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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.

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.

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.

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.

The use of Next Generation Sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.

The MHC class-I related receptor or neonatal Fc receptor (FcRn) protects IgG and albumin from degradation by rescuing them in endothelial cells in a pH dependent fashion and consequently increases their respective half-lives. Monoclonal antibody-based therapies are of increasing interest and characterizing the interaction with FcRn is important for the development of an antibody candidate. In order to facilitate the production of soluble FcRn suitable for interaction studies, we generated semi-stable pools co-expressing FcRn α-chain, β2-microglobulin, biotin ligase and EGFP using a dual promoter, multi-cistronic vector. Human and mouse FcRn were purified in the mg/L range of culture medium and a single purification step was sufficient to reach a high level of purity. The receptors were characterized by ELISA, flow cytometry and surface plasmon resonance and shown to be functional. The single site biotinylation facilitated the directional immobilization of FcRn on the sensor chip and significantly increased the response level of the surface compared to amine coupling used in previous studies. Using this system, the affinity constants of seven IgGs, from various species and isotypes, were determined for human and mouse FcRn, including two hamster isotypes. These results confirm the higher selectivity of the human receptor and the promiscuous binding of mFcRn to IgGs from different species.

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