CD40 is a central co-stimulatory receptor implicated in the development of productive anti-tumor immune responses across multiple cancers, including bladder cancer. Despite strong preclinical rationale, systemic administration of therapeutic agonistic antibodies targeting the CD40 pathway have demonstrated dose limiting toxicities with minimal clinical activity to date, emphasizing an important need for optimized CD40-targeted approaches, including rational combination therapy strategies. Here, we describe an important role for the endogenous IL-15 pathway in contributing to the therapeutic activity of CD40 agonism in orthotopic bladder tumors, with upregulation of trans-presented IL-15/IL-15Rα surface complexes, particularly by cross-presenting cDC1s, and associated enrichment of activated CD8 T cells within the bladder tumor microenvironment. In bladder cancer patient samples, we identify DCs as the primary source of IL-15, however, they lack high levels of IL-15Rα at baseline. Using humanized immunocompetent orthotopic bladder tumor models, we demonstrate the ability to therapeutically augment this interaction through combined treatment with anti-CD40 agonist antibodies and exogenous IL-15, including the fully-human Fc-optimized antibody 2141-V11 currently in clinical development for the treatment of bladder cancer. Combination therapy enhances the crosstalk between Batf3-dependent cDC1s and CD8 T cells, driving robust primary anti-tumor activity and further stimulating long-term systemic anti-tumor memory responses associated with circulating memory-phenotype T and NK cell populations. Collectively, these data reveal an important role for IL-15 in mediating anti-tumor CD40 agonist responses in bladder cancer and provide key proof-of-concept for combined use of Fc-optimized anti-CD40 agonist antibodies and agents targeting the IL-15 pathway. These data support expansion of ongoing clinical studies evaluating anti-CD40 agonist antibodies and IL-15-based approaches to evaluate combinations of these promising therapeutics for the treatment of patients with bladder cancer.

While anti-CD47 antibodies hold promise for cancer immunotherapy, early phase clinical trials have shown limited signs of clinical benefit, suggesting that blockade of CD47 alone may not be sufficient for effective tumor control. Here, we investigate the contributions of the Fc domain of anti-CD47 antibodies required for optimal in vivo antitumor activity across multiple species-matched models, providing new insights into the mechanisms underlying the efficacy of this emerging class of therapeutic antibodies. Using a novel mouse model humanized for CD47, SIRPα and FcγRs, we demonstrate that local administration of an Fc-engineered anti-CD47 antibody with enhanced binding to activating FcγRs modulates myeloid and T-cell subsets in the tumor microenvironment, resulting in improved long-term systemic antitumor immunity and minimal on-target off-tumor toxicity. Our results highlight the importance of Fc optimization in the development of effective anti-CD47 therapies and provide a novel approach for enhancing the antitumor activity of this promising immunotherapy.

Background The CD47/SIRPa axis plays a crucial role in cancer immunosurveillance.1 2 While anti-CD47 antibodies have shown promise in several preclinical models,1 3–8 results from early phase clinical trials have shown limited clinical benefit,9–11 suggesting that the sole blockade of CD47 by the antibody Fab domain might not be sufficient for effective tumor control. A critical question that remains to be answered is whether interactions between the antibody Fc and Fc gamma receptors (FcgRs) also contribute to their antitumor activity.12 13 Our study aims to investigate the role of the Fc domain in the in vivo antitumor activity of anti-CD47 antibodies using immunocompetent species-matched models, overcoming limitations of previous studies conducted in immunocompromised models or with interspecies differences between mouse (m) and human (h) CD47, SIRPa and FcγRs. Methods We modified the Fc domain of the anti-mCD47 antibody MIAP301 to generate antibodies with varying affinity to mFcgRs: 1) MIAP301-mIgG2a Fc, binding to preferentially to activating mFcgRs, 2) MIAP301-mIgG1 Fc, binding to the inhibitory mFcgRIIB, and 3) MIAP301-mIgG1-D265A Fc, which lacks binding to any mFcgRs. We evaluated the antitumor activity of these antibodies in MC38 and B16 tumor models in immunocompetent C57BL/6J mice and mice lacking activating FcgRs.15 Additionally, we generated a mouse humanized for the expression of hCD47, hSIRPa and hFcgRs by CRISPR/Cas9-mediated gene-targeting strategy, and by backcrossing to our hFcgR mice.16 We used this model to compare the antitumor activities of the anti-hCD47 antibody Magrolimab (5F9-hIgG4).14 and an Fc-optimized variant that enhances binding for all the activating hFcgRs (5F9-GAALIE) generated in the lab. Results The MIAP301-mIgG2a Fc variant led to the most significant reduction in tumor burden when compared to the control or other Fc variants in WT mice in both MC38 and B16 models. This therapeutic effect was abrogated in mice lacking activating FcgRs (figure 1). The CD47/SIRPa/FcgR humanized mice recapitulate the expression profile of CD47 and SIRPa found in human cells (figure 2). Furthermore, increasing dosing concentrations of both 5F9-hIgG4 and 5F9-GAALIE antibodies led to on-target anemia and thrombocytopenia in hCD47/hSIRPa/hFcgR mice, recapitulating results from clinical trials (figure 3). Intratumoral administration of the Fc optimized 5F9-GAALIE results in enhanced long-term antitumor immunity, abscopal antitumor effect, and minimal on-target toxicity when compared to 5F9-hIgG4 or control alone or in combination with PD-1 blockade (figure 4). Conclusions The antitumor activities of anti-CD47 antibodies require interactions with activating FcgRs, highlighting the importance of Fc optimization in the development of effective anti-CD47 therapies. Acknowledgements We thank Maria L. Baez, Alessandra E. Marino, and Carlo M. Sevilla for their excellent technical assistance. We also thank all the members of the J.V.R. Laboratory of Molecular Genetics and Immunology for helping discussions and sharing experiment materials. References Willingham SB, Volkmer JP, Gentles AJ, et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc Natl Acad Sci U S A 2012;109:6662–7. Adams S, van der Laan LJ, Vernon-Wilson E, et al. Signal-regulatory protein is selectively expressed by myeloid and neuronal cells. J Immunol 1998;161:1853–9. Chao MP, Alizadeh AA, Tang C, et al. Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma. Cell 2010;142:699–713. Majeti R, Chao MP, Alizadeh AA, et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell 2009;138:286–99. Jaiswal S, Jamieson CH, Pang WW, et al. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell 2009;138:271–85. Weiskopf K, Jahchan NS, Schnorr PJ, et al. CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer. J Clin Invest 2016;126:2610–20. Liu X, Pu Y, Cron K, et al. CD47 blockade triggers T cell-mediated destruction of immunogenic tumors. Nat Med 2015;21:1209–15. Liu Q, Wen W, Tang L, et al. Inhibition of SIRPalpha in dendritic cells potentiates potent antitumor immunity. Oncoimmunology 2016;5:e1183850. Bouwstra R, van Meerten T, Bremer E. CD47-SIRPalpha blocking-based immunotherapy: Current and prospective therapeutic strategies. Clin Transl Med 2022;12:e943. Uger R, Johnson L. Blockade of the CD47-SIRPalpha axis: a promising approach for cancer immunotherapy. Expert Opin Biol Ther 2020;20:5–8. Jalil AR, Andrechak JC, Discher DE. Macrophage checkpoint blockade: results from initial clinical trials, binding analyses, and CD47-SIRPalpha structure-function. Antib Ther 2020;3:80–94. Zhao XW, Matlung HL, Kuijpers TW, van den Berg TK. On the mechanism of CD47 targeting in cancer. Proc Natl Acad Sci U S A 2012;109:E2843; author reply E4–5. Zhao XW, Kuijpers TW, van den Berg TK. Is targeting of CD47-SIRPalpha enough for treating hematopoietic malignancy? Blood 2012;119:4333–4; author reply 4–5. Takai T, Li M, Sylvestre D, Clynes R, Ravetch JV. FcR gamma chain deletion results in pleiotrophic effector cell defects. Cell 1994;76:519–29. Smith P, DiLillo DJ, Bournazos S, Li F, Ravetch JV. Mouse model recapitulating human Fcgamma receptor structural and functional diversity. Proc Natl Acad Sci U S A 2012;109:6181–6. Advani R, Flinn I, Popplewell L, et al. CD47 Blockade by Hu5F9-G4 and Rituximab in Non-Hodgkin's Lymphoma. N Engl J Med 2018;379:1711–21.

Therapeutic human IgG antibodies are routinely tested in mouse models of oncologic, infectious, and autoimmune diseases. However, assessing the efficacy and safety of long-term administration of these agents has been limited by endogenous anti-human IgG immune responses that act to clear human IgG from serum and relevant tissues, thereby reducing their efficacy and contributing to immune complex–mediated pathologies, confounding evaluation of potential toxicity. For this reason, human antibody treatment in mice is generally limited in duration and dosing, thus failing to recapitulate the potential clinical applications of these therapeutics. Here, we report the development of a mouse model that is tolerant of chronic human antibody administration. This model combines both a human IgG1 heavy chain knock-in and a full recapitulation of human Fc receptor (FcγR) expression, providing a unique platform for in vivo testing of human monoclonal antibodies with relevant receptors beyond the short term. Compared to controls, hIgG1 knock-in mice mount minimal anti-human IgG responses, allowing for the persistence of therapeutically active circulating human IgG even in the late stages of treatment in chronic models of immune thrombocytopenic purpura and metastatic melanoma.

Given the role of myeloid cells in T cell activation and in the antitumor response, targeting checkpoint molecules expressed on this population represents a promising strategy to augment antitumor immunity. However, myeloid checkpoints that can be effectively used as immunotherapy targets are still lacking. Here, we demonstrate the therapeutic potential of targeting the myeloid receptors Siglec-7 and Siglec9 in vivo. By using a humanized immunocompetent murine model, we demonstrate that human Siglec-7 and Siglec-9, in addition to the murine homolog Siglec-E, inhibit the endogenous antitumor immune response, as well as the response to tumor-targeting and immune checkpoint inhibiting antibodies in vivo. The impact of these Siglecs on tumor progression is highly dependent on the anatomical distribution of the tumor and, as a consequence, the local tumor microenvironment, as tumors with a more immune-suppressive tumor microenvironment are less sensitive to Siglec perturbation. Finally, to assess the potential of these two receptors as targets for immunotherapy, we developed Fc engineered blocking antibodies to Siglec-7 and Siglec-9 and demonstrate that Siglec-7 and Siglec-9 blockade can significantly reduce tumor burden in vivo, demonstrating the therapeutic potential of targeting these two receptors.

The inhibitory FcγR FcγRIIB is widely expressed on B cells, dendritic cells (DCs), and myeloid effector cells and modulates a variety of Ab-driven in vivo functions. Although it has been established that FcγRIIB plays an important role in the maintenance of peripheral tolerance, the responsible cell-specific FcγRIIB expression remains to be determined. In this study, we generated mice with selective deletion of FcγRIIB in B cells, DCs, and myeloid effector cells and evaluated these novel strains in models of tolerance and autoimmune diseases. Our results demonstrate that mice with selective deletion of FcγRIIB expression in B cells and DCs have increased Ab and T cell responses, respectively, and display enhanced susceptibility to disease in distinct models, suggesting that FcγRIIB expression in distinct cellular populations contributes to the maintenance of peripheral tolerance through different mechanisms.

The in vivo biological activities of IgG antibodies result from their bifunctional nature, in which antigen recognition by the Fab is coupled to the effector and immunomodulatory diversity found in the Fc domain. This diversity, resulting from both amino acid and glycan heterogeneity, is translated into cellular responses through Fc gamma receptors (Fc gamma Rs), a structurally and functionally diverse family of cell surface receptors found throughout the immune system. Although many of the overall features of this system are maintained throughout mammalian evolution, species diversity has precluded direct analysis of human antibodies in animal species, and, thus, detailed investigations into the unique features of the human IgG antibodies and their Fc gamma Rs have been limited. We now report the development of a mouse model in which all murine Fc gamma Rs have been deleted and human Fc gamma Rs, encoded as transgenes, have been inserted into the mouse genome resulting in recapitulation of the unique profile of human Fc gamma R expression. These human Fc gamma Rs are shown to function to mediate the immunomodulatory, inflammatory, and cytotoxic activities of human IgG antibodies and Fc engineered variants and provide a platform for the detailed mechanistic analysis of therapeutic and pathogenic IgG antibodies.

Cellular Fcγ receptors are essential for IgG-dependent effector functions in vivo. There is convincing evidence that selective activating Fcγ receptors are responsible for the activity of individual IgG subclasses. Thus, IgG1 activity is absent in FcγRIII-deficient mice, and several studies suggest that the activity of the most potent IgG subclasses, IgG2a and IgG2b, might be dependent on either individual or a combination of activating FcγRs. To study the role of individual activating FcγRs for IgG subclass activity, we generated an FcγRIV-deficient mouse and showed that a variety of IgG2a- and IgG2b-dependent effector functions are impaired in the absence of this activating Fc receptor in models of autoimmunity and antibody-dependent cellular cytotoxicity.