"Innovative strategies for the generation of novel human therapeutic anti-tumor and immunomodulatory antibodies"

Immunotherapy, based on the use of novel human mAbs endowed with antitumor or immunomodulatory activity, is an increasingly important strategy for cancer therapy. Monoclonal antibodies can be directed against Tumor Associated Antigens (TAAs), to inhibit their oncogenic function, or against Immune Checkpoints (IC), to modulate specific T cell responses against cancer. Furthermore, ongoing clinical trials in oncology are currently testing combinatorial treatments of anti-TAA and immunomodulatory antibodies. In our laboratory novel human antitumor immunoagents have been successfully produced against the TAA ErbB2, which is a Tyrosine Kinase Receptor, overexpressed in breast cancer and several other carcinomas. In particular, a fully human single chain antibody fragment (scFv), named Erbicin, able to bind to an epitope of ErbB2 different from those recognized by the clinically validated mAbs Trastuzumab and Pertuzumab, was isolated by phage display selection on live cells. A human anti-ErbB2 “compact antibody” (100kDa) was also generated by the fusion of Erbicin with a human IgG1 Fc, which was found to efficiently inhibit ErbB2-positive tumor growth both in vitro and in vivo. As a further progress, a trispecific antibody derivative, named Tribody, targeting three noncompeting epitopes on the extracellular domain of ErbB2 was obtained by fusing 3 binding moieties derived from Erb-hcAb, Trastuzumab and Pertuzumab, respectively. The triparatopic Tribody significantly downregulates ErbB2 and inhibits the cell growth of tumor cells, including those resistant to Trastuzumab. We show here that the multiparatope tribody combines and potentiates the therapeutic effects of the 3 different antibodies in 1 single antibody construct, thus allowing for the reduction of costs of antibody production and overcoming the limits related to monotherapy associated drug resistance. On the other hand, we performed a massive parallel screening of phage antibody library to obtain a large repertoire of fully human immunomodulatory antibodies against several-immune regulatory checkpoints to be used in monotherapy or in combinatorial treatments for cancer therapy. We used for the first time an innovative selection strategy on human activated lymphocytes to generate a large collection of scFvs against 10 different IC, called “Immunome Library”, from which scFvs specifically recognizing a given receptor could be pulled out by subsequent affinity selection cycles on recombinant purified proteins used as baits. By Next Generation Sequencing and bioinformatic analysis we ranked individual scFvs in each collection and identified those with the highest level of enrichment. Human IgGs from three of these collections (i.e. PD-1, PD-L1 and LAG-3) were generated and tested for their binding and biological activity. In particular, they were found to specifically bind to their targets with high affinity, to efficiently activate T cell proliferation, induce cytokine secretion and inhibit in vivo tumor growth. Interestingly, the novel isolated mAbs have comparable or even better binding affinity and biological activity than the clinically validated anti-PD-1 mAb Nivolumab. Finally, in order to explore the possibility to enhance the antitumor effects of the anti-TAA and immunomodulatory antibodies we tested some combinatorial treatments. We found that the anti-ErbB2 antibody Erb-hcAb and the novel anti-PD-L1 mAb, generated in our laboratory, showed additive antitumor effects on a panel of ErbB2-positive tumor cells, thus suggesting novel therapeutic approaches for the therapy of breast cancer

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic <it>P. pastoris </it>yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs.</p> <p>Results</p> <p>Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and <it>P. pastoris </it>cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from <it>Pichia </it>supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in <it>Pichia </it>more efficient than most other previously described trispecific or trivalent molecules produced in <it>E. coli</it>.</p> <p>Conclusion</p> <p>Bi- and tribody molecules are efficiently produced in <it>P. pastoris</it>. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of <it>P. pastoris </it>as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.</p

Restructuring Biotecnol

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA – School of Business and EconomicsThis paper is a case-based essay on a biotechnology company – Biotecnol – which is undergoing a period of crucial decisions regarding its activity: to maintain its business as it is, meaning it will continue to provide services together with the development of proprietary products; or to decide to focus on only one area giving up on the other. In order to perform this project, I had several meetings at Biotecnol’s headquarters with both the CEO and the CFO. During these encounters we had long conversations where I had the opportunity to discuss and place all the questions and doubts regarding the history of the company in order to fully understand its background and business

CD20-targeting in B-cell malignancies: novel prospects for antibodies and combination therapies

Expression of CD20 antigen by the most of transformed B cells is believed to be the driving force for targeting this molecule by using anti-CD20 monoclonal antibodies. While it is true that most lymphoma/leukemia patients can be cured, these regimens are limited by the emergence of treatment resistance. Based on these observations, development of anti-CD20 monoclonal antibodies and combination therapies have been recently proposed, in particular with the aim to optimize the cytotoxic activity. Here we outline a range of new experimental agents concerning the CD20 positive B-cell tumors which provide high benefit from conventional therapy. © 2016, Springer Science+Business Media New York

Effektormechanismen eines rekombinanten, bispezifischen Antikörpers, [(CD20)2xCD16], im Tribody-Format

Monoklonale Antikörper, wie der bei Non-Hodgkin-Lymphomen therapeutisch eingesetzte CD20-Antikörper Rituximab, vermitteln in vitro verschiedene Effektormechanismen, wie Komplement-abhängige Zytotoxizität (CDC), Antikörper-abhängige zell-vermittelte Zytotoxizität (ADCC) und Proliferationsinhibition zur Eliminierung von Tumorzellen. Verschiedene Studien im Menschen und im Tiermodell weisen darauf hin, dass die effektive Rekrutierung Fcγ Rezeptor- (FcγR-)exprimierender Effektorzellen und damit die Vermittlung von ADCC in vivo einen wichtigen Wirkmechanismus darstellt. So wurde ein Zusammenhang zwischen der Expression bestimmter FcγR Allele, die Immunglobulin G mit höherer Affinität binden, und verbesserten Ansprechraten auf eine Therapie mit monoklonalen Antikörpern und damit verbunden ein verlängertes Überleben festgestellt. Neue Strategien zur Verstärkung der ADCC könnten somit zur Entwicklung von effizienteren Antikörper-basierten Therapien führen. In der vorliegenden Arbeit wurde ein bispezifischer Antikörper, [(CD20)2xCD16], konstruiert, der CD20 auf Lymphomzellen und FcγRIII (CD16) auf Effektorzellen bindet. In den durchgeführten Studien wurden dabei keine Unterschiede in den Affinitäten von [(CD20)2xCD16] zu den verschiedenen FcγRIIIa Allelen festgestellt, wie es für Rituximab beschrieben wurde. Durch Vermittlung von ADCC als einzigen Effektormechanismus induzierte [(CD20)2xCD16] effektive Lyse humaner Lymphomzelllinien und isolierter Tumorzellen von Patienten mit unterschiedlichen B-Zell Lymphomen. In Versuchen mit isolierten Effektorzellen zeigte sich, dass [(CD20)2xCD16] mit einer gegenüber Rituximab neunfach niedrigeren, halbmaximalen effektiven Konzentration Lyse von Tumorzellen induzierte. In komplexeren Analysen mit Vollblutproben, in welchen dem monoklonalen Antikörper auch CDC als Wirkmechanismus zur Verfügung stand, vermittelte [(CD20)2xCD16] durch die Aktivierung autologer Effektoren ebenfalls eine höhere Depletion der B-Zellen. Auch in vivo war [(CD20)2xCD16] in der Lage, die B-Zellzahl in Anwesenheit autologer Effektoren zu reduzieren. Die Ergebnisse der vorliegenden Arbeit demonstrieren, dass ein bispezifischer Antikörper im Tribody-Format, [(CD20)2xCD16], zur gezielten Vermittlung von ADCC als einzigen Wirkmechanismus effektiv humane Lymphomzellen lysieren und konventionellen CD20-Antikörpern überlegen sein kann. Da bisher nicht alle Patienten von der Behandlung mit monoklonalen Antikörpern profitieren, bietet das hier beschriebene Molekül möglicherweise eine attraktive Weiterentwicklung von Antikörper-basierten Tumortherapien

Targeting ADCC: A different approach to HER2 breast cancer in the immunotherapy era

The clinical outcome of patients with human epidermal growth factor receptor 2 (HER2) amplified breast carcinoma (BC) has improved with the development of anti-HER2 targeted therapies. However, patients can experience disease recurrence after curative intent and disease progression in the metastatic setting. In the current era of evolving immunotherapy agents, the understanding of the immune response against HER2 tumor cells developed by anti-HER2 antibodies (Abs) is rapidly evolving. Trastuzumab therapy promotes Natural Killer (NK) cell activation in patients with BC overexpressing HER2, indicating that the efficacy of short-term trastuzumab monotherapy, albeit direct inhibition of HER, could also be related with antibody-dependent cell-mediated cytotoxicity (ADCC). Currently, dual HER2 blockade using trastuzumab and pertuzumab is the standard of care in early and advanced disease as this combination could confer an additive effect in ADCC. In patients with disease relapse or progression, ADCC may be hampered by several factors such as FcγRIIIa polymorphism and an immunosuppressive environment, among others. Hence, new drug development strategies are being investigated aiming to boost the ADCC response triggered by anti-HER2 therapy. In this review, we summarize these strategies and the rationale, through mAbs engineering and combinatorial strategies, focusing on clinical results and ongoing trials.Fil: Mandó, Pablo. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Centro de Educación Médica e Investigaciones Clínicas "Norberto Quirno"; ArgentinaFil: Rivero, Sergio G.. Instituto Alexander Fleming.; ArgentinaFil: Rizzo, Manglio Miguel. Universidad Austral. Hospital Universitario Austral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pinkasz, Marina. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Levy, Estrella Mariel. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Therapeutic avenues for γδ T cells in cancer

© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.γδ T cells are regarded as promising effector lymphocytes for next-generation cancer immunotherapies. In spite of being relatively rare in human peripheral blood, γδ T cells are more abundant in epithelial tissues where many tumors develop, and have been shown to actively participate in anticancer immunity as cytotoxic cells or as "type 1" immune orchestrators. A major asset of γδ T cells for tackling advanced cancers is their independence from antigen presentation via the major histocompatibility complex, which clearly sets them apart from conventional αβ T cells. Here we discuss the main therapeutic strategies based on human γδ T cells. These include antibody-based bispecific engagers and adoptive cell therapies, either focused on the Vδ1+ or Vδ2+ γδ T-cell subsets, which can be expanded selectively and differentiated or engineered to maximize their antitumor functions. We review the preclinical data that supports each of the therapeutic strategies under development; and summarize the clinical trials being pursued towards establishing γδ T cell-based treatments for solid and hematological malignancies.This study was funded by Fundação para a Ciência e Tecnologia of the Portuguese Ministério da Ciência, Tecnologia e Ensino Superior (PTDC/MED-ONC/6829/2020 to BS-S and 2021.01953.CEECIND to SM).info:eu-repo/semantics/publishedVersio

Engineered antibodies: new possibilities for brain PET?

International audienceAlmost 50 million people worldwide are affected by Alzheimer's disease (AD), the most common neurodegenerative disorder. Development of disease-modifying therapies would benefit from reliable, non-invasive positron emission tomography (PET) biomarkers for early diagnosis, monitoring of disease progression, and assessment of therapeutic effects. Traditionally, PET ligands have been based on small molecules that, with the right properties, can penetrate the blood-brain barrier (BBB) and visualize targets in the brain. Recently a new class of PET ligands based on antibodies have emerged, mainly in applications related to cancer. While antibodies have advantages such as high specificity and affinity, their passage across the BBB is limited. Thus, to be used as brain PET ligands, antibodies need to be modified for active transport into the brain. Here, we review the development of radioligands based on antibodies for visualization of intrabrain targets. We focus on antibodies modified into a bispecific format, with the capacity to undergo transferrin receptor 1 (TfR1)-mediated transcytosis to enter the brain and access pathological proteins, e.g. amyloid-beta. A number of such antibody ligands have been developed, displaying differences in brain uptake, pharmacokinetics, and ability to bind and visualize the target in the brain of transgenic mice. Potential pathological changes related to neurodegeneration, e.g. misfolded proteins and neuroinflammation, are suggested as future targets for this novel type of radioligand. Challenges are also discussed, such as the temporal match of radionuclide half-life with the ligand's pharmacokinetic profile and translation to human use. In conclusion, brain PET imaging using bispecific antibodies, modified for receptor-mediated transcytosis across the BBB, is a promising method for specifically visualizing molecules in the brain that are difficult to target with traditional small molecule ligands