In this report, we identify a distinct mechanism of synergy between mAbs in cancer therapy leading to cures in the absence of chemotherapy. Specifically, we utilized a blocking anti-CD47 antibody in combination with the anti-CD20 antibody rituximab to eradicate human NHL through a mechanism of synergy involving FcR-independent enabling of phagocytosis by anti-CD47 antibody and FcR-dependent stimulation of phagocytosis by rituximab. In addition, the identification of CD47 expression as a prognostic factor could be incorporated into standard clinical prognostic considerations across multiple subtypes of NHL, and may be useful in risk-adapted therapy decision-making.
While it is thought that many therapeutic mAbs for human malignancies, including rituximab, function primarily through NK cell-mediated ADCC, several lines of evidence indicate that the therapeutic effect of anti-CD47 antibody alone or in combination with rituximab is mediated primarily through macrophage phagocytosis. First, synergistic macrophages phagocytosis was observed with combination anti-CD47 antibody and rituximab in vitro
, while no synergy was observed for direct apoptosis, ADCC, or CDC (, S6
, ). Second, phagocytosis of NHL cells in vivo
was observed with either anti-CD47 antibody or rituximab alone, and most importantly, significantly increased with combination therapy (). Third, the therapeutic effect of combination antibody treatment was similar in an NHL xenotransplant model using complement and NK cell-deficient NSG mice () as in complement and NK cell-competent SCID mice (Figure S4A,B
), suggesting that macrophages alone are sufficient to mediate the therapeutic effect. Fourth, depletion of macrophages, but not complement or NK cells abrogated the synergistic effect of anti-CD47 antibody in combination with rituximab (Figure S6E
). These studies highlight the importance of macrophages as effectors of anti-CD47 antibody therapy in human NHL.
This study describes a mechanism of antibody synergy in the elimination of NHL in the absence of chemotherapy. Combination antibody therapies for NHL have previously been investigated, mostly in combination with rituximab, with some progressing to clinical trials. These include a humanized antibody targeting the B cell antigen CD22 (epratuzumab) and galiximab, a chimeric antibody targeting the co-stimulatory ligand CD80. Phase I/II studies with either epratuzumab or galiximab in combination with rituximab demonstrate relative safety and clinical responses equal to or greater than single agent therapy alone (Leonard et al., 2005
; Leonard et al., 2007
; Leonard et al., 2008
). Based on these results, phase III trials are underway. Antibody combinations involving anti-CD20 antibodies and antibodies to pro-apoptotic receptors are also being explored in pre-clinically (Daniel et al., 2007
; Maddipatla et al., 2007
). These studies highlight the clinical potential of combination antibody approaches in NHL patients.
Combination therapy with two or more mAbs possesses several advantages compared to monotherapies in NHL or other malignancies. First, therapy solely with monoclonal antibodies targeting cancer-specific antigens could result in decreased off-target toxicity compared to current therapeutic regimens that utilize chemotherapy. Second, synergy between two distinct antibody effector mechanisms, FcR-independent and FcR-dependent as shown here, would result in increased therapeutic efficacy. Third, antibody targeting of two distinct cell-surface antigens would be more likely to eliminate cancer cells with pre-existing epitope variants or epitope loss, such as those reported in rituximab-refractory/resistant NHL patients (Foran et al., 2001
; Hiraga et al., 2009
; Kennedy et al., 2004
). Fourth, a bispecific FcR-engaging antibody with one arm binding and blocking CD47 and the other binding to a validated cancer antibody target (CD20) could reduce potential antibody toxicity, while retaining the synergy effect, especially since CD47 is expressed in multiple normal tissue types. Although we demonstrated that an anti-mouse CD47 antibody is relatively non-toxic to wild type mice (Majeti et al., 2009
), a clinical anti-human CD47 antibody may have a different human toxicity profile that could be overcome by a bispecific antibody.
In addition to its application in NHL, the reported mechanism of antibody synergy provides proof-of-principle that a blocking mAb directed against CD47 can synergize with an FcR-activating antibody to provide superior therapeutic efficacy. This finding raises the possibility of potential synergy between an anti-CD47 antibody and other clinically approved therapeutic antibodies that may activate FcRs on immune effector cells for the treatment of diverse human malignancies including: trastuzumab (Herceptin) for HER2-positive breast carcinomas, cetuximab (Erbitux) for colorectal carcinomas and head and neck squamous cell carcinomas, alemtuzumab (Campath) for CLL and T-cell lymphoma, and others in development (Finn, 2008
). To date, we have demonstrated effective anti-CD47 antibody targeting of several human cancers including AML (Majeti et al., 2009
), bladder cancer (Chan et al., 2009
), and now NHL, leading us to speculate that CD47 targeting will be effective against a wide range of human cancers.