Julien Villaudy

Interleukin-12 and -23 Control Plasticity of CD127+ Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria

Human group 1 ILCs consist of at least three phenotypically distinct subsets, including NK cells, CD127(+) ILC1, and intraepithelial CD103(+) ILC1. In inflamed intestinal tissues from Crohns disease patients, numbers of CD127(+) ILC1 increased at the cost of ILC3. Here we found that differentiation of ILC3 to CD127(+) ILC1 is reversible in vitro and in vivo. CD127(+) ILC1 differentiated to ILC3 in the presence of interleukin-2 (IL-2), IL-23, and IL-1β dependent on the transcription factor RORγt, and this process was enhanced in the presence of retinoic acid. Furthermore, we observed in resection specimen from Crohns disease patients a higher proportion of CD14(+) dendritic cells (DC), which in vitro promoted polarization from ILC3 to CD127(+) ILC1. In contrast, CD14(-) DCs promoted differentiation from CD127(+) ILC1 toward ILC3. These observations suggest that environmental cues determine the composition, function, and phenotype of CD127(+) ILC1 and ILC3 in the gut.

IL-1β, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs

Group 2 innate lymphoid cells (ILC2s) secrete type 2 cytokines, which protect against parasites but can also contribute to a variety of inflammatory airway diseases. We report here that interleukin 1β (IL-1β) directly activated human ILC2s and that IL-12 induced the conversion of these activated ILC2s into interferon-γ (IFN-γ)-producing ILC1s, which was reversed by IL-4. The plasticity of ILCs was manifested in diseased tissues of patients with severe chronic obstructive pulmonary disease (COPD) or chronic rhinosinusitis with nasal polyps (CRSwNP), which displayed IL-12 or IL-4 signatures and the accumulation of ILC1s or ILC2s, respectively. Eosinophils were a major cellular source of IL-4, which revealed cross-talk between IL-5-producing ILC2s and IL-4-producing eosinophils. We propose that IL-12 and IL-4 govern ILC2 functional identity and that their imbalance results in the perpetuation of type 1 or type 2 inflammation.

Critical assessment of human antibody generation in humanized mouse models

Immunodeficient mice reconstituted with human hematopoietic stem cells provide a small-animal model for the study of development and function of human hematopoietic cells in vivo. However, in the current models, the immune response, and especially the humoral response by the human immune cells is far from optimal. The B cells found in these mice exhibit an immature and abnormal phenotype correlating with a reduced capacity to produce antigen-specific affinity matured antibodies upon infection or immunization. Herein, we review the current state of knowledge of development, function and antibody production of human B cells and discuss the obstacles for the improvement of these models.

PO-436 Retargeting T-cell cytotoxicity to a unique sialylated epitope on CD43 expressed by acute myeloid leukaemia

Introduction From the B cell repertoire of an acute myeloid leukaemia (AML) patient in long-term remission after hematopoietic stem cell transplantation we recently identified the antibody AT1413. AT1413 binds CD43s, a unique sialylated epitope on CD43 present on AML and myeloid cells but not on B and T cells. Besides its therapeutic potential as a naked antibody, AT1413 provides an interesting candidate for a bispecific T-cell engaging antibody (bTCE). bTCEs have been clinically validated as a powerful tool for harnessing the cytotoxicity of polyclonal T cells against a hematologic cancer. Simultaneous binding to a cancer surface antigen and the T-cell surface protein CD3ε mediates cancer cell recognition and T-cell mediated killing independent of the T-cell receptor specificity. Material and methods To generate an AT1413 bTCE, we first modified AT1413 to abolish Fc-receptor interaction. Second, we assembled the bispecific by chemo-enzymatic linkage using a combination of a sortase-catalysed transpeptidation reaction and a subsequent cycloaddition reaction. AML target cell lysis by T-cells was assessed in vitro in a cytotoxicity assay. Up-regulation of T-cell activation markers CD69 and CD25 and cytokine production were monitored as indicators for T-cell activation. T-cell proliferation was assessed. In vivo , AT1413 bTCE was tested in two mouse models, one where human PBMCs were co-injected at the start of bTCE treatment and the other in which a human immune system (HIS) was engrafted at birth. Results and discussions AT1413 bTCE was confirmed to retain dual binding capacity for both AML cells and CD3ε-expressing Jurkat cells. In vitro, AT1413 bTCE successfully induced T-cell mediated cytotoxicity against different CD43s expressing AML cell lines as well as primary AML blasts. Endothelial cells that have a detectable but considerably lower binding capacity for AT1413 remained unaffected. Besides cytotoxicity, T-cell activation and T-cell proliferation were observed and were dependent on the presence of target-expressing AML cells. In vivo testing of AT1413 bTCE dosed at 2 mg/kg revealed potent AML tumour growth inhibition of 89%–99% in two mouse models when compared with a control bTCE. In the HIS model, normal human hematopoietic cells remained present in mice treated with AT1413 bTCE. Conclusion Our results indicate that CD43s is a potential new target for T-cell engaging antibodies. Consequently, AT1413 holds therapeutic potential not only as an unmodified antibody, but also in a bispecific, T-cell engaging format.

Abstract LB-200: A patient derived antibody targeting the tetraspanin CD9 synergistically inhibits tumor growth with an anti PD1 antibody

Background. It is generally accepted that immune reactions against cancer cells can be induced by immunotherapy. Here, we investigated the possibility that an antibody response has contributed to the success of the immunotherapy of a cancer patient. Methods. A patient with metastatic melanoma was successfully treated by adoptive transfer of ex vivo expanded autologous tumor reactive T cells1. This patient is still tumor free 9 years after treatment. Peripheral blood memory B cells were immortalized by ectopic Bcl-6 and Bcl-xL expression2 and analyzed for the presence of tumor-reactive B cells. Results. We isolated one B cell clone that produced an antibody, named AT1412, recognizing a novel cell surface epitope on the tetraspanin CD9 which is broadly expressed and involved in multiple cellular activities including proliferation and adherence. AT1412 bound more strongly to melanoma cells than to melanocytes and other healthy tissues indicating that the AT1412 epitope is overexpressed on tumor cells. In addition, AT1412 strongly reacted with other tumor types including colon, pancreas and, breast cancer. Further analysis revealed that AT1412 favors binding to a clustered state of CD9. CD9 clusters are dependent on palmitoylation and known to be present on metastatic cells3. Previously published high affinity mouse antibodies targeting human CD9 were found to trigger aggregation of platelets and thus unfit for therapeutic use4. In sharp contrast, AT1412 does not induce platelet aggregation. To address whether AT1412 fails to aggregate platelets because of its low affinity for CD9 we generated high affinity variants of AT1412 using our highly efficient affinity maturation platform. We obtained AT1412 variants with affinities up to 250 fold higher than the wild type AT1412 comparable to that of mouse anti-human CD9 antibodies. These high affinity variants still did not aggregate platelets indicating that the absence of platelet aggregation by AT1412 is due to the spatial arrangement of the AT1412 epitope present on CD9 and not its low affinity. The antibody was able to reduce growth of melanoma tumors and block metastasis in melanoma xenografted mice. A striking synergistic inhibition of tumor growth was observed in mice that were treated with our CD9 antibody in the presence of a clinically successful anti-PD1 checkpoint inhibitor antibody. Discussion. These data suggest that the antibody contributed to the success of the immunotherapy in this patient. This antibody could act together with tumor-reactive T cells in eradicating circulating tumor cells and/or preventing settlement of metastatic tumor cells. Importantly, no antibody-related adverse effects were observed during and after treatment of this patient indicating that the antibody is safe for use in humans. 1. Verdegaal, Cancer Immunol Immunother, 2011 2. Kwakkenbos, Nat Med, 2010 3. Yang, JBC, 2006 4. Boucheix, FEBS Letter, 1983 Citation Format: Remko Schotte, Pauline van Helden, Daniel Go, Christien Fatmawati, Els Verdegaal, Camile Bru, Julien Villaudy, Koen Wagner, Sjoerd van der Burg, Hergen Spits, Wouter Pos. A patient derived antibody targeting the tetraspanin CD9 synergistically inhibits tumor growth with an anti PD1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-200. doi:10.1158/1538-7445.AM2017-LB-200

Abstract A061: Acute myeloid leukemia patients cured after allogeneic hematopoietic stem cell transplantation generate tumor-specific cytotoxic antibodies that kill AML blasts

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) can cure acute myeloid leukemia (AML) when the donor immune system generates a potent graft versus leukemia (GvL) response. While the role of T cells and NK cells in GvL immune responses has been established, the contribution of B cells to GvL responses is less clear. Using SEREX and other techniques, the presence of antibodies directed against established tumor antigens following allogeneic HSCT has been demonstrated, but because these antibodies were not obtained in monoclonal format, the function of these antibodies could not be analyzed. Aim: To investigate the role of antibodies produced by donor-derived B cells in GvL responses. Methods: We selected five patients with high-risk AML who remained disease-free for more than 5 years after allogeneic HSCT and thus have mounted a potent GvL response. From the peripheral blood of these patients we isolated memory B cells that we transduced with Bcl-6 and Bcl-xL, to establish antibody-producing clonal B cell lines. Blood was obtained 2 years after allogeneic HSCT. B cell lines were screened for the production of antibodies that specifically bound to surface antigens on AML cell lines and AML blasts isolated from patients in our clinic. Target identification was performed by immunoprecipitation and mass-spectometry. Results: We identified patient derived clonal B cell lines producing antibodies that recognized antigens expressed on the cell surface of AML cells, but not on normal hematopoietic and non-hematopoietic cells. Antibodies were donor-derived, and a number of these antibodies recognized the U5 snRNP200 complex. The U5 snRNP200 complex is a component of the spliceosome that in normal cells is located in the nucleus but that is exposed on the cell membrane of AML cells. U5 snRNP200 complex-specific antibodies were specific for allogeneic HSCT recipients with AML, as they were found in in 4 out of 5 AML patients screened, but were not found in multiple myeloma patients who received an allogeneic HSCT or in healthy individuals. Strikingly, U5 snRNP200 complex-specific antibodies induced death of AML cells in vitro, and, in a human AML mouse model, in vivo. Cell death was induced in the absence of cytotoxic leukocytes or of complement, through a non-apoptotic process that depended on destabilization of the cytoskeleton as cell death could be blocked by incubation of the target cells with cytochalasin D, an actin polymerization inhibitor. Cytotoxicity of the U5 snRNP200 antibodies was present at 4°C and 37°C, suggesting that cell death was induced by a passive process. Indeed, interaction of the antibodies with their target cells did not induce a calcium flux. Cytotoxicity of the antibodies depended on the Fc region of the antibody, since recombinant U5 snRNP200 complex-specific antibodies with a defective Fc region were not cytotoxic. Summary and Conclusions: Allogeneic HSCT recipients with robust donor anti-AML immunity generate antibodies against a component of the spliceosome, the U5 snRNP200 complex, that is expressed on the membrane of AML blasts. U5 snRNP200 antibodies are cytotoxic in vivo and in vitro, demonstrating the potency of the humoral immune system in tumor immunology. Citation Format: Marijn Gillissen, Martijn Kedde, Greta De Jong, Etsuko Yasuda, Sophie Levie, Arjen Bakker, Yvonne Claassen, Koen Wagner, Julien Villaudy, Martino Bohne, Dave Speijer, Paul Hensbergen, Pauline van Helden, Tim Beaumont, Hergen Spits, Mette D. Hazenberg. Acute myeloid leukemia patients cured after allogeneic hematopoietic stem cell transplantation generate tumor-specific cytotoxic antibodies that kill AML blasts [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A061.

Abstract A026: Tumor-specific glycosylated CD43 is a novel and highly specific target for acute myeloid leukemia and myelodysplastic syndrome

Background: Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are high-risk diseases with a poor prognosis. Even with intensive treatment regimens less than 50% of patients can be cured, and for the majority of patients - those over 65 years of age and/or patients with comorbidities - such intensive regimens are not feasible. Novel therapeutic approaches such as immunotherapy directed against a highly specific tumor target are highly needed. Aims: The aim of our study was to identify novel therapeutic antibodies that are highly specific for AML and to discover novel tumor-specific antigens, widely expressed on AML and MDS but not on healthy hematopoietic and non-hematopoietic cells. Methods: For this we made use of the oldest human tumor immunology model with proven efficacy available: an allogeneic HSCT patient with a potent graft versus AML allo-immune response. From this patient we isolated CD27+ IgG+ memory B lymphocytes and transduced these cells with Bcl-6 and Bcl-xL, thereby generating pre-plasmablast B cell clones that produce abundant antibodies. Supernatants of these B cell clones were used to screen for binding to surface antigens on the AML cell line THP-1. Results: We identified an IgG1 antibody, AT14-013, that specifically interacted with AML cell lines THP-1, MOLM-13, SH-2 and others, and with leukemic blasts isolated from newly diagnosed AML and MDS patients from our clinic. AT14-013 did not interact with healthy hematopoietic and non-hematopoietic cells. This antibody was of donor origin and was antigen experienced as it contained 26/11 somatic hypermutations in the heavy and light chains, respectively. Target identification using mass spectrometry analysis and epitope mapping strategies with FLAG-tagged truncated variants of CD43 expressed by THP-1 that we created revealed CD43 as the target. CD43 is expressed by all hematopoietic cells, but AT14-013 targeted a specific, sialylated epitope on CD43 that is uniquely and widely expressed on all types of AML, as illustrated by its reactivity with blasts of each of 48 randomly selected AML and MDS patients in our clinic. AT14-013 induced antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity of AML cell lines and primary blasts. Summary and conclusion: We have identified onco-sialylated CD43 (CD43os) as a novel tumor-specific target that is widely expressed on AML and MDS blasts. Antibodies against this target have high potential as therapeutic antibodies, either as a naked antibody or manufactured into an antibody-drug conjugate, bispecific T cell engager or CAR (chimeric antigen receptor) T cell. Citation Format: Marijn Gillissen, Martijn Kedde, Etsuko Yasuda, Greta De Jong, Sophie Levie, Arjen Bakker, Paul Hensbergen, Julien Villaudy, Tim Beaumont, Pauline van Helden, Hergen Spits, Mette D. Hazenberg. Tumor-specific glycosylated CD43 is a novel and highly specific target for acute myeloid leukemia and myelodysplastic syndrome [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A026.