Publications by authors named "Dario Neri"

331 Publications

A novel antibody-interleukin-15 fusion protein selectively localizes to tumors, synergizes with TNF-based immunocytokine and inhibits metastasis.

Mol Cancer Ther 2021 Feb 25. Epub 2021 Feb 25.

Biology, Philochem AG

Interleukin-15 (IL15) is an immunostimulatory cytokine which holds promises for cancer therapy, but its performance (alone or as partner for fusion proteins) has often been limited by suboptimal accumulation in the tumor and very rapid clearance from circulation. Most recently, the Sushi Domain (SD, the shortest region of IL15 receptor alpha, capable of binding to IL15) has been fused to IL15-based anti-cancer products in order to increase its biological activity. Here, we describe two novel antibody fusion proteins (termed F8-F8-IL15 and F8-F8-SD-IL15), specific to the alternatively-spliced EDA domain of fibronectin (a marker of tumor neoangiogenisis, expressed in the majority of solid and hematological tumors, but absent in normal healthy tissues) and featuring the F8 antibody in single-chain diabody format (with a short linker between VH and VL, thus allowing the domains to pair with the complementary ones of another chain). Unlike previously described fusions of the F8 antibody with human IL15, F8-F8-IL15 and F8-F8-SD-IL15 exhibited a preferential uptake in solid tumors, as evidenced by quantitative biodistribution analysis with radioiodinated protein preparations. Both products were potently active in vivo against mouse metastatic colon carcinomas and in sarcoma lesion in combination with targeted tumor necrosis factor. The results may be of clinical significance, as F8-F8-IL15 and F8-F8-SD-IL15 are fully-human proteins, which recognize the cognate tumor-associated antigen with identical affinity in mouse and man.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0853DOI Listing
February 2021

A DNA-encoded chemical library based on peptide macrocycles.

Chemistry 2021 Feb 14. Epub 2021 Feb 14.

ETH Zürich: Eidgenossische Technische Hochschule Zurich, D-CHAB, SWITZERLAND.

We describe the synthesis and characterization of a novel DNA-encoded library of macrocyclic peptide derivatives, based on three sets of proteinogenic and non-proteinogenic amino acid building blocks and featuring the use of copper alkyne-azide cycloaddition (CuAAC) reaction for ring closure. The library (termed YO-DEL) which contains 1,254,838 compounds, was encoded with DNA in single-stranded format and was screened against target proteins of interest using affinity capture procedures and photocrosslinking. YO-DEL selections yielded specific binders against serum albumins, carbonic anhydrases and NKp46, a marker of activated Natural Killer cells.
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http://dx.doi.org/10.1002/chem.202005423DOI Listing
February 2021

Inference of molecular structure for characterization and improvement of clinical grade immunocytokines.

J Struct Biol 2021 Jan 23;213(1):107696. Epub 2021 Jan 23.

The Armenise-Harvard Laboratory of Structural Biology, Dept. Biology and Biotechnology, University of Pavia, Via Ferrata 9/A, 27100 Pavia Italy. Electronic address:

The use of immunomodulatory agents for the treatment of cancer is gaining a growing biopharmaceutical interest. Antibody-cytokine fusion proteins, namely immunocytokines, represent a promising solution for the regulation of the immune system at the site of disease. The three-dimensional arrangement of these molecules can profoundly influence their biological activity and pharmacokinetic properties. Structural techniques might provide important insight in the 3D arrangement of immunocytokines. Here, we performed structure investigations on clinical grade fusion proteins L19-IL2, IL12-L19L19 and L19L19-IL2 to elucidate their quaternary organization. Crystallographic characterization of the common L19 antibody fragment at a resolution of 2.0-Å was combined with low-resolution studies of the full-length chimeric molecules using small-angle synchrotron X-ray scattering (SAXS) and negative stain electron microscopy. Characterization of the full-length quaternary structures of the immunocytokines in solution by SAXS consistently supported the diabody structure in the L19-IL2 immunocytokine and allowed generation of low-resolution models of the chimeric proteins L19L19-IL2 and IL12-L19L19. Comparison with 3D reconstructions obtained from negative-stain electron microscopy revealed marked flexibility associated to the linker regions connecting the cytokine and the antibody components of the chimeric proteins. Collectively, our results indicate that low-resolution molecular structure characterizations provide useful complementary insights for the quality control of immunocytokines, constituting a powerful tool to guide the design and the subsequent optimization steps towards clinical enhancement of these chimeric protein reagents.
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http://dx.doi.org/10.1016/j.jsb.2021.107696DOI Listing
January 2021

Anti-CD117 immunotherapy to eliminate hematopoietic and leukemia stem cells.

Exp Hematol 2021 Mar 20;95:31-45. Epub 2021 Jan 20.

Department of Medical Oncology and Hematology, University Hospital Zurich and University of Zurich, Comprehensive Cancer Center Zurich (CCCZ), Zurich, Switzerland. Electronic address:

Precise replacement of diseased or dysfunctional organs is the goal of regenerative medicine and has appeared to be a distant goal for a long time. In the field of hematopoietic stem cell transplantation, this goal is now becoming tangible as gene-editing technologies and novel conditioning agents are entering the clinical arena. Targeted immunologic depletion of hematopoietic stem cells (HSCs), which are at the very root of the hematopoietic system, will enable more selective and potentially more effective hematopoietic stem cell transplantation in patients with hematological diseases. In contrast to current conditioning regimes based on ionizing radiation and chemotherapy, immunologic conditioning will spare mature hematopoietic cells and cause substantially less inflammation and unspecific collateral damage to other organs. Biological agents that target the stem cell antigen CD117 are the frontrunners for this purpose and have exhibited preclinical activity in depletion of healthy HSCs. The value of anti-CD117 antibodies as conditioning agents is currently being evaluated in early clinical trials. Whereas mild, antibody-based immunologic conditioning concepts might be appropriate for benign hematological disorders in which incomplete replacement of diseased cells is sufficient, higher efficacy will be required for treatment and elimination of hematologic stem cell malignancies such as acute myeloid leukemia and myelodysplastic syndrome. Antibody-drug conjugates, bispecific T-cell engaging and activating antibodies (TEAs), or chimeric antigen receptor (CAR) T cells might offer increased efficacy compared with naked antibodies and yet higher tolerability and safety compared with current genotoxic conditioning approaches. Here, we summarize the current state regarding immunologic conditioning concepts for the treatment of HSC disorders and outline potential future developments.
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http://dx.doi.org/10.1016/j.exphem.2021.01.003DOI Listing
March 2021

Antibody-based delivery of interleukin-9 to neovascular structures: Therapeutic evaluation in cancer and arthritis.

Exp Biol Med (Maywood) 2021 Jan 21:1535370220981578. Epub 2021 Jan 21.

Philochem AG, Libernstrasse 3, Otelfingen 8112, Switzerland.

Interleukin-9 is a cytokine with multiple functions, including the ability to activate group 2 innate lymphoid cells, which has been postulated to be therapeutically active in mouse models of arthritis. Similarly, interleukin-9 has been suggested to play an important role in tumor immunity. Here, we describe the cloning, expression, and characterization of three fusion proteins based on murine interleukin-9 and the F8 antibody, specific to the alternatively spliced EDA domain of fibronectin. EDA is strongly expressed in cancer and in various arthritic conditions, while being undetectable in the majority of healthy organs. Interleukin-9-based fusion proteins with an irrelevant antibody specific to hen egg lysozyme served as negative control in our study. The fusion proteins were characterized by quantitative biodistribution analysis in tumor-bearing mice using radioiodinated protein preparations. The highest tumor uptake and best tumor:organ ratios were observed for a format, in which the interleukin-9 moiety was flanked by two units of the F8 antibody in single-chain Fv format. Biological activity of interleukin-9 was retained when the payload was fused to antibodies. However, the targeted delivery of interleukin-9 to the disease site resulted in a modest anti-tumor activity in three different murine models of cancer (K1735M2, CT26, and F9), while no therapeutic benefit was observed in a collagen induced model of arthritis. Collectively, these results confirm the possibility to deliver interleukin-9 to the site of disease but cast doubts about the alleged therapeutic activity of this cytokine in cancer and arthritis, which has been postulated in previous publications.
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http://dx.doi.org/10.1177/1535370220981578DOI Listing
January 2021

Immunotherapy with Immunocytokines and PD-1 Blockade Enhances the Anticancer Activity of Small Molecule-Drug Conjugates Targeting Carbonic Anhydrase IX.

Mol Cancer Ther 2021 Mar 22;20(3):512-522. Epub 2020 Dec 22.

Philochem AG, Otelfingen, Zurich, Switzerland.

Small molecule-drug conjugates (SMDCs) represent an alternative to conventional antitumor chemotherapeutic agents, with the potential to improve the therapeutic window of cytotoxic payloads through active delivery at the site of the disease. In this article, we describe novel combination therapies consisting of anti-carbonic anhydrase IX SMDCs combined with different immunomodulatory products. The therapeutic effect of the SMDCs was potentiated by combination with PD-1 blockade and with tumor-homing antibody-cytokine fusions in mouse models of renal cell carcinoma and colorectal cancer. The combination with L19-IL12, a fusion protein specific to the alternatively spliced EDB domain of fibronectin containing the murine IL12 moiety, was also active against large established tumors. Analysis of the microscopic structures of healthy organs performed 3 months after tumor eradication confirmed absence of pathologic abnormalities in the healthy kidney, liver, lung, stomach, and intestine. Our findings may be of clinical significance as they provide motivation for the development of combinations based on SMDCs and immunotherapy for the treatment of renal cell carcinoma and hypoxic tumors.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0361DOI Listing
March 2021

Antibody-mediated delivery of LIGHT to the tumor boosts natural killer cells and delays tumor progression.

MAbs 2021 Jan-Dec;13(1):1868066

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich) , Zürich, Switzerland.

LIGHT is a member of the tumor necrosis factor superfamily, which has been claimed to mediate anti-tumor activity on the basis of cancer cures observed in immunocompetent mice bearing transgenic LIGHT-expressing tumors. The preclinical development of a LIGHT-based therapeutic has been hindered by the lack of functional stability exhibited by this protein. Here, we describe the cloning, expression, and characterization of five antibody-LIGHT fusion proteins, directed against the alternatively spliced extra domain A of fibronectin, a conserved tumor-associated antigen. Among the five tested formats, only the sequential fusion of the F8 antibody in single-chain diabody format, followed by the LIGHT homotrimer expressed as a single polypeptide, yielded a protein (termed "F8-LIGHT") that was not prone to aggregation. A quantitative biodistribution analysis in tumor-bearing mice, using radio-iodinated protein preparations, confirmed that F8-LIGHT was able to preferentially accumulate at the tumor site, with a tumor-to-blood ratio of ca. five to one 24 hours after intravenous administration. Tumor therapy experiments, performed in two murine tumor models (CT26 and WEHI-164), featuring different levels of lymphocyte infiltration into the neoplastic mass, revealed that F8-LIGHT could significantly reduce tumor-cell growth and was more potent than a similar fusion protein (KSF-LIGHT), directed against hen egg lysozyme and serving as negative control of irrelevant specificity in the mouse. At a mechanistic level, the activity of F8-LIGHT was mainly due to an intratumoral expansion of natural killer cells, whereas there was no evidence of expansion of CD8 + T cells, neither in the tumor, nor in draining lymph nodes. : CTLA-4: Cytotoxic T-lymphocytes-associated protein 4; EGFR: Epidermal growth factor receptor; HVEM: Herpesvirus entry mediator; IFNγ: Interferon-gamma; LIGHT: Lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes; LTβR: Lymphotoxin beta receptor; NF-κB: Nuclear factor "kappa-light-chain-enhancer" of activated B cells; NK: Natural killer cells; PD-1: Programmed cell death protein 1; PD-L1: Programmed death-ligand 1; TNF: Tumor necrosis factor.
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http://dx.doi.org/10.1080/19420862.2020.1868066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808322PMC
January 2021

A Single-Stranded DNA-Encoded Chemical Library Based on a Stereoisomeric Scaffold Enables Ligand Discovery by Modular Assembly of Building Blocks.

Adv Sci (Weinh) 2020 Nov 14;7(22):2001970. Epub 2020 Oct 14.

Department of Chemistry and Applied Biosciences ETH Zürich Zürich 8092 Switzerland.

A versatile and Lipinski-compliant DNA-encoded library (DEL), comprising 366 600 glutamic acid derivatives coupled to oligonucleotides serving as amplifiable identification barcodes is designed, constructed, and characterized. The GB-DEL library, constructed in single-stranded DNA format, allows de novo identification of specific binders against several pharmaceutically relevant proteins. Moreover, hybridization of the single-stranded DEL with a set of known protein ligands of low to medium affinity coupled to a complementary DNA strand results in self-assembled selectable chemical structures, leading to the identification of affinity-matured compounds.
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http://dx.doi.org/10.1002/advs.202001970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675038PMC
November 2020

An engineered 4-1BBL fusion protein with "activity on demand".

Proc Natl Acad Sci U S A 2020 12 25;117(50):31780-31788. Epub 2020 Nov 25.

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), CH-8093 Zürich, Switzerland;

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display "activity on demand" properties at the site of disease on antigen binding and reduce toxicity to normal tissues.
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http://dx.doi.org/10.1073/pnas.2013615117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749310PMC
December 2020

Targeting an engineered cytokine with interleukin-2 and interleukin-15 activity to the neovasculature of solid tumors.

Oncotarget 2020 Nov 3;11(44):3972-3983. Epub 2020 Nov 3.

Department of Chemistry and Applied Biosciences (D-CHAB), Institute for Pharmaceutical Sciences (IPW), 8093 Zurich, Switzerland.

There is a growing interest in the antibody-based delivery of cytokines to the tumor environment as a means to boost the anti-cancer activity of tumor-resident T cells and NK cells. Here, we describe the expression and characterization of fusion proteins, featuring the L19 antibody (specific to the alternatively-spliced EDB domain of fibronectin) and an engineered cytokine with interleukin-2 and interleukin-15 properties. The cytokine moiety was fused either at the N-terminal or at the C-terminal extremity and both fusion proteins showed a selective tumor accumulation in a quantitative biodistribution experiment. The N-terminal fusion inhibited tumor growth in immunocompetent mice bearing F9 carcinomas or WEHI-164 sarcomas when used as single agent. The anticancer activity was compared to the one of the same cytokine payload used as recombinant protein or fused to an anti-hen egg lysozyme antibody, serving as negative control of irrelevant specificity in the mouse. These results indicate that the antibody-based delivery of engineered cytokines to the tumor neovasculature may mediate a potent anticancer activity.
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http://dx.doi.org/10.18632/oncotarget.27772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646832PMC
November 2020

Novel human monoclonal antibodies specific to the alternatively spliced domain D of Tenascin C efficiently target tumors .

MAbs 2020 Jan-Dec;12(1):1836713

Biology department, Philochem AG , Otelfingen, Switzerland.

Antibody-based delivery of bioactive molecules represents a promising strategy for the improvement of cancer immunotherapy. Here, we describe the generation and characterization of R6N, a novel fully human antibody specific to the alternatively spliced domain D of Tenascin C, which is highly expressed in the stroma of primary tumors and metastasis. The R6N antibody recognized its cognate tumor-associated antigen with identical specificity in mouse and human specimens. Moreover, the antibody was able to selectively localize to solid tumors as evidenced by immunofluorescence-based biodistribution analysis. Encouraged by these results, we developed a novel fusion protein (termed mIL12-R6N) consisting of the murine interleukin 12 fused to the R6N antibody in homodimeric tandem single-chain variable fragment arrangement. mIL12-R6N exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. The experiments presented in this work provide a rationale for possible future applications for the R6N antibody for the treatment of cancer patients.
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http://dx.doi.org/10.1080/19420862.2020.1836713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646483PMC
November 2020

Special edition on DNA-Encoded chemical libraries.

Biochem Biophys Res Commun 2020 Dec;533(2):iii-iv

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http://dx.doi.org/10.1016/j.bbrc.2020.10.055DOI Listing
December 2020

A novel format for recombinant antibody-interleukin-2 fusion proteins exhibits superior tumor-targeting properties .

Oncotarget 2020 Oct 13;11(41):3698-3711. Epub 2020 Oct 13.

Philochem AG, Otelfingen, Switzerland.

The targeted delivery of interleukin-2 to the tumor is gaining attention as an avenue to potentiate the action of T and NK cells at the site of disease. We have previously described the fusion of the L19 antibody, specific to the EDB domain of fibronectin, with human interleukin-2, using a non-covalent homodimeric diabody format. Here, we describe four novel formats for the L19-IL2 fusion, featuring different arrangements of antibody and IL2. A comparative quantitative biodistribution analysis in tumor-bearing mice using radioiodinated proteins revealed that the novel format (L19L19-IL2, with the antibody in single-chain diabody format) exhibited the best biodistribution results. assays on peripheral blood mononuclear cells showed a decrease activation of regulatory T cells when single IL2 domain was used. , both L19-IL2 and L19L19-IL2 inhibited tumor growth in immunocompetent mouse models of cancer. T-cell analysis revealed similar levels of CD4 and FoxP3 cells, with an expansion of the CD8 T cell in mice treated with L19-IL2 and L19L19-IL2. The percentage of CD4 regulatory T cells was markedly decreased with L19L19-IL2 combined with a mouse-specific PD-1 blocker. Collectively, these data indicate that the new L19L19-IL2 format exhibits favorable tumor-homing properties and mediates a potent anti-cancer activity .
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http://dx.doi.org/10.18632/oncotarget.27726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566808PMC
October 2020

Sortase-Mediated Site-Specific Modification of Interleukin-2 for the Generation of a Tumor-Targeting Acetazolamide-Cytokine Conjugate.

ACS Omega 2020 Oct 30;5(40):26077-26083. Epub 2020 Sep 30.

Philochem AG, Libernstrasse 3, 8112 Otelfingen, Switzerland.

Small ligands specific to tumor-associated antigens can be used as alternatives to antibodies for the delivery of small payloads such as radionuclides, cytotoxic drugs, and fluorophores. Their use as a delivery moiety of bioactive proteins such as cytokines remains largely unexplored. Here, we describe the preparation and in vivo characterization of the first small molecule-cytokine conjugate targeting carbonic anhydrase IX (CAIX), a marker of renal cell carcinoma and hypoxia. Site-specific conjugation between interleukin-2 and acetazolamide was obtained by sortase A-mediated transpeptidation. Binding of the conjugate to the cognate CAIX antigen was confirmed by surface plasmon resonance. The in vivo targeting of structures expressing carbonic anhydrase IX was assessed by biodistribution experiments in tumor-bearing mice. Optimization of manufacturability and tumor-targeting performance of acetazolamide-cytokine products will be required in order to enable industrial applications.
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http://dx.doi.org/10.1021/acsomega.0c03592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558062PMC
October 2020

Immunocytokines are a promising immunotherapeutic approach against glioblastoma.

Sci Transl Med 2020 10;12(564)

Department of Neurology and Brain Tumor Center, University Hospital Zurich and University of Zurich, CH-8091 Zurich, Switzerland.

Glioblastoma is a poorly immunogenic cancer, and the successes with recent immunotherapies in extracranial malignancies have, so far, not been translated to this devastating disease. Therefore, there is an urgent need for new strategies to convert the immunologically cold glioma microenvironment into a hot one to enable effective antitumor immunity. Using the L19 antibody, which is specific to a tumor-associated epitope of extracellular fibronectin, we developed antibody-cytokine fusions-immunocytokines-with interleukin-2 (IL2), IL12, or tumor necrosis factor (TNF). We showed that L19 accumulated in the tumor microenvironment of two orthotopic immunocompetent mouse glioma models. Furthermore, intravenous administration of L19-mIL12 or L19-mTNF cured a proportion of tumor-bearing mice, whereas L19-IL2 did not. This therapeutic activity was abolished in RAG mice or upon depletion of CD4 or CD8 T cells, suggesting adaptive immunity. Mechanistically, both immunocytokines promoted tumor-infiltrating lymphocytes and increased the amounts of proinflammatory cytokines within the tumor microenvironment. In addition, L19-mTNF induced tumor necrosis. Systemic administration of the fully human L19-TNF fusion protein to patients with glioblastoma (NCT03779230) was safe, decreased regional blood perfusion within the tumor, and was associated with increasing tumor necrosis and an increase in tumor-infiltrating CD4 and CD8 T cells. The extensive preclinical characterization and subsequent clinical translation provide a robust basis for future studies with immunocytokines to treat malignant brain tumors.
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http://dx.doi.org/10.1126/scitranslmed.abb2311DOI Listing
October 2020

Ubiquitin Phosphorylation at Thr12 Modulates the DNA Damage Response.

Mol Cell 2020 11 5;80(3):423-436.e9. Epub 2020 Oct 5.

Institute of Molecular Cancer Research, University of Zurich, 8057 Zurich, Switzerland. Electronic address:

The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes.
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http://dx.doi.org/10.1016/j.molcel.2020.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655664PMC
November 2020

An Antibody-Tumor Necrosis Factor Fusion Protein that Synergizes with Oxaliplatin for Treatment of Colorectal Cancer.

Mol Cancer Ther 2020 12 30;19(12):2554-2563. Epub 2020 Sep 30.

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zürich, Switzerland.

We have cloned and characterized a novel fusion protein (Sm3E-TNF), consisting of the mAb, S 6m3E, in single-chain Fv fragment format, fused to murine TNF. The protein, which was expressed in mammalian cells and purified as a noncovalent stable homotrimer, bound to the cognate carcinoembryonic antigen (CEA) and retained TNF activity. A quantitative biodistribution experiment, performed in immunocompetent mice with CT26 colon carcinomas transfected with human CEA, revealed that Sm3E-TNF was able to preferentially accumulate in the tumors with excellent selectivity (tumor:blood ratio = 56:1, 24 hours after intravenous administration). The fusion protein mediated a rapid hemorrhagic necrosis of a large portion of the tumor mass, but a rim survived and eventually regrew. Surprisingly, the combination of Sm3E-TNF with 5-fluorouracil led to a reduction of therapeutic activity, while a combination with oxaliplatin led to a prolonged stabilization, with complete tumor eradication in 40% of treated mice. These therapy results were confirmed in a second immunocompetent mouse model of colorectal cancer (CEA-transfected C51 tumors) and provide a rationale for the possible clinical use of oxaliplatin in combination with fully human antibody-TNF fusions.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0729DOI Listing
December 2020

The targeted delivery of interleukin-12 to the carcinoembryonic antigen increases the intratumoral density of NK and CD8 T cell in an immunocompetent mouse model of colorectal cancer.

J Gastrointest Oncol 2020 Aug;11(4):803-811

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zürich, Switzerland.

The recent success achieved by immune checkpoint inhibitors in the field of immuno-oncology has been less evident for the treatment of metastatic colorectal cancer (mCRC) patients. To date, cancer immunotherapy has been efficacious only in few patients bearing high mutational burden (less than 25%) mCRCs. In this Communication, we report the generation of a novel antibody cytokine fusion protein (termed Sm3E-mIL12) targeting the CRC-associated carcinoembryonic antigen (CEA). The antibody moiety bound avidly to CEA when immobilized on solid supports, and selectively stained C51 tumor cells transfected with the antigen (C51-CEA). The cytokine payload retained full activity , as compared to the parental recombinant interleukin-12 (IL12). microscopic analyses revealed a homogenous distribution of Sm3E-mIL12 in the neoplastic mass upon intravenous administration. , Sm3E-mIL12 was well tolerated up to 180 µg per mouse. The targeted delivery of IL12 to CEA-expressing C51 carcinomas led to durable complete responses in 60% of the treated mice. The intratumoral density of immune effector cells was markedly increased after the third injection of Sm3E-mIL12, in keeping with the progressive regression of the neoplastic mass. The data suggest that a fully human analogue may be considered for the treatment of patients with mCRC.
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http://dx.doi.org/10.21037/jgo.2020.04.02DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7475332PMC
August 2020

Selective Fragments for the CREBBP Bromodomain Identified from an Encoded Self-assembly Chemical Library.

ChemMedChem 2020 Sep 19;15(18):1752-1756. Epub 2020 Aug 19.

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 4, 8093, Zürich, Switzerland.

DNA-encoded chemical libraries (DECLs) are collections of chemical moieties individually coupled to distinctive DNA barcodes. Compounds can be displayed either at the end of a single DNA strand (i. e., single-pharmacophore libraries) or at the extremities of two complementary DNA strands (i. e., dual-pharmacophore libraries). In this work, we describe the use of a dual-pharmacophore encoded self-assembly chemical (ESAC) library for the affinity maturation of a known 4,5-dihydrobenzodiazepinone ring (THBD) acetyl-lysine (KAc) mimic for the cyclic-AMP response element binding protein (CREB) binding protein (CREBBP or CBP) bromodomain. The new pair of fragments discovered from library selection showed a sub-micromolar affinity for the CREBBP bromodomain in fluorescence polarization and ELISA assays, and selectivity against BRD4(1).
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http://dx.doi.org/10.1002/cmdc.202000528DOI Listing
September 2020

A Microdosing Study with Tc-PHC-102 for the SPECT/CT Imaging of Primary and Metastatic Lesions in Renal Cell Carcinoma Patients.

J Nucl Med 2021 Mar 17;62(3):360-365. Epub 2020 Jul 17.

Department of Biomedical Imaging and Image-Guided Therapy, Division of Nuclear Medicine, Medical University of Vienna, Vienna, Austria

Tc-PHC-102 is a Tc-labeled derivative of acetazolamide, a high-affinity small organic ligand of carbonic anhydrase IX (CAIX). Tc-PHC-102 has previously shown favorable in vivo biodistribution properties in mouse models of CAIX-positive clear cell renal cell carcinoma (ccRCC) and colorectal cancer. In this study, we aimed to explore the targeting performance of Tc-PHC-102 in SPECT in patients with renal cell carcinoma while also assessing the safety and tolerability of the radiotracer. We studied 5 patients with localized or metastatic ccRCC in a microdosing regimen, after the administration of a 50-μg total of CAIX ligand and 600-800 MBq of Tc-PHC-102. Tissue distribution and residence time in normal organs and tumors were analyzed by serial SPECT/CT scans at 3 time points (30 min, 2 h, and 6 h) after intravenous administration. In the 5 patients studied, Tc-PHC-102 was well tolerated and no study drug-related adverse events were recorded. In the stomach, kidneys, and gallbladder, the radiotracer showed a rapid initial uptake, which cleared over time. Localization of the study drug in primary tumors of 5 patients was observed, with favorable tumor-to-background ratios. Tc-PHC-102 SPECT/CT allowed the identification of 4 previously unknown lung and lymph node metastases in 2 patients. Tc-PHC-102 is a promising SPECT tracer for the imaging of patients with ccRCC. This tracer has the potential to identify primary and metastatic lesions in different anatomic locations. Tc-PHC-102 might also serve as a companion diagnostic agent for future CAIX-targeting therapeutics.
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http://dx.doi.org/10.2967/jnumed.120.245530DOI Listing
March 2021

Complexation with a Cognate Antibody Fragment Facilitates Affinity Measurements of Fluorescein-Linked Small Molecule Ligands.

Anal Chem 2020 08 15;92(15):10822-10829. Epub 2020 Jul 15.

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland.

The availability of reliable methods for the characterization of the binding of small molecule ligands to protein targets is crucially important for drug discovery. We have adapted a method, routinely used for the characterization of monoclonal antibodies (enzyme-linked immunosorbent assay, or "ELISA"), to small molecule ligands, using fluorescein conjugates and antifluorescein antibodies as detection reagents. The new small molecule-ELISA methodology was tested using a panel of binders specific to carbonic anhydrase II, with dissociation constants ranging between 6 μM and 14 nM. An excellent agreement was found between ELISA measurements and fluorescence polarization results. The methodology was also extended to BIAcore measurements and implemented for ligands coupled to oligonucleotides. Small molecule-ELISA procedures are particularly useful in the context of DNA-encoded libraries, for which hit validation procedures need to be performed on dozens of candidate molecules and hit compounds can be conveniently resynthesized on DNA.
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http://dx.doi.org/10.1021/acs.analchem.0c02304DOI Listing
August 2020

Stereotactic ablative body radiotherapy (SABR) combined with immunotherapy (L19-IL2) versus standard of care in stage IV NSCLC patients, ImmunoSABR: a multicentre, randomised controlled open-label phase II trial.

BMC Cancer 2020 Jun 15;20(1):557. Epub 2020 Jun 15.

The D-Lab and The M-Lab, Department of Precision Medicine, GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands.

Background: About 50% of non-small cell lung cancer (NSCLC) patients have metastatic disease at initial diagnosis, which limits their treatment options and, consequently, the 5-year survival rate (15%). Immune checkpoint inhibitors (ICI), either alone or in combination with chemotherapy, have become standard of care (SOC) for most good performance status patients. However, most patients will not obtain long-term benefit and new treatment strategies are therefore needed. We previously demonstrated clinical safety of the tumour-selective immunocytokine L19-IL2, consisting of the anti-ED-B scFv L19 antibody coupled to IL2, combined with stereotactic ablative radiotherapy (SABR).

Methods: This investigator-initiated, multicentric, randomised controlled open-label phase II clinical trial will test the hypothesis that the combination of SABR and L19-IL2 increases progression free survival (PFS) in patients with limited metastatic NSCLC. One hundred twenty-six patients will be stratified according to their metastatic load (oligo-metastatic: ≤5 or poly-metastatic: 6 to 10) and randomised to the experimental-arm (E-arm) or the control-arm (C-arm). The C-arm will receive SOC, according to the local protocol. E-arm oligo-metastatic patients will receive SABR to all lesions followed by L19-IL2 therapy; radiotherapy for poly-metastatic patients consists of irradiation of one (symptomatic) to a maximum of 5 lesions (including ICI in both arms if this is the SOC). The accrual period will be 2.5-years, starting after the first centre is initiated and active. Primary endpoint is PFS at 1.5-years based on blinded radiological review, and secondary endpoints are overall survival, toxicity, quality of life and abscopal response. Associative biomarker studies, immune monitoring, CT-based radiomics, stool collection, iRECIST and tumour growth rate will be performed.

Discussion: The combination of SABR with or without ICI and the immunocytokine L19-IL2 will be tested as 1st, 2nd or 3rd line treatment in stage IV NSCLC patients in 14 centres located in 6 countries. This bimodal and trimodal treatment approach is based on the direct cytotoxic effect of radiotherapy, the tumour selective immunocytokine L19-IL2, the abscopal effect observed distant from the irradiated metastatic site(s) and the memory effect. The first results are expected end 2023.

Trial Registration: ImmunoSABR Protocol Code: NL67629.068.18; EudraCT: 2018-002583-11; Clinicaltrials.gov: NCT03705403; ISRCTN ID: ISRCTN49817477; Date of registration: 03-April-2019.
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http://dx.doi.org/10.1186/s12885-020-07055-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296663PMC
June 2020

Impact of Ligand Size and Conjugation Chemistry on the Performance of Universal Chimeric Antigen Receptor T-Cells for Tumor Killing.

Bioconjug Chem 2020 07 23;31(7):1775-1783. Epub 2020 Jun 23.

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), 8093 Zurich, Switzerland.

All Universal Chimeric Antigen Receptor T-cells (UniCAR T-cells) are T-cells which have been engineered to recognize a haptenated ligand. Due to this feature, UniCAR T-cells have the potential to mediate a potent and selective tumor killing only in the presence of a haptenated tumor ligand, thus avoiding the long-lasting biocidal effects of conventional CAR T-cells. We have used fluorescein-labeled versions of small organic ligands and different antibody formats specific to carbonic anhydrase IX (a tumor-associated antigen) in order to assess whether the killing potential of UniCAR T-cells depended on the molecular features of the haptenated molecule. Both small molecule ligands and larger antibody fragments were potent in mediating tumor cell killing over a broad concentration range. Antibodies could be conveniently used both in IgG format and as smaller diabody fragments. Importantly, the use of site-specific chemical modification strategies for the antibody coupling to fluorescein led to a substantial improvement of tumor cell killing performance, compared to the random modification of primary amino groups on the antibody surface.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00258DOI Listing
July 2020

Immunotherapy of CT26 murine tumors is characterized by an oligoclonal response of tissue-resident memory T cells against the AH1 rejection antigen.

Eur J Immunol 2020 10 9;50(10):1591-1597. Epub 2020 Jun 9.

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Zürich, Switzerland.

Mice bearing CT26 tumors can be cured by administration of L19-mIL12 or F8-mTNF, two antibody fusion proteins which selectively deliver their cytokine payload to the tumor. In both settings, cancer cures crucially depended on CD8 T cells and the AH1 peptide (derived from the gp70 protein of the murine leukemia virus) acted as the main tumor-rejection antigen, with ∼50% of CD8 T cells in the neoplastic mass being AH1-specific after therapy. In order to characterize the clonality of the T cell response, its phenotype, and activation status, we isolated CD8 T cells from tumors and secondary lymphoid organs and submitted them to T cell receptor (TCR) and total mRNA sequencing. We found an extremely diverse repertoire of more than 40 000 unique TCR sequences, but the ten most abundant TCRs accounted for >60% of CD8 T-cell clones in the tumor. AH1-specific TCRs were consistently found among the most abundant sequences. AH1-specific T cells in the tumor had a tissue-resident memory phenotype. Treatment with L19-mIL12 led to overexpression of IL-12 receptor and of markers of cell activation and proliferation. These data suggest that the antitumor response driven by antibody-cytokine fusions proceeds through an oligoclonal expansion and activation of tumor-infiltrating CD8 T cells.
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http://dx.doi.org/10.1002/eji.201948433DOI Listing
October 2020

Reflections on DNA-encoded chemical libraries.

Biochem Biophys Res Commun 2020 06 18;527(3):757-759. Epub 2020 May 18.

Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog Weg 4, CH, 8093 Zurich, Switzerland. Electronic address:

In this short Editorial, we reflect on certain milestones that have led to the development of DNA-Encoded Chemical Libraries, while also providing a glimpse to future challenges and opportunities.
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http://dx.doi.org/10.1016/j.bbrc.2020.04.080DOI Listing
June 2020

Comparative evaluation of DNA-encoded chemical selections performed using DNA in single-stranded or double-stranded format.

Biochem Biophys Res Commun 2020 Dec 5;533(2):223-229. Epub 2020 May 5.

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 4, CH-8093, Zürich, Switzerland. Electronic address:

DNA-encoded chemical libraries (DEL) are increasingly being used for the discovery and optimization of small organic ligands to proteins of biological or pharmaceutical interest. The DNA fragments, that serve as amplifiable identification barcodes for individual compounds in the library, are typically used in double-stranded DNA format. To the best of our knowledge, a direct comparison of DEL selections featuring DNA in either single- or double-stranded DNA format has not yet been reported. In this article, we describe a comparative evaluation of selections with two DEL libraries (named GB-DEL and NF-DEL), based on different chemical designs and produced in both single- and double-stranded DNA format. The libraries were selected in identical conditions against multiple protein targets, revealing comparable and reproducible fingerprints for both types of DNA formats. Surprisingly, selections performed with single-stranded DNA barcodes exhibited improved enrichment factors compared to double-stranded DNA. Using high-affinity ligands to carbonic anhydrase IX as benchmarks for selection performance, we observed an improved selectivity for the NF-DEL library (on average 2-fold higher enrichment factors) in favor of single-stranded DNA. The enrichment factors were even higher for the GB-DEL selections (approximately 5-fold), compared to the same library in double-stranded DNA format. Collectively, these results indicate that DEL libraries can conveniently be synthesized and screened in both single- and double-stranded DNA format, but single-stranded DNA barcodes typically yield enhanced enrichment factors.
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http://dx.doi.org/10.1016/j.bbrc.2020.04.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116422PMC
December 2020

On-DNA hit validation methodologies for ligands identified from DNA-encoded chemical libraries.

Biochem Biophys Res Commun 2020 Dec 30;533(2):235-240. Epub 2020 Apr 30.

Philochem AG, Libernstrasse 3, CH-8112, Otelfingen, Switzerland. Electronic address:

DNA-encoded chemical libraries (DECLs) are large compound collections attached to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of pharmaceutical interest. In DECL selections, ligands are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified using this procedure need to be validated by resynthesis and by performing affinity measurements. Here we report novel on-DNA hit validation strategies, which enable the facile confirmation of ligand-protein interaction as well as the determination of equilibrium and kinetic binding constants. The experimental procedures, which had been inspired by enzyme-linked immunosorbent assays (ELISA), were validated using ligands of different affinity to carbonic anhydrase II and IX.
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http://dx.doi.org/10.1016/j.bbrc.2020.04.030DOI Listing
December 2020

Anti-human CD117 CAR T-cells efficiently eliminate healthy and malignant CD117-expressing hematopoietic cells.

Leukemia 2020 10 1;34(10):2688-2703. Epub 2020 May 1.

Department of Medical Oncology and Hematology, University Hospital Zurich and University of Zurich, Comprehensive Cancer Center Zurich (CCCZ), Zurich, Switzerland.

Acute myeloid leukemia (AML) initiating and sustaining cells maintain high cell-surface similarity with their cells-of-origin, i.e., hematopoietic stem and progenitor cells (HSPCs), and identification of truly distinguishing leukemia-private antigens has remained elusive to date. To nonetheless utilize surface antigen-directed immunotherapy in AML, we here propose targeting both, healthy and malignant human HSPC, by chimeric antigen receptor (CAR) T-cells with specificity against CD117, the cognate receptor for stem cell factor. This approach should spare most mature hematopoietic cells and would require CAR T termination followed by subsequent transplantation of healthy HSPCs to rescue hematopoiesis. We successfully generated anti-CD117 CAR T-cells from healthy donors and AML patients. Anti-CD117 CAR T-cells efficiently targeted healthy and leukemic CD117-positive cells in vitro. In mice xenografted with healthy human hematopoiesis, they eliminated CD117-expressing, but not CD117-negative human cells. Importantly, in mice xenografted with primary human CD117-positive AML, they eradicated disease in a therapeutic setting. Administration of ATG in combination with rituximab, which binds to the co-expressed CAR T-cell transduction/selection marker RQR8, led to CAR T-cell depletion. Thus, we here provide the first proof of concept for the generation and preclinical efficacy of CAR T-cells directed against CD117-expressing human hematopoietic cells.
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http://dx.doi.org/10.1038/s41375-020-0818-9DOI Listing
October 2020

The immunocytokine L19-TNF eradicates sarcomas in combination with chemotherapy agents or with immune check-point inhibitors.

Anticancer Drugs 2020 09;31(8):799-805

Philochem AG, Otelfingen.

Antibody-cytokine fusion proteins (also called 'immunocytokines') represent an emerging class of biopharmaceutical products, which are being considered for cancer immunotherapy. When used as single agents, pro-inflammatory immunocytokines are rarely capable of inducing complete and durable cancer regression in mouse models and in patients. However, the combination treatment with conventional chemotherapy or with other immune-stimulatory agents typically increases the therapeutic efficacy of immunocytokines. In this article, we describe combination treatments of a tumor-targeting antibody-cytokine fusion protein based on the L19 antibody (specific to a splice isoform of fibronectin) fused to murine tumor necrosis factor with standard chemotherapy (dacarbazine, trabectedin or melphalan) or with an immune check-point inhibitor (anti-PD-1) in a BALB/c derived immunocompetent murine model of sarcoma (WEHI-164). All combination treatments led to improved tumor remission compared to single-agent treatments, suggesting that these combination partners may be suitable for further clinical development in sarcoma patients.
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http://dx.doi.org/10.1097/CAD.0000000000000938DOI Listing
September 2020