Genoway (France)

Abstract B-cell maturation antigen (BCMA) targeting gained a rapid approval for multiple myeloma, and is currently investigated in B-cell lymphomas. Here, we report that the trans Golgi network (TGN) retained BCMA due to a motif in its transmembrane/cytoplasmic domain in diffuse large B-cell lymphomas (DLBCL). To achieve signaling, one of its ligands, a proliferation inducing ligand (APRIL), bound to surface heparan sulfate proteoglycans (HSPGs), got endocytosed by the calveolin pathway and trafficked to the TGN via the retrograde route. In the TGN, APRIL/BCMA interactions activated the NF-κB pathway. BCMA ubiquitination followed by proteosomal degradation mediated signal termination. Growth impairment of DLBCL xenografts in APRIL -deficient animals confirmed the in vivo activity of TGN BCMA. Our study constitutes the first description for a signal activity from the Golgi apparatus among the TNF receptor family.

BackgroundCurrent therapeutic approaches involving gd T cell include adoptive cell transfer, in vivo stimulation and combined therapies. While preliminary results are promising, investigation of such therapies in preclinical models is challenging, because γδ T cells are not developed at satisfactory levels in most of the humanized mouse models.MethodsHere we described the presence and functionality of γδ T cell in BRGSF (Balb/C Rag2-/-, IL2Rγ-/-, SIRPαNOD and Flt3-/-), a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year1 and mice do not develop GvHD.Resultsγδ T cells develop in BRGSF-HIS and are present in blood, spleen, lung, thymus and bone marrow. In blood, TCRγδ+ cells frequency in blood is variable (intra and inter-donor) and represent 3–5% of CD3+ cells in average. Vδ1, Vδ2 and Vδ3 are detected in blood and organs, and, as expected, Vδ1 and Vδ3 frequencies are increased in tissues compared to blood. A Vδ1-targeting agent induced ex vivo increase on activation markers CD25, 4-1BB, and CD69 expression, while decreasing CD27, a naïve gd T marker, suggesting that Vδ1 T cells developed in the model are functional. Similarly, an ‘Expander’ targeting Vγ9 significantly increased Vγ9 expressing cells frequencies. Additionally, the expression of cell activation marker CD25 increased, as well as the serum levels of perforin and INF-γ in mice treated with the expander. Interestingly, γδ T cells were recruited into the tumor microenvironment of the triple negative breast cancer line MDA-MB-231 and the colorectal adenocarcinoma HT29. The recruitment of γδ T cells into the TME suggests that therapies designed to expand and activate γδ T cells could be assessed in BRGSF-HIS mice. Investigation of efficacy of γδ T cells still remains to be investigated in the model.ConclusionsThe development of functional γδ T cells in BRGSF-HIS mice brings a new perspective on the assessment of therapies targeting this cell population in humanized mouse models.ReferenceLabarthe L, Henriquez S, Lambotte O, Di Santo JP, Le Grand R, Pflumio F, ArcangeliM-L, Legrand N, Bourgeois C. Exhaustion and senescence marker profiles on human T cells in BRGSF-A2 humanized mice resemble those in human samples. J Leukoc Biol 2020;107(1):27–42.Ethics ApprovalThis study was conducted in compliance with CEE directive 86/609 of 24th November 1986 on the harmonization of laws, regulations or administrative provisions relating to the protection of animals used for experimental or other scientific purposes. It was also conducted in compliance with the French law, décret n° 87.848 of 19th October 1987 modified by the décret n° 2001-464 of 29th May 2001 and the décret n° 2001-486 of the 6th June 2001. From 1st February 2013, the CEE directive 2010/63/UE of 22nd September 2010 and the French décret n° 2013-118 of 1st February 2013 were applied. The study was performed in non-GLP conditions.

BackgroundThe relevance of preclinical models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. BRGSF (Balb/C Rag2-/-, IL2Rγ-/-, SIRPαNOD and Flt3-/-) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year1 and mice do not develop GvHD. Additionally, myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models which overexpress human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment.MethodsWe describe here the immune cell infiltration in the tumor microenvironment (TME) following engraftment of cell lung cancer A549, human triple negative cancer (TNBC) cell MDA-MB-231, and human pancreatic adenocarcinoma HPAFII in BRGSF-HIS mice.ResultsEffect of Flt3L-induced boost of myeloid compartment is seen in an A549 tumor model. A single boost prior to the tumor cell inoculation associates with increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, and reduced tumor-infiltrating NK cells in the TME. MDA-MB-231 and HPAFII cell lines are widely used in cancer research and drug development.2 We show that upon implantation in BRGSF-HIS mice, in vivo growth of human cell line-derived xenograft is CD34+ donor independent and the TME composition varies according to the cell line used. As seen in TNBC patients (Zheng et al., 2021); TME of MDA-MB-231-bearing BRGSF-HIS mice is enriched in myeloid cells, mostly CD206+/CD163+ M2 macrophages. CD163-expressing M2 macrophages represent the main tumor-associated macrophages, and are known to promote breast cancer initiation, angiogenesis, invasion, and metastasis by generating an immunosuppressive TME. Interestingly, TME composition evolves over time, with increased T cells frequency in later stage (tumor volume over 1000 mm3 compared to 500 mm3) depicting cancer cell plasticity. Conversely, TME of HPAFII-bearing mice is mainly composed of T cells enabling T cells-based therapies.ConclusionsThese data suggest that BRGFS-HIS mice is a valuable tool to investigate immune cell infiltration in the TME, enabling a translatable assessment of mechanism of action of immunotherapies.ReferencesLabarthe L, Henriquez S, Lambotte O, Di Santo JP, Le Grand R, Pflumio F, ArcangeliM-L, Legrand N, Bourgeois C. Exhaustion and senescence marker profiles on human T cells in BRGSF-A2 humanized mice resemble those in human samples. J Leukoc Biol 2020;107(1):27–42.Zheng H, Siddharth S, Parida S, Wu X, Sharma D. Tumor microenvironment: key players in triple negative breast cancer immunomodulation. Cancers 2021;13(13):3357.Ethics ApprovalThis study was conducted in compliance with CEE directive 86/609 of 24th November 1986 on the harmonization of laws, regulations or administrative provisions relating to the protection of animals used for experimental or other scientific purposes. It was also conducted in compliance with the French law, décret n° 87.848 of 19th October 1987 modified by the décret n° 2001-464 of 29th May 2001 and the décret n° 2001-486 of the 6th June 2001. From 1st February 2013, the CEE directive 2010/63/UE of 22nd September 2010 and the French décret n° 2013-118 of 1st February 2013 were applied. The study was performed in non-GLP conditions.

Abstract Agonist antibodies targeting CD28 have proven to be effective against cancer, but also faced challenges due to severe adverse events triggered by its activation. Human (hCD28) and mouse CD28 (mCD28) have different signaling responses, with CD28 ligands and superagonists inducing pro-inflammatory cytokines upon stimulation in absence of TCR ligation in humans, but not in mice. Expression of CD28i amplifier isoform, which is thought to enhanced the production of cytokines in humans, could partially explain this difference, as it is not expressed in mice. In addition, evidence suggests that the different signaling between hCD28 and mCD28 relies on one amino acid change in the intracellular domain (ICD)[1]. Herein, we describe a CD28 humanized mouse model for assessment of CD28-targeting agents. To improve translatability, we decided to keep the expression of both canonical and CD28i human isoforms to avoid undermining the biological effects of the testing agents. Although keeping the human ICD could favor the evaluation of cytokine production and therefore the safety of the test agents, we decided to keep the mouse ICD to enable a proper interaction of CD28 with its signaling partners, allowing a physiological stimulation of CD28 in efficacy studies. The hCD28 model expresses a chimeric protein, with the whole human extracellular domain, and mouse intracellular domain. Human CD28 is expressed at physiological levels on T cells, and these cells can be activated and proliferate in response to hCD28 ligands and agonist antibodies. Tumor growth inhibition and complete tumor regressions are achieved in a MC38-TAA colorectal cancer model following prophylactic treatment with CD28-TAA bispecific antibody in combination with anti-PD-1. Extension of this study and re-challenged of the tumor-free surviving animals shows that re-challenged hCD28 mice have enhanced survival compared to naïve hCD28 mice inoculated with MC38-TAA, suggesting tumor immunological memory. Furthermore, TGN1412 administration in CD28 mice induces cytokine release, and a slightly body weight loss, but no major change in body temperature. Although the model does not fully reproduce the ability of human CD28 to produce cytokines upon activation due to the mouse ICD, the expression of the amplifier isoform CD28i might contribute to the response to TGN1412. Altogether, data suggest that hCD28 model enables assessment of efficacy and safety of CD28-targeting agents. The hCD28 model was intercrossed with CD3 humanized models to enable assessment of combination therapies and bispecific antibodies targeting both CD3 and CD28. [1] Porciello N, Grazioli P, Campese AF, et al. A non-conserved amino acid variant regulates differential signalling between human and mouse CD28. Nat Commun 2018; 9:1-16 Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Yacine Cherifi, Patricia Isnard-Petit, Kader Thiam. CD28 humanized mouse model for efficacy and safety assessment of CD28-targeting therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5292.

Abstract Gliomas are the most common neoplasm in the central nervous system (CNS). Although immunotherapy has shown efficacy in several tumor types, no immunotherapies are approved by the US Food and Drug Administration for treatment of gliomas. One of the biggest challenges in using immunotherapies in the CNS is the existence of the blood-brain barrier (BBB), which limits drug delivery and the subsequent therapeutic drug efficacy. In addition to biodistribution, assessment of PK in preclinical models do not reflect the clinical findings. Conjugation of therapeutic agents with molecules binding to TRFC expressed along the BBB are being used as one of the alternative approaches to deliver drugs into the CNS. Herein, we describe a novel TFRC humanized mouse model (hTFRC), developed to enable assessment of compounds targeting human TFRC and aiming to deliver drug through the BBB. hTFRC model was developed by knock-in at the mouse endogenous locus to enable a physiological expression of TFRC. Splenocytes from hTFRC mice stimulated with labelled pHrodo Red human transferrin show internalization of human transferrin, suggesting that humanization of the transferrin receptor does not change the endocytosis process. In vivo, treatment of hTFRC mice with anti-BACE1/anti-TFRC antibodies enables the transfer of anti-BACE1 to the brain, while anti-BACE1 antibody is not. As a consequence of the drug delivery, mice treated with anti-BACE1/anti-TFRC show a marked reduction in Amyloid-β 1-40 levels in the brain. Altogether, the data suggest that hTFRC is expressed in the BBB and enables the efficient shuttling of therapeutic antibodies to the brain. While assessment of TFRC-targeting antibodies in a glioma model remains to be performed, the hTFRC model is a promising tool to assess delivery and efficacy of immunotherapies in gliomas. Moreover, the hTFRC model is also being intercrossed with the well-established hSA/FcRn humanized mouse model (Viuff et al., 2016), to enable a more translatable PK and biodistribution of compounds targeting TFRC. Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Burak Ozgür, Peter Ravn, Yacine Cherifi, Florence Sotty, Patricia Isnard-Petit, Kader Thiam. Novel TFRC humanized mouse model for drug delivery to the brain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2045.

In autoimmune diseases, autoreactive B cells comprise only the 0.1-0.5% of total circulating B cells. However, current first-line treatments rely on non-specific and general suppression of the immune system, exposing patients to severe side effects. For this reason, identification of targeted therapies for autoimmune diseases is an unmet clinical need. Here, we designed a novel class of immunotherapeutic molecules, Bi-specific AutoAntigen-T cell Engagers (BiAATEs), as a potential approach for targeting the small subset of autoreactive B cells. To test this approach, we focused on a prototype autoimmune disease of the kidney, membranous nephropathy (MN), in which phospholipase A2 receptor (PLA2R) serves as primary nephritogenic antigen. Specifically, we developed a BiAATE consisting of the immunodominant Cysteine-Rich (CysR) domain of PLA2R and the single-chain variable fragment (scFv) of an antibody against the T cell antigen CD3, connected by a small flexible linker. BiAATE creates an immunological synapse between autoreactive B cells bearing an CysR-specific surface Ig+ and T cells. Ex vivo, the BiAATE successfully induced T cell-dependent depletion of PLA2R-specific B cells isolated form MN patients, sparing normal B cells. Systemic administration of BiAATE to mice transgenic for human CD3 reduced anti-PLA2R antibody levels following active immunization with PLA2R. Should this approach be confirmed for other autoimmune diseases, BiAATEs could represent a promising off-the-shelf therapy for precision medicine in virtually all antibody-mediated autoimmune diseases for which the pathogenic autoantigen is known, leading to a paradigm shift in the treatment of these diseases.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy that reduces cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by an FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 32.8 nM and NB-ACE2 KD = 31.7 nM) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ β = 13.5 h) than soluble ACE2 (t½ β = 2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. Statement of significanceThe COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHA-ACE2), that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19.

Abstract T cell engagers show high efficacy in B cells malignances. High risk of immune-related adverse events, including cytokine release syndrome (CRS), is reported in patients due to on-target off-site effects of T cell engagers. Thus, reliable and translational mouse models are required to predict potential safety issues and investigate their rescue. Here we describe two preclinical models mimicking to some extent CRS induction upon activation with anti-CD3 antibodies.BRGSF (Balb/C Rag2-/-, IL2Rγ-/-, SIRPαNOD and Flt3-/-) is a highly immunodeficient mouse featuring reduced murine myeloid cells. Development of human lymphoid (B and T cells) and myeloid (NK, cDC, pDC and monocytes) compartments upon CD34+ HSC-engraftment are observed in blood, spleen and bone marrow and are stable for over a year, without side effects. Anti-hCD3 mAb, OKT3, administration in BRGSF-HIS mice induced a rapid release of human cytokines (i.e., IL-6, TNF-α, IFN-γ, IL-2) in serum. Pretreatment with Flt3L, enhancing the human myeloid compartment, boosted the production of cytokines. CRS clinical signs such as hypothermia and weight lost were reproduced in OKT3-injected BRGSF-HIS mice. Pretreatment with Tocilizumab (an anti-IL-6R) reduced cytokine production, suggesting a strong role of myeloid cells in the induced-CRS pathogenesis. The main limitation of this model is the lack of a fully functional crosstalk between human immune cells and mouse stroma, which can undermine endothelial cells contribution in CRS. Alternatively, the CD3ε N-terminal epitope knock-in mice expressing the humanized epitope of anti-CD3 clone SP34 (hCD3ε mice) enables the investigation of endothelial versus immune cells dialogue in CRS pathogenesis. hCD3ε mice display a functional CD3-TCR complex and a functional T and B cooperation, as shown by T cell proliferation upon SP34 activation and antigen-specific antibodies production, respectively. Cytotoxic responses were induced by various bispecific antibodies, ex vivo and in vivo. Splenocytes from hCD3ε mice activated with BsAb1 bispecific antibody targeting CD3ε and a tumor-associated antigen (TAA) induced mouse cytokines production (IFN-γ, TNF-α, IL-6, IL-1β, IL-10) in a concentration-dependent manner. A 2nd BSAb targeting CD3ε and an irrelevant TAA didn’t induce any cytokine release, confirming the specificity of the previous cytokine induction.These data suggest that both BRGSF-HIS and hCD3ε models could be used to assess T cell engager-induced CRS in a complementary setting: while BRGSF-HIS mice enable monitoring of any anti-hCD3 agent, the model might undermine the contribution of mouse endothelial cells due to specie specificity barrier of human cytokines and mouse cells. In contrast, hCD3ε mice enable monitoring of cytokine induction upon activation with SP34-derived anti-hCD3 taking into consideration a full crosstalk between immune and endothelial cells. Citation Format: Perrine Martin-Jeantet, Florence Renart-Depontieu, Ludovic Bourre, Gaëlle Martin, Angela Pappalardo, Dean O. Campbell, Astrid Doerner, Yacine Cherifi, Fabiane Sônego, Kader Thiam. BRGSF-HIS and CD3Ε humanized mice: Translatable preclinical mouse models for assessment of T-cell engagers-induced CRS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4036.

BackgroundThe breakthrough of immunotherapies has unleashed new hope and new success for cancer therapy. However, the choice of a preclinical model is one of the main challenges as they are important for evaluation of translatability to help support testing in clinical studies, including potential efficacy and tolerability of immunotherapies during preclinical development.The development of mouse models featuring a human immune system (HIS) provides new paths for the investigation of the efficacy of immunotherapies in preclinical models engrafted with human tumors. Although these models provided a breakthrough in the assessment of immune targeting agents, they also come with a few significant caveats. These include: a lack of a mature human myeloid compartment in the mouse, and a short life span of the model when this compartment is promoted at non-physiological levels via the over-expression of human cytokines. Here, we report a novel mouse model (BRGSF-HIS), featuring functional human lymphoid and myeloid compartments. The human immune response of this model was assessed through examination of the immune cells composition in the tumor microenvironment (TME), and its ability to respond to biologics known to trigger cytokine release syndrome (CRS).MethodsBRGSF (balb/C Rag2-/-, IL2Rg-/-, SIRPaNOD and Flt3-/-) is a highly immunodeficient mouse, with reduced murine myeloid cells, which allows long term CD34+ HSC-engraftment and development of human lymphoid and myeloid compartments (human CD141+ and CD1c+ DC subsets, CD123+ pDC, CD14+ monocytes), in blood, spleen and bone marrow. The engraftment is stable for over twelve months with no side effects. The effect of exogenous human Flt3 ligand (Flt3L) on the composition of TME in A549 model, and an anti-CD3 antibody (OKT3)-induced CRS, were assessed.ResultsExogenous human Flt3L significantly and transiently increased the proportion of human myeloid cells. This can be recalled by continuous dosing of Flt3L. Assessment of tumor immunobiology in A549 model, showed increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, while tumor-infiltrating NK cells were decreased. The presence of myeloid cells provides a new opportunity for assessment of myeloid targeted therapies, as proven using pDC-depleting antibodies. OKT3 administration resulted in CRS symptoms including a temperature drop, body weight loss and a change in serum cytokine levels. Symptoms were mitigated upon administration of tocilizumab, suggesting the contribution of the myeloid compartment in the response observed.ConclusionsThese data demonstrate that BRGSF-HIS mice support development of functional human myeloid cells and that this mouse model enables preclinical evaluation of cancer immunotherapy in vivo.