eugène Boeckel, 67000 Strasbourg, France.

Xanthurenic acid (XA) is a metabolite of the kynurenine pathway (KP) synthetized in the brain from dietary or microbial tryptophan that crosses the blood-brain barrier through carrier-mediated transport. XA and kynurenic acid (KYNA) are two structurally related compounds of KP occurring at micromolar concentrations in the CNS and suspected to modulate some pathophysiological mechanisms of neuropsychiatric and/or neurodegenerative diseases. Particularly, various data including XA cerebral distribution (from 1 µM in olfactory bulbs and cerebellum to 0.1-0.4 µM in A and A), its release, and interactions with G protein-dependent XA-receptor, glutamate transporter and metabotropic receptors, strongly support a signaling and/or neuromodulatory role for XA. However, while the parent molecule KYNA is considered as potentially involved in neuropsychiatric disorders because of its inhibitory action on dopamine release in the striatum, the effect of XA on brain dopaminergic activity remains unknown. Here, we demonstrate that acute local/microdialysis-infusions of XA dose-dependently stimulate dopamine release in the rat prefrontal cortex (four-fold increase in the presence of 20 µM XA). This stimulatory effect is blocked by XA-receptor antagonist NCS-486. Interestingly, our results show that the peripheral/intraperitoneal administration of XA, which has been proven to enhance intra-cerebral XA concentrations (about 200% increase after 50 mg/kg XA i.p), also induces a dose-dependent increase of dopamine release in the cortex and striatum. Furthermore, our in vivo electrophysiological studies reveal that the repeated/daily administrations of XA reduce by 43% the number of spontaneously firing dopaminergic neurons in the ventral tegmental area. In the substantia nigra, XA treatment does not change the number of firing neurons. Altogether, our results suggest that XA may contribute together with KYNA to generate a KYNA/XA ratio that may crucially determine the brain normal dopaminergic activity. Imbalance of this ratio may result in dopaminergic dysfunctions related to several brain disorders, including psychotic diseases and drug dependence.
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http://dx.doi.org/10.3390/ijms22136974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268472PMC
June 2021

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay.

SLAS Discov 2021 10 19;26(9):1189-1199. Epub 2021 Jun 19.

Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology IPMB, Heidelberg University, Heidelberg, Germany.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (M). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 M. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance and characterized Förster resonance energy transfer (FRET) substrates with a preference for 2-Abz/Tyr(3-NO) FRET pairs. The substrates 2-AbzSAVLQSGTyr(3-NO)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and 2-AbzVVTLQSGTyr(3-NO)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at position P5 improved the catalytic efficiency. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.
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http://dx.doi.org/10.1177/24725552211020681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8458682PMC
October 2021

Diversity-oriented synthesis of peptide-boronic acids by a versatile building-block approach.

Chem Sci 2020 Aug 21;11(36):9898-9903. Epub 2020 Aug 21.

Department of Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University Im Neuenheimer Feld 364 69120 Heidelberg Germany

A new strategy for the synthesis of peptide-boronic acids (PBAs) is presented. 20 Fmoc-protected natural amino acids with orthogonal side-chain protection were straightforwardly converted into their corresponding boron analogues in three simple steps. Subsequent immobilisation on commercially available 1-glycerol polystyrene resin and on-resin transformations yielded a diversity of sequences in high purity. The strategy eliminates various synthetic obstacles such as multi-step routes, low yields, and inseparable impurities. The described method comprises great potential to be implemented in automated combinatorial approaches by markedly facilitating the access to a variety of PBAs. The coupling of amino acids or other building blocks with α-aminoboronates allows the creation of hybrid molecules with significant potential in various scientific disciplines, such as medicinal chemistry, structural biology, and materials science.
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http://dx.doi.org/10.1039/d0sc03999cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8162117PMC
August 2020

A quantitative assessment of silicone and PTFE-based stamp techniques for restoring occlusal anatomy using resin-based composites.

Clin Oral Investig 2021 May 28. Epub 2021 May 28.

Department of Conservative Dentistry, Periodontology and Endodontology, University Centre of Dentistry, Oral Medicine and Maxillofacial Surgery, University Hospital Tübingen, 72076, Tübingen, Germany.

Objectives: Publications on stamp techniques for placing resin-based composite (RBC) restorations consist mainly of case studies. Furthermore, comparative studies are rare and no longer relevant to the materials tested today. Thus, two general techniques were investigated in this study.

Materials And Methods: Standardized occlusion class I cavities were prepared in twenty-eight extracted caries-free wisdom teeth with unimpaired occlusal surfaces and restored with the RBC material Grandio. Light curing of the final layer was performed either after removal of the stamp isolated with PTFE tape or by leaving a stamp made of transparent polysiloxane in place. CEREC scans of the RBC restorations placed (follow-up) were superimposed on scans of the unimpaired occlusal surface (baseline) and quantitatively analyzed with the software OraCheck with regard to volume change and gain or loss of layer thickness in six sectional planes.

Results: Assessing the excess material, there was no difference (p = 0.31) between the silicone technique (0.26 mm ± 0.02) and the PTFE technique (0.22 mm ± 0.02 mm). Nevertheless, the loss of tooth substance was significantly greater (p < 0.001) with the silicone technique (-0.29 mm ± 0.02 mm) than with the PTFE technique (-0.15 mm ± 0.02 mm).

Conclusions: With the PTFE stamp technique, less healthy tooth structure was removed during the finishing procedure and the stamp was more dimensionally stable.

Clinical Relevance: The study shows the advantages and disadvantages of the investigated stamp techniques and helps the practitioner to choose an appropriate technique.
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http://dx.doi.org/10.1007/s00784-021-03992-8DOI Listing
May 2021

Simlukafusp alfa (FAP-IL2v) immunocytokine is a versatile combination partner for cancer immunotherapy.

MAbs 2021 Jan-Dec;13(1):1913791

Roche Innovation Center Zurich, Zurich, Switzerland.

Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rβγ > IL-2 Rβγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rβγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the and activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.
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http://dx.doi.org/10.1080/19420862.2021.1913791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115765PMC
May 2021

Improved Protein and PTM Characterization with a Practical Electron-Based Fragmentation on Q-TOF Instruments.

J Am Soc Mass Spectrom 2021 Aug 29;32(8):2081-2091. Epub 2021 Apr 29.

Agilent Technologies, Inc Santa Clara, California 95051, United States.

Electron-based dissociation (ExD) produces uncluttered mass spectra of intact proteins while preserving labile post-translational modifications. However, technical challenges have limited this option to only a few high-end mass spectrometers. We have developed an efficient ExD cell that can be retrofitted in less than an hour into current LC/Q-TOF instruments. Supporting software has been developed to acquire, process, and annotate peptide and protein ExD fragmentation spectra. In addition to producing complementary fragmentation, ExD spectra enable many isobaric leucine/isoleucine and isoaspartate/aspartate pairs to be distinguished by side-chain fragmentation. The ExD cell preserves phosphorylation and glycosylation modifications. It also fragments longer peptides more efficiently to reveal signaling cross-talk between multiple post-translational modifications on the same protein chain and cleaves disulfide bonds in cystine knotted proteins and intact antibodies. The ability of the ExD cell to combine collisional activation with electron fragmentation enables more complete sequence coverage by disrupting intramolecular electrostatic interactions that can hold fragments of large peptides and proteins together. These enhanced capabilities made possible by the ExD cell expand the size of peptides and proteins that can be analyzed as well as the analytical certainty of characterizing their post-translational modifications.
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http://dx.doi.org/10.1021/jasms.0c00482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8343505PMC
August 2021

Beyond Basicity: Discovery of Nonbasic DENV-2 Protease Inhibitors with Potent Activity in Cell Culture.

J Med Chem 2021 04 14;64(8):4567-4587. Epub 2021 Apr 14.

Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology IPMB, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.

The viral serine protease NS2B-NS3 is one of the promising targets for drug discovery against dengue virus and other flaviviruses. The molecular recognition preferences of the protease favor basic, positively charged moieties as substrates and inhibitors, which leads to pharmacokinetic liabilities and off-target interactions with host proteases such as thrombin. We here present the results of efforts that were aimed specifically at the discovery and development of noncharged, small-molecular inhibitors of the flaviviral proteases. A key factor in the discovery of these compounds was a cellular reporter gene assay for the dengue protease, the DENV2proHeLa system. Extensive structure-activity relationship explorations resulted in novel benzamide derivatives with submicromolar activities in viral replication assays (EC 0.24 μM), selectivity against off-target proteases, and negligible cytotoxicity. This structural class has increased drug-likeness compared to most of the previously published active-site-directed flaviviral protease inhibitors and includes promising candidates for further preclinical development.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02042DOI Listing
April 2021

Frailty as a predictive factor for survival after liver transplantation, especially for patients with MELD≤15-a prospective study.

Langenbecks Arch Surg 2021 Sep 13;406(6):1963-1969. Epub 2021 Apr 13.

Department of General, Visceral and Transplantation Surgery, University Hospital of Essen, Hufelandstraße 55, 45147, Essen, Germany.

Introduction: Frailty has been discussed as a predictor of morbidity and mortality for liver cirrhosis. The aim of our study is to evaluate the role of frailty in liver transplantation, particularly for patients with MELD scores < 15.

Methods: All patients listed for liver transplantation between September 2015 and November 2018 were prospectively included in the study. Frailty was assessed by Fried's frailty classification. Pre-, intra-, and postoperative data were prospectively recorded. Univariate and multivariate regression analyses were performed. The ethical approval of the institutional board review was obtained for the study.

Results: There were 114 patients included in the study, and their median MELD score was 16. Of these, 86 patients were defined as frail (75.4%). A total of 62 patients (54.4%) underwent liver transplantation, 11 (17.7%) died postoperatively, and 24 patients (21.0%) died while on the waitlist. All postoperative mortality cases were frail, and only 3 patients (12.5%) were non-frail in the waitlist mortality group. There were 14 patients who had MELD scores of <15 (58.3%). The overall survival of non-frail patients was significantly better than that of frail patients. The multivariate regression analyses identified frailty criteria, including unintended weight loss and low hand grip strength, and platelet count and being married or living in a solid partnership were prognostic factors for survival in all patients.

Conclusion: The addition of frailty assessment can be beneficial for predicting mortality after liver transplantation, especially in patients with low MELD score. Frail patients on the waitlist have significant risk for mortality even with low MELD score.
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http://dx.doi.org/10.1007/s00423-021-02109-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8481213PMC
September 2021

How well can today's tooth-colored dental restorative materials reproduce the autofluorescence of human teeth? - Ambition and reality!

J Esthet Restor Dent 2021 Jul 24;33(5):720-738. Epub 2021 Mar 24.

Department of Conservative Dentistry, Periodontology and Endodontology, University Centre of Dentistry, Oral Medicine and Maxillofacial Surgery, University Hospital Tübingen, Tübingen, Germany.

Objectives: The autofluorescence of dental hard tissues has been known for over 100 years. Thus, manufacturers add fluorophores to dental restorative materials to improve the esthetic properties of these materials. So far, there has been no study evaluating the ability of these fluorophores to reproduce the autofluorescence of dental hard tissues.

Materials And Methods: A total of 240 different color shades representing 17 different brands of fluorescent light-curing RBC and CAD/CAM restorative materials were analyzed with a monochromator-based microplate reader. Additionally, combined enamel-dentin specimens (n = 11) were analyzed as "gold standard". The total fluorescence (TF) and the physiologically relevant luminous efficiency function adjusted total fluorescence (TF ) were determined. The differences between the brands and the enamel-dentin specimens were further evaluated and visualized as contour plots.

Results: Merely the TF of the brands CERASMART™, Filtek Supreme XTE™, KZR-CAD HD 2, and LuxaCam composite were not significantly different to the enamel-dentin specimens. The analysis of the contour plots revealed that even these four materials showed a fluorescence excess for the excitation wavelengths below about 400 nm and a deficit above this wavelength.

Conclusion: None of the materials analyzed in this study were able to reproduce the natural fluorescence spectrum of the enamel-dentin specimens.

Clinical Significance: Unlike the statements and images of blue fluorescent materials in the manufacturers' brochures, none of the materials examined here is fully capable of reproducing the natural autofluorescence of teeth.
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http://dx.doi.org/10.1111/jerd.12729DOI Listing
July 2021

Stromal FAP is an independent poor prognosis marker in non-small cell lung adenocarcinoma and associated with p53 mutation.

Lung Cancer 2021 05 25;155:10-19. Epub 2021 Feb 25.

Karolinska Institutet, Cancer Center Karolinska, Department of Oncology-Pathology, Stockholm, Sweden. Electronic address:

Objectives: Fibroblasts regulate tumor growth and immune surveillance. Here, we study FAP, PDGFβR and α-SMA fibroblast markers in a well-annotated clinical cohort of non-small-cell lung cancer (NSCLC) for analyses of associations with immune cell infiltration, mutation status and survival.

Materials And Methods: A well-annotated NSCLC cohort was subjected to IHC analyses of stromal expression of FAP, PDGFβR and α-SMA and of stromal CD8 density. Fibroblast markers-related measurements were analyzed with regard to potential associations with CD8 density, cancer genetic driver mutations, survival and PD-L1 expression in the whole NSCLC cohort and in subsets of patients.

Results: High stromal FAP expression was identified as an independent poor prognostic marker in the whole study population (HR 1.481; 95 % CI, 1.012-2.167, p = 0.023) and in the histological subset of adenocarcinoma (HR 1.720; 95 % CI, 1.126-2.627, p = 0.012). Among patients with adenocarcinoma, a particularly strong association of FAP with poor survival was detected in patients with low stromal CD8 infiltration, and in other subpopulations identified by specific clinical characteristics; elderly patients, females, non-smokers and patients with normal ECOG performance status. α-SMA expression was negatively associated with CD8 infiltration in non-smokers, but none of the fibroblast markers expression was associated with CD8 density in the whole study population. Significant associations were detected between presence of p53 mutations and high α-SMA (p = 0.003) and FAP expression (p < 0.001).

Conclusion: The study identifies FAP intensity as a candidate independent NSCLC prognostic biomarker. The study also suggests continued analyses of the relationships between genetic driver mutations and the composition of tumor stroma, as well as continued probing of marker-defined fibroblasts as NSCLC subset-specific modifiers of immune surveillance and outcome.
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http://dx.doi.org/10.1016/j.lungcan.2021.02.028DOI Listing
May 2021

A human receptor occupancy assay to measure anti-PD-1 binding in patients with prior anti-PD-1.

Cytometry A 2021 Aug 18;99(8):832-843. Epub 2021 Mar 18.

Roche Pharma Research and Early Development, Early Biomarker Development Oncology, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland.

Receptor occupancy (RO) assessment by flow cytometry is an important pharmacodynamic (PD) biomarker in the clinical development of large molecules such as monoclonal therapeutic antibodies (mAbs). The total-drug-bound RO assay format directly assesses mAb binding to cell surface targets using anti-drug detection antibodies. Here, we generated a flow cytometry detection antibody specifically binding to mAbs of the IgG P329GLALA backbone. Using this reagent, we developed a total-drug-bound RO assay format for RG7769, a bi-specific P329GLALA containing mAb targeting PD-1 and TIM3 on T cells. In its fit-for-purpose validated version, this RO assay has been used in the Phase-I dose escalation study of RG7769, informing on peripheral T cell RO and RG7769 antibody binding capacity (ABC). We assessed RG7769 RO in checkpoint-inhibitor (CPI) naïve patients and anti-PD-1 CPI experienced patients using our novel assay. Here, we show that in both groups, complete T cell RO can be achieved (~100%). However, we found that the maximum number of T cell binding sites for RG7769 pre-dosing was roughly twofold lower in patients recently having undergone anti-PD-1 treatment. We show that this is due to steric hindrance exerted by competing mAbs masking the available drug binding sites. Our findings highlight the importance of quantitative mAb assessment in addition to relative RO especially in the context of patients who have previously received anti-PD-1 treatment.
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http://dx.doi.org/10.1002/cyto.a.24334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8451911PMC
August 2021

Acquired cancer cell resistance to T cell bispecific antibodies and CAR T targeting HER2 through JAK2 down-modulation.

Nat Commun 2021 02 23;12(1):1237. Epub 2021 Feb 23.

Preclinical Research Program, Vall d'Hebron Institute of Oncology (VHIO), Vall d'Hebron Barcelona Hospital Campus, Barcelona, 08035, Spain.

Immunotherapy has raised high expectations in the treatment of virtually every cancer. Many current efforts are focused on ensuring the efficient delivery of active cytotoxic cells to tumors. It is assumed that, once these active cytotoxic cells are correctly engaged to cancer cells, they will unfailingly eliminate the latter, provided that inhibitory factors are in check. T cell bispecific antibodies (TCBs) and chimeric antigen receptors (CARs) offer an opportunity to test this assumption. Using TCB and CARs directed against HER2, here we show that disruption of interferon-gamma signaling confers resistance to killing by active T lymphocytes. The kinase JAK2, which transduces the signal initiated by interferon-gamma, is a component repeatedly disrupted in several independently generated resistant models. Our results unveil a seemingly widespread strategy used by cancer cells to resist clearance by redirected lymphocytes. In addition, they open the possibility that long-term inhibition of interferon-gamma signaling may impair the elimination phase of immunoediting and, thus, promote tumor progression.
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http://dx.doi.org/10.1038/s41467-021-21445-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902842PMC
February 2021
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