Nathalie Fournier

Chronic lymphocytic leukaemia cells are efficiently killed by an anti‐CD20 monoclonal antibody selected for improved engagement of FcγRIIIA/CD16

Patients with chronic lymphocytic leukaemia (CLL) treated with a combination of fludarabine, cyclophosphamide and rituximab show a high response rate. However, only a poor response is observed following rituximab monotherapy. The use of chemo‐immunotherapy is often associated with haematological and infectious complications. Thus, an antibody with an enhanced ability to kill CLL cells could lead to better clinical responses to antibody monotherapy and the possibility of lowering drug doses during chemo‐immunotherapy. We generated a chimeric anti‐CD20 monoclonal antibody (mAb), EMAB‐6, which has a low fucose content. Apoptosis and complement activities for EMAB‐6 were similar to those seen for rituximab. By contrast, EMAB‐6 mAb showed improved Fcγ receptor IIIA (FcγRIIIA)/CD16 binding and FcγRIIIA‐dependent effector functions. It induced a higher in vitro antibody‐dependent cellular cytotoxicity against CLL cells and a higher FcγRIIIA‐mediated interleukin‐2 production by FcγRIIIA+ Jurkat cells in the presence of CLL cells at both low and maximally saturating concentrations. Comparative studies between CLL and lymphoma cells coated with EMAB‐6 or rituximab indicated that the difference of efficacy was more pronounced at low doses and when target cells expressed fewer CD20 molecules. Thus, EMAB‐6 mAb represents a promising drug candidate for the treatment of CLL by inducing a strong cytotoxicity against tumour cells that express low CD20 levels.

Rise and Fall of an Anti-MUC1 Specific Antibody

Background So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate. Results A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10−10 M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells. Conclusions Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these “best in class” binding parameters, the therapeutic success of this antibody was prevented by the target biology.

Selection of IgG Variants with Increased FcRn Binding Using Random and Directed Mutagenesis: Impact on Effector Functions

Despite the reasonably long half-life of immunoglogulin G (IgGs), market pressure for higher patient convenience while conserving efficacy continues to drive IgG half-life improvement. IgG half-life is dependent on the neonatal Fc receptor (FcRn), which among other functions, protects IgG from catabolism. FcRn binds the Fc domain of IgG at an acidic pH ensuring that endocytosed IgG will not be degraded in lysosomal compartments and will then be released into the bloodstream. Consistent with this mechanism of action, several Fc-engineered IgG with increased FcRn affinity and conserved pH dependency were designed and resulted in longer half-life in vivo in human FcRn-transgenic mice (hFcRn), cynomolgus monkeys, and recently in healthy humans. These IgG variants were usually obtained by in silico approaches or directed mutagenesis in the FcRn-binding site. Using random mutagenesis, combined with a pH-dependent phage display selection process, we isolated IgG variants with improved FcRn-binding, which exhibited longer in vivo half-life in hFcRn mice. Interestingly, many mutations enhancing Fc/FcRn interaction were located at a distance from the FcRn-binding site validating our random molecular approach. Directed mutagenesis was then applied to generate new variants to further characterize our IgG variants and the effect of the mutations selected. Since these mutations are distributed over the whole Fc sequence, binding to other Fc effectors, such as complement C1q and FcγRs, was dramatically modified, even by mutations distant from these effectors’ binding sites. Hence, we obtained numerous IgG variants with increased FcRn-binding and different binding patterns to other Fc effectors, including variants without any effector function, providing distinct “fit-for-purpose” Fc molecules. We therefore provide evidence that half-life and effector functions should be optimized simultaneously as mutations can have unexpected effects on all Fc receptors that are critical for IgG therapeutic efficacy.

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGenTM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.

Improved in vitro and in vivo activity against CD303-expressing targets of the chimeric 122A2 antibody selected for specific glycosylation pattern

ABSTRACT Plasmacytoid dendritic cells (pDCs) play a central role for both innate and adaptive antiviral responses, as they direct immune responses through their unique ability to produce substantial concentrations of type I interferon (IFNs) upon viral encounter while also activating multiple immune cells, including macrophages, DCs, B, natural killer and T cells. Recent evidence clearly indicates that pDCs also play a crucial role in some cancers and several auto-immune diseases. Although treatments are currently available to patients with such pathologies, many are not fully efficient. We are proposing here, as a new targeted-based therapy, a novel chimeric monoclonal antibody (mAb) that mediates a strong cellular cytotoxicity directed against a specific human pDC marker, CD303. This antibody, ch122A2 mAb, is characterized by low fucose content in its human IgG1 constant (Fc) region, which induces strong in vitro and in vivo activity against human pDCs. We demonstrated that this effect relates in part to its specific Fc region glycosylation pattern, which increased affinity for CD16/FcγRIIIa. Importantly, ch122A2 mAb induces the down-modulation of CpG-induced IFN-α secretion by pDCs. Additionally, ch122A2 mAb shows in vitro high pDC depletion mediated by antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis. Remarkably, in vivo ch122A2 mAb efficacy is also demonstrated in humanized mice, resulting in significant pDC depletion in bloodstream and secondary lymphoid organs such as spleen. Together, our data indicates that ch122A2 mAb could represent a promising cytotoxic mAb candidate for pathologies in which decreasing type I IFNs or pDCs depleting may improve patient prognosis.

Abstract 2528: 3C23K: an anti-human Müllerian inhibiting substance type II receptor humanized monoclonal antibody for ovarian cancer targeted therapy

Asymptomatic in early stages, ovarian cancer is a “silent killer” representing the fifth leading cause of female deaths in western countries. Every year 56,967 women in Europe and USA die as a consequence of this disease. The incidence of ovarian cancer is forecast to undergo a 13 % increase in the next eight years in the seven major developed countries to reach about 72,000 annual cases in 2019. Due to the limitations of the current therapeutic approaches, there is a strong need for novel, more efficient, therapies. For this reason, we have produced a humanized monoclonal antibody 3C23K targeting the human Mullerian Inhibiting Substance type II Receptor (MISRII), expressed on most ovarian cancer subtypes, including epithelial ovarian cancer (EOC) representing more than 90% of ovarian cancers. This monoclonal antibody derives from the murine monoclonal antibody 12G4 and displays a particular glycosylation profile known to favor effector recruitment (EMABling®) as previously demonstrated in vitro. In vivo, we also showed that 3C23K exhibited a significant effect on tumor growth against several ovarian tumor xenografted models derived from patient primary EOC tumors. In this study, we first confirmed by peptide microarray that the epitope of 3C23K antibody was strictly identical to that of 12G4 antibody and a 3D-model of the full MISRII molecule was generated in order to better localize the 3C23K epitope. Furthermore, SPR studies demonstrated cross-reactivity of 3C23K with MISRII of rabbit, dog, pig, cow and primate. In vivo, antitumor activity of 3C23K against xenografted EOC tumor models was confirmed with various concentrations and treatment schedules. Moreover, interestingly, we constructed a mutant form of 3C23K harboring two mutations in the Fc region (G236R/L328R) in order to prevent binding to both murine and human FcαRs, and demonstrated that such a modification abolished antitumor activity. This data confirmed in vivo that recruitment of effectors is essential for 3C23K efficacy. Finally, in order to anticipate clinical treatment of ovarian cancers, 3C23K was tested in vivo in combination with carboplatin. Combination, when compared with treatment of each product alone, resulted in more than additive antitumor activity. Altogether these data showed that humanized monoclonal antibody 3C23K represents a promising candidate for ovarian cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2528. doi:1538-7445.AM2012-2528

Abstract 4574: Towards a new targeted therapy for ovarian cancer : Development of an anti-human müllerian inhibiting substance type II receptor humanized antibody

Asymptomatic in early stages, ovarian cancer is a “silent killer” corresponding to the 5th cause of female deaths in US. In 2010 it is estimated that 21,880 women in US will be diagnosed with ovarian cancer. Due to the limitations of the current therapeutic approaches, there is a strong need for novel, more efficient, therapies. We describe here a humanized monoclonal antibody 3C23K targeting the human Mullerian Inhibiting Substance type II Receptor (MISRII), expressed on most ovarian cancer subtypes, including epithelial ovarian cancer (EOC) representing more than 90% of ovarian cancers. Starting from murine monoclonal antibody 12G4, 3C23K was obtained following chimerization, humanization by CDR grafting and affinity maturation. Moreover, 3C23K displays a particular glycosylation profile known to favor effector recruitment in vitro and in vivo (EMABling®). Assessed on MISRII transfected cells (cov434-MISRII), 3C23K was characterized by an affinity constant to the antigen increased by 3 fold as compared to murine 12G4. In order to evaluate 3C23K efficacy in vitro and in vivo, we have established several MISRII expressing cell lines derived from patient primary EOC tumors. Functional characterization of 3C23K has been carried out in vitro, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity, apoptosis and intracellular signaling. In particular, 3C23K was found to display a higher ADCC activity on MISRII expressing cell lines as compared to the same sequence expressed in CHO cells. In vivo, 3C23K exhibited a significant effect on tumor growth against several ovarian tumor xenografted models derived from patient primary EOC tumors. Altogether these data strongly suggest that humanized monoclonal antibody 3C23K may represent a promising candidate for ovarian cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4574. doi:10.1158/1538-7445.AM2011-4574