Multi-targeting of the HER (ErbB) family in pancreatic cancer : validation of a novel therapy based on tetravalent bispecific antibodies and their comparison in vitro and in vivo with monospecific parental antibody combinations

Le cancer du pancréas est la quatrième cause de décès par cancer en Europe, avec un nombre croissant de cas chaque année. Si aucun progrès thérapeutique n'est réalisé, ce cancer sera la troisième cause de décès par cancer après le cancer du sein en 2030. Actuellement, l'adénocarcinome pancréatique (90 % des cancers du pancréas) est traité soit par chirurgie, soit par chimiothérapie, soit par une combinaison de radiothérapie et de chimiothérapie. La famille des récepteurs tyrosine kinases HER (ErbB) joue un rôle essentiel pour réguler la prolifération, l'invasion, la différenciation et la survie cellulaire. Suite à l'activation par de nombreux ligands, les récepteurs HER modifient leur structure tridimensionnelle en une conformation active capable de former des homo- ou hétéro-dimères hautement mitogènes modulant la signalisation et la carcinogenèse. Une signalisation HER anormale dans les tumeurs peut être causée par une surexpression du récepteur, une activité constitutive mutationnelle ou une surexpression des ligands. Elle entraîne une prolifération cellulaire et une dépendance à l'oncogène, rendant ainsi ces récepteurs, impliqués dans la transduction du signal, intéressants pour une thérapie ciblée en cancérologie. Le récepteur EGFR est exprimé dans 45 à 95% des cancers du pancréas et son expression est généralement corrélée à un mauvais pronostic dans les cancers du pancréas après résection. La surexpression de HER2 a également été décrite dans 7-58% des cancers du pancréas. L'expression de HER3, moins importante que celles d’EGFR et de HER2, est corrélée à la progression tumorale et à la survie réduite des patients.À ce jour, plusieurs anticorps monoclonaux (MAbs) dirigés contre les monomères EGFR et HER2 et HER3 ont été autorisés et sont utilisés dans divers types de cancer, ou évalués en phase I/II chez des patients. Il a été démontré que les mélanges de MAbs ciblant EGFR, HER2 ou HER3 améliorent l'inhibition de la croissance tumorale par une meilleure internalisation et dégradation des récepteurs. Notre équipe a démontré l'intérêt de cibler les hétérodimères EGFR/HER2, par des combinaisons de MAbs pour inhiber la croissance tumorale pancréatique. Enfin, un mélange d'anticorps (Pan-HER), qui cible simultanément EGFR, HER2 et HER3, s'est révélé efficace pour surmonter l'hétérogénéité tumorale. Les succès cliniques obtenus par les MAbs ainsi que leurs limites ont conduit au développement d’une deuxième génération d'anticorps thérapeutiques incluant les anticorps bispécifiques (BsAbs). Ces BsAbs ciblent doublement les récepteurs membranaires cellulaires impliqués dans la progression du cancer. La famille HER se prête bien à cette deuxième approche BsAb avec des molécules en études cliniques.Dans ce projet, nous utiliserons un format d’anticorps bispécifique innovant développé par Biomunex Pharmaceuticals. Ces BsAbs sont des IgG1 tétravalentes de 250 kDa, possédant 2 sites de liaison (Fabs) pour chacun des 2 antigènes différents, et un domaine Fc fonctionnel. Le choix des Fabs ciblant simultanément les dimères HER s’est fait à partir de deux anticorps anti-EGFR (cetuximab et matuzumab), deux anti-HER2 (trastuzumab et pertuzumab) et deux anti-HER3 (patritumab et elgemtumab). Mes travaux de thèse avaient pour objectifs (1) de sélectionner le BsAb le plus efficace dans le cancer du pancréas, (2) d’étudier in vitro et in vivo des effets biologiques des BsAbs en comparaison avec les MAbs parentaux et les combinaisons correspondantes, (3) de mieux connaitre la biologie des BsAbs, (4) d’apporter des connaissances innovantes sur la biologie des systèmes de la famille HER, intégrant à la fois des données sur la signalisation, la dégradation des récepteurs, la prolifération et l’apoptose, avec les effets sur la réponse immunitaire.Pancreatic cancer is the fourth leading cause of cancer death in Europe, with an increasing number of cases each year. If no therapeutic progress is made, this cancer will be the third leading cause of cancer death after breast cancer by 2030. Currently, pancreatic adenocarcinoma (90% of pancreatic cancers) is treated either by surgery, chemotherapy or a combination of radiation and chemotherapy. The HER family of receptor tyrosine kinases (ErbB) plays an essential role in regulating cell proliferation, invasion, differentiation and survival. Upon activation by multiple ligands, HER receptors change their three-dimensional structure into an active conformation capable of forming highly mitogenic homo- or heterodimers modulating signaling and carcinogenesis. Abnormal HER signaling in tumors can be caused by receptor overexpression, mutational constitutive activity or ligand overexpression. It leads to cell proliferation and oncogene dependence, making these receptors, involved in signal transduction, interesting for targeted therapy in cancer. The EGFR receptor is expressed in 45-95% of pancreatic cancers and its expression is generally correlated with a poor prognosis in pancreatic cancers after resection. HER2 overexpression has also been described in 7-58% of pancreatic cancers. HER3 expression, which is less important than EGFR and HER2 expression, is correlated with tumor progression and reduced patient survival.To date, several monoclonal antibodies (MAbs) directed against EGFR and HER2 and HER3 monomers have been approved and are being used in various types of cancer or evaluated in phase I/II patients. Mixtures of MAbs targeting EGFR, HER2 or HER3 have been shown to improve tumor growth inhibition through enhanced receptor internalization and degradation. Our team has demonstrated the interest of targeting EGFR/HER2 heterodimers by combinations of MAbs to inhibit pancreatic tumor growth. Finally, a combination of antibodies (Pan-HER), which simultaneously targets EGFR, HER2 and HER3, has been shown to overcome tumor heterogeneity. The clinical success of MAbs and their limitations have led to the development of a second generation of therapeutic antibodies including bispecific antibodies (BsAbs). These BsAbs target dual cell membrane receptors involved in cancer progression. The HER family is well suited for this second BsAb approach with molecules in clinical trials.In this project, we will use an innovative bispecific antibody format developed by Biomunex Pharmaceuticals. These BsAbs are tetravalent IgG1 of 250 kDa, with two binding sites (Fabs) for each of the two different antigens, and a functional Fc domain. The choice of Fabs simultaneously targeting HER dimers was made from two anti-EGFR antibodies (cetuximab and matuzumab), two anti-HER2 antibodies (trastuzumab and pertuzumab) and two anti-HER3 antibodies (patritumab and elgemtumab). The objectives of my thesis were (1) to select the most effective BsAb in pancreatic cancer, (2) to study in vitro and in vivo biological effects of BsAbs in comparison with parental MAbs and corresponding combinations, (3) to better understand the biology of BsAbs, (4) to provide innovative knowledge on the biology of HER family systems, integrating data on signaling, receptor degradation, proliferation and apoptosis, with effects on the immune respons

Anti-tumoral activity of the Pan-HER (Sym013) antibody mixture in gemcitabine-resistant pancreatic cancer models

International audienc

Presence of T cells directed against CD20-derived peptides in healthy individuals and lymphoma patients

International audiencePreclinical and clinical studies have suggested that cancer treatment with antitumor antibodies induces a specific adaptive T cell response. A central role in this process has been attributed to CD4+ T cells, but the relevant T cell epitopes, mostly derived from non-mutated self-antigens, are largely unknown. In this study, we have characterized human CD20-derived epitopes restricted by HLA-DR1, HLA-DR3, HLA-DR4, and HLA-DR7, and investigated whether T cell responses directed against CD20-derived peptides can be elicited in human HLA-DR-transgenic mice and human samples. Based on in vitro binding assays to recombinant human MHC II molecules and on in vivo immunization assays in H-2 KO/HLA-A2+-DR1+ transgenic mice, we have identified 21 MHC II-restricted long peptides derived from intracellular, membrane, or extracellular domains of the human non-mutated CD20 protein that trigger in vitro IFN-γ production by PBMCs and splenocytes from healthy individuals and by PBMCs from follicular lymphoma patients. These CD20-derived MHC II-restricted peptides could serve as a therapeutic tool for improving and/or monitoring anti-CD20 T cell activity in patients treated with rituximab or other anti-CD20 antibodies

Image_4_Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.tif

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.</p

Table_5_Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.xlsx

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.</p

Table_1_Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.xlsx

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.</p

Table_4_Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.xlsx

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.</p

Image_1_Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.tif

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.</p