Abstract INTRODUCTION: 4-1BB (CD137) is an inducible co-stimulatory molecule that plays crucial roles in immune activation. Upon ligand binding, 4-1BB forms trimeric signaling complexes triggering receptor activation. However, therapeutic monoclonal antibodies targeting 4-1BB often face challenges due to peripheral toxicities arising from systemic receptor activation. To address this, bispecific antibodies (bsAbs) have been developed to selectively activate 4-1BB in a tumor-associated antigen (TAA)-dependent manner. This report investigates whether a combination of bsAbs, pairing 4-1BB and TAA targeting arms, can further enhance 4-1BB clustering and activation in the tumor microenvironment. METHODS: A panel of Fc-silent bsAbs targeting different domains on 4-1BB and HER2 were generated and tested in combination for optimal 4-1BB signaling. For that purpose, 4-1BB reporter cells were co-cultured with HER2-expressing tumor cells. Functional in vitro assays were conducted using primary human T cells isolated from peripheral blood mononuclear cells to assess cytokine production and their tumoricidal activity. In vivo efficacy was evaluated in PBMC-engrafted NOG mice inoculated with JIMT-1, a HER2-expressing tumor cell line. RESULTS: The simultaneous binding of two bsAbs to the same epitope on 4-1BB, while targeting distinct epitopes on HER2, markedly enhances 4-1BB signaling, compared to the single bsAbs. 4-1BB signaling triggered by the bsAbs is driven by HER2 expression, with strong IL-2 release observed only in the presence of HER2-positive cells. In JIMT-1 xenograft models, combination of the bsAb pair with a T cell engager (TCE) resulted in superior antitumor activity and enhanced CD4+ and CD8+ T-cell infiltration within the tumor microenvironment, compared to either single bsAb plus TCE or the TCE alone. CONCLUSIONS: This study introduces a novel bsAb combination strategy that amplifies TAA-driven activation of 4-1BB+ T cells and may unlock similar opportunities to other TNFR superfamily targets such as OX40 and CD40. Citation Format: Lucie Diby, Pauline Malinge, Valery Moine, Lise Nouveau, Laurence Chatel, Krzysztof Masternak, Limin Shang, Walter Ferlin, Nicolas Fischer, Jose Saro, Mikael Pittet, Vanessa Buatois, Eric Hatterer. Tumor-selective activation of 4-1BB receptor via bispecific antibody combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5553.

Background Owing to their roles in promoting T cell and natural killer (NK) cell activation and proliferation, interleukins-2 (IL-2) and interleukins-15 (IL-15) have been pursued as promising pathways to target in cancer immunotherapy. Nonetheless, their wider therapeutic application has been hampered by severe dose-limiting toxicities including systemic cytokine release and organ edema for IL-2, and inconvenient intratumoral administration for IL-15. To address these safety issues, we generated IL-2R/IL-15R×TAA (tumor-associated antigen) bispecific antibody (bsAb) pairs to selectively activate IL-2R signaling in the tumor microenvironment.Methods Each bsAb pair is composed of one bsAb targeting CD122 and a TAA epitope, and the other bsAb targeting CD132 and the same or a different TAA epitope. In vitro assays were performed to characterize the IL-2R/IL-15R agonistic activity of the bsAb pairs, as well as their capacity to enhance T-cell-mediated killing of TAA+ malignant cells. Using a syngeneic mouse tumor model, in vivo biological activity and systemic toxicity of the bsAb pairs were assessed in comparison with IL-2. The in vivo antitumor activity was assessed in combination with an anti-mouse programmed cell death protein 1 (mPD-1) monoclonal antibody.Results We demonstrated with two different TAAs (human epidermal growth factor receptor 2 (HER2) and mesothelin (MSLN)) that the CD122×TAA/CD132×TAA bsAb pairs mediate effective activation of immune cells exclusively in the presence of TAA+ tumor cells. In syngeneic hMSLN-MC38 tumor-bearing mice, the CD122×MSLN-1/CD132×MSLN-2 bsAb pair promotes selective activation and expansion of NK cells and central memory CD8+ T cells inside the tumor without inducing organ edema or systemic cytokine release, two well-known manifestations of IL-2 associated toxicity. In combination with checkpoint inhibitor anti-mPD-1, the bsAb pair boosts the accumulation of CD8+ effector T cells and NK cells, leading to a favorable CD8+ T cell to CD4+ regulatory T cell ratio for a more robust inhibition of tumor growth.Conclusions Overall, the findings suggest that this innovative therapeutic approach effectively leverages the antitumor activity of IL-2 and IL-15 pathways while minimizing their associated systemic toxicities. This dual bsAb format holds potential for broader application in other immune-activating pathways.

BackgroundBlocking the CD47 “don’t eat me”-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells.MethodsWe present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format).ResultsNILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses.ConclusionNILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.

Background T-cell retargeting to eliminate CEACAM5-expressing cancer cells via CEACAM5xCD3 bispecific antibodies (BsAbs) showed limited clinical activity so far, mostly due to insufficient T-cell activation, dose-limiting toxicities, and formation of anti-drug antibodies (ADA). Methods We present here the generation and preclinical development of NILK-2301, a BsAb composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). Results NILK-2301 binds CD3ɛ on T-cells with its lambda light chain arm with an affinity of ≈100 nM, and the CEACAM5 A2 domain on tumor cells by its kappa light chain arm with an affinity of ≈5 nM. FcγR-binding is abrogated by the “LALAPA” mutation (Leu234Ala, Leu235Ala, Pro329Ala). NILK-2301 induced T-cell activation, proliferation, cytokine release, and T-cell dependent cellular cytotoxicity of CEACAM5-positive tumor cell lines (5/5 colorectal, 2/2 gastric, 2/2 lung), e.g., SK-CO-1 ( E max = 89%), MKN-45 ( E max = 84%), and H2122 ( E max = 97%), with EC 50 ranging from 0.02 to 0.14 nM. NILK-2301 binds neither to CEACAM5-negative or primary colon epithelial cells nor to other CEACAM family members. NILK-2301 alone or in combination with checkpoint inhibition showed activity in organotypic tumor tissue slices and colorectal cancer organoid models. In vivo, NILK-2301 at 10 mg/kg significantly delayed tumor progression in colon- and a pancreatic adenocarcinoma model. Single-dose pharmacokinetics (PK) and tolerability in cynomolgus monkeys at 0.5 or 10 mg/kg intravenously or 20 mg subcutaneously showed dose-proportional PK, bioavailability ≈100%, and a projected half-life in humans of 13.1 days. NILK-2301 was well-tolerated. Data were confirmed in human FcRn TG32 mice. Conclusions In summary, NILK-2301 combines promising preclinical activity and safety with lower probability of ADA-generation due to its format compared to other molecules and is scheduled to enter clinical testing at the end of 2023.

To enhance efficacy of anti-PD-1/PD-L1 antibodies, many combinations with various therapeutic agents are being investigated. Blocking the CD47/SIRPα myeloid checkpoint with monoclonal antibodies (mAbs) or decoy receptors is emerging as an effective approach to mobilize dendritic cells and macrophages to support T-cell mediated antitumor responses. The benefit of combining CD47/SIRPα and PD-1/PD-L1 blockade to improve tumor control has been convincingly demonstrated in preclinical models and is now being explored in patients. However, CD47 mAbs are hindered by ubiquitous CD47 expression, leading to pharmacokinetic (PK) and safety issues.NI-2901, an IgG4 CD47xPD-L1 bispecific antibody (bsAb), was generated using the κλ-body platform. In vitro assays were used to characterize its binding profile and checkpoint inhibition as well as its capacity to enhance T-cell activation and macrophage-mediated phagocytosis of tumor cells. PD-L1-independent CD47 antitumor activity was assessed in vivo in a PD-L1-negative xenograft model and compared to the anti-CD47 magrolimab analog. PK and tolerability of NI-2901 were evaluated in non-human primates (NHP), allowing for translational modeling to predict PK and dosing regimens in humans. Consistent with its intermediate affinity to CD47, NI-2901 shows lower binding to RBC as compared to magrolimab analog and is still able to induce CD47/SIRPα blockade on PD-L1-negative tumor cells, that is significantly enhanced once PD-L1 is expressed. As a result, the bsAb is able to enhance the phagocytosis of PD-L1-negative and -positive tumor cell lines induced by mAbs targeting tumor-associated antigens (e.g. rituximab, trastuzumab and anti-CD19) and demonstrates in vivo activity in the Raji B-cell lymphoma xenograft model. Given its high affinity for PD-L1, NI-2901 triggers an effective blockade of the PD-1/PD-L1 interaction, inducing T-cell activation in vitro to a degree similar to anti-PD-L1 benchmark antibodies atezolizumab and avelumab. In immunocompetent huCD47/huSIRPα-transgenic mice engrafted with MC38 cells engineered to express human PD-L1 and CD47, NI-2901 displayed significant anti-tumor activity. In a NHP study, NI-2901 was well-tolerated after four weekly injections at 30mg/kg, showing no signs of hemotoxicity. In contrast, the magrolimab analog induced a significant drop in RBC already after a single injection at 10mg/kg. PK modeling and simulations in humans suggest a more favorable dosing regimen as compared to CD47 targeted approaches. In conclusion, NI-2901, a dual immune checkpoint inhibitor, triggered effective T-cell activation and enhanced phagocytosis of tumor cells. Also, NI-2901 demonstrated significant antitumor activity in vivo and is therefore expected to show improved clinical efficacy over PD-1/PD-L1 blockade alone. The bsAb was well-tolerated in NHP without inducing RBC or platelet depletion. Citation Format: Xavier Chauchet, Sebastien Calloud, Pauline LLoveras, Nicolas Bosson, Margaux Legrand, Laurence Chatel, Laura Cons, Adeline Lesnier, Pauline Malinge, Guillemette Pontini, Christophe Guillamo, Dmitry Shchelokov, Oleg Demin, Ulla Ravn, Valéry Moine, Bruno Daubeuf, Giovanni Magistrelli, Yves Poitevin, Susana Salgado-Pires, Limin Shang, Nicolas Fischer, Walter Ferlin, Krzysztof Masternak. NI-2901, an affinity-optimized CD47xPD-L1 bispecific antibody for dual immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2951.

the CODEX ® instrument and readily deployable on commercially available fluorescence microscopy systems. Using a 30+ antibody CODEX ® panel, we compared formalin-fixed paraffin embedded (FFPE) human breast cancer tissues at different stages of disease progression with normal breast tissues. Our antibody panel was designed to detect cancer cells as well as non-malignant cells in order to comprehensively survey the tumor microenvironment and normal control tissues. Data were analyzed using the CODEX ® software suite to identify key cell types and analyze spatial associations. Results Our analyses revealed more than 20 distinct cell types in human breast cancer and normal tissues. Cell populations, biomarker expression and cellular spatial distributions differed distinctly between cancerous and normal breast tissues. Differences were robust, repeatedly observed and indicative of altered cellular milieus in normal versus cancerous breast tissues. Conclusions Collectively, these data establish CODEX ® as a readily deployable and practical tool for spatially-resolved, highly multiplexed biomarker analysis of human FFPE samples. Disclosure Information O. Braubach: A. Employment (full or part-time); Significant; Akoya Biosciences. S. Basak: A. Employment (full or part-time); Significant; Akoya Biosciences. M. Gallina: A. Employment (full or part-time); Significant; Akoya Biosciences. W. Lee: A. Employment (full or part-time); Significant; Akoya Biosciences. J. Kim: A. Employment (full or part-time); Significant; Akoya Biosciences. C. Hempel: A. Employment (full or part-time); Significant; Akoya Biosciences. E. Williams: A. Employment (full or part-time); Significant; Akoya Biosciences. O. Shang: A. Employment (full or parttime); Significant; Akoya Biosciences. B. Cheung: A. Employment (full or part-time); Significant; Akoya Biosciences. J. Kennedy-Darling: A. Employment (full or part-time); Significant; Akoya Biosciences.

To enhance efficacy of anti-PD-1/PD-L1 antibodies, many combinations with various therapeutic agents are being investigated. Blocking the CD47/SIRP a checkpoint with monoclonal antibodies (mAbs) or decoy receptors is emerging as an effective approach to mobilize myeloid cells and support T-cell mediated antitumor responses. The benefit of combining CD47/SIRP a and PD-1/PD-L1 blockade to improve tumor control has been demonstrated in preclinical models and is being explored in patients. However, CD47 mAbs are hin-dered by ubiquitous CD47 expression leading to pharmacoki-netic (PK) and safety issues.

Blocking the CD47/SIRPα checkpoint has recently emerged as an effective approach to mobilize the myeloid cell compartment and to improve antitumor responses in the clinic. Preclinical models have demonstrated the synergistic benefit of combined CD47/SIRPα and PD-1/PD-L1 blockade. Combinations of monoclonal antibodies (mAbs) targeting these two checkpoint pathways are being explored in the clinic. CD47xPD-L1 bispecific antibodies (bsAbs) stand as an attractive alternative to mAb combinations, even more so as they provide a potential solution to improve the pharmacokinetic profile and safety issues faced by CD47 targeted-mAbs and SIRPα-Fc fusion proteins. CD47xPD-L1 bsAbs are expected to preferentially inhibit CD47 on PD-L1 expressing cells, displaying improved safety and pharmacokinetics, but also superior tumor microenvironment targeting capabilities. With the objective of finding the optimal CD47xPD-L1 bsAb, an array of bispecific antibodies (bsAbs) was generated associating a high affinity PD-L1 arm to CD47 arms with varying affinities. The CD47xPD-L1 bsAbs of human IgG4 isotype were generated using our fully human κλ body antibody platform. The candidate molecules were screened for binding and receptor-blocking activity and tested for their capacity to enhance T-cell activation and phagocytosis of tumor cells in the presence of anti-HER-2 mAb, trastuzumab. Selected bsAbs were also evaluated in a xenograft mouse model. The CD47xPD-L1 bsAbs demonstrated an effective blockade of the PD-1/PD-L1 interaction, being able to induce T-cell activation in vitro similar to the anti-PD-L1 clinical benchmark, atezolizumab. Consistent with their CD47 affinities, the bsAbs showed varying levels of CD47 blockade on PD-L1-negative cells and a low binding capacity to red blood cells. Nonetheless, trastuzumab-mediated phagocytosis of tumor cells expressing low levels of PD-L1 could be significantly enhanced by these bsAbs, confirming the PD-L1-independent activity of the CD47 blocking arms. The latter findings were corroborated in vivo using PD-L1-negative Raji cells in a xenograft mouse model. Selected bsAbs will be now tested for tolerability and pharmacokinetic profiles in human-CD47/human-SIRPα transgenic mice. Lead candidate(s) will be evaluated further for PK and safety attributes in non-human primates in early Q1, 2022. Citation Format: Xavier Chauchet, Sébastien Calloud, Margaux Legrand, Laura Cons, Laurence Chatel, Pauline Lloveras, Coline Burnet-Merlin, Louis Hellequin, Nicolas Bosson, Pauline Malinge, Nicolas Pleche, Jérémie Bourguignon, Guillemette Pontini, Christophe Guillamo, Ulla Ravn, Valéry Moine, Bruno Daubeuf, Yves Poitevin, Giovanni Magistrelli, Limin Shang, Walter Ferlin, Krzysztof Masternak. NI-2901, a CD47xPD-L1 bispecific antibody for dual immune checkpoint blockade with fine-tuned affinity to reduce erythrocyte binding and improve biodistribution [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 3428.

CD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model. The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments. We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells. The study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).

The aberrant release of inflammatory mediators often referred to as a cytokine storm or cytokine release syndrome (CRS), is a common and sometimes fatal complication in acute infectious diseases including Ebola, dengue, COVID-19, and influenza. Fatal CRS occurrences have also plagued the development of highly promising cancer therapies based on T-cell engagers and chimeric antigen receptor (CAR) T cells. CRS is intimately linked with dysregulated and excessive cytokine release, including IFN-γ, TNF-α, IL 1, IL-6, and IL-10, resulting in a systemic inflammatory response leading to multiple organ failure. Here, we show that mice intravenously administered the agonistic hamster anti-mouse CD3ε monoclonal antibody 145-2C11 develop clinical and laboratory manifestations seen in patients afflicted with CRS, including body weight loss, hepatosplenomegaly, thrombocytopenia, increased vascular permeability, lung inflammation, and hypercytokinemia. Blood cytokine levels and gene expression analysis from lung, liver, and spleen demonstrated a hierarchy of inflammatory cytokine production and infiltrating immune cells with differentiating organ-dependent kinetics. IL-2, IFN-γ, TNF-α, and IL-6 up-regulation preceded clinical signs of CRS. The co-treatment of mice with a neutralizing anti-cytokine antibody cocktail transiently improved early clinical and laboratory features of CRS. We discuss the predictive use of this model in the context of new anti-cytokine strategies to treat human CRS.

Mesothelin (MSLN) is a cell surface glycoprotein overexpressed in several solid malignancies, including gastric, lung, mesothelioma, pancreatic and ovarian cancers. While several MSLN-targeting therapeutic approaches are in development, only limited efficacy has been achieved in patients. A potential shortcoming of several described antibody-based approaches is that they target the membrane distal region of MSLN and, additionally, are known to be handicapped by the high levels of circulating soluble MSLN in patients. We show here, using monoclonal antibodies (mAbs) targeting different MSLN-spanning epitopes, that the membrane-proximal region resulted in more efficient killing of MSLN-positive tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Surprisingly, no augmented killing was observed in antibody-dependent cellular phagocytosis (ADCP) by mAbs targeting this membrane-proximal region. To further increase the ADCP potential, we, therefore, generated bispecific antibodies (bsAbs) coupling a high-affinity MSLN binding arm to a blocking CD47 arm. Here, targeting the membrane-proximal domain of MSLN demonstrated enhanced ADCP activity compared to membrane-distal domains when the bsAbs were used in in vitro phagocytosis killing assays. Importantly, the superior anti-tumor activity was also translated in xenograft tumor models. Furthermore, we show that the bsAb approach targeting the membrane-proximal epitope of MSLN optimized ADCC activity by augmenting FcγR-IIIA activation and enhanced ADCP via a more efficient blockade of the CD47/SIRPα axis.

Background Mesothelin (MSLN) is recognized as a relevant tumor-associated antigen for cancer immunotherapy, because of its overexpression on various solid tumors, including mesothelioma, pancreatic, lung, gastric and ovarian carcinoma. However, an anti-MSLN monoclonal antibody (mAb), amatuximab, has demonstrated only limited efficacy in clinical trials. It has been already demonstrated that the targeting of a membrane-distal domain of an antigen with a mAb is suboptimal at inducing Fc-related effector functions. As amatuximab targets a membrane-distal domain of MSLN, we investigated whether mAbs targeting different epitopes would bestow a better efficacy. Furthermore, in order to incorporate novel modalities to enhance tumor-killing, we have paired these MSLN targeting arms with an anti-CD47 arm to generate bispecific antibodies (bsAb). Indeed, the ‘don’t eat me signal’ CD47 is a promising target in cancer and therapeutic blockade has recently showed clinical evidence of efficacy. Therefore, we investigated the contribution of a CD47 arm and the impact of the different anti-MSLN targeting arms on the tumoricidal activities of CD47xMSLN bsAbs. Materials and Methods A panel of anti-MSLN mAbs and CD47xMSLN biAbs carrying the same anti-CD47 arm and different anti-MSLN arms were generated and characterized for their epitope specificity. Their tumor cell killing efficacy in vitro and in vivo was analyzed using cell-based assays, xenograft models and various MSLN+ human malignant cell lines originated from different tissues (e.g., lung, gastric and hepatic origin). Results Our data revealed that all CD47xMSLN bsAbs, regardless of the recognized MSLN epitope, showed higher activity than the corresponding anti-MSLN mAbs in tumor-cell killing assays and demonstrated superior anti-tumor activity in a xenograft model. Targeting a membrane-proximal epitope rendered an anti-MSLN mAb more effective in mediating antibody-dependent cell-mediated cytotoxicity (ADCC) but did not optimize antibody dependent cellular phagocytosis (ADCP) activity. However, targeting the membrane-proximal epitope of MSLN afforded the CD47xMSLN bsAb enhanced ADCC and ADCP activity, resulting in superior activity in vivo. Mechanistically, engaging a MSLN membrane proximal region with a CD47-bsAb format not only enhanced FcγR-IIIA signaling but also interestingly disrupted more efficiently the CD47/SIRPα axis, resulting in optimized phagocytosis of tumor cells. Finally, we showed that treatment with CD47xMSLN bsAb targeting membrane proximal MSLN epitope induced an accumulation of myeloid cells and NK cells in the tumor microenvironment. Conclusions This study demonstrated that when designing antibody-based molecules, the targeted region on a tumor-associated antigen needs to be carefully considered to ensure maximal effector function. In the context of MSLN-positive solid tumors, we showed that an approach targeting a membrane-proximal epitope coupled to a CD47-blocking arm afforded an improved ADCC and ADCP profile, translating into increased in vivo efficacy. Disclosure Information E. Hatterer: None. X. Chauchet: None. F. Richard: None. L. Barba: None. V. Moine: None. L. Chatel: None. N. Fischer: None. W. Ferlin: None. V. Buatois: None. K. Masternak: None. L. Shang: None.

Up-regulation of CD47 is an immune evasion mechanism used by different cancers to evade immune surveillance. Through its interaction with signal-regulatory protein alpha (SIRPα) on myeloid cells, CD47 delivers a universal “don’t eat me” signal to phagocytes, which prevents immune cells from efficiently eliminating tumor cells. Blockade of the SIRPα–CD47 innate immune checkpoint has therefore emerged as a new way to treat cancer. Several CD47-targeting molecules are in development with encouraging results obtained with monoclonal antibodies (mAb). However, the pharmacologic properties and the safety profile of molecules indiscriminately blocking CD47 can be improved by selectively inhibiting CD47 only on tumor cells. For this purpose, we generated bispecific antibodies (bsAbs) capable of targeting blockade of CD47 specifically to malignanT-cells through the co-engagement of a tumor-associated antigen (TAA). The bsAb NI-1801 specifically targets mesothelin (MSLN)-positive tumors. NI-1801 was shown to bind to MSLN-positive tumor cells, but not to MSLN-negative cells expressing physiologic levels of CD47 (e.g., leukocytes, erythrocytes, platelets). NI-1801 blocks the CD47-SIRPα interaction in a MSLN-dependent manner and thus minimizes the side effects related to a nonspecific blockade of CD47 on healthy cells. Studying antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) of various MSLN-positive human tumor cell lines revealed that NI-1801 markedly enhanced killing as compared to amatuximab (an anti-MSLN mAb in clinical trials) and to the corresponding anti-MSLN mAb, exemplifying the role of blocking the “don’t eat me” signal to target cancer. NI-1801 also showed efficacy in various xenograft tumor models and analysis of the tumor microenvironment (TME) revealed a significant increase in leukocyte subpopulations (macrophages/monocytes and NK cells) of NI-1801 treated mice, suggesting that NI-1801 mediates the recruitment of monocytes from blood. Additionally, NI-1801 treatment affected the ratio between MHC-II-low and MHC-II-high macrophages in the TME. Finally, nonhuman primate studies with NI-1801 demonstrated a linear elimination profile, minimal target-mediated drug disposition and no hematologic toxicity. Taken together, these results illustrate that this strategy possesses potent anticancer activities both in vitro and in vivo in conjunction with favorable pharmacologic and toxicologic profiles. Citation Format: Stefano Majocchi, Valery Moine, Xavier Chauchet, Lucile Broyer, Laura Cons, Laurence Chatel, Eric Hatterer, Vanessa Buatois, Hasnaa Haddouk, Gerard Didelot, Giovanni Magistrelli, Yves Poitevin, Ulla Ravn, Anne Papaioannou, Francoise Richard, Limin Shang, Marie H. Kosco-Vilbois, Nicolas Fischer, Walter G. Ferlin, Krzysztof Masternak. Selective blockage of the innate immune checkpoint receptor CD47 on mesothelin (MSLN) positive solid tumor cells via dual targeting bispecific antibodies alters the tumor microenvironment to control tumor growth [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A088.

ABSTRACT Introduction CD47 is a membrane protein overexpressed on tumour cells and considered as an innate immune checkpoint. CD47 interacts with SIRPα on myeloid cells and induces a ‘don’t eat me’ signal limiting cell clearance by phagocytes. Blocking the CD47-SIRPα axis is thus an attractive approach to foster tumour cell killing by tumor-associated macrophages (TAMs) and boost cross-priming of anti-tumour T cells by dendritic cells (DC). However, CD47 targeting is hindered by haematological toxicity due to its ubiquitous expression. To focus CD47 blockade on tumour cells, we generated two bispecific antibodies (biAbs) pairing a high affinity arm targeting a tumour associated antigen (TAA), i.e., CD19 or mesothelin (MSLN), to an optimised lower affinity arm targeting CD47 and investigated their efficacy and mechanism of action (MoA) in xenograft tumour models. Material and methods BiAbs targeting CD47xCD19 or CD47xMSLN were tested in immunodeficient mice implanted with the following human tumour cell lines: Raji (CD19+, lymphoma), OVCAR-3 (MSLN+, ovarian) or MSLN-transfected HepG2 (hepatic). Analysis by flow cytometry was used to evaluate the impact of the biAbs on the tumour microenvironment (TME). The contribution of macrophages to tumour growth inhibition was addressed by clodronate-mediated depletion. Finally, biAbs-stimulated cross-priming of anti-tumour T-cells by DC was assessed in vitro. Results and discussions BiAbs controlled the growth of CD19 +lymphomas and MSLN +tumours. In the Raji xenograft model, treatment with the CD47xCD19 biAb did not increase the number of TAMs, but enhanced their tumoricidal activity (i.e., more macrophages engulfing tumour cells). The treatment also reduced the proportion of CD11b+Ly6g+ granulocytic myeloid-derived suppressor cells. In contrast, in the MSLN-transfected HepG2 xenograft model, treatment with the CD47xMSLN biAb induced a significant increase of macrophages in the tumour. Depletion of phagocytic cells with clodronate in both models impaired the anti-tumour activity of the biAbs, highlighting the role of macrophages in the MoA. In the antigen-cross presentation assay, the biAbs promoted cross-priming of tumour antigen-specific T-cells, suggesting the potential of CD47xTAA biAbs to trigger anti-tumour T cell responses. Conclusion CD47 targeting biAbs, tethered to cancer cells by their TAA binding arm, remodel the TME and control the lymphoma and MSLN +tumours. Furthermore, the observed induction of the adaptive immune response may further enhance the antitumor efficacy of such biAbs in patients.

CD47 serves as an anti-phagocytic receptor that is upregulated by cancer to promote immune escape. As such, CD47 is the focus of intense immuno-oncology drug development efforts. However, as CD47 is expressed ubiquitously, clinical development of conventional drugs, e.g., monoclonal antibodies, is confronted with patient safety issues and poor pharmacology due to the widespread CD47 "antigen sink". A potential solution is tumor-directed blockade of CD47, which can be achieved with bispecific antibodies (biAbs). Using mouse CD47-blocking biAbs in a syngeneic tumor model allowed us to evaluate the efficacy of tumor-directed blockade of CD47 in the presence of the CD47 antigen sink and a functional adaptive immune system. We show here that CD47-targeting biAbs inhibited tumor growth in vivo, promoting durable antitumor responses and stimulating CD8(+) T cell activation in vitro. In vivo efficacy of the biAbs could be further enhanced when combined with chemotherapy or PD-1/PD-L1 immune checkpoint blockade. We also show that selectivity and pharmacological properties of the biAb are dependent on the affinity of the anti-CD47 arm. Taken together, our study validates the approach to use CD47-blocking biAbs either as a monotherapy or part of a multi-drug approach to enhance antitumor immunity.

Background: The pathogenesis of macrophage activation syndrome (MAS) is not clearly understood: a large body of evidence supports the involvement of mechanisms similar to those implicated in the setting of primary hemophagocytic lymphohistiocytosis. Objective: We sought to investigate the pathogenic role of IFN-gamma and the therapeutic efficacy of IFN-gamma neutralization in an animal model of MAS. Methods: We used an MAS model established in mice transgenic for human IL-6 (IL-6TG mice) challenged with LPS (MAS mice). Levels of IFN-gamma and IFN-gamma-inducible chemokines were evaluated by using real-time PCR in the liver and spleen and by means of ELISA in plasma. IFN-gamma neutralization was achieved by using the anti-IFN-gamma antibody XMG1.2 in vivo. Results: Mice with MAS showed a significant upregulation of the IFN-gamma pathway, as demonstrated by increased mRNA levels of Ifng and higher levels of phospho-signal transducer and activator of transcription 1 in the liver and spleen and increased expression of the IFN-gamma-inducible chemokines Cxcl9 and Cxcl10 in the liver and spleen, as well as in plasma. A marked increase in Il12a and Il12b expression was also found in livers and spleens of mice with MAS. In addition, mice with MAS had a significant increase in numbers of liver CD68(+) macrophages. Mice with MAS treated with an anti-IFN-gamma antibody showed a significant improvement in survival and body weight recovery associated with a significant amelioration of ferritin, fibrinogen, and alanine aminotransferase levels. In mice with MAS, treatment with the anti-IFN-gamma antibody significantly decreased circulating levels of CXCL9, CXCL10, and downstream proinflammatory cytokines. The decrease in CXCL9 and CXCL10 levels paralleled the decrease in serum levels of proinflammatory cytokines and ferritin. Conclusion: These results provide evidence for a pathogenic role of IFN-gamma in the setting of MAS.