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.

Abstract INTRODUCTION: The CD47-SIRPα axis is a crucial immune checkpoint, allowing tumor cells to evade immune destruction. Initial efforts to target CD47 with monoclonal antibodies and SIRPa-Fc fusion proteins encountered significant hurdles due to ubiquitous and abundant CD47 expression in particular on blood cells (including RBC and platelets). To overcome the limitation of mono-specific CD47 targeting, we generate bispecific antibodies (bsAbs) that block CD47 selectively on tumor cells, upon co-engagement of a tumor-associated antigen (TAA) on the cell surface. Harboring an immunologically active Fc portion (human IgG1 Fc) these TAA/CD47 bsAbs mediate efficient tumor cell killing through antibody-dependent cellular phagocytosis (ADCP) and antibody-directed cellular cytotoxicity (ADCC). Importantly, this strategy has now been clinically validated: NI-1801, our mesothelin/CD47 bsAb, has demonstrated selective and safe CD47 targeting together with encouraging antitumor activity in heavily pretreated ovarian cancer patients (1). METHODS: EGFR/CD47 and HER2/CD47 bispecific antibodies were assembled using the κλ-body technology/platform (2). For that goal, high affinity antibody arms specific to different regions of the extracellular domains of EGFR or HER2 were paired with a low affinity anti-CD47 arm. The resulting hIgG1 bsAbs were tested in vitro for activity in ADCP assays with TAA-positive tumor cells and monocyte-derived macrophages as effector cells, and in ADCC assays using unfractionated PBMCs as effector cells. RESULTS: The EGFR/CD47 and HER2/CD47 bsAbs efficiently induce ADCP and ADCC of tumor cell expressing varying levels of EGFR or HER2, respectively, including low level-expressing cancer cell lines. Interestingly, we could observe that the killing activity depended not only on the affinity of the TAA arm, but also on the region of the ECD bound by the TAA arm, suggesting that the geometry of the antigen-co-engagement on tumor cell surface may affect the efficiency of the immunological synapse. This was particularly apparent when comparing the killing activity mediated by different HER2xCD47 bsAbs. CONCLUSION: The “unbalanced affinities” design of TAA/CD47 bispecific antibodies effectively addresses the safety and pharmacokinetic liabilities observed with monospecific CD47 antibodies. Importantly, our clinical experience with NI-1801 (MSLN×CD47) validates both the safety and the potential clinical benefit of this tumor-restricted CD47-blocking strategy (1). Together, these findings support TAA/CD47 bsAbs as a valuable addition to the anti-cancer armamentarium, with the EGFR/CD47 and HER2/CD47 bsAbs described here representing promising development candidates. (1) De La Motte Rouge T. ESMO 2025 (Abstract #1512O) (2): Fischer N. Nat Commun, 2015 Feb 12:6:6113 Citation Format: Eric Hatterer, Vanessa Buatois, Alizée Viandier, Emeline Rousset, Cécile Raymond, Adeline Lesnier, Pauline Lloveras, Christelle Mougin, Ulla Ravn, Pauline Malinge, Frederic Bollin, Limin Shang, Walter Ferlin, Nicolas Fischer, Jose Saro, Krzysztof Masternak, . Bispecific antibodies targeting EGFR and HER2 for effective killing of solid tumor cells via a safe CD47-SIRPA modulation [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 5532.

Tau immunotherapy has recently shown clinical promise but required high dosing. We developed NIDB-3101, a novel third-generation, high-affinity anti-tau biparatopic antibody designed for superior tau binding, aggregation inhibition, and extended half-life. NIDB-3101 binds tau’s microtubule-binding region and C-terminal domains. Various binding and cellular functional assays using recombinants, but more importantly human AD extracts were used to assess NIDB-3101 benefits. Half-life mutations impact was assessed via FcRn binding and cellular assays recycling. NIDB-3101 exhibited sub-nanomolar affinity, binding a broad spectrum of pathological tau species in AD homogenates, inhibited AD extracts-induced cellular effect compared to benchmark antibodies. Mutations enhanced hFcRn-mediated cellular recycling. NIDB-3101 captures a broad spectrum of pathological tau species leading to strong cellular efficacy using human AD extracts, supporting further clinical development as a potential disease-modifying therapy for AD and related tauopathies.

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.

Supplementary Table S5 shows the hematological results of cynomolgus monkeys treated with vehicle (Ctr.) or NI-1701 (Treat.) at 30 and 100 mg/kg over 4 weeks.

Supplementary Table S2 shows the cell surface density (expressed as number of receptors per cell) for CD19, CD47, CD20 on different human tumor cell lines.

Supplementary Table S4 shows the mean Cmax values from the DRF and the single dose study.

Supplementary Table S3 shows the ratio of AUC0-inf low/high dose for the PK single dose study.

Supplementary Figure S1 shows shows the percentage of in vitro phagocytosis of Raji cells by macrophages with NI-1701 or CD19/CD47hi biAb.

Supplementary Figure S2 shows in vitro hemagglutination in human or cynomolgus monkeys whole blood, or platelet aggregation in human platelet rich-plasma (PRP)in presence of NI-1701

Supplementary material and methods

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.

Video 1 shows the real time phagocytosis of cancer cells in presence of NI-1701

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.

The CD47- signal regulatory protein α (SIRPα) axis, originally discovered as a mechanism of self-recognition, has emerged as a novel innate immune check-point employed by cancer cells to escape immune surveillance. Over-expression of CD47 on a plethora of hematologic and solid cancers has been shown to be associated with poor prognosis. CD47 blockade is thus considered as an attractive strategy to retune the host immune system toward eliminating cancer cells. Clinical efficacy has been achieved in patients with Non-Hodgkin lymphoma (NHL) treated with a combination of the anti-CD20 monoclonal antibody (mAb), rituximab, and the anti-CD47 mAb, Hu5F9-G4. However, in parallel, due to the ubiquitous expression of CD47 on healthy cells, toxicity is observed limiting exposure which impairs clinical development of anti-CD47 mAbs. To harness the tumoricidal potential and avoid the liabilities of CD47 blockade, we have developed bispecific antibodies (bsAbs), co-targeting CD47 and a tumor associated antigen (TAA), for selective blockade of CD47 on malignant cells. Mesothelin (MSLN) has been selected as one of these TAAs as it is over-expressed on multiple types of solid tumors. An anti-CD47xMSLN bsAb with a fully functional IgG1 Fc domain has been generated and tested for efficacy and safety in vitro and in vivo using cell-based assays and animal models. With a panel of human MSLN+ target cells, the bsAb kills more efficiently through Ab-dependent cellular phagocytosis (ADCP) and cell-mediated cytotoxicity (ADCC) as compared to the anti-MSLN mAb, amatuximab. Efficacy of the bsAb was minimally affected by soluble MSLN as compared to amatuximab. Mechanistic studies demonstrated that the increased ADCP and ADCC were due to co-engagement-mediated blockade of both target proteins. In vivo using xenograft models, whereas amatuximab was unable, the bsAb inhibited tumor growth of OVCAR3 and MSLN-transfected HepG2 cell lines. Phenotypic analysis showed that treatment with the bsAb induced the accumulation of myeloid cells with increased F4/80 expression in the tumor microenvironment. Importantly, the bsAb was well-tolerated in an exploratory four-week repeated dose study at the highest dose tested (10 mg/kg) in cynomolgus monkeys. Conclusion: Selective CD47 targeting on tumor cells with an anti-CD47xMSLN bsAb showed efficient killing of MSLN+ tumor cells in vitro and in vivo. A study in non-human primates administered weekly a therapeutically relevant dose over 28 days was well tolerated demonstrating no adverse hematological profiles. Citation Format: Limin Shang, Vanessa Buatois, Eric Hatterer, Xavier Chauchet, Hasnaà Haddouk, Stefano Majocchi, Krzysztof Masternak, Marie H. Kosco-Vilbois, Nicolas Fischer, Walter G. Ferlin. Selectively targeting CD47 with bispecific antibody to efficiently eliminate mesothelin-positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 546.

AbstractCD47, an ubiquitously expressed innate immune checkpoint receptor that serves as a universal “don't eat me” signal of phagocytosis, is often upregulated by hematologic and solid cancers to evade immune surveillance. Development of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hemotoxicity including anemia. To overcome such liabilities, we have developed a fully human bispecific antibody, NI-1701, designed to coengage CD47 and CD19 selectively on B cells. NI-1701 demonstrates favorable elimination kinetics with no deleterious effects seen on hematologic parameters following single or multiple administrations to nonhuman primates. Potent in vitro and in vivo activity is induced by NI-1701 to kill cancer cells across a plethora of B-cell malignancies and control tumor growth in xenograft mouse models. The mechanism affording maximal tumor growth inhibition by NI-1701 is dependent on the coengagement of CD47/CD19 on B cells inducing potent antibody-dependent cellular phagocytosis of the targeted cells. NI-1701–induced control of tumor growth in immunodeficient NOD/SCID mice was more effective than that achieved with the anti-CD20 targeted antibody, rituximab. Interestingly, a synergistic effect was seen when tumor-implanted mice were coadministered NI-1701 and rituximab leading to significantly improved tumor growth inhibition and regression in some animals. We describe herein, a novel bispecific antibody approach aimed at sensitizing B cells to become more readily phagocytosed and eliminated thus offering an alternative or adjunct therapeutic option to patients with B-cell malignancies refractory/resistant to anti-CD20–targeted therapy. Mol Cancer Ther; 17(8); 1739–51. ©2018 AACR.

e15126 Background: Mesothelin (MSLN) is a cell surface glycoprotein associated with poor prognosis in many cancers, with elevated soluble MSLN (sMSLN) serum levels considered a biomarker of disease severity. Monoclonal antibodies (mAbs) targeting MSLN have demonstrated limited clinical efficacy. CD47, a checkpoint for innate immune cells, provides a ‘don’t eat me’ signal allowing healthy cells to limit elimination by phagocytes. High CD47 expression correlates with poor prognosis yet mAbs blocking CD47 are hindered by CD47’s ubiquitous expression. We developed a bispecific antibody (biAb) that: 1. targets checkpoint inhibition to the tumor via a high affinity anti-MSLN arm, and 2. avoids binding to non-cancer cells by an anti-CD47 arm of an optimized affinity. Methods: BiAbs coupling MSLN-targeting arms with a CD47-targeting arm were tested in antibody dependent cellular phagocytosis (ADCP) in vitro and xenograft tumor models. Human tumor cell lines used: NCI-N87 (gastric), HPAC (pancreatic), OVCAR-3 and Caov-3 (ovarian) and MSLN-transfected HepG2 (hepatic). Results: MSLNxCD47 biAbs significantly enhance macrophage-mediated ADCP of NCI-N87, HPAC, OVCAR-3 and Caov-3 as compared to the anti-MSLN mAb, Amatuximab. As compared to Amatuximab, the impact of sMSLN on biAb activity is limited. In a hepatic xenograft tumor model, MSLN/CD47 biAbs significantly control tumor growth, while Amatuximab and B6H12, an anti-CD47 mAb, demonstrate minimal anti-tumor activity. Tumor control in vivo is also observed for ovarian and gastric tumor cell lines. Dissecting the mechanism affording tumor control in vivo, macrophages are mobilized into the tumors in MSLN/CD47 biAb treated mice as compared to the isotype control. These recruited macrophages were shown to be critical for control of tumor growth. Conclusions: MSLN/CD47 targeting biAbs potentially offer a superior strategy to MSLN-targeted mAbs in managing solid tumors by remodeling the immune phenotype of the tumor microenvironment.

CD19 is a B cell-specific receptor that regulates the threshold of B cell receptor (BCR)-mediated cell proliferation. A CD47xCD19 bispecific antibody (biAb) was generated to target and deplete B cells via multiple antibody-mediated mechanisms. Interestingly, the biAb, constructed of a CD19 binding arm and a CD47 binding arm, inhibited BCR-mediated B-cell proliferation with an effect even more potent than a CD19 monoclonal antibody (mAb). The inhibitory effect of the biAb was not attributable to CD47 binding because a monovalent or bivalent anti-CD47 mAb had no effect on B cell proliferation. Fluorescence resonance energy transfer analysis demonstrated that co-engaging CD19 and CD47 prevented CD19 clustering and its migration to BCR clusters, while only engaging CD19 (with a mAb) showed no impact on either CD19 clustering or migration. The lack of association between CD19 and the BCR resulted in decreased phosphorylation of CD19 upon BCR activation. Furthermore, the biAb differentially modulated BCR-induced gene expression compared to a CD19 mAb. Taken together, this unexpected role of CD47xCD19 co-ligation in inhibiting B cell proliferation illuminates a novel approach in which two B cell surface molecules can be tethered, to one another in order, which may provide a therapeutic benefit in settings of autoimmunity and B cell malignancies.