- Email: [email protected]
Dual Targeting of Innate and Adaptive Checkpoints on Tumor Cells Limits Immune Evasion
Article
Dual Targeting of Innate and Adaptive Checkpoints on Tumor Cells Limits Immune Evasion Graphical Abstract
Authors Xiaojuan Liu, Longchao Liu, Zhenhua Ren, ..., Hua Peng, Mingzhao Zhu, Yang-Xin Fu
Correspondence [email protected] (M.Z.), [email protected] (Y.X.F.)
In Brief CD47 and PD-L1 serve as critical innate and adaptive checkpoints, respectively. Liu et al. show CD47 and PD-L1 coordinate in tumor cells to evade immune response. Furthermore, a bispecific antibody design enables better targeting on tumor cells, but less on nontumor cells, and enhanced therapeutic efficacy.
Highlights d
CD47 and PD-L1 checkpoints on tumor cells coordinate to suppress immune sensing
d
The bispecific antibody enables better targeting on tumor cells than on non-tumor cells
d
Dual targeting increases cytosolic DNA sensing in DCs for an anti-tumor T cell response
d
Chemotherapy induces danger signals that synergize with bispecific antibody
Liu et al., 2018, Cell Reports 24, 2101–2111 August 21, 2018 ª 2018 The Authors. https://doi.org/10.1016/j.celrep.2018.07.062
Cell Reports
Article Dual Targeting of Innate and Adaptive Checkpoints on Tumor Cells Limits Immune Evasion Xiaojuan Liu,1,2,5 Longchao Liu,3,5 Zhenhua Ren,3 Kaiting Yang,1 Hairong Xu,1 Yan Luan,4 Kai Fu,4 Jingya Guo,1 Hua Peng,1 Mingzhao Zhu,1,2,* and Yang-Xin Fu1,3,6,* 1Chinese
Academy of Sciences Key Laboratory of Infection and Immunity, IBP-UTSW Joint Immunotherapy Group, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China 3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA 4DingFu Biotarget Co. Ltd., Suzhou, Jiangsu 215125, China 5These authors contributed equally 6Lead Contact *Correspondence: [email protected] (M.Z.), [email protected] (Y.-X.F.) https://doi.org/10.1016/j.celrep.2018.07.062
SUMMARY
CD47 on tumor cells protects from phagocytosis, while PD-L1 dampens T cell-mediated tumor killing. However, whether and how CD47 and PD-L1 coordinate is poorly understood. We reveal that CD47 and PD-L1 on tumor cells coordinately suppress innate and adaptive sensing to evade immune control. Targeted blockade of both CD47 and PD-L1 on tumor cells with a bispecific anti-PD-L1-SIRPa showed significantly enhanced tumor targeting and therapeutic efficacy versus monotherapy. Mechanistically, systemic delivery of the dual-targeting heterodimer significantly increased DNA sensing, DC cross-presentation, and anti-tumor T cell response. In addition, chemotherapy that increases ‘‘eat me’’ signaling further synergizes with the bispecific reagent for better tumor control. Our data indicate that tumor cells evolve to utilize both innate and adaptive checkpoints to evade anti-tumor immune responses and that tumor cell-specific dualtargeting of both checkpoints represents an improved strategy for tumor immunotherapy. INTRODUCTION CD47, ubiquitously expressed on the membrane of all cells in mice and humans, is an essential ‘‘don’t eat me’’ signal (Barclay and Van den Berg, 2014; Brown and Frazier, 2001; Okazawa et al., 2005). By interacting with its receptor SIRPa, which is primarily expressed on phagocytic macrophages and dendritic cells (DCs), it inhibits myosin-IIA accumulation at the phagocytic synapse and, consequently, prevents phagocytosis (Oldenborg et al., 2000; Tsai and Discher, 2008). Although the CD47-SIRPa interaction plays essential roles in self-recognition and selfhomeostasis, it appears to have been hijacked by tumor cells during evolution. Increased expression of CD47 has been observed in nearly all types of tumors examined (Baccelli et al.,
2013, 2014; Cioffi et al., 2015; Jaiswal et al., 2009). This becomes a potentially important strategy for tumor cells to evade phagocytosis and clearance. Taking advantage of this principle, systemic blockade of the CD47-SIRPa interaction has demonstrated encouraging efficacy in limiting tumor growth in xenograft studies (Chao et al., 2010a; Edris et al., 2012; Lo et al., 2016; Weiskopf et al., 2013; Willingham et al., 2012; Xiao et al., 2015; Yoshida et al., 2015). However, in syngeneic grafts, given the broad expression of CD47, the anti-CD47 antibody has been shown to bind to non-tumor cells, especially red blood cells, thus limiting its systemic use (Dheilly et al., 2017; Liu et al., 2015a; Piccione et al., 2016; Ponce et al., 2017). Thus, preferentially targeting tumor tissues with a CD47-blocking reagent remains a challenge. PD-L1 is another membrane protein that is highly expressed on some tumor cells and tumor infiltrating leukocytes (Dong et al., 2002; Green et al., 2010; Taube et al., 2012; Thompson et al., 2004). By interacting with its receptor, PD-1, which is mainly expressed on T cells, PD-L1 inhibits T cell activation, proliferation, and effector function; causes T cell exhaustion; and dampens anti-tumor adaptive immune responses (Blackburn et al., 2009; Brown et al., 2003; Freeman et al., 2000). Interruption of the PD-L1-PD-1 signaling pathway can rescue exhausted T cells and restore anti-tumor responses. This strategy has achieved initial success in clinics in recent years (Brahmer et al., 2010; Brahmer et al., 2012; Topalian et al., 2012; Wolchok et al., 2013). It suggests that PD-L1 is a key molecule for tumor evasion of the immune system. Since both CD47 and PD-L1 are highly expressed on tumor cells and serve as critical innate and adaptive checkpoints, respectively, whether and how they coordinate for tumor immune evasion becomes intriguing. This is further emphasized by a recent study demonstrating the possibility of oncogenicdriven co-expression of CD47 and PD-L1 for tumorigenesis (Casey et al., 2016). In the present study, we revealed that CD47 and PD-L1, expressed on tumor cells, serve as two-step checkpoints in tumor immune evasion through the DNA sensing pathway. Dual-targeting blockade of both molecules using a newly engineered bispecific heterodimer protein, anti-PD-L1SIRPa, shows better tumor-cell targeting, exerts a stronger therapeutic effect than either single blockade alone and has no
Cell Reports 24, 2101–2111, August 21, 2018 ª 2018 The Authors. 2101 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Figure 1. CD47 and PD-L1 on Tumor Cells Coordinate to Evade Immune Control (A) C57BL/6 female WT mice (n = 10/group) were inoculated s.c. with 1 3 105 cells of the colorectal adenocarcinoma cell line MC38 or MC38-CD47KO. Tumor growth was measured and compared once a week starting on day 0. Statistical significance was determined by a Student’s t test. (B) C57BL/6 female WT mice (n = 9–10/group) were inoculated s.c. with 1 3 105 cells of MC38 or MC38-CD47KO. CD8-depleting antibody (TIB210, 200 mg/mouse) was administered twice a week, starting on day 1. Tumor growth was measured and compared once a week starting on day 0. Statistical significance was determined by a Student’s t test. (C) C57BL/6 female WT mice (n = 3/group) were inoculated s.c. with 1 3 105 MC38 or MC38CD47KO tumor cells. The PD-L1 expression on tumor cells was assessed by flow cytometry on day 20. Staining control is shown in gray. (D) C57BL/6 female WT mice (n = 5 or 10) were inoculated s.c. with 2 3 105 cells of MC38, MC38-CD47KO, MC38-PD-L1KO or MC38-CD47/ PD-L1DKO. Tumor growth was measured and compared twice a week starting on day 0. Statistical significance was determined by a Student’s t test. (E) C57BL/6 female WT mice and Rag1/ mice (n = 5/group) were inoculated s.c. with 5 3 105 cells of MC38 or MC38-CD47/PD-L1DKO. Tumor growth was measured. Statistical significance was determined by a Student’s t test. (F) C57BL/6 female WT mice (n = 6/group) were inoculated s.c. with 2 3 105 cells of MC38 or MC38CD47/PD-L1DKO. CD8-depleting antibody was administered twice a week, starting on day 1. Tumor growth was measured and compared twice a week starting on day 0. Statistical significance was determined by a Student’s t test. See also Figures S1–S3.
detectable side effects. Thus, this study provides insight into how tumor cells evade or limit immune attack through coordinative innate and adaptive checkpoints as well as how both innate and adaptive checkpoints can be simultaneously targeted for enhanced tumor therapy. RESULTS CD47 and PD-L1 on Tumor Cells Coordinate to Evade Immune Control To test whether CD47 is required for tumor cell immune evasion, we generated a CD47-deficient colorectal tumor cell line named ‘‘MC38-CD47 knockout’’ (MC38-CD47KO) (Figure S1A). MC38-CD47KO tumor cells grew equally well in vitro compared with parental cells (data not shown). When inoculated subcutaneously (s.c.) into immune competent wildtype (WT) animals, they grew slower than parental cells but were not completely arrested (Figure 1A). This result suggested that CD47 plays only a partial role in tumor cell immune evasion and other mechanisms work together with CD47 or independently for tumor cell immune evasion. We previously reported
2102 Cell Reports 24, 2101–2111, August 21, 2018
that MC38 growth is under adaptive T cell control and that T cell depletion enhances tumorigenesis (Liu et al., 2015b). Similarly, the growth of MC38-CD47KO is also under T cell control (Figures 1B and S2). In line with this finding, under T cell pressure, the PD-L1 expression on MC38-CD47KO tumor cells was upregulated (Figure 1C). This result raises the possibility that PD-L1-mediated adaptive immune suppression may contribute to tumor growth in the absence of CD47-mediated innate immune escape. We then wondered whether further ablation of PD-L1 signaling would completely inhibit tumorigenesis. To test this, we further knocked out PD-L1 on MC38-CD47KO cells to obtain MC38-CD47/PD-L1DKO cells (Figure S1A). MC38-PD-L1KO cells were also generated as control (Figure S1A). In stark contrast with MC38-CD47KO and MC38-PD-L1KO cells, MC38-CD47/PD-L1DKO cells showed almost complete growth arrest (Figures 1D and S1B). This coordinated role of CD47 and PD-L1 on tumor growth appears general since it is also replicated in the B16F10 tumor model (Figure S3). The growth arrest of MC38-CD47/PD-L1DKO depends on host CD8+ T cells as they grew at a comparable level as WT MC38 tumor cells in Rag1/ mice or in WT mice
Figure 2. Innate Sensing by DCs Is Required for CD47 and PD-L1-Mediated Evasion (A) C57BL/6 female WT mice and Ifnar/ mice (n = 4 or 5/group) were inoculated s.c. with 5 3 105 MC38 or MC38-CD47/PD-L1DKO tumor cells. Tumor volume was measured at the indicated time points. (B) C57BL/6 female WT mice and Sting/ mice (n = 5/group) were inoculated s.c. with 5 3 105 MC38 or MC38-CD47/PD-L1DKO tumor cells. Tumor volume was measured at the indicated time points. (C) C57BL/6 female WT mice and CD11c-cre 3 Stingfl/fl mice (n = 5/group) were inoculated s.c. with 5 3 105 WT MC38 or MC38-CD47/PD-L1DKO tumor cells. Tumor volume was measured at the indicated time points. For all graphs, data represent mean ± SEM and statistical analysis was performed by Student’s t test.
depleted of CD8+ T cells (Figures 1E and 1F). Together, these data suggest that CD47 and PD-L1 on tumor cells coordinate for tumor immune escape. Innate Sensing by DCs Is Required for CD47- and PD-L1-Mediated Evasion To evaluate whether type I interferons (IFNs) are required for CD47- and PD-L1-mediated tumor immune evasion, we inoculated MC38-CD47/PD-L1DKO tumor cells into Ifnar/ mice. In stark contrast to the tumor growth arrest of MC38-CD47/PDL1DKO in WT mice, MC38-CD47/PD-L1DKO in Ifnar/ mice grew comparably as WT MC38 tumor cells in WT hosts (Figure 2A). The cGAS/STING pathway in DCs is essential for tumor-cell-derived cytosolic DNA sensing (Ishikawa and Barber, 2008; Ishikawa et al., 2009; Li et al., 2013), and CD47 on tumors can suppress this pathway (Liu et al., 2015b). To determine whether this DNA sensing pathway in DCs is essential for inhibiting CD47- and PD-L1-mediated tumor immune evasion, we measured MC38-CD47/PD-L1DKO tumor growth in STING-deficient mice and further in DC-specific STING-deficient mice. In stark contrast to the comparable growth of WT MC38 tumors in WT, STING-deficient, or DC-specific STING-deficient mice, MC38-CD47/PD-L1DKO tumor cells, which fail to grow in WT mice, grew in STING-deficient or DC-specific STING-deficient mice at a comparable level as WT MC38 tumor cells in WT hosts (Figures 2B and 2C). Therefore, these results suggest that the DC-specific STING signaling and IFNa production play an essential role in inhibiting CD47- and PD-L1-mediated tumor immune evasion. Bispecific Anti-PD-L1-SIRPa Maintains Binding to Tumor Cells, while Greatly Reducing Binding to Red Blood Cells Given the broad expression of CD47, a general concern for systemic administration of current CD47 targeting reagents is offtargeting which results in impaired therapeutic efficiency and generation of side effects. In fact, with the same dose of antiCD47 or SIRPa-hFc, intratumoral administration generated a much better anti-tumor effect than did systemic treatment
(Figures S4A and S4B). Since both CD47 and PD-L1 are highly expressed on tumor cells, and more importantly, CD47 and PD-L1 ablation on tumor cells dramatically suppressed tumorigenesis, we wondered whether a bispecific format with the dual targeting of tumor-specific CD47 and PD-L1 would enable enhanced tumor-targeting specificity and therapeutic effect while reducing the toxicity. Therefore, based on the Fc heterodimer technique, we generated a fusion protein targeting both PD-L1 and CD47 (Figure 3A). Size-exclusion chromatography assay confirmed its heterogenicity (Figure 3A). The anti-PDL1-SIRPa fusion protein preferentially bound CD47 and PD-L1 on MC38 cells, with a major contribution from CD47 and a minor one from PD-L1 (Figure 3B). To evaluate the hematological safety, we first tested the binding of anti-PD-L1-SIRPa to red blood cells (RBCs) in vitro. SIRPa-hFc showed a strong binding with RBCs, whereas anti-PD-L1-SIRPa had little, if any, binding to RBCs (Figure 3C). In vivo, administration of 400 mg of antiPD-L1-SIRPa did not show any significant reduction of RBCs, platelets (PLTs), or hemoglobin (HGB) and increase of Hemin in serum, different from SIRPa-hFc treatment (Figures S5A– S5D). To further determine whether anti-PD-L1-SIRPa could remain tumor cell targeting efficacy in the presence of a large antigen sink, WT MC38 tumor cells were mixed with a 15-fold excess of RBCs and then incubated with fusion proteins. Compared with SIRPa-hFc, anti-PD-L1-SIRPa selectively bound tumor cells (Figure 3D). The binding selectivity of the bispecific fusion protein was also confirmed in human tumor cells and human RBCs and PLTs using corresponding human reagents (data not shown). Therefore, these data suggest that anti-PD-L1-SIRPa could preferentially bind tumor cells without off-tumor toxicity after systemic delivery. Finally, we performed a single-dose PK study in which log-fold-increasing doses of anti-PD-L1-SIRPa were administered intraperitoneally (i.p.). Serum was collected at different time points and human immunoglobulin G (hIgG) serum concentration was determined by ELISA (Figures S6A–S6C). Compared to SIRPa-hFc, significantly higher level of the anti-PD-L1-SIRPa fusion protein was maintained in the blood circulation suggesting a decreased antigen sink effect.
Cell Reports 24, 2101–2111, August 21, 2018 2103
Figure 3. Bispecific Anti-PD-L1-SIRPa Maintains Binding to Tumor Cells, while Greatly Reducing Binding to RBCs (A) Schematic of anti-PD-L1-SIRPa. The wild-type mouse SIRPa ECD (red) was linked with the anti-PD-L1 (blue) antibody. Constant regions were human IgG1 isotype (green). (B) MC38, MC38-CD47KO, MC38-PD-L1KO, or MC38-CD47/PDL1DKO tumor cells were incubated with 10 mg/mL of anti-PD-L1-SIRPa prior to staining with PE anti-human IgG secondary antibody and detected by flow cytometry. (C) Whole blood from a C57BL/6 female WT mouse was incubated with the indicated antibodies pre-labeled with a PE anti-human IgG secondary antibody and assessed by flow cytometry, and the result was expressed as mean fluorescence intensity. (D) MC38 tumor cells were mixed with a 15-fold excess of mouse blood cells. The cell mixture was incubated with 5 mg/mL of SIRPa-hFc or anti-PD-L1-SIRPa prior to staining with PE anti-human IgG secondary antibody and detected by flow cytometry. Red, hFc isotype; blue, SIRPa-hFc or anti-PD-L1-SIRPa. See also Figures S4–S6.
Bispecific Anti-PD-L1-SIRPa Inhibits Tumor Growth via Co-targeting Tumor Cells Next, we aimed to determine whether simultaneous blockade of PD-L1 and CD47 on tumor cells by anti-PD-L1-SIRPa could generate an enhanced anti-tumor effect compared with single blockades. WT C57BL/6 mice were inoculated with MC38 tumor cells. When tumors were well established, mice were intratumorally (i.t.) administered with human IgG isotype control, anti-PDL1, SIRPa-hFc, a mixture of anti-PD-L1 and SIRPa-hFc or antiPD-L1-SIRPa, and the tumor growth was monitored. Compared with the isotype control and the single treatments, both mixtures of anti-PD-L1 and SIRPa-hFc and anti-PD-L1-SIRPa showed
2104 Cell Reports 24, 2101–2111, August 21, 2018
significantly enhanced tumor inhibition and extended mouse survival (Figures 4A and 4B). To evaluate the efficacy of systemic treatment, we i.p. treated established tumor-bearing mice with reagents as described above. Different from intratumor therapy, a mixture of anti-PD-L1 and SIRPa-hFc lost its therapeutic efficacy, while anti-PD-L1-SIRPa treatment still significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice (Figures 4C and 4D). The therapeutic effect of the bispecific anti-PD-L1-SIRPa during systemic administration was also shown in a CT26 tumor model, a commonly used model for PDL1 study in BALB/c mice (Figure 4E). Thus, the anti-PD-L1-SIRPa strategy may have broad application in multiple tumors.
Figure 4. Bispecific Anti-PD-L1-SIRPa Inhibits Tumor Growth via Co-targeting Tumor Cells (A) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.t. with 50 mg hIgG isotype control, 25 mg of anti-PD-L1 or SIRPa-hFc, 25 mg of anti-PD-L1 + 25 mg of SIRPa-hFc, or 50 mg of anti-PD-L1-SIRPa on days 11 and 14. Tumor growth was measured every 4 days starting on day 0. Data represent mean ± SEM and statistical significance was determined by a Student’s t test. (B) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.t. with 50 mg hIgG isotype control, 25 mg of anti-PD-L1 or SIRPa-hFc, 25 mg of anti-PD-L1 + 25 mg of SIRPa-hFc, or 50 mg of anti-PD-L1-SIRPa on days 11 and 14. Tumor growth was measured every 4 days starting on day 0. Statistical significance was determined by a log rank (Mantel-Cox) test. (C) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control, 50 mg of anti-PDL1 or SIRPa-hFc, 50 mg of anti-PD-L1 + 50 mg of SIRPa-hFc, or 100 mg of anti-PD-L1-SIRPa on days 10 and 14. Tumor growth was measured every 4 days starting on day 0. Data represent mean ± SEM and statistical significance was determined by a Student’s t test. (D) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control, 50 mg of anti-PDL1 or SIRPa-hFc, 50 mg of anti-PD-L1 + 50 mg of SIRPa-hFc, or 100 mg of anti-PD-L1-SIRPa on days 10 and 14. Tumor growth was measured every 4 days starting on day 0. Statistical significance was determined by a log rank (Mantel-Cox) test. (E) BALB/c female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 CT26 tumor cells and treated i.p. with 100 mg hIgG isotype control, 50 mg of anti-PD-L1 or SIRPa-hFc, 50 mg of anti-PD-L1 + 50 mg of SIRPa-hFc, or 100 mg of anti-PD-L1-SIRPa on days 10 and 14. Tumor growth was measured every 4 days starting on day 0. Statistical significance was determined by a log rank (Mantel-Cox) test.
(legend continued on next page)
Cell Reports 24, 2101–2111, August 21, 2018 2105
To determine whether tumor-expressing CD47 and PD-L1 are required for the therapeutic effect of anti-PD-L1-SIRPa, we intratumorally administered hIgG isotype control, anti-PDL1, SIRPa-hFc or anti-PD-L1-SIRPa in MC38-CD47KO or MC38-PD-L1KO tumor-bearing mice. The therapeutic effect of anti-PD-L1-SIRPa disappeared in the absence of either CD47 or PD-L1 on tumor cells (Figures 4F and 4G), probably due to reduced tumor cell targeting by anti-PD-L1-SIRPa when one target is missing. Furthermore, PD-L1 deficiency on the host did not diminish the therapeutic effect of anti-PD-L1-SIRPa (Figure 4H). These results indicate that the simultaneous targeting of both PD-L1 and CD47 on tumor cells, but not on host cells, is required for the anti-PD-L1-SIRPa-mediated therapeutic effect. Intratumoral CD8+ T Cells Are Essential for the Therapeutic Effect of Anti-PD-L1-SIRPa To determine the extent to which the anti-tumor effect of antiPD-L1-SIRPa depends on host T cells, Rag1/ mice were inoculated with MC38 tumor cells and treated with anti-PD-L1-SIRPa as before. However, no therapeutic effect was observed, suggesting an essential role for host T cells (Figure 5A). To further elucidate which T cell subset is required for anti-PD-L1-SIRPamediated tumor control, we systemically treated MC38 tumorbearing C57BL/6 mice with anti-PD-L1-SIRPa together with CD8+ or CD4+ T cell-depleting antibodies. In the absence of CD8+ T cells, the therapeutic effect of anti-PD-L1-SIRPa was completely abrogated (Figure 5B), whereas depletion of CD4+ T cells had no effect (Figure 5C). To determine whether antiPD-L1-SIRPa treatment enhances the tumor antigen-specific T cell response, mice were inoculated with MC38-OT-I tumor cells and treated with human IgG, anti-PD-L1, SIRPa-hFc or anti-PD-L1-SIRPa i.p. 11 days after antibody treatment, cells from the tumor-draining lymph nodes were restimulated in vitro with SIINFEKL peptide, and IFN-g production was measured by enzyme-linked immunospot (ELISPOT) assay. Compared with the isotype control and single treatments, significantly more IFN-g-producing cells were detected in the anti-PD-L1SIRPa-treated group (Figure 5D). To further determine the true anti-tumor T cell response, but not to model tumor antigen OVA, we treated MC38 tumors, and irradiated MC38 tumor cells were used for restimulation for ELISPOT assay. Again, more tumor-specific IFN-g-producing cells were determined in the antiPD-L1-SIRPa-treated group compared with isotype control and single treatments (Figure 5E). To further determine whether antiPD-L1-SIRPa also affects intratumor antigen-specific T cell response, tumor-bearing mice were treated with FTY720 before fusion protein treatment to inhibit T cell circulation. Interestingly, FTY720 did not diminish the therapeutic effect of anti-PD-L1-
SIRPa (Figure 5F). Together, these results suggest that intratumor CD8+ T cell response is enhanced by anti-PD-L1-SIRPa and essential for its therapeutic efficacy. Anti-PD-L1-SIRPa Enhances Intratumoral DC Innate Sensing Intratumor antigen-presenting cells, such as DCs, are required for T cell response generation. To evaluate whether anti-PDL1-SIRPa enhances intratumor DC cross-presentation, we collected DCs from the tumor microenvironment of MC38OT-I-bearing mice after treatment and co-cultured them with OVA-specific OT-I T cells. Compared with the isotype control and single treatments, DCs harvested from the tumors treated with anti-PD-L1-SIRPa showed significantly higher cross-presentation function than those from the other groups, as indicated by the increased number of IFN-g-producing cells (Figure 6A). Given the important role of DC-producing IFNa in the arrest of MC38-CD47/PD-L1DKO tumor growth, we evaluated whether type I IFN was required for the therapeutic efficacy of anti-PDL1-SIRPa. Indeed, intratumor IFNAR blockade significantly reduced the efficacy of anti-PD-L1-SIRPa (Figure 6B). To detect whether the DC-specific STING pathway is essential for initiating IFNa production by anti-PD-L1-SIRPa treatment, we tested the efficacy of anti-PD-L1-SIRPa treatment in STING-deficient and CD11c-STING-deficient mice. As expected, the therapeutic efficacy of anti-PD-L1-SIRPa was primarily abolished in such mice (Figures 6C and 6D). Thus, these results suggest the critical role of the DC-specific STING pathway in this treatment. Chemotherapy Induces More ‘‘Eat Me’’ Signals and Synergizes with Anti-PD-L1-SIRPa for Tumor Eradication At the clinic, chemotherapy is often a first-line therapy. Certain chemo-drugs enhance calreticulin (CRT) exposure on tumor cells (Obeid et al., 2007), which is an important ‘‘eat me’’ signal that antagonizes with the CD47 ‘‘don’t eat me’’ signal (Chao et al., 2010b). We therefore hypothesized that pretreatment with chemotherapy might synergizes with anti-PD-L1-SIRPa to further enhance tumor eradication. Doxorubicin (DOX) has been reported to induce CRT exposure on tumor cells (Obeid et al., 2007). We first confirmed that treatment of MC38 tumor cells with DOX indeed induced CRT exposure on the cell surface (Figure 7A). Then we tested whether DOX would synergize with anti-PD-L1-SIRPa treatment. Mice were inoculated with MC38 tumor cells, and, 11 days later, DOX was administered intratumorally 1 day before antibody treatments. Compared with the isotype control and single treatments, DOX plus anti-PD-L1SIRPa treatment increased tumor inhibition and prolonged the survival of tumor-bearing mice (Figures 7B and 7C). To verify
(F) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38-CD47KO tumor cells and treated i.t. with 50 mg hIgG isotype control, anti-PD-L1, SIRPa-hFc, or anti-PD-L1-SIRPa on days 10 and 15. Tumor growth was measured every 4 days starting on day 0. Data represent mean ± SEM and statistical significance was determined by a Student’s t test. (G) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38-PD-L1KO tumor cells and treated i.t. with 50 mg hIgG isotype control, anti-PDL1, SIRPa-hFc, or anti-PD-L1-SIRPa on days 10 and 15. Tumor growth was measured every 4 days starting on day 0. Data represent mean ± SEM and statistical significance was determined by a Student’s t test. (H) C57BL/6 female WT or PD-L1 knockout mice (n = 4–6) were inoculated s.c. with 1 3 106 MC38 and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1SIRPa on days 8 and 11. Tumor growth was measured twice per week. Data represent mean ± SEM and statistical significance was determined a by Student’s t test.
2106 Cell Reports 24, 2101–2111, August 21, 2018
Figure 5. CD8+ T Cells Are Essential for the Therapeutic Effect of Anti-PD-L1-SIRPa (A) C57BL/6 female Rag1/ mice (n = 5or 6/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1-SIRPa on days 10 and 14. Tumor growth was measured every 4 days starting on day 0. (B) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1-SIRPa on days 10 and 14. CD8depleting antibody was administered twice a week starting on day 1. The tumor growth was measured and compared twice a week starting on day 0. (C) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1-SIRPa on days 8 and 11. CD4depleting antibody was administered twice a week starting on day 1. The tumor growth was measured and compared twice a week starting on day 0. (D) C57BL/6 female WT mice (n = 3 or 4/group) were inoculated s.c. with 2 3 105 MC38-OT-I tumor cells and treated i.p. with 50 mg hIgG isotype control, 25 mg of anti-PD-L1 or SIRPa-hFc, or 50 mg of anti-PD-L1-SIRPa on days 10 and 14. Lymphocytes from DLNs were isolated on day 21 and stimulated with 10 mg/mL OT-I peptide. IFNg-producing cells were enumerated by ELISPOT assay. (E) C57BL/6 female WT mice (n = 7/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 50 mg hIgG isotype control, 25 mg of anti-PD-L1 or SIRPa-hFc, or 50 mg of anti-PD-L1-SIRPa on days 10 and 14. Lymphocytes from DLNs were isolated on day 21 and stimulated with 60-Gy-irradiated MC38 tumor cells. The ratio of DLN cells to tumor cells is 5:1. IFN-g-producing cells were enumerated by ELISPOT assay after 2 days. (F) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1-SIRPa on days 10 and 14. 25 mg of FTY720 was administered i.p. on the day 1 and 5 mg of FTY720 on days 0–2. The tumor growth was measured and compared twice a week starting on day 0. For all graphs, data represent mean ± SEM and statistical significance was determined by a Student’s t test.
whether the synergy between DOX and anti-PD-L1-SIRPa was due to enhanced exposure of the ‘‘eat me’’ molecule CRT, we treated mice with Z-VAD-FMK, a caspase inhibitor that results in reduced expression of membrane CRT (Casares et al., 2005; Obeid et al., 2007), before DOX and fusion protein treatment. As speculated, the synergy between DOX and anti-PD-L1-SIRPa disappeared with Z-VAD-FMK treatment (Figure 7D). Thus, these data indicate that DOX, which induces more ‘‘eat me’’ signaling, has a synergistic role when used in combination with anti-PD-L1SIRPa. DISCUSSION In this study, we showed that CD47 and PD-L1 on tumor cells coordinate for two-step evasion of innate and adaptive immunity. In
a previous study, Dheilly et al. demonstrated that anti-mesothelin (or CD19)/anti-CD47 bsAb overcome tolerability and ‘‘antigen sink’’ issues by selectively blocking the CD47-SIRPa interaction on malignant cells expressing a specific tumor-associated antigen (Dheilly et al., 2017). However, given the immunodeficient and xenograft model used, the targeting efficiency, the role of T cells in anti-tumor efficacy and the side effect have not been adequately evaluated in the previous study. Both PD-L1 and CD47 are highly expressed on tumor cells, whereas most normal tissues, including RBCs, lack PD-L1 expression, so we used a higher-affinity anti-PD-L1 and the lower-affinity SIRPa as heterodimer partners in our current study. Furthermore, by using immunocompetent synergistic tumor model, we showed that the bispecific heterodimer anti-PD-L1-SIRPa fusion protein target tumor cells efficiently while only weakly bind RBCs,
Cell Reports 24, 2101–2111, August 21, 2018 2107
Figure 6. Anti-PDL1-SIRPa Enhances Intratumoral DC Innate Sensing (A) C57BL/6 female WT mice (n = 8/group) were inoculated s.c. with 2 3 105 MC38-OT-I tumor cells and treated i.t. with 50 mg hIgG isotype control, 25 mg of anti-PD-L1 or SIRPa-hFc, or 50 mg of anti-PD-L1-SIRPa on days 10 and 14. Three days after the second treatment, CD45+CD11c+MHCII+ DC cells were isolated from tumor and cocultured with isolated CD8+ T cells from naive OT-I mice for 48 hr. IFN-g-producing cells were enumerated by ELISPOT assay. (B) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1-SIRPa on days 10 and 14. 50 mg antiIFNAR1 antibody was administered i.t. on the days 1, 4, and 8. Tumor growth was measured. (C) C57BL/6 female WT and Sting/ mice (n = 5/ group) were inoculated s.c. with 4 3 105 MC38 tumor cells and treated i.p. with 200 mg hIgG isotype control or anti-PD-L1-SIRPa on days 11 and 14. Tumor growth was measured. (D) C57BL/6 male WT or CD11c-cre 3 Stingfl/fl mice (n = 5/group) were inoculated s.c. with 5 3 105 MC38 tumor cells and treated i.p. with 200 mg hIgG isotype control or anti-PD-L1-SIRPa on days 13 and 16. Tumor growth was measured. For all graphs, data represent mean ± SEM and statistical significance was determined by a Student’s t test.
compared with SIRPa-Fc. Consequently, the enhanced tumor targeting by the bispecific fusion protein generated much better anti-tumor efficacy than single targeting or cocktail. This is also consistent with the data showing that both CD47 and PD-L1 on tumor cells are necessary for immune evasion. On one hand, the generation of T cell response and PD-L1 upregulation by CD47-SIRPa blockade create a better situation for anti-PD-L1 to target. On the other hand, type I interferons generated during CD47 blockade may enhance DC co-stimulatory function, which is required for full rejuvenation of intratumor exhausted T cells during anti-PD-L1 blockade (Kelly, 2017). In addition to the synergistic effect of CD47 and PD-L1 blockade for adaptive T cell responses, they may also cooperate in reducing the tumor burden at the innate level. Recent studies have reported that PD-1 signaling inhibits phagocytosis of tumor-associated macrophages (Gordon et al., 2017). It would be interesting to investigate whether the dual blockade of CD47 and PD-L1 on tumor cells would create an additional bridge promoting macrophage phagocytosis of tumor cells. Recent work has shown a critical role for DCs in capturing tumor antigen and mitochondrial DNA during CD47 blockade (Xu et al., 2017). Whether PD-L1 blockade would simultaneously facilitate DC capture remains an intriguing question. In addition, given the possibility of PD-L1 reverse signaling, it would also be interesting to study whether PD-L1 blockade modulates tumor cells themselves for enhanced mitochondrial DNA release. Some chemotherapy drugs can directly kill tumor cells, while others may additionally exert stress on tumor cells and induce ‘‘eat me’’ signaling. CRT exposure is one of the major ‘‘eat me’’ signals. CRT enhances tumor cell phagocytosis by macro-
2108 Cell Reports 24, 2101–2111, August 21, 2018
phages/DCs, further leading to the generation of anti-tumor T cell response. This may be especially synergistic with the bispecific anti-PD-L1-SIRPa fusion protein. Indeed, DOX pretreatment significantly enhances the anti-tumor effect of the antiPD-L1-SIRPa fusion protein. However, it should be noted that not all chemotherapy drugs are proficient at inducing immunogenetic cell death. Therefore, it remains to be determined whether the anti-PD-L1-SIRPa fusion protein has synergistic effects with those chemotherapy drugs. In conclusion, our study has revealed the critical coordination of tumor-cell-specific CD47 and PD-L1 for tumor evasion. Based on this finding, we have developed an improved strategy that simultaneously targets both CD47 and PD-L1 on tumor cells, while reducing their off-target binding to healthy cells. This strategy results in significantly enhanced anti-tumor effects compared with either single CD47 or PD-L1 blockade alone and shows synergistic effects of chemotherapy, such as DOX. We believe this dual-targeting strategy will provide insight into tumor immunotherapy for better tumor control.
EXPERIMENTAL PROCEDURES Mice Six- to eight-week-old female C57BL/6J and BALB/c mice were purchased from Beijing Vital River Laboratory in China. Rag1/, Ifnar/, Sting/, OT-I CD8+ T cell receptor (TCR)-Tg, CD11c-Cre-Tg, and Stingfl/fl mice were obtained from an internal breeding facility. All mice were maintained under specific pathogen-free conditions, and age- and sex-matched mice were used between 6–12 weeks of age, in accordance with the experimental animal guidelines set by the Institutional Animal Care and Use Committee of the
Figure 7. Chemotherapy Induces More ‘‘Eat Me’’ Signals and Synergizes with Anti-PDL1-SIRPa for Tumor Eradication (A) 1 3 106 MC38 tumor cells were cocultured with 2 mg/mL DOX for 12 hr, and calreticulin was measured by flow cytometry. (B) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1SIRPa on days 10 and 14. 50 mg of DOX was administered i.t. 1 day before anti-PD-L1-SIRPa. Tumor volume was measured. Data represent mean ± SEM and statistical significance was determined by a Student’s t test. (C) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1SIRPa on days 10 and 14. 50 mg of DOX was administered i.t. 1 day before anti-PD-L1-SIRPa. Tumor volume was measured. Statistical significance was determined by a log rank (Mantel-Cox) test. (D) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1SIRPa on days 10 and 14. 50 mg of DOX was administered i.t. 1 day before anti-PD-L1-SIRPa. 40 mM 3 50 mL of V-ZAD-FMK was administered i.t. every 4 days from 1 day before anti-PD-L1-SIRPa treatment. Tumor volume was measured. Statistical significance was determined by a log rank (Mantel-Cox) test.
Institute of Biophysics, CAS, and the University of Texas Southwestern Medical Center at Dallas.
were produced in-house. The endotoxin level for Ab and fusion protein was lower than 0.2 EU (endotoxin units)/mg of protein.
Cell Lines and Reagents All cell lines were free of mycoplasma contamination. MC38 and CT26 are murine colon adenocarcinoma cell lines. MC38-OTIp was sorted and subcloned after MC38 cells were stably transduced with the retrovirus-expressing mouse CCR7-OTIp (peptide epitope for OTI). MC38-CD47KO, MC38-PD-L1KO, and MC38-CD47/PD-L1DKO tumor cells were acquired using CRISPR/Cas9 technology. The single guide RNA (sgRNA) sequences were as follows: sgCD47-F 50 -TTGGCGGCGGCGCTGTTGCT-30 and sgCD47-R 50 -CCCTT GCATCGTCCGTAATG-30 ; sgPD-L1-F 50 -GGCTCCAAAGGACTTGTACG-30 ; and sgPD-L1-R 50 -GACTTGTACGTGGTGGAGTA-30 . B16F10 is a murine melanoma cell line. B16F10-CD47KO, B16F10-PD-L1KO, and B16F10-CD47/PDL1DKO tumor cells were acquired using CRISPR/Cas9 technology. Fluorescein isothiocyanate (FITC)-conjugated anti-mCD47, PE-conjugated anti-PD-L1, PE-conjugated anti-human IgG, APC-conjugated anti-CD45.2, PE-Cy7-conjugated anti-CD11c, FITC-conjugated anti-MHCII, Percp-Cy5.5-conjugated anti-CD11b, and PE-conjugated anti-F4/80 were purchased from BioLegend Company and FTY720 was purchased from Sigma-Aldrich. The chemotherapeutic reagents DOX and Z-VAD-FMK were purchased from Sigma-Aldrich and prepared in accordance with the manufacturer’s recommendations. Anti-mIFNAR1-neutralizing monoclonal antibody (mAb) (clone MAR1-5A3) was purchased from BioXcell (West Lebanon, NH, USA). The variable regions of light chain and heavy chain of anti-PD-L1 Ab sequence were synthesized according to patent (patent no.: US 8,217,149 B2, YW243.55.S70). Anti-PDL1, SIRPa-hFc, and anti-PD-L1-SIRPa fusion proteins; anti-CD8-depleting antibody (clone TIB210); and anti-CD4-depleting antibody (clone GK1.5)
Tumor Inoculation, Treatment, and Digestion For tumor growth observation, we s.c. injected MC38, MC38-CD47KO, MC38PD-L1KO, MC38-CD47/PD-L1DKO, or B16F10 and B16F10-CD47KO, B16F10PD-L1KO and B16F10-CD47/PD-L1DKO tumor cells into the flanks of mice. Tumor volumes were measured by the length (a), width (b), and height (h) and calculated as follows: tumor volume = a*b*h/2. For fusion protein treatment, we s.c. injected MC38, CT26, or MC38-OTIp tumor cells into the flanks of mice. Tumors, which were allowed to grow for 10–14 days to reach 80 mm3, were treated with fusion proteins intratumorally or i.p., and the tumor volume was measured. For CD8 or CD4 depletion, anti-CD8 (clone TIB210) or anti-CD4 (clone GK1.5) antibody was injected i.p. To block the migration of activated T cells, FTY720 was administered i.p. For type I IFN blockade experiments, anti-IFNAR1 mAb was intratumorally injected. For DOX and Z-VADFMK treatment, DOX or Z-VAD-FMK was administered intratumorally. Tumor tissues were excised and digested with 1 mg/mL collagenase IV (Sigma) in the incubator. After 40 min, tumors were passed through a 70-mm cell strainer to remove large pieces of the undigested tumor. Tumor-infiltrating cells were then washed twice with PBS. Production of Anti-PD-L1, SIRPa-hIg, or Anti-PD-L1-SIRPa Fusion Protein Anti-PD-L1 or SIRPa-hFc was generated as previously described (Tang et al., 2016). Briefly, a plasmid encoding the DNA sequence of anti-PD-L1 or SIRPa IgV-like domain was cloned into the pEE12.4 expression plasmid (Lonza, Basel, Switzerland) between the IgG IgGk leading sequence and the human
Cell Reports 24, 2101–2111, August 21, 2018 2109
IgG1 Fc sequence using BsiWI and BstBI. A heterodimer of anti-PD-L1-SIRPa was achieved by the knobs-into-holes approach. The fusion proteins were transiently expressed in FreeStyle 293-F cells and purified by a protein A column in accordance with the manufacturer’s instructions. ELISPOT Assay For tumor-specific CD8+ T cell functional assays in the MC38-OT-I model, tumor-draining lymph nodes (DLNs) were removed 11 days after fusion protein treatment. 3 3 105 DLN cells were then restimulated with 10 mg/mL OT-I peptide (SIINFEKL). For tumor-specific CD8+ T cell functional assays in the MC38 model, tumor DLNs were removed 11 days after fusion protein treatment. 5 3 105 DLN cells were then restimulated with 1 3 105 MC38 tumor cells irradiated with 60 Gy. A 96-well HTS-IP plate (BD PharMingen) was pre-coated with the anti-IFN-g antibody (BD PharMingen) at a 1:250 dilution overnight at 4 C. After co-culture for 48 hr, cells were removed, 2 mg/mL biotinylated anti-IFN-g antibody (BD PharMingen) at a 1:250 dilution was added, and the plate was incubated for 2 hr at room temperature. Avidin-horseradish peroxidase (BD PharMingen) at a 1:1,000 dilution was then added, and the plate was incubated for 1 hr at room temperature. The cytokine spots of IFN-g were developed in accordance with the manufacturer’s protocol (BD PharMingen). Ex Vivo DC Cross-Presentation Assay MC38-OT-I-bearing mice were treated with fusion proteins by intratumoral injection on days 11 and 14. Three days after the second treatment, tumors were digested, and DCs were purified by fluorescence-activated cell sorting (FACS). Approximately 5 3 104 DCs were mixed together with purified 5 3 105 OT-I T cells, which were sorted on a FACSAria II Cell Sorter (BD) from lymph nodes of 6- to 12-week-old OT-I transgenic mice. After 48 hr, IFN-g-producing cells were enumerated by ELISPOT assay (BD PharMingen). Flow Cytometric Sorting and Analysis Single-cell suspensions were blocked with anti-FcR (clone 2.4G2, BioXcell) and then stained with antibodies (1:500 dilution) against PD-L1, CD47, CD3, CD8, Va2, CD11c, MHCII, CD11b, F4/80, CD45, and DAPI. Cells were analyzed on Fortessa cytometer or sorted on a FACSAria II Cell Sorter (BD). Data were analyzed with FlowJo Software. Statistical Analysis Data were analyzed using Prism 6.0 software (GraphPad) and are presented as the mean ± SEM. p values were assessed using the two-tailed unpaired Student’s t test or two-way analysis of variance, with p values considered significant as follows: *p < 0.05; **p < 0.01; and ***p < 0.001. For tumor-free mice frequencies, statistics were performed with the log rank (Mantel-Cox) test. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and six figures and can be found with this article online at https://doi.org/10. 1016/j.celrep.2018.07.062. ACKNOWLEDGMENTS We thank Ting Xu, Yan Luan, Kai Fu, Shuli Ma, and Jianjian Peng for providing the heterodimeric Fc sequence. This work was, in part, supported by grants from the National Key R&D Program of China (2016YFC1303405 to Y.-X.F.), the Chinese Academy of Sciences (XDA12020212 to M.Z. and Y.-X.F.), and the U.S. NIH through the National Cancer Institute (CA141975 to Y.-X.F.). AUTHOR CONTRIBUTIONS X.L., M.Z., and Y.-X.F. designed the experiments and analyzed the data. X.L., L.L, Z.R., K.Y., Y.L., and K.F. performed the experiments. H.X., J.G., and H.P. provided the reagents. X.L., M.Z., and Y.-X.F. wrote the manuscript. Y.-X.F. supervised the project.
2110 Cell Reports 24, 2101–2111, August 21, 2018
DECLARATION OF INTERESTS The authors declare no competing interests. Received: September 11, 2017 Revised: June 1, 2018 Accepted: July 17, 2018 Published: August 21, 2018 REFERENCES Baccelli, I., Schneeweiss, A., Riethdorf, S., Stenzinger, A., Schillert, A., Vogel, V., Klein, C., Saini, M., Ba¨uerle, T., Wallwiener, M., et al. (2013). Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nat. Biotechnol. 31, 539–544. Baccelli, I., Stenzinger, A., Vogel, V., Pfitzner, B.M., Klein, C., Wallwiener, M., Scharpff, M., Saini, M., Holland-Letz, T., Sinn, H.P., et al. (2014). Co-expression of MET and CD47 is a novel prognosticator for survival of luminal breast cancer patients. Oncotarget 5, 8147–8160. Barclay, A.N., and Van den Berg, T.K. (2014). The interaction between signal regulatory protein alpha (SIRPa) and CD47: structure, function, and therapeutic target. Annu. Rev. Immunol. 32, 25–50. Blackburn, S.D., Shin, H., Haining, W.N., Zou, T., Workman, C.J., Polley, A., Betts, M.R., Freeman, G.J., Vignali, D.A., and Wherry, E.J. (2009). Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat. Immunol. 10, 29–37. Brahmer, J.R., Drake, C.G., Wollner, I., Powderly, J.D., Picus, J., Sharfman, W.H., Stankevich, E., Pons, A., Salay, T.M., McMiller, T.L., et al. (2010). Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J. Clin. Oncol. 28, 3167–3175. Brahmer, J.R., Tykodi, S.S., Chow, L.Q., Hwu, W.J., Topalian, S.L., Hwu, P., Drake, C.G., Camacho, L.H., Kauh, J., Odunsi, K., et al. (2012). Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455–2465. Brown, E.J., and Frazier, W.A. (2001). Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 11, 130–135. Brown, J.A., Dorfman, D.M., Ma, F.R., Sullivan, E.L., Munoz, O., Wood, C.R., Greenfield, E.A., and Freeman, G.J. (2003). Blockade of programmed death1 ligands on dendritic cells enhances T cell activation and cytokine production. J. Immunol. 170, 1257–1266. Casares, N., Pequignot, M.O., Tesniere, A., Ghiringhelli, F., Roux, S., Chaput, N., Schmitt, E., Hamai, A., Hervas-Stubbs, S., Obeid, M., et al. (2005). Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J. Exp. Med. 202, 1691–1701. Casey, S.C., Tong, L., Li, Y., Do, R., Walz, S., Fitzgerald, K.N., Gouw, A.M., €tgemann, I., Eilers, M., and Felsher, D.W. (2016). MYC regulates Baylot, V., Gu the antitumor immune response through CD47 and PD-L1. Science 352, 227–231. Chao, M.P., Alizadeh, A.A., Tang, C., Myklebust, J.H., Varghese, B., Gill, S., Jan, M., Cha, A.C., Chan, C.K., Tan, B.T., et al. (2010a). Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma. Cell 142, 699–713. Chao, M.P., Jaiswal, S., Weissman-Tsukamoto, R., Alizadeh, A.A., Gentles, A.J., Volkmer, J., Weiskopf, K., Willingham, S.B., Raveh, T., Park, C.Y., et al. (2010b). Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47. Sci. Transl. Med. 2, 63ra94. Cioffi, M., Trabulo, S., Hidalgo, M., Costello, E., Greenhalf, W., Erkan, M., Kleeff, J., Sainz, B., Jr., and Heeschen, C. (2015). Inhibition of CD47 effectively targets pancreatic cancer stem cells via dual mechanisms. Clin. Cancer Res. 21, 2325–2337. Dheilly, E., Moine, V., Broyer, L., Salgado-Pires, S., Johnson, Z., Papaioannou, A., Cons, L., Calloud, S., Majocchi, S., Nelson, R., et al. (2017). Selective
blockade of the ubiquitous checkpoint receptor CD47 is enabled by dual-targeting bispecific antibodies. Mol. Ther. 25, 523–533. Dong, H., Strome, S.E., Salomao, D.R., Tamura, H., Hirano, F., Flies, D.B., Roche, P.C., Lu, J., Zhu, G., Tamada, K., et al. (2002). Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat. Med. 8, 793–800. Edris, B., Weiskopf, K., Volkmer, A.K., Volkmer, J.P., Willingham, S.B., Contreras-Trujillo, H., Liu, J., Majeti, R., West, R.B., Fletcher, J.A., et al. (2012). Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma. Proc. Natl. Acad. Sci. USA 109, 6656–6661. Freeman, G.J., Long, A.J., Iwai, Y., Bourque, K., Chernova, T., Nishimura, H., Fitz, L.J., Malenkovich, N., Okazaki, T., Byrne, M.C., et al. (2000). Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 192, 1027–1034. Gordon, S.R., Maute, R.L., Dulken, B.W., Hutter, G., George, B.M., McCracken, M.N., Gupta, R., Tsai, J.M., Sinha, R., Corey, D., et al. (2017). PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity. Nature 545, 495–499. Green, M.R., Monti, S., Rodig, S.J., Juszczynski, P., Currie, T., O’Donnell, E., Chapuy, B., Takeyama, K., Neuberg, D., Golub, T.R., et al. (2010). Integrative analysis reveals selective 9p24.1 amplification, increased PD-1 ligand expression, and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma. Blood 116, 3268–3277. Ishikawa, H., and Barber, G.N. (2008). STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678. Ishikawa, H., Ma, Z., and Barber, G.N. (2009). STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792. Jaiswal, S., Jamieson, C.H., Pang, W.W., Park, C.Y., Chao, M.P., Majeti, R., Traver, D., van Rooijen, N., and Weissman, I.L. (2009). CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell 138, 271–285. Kelly, P.N. (2017). CD28 is a critical target for PD-1 blockade. Science 355, 1386. Li, X.D., Wu, J., Gao, D., Wang, H., Sun, L., and Chen, Z.J. (2013). Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341, 1390–1394. Liu, J., Wang, L., Zhao, F., Tseng, S., Narayanan, C., Shura, L., Willingham, S., Howard, M., Prohaska, S., Volkmer, J., et al. (2015a). Pre-clinical development of a humanized anti-CD47 antibody with anti-cancer therapeutic potential. PLoS ONE 10, e0137345. Liu, X., Pu, Y., Cron, K., Deng, L., Kline, J., Frazier, W.A., Xu, H., Peng, H., Fu, Y.X., and Xu, M.M. (2015b). CD47 blockade triggers T cell-mediated destruction of immunogenic tumors. Nat. Med. 21, 1209–1215. Lo, J., Lau, E.Y., So, F.T., Lu, P., Chan, V.S., Cheung, V.C., Ching, R.H., Cheng, B.Y., Ma, M.K., Ng, I.O., and Lee, T.K. (2016). Anti-CD47 antibody suppresses tumour growth and augments the effect of chemotherapy treatment in hepatocellular carcinoma. Liver Int. 36, 737–745.
Oldenborg, P.A., Zheleznyak, A., Fang, Y.F., Lagenaur, C.F., Gresham, H.D., and Lindberg, F.P. (2000). Role of CD47 as a marker of self on red blood cells. Science 288, 2051–2054. Piccione, E.C., Juarez, S., Tseng, S., Liu, J., Stafford, M., Narayanan, C., Wang, L., Weiskopf, K., and Majeti, R. (2016). SIRPalpha-antibody fusion proteins selectively bind and eliminate dual antigen-expressing tumor cells. Clin. Cancer Res. 22, 5109–5119. Ponce, L.P., Fenn, N.C., Moritz, N., Krupka, C., Kozik, J.H., Lauber, K., Subklewe, M., and Hopfner, K.P. (2017). SIRPa-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells. Oncotarget 8, 11284–11301. Tang, H., Wang, Y., Chlewicki, L.K., Zhang, Y., Guo, J., Liang, W., Wang, J., Wang, X., and Fu, Y.X. (2016). Facilitating T cell infiltration in tumor microenvironment overcomes resistance to PD-L1 blockade. Cancer Cell 29, 285–296. Taube, J.M., Anders, R.A., Young, G.D., Xu, H., Sharma, R., McMiller, T.L., Chen, S., Klein, A.P., Pardoll, D.M., Topalian, S.L., and Chen, L. (2012). Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci. Transl. Med. 4, 127ra37. Thompson, R.H., Gillett, M.D., Cheville, J.C., Lohse, C.M., Dong, H., Webster, W.S., Krejci, K.G., Lobo, J.R., Sengupta, S., Chen, L., et al. (2004). Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target. Proc. Natl. Acad. Sci. USA 101, 17174– 17179. Topalian, S.L., Hodi, F.S., Brahmer, J.R., Gettinger, S.N., Smith, D.C., McDermott, D.F., Powderly, J.D., Carvajal, R.D., Sosman, J.A., Atkins, M.B., et al. (2012). Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med. 366, 2443–2454. Tsai, R.K., and Discher, D.E. (2008). Inhibition of ‘‘self’’ engulfment through deactivation of myosin-II at the phagocytic synapse between human cells. J. Cell Biol. 180, 989–1003. Weiskopf, K., Ring, A.M., Ho, C.C., Volkmer, J.P., Levin, A.M., Volkmer, A.K., Ozkan, E., Fernhoff, N.B., van de Rijn, M., Weissman, I.L., and Garcia, K.C. (2013). Engineered SIRPa variants as immunotherapeutic adjuvants to anticancer antibodies. Science 341, 88–91. Willingham, S.B., Volkmer, J.P., Gentles, A.J., Sahoo, D., Dalerba, P., Mitra, S.S., Wang, J., Contreras-Trujillo, H., Martin, R., Cohen, J.D., et al. (2012). The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc. Natl. Acad. Sci. USA 109, 6662–6667. Wolchok, J.D., Kluger, H., Callahan, M.K., Postow, M.A., Rizvi, N.A., Lesokhin, A.M., Segal, N.H., Ariyan, C.E., Gordon, R.A., Reed, K., et al. (2013). Nivolumab plus ipilimumab in advanced melanoma. N. Engl. J. Med. 369, 122–133. Xiao, Z., Chung, H., Banan, B., Manning, P.T., Ott, K.C., Lin, S., Capoccia, B.J., Subramanian, V., Hiebsch, R.R., Upadhya, G.A., et al. (2015). Antibody mediated therapy targeting CD47 inhibits tumor progression of hepatocellular carcinoma. Cancer Lett. 360, 302–309.
Obeid, M., Tesniere, A., Ghiringhelli, F., Fimia, G.M., Apetoh, L., Perfettini, J.L., Castedo, M., Mignot, G., Panaretakis, T., Casares, N., et al. (2007). Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 13, 54–61.
Xu, M.M., Pu, Y., Han, D., Shi, Y., Cao, X., Liang, H., Chen, X., Li, X.D., Deng, L., Chen, Z.J., et al. (2017). Dendritic cells but not macrophages sense tumor mitochondrial DNA for cross-priming through signal regulatory protein a signaling. Immunity 47, 363–373.e5.
Okazawa, H., Motegi, S., Ohyama, N., Ohnishi, H., Tomizawa, T., Kaneko, Y., Oldenborg, P.A., Ishikawa, O., and Matozaki, T. (2005). Negative regulation of phagocytosis in macrophages by the CD47-SHPS-1 system. J. Immunol. 174, 2004–2011.
Yoshida, K., Tsujimoto, H., Matsumura, K., Kinoshita, M., Takahata, R., Matsumoto, Y., Hiraki, S., Ono, S., Seki, S., Yamamoto, J., and Hase, K. (2015). CD47 is an adverse prognostic factor and a therapeutic target in gastric cancer. Cancer Med. 4, 1322–1333.
Cell Reports 24, 2101–2111, August 21, 2018 2111
Dual Targeting of Innate and Adaptive Checkpoints on Tumor Cells Limits Immune Evasion Graphical Abstract
Authors Xiaojuan Liu, Longchao Liu, Zhenhua Ren, ..., Hua Peng, Mingzhao Zhu, Yang-Xin Fu
Correspondence [email protected] (M.Z.), [email protected] (Y.X.F.)
In Brief CD47 and PD-L1 serve as critical innate and adaptive checkpoints, respectively. Liu et al. show CD47 and PD-L1 coordinate in tumor cells to evade immune response. Furthermore, a bispecific antibody design enables better targeting on tumor cells, but less on nontumor cells, and enhanced therapeutic efficacy.
Highlights d
CD47 and PD-L1 checkpoints on tumor cells coordinate to suppress immune sensing
d
The bispecific antibody enables better targeting on tumor cells than on non-tumor cells
d
Dual targeting increases cytosolic DNA sensing in DCs for an anti-tumor T cell response
d
Chemotherapy induces danger signals that synergize with bispecific antibody
Liu et al., 2018, Cell Reports 24, 2101–2111 August 21, 2018 ª 2018 The Authors. https://doi.org/10.1016/j.celrep.2018.07.062
Cell Reports
Article Dual Targeting of Innate and Adaptive Checkpoints on Tumor Cells Limits Immune Evasion Xiaojuan Liu,1,2,5 Longchao Liu,3,5 Zhenhua Ren,3 Kaiting Yang,1 Hairong Xu,1 Yan Luan,4 Kai Fu,4 Jingya Guo,1 Hua Peng,1 Mingzhao Zhu,1,2,* and Yang-Xin Fu1,3,6,* 1Chinese
Academy of Sciences Key Laboratory of Infection and Immunity, IBP-UTSW Joint Immunotherapy Group, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China 3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA 4DingFu Biotarget Co. Ltd., Suzhou, Jiangsu 215125, China 5These authors contributed equally 6Lead Contact *Correspondence: [email protected] (M.Z.), [email protected] (Y.-X.F.) https://doi.org/10.1016/j.celrep.2018.07.062
SUMMARY
CD47 on tumor cells protects from phagocytosis, while PD-L1 dampens T cell-mediated tumor killing. However, whether and how CD47 and PD-L1 coordinate is poorly understood. We reveal that CD47 and PD-L1 on tumor cells coordinately suppress innate and adaptive sensing to evade immune control. Targeted blockade of both CD47 and PD-L1 on tumor cells with a bispecific anti-PD-L1-SIRPa showed significantly enhanced tumor targeting and therapeutic efficacy versus monotherapy. Mechanistically, systemic delivery of the dual-targeting heterodimer significantly increased DNA sensing, DC cross-presentation, and anti-tumor T cell response. In addition, chemotherapy that increases ‘‘eat me’’ signaling further synergizes with the bispecific reagent for better tumor control. Our data indicate that tumor cells evolve to utilize both innate and adaptive checkpoints to evade anti-tumor immune responses and that tumor cell-specific dualtargeting of both checkpoints represents an improved strategy for tumor immunotherapy. INTRODUCTION CD47, ubiquitously expressed on the membrane of all cells in mice and humans, is an essential ‘‘don’t eat me’’ signal (Barclay and Van den Berg, 2014; Brown and Frazier, 2001; Okazawa et al., 2005). By interacting with its receptor SIRPa, which is primarily expressed on phagocytic macrophages and dendritic cells (DCs), it inhibits myosin-IIA accumulation at the phagocytic synapse and, consequently, prevents phagocytosis (Oldenborg et al., 2000; Tsai and Discher, 2008). Although the CD47-SIRPa interaction plays essential roles in self-recognition and selfhomeostasis, it appears to have been hijacked by tumor cells during evolution. Increased expression of CD47 has been observed in nearly all types of tumors examined (Baccelli et al.,
2013, 2014; Cioffi et al., 2015; Jaiswal et al., 2009). This becomes a potentially important strategy for tumor cells to evade phagocytosis and clearance. Taking advantage of this principle, systemic blockade of the CD47-SIRPa interaction has demonstrated encouraging efficacy in limiting tumor growth in xenograft studies (Chao et al., 2010a; Edris et al., 2012; Lo et al., 2016; Weiskopf et al., 2013; Willingham et al., 2012; Xiao et al., 2015; Yoshida et al., 2015). However, in syngeneic grafts, given the broad expression of CD47, the anti-CD47 antibody has been shown to bind to non-tumor cells, especially red blood cells, thus limiting its systemic use (Dheilly et al., 2017; Liu et al., 2015a; Piccione et al., 2016; Ponce et al., 2017). Thus, preferentially targeting tumor tissues with a CD47-blocking reagent remains a challenge. PD-L1 is another membrane protein that is highly expressed on some tumor cells and tumor infiltrating leukocytes (Dong et al., 2002; Green et al., 2010; Taube et al., 2012; Thompson et al., 2004). By interacting with its receptor, PD-1, which is mainly expressed on T cells, PD-L1 inhibits T cell activation, proliferation, and effector function; causes T cell exhaustion; and dampens anti-tumor adaptive immune responses (Blackburn et al., 2009; Brown et al., 2003; Freeman et al., 2000). Interruption of the PD-L1-PD-1 signaling pathway can rescue exhausted T cells and restore anti-tumor responses. This strategy has achieved initial success in clinics in recent years (Brahmer et al., 2010; Brahmer et al., 2012; Topalian et al., 2012; Wolchok et al., 2013). It suggests that PD-L1 is a key molecule for tumor evasion of the immune system. Since both CD47 and PD-L1 are highly expressed on tumor cells and serve as critical innate and adaptive checkpoints, respectively, whether and how they coordinate for tumor immune evasion becomes intriguing. This is further emphasized by a recent study demonstrating the possibility of oncogenicdriven co-expression of CD47 and PD-L1 for tumorigenesis (Casey et al., 2016). In the present study, we revealed that CD47 and PD-L1, expressed on tumor cells, serve as two-step checkpoints in tumor immune evasion through the DNA sensing pathway. Dual-targeting blockade of both molecules using a newly engineered bispecific heterodimer protein, anti-PD-L1SIRPa, shows better tumor-cell targeting, exerts a stronger therapeutic effect than either single blockade alone and has no
Cell Reports 24, 2101–2111, August 21, 2018 ª 2018 The Authors. 2101 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Figure 1. CD47 and PD-L1 on Tumor Cells Coordinate to Evade Immune Control (A) C57BL/6 female WT mice (n = 10/group) were inoculated s.c. with 1 3 105 cells of the colorectal adenocarcinoma cell line MC38 or MC38-CD47KO. Tumor growth was measured and compared once a week starting on day 0. Statistical significance was determined by a Student’s t test. (B) C57BL/6 female WT mice (n = 9–10/group) were inoculated s.c. with 1 3 105 cells of MC38 or MC38-CD47KO. CD8-depleting antibody (TIB210, 200 mg/mouse) was administered twice a week, starting on day 1. Tumor growth was measured and compared once a week starting on day 0. Statistical significance was determined by a Student’s t test. (C) C57BL/6 female WT mice (n = 3/group) were inoculated s.c. with 1 3 105 MC38 or MC38CD47KO tumor cells. The PD-L1 expression on tumor cells was assessed by flow cytometry on day 20. Staining control is shown in gray. (D) C57BL/6 female WT mice (n = 5 or 10) were inoculated s.c. with 2 3 105 cells of MC38, MC38-CD47KO, MC38-PD-L1KO or MC38-CD47/ PD-L1DKO. Tumor growth was measured and compared twice a week starting on day 0. Statistical significance was determined by a Student’s t test. (E) C57BL/6 female WT mice and Rag1/ mice (n = 5/group) were inoculated s.c. with 5 3 105 cells of MC38 or MC38-CD47/PD-L1DKO. Tumor growth was measured. Statistical significance was determined by a Student’s t test. (F) C57BL/6 female WT mice (n = 6/group) were inoculated s.c. with 2 3 105 cells of MC38 or MC38CD47/PD-L1DKO. CD8-depleting antibody was administered twice a week, starting on day 1. Tumor growth was measured and compared twice a week starting on day 0. Statistical significance was determined by a Student’s t test. See also Figures S1–S3.
detectable side effects. Thus, this study provides insight into how tumor cells evade or limit immune attack through coordinative innate and adaptive checkpoints as well as how both innate and adaptive checkpoints can be simultaneously targeted for enhanced tumor therapy. RESULTS CD47 and PD-L1 on Tumor Cells Coordinate to Evade Immune Control To test whether CD47 is required for tumor cell immune evasion, we generated a CD47-deficient colorectal tumor cell line named ‘‘MC38-CD47 knockout’’ (MC38-CD47KO) (Figure S1A). MC38-CD47KO tumor cells grew equally well in vitro compared with parental cells (data not shown). When inoculated subcutaneously (s.c.) into immune competent wildtype (WT) animals, they grew slower than parental cells but were not completely arrested (Figure 1A). This result suggested that CD47 plays only a partial role in tumor cell immune evasion and other mechanisms work together with CD47 or independently for tumor cell immune evasion. We previously reported
2102 Cell Reports 24, 2101–2111, August 21, 2018
that MC38 growth is under adaptive T cell control and that T cell depletion enhances tumorigenesis (Liu et al., 2015b). Similarly, the growth of MC38-CD47KO is also under T cell control (Figures 1B and S2). In line with this finding, under T cell pressure, the PD-L1 expression on MC38-CD47KO tumor cells was upregulated (Figure 1C). This result raises the possibility that PD-L1-mediated adaptive immune suppression may contribute to tumor growth in the absence of CD47-mediated innate immune escape. We then wondered whether further ablation of PD-L1 signaling would completely inhibit tumorigenesis. To test this, we further knocked out PD-L1 on MC38-CD47KO cells to obtain MC38-CD47/PD-L1DKO cells (Figure S1A). MC38-PD-L1KO cells were also generated as control (Figure S1A). In stark contrast with MC38-CD47KO and MC38-PD-L1KO cells, MC38-CD47/PD-L1DKO cells showed almost complete growth arrest (Figures 1D and S1B). This coordinated role of CD47 and PD-L1 on tumor growth appears general since it is also replicated in the B16F10 tumor model (Figure S3). The growth arrest of MC38-CD47/PD-L1DKO depends on host CD8+ T cells as they grew at a comparable level as WT MC38 tumor cells in Rag1/ mice or in WT mice
Figure 2. Innate Sensing by DCs Is Required for CD47 and PD-L1-Mediated Evasion (A) C57BL/6 female WT mice and Ifnar/ mice (n = 4 or 5/group) were inoculated s.c. with 5 3 105 MC38 or MC38-CD47/PD-L1DKO tumor cells. Tumor volume was measured at the indicated time points. (B) C57BL/6 female WT mice and Sting/ mice (n = 5/group) were inoculated s.c. with 5 3 105 MC38 or MC38-CD47/PD-L1DKO tumor cells. Tumor volume was measured at the indicated time points. (C) C57BL/6 female WT mice and CD11c-cre 3 Stingfl/fl mice (n = 5/group) were inoculated s.c. with 5 3 105 WT MC38 or MC38-CD47/PD-L1DKO tumor cells. Tumor volume was measured at the indicated time points. For all graphs, data represent mean ± SEM and statistical analysis was performed by Student’s t test.
depleted of CD8+ T cells (Figures 1E and 1F). Together, these data suggest that CD47 and PD-L1 on tumor cells coordinate for tumor immune escape. Innate Sensing by DCs Is Required for CD47- and PD-L1-Mediated Evasion To evaluate whether type I interferons (IFNs) are required for CD47- and PD-L1-mediated tumor immune evasion, we inoculated MC38-CD47/PD-L1DKO tumor cells into Ifnar/ mice. In stark contrast to the tumor growth arrest of MC38-CD47/PDL1DKO in WT mice, MC38-CD47/PD-L1DKO in Ifnar/ mice grew comparably as WT MC38 tumor cells in WT hosts (Figure 2A). The cGAS/STING pathway in DCs is essential for tumor-cell-derived cytosolic DNA sensing (Ishikawa and Barber, 2008; Ishikawa et al., 2009; Li et al., 2013), and CD47 on tumors can suppress this pathway (Liu et al., 2015b). To determine whether this DNA sensing pathway in DCs is essential for inhibiting CD47- and PD-L1-mediated tumor immune evasion, we measured MC38-CD47/PD-L1DKO tumor growth in STING-deficient mice and further in DC-specific STING-deficient mice. In stark contrast to the comparable growth of WT MC38 tumors in WT, STING-deficient, or DC-specific STING-deficient mice, MC38-CD47/PD-L1DKO tumor cells, which fail to grow in WT mice, grew in STING-deficient or DC-specific STING-deficient mice at a comparable level as WT MC38 tumor cells in WT hosts (Figures 2B and 2C). Therefore, these results suggest that the DC-specific STING signaling and IFNa production play an essential role in inhibiting CD47- and PD-L1-mediated tumor immune evasion. Bispecific Anti-PD-L1-SIRPa Maintains Binding to Tumor Cells, while Greatly Reducing Binding to Red Blood Cells Given the broad expression of CD47, a general concern for systemic administration of current CD47 targeting reagents is offtargeting which results in impaired therapeutic efficiency and generation of side effects. In fact, with the same dose of antiCD47 or SIRPa-hFc, intratumoral administration generated a much better anti-tumor effect than did systemic treatment
(Figures S4A and S4B). Since both CD47 and PD-L1 are highly expressed on tumor cells, and more importantly, CD47 and PD-L1 ablation on tumor cells dramatically suppressed tumorigenesis, we wondered whether a bispecific format with the dual targeting of tumor-specific CD47 and PD-L1 would enable enhanced tumor-targeting specificity and therapeutic effect while reducing the toxicity. Therefore, based on the Fc heterodimer technique, we generated a fusion protein targeting both PD-L1 and CD47 (Figure 3A). Size-exclusion chromatography assay confirmed its heterogenicity (Figure 3A). The anti-PDL1-SIRPa fusion protein preferentially bound CD47 and PD-L1 on MC38 cells, with a major contribution from CD47 and a minor one from PD-L1 (Figure 3B). To evaluate the hematological safety, we first tested the binding of anti-PD-L1-SIRPa to red blood cells (RBCs) in vitro. SIRPa-hFc showed a strong binding with RBCs, whereas anti-PD-L1-SIRPa had little, if any, binding to RBCs (Figure 3C). In vivo, administration of 400 mg of antiPD-L1-SIRPa did not show any significant reduction of RBCs, platelets (PLTs), or hemoglobin (HGB) and increase of Hemin in serum, different from SIRPa-hFc treatment (Figures S5A– S5D). To further determine whether anti-PD-L1-SIRPa could remain tumor cell targeting efficacy in the presence of a large antigen sink, WT MC38 tumor cells were mixed with a 15-fold excess of RBCs and then incubated with fusion proteins. Compared with SIRPa-hFc, anti-PD-L1-SIRPa selectively bound tumor cells (Figure 3D). The binding selectivity of the bispecific fusion protein was also confirmed in human tumor cells and human RBCs and PLTs using corresponding human reagents (data not shown). Therefore, these data suggest that anti-PD-L1-SIRPa could preferentially bind tumor cells without off-tumor toxicity after systemic delivery. Finally, we performed a single-dose PK study in which log-fold-increasing doses of anti-PD-L1-SIRPa were administered intraperitoneally (i.p.). Serum was collected at different time points and human immunoglobulin G (hIgG) serum concentration was determined by ELISA (Figures S6A–S6C). Compared to SIRPa-hFc, significantly higher level of the anti-PD-L1-SIRPa fusion protein was maintained in the blood circulation suggesting a decreased antigen sink effect.
Cell Reports 24, 2101–2111, August 21, 2018 2103
Figure 3. Bispecific Anti-PD-L1-SIRPa Maintains Binding to Tumor Cells, while Greatly Reducing Binding to RBCs (A) Schematic of anti-PD-L1-SIRPa. The wild-type mouse SIRPa ECD (red) was linked with the anti-PD-L1 (blue) antibody. Constant regions were human IgG1 isotype (green). (B) MC38, MC38-CD47KO, MC38-PD-L1KO, or MC38-CD47/PDL1DKO tumor cells were incubated with 10 mg/mL of anti-PD-L1-SIRPa prior to staining with PE anti-human IgG secondary antibody and detected by flow cytometry. (C) Whole blood from a C57BL/6 female WT mouse was incubated with the indicated antibodies pre-labeled with a PE anti-human IgG secondary antibody and assessed by flow cytometry, and the result was expressed as mean fluorescence intensity. (D) MC38 tumor cells were mixed with a 15-fold excess of mouse blood cells. The cell mixture was incubated with 5 mg/mL of SIRPa-hFc or anti-PD-L1-SIRPa prior to staining with PE anti-human IgG secondary antibody and detected by flow cytometry. Red, hFc isotype; blue, SIRPa-hFc or anti-PD-L1-SIRPa. See also Figures S4–S6.
Bispecific Anti-PD-L1-SIRPa Inhibits Tumor Growth via Co-targeting Tumor Cells Next, we aimed to determine whether simultaneous blockade of PD-L1 and CD47 on tumor cells by anti-PD-L1-SIRPa could generate an enhanced anti-tumor effect compared with single blockades. WT C57BL/6 mice were inoculated with MC38 tumor cells. When tumors were well established, mice were intratumorally (i.t.) administered with human IgG isotype control, anti-PDL1, SIRPa-hFc, a mixture of anti-PD-L1 and SIRPa-hFc or antiPD-L1-SIRPa, and the tumor growth was monitored. Compared with the isotype control and the single treatments, both mixtures of anti-PD-L1 and SIRPa-hFc and anti-PD-L1-SIRPa showed
2104 Cell Reports 24, 2101–2111, August 21, 2018
significantly enhanced tumor inhibition and extended mouse survival (Figures 4A and 4B). To evaluate the efficacy of systemic treatment, we i.p. treated established tumor-bearing mice with reagents as described above. Different from intratumor therapy, a mixture of anti-PD-L1 and SIRPa-hFc lost its therapeutic efficacy, while anti-PD-L1-SIRPa treatment still significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice (Figures 4C and 4D). The therapeutic effect of the bispecific anti-PD-L1-SIRPa during systemic administration was also shown in a CT26 tumor model, a commonly used model for PDL1 study in BALB/c mice (Figure 4E). Thus, the anti-PD-L1-SIRPa strategy may have broad application in multiple tumors.
Figure 4. Bispecific Anti-PD-L1-SIRPa Inhibits Tumor Growth via Co-targeting Tumor Cells (A) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.t. with 50 mg hIgG isotype control, 25 mg of anti-PD-L1 or SIRPa-hFc, 25 mg of anti-PD-L1 + 25 mg of SIRPa-hFc, or 50 mg of anti-PD-L1-SIRPa on days 11 and 14. Tumor growth was measured every 4 days starting on day 0. Data represent mean ± SEM and statistical significance was determined by a Student’s t test. (B) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.t. with 50 mg hIgG isotype control, 25 mg of anti-PD-L1 or SIRPa-hFc, 25 mg of anti-PD-L1 + 25 mg of SIRPa-hFc, or 50 mg of anti-PD-L1-SIRPa on days 11 and 14. Tumor growth was measured every 4 days starting on day 0. Statistical significance was determined by a log rank (Mantel-Cox) test. (C) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control, 50 mg of anti-PDL1 or SIRPa-hFc, 50 mg of anti-PD-L1 + 50 mg of SIRPa-hFc, or 100 mg of anti-PD-L1-SIRPa on days 10 and 14. Tumor growth was measured every 4 days starting on day 0. Data represent mean ± SEM and statistical significance was determined by a Student’s t test. (D) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control, 50 mg of anti-PDL1 or SIRPa-hFc, 50 mg of anti-PD-L1 + 50 mg of SIRPa-hFc, or 100 mg of anti-PD-L1-SIRPa on days 10 and 14. Tumor growth was measured every 4 days starting on day 0. Statistical significance was determined by a log rank (Mantel-Cox) test. (E) BALB/c female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 CT26 tumor cells and treated i.p. with 100 mg hIgG isotype control, 50 mg of anti-PD-L1 or SIRPa-hFc, 50 mg of anti-PD-L1 + 50 mg of SIRPa-hFc, or 100 mg of anti-PD-L1-SIRPa on days 10 and 14. Tumor growth was measured every 4 days starting on day 0. Statistical significance was determined by a log rank (Mantel-Cox) test.
(legend continued on next page)
Cell Reports 24, 2101–2111, August 21, 2018 2105
To determine whether tumor-expressing CD47 and PD-L1 are required for the therapeutic effect of anti-PD-L1-SIRPa, we intratumorally administered hIgG isotype control, anti-PDL1, SIRPa-hFc or anti-PD-L1-SIRPa in MC38-CD47KO or MC38-PD-L1KO tumor-bearing mice. The therapeutic effect of anti-PD-L1-SIRPa disappeared in the absence of either CD47 or PD-L1 on tumor cells (Figures 4F and 4G), probably due to reduced tumor cell targeting by anti-PD-L1-SIRPa when one target is missing. Furthermore, PD-L1 deficiency on the host did not diminish the therapeutic effect of anti-PD-L1-SIRPa (Figure 4H). These results indicate that the simultaneous targeting of both PD-L1 and CD47 on tumor cells, but not on host cells, is required for the anti-PD-L1-SIRPa-mediated therapeutic effect. Intratumoral CD8+ T Cells Are Essential for the Therapeutic Effect of Anti-PD-L1-SIRPa To determine the extent to which the anti-tumor effect of antiPD-L1-SIRPa depends on host T cells, Rag1/ mice were inoculated with MC38 tumor cells and treated with anti-PD-L1-SIRPa as before. However, no therapeutic effect was observed, suggesting an essential role for host T cells (Figure 5A). To further elucidate which T cell subset is required for anti-PD-L1-SIRPamediated tumor control, we systemically treated MC38 tumorbearing C57BL/6 mice with anti-PD-L1-SIRPa together with CD8+ or CD4+ T cell-depleting antibodies. In the absence of CD8+ T cells, the therapeutic effect of anti-PD-L1-SIRPa was completely abrogated (Figure 5B), whereas depletion of CD4+ T cells had no effect (Figure 5C). To determine whether antiPD-L1-SIRPa treatment enhances the tumor antigen-specific T cell response, mice were inoculated with MC38-OT-I tumor cells and treated with human IgG, anti-PD-L1, SIRPa-hFc or anti-PD-L1-SIRPa i.p. 11 days after antibody treatment, cells from the tumor-draining lymph nodes were restimulated in vitro with SIINFEKL peptide, and IFN-g production was measured by enzyme-linked immunospot (ELISPOT) assay. Compared with the isotype control and single treatments, significantly more IFN-g-producing cells were detected in the anti-PD-L1SIRPa-treated group (Figure 5D). To further determine the true anti-tumor T cell response, but not to model tumor antigen OVA, we treated MC38 tumors, and irradiated MC38 tumor cells were used for restimulation for ELISPOT assay. Again, more tumor-specific IFN-g-producing cells were determined in the antiPD-L1-SIRPa-treated group compared with isotype control and single treatments (Figure 5E). To further determine whether antiPD-L1-SIRPa also affects intratumor antigen-specific T cell response, tumor-bearing mice were treated with FTY720 before fusion protein treatment to inhibit T cell circulation. Interestingly, FTY720 did not diminish the therapeutic effect of anti-PD-L1-
SIRPa (Figure 5F). Together, these results suggest that intratumor CD8+ T cell response is enhanced by anti-PD-L1-SIRPa and essential for its therapeutic efficacy. Anti-PD-L1-SIRPa Enhances Intratumoral DC Innate Sensing Intratumor antigen-presenting cells, such as DCs, are required for T cell response generation. To evaluate whether anti-PDL1-SIRPa enhances intratumor DC cross-presentation, we collected DCs from the tumor microenvironment of MC38OT-I-bearing mice after treatment and co-cultured them with OVA-specific OT-I T cells. Compared with the isotype control and single treatments, DCs harvested from the tumors treated with anti-PD-L1-SIRPa showed significantly higher cross-presentation function than those from the other groups, as indicated by the increased number of IFN-g-producing cells (Figure 6A). Given the important role of DC-producing IFNa in the arrest of MC38-CD47/PD-L1DKO tumor growth, we evaluated whether type I IFN was required for the therapeutic efficacy of anti-PDL1-SIRPa. Indeed, intratumor IFNAR blockade significantly reduced the efficacy of anti-PD-L1-SIRPa (Figure 6B). To detect whether the DC-specific STING pathway is essential for initiating IFNa production by anti-PD-L1-SIRPa treatment, we tested the efficacy of anti-PD-L1-SIRPa treatment in STING-deficient and CD11c-STING-deficient mice. As expected, the therapeutic efficacy of anti-PD-L1-SIRPa was primarily abolished in such mice (Figures 6C and 6D). Thus, these results suggest the critical role of the DC-specific STING pathway in this treatment. Chemotherapy Induces More ‘‘Eat Me’’ Signals and Synergizes with Anti-PD-L1-SIRPa for Tumor Eradication At the clinic, chemotherapy is often a first-line therapy. Certain chemo-drugs enhance calreticulin (CRT) exposure on tumor cells (Obeid et al., 2007), which is an important ‘‘eat me’’ signal that antagonizes with the CD47 ‘‘don’t eat me’’ signal (Chao et al., 2010b). We therefore hypothesized that pretreatment with chemotherapy might synergizes with anti-PD-L1-SIRPa to further enhance tumor eradication. Doxorubicin (DOX) has been reported to induce CRT exposure on tumor cells (Obeid et al., 2007). We first confirmed that treatment of MC38 tumor cells with DOX indeed induced CRT exposure on the cell surface (Figure 7A). Then we tested whether DOX would synergize with anti-PD-L1-SIRPa treatment. Mice were inoculated with MC38 tumor cells, and, 11 days later, DOX was administered intratumorally 1 day before antibody treatments. Compared with the isotype control and single treatments, DOX plus anti-PD-L1SIRPa treatment increased tumor inhibition and prolonged the survival of tumor-bearing mice (Figures 7B and 7C). To verify
(F) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38-CD47KO tumor cells and treated i.t. with 50 mg hIgG isotype control, anti-PD-L1, SIRPa-hFc, or anti-PD-L1-SIRPa on days 10 and 15. Tumor growth was measured every 4 days starting on day 0. Data represent mean ± SEM and statistical significance was determined by a Student’s t test. (G) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38-PD-L1KO tumor cells and treated i.t. with 50 mg hIgG isotype control, anti-PDL1, SIRPa-hFc, or anti-PD-L1-SIRPa on days 10 and 15. Tumor growth was measured every 4 days starting on day 0. Data represent mean ± SEM and statistical significance was determined by a Student’s t test. (H) C57BL/6 female WT or PD-L1 knockout mice (n = 4–6) were inoculated s.c. with 1 3 106 MC38 and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1SIRPa on days 8 and 11. Tumor growth was measured twice per week. Data represent mean ± SEM and statistical significance was determined a by Student’s t test.
2106 Cell Reports 24, 2101–2111, August 21, 2018
Figure 5. CD8+ T Cells Are Essential for the Therapeutic Effect of Anti-PD-L1-SIRPa (A) C57BL/6 female Rag1/ mice (n = 5or 6/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1-SIRPa on days 10 and 14. Tumor growth was measured every 4 days starting on day 0. (B) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1-SIRPa on days 10 and 14. CD8depleting antibody was administered twice a week starting on day 1. The tumor growth was measured and compared twice a week starting on day 0. (C) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1-SIRPa on days 8 and 11. CD4depleting antibody was administered twice a week starting on day 1. The tumor growth was measured and compared twice a week starting on day 0. (D) C57BL/6 female WT mice (n = 3 or 4/group) were inoculated s.c. with 2 3 105 MC38-OT-I tumor cells and treated i.p. with 50 mg hIgG isotype control, 25 mg of anti-PD-L1 or SIRPa-hFc, or 50 mg of anti-PD-L1-SIRPa on days 10 and 14. Lymphocytes from DLNs were isolated on day 21 and stimulated with 10 mg/mL OT-I peptide. IFNg-producing cells were enumerated by ELISPOT assay. (E) C57BL/6 female WT mice (n = 7/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 50 mg hIgG isotype control, 25 mg of anti-PD-L1 or SIRPa-hFc, or 50 mg of anti-PD-L1-SIRPa on days 10 and 14. Lymphocytes from DLNs were isolated on day 21 and stimulated with 60-Gy-irradiated MC38 tumor cells. The ratio of DLN cells to tumor cells is 5:1. IFN-g-producing cells were enumerated by ELISPOT assay after 2 days. (F) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1-SIRPa on days 10 and 14. 25 mg of FTY720 was administered i.p. on the day 1 and 5 mg of FTY720 on days 0–2. The tumor growth was measured and compared twice a week starting on day 0. For all graphs, data represent mean ± SEM and statistical significance was determined by a Student’s t test.
whether the synergy between DOX and anti-PD-L1-SIRPa was due to enhanced exposure of the ‘‘eat me’’ molecule CRT, we treated mice with Z-VAD-FMK, a caspase inhibitor that results in reduced expression of membrane CRT (Casares et al., 2005; Obeid et al., 2007), before DOX and fusion protein treatment. As speculated, the synergy between DOX and anti-PD-L1-SIRPa disappeared with Z-VAD-FMK treatment (Figure 7D). Thus, these data indicate that DOX, which induces more ‘‘eat me’’ signaling, has a synergistic role when used in combination with anti-PD-L1SIRPa. DISCUSSION In this study, we showed that CD47 and PD-L1 on tumor cells coordinate for two-step evasion of innate and adaptive immunity. In
a previous study, Dheilly et al. demonstrated that anti-mesothelin (or CD19)/anti-CD47 bsAb overcome tolerability and ‘‘antigen sink’’ issues by selectively blocking the CD47-SIRPa interaction on malignant cells expressing a specific tumor-associated antigen (Dheilly et al., 2017). However, given the immunodeficient and xenograft model used, the targeting efficiency, the role of T cells in anti-tumor efficacy and the side effect have not been adequately evaluated in the previous study. Both PD-L1 and CD47 are highly expressed on tumor cells, whereas most normal tissues, including RBCs, lack PD-L1 expression, so we used a higher-affinity anti-PD-L1 and the lower-affinity SIRPa as heterodimer partners in our current study. Furthermore, by using immunocompetent synergistic tumor model, we showed that the bispecific heterodimer anti-PD-L1-SIRPa fusion protein target tumor cells efficiently while only weakly bind RBCs,
Cell Reports 24, 2101–2111, August 21, 2018 2107
Figure 6. Anti-PDL1-SIRPa Enhances Intratumoral DC Innate Sensing (A) C57BL/6 female WT mice (n = 8/group) were inoculated s.c. with 2 3 105 MC38-OT-I tumor cells and treated i.t. with 50 mg hIgG isotype control, 25 mg of anti-PD-L1 or SIRPa-hFc, or 50 mg of anti-PD-L1-SIRPa on days 10 and 14. Three days after the second treatment, CD45+CD11c+MHCII+ DC cells were isolated from tumor and cocultured with isolated CD8+ T cells from naive OT-I mice for 48 hr. IFN-g-producing cells were enumerated by ELISPOT assay. (B) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1-SIRPa on days 10 and 14. 50 mg antiIFNAR1 antibody was administered i.t. on the days 1, 4, and 8. Tumor growth was measured. (C) C57BL/6 female WT and Sting/ mice (n = 5/ group) were inoculated s.c. with 4 3 105 MC38 tumor cells and treated i.p. with 200 mg hIgG isotype control or anti-PD-L1-SIRPa on days 11 and 14. Tumor growth was measured. (D) C57BL/6 male WT or CD11c-cre 3 Stingfl/fl mice (n = 5/group) were inoculated s.c. with 5 3 105 MC38 tumor cells and treated i.p. with 200 mg hIgG isotype control or anti-PD-L1-SIRPa on days 13 and 16. Tumor growth was measured. For all graphs, data represent mean ± SEM and statistical significance was determined by a Student’s t test.
compared with SIRPa-Fc. Consequently, the enhanced tumor targeting by the bispecific fusion protein generated much better anti-tumor efficacy than single targeting or cocktail. This is also consistent with the data showing that both CD47 and PD-L1 on tumor cells are necessary for immune evasion. On one hand, the generation of T cell response and PD-L1 upregulation by CD47-SIRPa blockade create a better situation for anti-PD-L1 to target. On the other hand, type I interferons generated during CD47 blockade may enhance DC co-stimulatory function, which is required for full rejuvenation of intratumor exhausted T cells during anti-PD-L1 blockade (Kelly, 2017). In addition to the synergistic effect of CD47 and PD-L1 blockade for adaptive T cell responses, they may also cooperate in reducing the tumor burden at the innate level. Recent studies have reported that PD-1 signaling inhibits phagocytosis of tumor-associated macrophages (Gordon et al., 2017). It would be interesting to investigate whether the dual blockade of CD47 and PD-L1 on tumor cells would create an additional bridge promoting macrophage phagocytosis of tumor cells. Recent work has shown a critical role for DCs in capturing tumor antigen and mitochondrial DNA during CD47 blockade (Xu et al., 2017). Whether PD-L1 blockade would simultaneously facilitate DC capture remains an intriguing question. In addition, given the possibility of PD-L1 reverse signaling, it would also be interesting to study whether PD-L1 blockade modulates tumor cells themselves for enhanced mitochondrial DNA release. Some chemotherapy drugs can directly kill tumor cells, while others may additionally exert stress on tumor cells and induce ‘‘eat me’’ signaling. CRT exposure is one of the major ‘‘eat me’’ signals. CRT enhances tumor cell phagocytosis by macro-
2108 Cell Reports 24, 2101–2111, August 21, 2018
phages/DCs, further leading to the generation of anti-tumor T cell response. This may be especially synergistic with the bispecific anti-PD-L1-SIRPa fusion protein. Indeed, DOX pretreatment significantly enhances the anti-tumor effect of the antiPD-L1-SIRPa fusion protein. However, it should be noted that not all chemotherapy drugs are proficient at inducing immunogenetic cell death. Therefore, it remains to be determined whether the anti-PD-L1-SIRPa fusion protein has synergistic effects with those chemotherapy drugs. In conclusion, our study has revealed the critical coordination of tumor-cell-specific CD47 and PD-L1 for tumor evasion. Based on this finding, we have developed an improved strategy that simultaneously targets both CD47 and PD-L1 on tumor cells, while reducing their off-target binding to healthy cells. This strategy results in significantly enhanced anti-tumor effects compared with either single CD47 or PD-L1 blockade alone and shows synergistic effects of chemotherapy, such as DOX. We believe this dual-targeting strategy will provide insight into tumor immunotherapy for better tumor control.
EXPERIMENTAL PROCEDURES Mice Six- to eight-week-old female C57BL/6J and BALB/c mice were purchased from Beijing Vital River Laboratory in China. Rag1/, Ifnar/, Sting/, OT-I CD8+ T cell receptor (TCR)-Tg, CD11c-Cre-Tg, and Stingfl/fl mice were obtained from an internal breeding facility. All mice were maintained under specific pathogen-free conditions, and age- and sex-matched mice were used between 6–12 weeks of age, in accordance with the experimental animal guidelines set by the Institutional Animal Care and Use Committee of the
Figure 7. Chemotherapy Induces More ‘‘Eat Me’’ Signals and Synergizes with Anti-PDL1-SIRPa for Tumor Eradication (A) 1 3 106 MC38 tumor cells were cocultured with 2 mg/mL DOX for 12 hr, and calreticulin was measured by flow cytometry. (B) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1SIRPa on days 10 and 14. 50 mg of DOX was administered i.t. 1 day before anti-PD-L1-SIRPa. Tumor volume was measured. Data represent mean ± SEM and statistical significance was determined by a Student’s t test. (C) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1SIRPa on days 10 and 14. 50 mg of DOX was administered i.t. 1 day before anti-PD-L1-SIRPa. Tumor volume was measured. Statistical significance was determined by a log rank (Mantel-Cox) test. (D) C57BL/6 female WT mice (n = 5/group) were inoculated s.c. with 2 3 105 MC38 tumor cells and treated i.p. with 100 mg hIgG isotype control or anti-PD-L1SIRPa on days 10 and 14. 50 mg of DOX was administered i.t. 1 day before anti-PD-L1-SIRPa. 40 mM 3 50 mL of V-ZAD-FMK was administered i.t. every 4 days from 1 day before anti-PD-L1-SIRPa treatment. Tumor volume was measured. Statistical significance was determined by a log rank (Mantel-Cox) test.
Institute of Biophysics, CAS, and the University of Texas Southwestern Medical Center at Dallas.
were produced in-house. The endotoxin level for Ab and fusion protein was lower than 0.2 EU (endotoxin units)/mg of protein.
Cell Lines and Reagents All cell lines were free of mycoplasma contamination. MC38 and CT26 are murine colon adenocarcinoma cell lines. MC38-OTIp was sorted and subcloned after MC38 cells were stably transduced with the retrovirus-expressing mouse CCR7-OTIp (peptide epitope for OTI). MC38-CD47KO, MC38-PD-L1KO, and MC38-CD47/PD-L1DKO tumor cells were acquired using CRISPR/Cas9 technology. The single guide RNA (sgRNA) sequences were as follows: sgCD47-F 50 -TTGGCGGCGGCGCTGTTGCT-30 and sgCD47-R 50 -CCCTT GCATCGTCCGTAATG-30 ; sgPD-L1-F 50 -GGCTCCAAAGGACTTGTACG-30 ; and sgPD-L1-R 50 -GACTTGTACGTGGTGGAGTA-30 . B16F10 is a murine melanoma cell line. B16F10-CD47KO, B16F10-PD-L1KO, and B16F10-CD47/PDL1DKO tumor cells were acquired using CRISPR/Cas9 technology. Fluorescein isothiocyanate (FITC)-conjugated anti-mCD47, PE-conjugated anti-PD-L1, PE-conjugated anti-human IgG, APC-conjugated anti-CD45.2, PE-Cy7-conjugated anti-CD11c, FITC-conjugated anti-MHCII, Percp-Cy5.5-conjugated anti-CD11b, and PE-conjugated anti-F4/80 were purchased from BioLegend Company and FTY720 was purchased from Sigma-Aldrich. The chemotherapeutic reagents DOX and Z-VAD-FMK were purchased from Sigma-Aldrich and prepared in accordance with the manufacturer’s recommendations. Anti-mIFNAR1-neutralizing monoclonal antibody (mAb) (clone MAR1-5A3) was purchased from BioXcell (West Lebanon, NH, USA). The variable regions of light chain and heavy chain of anti-PD-L1 Ab sequence were synthesized according to patent (patent no.: US 8,217,149 B2, YW243.55.S70). Anti-PDL1, SIRPa-hFc, and anti-PD-L1-SIRPa fusion proteins; anti-CD8-depleting antibody (clone TIB210); and anti-CD4-depleting antibody (clone GK1.5)
Tumor Inoculation, Treatment, and Digestion For tumor growth observation, we s.c. injected MC38, MC38-CD47KO, MC38PD-L1KO, MC38-CD47/PD-L1DKO, or B16F10 and B16F10-CD47KO, B16F10PD-L1KO and B16F10-CD47/PD-L1DKO tumor cells into the flanks of mice. Tumor volumes were measured by the length (a), width (b), and height (h) and calculated as follows: tumor volume = a*b*h/2. For fusion protein treatment, we s.c. injected MC38, CT26, or MC38-OTIp tumor cells into the flanks of mice. Tumors, which were allowed to grow for 10–14 days to reach 80 mm3, were treated with fusion proteins intratumorally or i.p., and the tumor volume was measured. For CD8 or CD4 depletion, anti-CD8 (clone TIB210) or anti-CD4 (clone GK1.5) antibody was injected i.p. To block the migration of activated T cells, FTY720 was administered i.p. For type I IFN blockade experiments, anti-IFNAR1 mAb was intratumorally injected. For DOX and Z-VADFMK treatment, DOX or Z-VAD-FMK was administered intratumorally. Tumor tissues were excised and digested with 1 mg/mL collagenase IV (Sigma) in the incubator. After 40 min, tumors were passed through a 70-mm cell strainer to remove large pieces of the undigested tumor. Tumor-infiltrating cells were then washed twice with PBS. Production of Anti-PD-L1, SIRPa-hIg, or Anti-PD-L1-SIRPa Fusion Protein Anti-PD-L1 or SIRPa-hFc was generated as previously described (Tang et al., 2016). Briefly, a plasmid encoding the DNA sequence of anti-PD-L1 or SIRPa IgV-like domain was cloned into the pEE12.4 expression plasmid (Lonza, Basel, Switzerland) between the IgG IgGk leading sequence and the human
Cell Reports 24, 2101–2111, August 21, 2018 2109
IgG1 Fc sequence using BsiWI and BstBI. A heterodimer of anti-PD-L1-SIRPa was achieved by the knobs-into-holes approach. The fusion proteins were transiently expressed in FreeStyle 293-F cells and purified by a protein A column in accordance with the manufacturer’s instructions. ELISPOT Assay For tumor-specific CD8+ T cell functional assays in the MC38-OT-I model, tumor-draining lymph nodes (DLNs) were removed 11 days after fusion protein treatment. 3 3 105 DLN cells were then restimulated with 10 mg/mL OT-I peptide (SIINFEKL). For tumor-specific CD8+ T cell functional assays in the MC38 model, tumor DLNs were removed 11 days after fusion protein treatment. 5 3 105 DLN cells were then restimulated with 1 3 105 MC38 tumor cells irradiated with 60 Gy. A 96-well HTS-IP plate (BD PharMingen) was pre-coated with the anti-IFN-g antibody (BD PharMingen) at a 1:250 dilution overnight at 4 C. After co-culture for 48 hr, cells were removed, 2 mg/mL biotinylated anti-IFN-g antibody (BD PharMingen) at a 1:250 dilution was added, and the plate was incubated for 2 hr at room temperature. Avidin-horseradish peroxidase (BD PharMingen) at a 1:1,000 dilution was then added, and the plate was incubated for 1 hr at room temperature. The cytokine spots of IFN-g were developed in accordance with the manufacturer’s protocol (BD PharMingen). Ex Vivo DC Cross-Presentation Assay MC38-OT-I-bearing mice were treated with fusion proteins by intratumoral injection on days 11 and 14. Three days after the second treatment, tumors were digested, and DCs were purified by fluorescence-activated cell sorting (FACS). Approximately 5 3 104 DCs were mixed together with purified 5 3 105 OT-I T cells, which were sorted on a FACSAria II Cell Sorter (BD) from lymph nodes of 6- to 12-week-old OT-I transgenic mice. After 48 hr, IFN-g-producing cells were enumerated by ELISPOT assay (BD PharMingen). Flow Cytometric Sorting and Analysis Single-cell suspensions were blocked with anti-FcR (clone 2.4G2, BioXcell) and then stained with antibodies (1:500 dilution) against PD-L1, CD47, CD3, CD8, Va2, CD11c, MHCII, CD11b, F4/80, CD45, and DAPI. Cells were analyzed on Fortessa cytometer or sorted on a FACSAria II Cell Sorter (BD). Data were analyzed with FlowJo Software. Statistical Analysis Data were analyzed using Prism 6.0 software (GraphPad) and are presented as the mean ± SEM. p values were assessed using the two-tailed unpaired Student’s t test or two-way analysis of variance, with p values considered significant as follows: *p < 0.05; **p < 0.01; and ***p < 0.001. For tumor-free mice frequencies, statistics were performed with the log rank (Mantel-Cox) test. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and six figures and can be found with this article online at https://doi.org/10. 1016/j.celrep.2018.07.062. ACKNOWLEDGMENTS We thank Ting Xu, Yan Luan, Kai Fu, Shuli Ma, and Jianjian Peng for providing the heterodimeric Fc sequence. This work was, in part, supported by grants from the National Key R&D Program of China (2016YFC1303405 to Y.-X.F.), the Chinese Academy of Sciences (XDA12020212 to M.Z. and Y.-X.F.), and the U.S. NIH through the National Cancer Institute (CA141975 to Y.-X.F.). AUTHOR CONTRIBUTIONS X.L., M.Z., and Y.-X.F. designed the experiments and analyzed the data. X.L., L.L, Z.R., K.Y., Y.L., and K.F. performed the experiments. H.X., J.G., and H.P. provided the reagents. X.L., M.Z., and Y.-X.F. wrote the manuscript. Y.-X.F. supervised the project.
2110 Cell Reports 24, 2101–2111, August 21, 2018
DECLARATION OF INTERESTS The authors declare no competing interests. Received: September 11, 2017 Revised: June 1, 2018 Accepted: July 17, 2018 Published: August 21, 2018 REFERENCES Baccelli, I., Schneeweiss, A., Riethdorf, S., Stenzinger, A., Schillert, A., Vogel, V., Klein, C., Saini, M., Ba¨uerle, T., Wallwiener, M., et al. (2013). Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nat. Biotechnol. 31, 539–544. Baccelli, I., Stenzinger, A., Vogel, V., Pfitzner, B.M., Klein, C., Wallwiener, M., Scharpff, M., Saini, M., Holland-Letz, T., Sinn, H.P., et al. (2014). Co-expression of MET and CD47 is a novel prognosticator for survival of luminal breast cancer patients. Oncotarget 5, 8147–8160. Barclay, A.N., and Van den Berg, T.K. (2014). The interaction between signal regulatory protein alpha (SIRPa) and CD47: structure, function, and therapeutic target. Annu. Rev. Immunol. 32, 25–50. Blackburn, S.D., Shin, H., Haining, W.N., Zou, T., Workman, C.J., Polley, A., Betts, M.R., Freeman, G.J., Vignali, D.A., and Wherry, E.J. (2009). Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat. Immunol. 10, 29–37. Brahmer, J.R., Drake, C.G., Wollner, I., Powderly, J.D., Picus, J., Sharfman, W.H., Stankevich, E., Pons, A., Salay, T.M., McMiller, T.L., et al. (2010). Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J. Clin. Oncol. 28, 3167–3175. Brahmer, J.R., Tykodi, S.S., Chow, L.Q., Hwu, W.J., Topalian, S.L., Hwu, P., Drake, C.G., Camacho, L.H., Kauh, J., Odunsi, K., et al. (2012). Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455–2465. Brown, E.J., and Frazier, W.A. (2001). Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 11, 130–135. Brown, J.A., Dorfman, D.M., Ma, F.R., Sullivan, E.L., Munoz, O., Wood, C.R., Greenfield, E.A., and Freeman, G.J. (2003). Blockade of programmed death1 ligands on dendritic cells enhances T cell activation and cytokine production. J. Immunol. 170, 1257–1266. Casares, N., Pequignot, M.O., Tesniere, A., Ghiringhelli, F., Roux, S., Chaput, N., Schmitt, E., Hamai, A., Hervas-Stubbs, S., Obeid, M., et al. (2005). Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J. Exp. Med. 202, 1691–1701. Casey, S.C., Tong, L., Li, Y., Do, R., Walz, S., Fitzgerald, K.N., Gouw, A.M., €tgemann, I., Eilers, M., and Felsher, D.W. (2016). MYC regulates Baylot, V., Gu the antitumor immune response through CD47 and PD-L1. Science 352, 227–231. Chao, M.P., Alizadeh, A.A., Tang, C., Myklebust, J.H., Varghese, B., Gill, S., Jan, M., Cha, A.C., Chan, C.K., Tan, B.T., et al. (2010a). Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma. Cell 142, 699–713. Chao, M.P., Jaiswal, S., Weissman-Tsukamoto, R., Alizadeh, A.A., Gentles, A.J., Volkmer, J., Weiskopf, K., Willingham, S.B., Raveh, T., Park, C.Y., et al. (2010b). Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47. Sci. Transl. Med. 2, 63ra94. Cioffi, M., Trabulo, S., Hidalgo, M., Costello, E., Greenhalf, W., Erkan, M., Kleeff, J., Sainz, B., Jr., and Heeschen, C. (2015). Inhibition of CD47 effectively targets pancreatic cancer stem cells via dual mechanisms. Clin. Cancer Res. 21, 2325–2337. Dheilly, E., Moine, V., Broyer, L., Salgado-Pires, S., Johnson, Z., Papaioannou, A., Cons, L., Calloud, S., Majocchi, S., Nelson, R., et al. (2017). Selective
blockade of the ubiquitous checkpoint receptor CD47 is enabled by dual-targeting bispecific antibodies. Mol. Ther. 25, 523–533. Dong, H., Strome, S.E., Salomao, D.R., Tamura, H., Hirano, F., Flies, D.B., Roche, P.C., Lu, J., Zhu, G., Tamada, K., et al. (2002). Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat. Med. 8, 793–800. Edris, B., Weiskopf, K., Volkmer, A.K., Volkmer, J.P., Willingham, S.B., Contreras-Trujillo, H., Liu, J., Majeti, R., West, R.B., Fletcher, J.A., et al. (2012). Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma. Proc. Natl. Acad. Sci. USA 109, 6656–6661. Freeman, G.J., Long, A.J., Iwai, Y., Bourque, K., Chernova, T., Nishimura, H., Fitz, L.J., Malenkovich, N., Okazaki, T., Byrne, M.C., et al. (2000). Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 192, 1027–1034. Gordon, S.R., Maute, R.L., Dulken, B.W., Hutter, G., George, B.M., McCracken, M.N., Gupta, R., Tsai, J.M., Sinha, R., Corey, D., et al. (2017). PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity. Nature 545, 495–499. Green, M.R., Monti, S., Rodig, S.J., Juszczynski, P., Currie, T., O’Donnell, E., Chapuy, B., Takeyama, K., Neuberg, D., Golub, T.R., et al. (2010). Integrative analysis reveals selective 9p24.1 amplification, increased PD-1 ligand expression, and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma. Blood 116, 3268–3277. Ishikawa, H., and Barber, G.N. (2008). STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678. Ishikawa, H., Ma, Z., and Barber, G.N. (2009). STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792. Jaiswal, S., Jamieson, C.H., Pang, W.W., Park, C.Y., Chao, M.P., Majeti, R., Traver, D., van Rooijen, N., and Weissman, I.L. (2009). CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell 138, 271–285. Kelly, P.N. (2017). CD28 is a critical target for PD-1 blockade. Science 355, 1386. Li, X.D., Wu, J., Gao, D., Wang, H., Sun, L., and Chen, Z.J. (2013). Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341, 1390–1394. Liu, J., Wang, L., Zhao, F., Tseng, S., Narayanan, C., Shura, L., Willingham, S., Howard, M., Prohaska, S., Volkmer, J., et al. (2015a). Pre-clinical development of a humanized anti-CD47 antibody with anti-cancer therapeutic potential. PLoS ONE 10, e0137345. Liu, X., Pu, Y., Cron, K., Deng, L., Kline, J., Frazier, W.A., Xu, H., Peng, H., Fu, Y.X., and Xu, M.M. (2015b). CD47 blockade triggers T cell-mediated destruction of immunogenic tumors. Nat. Med. 21, 1209–1215. Lo, J., Lau, E.Y., So, F.T., Lu, P., Chan, V.S., Cheung, V.C., Ching, R.H., Cheng, B.Y., Ma, M.K., Ng, I.O., and Lee, T.K. (2016). Anti-CD47 antibody suppresses tumour growth and augments the effect of chemotherapy treatment in hepatocellular carcinoma. Liver Int. 36, 737–745.
Oldenborg, P.A., Zheleznyak, A., Fang, Y.F., Lagenaur, C.F., Gresham, H.D., and Lindberg, F.P. (2000). Role of CD47 as a marker of self on red blood cells. Science 288, 2051–2054. Piccione, E.C., Juarez, S., Tseng, S., Liu, J., Stafford, M., Narayanan, C., Wang, L., Weiskopf, K., and Majeti, R. (2016). SIRPalpha-antibody fusion proteins selectively bind and eliminate dual antigen-expressing tumor cells. Clin. Cancer Res. 22, 5109–5119. Ponce, L.P., Fenn, N.C., Moritz, N., Krupka, C., Kozik, J.H., Lauber, K., Subklewe, M., and Hopfner, K.P. (2017). SIRPa-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells. Oncotarget 8, 11284–11301. Tang, H., Wang, Y., Chlewicki, L.K., Zhang, Y., Guo, J., Liang, W., Wang, J., Wang, X., and Fu, Y.X. (2016). Facilitating T cell infiltration in tumor microenvironment overcomes resistance to PD-L1 blockade. Cancer Cell 29, 285–296. Taube, J.M., Anders, R.A., Young, G.D., Xu, H., Sharma, R., McMiller, T.L., Chen, S., Klein, A.P., Pardoll, D.M., Topalian, S.L., and Chen, L. (2012). Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci. Transl. Med. 4, 127ra37. Thompson, R.H., Gillett, M.D., Cheville, J.C., Lohse, C.M., Dong, H., Webster, W.S., Krejci, K.G., Lobo, J.R., Sengupta, S., Chen, L., et al. (2004). Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target. Proc. Natl. Acad. Sci. USA 101, 17174– 17179. Topalian, S.L., Hodi, F.S., Brahmer, J.R., Gettinger, S.N., Smith, D.C., McDermott, D.F., Powderly, J.D., Carvajal, R.D., Sosman, J.A., Atkins, M.B., et al. (2012). Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med. 366, 2443–2454. Tsai, R.K., and Discher, D.E. (2008). Inhibition of ‘‘self’’ engulfment through deactivation of myosin-II at the phagocytic synapse between human cells. J. Cell Biol. 180, 989–1003. Weiskopf, K., Ring, A.M., Ho, C.C., Volkmer, J.P., Levin, A.M., Volkmer, A.K., Ozkan, E., Fernhoff, N.B., van de Rijn, M., Weissman, I.L., and Garcia, K.C. (2013). Engineered SIRPa variants as immunotherapeutic adjuvants to anticancer antibodies. Science 341, 88–91. Willingham, S.B., Volkmer, J.P., Gentles, A.J., Sahoo, D., Dalerba, P., Mitra, S.S., Wang, J., Contreras-Trujillo, H., Martin, R., Cohen, J.D., et al. (2012). The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc. Natl. Acad. Sci. USA 109, 6662–6667. Wolchok, J.D., Kluger, H., Callahan, M.K., Postow, M.A., Rizvi, N.A., Lesokhin, A.M., Segal, N.H., Ariyan, C.E., Gordon, R.A., Reed, K., et al. (2013). Nivolumab plus ipilimumab in advanced melanoma. N. Engl. J. Med. 369, 122–133. Xiao, Z., Chung, H., Banan, B., Manning, P.T., Ott, K.C., Lin, S., Capoccia, B.J., Subramanian, V., Hiebsch, R.R., Upadhya, G.A., et al. (2015). Antibody mediated therapy targeting CD47 inhibits tumor progression of hepatocellular carcinoma. Cancer Lett. 360, 302–309.
Obeid, M., Tesniere, A., Ghiringhelli, F., Fimia, G.M., Apetoh, L., Perfettini, J.L., Castedo, M., Mignot, G., Panaretakis, T., Casares, N., et al. (2007). Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 13, 54–61.
Xu, M.M., Pu, Y., Han, D., Shi, Y., Cao, X., Liang, H., Chen, X., Li, X.D., Deng, L., Chen, Z.J., et al. (2017). Dendritic cells but not macrophages sense tumor mitochondrial DNA for cross-priming through signal regulatory protein a signaling. Immunity 47, 363–373.e5.
Okazawa, H., Motegi, S., Ohyama, N., Ohnishi, H., Tomizawa, T., Kaneko, Y., Oldenborg, P.A., Ishikawa, O., and Matozaki, T. (2005). Negative regulation of phagocytosis in macrophages by the CD47-SHPS-1 system. J. Immunol. 174, 2004–2011.
Yoshida, K., Tsujimoto, H., Matsumura, K., Kinoshita, M., Takahata, R., Matsumoto, Y., Hiraki, S., Ono, S., Seki, S., Yamamoto, J., and Hase, K. (2015). CD47 is an adverse prognostic factor and a therapeutic target in gastric cancer. Cancer Med. 4, 1322–1333.
Cell Reports 24, 2101–2111, August 21, 2018 2111