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CD47 is an important innate immune checkpoint through its interaction with its inhibitory receptor on macrophages, signal-regulatory protein α (SIRPα). Therapeutic blockade of CD47–SIRPα interactions is a promising immuno-oncology treatment that promotes clearance of cancer cells. However, CD47–SIRPα interactions also maintain homeostatic lymphocyte levels. In this study, we report that the mouse splenic marginal zone B cell population is dependent on intact CD47–SIRPα interactions and blockade of CD47 leads to the loss of these cells. This depletion is accompanied by elevated levels of monocyte-recruiting chemokines CCL2 and CCL7 and infiltration of CCR2+Ly6Chi monocytes into the mouse spleen. In the absence of CCR2 signaling, there is no infiltration and reduced marginal zone B cell depletion. These data suggest that CD47 blockade leads to clearance of splenic marginal zone B cells.

BackgroundCEA (CEACAM5) is a well-validated cell-surface antigen that is highly expressed in multiple solid tumors. Bolt's immune-stimulating antibody conjugates (ISACs) direct a TLR7/8 agonist into tumors to activate tumor-infiltrating myeloid cells and initiate a broad innate and adaptive anti-tumor immune response.1 The favorable properties of CEA, including robust cell surface expression, low internalization rate, and limited normal tissue expression, suggest that the antigen may be a suitable ISAC target. We are evaluating an anti-CEA ISAC, BDC-2034, as a multi-functional approach to treat CEA-expressing cancers.MethodsAnti-CEA antibodies were tested for binding affinity and specificity, CEA-targeted antibody-dependent cellular phagocytosis (ADCP), and myeloid-mediated tumor cell killing. Selected antibodies were conjugated to proprietary TLR7/8 agonists, and the resulting CEA ISACs were evaluated for in vitro myeloid activation and in vivo efficacy against xenograft tumors.ResultsAntibody CEA1 binds to the CEA protein with high affinity (EC50 = 0.25 nM), binds selectively to CEA-positive tumor cell lines, and mediates ADCP more efficiently than a reference anti-CEA antibody, labetuzumab (figure 1). We generated BDC-2034 by conjugating a potent TLR7/8 agonist to CEA1. BDC-2034 tumor cell binding drives myeloid effector cell ADCP, agonist delivery to TLR7 and TLR8 in endosomes, and secretion of cytokines critical for innate and adaptive immunity (including IL-12p70, CXCL10, and TNFa). In the HPAF II + cDC co-culture model, IL-12p70 is induced with EC50 = 1.2 nM, and the level of induction is at least ten-fold higher than with ISACs using labetuzumab (figure 2). Potent cellular activity is strictly dependent on tumor cell CEA expression; in whole blood, in the absence of CEA-expressing tumor cells, cytokine induction was only observed at approximately 100-fold higher concentrations. BDC-2034 inhibits the growth of HPAF II xenograft tumors in SCID/beige mice with a minimal efficacious dose (MED) of 1 mg/kg, demonstrating anti-tumor activity solely through innate immune activation (figure 3). The TLR7/8 agonist in BDC-2034 has relatively poor activity in mice; a surrogate CEA1 ISAC with a mouse TLR7-activating agonist achieved MED = 0.5 mg/kg in the HPAF II model, with eradication of all tumors at the 5 mg/kg dose.ConclusionsThese pre-clinical data demonstrate the potential of BDC-2034 to treat CEA-expressing human cancers. Most importantly, the antigen-dependent induction of immune-stimulating cytokines promises a robust immune response that combines the activation of innate and adaptive arms.ReferenceAckerman S, Pearson C, Gregorio J. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nature Cancer 2021;2:18–33. https://doi.org/10.1038/s43018-020-00136-xAbstract 784 Figure 1ADCP. Anti-CEA antibody CEA1 is an efficient inducer of ADCP of Raji/CEA cells by M-CSF differentiated monocyte-derived macrophagesAbstract 784 Figure 2Tumor-dependent dendritic cell activation. BDC-2034 induces IL-12p70 secretion from primary dendritic cells (cDC); native CEA1 antibody and reference anti-CEA ISAC are ineffectiveAbstract 784 Figure 3Efficacy against xenograft tumors. BDC-2034 inhibits the growth of HPAF II tumors in SCID/beige mice; native CEA1 antibody and isotype ISAC are ineffective

Significance Novel therapies are urgently needed that can ameliorate the clinical syndromes of cerebral malaria, the most severe consequences of Plasmodium infection, and thereby reduce malaria fatality. Monoclonal antibodies that target CD47, a “don’t eat me” signal, have been demonstrated to enhance cellular clearance of cancer cells by promoting macrophage phagocytosis. We sought to adopt this therapeutic strategy to ameliorate the clinical syndromes associated with cerebral malaria with the goals of reducing disease-associated morbidity and mortality. We demonstrate that CD47 blockade by anti-CD47 injection leads to survival from cerebral malaria in mice., CD47 is an antiphagocytic “don’t eat me” signal that inhibits programmed cell removal of self. As red blood cells (RBCs) age they lose CD47 expression and become susceptible to programmed cell removal by macrophages. CD47−/− mice infected with Plasmodium yoelii, which exhibits an age-based preference for young RBCs, were previously demonstrated to be highly resistant to malaria infection. Our study sought to test the therapeutic benefit of CD47 blockade on ameliorating the clinical syndromes of experimental cerebral malaria (ECM), using the Plasmodium berghei ANKA (Pb-A) murine model. In vitro we tested the effect of anti-CD47 mAb on Plasmodium-infected RBC phagocytosis and found that anti-CD47 treatment significantly increased clearance of Plasmodium-infected RBCs. Infection of C57BL/6 mice with Pb-A is lethal and mice succumb to the clinical syndromes of CM between days 6 and 10 postinfection. Strikingly, treatment with anti-CD47 resulted in increased survival during the cerebral phase of Pb-A infection. Anti-CD47–treated mice had increased lymphocyte counts in the peripheral blood and increased circulating levels of IFN-γ, TNF-α, and IL-22. Despite increased circulating levels of inflammatory cytokines, anti-CD47–treated mice had reduced pathological features in the brain. Survival of ECM in anti-CD47–treated mice was correlated with reduced cellular accumulation in the cerebral vasculature, improved blood–brain barrier integrity, and reduced cytotoxic activity of infiltrating CD8+ T cells. These results demonstrate the therapeutic benefit of anti-CD47 to reduce morbidity in a lethal model of ECM, which may have implications for preventing mortality in young African children who are the highest casualties of CM.

Lyme disease, caused by the bacteria Borrelia burgdorferi, is the most common and rapidly growing vector-borne infectious disease in the United States and Europe. High variability in disease burden among Lyme patients suggests that individual immune responses may be key drivers of clinical presentation and patient outcomes. Use of high resolution flow-based immunosorbent profiling revealed that a subset of Lyme patients with persistent symptoms were producing high concentrations of IgE specific to B. burgdorferi. Comparing C57B/6 mice, which are tolerant to B. burgdorferi, and C3H/HeJ mice, which are susceptible to disease, we find high levels of IgE specific for B. burgdorferi in C3H/HeJ but not C57B/6 mice. Furthermore, IgE was found to target Borrelia peptidoglycan in both acute and chronic infection models. Histologic analysis of mouse Lyme arthritic ankle tissue showed mast cells, which release highly immunogenic effectors upon activation by bound IgE, degranulating at significantly higher rates compared to uninfected controls. Forced mast cell degranulation exacerbated Lyme arthritis in infected mice. This data suggests that a subset of Lyme patients with persistent symptoms may have developed an allergic response to conserved bacterial antigens from a B. burgdorferi infection, as opposed to an autoimmune type response. Inclusion of IgE reactivity in diagnostic testing and examination of pathological immune responses to bacterial antigens could assist clinicians in patient care and effective treatments. Research reported in this publication was supported by the Fairbairn family foundation; Bay Area Lyme Foundation; the Younger family foundation; the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation; the Virginia and D. K. Ludwig Fund for Cancer Research; M.C.T. was supported by Stanford Immunology training grant 5T32AI007290, and the NIH NRSA 1 F32 AI124558-01 award. L.B.T.D. was supported by a Stanford Diversifying Academia Recruiting Excellence fellowship. S.D.G was supported by the California Institute for Regenerative Medicine Bridges 2.0 Training Program grant EDUC2-08397. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Borrelia burgdorferi is a bacterial spirochete that can cause Lyme disease (LD) after infecting a susceptible host. Immune responses to the bacteria are highly variable and host specific. The murine substrain, C3H/HeJ, is a frequently utilized model for LD. Interestingly, over a prolonged infection, mice develop dermatitis on tail skin, which shares critical features with human skin. Female C3H/HeJ mice aged 5–8 weeks, 1 year, or 2 years were infected intraperitoneally with 105 B. burgdorferi. Dermatitis was evaluated by gross examination and histology. Dermatitis worsened over the course of untreated infection, with ulceration, hemorrhaging, flaking, hair loss, and dark lesions as well as spongiosis and acanthosis. These features of dermatitis were present in infected mice after 1 year of age. This relationship among LD, atopic dermatitis, and host age seen in the C3H/HeJ mouse model is consistent with a large pool (342,499) of human epidemiological data from Finland. We identified 5,248 individuals with LD and 17,233 with atopic dermatitis in FinnGen. Retrospective analysis shows LD is associated with atopic dermatitis (OR = 1.91 [1.68 −2.37], P < 2e−16). More visits due to LD complications (3 or more visits versus 1 visit) were associated with atopic dermatitis (OR = 2.19 [1.35–3.55], P = 0.0014) and risk of developing atopic dermatitis over time (HR = 2.26 [1.54–3.95], P = 0.0017). Data from mice and humans reveal a novel relationship among LD, age, and atopic dermatitis. Through defined pathological scoring, we demonstrate the onset of murine atopic dermatitis with B. burgdorferi infection, which is further exacerbated by host age at time of infection, and likewise report a similar association in human epidemiological data from FinnGen. Research was supported by the Fairbairn Family foundation; Bay Area Lyme Foundation; the Younger family foundation; the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation; the Virginia and D. K. Ludwig Fund for Cancer Research; AML grant R01CA086017; the PCBC from NIHLB U01HL099999; as well as grant U19AI109662. M.C.T. was supported by Stanford Immunology training grant 5T32AI007290, and the NIH NRSA 1 F32 AI124558-01 award. L.B.T.D. was supported by a Stanford Diversifying Academia Recruiting Excellence fellowship. S.G. was supported by the California Institute for Regenerative Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile., It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 “don’t eat me” signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis. Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.

SUMMARY Paradoxically, early host responses to infection include the upregulation of the antiphagocytic molecule, CD47. This suggests that CD47 blockade could enhance antigen presentation and subsequent immune responses. Indeed, mice treated with anti-CD47 monoclonal antibody following lymphocytic choriomeningitis virus infections show increased activation of both macrophages and dendritic cells (DCs), enhancement of the kinetics and potency of CD8+ T cell responses, and significantly improved virus control. Treatment efficacy is critically dependent on both APCs and CD8+ T cells. In preliminary results from one of two cohorts of humanized mice infected with HIV-1 for 6 weeks, CD47 blockade reduces plasma p24 levels and restores CD4+ T cell counts. The results indicate that CD47 blockade not only enhances the function of innate immune cells but also links to adaptive immune responses through improved APC function. As such, immunotherapy by CD47 blockade may have broad applicability to treat a wide range of infectious diseases., Graphical Abstract, In Brief Cham et al. describe a way to enhance natural immune responses to infections by blocking interactions between two molecules (CD47 and SIRPα) that normally put brakes on the immune system. Since this therapy targets the immune system, it could have broad applicability against a wide range of infectious agents.