Alexandra Jacquet

Antigen-specific T-T interactions regulate CD4 T-cell expansion

The regulation of CD4 T-cell numbers during an immune response should take account of the amount of antigen (Ag), the initial frequency of Ag-specific T cells, the mix of naive versus experienced cells, and (ideally) the diversity of the repertoire. Here we describe a novel mechanism of T-cell regulation that potentially deals with all of these parameters. We found that CD4 T cells establish a negative feedback loop by capturing their cognate major histocompatibility class (MHC)/peptide complexes from Ag-presenting cells and presenting them to Ag-experienced CD4 T cells, thereby inhibiting their recruitment into the response while allowing recruitment of naive T cells. The inhibition is Ag specific, begins at day 2 (long before Ag disappearance), and cannot be overcome by providing new Ag-loaded dendritic cells. In this way, CD4 T-cell proliferation is regulated in a functional relationship to the amount of Ag, while allowing naive T cells to generate repertoire variety.

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.

Preclinical study of Ublituximab, a Glycoengineered anti-human CD20 antibody, in murine models of primary cerebral and intraocular B-cell lymphomas.

PURPOSE Primary cerebral lymphoma (PCL) and primary intraocular lymphoma (PIOL) belong to the systemic diffuse large B-cell lymphoma family and are characterized by the presence of CD20(+) lymphoma B cells in the brain or the eye. These highly aggressive malignancies have a poor prognosis and no specific therapy. The presence of effector immune cells in the damaged brain and vitreous suggests that treatment with anti-human CD20 (hCD20) monoclonal antibodies might be effective. We developed murine models of PCL and PIOL to assess the intracerebral and intraocular antitumor effect of ublituximab, a promising glycoengineered anti-hCD20 mAb with a high affinity for FcγRIIIa (CD16) receptors. METHODS The murine lymphoma B-cell line A20.IIA-GFP-hCD20 (H-2(d)) was injected into the right cerebral striatum or the vitreous of immunocompetent adult BALB/c mice (H-2(d)). Four to 7 days later, ublituximab was injected intracerebrally or intravitreously into the tumor site. Rituximab was the reference compound. Survival was monitored for injected mice; histopathological and flow cytometric analyses were performed to study tumor growth and T-cell infiltration. RESULTS Single doses of ublituximab, injected intracerebrally or intravitreously, had a marked antitumor effect, more pronounced than that obtained with the same dose of rituximab in these conditions. The reduction in tumor cells was correlated with an increased proportion of CD8(+) T cells. This efficacy was observed only against lymphoma B cells expressing hCD20. CONCLUSIONS These in vivo results confirm the potential of the glycoengineered anti-hCD20 mAb ublituximab as an innovative therapeutic approach to treat primary central nervous system lymphoma and other B-cell lymphomas.

Long Peptide Vaccination Can Lead to Lethality through CD4+ T Cell-Mediated Cytokine Storm

The optimization of anticancer therapeutic vaccines can lead to better efficacy but also to stronger adverse effects. In a mouse model of antitumor vaccination using a long peptide (LP), which included MHC class I- and II-restricted male (H-Y) epitopes, we observed unexpected mortality. Mice with an increased frequency of anti–H-Y CD4 T cells were primed with LP+CpG and boosted 10 d later. Within hours of boost, they displayed shock-like signs with high mortality. Serum cytokine levels were high. TNF-α secreted by the CD4 T cells was identified as the key effector molecule. Priming with a short peptide (SP), which included the MHC class II-restricted epitope, was a more efficient primer than LP, but did not lead to mortality when used as boost. The high mortality induced by LP compared with SP was probably related to its specific ability to be presented by B cells. Finally, targeting the LP sequence to dendritic cells allowed tumor protection without side effects. Our data: 1) confirm that the immune system can be very dangerous; 2) caution against the use of systemic activation of high-frequency Ag-specific T cells as induced by high doses of LP; and 3) underline the benefit of targeting Ag to dendritic cells.

The Dendritic Cell–like Functions of IFN-Producing Killer Dendritic Cells Reside in the CD11b+ Subset and Are Licensed by Tumor Cells

IFN producing killer dendritic cells (IKDC) were originally defined as CD11c(int) B220(+)NK1.1(+) (or CD49b(+)) cells that exert a potent tumoricidal activity in animals lacking B, T, and conventional natural killer effectors. MHC class II expression on tumor infiltrating IKDC prompted us to investigate their putative antigen presenting function. Here, we show that tumor cells license IKDC to acquire the properties of antigen presenting cells, i.e., expression of MHC class II and costimulatory CD86 molecules. We show that the CD11b(+) subset of IKDC are able to prime naïve CD4(+) T cells and cross-prime naïve CD8(+) T lymphocytes. Licensing of IKDC by tumor cells was mandatory for the full differentiation of T cells into polarized effectors. IKDC could engulf and process soluble Ova protein in a CD206-dependent manner. Finally, we show that CD11b(+)IKDC is selectively endowed with CTLA4Ig-inhibitable antigen presenting capacities and that targeting this subset with the detoxified adenylate cyclase toxin of Bordetella pertussis fused to antigen resulted in efficient cross-presentation of antigen by IKDC to specific TCR transgenic CD8(+)T cells in vivo. Collectively, our data indicate that upon exposure to tumor cells, IKDC subserve DC-like functions.

Intratumor CD4 T-Cell Accumulation Requires Stronger Priming than for Expansion and Lymphokine Secretion

T cells need to migrate to and accumulate inside tumors before mediating rejection of the tumor. The number of specific T cells inside tumors may depend on the efficiency of priming in the draining lymph node (DLN), intratumor deletion, suppressive phenomena, or both. We used monoclonal anti-male antigen CD4 (Marilyn) T cells and tumor cell lines expressing or not the corresponding antigen (Dby) to analyze CD4 T-cell accumulation in tumors. Priming by MHC II(+) or MHC II(-) male splenocytes or Dby(+) tumor cells induced similar Marilyn T-cell expansion in the DLN and recirculation in other lymph nodes and capacity to produce IFN-gamma. However, intratumor accumulation was different for each priming condition. In mice with Dby(-) tumors, MHC II(+) male splenocyte priming induced greater, although not statistically significant, Marilyn T-cell accumulation in the tumors than MHC II(-) male splenocyte priming. In mice with Dby(+) tumors, priming in the tumor DLN induced less Marilyn T-cell intratumor accumulation than priming by MHC II(+) male splenocytes. We saw comparable differences for Marilyn T-cell accumulation in gut lamina propria, suggesting that priming affects effector T-cell accumulation in inflamed tissues. Mature dendritic cells were loaded with graded doses of Dby peptide to control for antigen-presenting cell characteristics during priming. We observed similar proliferation, with higher concentrations inducing higher intratumor accumulation. Thus, intratumor accumulation requires stronger stimulation than for proliferation or the capacity to secrete lymphokines. In this system, priming intensity alone can explain the number of intratumor T cells without having to call for intratumor deletion or suppression phenomena.

Modeling the Specific CD4+ T Cell Response against a Tumor Neoantigen

The antitumor activity of CD4+ T cells is increasingly acknowledged in both humans and mice. The involved mechanisms have been mostly studied using transplanted tumor mouse systems. In these models, many tumor cells die at the time of implantation leading to the release of Ag in an inflammatory context contrasting with the slow and nondestructive growth of early-stage human tumors. In this study, we show that the presentation of a MHC class II–restricted model Ag (male, DBY) released by dying tumor cells may last more than 4 wk. The duration of Ag presentation varies according to the way the cells are killed before implantation. To avoid this artifactual early priming of the host precluding the study of the interactions between the immune system and tumors at the steady state, we generated a cell line expressing the DBY Ag in an inducible manner. Ag expression can be efficiently induced in vivo several days after tumor implantation. We show that the Ag reaches the lymph node and activates naive CD4+ T cells to proliferate and recirculate. We did not observe de novo induction of tumor-specific regulatory T cells. However, we observed Th1/Th17 effector cells in the tumor draining lymph node and tumors. Thus, when a neoantigen appears in established tumors, the immune system is not ignorant and naive CD4+ T cells are not tolerized. This opens up the possibility of therapeutic vaccines improving the immune response toward tumor-specific neoantigens.

Correction: Modeling the Specific CD4+ T cell Response against a Tumor Neoantigen

Flament, H., R. A. Ramirez, V. Premel, N. T. Joncker, A. Jacquet, S. Scholl, and O. Lantz. 2015. Modeling the specific CD4+ T cell response against a tumor neoantigen. J. Immunol. 194: [3501–3512][1]. The authors wish to correct an error made in the preparation of Fig. 6. A panel from Fig. 6B

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