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CD47, 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.

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 material and methods

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

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

Video 2 shows the real time phagocytosis of cancer cells in presence of hIgG1 control

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., A model of acute CRS replicating laboratory and clinical features observed in severe COVID‐19 conditions and cancer patients treated with immune agonists. Benefits of anti‐cytokine antibodies in early phase disease management was demonstrated, thus, offering a potential opportunity to consider additional therapeutic options.

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.

Background: Lymphoma is the most common hematological malignancy with non-Hodgkin B-cell lymphomas (NHL), including those with high unmet clinical need such as relapsed/refractory Diffuse Large B Cell Lymphoma (DLBCL), and Mantle-Cell Lymphoma (MCL), representing 90% of all lymphoma cases. CD19 is an attractive target for antibody-directed lymphoma therapies as it is expressed in most B cell malignancies, with normal tissue expression limited to B-cells thus reducing any on-target toxicity concerns. Antibody Drug Conjugates (ADCs) are the focus of intense interest as a means to provide selective tumor killing with increased efficacy and less toxicity than standard of care chemotherapies. IKS03 is an ADC comprised of an anti-CD19 antibody conjugated to a proprietary DNA-crosslinking PBD prodrug and includes a tumor-selective glucuronide-trigger technology for payload release and activation. ADC activity requires processing by beta-glucuronidase, a lysosomal enzyme often upregulated in tumor cells, while normal tissues with low levels of the enzyme are less able to process the ADC and are differentially spared. Methods: IKS03 was generated via chemo-enzymatic bioconjugation at defined sites on the antibody yielding an ADC with a drug to antibody ratio of 2. In vivo efficacy was evaluated in CD19-expressing cell line-derived xenograft models in mice including Farage (DLBCL, Germinal Centre B-cell subtype), OCI-LY10 (DLBCL, Activated B-Cell subtype), Granta-519 (MCL) and Ramos (Burkitt’s lymphoma). Activity was compared to benchmark ADCs known to have demonstrated clinical efficacy. Efficacy was also assessed in low passage DLBCL patient-derived xenograft models of known genomic profile. Toxicology studies were conducted in cynomolgus monkeys with immunophenotyping by flow cytometry included to quantify the level of normal B-cells following IKS03 administration. Results: IKS03 is highly effective in causing tumor regressions in DLBCL models at doses that are well tolerated. Complete regressions were observed with a single dose of 0.1 mg/kg in the Farage xenograft model. IKS03 is also highly active in the Granta-519 MCL model, with complete regressions observed with a single dose of 0.1 mg/kg. Tumor regression was observed in a high-grade triple-hit lymphoma PDX model with a single 0.3 mg/kg dose. IKS03 was tolerated in monkeys at the highest dose tested of 1.5 mg/kg (single dose). IKS03 cross reacts with monkey CD19 and reduced B-cell depletion was observed in relation to comparator agents, even at doses much greater (1.5 mg/kg) than that needed for complete responses in B-cell lymphoma mouse models (0.1 mg/kg). Conclusion: Preclinical data demonstrates that IKS03’s advanced ADC design results in an increased therapeutic margin compared to traditional ADCs with DNA-crosslinking payloads, with increased efficacy and decreased toxicity. Citation Format: Jenny Thirlway, Adam Lodge, Daniel J. Williamson, Justyna Mysliwy, Jutta Deckert, Xavier Chauchet, Laura Cons, Yun-Hee Park, Hyun-Min Ryu, Na Ra Han, Ho Young Song, Chul-Woong Chung, Robert J. Lutz. IKS03, a CD19-targeted antibody drug conjugate with enhanced efficacy and tolerability for treatment of B-cell lymphomas [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 1755.

PD-1/PD-L1 blockade has improved survival across many types of cancer, but only in a minority of patients. Co-targeting PD-1/PD-L1 and the CD47/SIRPα myeloid checkpoint with monoclonal antibody (mAb) combinations showed increased antitumor responses in preclinical studies. However, CD47 mAbs are hindered by ubiquitous CD47 expression leading to rapid target-mediated clearance and safety concerns, including anemia and thrombocytopenia. Consequently, dual-targeting CD47xPD-L1 bispecific antibodies (bsAbs) enabling selective inhibition of CD47 on PD-L1-positive tumors offer an alternative approach. A fully human bsAb pairing a high affinity PD-L1 arm to a low affinity CD47 arm was generated using the κλ-body platform. The latter is also used in our CD47xCD19 bispecific antibody NI-1701/TG-1801, currently in phase I clinical trials (NCT03804996, NCT04806035). The resulting CD47xPD-L1 bsAb of human IgG1 isotype (NI-2601) was evaluated in various binding and receptor-blocking assays, and then tested for its capacity to enhance T-cell activation in vitro and induce Fc-mediated killing of tumor cells through phagocytosis (ADCP) and antibody-dependent cell cytotoxicity (ADCC). A surrogate bsAb was also evaluated in vivo in a syngeneic mouse model. NI-2601 demonstrated effective blockade of PD-1/PD-L1 interaction, and T-cell activation in vitro, similar to the anti-PD-L1 clinical benchmarks atezolizumab and avelumab. Consistent with its low-affinity CD47 arm, the bsAb did not bind to red blood cells (RBC) and CD47 blockade was driven by PD-L1 co-engagement. Using a panel of tumor cell lines, expressing various PD-L1 levels, NI-2601 showed superior activity in ADCP and ADCC as compared to the anti-PD-L1 IgG1 mAb, avelumab. The anti-tumor activity of this approach using surrogate CD47xPD-L1 bsAb was confirmed in a syngeneic MC38 colon carcinoma model. Thus, NI-2601 is able to harness Fc-effector function to eliminate PD-L1-positive tumor cells while sparing PD-L1-negative cells, such as RBC or platelets. Pharmacokinetic and tolerability studies in non-human primate are planned for 2022. Citation Format: Xavier Chauchet, Nicolas Bosson, Margaux Legrand, Laura Cons, Sébastien Calloud, Alizée Viandier, Françoise Richard, Pauline Malinge, Tereza Bautzova, Jérémie Bourguignon, Guillemette Pontini, Elise Penarrieta, Mengzhu Sun, Ulla Ravn, Valéry Moine, Giovanni Magistrelli, Yves Poitevin, Stéphanie Hugues, Limin Shang, Walter Ferlin, Krzysztof Masternak. NI-2601, an Fc-active CD47xPD-L1 bispecific antibody that selectively targets CD47 on PD-L1-positive tumors [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 3429.

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.

BackgroundPD-1/PD-L1 blockade can significantly improve survival across many types of cancer, but only in a minority of patients. To broaden its therapeutic efficacy, several combination partners are now being evaluated together with PD-1/PD-L1 blockade. Agents blocking CD47/SIRPα innate immune checkpoint are one such example, and co-targeting PD-1/PD-L1 and CD47 with monoclonal antibody (mAb) combinations showed increased antitumor responses in preclinical studies. However, CD47 mAbs are hindered by ubiquitous CD47 expression leading to rapid target-mediated clearance and safety concerns. Consequently, dual-targeting CD47xPD-L1 bispecific antibodies (bsAbs) enabling preferential inhibition of CD47 on PD-L1-positive cells are being tested as an alternative approach. We compare here two distinct bsAbs, based on a common PD-L1 antibody arm, with differing FcgR-enabling effector functions and CD47-binding arm affinities.MethodsAn array of fully human bsAbs associating a high affinity PD-L1 arm to CD47 arms with varying affinities were generated using the κλ-body platform.1 CD47xPD-L1 bsAbs of human IgG1 isotype (CD47 low affinities) or IgG4 isotype (CD47 high affinities) were screened in various binding assays (including to red blood cells (RBC)) and in receptor-blocking assays, and then tested for their Fc-mediated killing and T-cell activation activity (SEA-stimulated PBMC assay). Selected molecules were evaluated in vivo.ResultsBoth bsAb approaches demonstrated strong blockade of PD-1/PD-L1 interaction and significantly enhanced T-cell activation in vitro. CD47lowxPD-L1 IgG1 bsAbs did not bind to RBC and showed PD-L1-guided inhibition of CD47. ADCP and ADCC experiments with a panel of tumor cell lines expressing various target levels showed superior killing activity with CD47lowxPD-L1 IgG1 bsAbs as compared to the anti-PD-L1 IgG1 mAb, avelumab. On the other hand, CD47highxPD-L1 IgG4 bsAbs showed residual RBC binding and PD-L1-independent blocking of CD47/SIRPα. These CD47high IgG4 bsAbs were able to enhance the anti-tumor activity of anti-tumor-associated antigen (TAA) mAbs in vitro (phagocytosis), and in vivo (Raji lymphoma xenograft model). In addition, anti-tumor activity of mouse CD47xPD-L1 bsAbs in a syngeneic MC38 colon carcinoma model was demonstrated.ConclusionsWith the objective of finding the optimal CD47xPD-L1 bsAb design, two approaches targeting CD47 and PD-L1 inhibition were tested. Both the CD47lowxPD-L1 IgG1 bsAbs and CD47highxPD-L1 IgG4 bsAbs were able to mediate enhanced antitumor responses, the former as a standalone treatment, the latter in conjunction with an anti-TAA mAb. To further characterize the CD47lowxPD-L1 and CD47highxPD-L1 bsAbs, lead candidates will be tested in PK and tolerability studies in non-human primates.ReferencesFischer N, Elson G, Magistrelli G, Dheilly E, Fouque N, Laurendon A, et al. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Nat Commun 2015 May;6(1):6113.

CD47 is a ubiquitously expressed immune checkpoint receptor that is often upregulated in cancer. CD47 interacts with its counter-receptor SIRPα on macrophages and other myeloid cells to inhibit cancer cell phagocytosis and drive immune evasion. To overcome tolerability and “antigen sink” issues arising from widespread CD47 expression, we generated dual-targeting bispecific antibodies that selectively block the CD47-SIRPα interaction on malignant cells expressing a specific tumor-associated antigen; e.g., CD19 or mesothelin. These bispecific κλ bodies are fully human, native IgG1 molecules, combining tumor targeting and selective CD47 blockade with immune activating mechanisms mediated by the Fc portion of the antibody. CD47-neutralizing κλ bodies efficiently kill cancer cells in vitro and in vivo but interact only weakly with healthy cells expressing physiological levels of CD47. Accordingly, a κλ body administered to non-human primates showed a typical IgG pharmacokinetic profile and was well tolerated. Importantly, κλ bodies preserve their tumoricidal capabilities in the presence of a CD47 antigen sink. Thus, dual-targeting κλ bodies allow for efficacious yet safe targeting of CD47 in cancer. Such a bispecific design could be applied to limit the extent of neutralization of other ubiquitously expressed therapeutic targets., Graphical Abstract, CD47 is a widely expressed anti-phagocytic “don’t eat me” signal. Cancer cells upregulate CD47 expression to escape phagocytic clearance and immune surveillance. Dheilly et al. show how bispecific antibodies can be used as an efficient and safe means for therapeutic targeting of CD47 in cancer.

CD47, 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.

Life-threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon γ (IFNγ) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection-driven model of sHLH in mice, we observed that the tissue-derived levels of IFNγ are actually 500- to 2000-fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFNγ gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFNγ activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFNγ-biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFNγ activity in patients with sHLH.

Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies., Bispecific antibodies allow for novel therapeutic approaches but industrial-scale production and immunogenicity represent significant challenges. Here Fischer et al. describe a unique human bispecific antibody format that exploits differing light chains to overcome these obstacles.

B cells play a major role in the pathogenesis of many autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes mellitus, as indicated by the efficacy of B cell–targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell–targeted therapy, anti-CD20 mAb, are contingent upon long-term depletion of peripheral B cells. In this article, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell AgR. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAbs do not require cell depletion; rather, they act by inducing an anergic-like state. Thus, we describe a novel B cell–targeted approach predicated on the induction of B cell anergy.

Heterogeneity in the N-glycans on therapeutic proteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. This heterogeneity arises from the multistep process of mammalian complex-type N-glycan synthesis. Here we report a glycoengineering strategy--which we call GlycoDelete--that shortens the Golgi N-glycosylation pathway in mammalian cells. This shortening results in the expression of proteins with small, sialylated trisaccharide N-glycans and reduced complexity compared to native mammalian cell glycoproteins. GlycoDelete engineering does not interfere with the functioning of N-glycans in protein folding, and the physiology of cells modified by GlycoDelete is similar to that of wild-type cells. A therapeutic human IgG expressed in GlycoDelete cells had properties, such as reduced initial clearance, that might be beneficial when the therapeutic goal is antigen neutralization. This strategy for reducing N-glycan heterogeneity on mammalian proteins could lead to more consistent performance of therapeutic proteins and modulation of biopharmaceutical functions.

Fc-modified anti-human CD3ε monoclonal antibodies (mAbs) are in clinical development for the treatment of autoimmune diseases. These next generation mAbs have completed clinical trials in patients with type-1 diabetes and inflammatory bowel disease demonstrating a narrow therapeutic window. Lowered doses are ineffective, yet higher pharmacologically-active doses cause an undesirable level of adverse events. Thus, there is a critical need for a return to bench research to explore ways of improving clinical outcomes. Indeed, we recently reported that a short course of treatment affords synergy, providing long-term disease amelioration when combining anti-mouse CD3 and anti-mouse tumor necrosis factor mAbs in experimental arthritis. Such strategies may widen the window between risk and benefit; however, to more accurately assess experimentally the biology and pharmacology, reagents that mimic the current development candidates were required. Consequently, we engineered an Fc-modified anti-mouse CD3ε mAb, 2C11-Novi. Here, we report the functional characterization of 2C11-Novi demonstrating that it does not bind FcγR in vitro and elicits little cytokine release in vivo, while maintaining classical pharmacodynamic effects (CD3-TCR downregulation and T cell killing). Furthermore, we observed that oral administration of 2C11-Novi ameliorated progression of remitting-relapsing experimental autoimmune encephalitis in mice, significantly reducing the primary acute and subsequent relapse phase of the disease. With innovative approaches validated in two experimental models of human disease, 2C11-Novi represents a meaningful tool to conduct further mechanistic studies aiming at exploiting the immunoregulatory properties of Fc-modified anti-CD3 therapies via combination therapy using parenteral or oral routes of administration.

Aims IFN γ mediates disease in the murine model of primary HLH. Similar to HLH, patients with MAS/sHLH often present with a cytokine storm, overwhelming inflammation and multiorgan dysfunction. Thus, to assess the potential implication of IFN γ in MAS, two variants of the murine MAS model were used. Administration of an anti-mouse IFN γ monoclonal antibody (MAb) in both models allowed us to determine the level and pivotal role of this proinflammatory cytokine. Methods C57BL/6 mice received repeated injections on days 0, 2, 4, 7 and 9 of the TLR9 agonist, CpG oligodeoxynucleotides (CpG ODN). To mimic a more severe, fulminant disease, along with CpG ODN, the anti-IL-10 Receptor mAb, 1B1.3A, was coadministered at 200 μ g/mouse on days 0, 2, 4 and 6. To assess the significance of IFN γ on disease, the mAb, XMG1.2, was injected 100 mg/kg) on days 1, 3 and 6. Levels of total IFN γ werequantified by a fit-for-purpose ELISA. Results In these models, IFN γ neutralization lead to disease amelioration with parameters such as weight loss, splenomegaly, white blood cell counts, hyperferritinemia, hypercytokinemia and anemia successfully addressed. Post XMG1.2 administration, accumulation of immune-complexed IFN γ reached a steady state level of 250 ng/ml in both models. IFN γ -induced inflammatory genes, found to be upregulated locally in the spleen and liver, were normalized by IFN γ -blockade. Conclusion Neutralization of IFN γ improves clinical and laboratory features in even the more severe, fulminant model of MAS. These data suggest that IFN γ , present as one of the cytokines in MAS, is central to disease and therefore provides a rationale for the neutralization of IFN γ as a potential targeted therapeutic approach in patients with severe form of MAS.

Neutralizing CD47, the ‘don't eat me signal’ hijacked by different tumor types, is a novel generally applicable therapeutic strategy. Because of a distinct mechanism of action and the ability to stimulate the innate anti-tumor immunity, CD47-neutralizing agents are poised as attractive candidates for combination therapies in association with other immunotherapies. However, the development of general CD47 antagonists could be hindered by the ubiquitous and abundant expression of CD47 on virtually all healthy cells. To overcome potential pharmacological and clinical liabilities of a general CD47 antagonist, we have developed bispecific kappa/lambda bodies, which selectively target CD47 in cancer cells. These kappa/lambda bodies: (i) are full-length bispecific IgGs, (ii) bind with high affinity and neutralize the CD47-SIRP alpha interaction in cancer cells expressing a tumor-associated antigen (TAA), and (iii) mediate efficient cell killing of TAA-positive cancer cells in vitro through Fc-dependent mechanisms such as ADCP (antibody mediated cellular phagocytosis) and ADCC (antibody mediated cellular cytotoxicity). We are currently developing two molecules of this type, one targeting CD47 and CD19 (for B cell malignancies), the other targeting CD47 and mesothelin (for various mesothelin-positive solid tumors). The efficacy of the CD47/CD19 kappa/lambda body was demonstrated in vivo, using two B-cell lymphoma xenograft models in NOD/SCID mice. We also performed a pharmacokinetics study in non-human primates with the CD47/CD19 lead candidate, with the objective of assessing the potential “antigen sink” effect related to ubiquitous CD47 expression on erythrocytes, platelets and other cells. Encouragingly, the CD47/CD19 kappa/lambda body administered in a single dose to cynomolgus monkeys, at 0.5 and 10 mg/kg, showed an acceptable pharmacokinetic profile and the absence of hematological toxicities. The example of the CD47/CD19 kappa/lambda body illustrates the power of the dual-targeting approach for addressing a ubiquitous cell surface receptor such as CD47. Citation Format: Krzysztof Masternak, Lucile Broyer, Elie Dheilly, Stefano Majocchi, Valéry Moine, Giovanni Magistrelli, François Rousseau, Ulla Ravn, Franck Gueneau, Pauline Malinge, Sébastien Calloud, Maud Charreton-Galby, Mireille Guerrier, Nessie Costes, Nicolas Bosson, Gérard Didelot, Lucie Bernard, Vanessa Buatois, Laura Cons, Laurence Chatel, Anne Papaioannou, Zoë Johnson, Walter Ferlin, Marie Kosco-Vilbois, Nicolas Fischer. Neutralizing CD47 in cancer cells with dual targeting kappa/lambda bodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2482. doi:10.1158/1538-7445.AM2015-2482

Introduction Repeated TLR9 stimulation was recently found to result in a macrophage activation syndrome (MAS)-like disease in mice [1] . Mice administered CpG (TLR9-ligand) developed cytopenia, anemia, thrombocytopenia, splenomegaly, hepatitis, and hyperferritemia. Administration of the anti-mouse IFNg monoclonal antibody (mAb), XMG1.2, was found to ameliorate disease. Our aim was to extend these studies by performing a comprehensive biomarker study to correlate the kinetics of IFNg production to disease endpoints. Methods Mice were administered CpG on days 0, 2, 4, 7 and 9. Tissue and blood were obtained at numerous time-points. Gene analysis and histology were performed on spleen and liver. Serum cytokines were analysed using the multiplex Luminex technology and leukocyte, red blood cell and haemoglobin counts were assessed using a haematological counter. Results Our results confirmed a multi-phasic production of IFNg in serum, spleen and liver following injection of CpG. The initial peak of IFNg production correlated with a rapid appearance of cytopenia and thrombocytopenia followed by the other features of MAS (e.g., anemia and splenomegaly). Gene analysis confirmed the induction of IFNg-related signature molecules in the spleen and liver. Conclusion In mice, repeated CpG injection leads to a MAS-like disease that is manifested by a multi-phasic production of IFNg. Disease parameters including anemia, decreased hemoglobin, splenomegaly, hypercytokinemia and ferritinemia were consistent with IFNg production. Finally, IFNg-induced biomarkers were also established in tissues from MAS-mice (e.g. CXCL10 and Class II major histocompatibility complex transactivator (CIITA)). The use of XMG1.2 will be used in the aim to correlate disease biomarkers and IFNg production at different time-points of disease progression. Our study, thus, allows us to more fully understand the physiologically-based pharmacokinetic (PK)/pharmacodynamics (PD) relationships based on IFNg -neutralization in the murine model of MAS.

IFNg is the pivotal mediator in the murine model of primary HLH. Mice given repeated injection with the TLR9 agonist, CpG-containing oligodeoxynucleotides, develop pathology resembling the human disease, sHLH. Coadministration of an anti-IL-10 Receptor (R) monoclonal antibody (mAb) with CpG induces more severe disease characterized also by hemophagocytosis (fulminant sHLH).