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. Author manuscript; available in PMC: 2024 Dec 1.
Published in final edited form as: Adv Ther (Weinh). 2023 Sep 15;6(12):2300114. doi: 10.1002/adtp.202300114

Discovery of Anti-CD47 Peptides as Innate Immune Checkpoint Inhibitors

Bahaa Mustafa 1, John Fetse 1, Sashi Kandel 1, Chien-Yu Lin 1, Pratik Adhikary 1, Umar-Farouk Mamani 1, Yanli Liu 1, Mohammed Nurudeen Ibrahim 1, Mohammed Alahmari 1, Kun Cheng 1,*
PMCID: PMC11034909  NIHMSID: NIHMS1935351  PMID: 38655206

Abstract

Cancer immunotherapy targeting adaptive immune cells has been attracting considerable interest due to its great success in treating multiple cancers. Recently, there is also increasing interest in agents that can stimulate innate immune cell activities. Immune checkpoint inhibitors targeting innate immune cells can block inhibitory interactions (‘don’t eat me’ signals) between tumor cells and phagocytes. CD47 is a transmembrane protein overexpressed in various cancers and acts as a potent ‘do not eat me’ signal that contributes to the immune evasion of cancer cells. Anti-CD47 peptides that can bind to CD47 and block CD47/SIRPα interaction were discovered using a novel phage display biopanning strategy. Anti-CD47 peptides enhanced the macrophage-mediated phagocytosis of NCI-H82 tumor cells in vitro. Unlike anti-CD47 antibodies, these peptides do not induce the agglutination of RBCs. Moreover, anti-CD47 peptides exhibit high specificity for MC-38 cancer cells expressing CD47. CMP-22 peptide showed the ability to increase the antitumor activity of doxorubicin and extends the survival of CT26 tumor-bearing mice. The discovered anti-CD47 peptides can be considered potential candidates for cancer immunotherapy by blocking the CD47/SIRPα interaction, especially in combination with chemotherapy, to elicit synergistic effects.

Keywords: CD47, SIRPα, Peptide, Phage display, Phagocytosis

Graphical Abstract

CD47/SIRPα is an innate immune checkpoint pathway. Cancer cells overexpress CD47 on their surface. Through phage display biopanning, three anti-CD47 peptides have been discovered that can block the binding of CD47 to SIRPα. This re-activates macrophages to phagocytose cancer cells. The discovered anti-CD47 peptides have the potential to be used as immune checkpoint inhibitors in combination with chemotherapy.

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1. Introduction

Cancer immunotherapy targeting adaptive immune cells has been attracting considerable interest due to its great success in treating multiple hematological and solid tumors 1. However, there is still a need for combination immunotherapies because suboptimal response rates are reported in some patients 2. Recently, there is increasing interest in agents that can stimulate innate immune cell activities 3. The innate immune system serves as the first line of nonspecific defense in the body, and macrophages have potent phagocytosis and antigen-presenting capabilities. In addition, macrophages are a major component of the tumor microenvironment, and some studies have shown that they can be recruited at a higher percentage than T cells in various cancers 4.

CD47, also known as integrin-associated protein, is an integral membrane glycoprotein now recognized as an important phagocytosis checkpoint that is expressed in almost all normal cells in the body. Overexpression of CD47 has been observed in various blood and solid tumors. Signal regulatory protein alpha (SIRPα, also known as CD172α) is a member of the signal regulatory protein family of immunoreceptors and is considered an inhibitory receptor that interacts with CD47. In contrast to CD47, the expression of SIRPα is more restricted to myeloid cells (monocytes, macrophages, and dendritic cells). The CD47/SIRPα interaction transmits a strong antiphagocytic signal through the deactivation of the myosin-II-associated machinery required for engulfment 5. Blockade of the CD47/SIRPα interaction has thus emerged as a promising therapeutic approach, and multiple studies have shown that anti-CD47 blockade promotes antitumor activity against various cancers mainly via macrophage-mediated phagocytosis of tumor cells 6, 7.

Here, we used peptide phage display to discover anti-CD47 peptides that not only bind to CD47 but also block the CD47/SIRPα interaction. These anti-CD47 peptides facilitated macrophage-mediated phagocytosis of NCI-H82 lung cancer cells. Moreover, the anti-CD47 peptides showed high specificity for Murine colon cancer MC-38 colon cancer cells expressing CD47. The discovered peptides showed a favorable hematological safety profile and did not induce hemagglutination of human RBCs. Using the CT26 syngeneic mouse model, the anti-CD47 peptide CMP-22 inhibited the growth of CT26 cells and exerted a synergistic effect when combined with doxorubicin. Our findings suggest that these anti-CD47 peptides represent potential innate immune checkpoint inhibitors for cancer immunotherapy.

2. Material and Methods

2.1. Cell culture

MC38 cells were purchased from Kerafast (Boston, MA). MC38-CD47 knockout MC38-CD47KO cell line was kindly provided by Dr. Yang-Xin Fu at the University of Texas Southwestern Medical Center. The level of CD47 in MC38-CD47KO cells was verified in Dr. Fu’s report.8 These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, 100 units/mL penicillin, 1% nonessential amino acid (NEAA) (Gibco, GrandsIsland, NY), 1% sodium pyruvate, 1% glutamine, 1% HEPES and 50 μg/mL gentamicin. NCI-H82 and A20 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Roswell Park Memorial Institute Medium (RPMI1640) with 10% FBS, 100 μg/mL streptomycin and 100 units/mL penicillin. Human peripheral blood mononuclear cells (PBMCs) and human packed RBCs were purchased from Zen-Bio Inc. (Research Triangle Park, NC) and cultured in RPMI 1640 with 10% FBS, 100 μg/mL streptomycin and 100 units/mL penicillin. All cells were grown at 37 °C in a humidified atmosphere containing 5% CO2.

2.2. Discovery of anti-CD47 peptides using phage display biopanning

The biopanning was performed in a 96-well plate by directly coating two wells with 100 ng of recombinant human CD47 protein (Catalog# ab174029, Abcam, Cambridge, MA) at 4 °C overnight. After washing with phosphate buffered saline (PBS), the first well was blocked with 2% BSA for 2 h, followed by incubation with 5000 ng of recombinant human SIRPα (Catalog# 71138, BPS Bioscience, San Diego, CA) for 1 h. The Ph.D.™-12 Phage Display Peptide Library (catalog # E81102, New England BioLabs, Ipswich, MA) was added into the first well and incubated at room temperature for 1 h. The unbound phages were then transferred from the first well to the second well coated with CD47 and incubated for 1 h. From the second well, the bound phages were eluted and amplified for the next round of biopanning. After completing five rounds of biopanning, phage plaques were randomly selected for DNA sequencing. The encoded peptides were either purchased from Genscript (Piscataway, NJ) or synthesized using a PurePep Chorus peptide synthesizer (Gyros Protein Technologies, Tucson, AZ) and then purified by HPLC.

2.3. Competition surface plasmon resonance (SPR) binding assay

Competition SPR was used to evaluate the ability of the anti-CD47 peptides to block the interaction between human CD47 (Catalog# ab174029, Abcam, Cambridge, MA) and biotinylated human SIRPα (Catalog# 71138, BPS Bioscience, San Diego, CA). The binding affinity of the anti-CD47 peptides to human CD47 was determined using a five-channel BI-4500 SPR (Biosensing Instrument Inc., Tempe, AZ). PBS (pH 7.4) was used as the running buffer, and 10 mM NaOH was used for regeneration following each binding interaction. The flow rate was set at 60 μL/min. A CM dextran chip pre-functionalized with streptavidin was mounted, and approximately 750 response units (RU) of biotinylated human SIRPα was immobilized onto the chip. Free human CD47 solution (5 nM) was injected as a control. Peptides with different concentrations were incubated with human CD47 protein (30nM) at 37°C for 30 min before injection into the coated chip. The results were analyzed with SPR Data Analysis software Version 3.8.4 (Biosensing Instrument Inc., Tempe, AZ).

2.4. Evaluation of the binding specificity of the anti-CD47 peptides

A flow cytometry-based binding assay was used to evaluate the specificity of the anti-CD47 peptides to CD47-expressing MC38 cells and MC38-CD47KO cells. The cells were dissociated using nonenzymatic cell dissociation solution (Gibco, Waltham, MA) and diluted to a density of 1×106 cells/mL in DMEM containing 10% FBS. 5-FAM-labeled anti-CD47 peptides with various concentrations (2.5–10 μM) were incubated with 0.5 mL of the suspended cells at 37°C for 2 h under shaking. The cells were washed with PBS and then analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).

2.5. Blocking of the CD47/SIRPα interaction

One hundred nanograms of recombinant human CD47 protein (Catalog# ab174029, Abcam, Cambridge, MA) was coated per well on a 96-well plate overnight at 4°C. The plate was blocked with 2% BSA at room temperature for 2 h. Various concentrations of the anti-CD47 peptides were added to the wells and incubated for 2 h at room temperature. Biotinylated recombinant human SIRPα (500 ng, Catalog# 71138, BPS Bioscience, San Diego, CA) was then added and incubated for 1 h. Streptavidin-HRP (R&D Systems, Minneapolis, MN) and chromogenic substrate reagent (R&D Systems, Minneapolis, MN) were added to each well, and the reaction was stopped after 20 min. The OD450 was recorded and referenced to OD540.

2.6. Cytotoxicity of the anti-CD47 peptides in NCI-H82 cells

The cytotoxicity of anti-CD47 peptides in NCI-H82 cells was evaluated using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) following the manufacturer’s protocol. NCI-H82 cells were seeded in a 96-well plate at a density of 5,000 cells/well and incubated in a humidified atmosphere with 5% CO2 at 37 °C. After 24 h, the medium was replaced with fresh medium containing various concentrations of anti-CD47 peptides (0.5, 1, 5, and 10 μM). After incubation for another 24 h, the medium was removed, and 80 μL of CellTiter-Glo Reagent was added to each well. The plate was mixed on an orbital shaker for 2 min to induce cell lysis. Finally, the plate was incubated at room temperature for 10 min, and luminescence was measured.

2.7. In vitro hemagglutination assay

A hemagglutination assay was performed to investigate the effect of anti-CD47 peptides on human RBCs as described 7. Human packed RBCs were incubated with 1 μM and 10 μM anti-CD47 peptides overnight at 37°C. Anti-human CD47 antibodies CC2C6 (Catalog#323102, BioLegend, San Diego, CA) and B6H12 (Catalog#5012617, Invitrogen, Waltham, MA) were used as controls at 1 μM. A blood smear was prepared by smearing a drop of blood onto a glass slide using a glass spreader slide. Smears were air-dried and imaged with an optical microscope. Qualitative analysis of the images was used for the evaluation of RBC agglutination.

2.8. In vitro phagocytosis assay

The phagocytosis assay was performed as described with some modifications 9. Human PBMCs were cultured in RPMI 1640 (2 mM glutamine, penicillin 100 μg/mL, streptomycin 100 μg/mL, 0.1 mM sodium pyruvate, 1% NEAA, 10% FBS, 10 mM HEPES, 25 μg/mL gentamicin) for 2 h at 37 oC for the plastic adhesion of monocytes. After 2 h, the monocytes were examined for attachment, and the non-adherent cells were removed. Macrophages were generated by culturing the monocytes in RPMI 1640 containing 40 ng/mL human macrophage colony stimulating factor (M-CSF) (PeproTech, Hamburg, Germany), 2mM glutamine, 100 μg/mL penicillin, 100 μg/mL streptomycin, 0.1 mM sodium pyruvate, 1% NEAA, 10% FBS, 10 mM HEPES, 25 μg/mL gentamicin for 10 days. The nonadherent cells were removed, and the adherent cells representing macrophages were detached by incubation with TrypLE cell dissociation buffer (Gibco, Carlsbad, CA). Differentiation was confirmed by morphological observation of adherent cells under an optical microscope. NCI-H82 cells were labeled with 2.5 μM CFSE (Invitrogen, Carlsbad, CA). A flat bottom 24-well plate was used and in each well macrophages (5×104) at a 1: 4 effector/target ratio were cocultured with CFSE-labelled NCI-H82 cells (2×105) and the anti-CD47 peptide (10 μM) or anti-CD47 mAb B6H12 (20 μg/mL) for 4 h in RPMI 1640 serum-free medium. The macrophages were washed and subsequently imaged using an inverted fluorescence microscope (Leica DMI3000B, Germany) and analyzed using ImageJ software.

2.9. In vivo antitumor activity of the anti-CD47 peptides

Eight-week-old male and female Balb/c mice were purchased from Charles Rivers Laboratories (Wilmington, Massachusetts) and housed in a temperature- and humidity-controlled room on a 12-h light-dark cycle. For the CT26 colon carcinoma model, 1×106 CT26 cells were subcutaneously implanted into the right flank of mice. For the A20 B-cell lymphoma model, 1×106 A20 cells were subcutaneously implanted into the right flank of female mice. The tumor size was assessed with a digital caliper and calculated with the formula (length×width2)/2. For the CT26 model, once the tumor size reached 50–100 mm3, the mice were randomly divided into four different treatment groups (10 mice/group, 50% female, 50% male). The mice in the first group were intraperitoneally injected with 5 mg/kg CMP-22 peptide daily for a total of 12 injections. The mice in the second group were intravenously injected with 5 mg/kg doxorubicin (LC Laboratories, Woburn, MA) on days 6, 9, and 12 after tumor implantation 10. The mice in the third group were injected with a combination of 5 mg/kg CMP-22 daily and 5 mg/kg doxorubicin on days 6, 9, and 12. Saline was used as a negative control in this study. Mouse body weights were monitored daily throughout the treatment period. For the A20 model, the mice were randomly divided into four different treatment groups. The mice in the first group were intraperitoneally injected with 5 mg/kg CMP-22 peptide daily for a total of 14 injections. The mice in the second group were intraperitoneally injected with 5 mg/kg pep-20 peptide daily for a total of 14 injections. The mice in the third group were injected with the anti-mouse CD47 antibody MIAP-301 20 mg/kg 11 intraperitonially every other day for a total of 7 injections. Saline was also used as a control in this study.

2.10. Statistical analyses

Data were presented as the mean ± standard deviation (SD) except for the animal studies the data were presented as mean ± standard error of the mean (SEM). An independent Student’s t test was used to compare differences if there were only two groups. One-way ANOVA followed by Dunnett’s test was used to compare differences when there were more than two groups of data. All tests were two-sided, and p values below the 5% level were regarded as significant. All the data were analyzed using GraphPad Prism (version 8).

3. Results

3.1. Discovery of anti-CD47 peptides using phage display biopanning

To identify anti-CD47 peptides that not only bind to human CD47 but also inhibit the CD47/SIRPα interaction (Figure 1a), we screened the Ph.D.-12 phage display peptide library using a novel biopanning strategy that we developed earlier 12, 13. Through this biopanning strategy, we can identify peptides that bind to specific residues of the target protein. As shown in Figure 1b, phage enrichment was achieved after five rounds of biopanning. Seventy-five phage plaques were randomly selected for DNA sequencing, and 26 different peptide sequences were discovered (Figure 1c).

Figure 1. Discovery of anti-CD47 peptides using phage display biopanning.

Figure 1.

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a) Anti-CD47 peptides block the CD47/SIRPα axis to activate macrophage-mediated phagocytosis of tumor cells. b) The number of recovered phages after each round of biopanning. Results are represented as the mean ± SD (n = 3). c) Sequences of the anti-CD47 peptides.

3.2. Anti-CD47 peptides block the CD47/SIRPα interaction

To evaluate the ability of the peptide candidates to block the human CD47/SIRPα interaction, we first screened the blocking efficiency of these peptides at 10 μM. Among the 26 peptides, the peptides GFM, CMP-21, and CMP-22 showed the highest blocking effect on the CD47/SIRPα interaction as illustrated in Figure 2a. The anti-human CD47 antibody (Catalog#MAB4670, R&D Systems) was used as a positive control to calibrate this assay. Next, we examined the dose-dependent effect of anti-CD47 peptides by determining the half-maximal inhibitory concentration (IC50) of the anti-CD47 antibody, CMP-22 peptide, CMP-21 peptide, and GFM peptide (Figure 2b). The anti-CD47 antibody blocked the CD47/SIRPα interaction with an IC50 of 0.537 nM. Among the discovered anti-CD47 peptides, CMP-22 peptide showed the greatest dose-dependent effect with and IC50 of 276 nM. The IC50 values of the peptides CMP-21 and GFM are 387 nM and 361 nM, respectively. These results suggest that the discovered anti-CD47 peptides can block the CD47/SIRPα interaction and will be screened for subsequent activity studies.

Figure 2. The CD47/SIRPα interaction is blocked by the anti-CD47 peptides and antibody.

Figure 2.

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a) Blocking efficiency of the anti-CD47 peptides (10 μM) and the anti-human CD47 antibody (100 nM) against the human CD47/SIRPα interaction. b) IC50 of the anti-CD47 peptides in blocking the CD47/SIRPα interaction. All results are represented as the mean ± SD (n = 3).

3.3. SPR-based competition assay

SPR is an important technique for studying the interaction between two different proteins. However, SPR has some limitations when evaluating the binding of small molecules, such as small peptides and a protein, due to the low responses. Therefore, we performed a competition SPR assay to test whether the discovered anti-CD47 peptides can bind CD47 and inhibit the human CD47/SIRPα interaction using a five-channel BI-4500 SPR. Approximately 750 RU of biotinylated recombinant human SIRPα was immobilized on a streptavidin pre-functionalized CM Dextran Chip. Anti-CD47 peptides ranging from 10 nM to 10 μM were incubated with human CD47 protein (30 nM) for 30 min before injecting into the chip. Free human CD47 protein (30 nM) was used as a control. As illustrated in Figure 3a–c, the SPR responses were significantly reduced by injecting different concentrations of the anti-CD47 peptide CMP-22, suggesting a concentration-dependent binding of the CMP-22 peptide to CD47 protein. Similar results were observed for the peptides CMP-21 and GFM (Figure 3 d–i). The findings from the SPR competition assay were consistent with the blocking assay in Figure 2, and the CMP-22 peptide showed the highest potency among the discovered peptides.

Figure 3. Evaluation of the binding affinity and blocking efficiency of the anti-CD47 peptides using competition SPR.

Figure 3.

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SPR sensograms (a), response curve (b), and blocking curve (c) of the CMP-22 peptide. SPR sensograms (d), response curve (e), and blocking curve (f) of the CMP-21 peptide. SPR sensograms (g), response curve (h), and blocking curve (i) of the GFM peptide. All results are represented as the mean ± SD (n = 3).

3.4. Binding specificity of the anti-CD47 peptides

After showing that the anti-CD47 peptides can competitively bind to CD47 and block its binding to SIRPα on the SPR sensor chip, we next examined the specificity of these peptides toward CD47 expressed on MC38 colon adenocarcinoma cells. CD47 knockout MC38 cells (MC38-CD47KO) were used as a negative control in this study. Binding affinities of FAM-labeled anti-CD47 peptides toward MC38 and MC38-CD47KO cells were compared using flow cytometry. As shown in Figure 4, the binding of all peptides to MC38 cells was significantly higher than that to MC38-CD47KO cells, indicating a good specificity of the peptides to murine CD47 protein expressed on MC38 cells. In particular, the peptides GFM and CMP-22 exhibited slightly higher specificity compared to the peptide CMP-21. Moreover, these peptides were incubated with the cells in the presence of 10% FBS, suggesting that FBS does not compromised the binding affinity and specificity of these peptides to CD47. All these results suggest that the anti-CD47 peptides can specifically bind to CD47-expressing cancer cells.

Figure 4. Binding specificity of anti-CD47 peptides to CD47-overexpressing cancer cells.

Figure 4.

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Percent of MC38 and MC38-CD47KO cells that bind to FAM-labeled peptides GFM (a), CMP-21 (b), and CMP-22 (c) at different concentrations and their corresponding histogram plots. Statistical significance was determined by two-tailed Student’s t-test (**P < 0.01). All results are presented as the mean ± SD (n=3).

3.5. Macrophage-mediated phagocytosis of cancer cells

Multiple studies have shown that the major mechanism of anti-CD47 therapies is the activation of macrophage-mediated phagocytosis of tumor cells 7, 9. Thus, we investigated the in vitro capability of the anti-CD47 peptides to facilitate macrophage-mediated phagocytosis of NCI-H82 human cancer cells. NCI-H82 cells are small cell lung carcinoma cells that express high levels of CD47 on the cell surface. CFSE-labeled NCI-H82 cells (green) were cocultured with human macrophages differentiated from human PBMCs (Figure 5a). The anti-CD47 antibody B6H12 (20 μg/mL) was used as a positive control as reported 14. In contrast to cells treated with PBS, CFSE-labeled NCI-H82 cells (green) treated with 10 μM anti-CD47 peptides (CMP-21, CMP-22, and GFM) for 4 h were efficiently phagocytosed by human macrophages (Figure 5b). The CMP-22 peptide showed better activity compared to the peptides CMP-21 and GFM. This is in accordance with the specificity result in Figure 4.

Figure 5. Anti-CD47 peptides promote macrophage-mediated phagocytosis of NCI-H82 cells.

Figure 5.

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a) Differentiation of human monocytes into macrophages. b) In-vitro phagocytosis assay was performed by co-culturing NCI-H82 cells with human peripheral blood monocyte-derived macrophages at a 1:4 ratio in serum-free medium at 37°C for 4h in the presence of 10 μM anti-CD47 peptides or 20 μg/mL anti-human CD47 antibody (B6H12). c). Cytotoxicity of GFM, CMP-21, and CMP-22 peptides in NCI-H82 cells at different concentrations. Triton X-100 was used as a positive control. Data are presented as the mean ± SD (n = 3).

We next examined whether the anti-CD47 peptides affected the viability of NCI-H82 cells. As shown in Figure 5c, all the three anti-CD47 peptides at concentrations up to 10 μM did not induce cytotoxicity in NCI-H82 cells after 24 h incubation. These data indicate that increased phagocytosis of NCI-H82 cells by anti-CD47 peptides is not due to cell death caused by direct cytotoxicity but rather due to the blockade of the CD47/SIRPα interaction.

3.6. In vitro hemagglutination study

A hemagglutination assay was applied to investigate the effect of anti-CD47 peptides (CMP-21, CMP-22, GFM) on human RBCs. Because CD47 is expressed on RBCs, molecules that bind to CD47 may potentially induce hemagglutination. As shown in Figure 6, anti-CD47 peptides had no effect on the aggregation of RBCs at concentrations of 1 and 10 μM. In contrast, two anti-CD47 antibodies (B6H12 and CC2C6) were used as positive controls at a comparable concentration of 1 μM induced hemagglutination as reported 15. Both B6H12 and CC2C6 antibodies caused significant agglutination of human RBCs (Figure 6). Hemagglutination is considered a type of homotypic interaction, in which two CD47 expressing cells such as RBCs aggregate when treated with bivalent CD47 binding molecules such as anti-CD47 antibodies 16. Our findings showed that these anti-CD47 peptides (CMP-21, CMP-22, and GFM) displayed a distinct RBC-sparing safety without inducing hemagglutination.

Figure 6. Hemagglutination assay of the anti-CD47 peptides.

Figure 6.

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The effect of anti-CD47 peptides on RBC agglutination was evaluated. Human RBCs were incubated with PBS, anti-CD47 peptides, and anti-CD47 antibodies at 37°C overnight. Blood smears were analyzed using an optical microscope. The experiment was performed in triplicate, and a representative image from each treatment was presented.

3.7. In vivo antitumor activity of the anti-CD47 peptide CMP-22

Among the three anti-CD47 peptides, the peptide CMP-22 showed the highest affinity (Figure 3), specificity (Figure 4), and in vitro phagocytosis activity (Figure 5). We, therefore, evaluated whether the CMP-22 peptide could functionally inhibit the CD47/SIRPα interaction and slow tumor growth in syngeneic mouse models. We first subcutaneously implanted A20 cells into Balb/c mice. Ten days after tumor implantation, we intraperitoneally administered CMP-22 peptide and monitored tumor growth. We used the anti-CD47 peptide pep-20 to compare the therapeutic effectiveness with the CMP-22 peptide 6. We also used anti-mouse CD47 MIAP-301 as a positive control in this study 11. There is no significant difference among the four groups, suggesting that monotherapy with the CMP-22 peptide, pep-20 peptide, or anti-CD47 antibody MIP-301 cannot inhibit tumor growth in A20 tumor-bearing mice (Figure 7a). This outcome contrasts with Liu’s study, in which the anti-CD47 antibody (MIAP-301) by itself demonstrated antitumor activity in the A20 mouse model 11. However, our result is consistent with other studies in which anti-CD47 monotherapy did not slow tumor growth in several syngeneic models 17, 18.

Figure 7. Anti-tumor activity of the CMP-22 peptide and other anti-CD47 inhibitors in an A20 B-cell lymphoma mouse model.

Figure 7.

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(a) A20 tumor-bearing Balb/c mice (n=8, 4 males and 4 females) were intraperitoneally injected daily with the CMP-22 peptide (5 mg/Kg), pep-20 peptide (5 mg/Kg) for a total of 12 injections and the anti-mouse CD47 antibody (20 mg/Kg) every other day for a total of 7 injections. Tumor volumes are presented as the mean ± SEM (n = 8). (b) Tumor growth curves of individual mice in each group.

To evaluate the combined effect of CD47 blockade with conventional chemotherapy, we next investigated whether the anti-CD47 peptide CMP-22 enhances the suppression of tumor growth by doxorubicin in vivo. Several studies have shown the synergistic effect of combining anti-CD47 treatment with doxorubicin in the CT26 syngeneic mouse model 19. Moreover, TTI-621, an anti-CD47 fusion protein comprised of the N-terminal V domain of human SIRPα linked to the human IgG1 Fc region is currently studied in a clinical trial for the treatment of leiomyosarcoma in combination with doxorubicin (NCT04996004).

CT26 murine colorectal carcinoma cell line highly expresses CD47 20 and is widely used for investigating the CD47/SIRPα blockade activity in vivo 21. In addition, the combination anti-CD47 antibody and doxorubicin showed synergistic activity in a hepatocellular carcinoma xenograft model. The anti-CD47 B6H12 antibody sensitized Hepatocellular carcinoma (HCC) to the effect of doxorubicin via inhibitory signaling through CTSS/PAR2 22. In another study, doxorubicin induced CD47 expression in triple negative breast cancer which may explain the additive effect of CD47 blockade in combination with doxorubicin 23. As illustrated in Figure 8, treatment with CMP-22 alone had little effect on the growth of tumors, but the peptide significantly enhanced the inhibitory effect of doxorubicin on tumor growth compared to the saline group. We found that the combination of the anti-CD47 peptide CMP-22 with doxorubicin significantly enhanced the inhibition of tumor growth compared to mice treated with doxorubicin or CMP-22 alone. These results are consistent with previous studies in which inhibition of CD47 alone through CD47/SIRPα blockade or silencing of CD47 expression showed minimal activity, enhanced the efficacy of doxorubicin against CT26 tumors 19, 24. It is worth mentioning that although the mouse CD47 protein shares 60–70% amino acid sequence identity with human CD47 25 and these anti-CD47 peptides were screened against the human CD47 protein, the peptide CMP-22 showed good antitumor activity in combination with doxorubicin.

Figure 8. Antitumor activity of the CMP-22 peptide and doxorubicin in a CT26 colon cancer mouse model.

Figure 8.

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CT26 tumor-bearing Balb/c mice (n=10, 5 males and 5 females) were intraperitoneally injected daily with the CMP-22 peptide (5 mg/Kg) for a total of 12 injections and intravenously injected with doxorubicin (5 mg/Kg) on days 6,9 and 12 after tumor implantation. (a) Tumor volume. The results were presented as the mean ± SEM (n=10). (b) Changes in body weight of tumor-bearing mice treated with different treatment. (c) Survival curves. Comparison of two survival curves was conducted using the Gehan-Breslow-Wilcoxon test. GraphPad Prism (version 8) was used for statistical analysis. (d) Tumor growth curves of individual mice in each group. (* P<0.05, ** P<0.01, *** P<0.001)

The body weight changes of the mice are shown in Figure 8d. No obvious weight loss was found in the CMP-22 compared to saline group. In addition, no further weight loss was found in the CMP-22/doxorubicin combination group compared to the doxorubicin alone group, indicating that CMP-22 did not cause additional systemic toxicity while enhancing the therapeutic efficacy. Doxorubicin at its maximum tolerated dose (5 mg/kg) 26 led to slight systemic toxicity in mice manifested by weight loss in mice in both the combination group and the doxorubicin alone group.

For the survival study, the mice were intraperitoneally injected with the CMP-22 peptide (5 mg/kg) daily from day 6 to day 17 or intravenously injected with doxorubicin (5 mg/kg) at days 6,9, and 12. As Figure 8c revealed, CMP-22 inhibited tumor growth and extended the survival of CT26 tumor-bearing mice compared with control animals. Moreover, CMP-22 in combination with doxorubicin extended the survival of CT26 bearing mice compared to those treated with doxorubicin alone or normal saline. As shown in Figure 8d, all mice in the saline group died by day 34. In contrast, 90% of the mice in the CMP-22 group died by day 34. Eight mice showed a response to CMP-22/doxorubicin combination, compared to CMP-22 or doxorubicin alone (1 and 5 mice, respectively).

4. Discussion

The CD47/SIRPα interaction can be blocked on either side to sufficiently activate the phagocytosis of target cells. From a safety standpoint, targeting SIRPα would be the preferred strategy due to the restricted expression pattern of SIRPα. Unlike CD47, targeting SIRPɑ avoids the “antigen sink” effect to a large extent in vivo. However, as CD47 is overexpressed in multiple cancers, targeting CD47 can limit off-target side effects in vivo, such as the binding of anti-SIRPɑ agents to neurons and muscles.

Peptides have some advantages compared to small molecules. Traditional small molecules are not readily excreted, which enhances their accumulation in organs alongside their toxic metabolites, causing side effects. On the other hand, peptides are metabolized to amino acids in the body and thus have a rare incidence of side effects. Moreover, when compared to protein-based biopharmaceuticals, peptides have a lower production complexity and, as a result, lower production costs. At the same time, peptides have some disadvantages that limit their application in drug development such as low oral bioavailability, low plasma stability, and short circulation time.

The mechanism of anti-CD47 therapies was investigated by multiple studies, and the results showed that anti-CD47 therapies mainly activate macrophage-mediated phagocytosis to treat various cancers due to specific intrinsic functions of CD47 27. Our results indicated that anti-CD47 peptides (CMP-21, CMP-22 and GFM) could enable engulfment of NCI-H82 tumor cells by macrophages in vitro by blocking the CD47/SIRPα interaction. This result is consistent with other studies showing that the blockade effects of disrupting the CD47/SIRPα interaction elicit direct macrophage-mediated phagocytosis rather than antibody-dependent cell-mediated cytotoxicity (ADCC) 28. In addition, the results of the cytotoxicity assay (Figure 5c) showed that the anti-CD47 peptides (CMP-21, CMP-22 and GFM) did not induce direct cytotoxicity to NCI-H82 cells. These data indicate that increased phagocytosis of NCI-H82 cells by anti-CD47 peptides does not result from cell death caused by direct cytotoxicity, and macrophage-mediated phagocytosis is the major mechanism of the therapeutic activity of the peptides.

Because CD47 is widely expressed on normal cells especially RBCs, molecules that bind CD47 could potentially cause hematological side effects. These adverse effects are associated with the expression of CD47, particularly senescent RBCs and blockade of CD47 on RBCs can lead to macrophage-mediated phagocytic removal and hemagglutination 7. Our results showed that the anti-CD47 peptides did not induce hemagglutination at a comparable concentration to anti-CD47 antibodies (B6H12 and CC2C6), which induced serious RBC hemagglutination. The results were consistent with previous reports in which anti-CD47 antibodies (B6H12 and CC2C6) induced RBC hemagglutination at concentrations up to 500 nM 15.

We first evaluated the antitumor activity of the CMP-22 peptide in an A20 syngeneic mouse model (Figure 7). We selected peptide CMP-22 for the in vivo studies based on the favorable blocking efficiency and in vitro phagocytosis activity over peptides (CMP-21 and GFM). Inconsistent with the results of Liu et al., we did not observe the therapeutic effect of the anti-CD47 antibody (MIAP-301) in the A20 model 11. In addition, CMP-22 and pep-20 peptides did not inhibit tumor growth in A20 tumor-bearing mice. However, our results were consistent with other studies in which anti-CD47 monotherapy did not slow tumor growth in several syngeneic models 17, 18. Many wild-type tumors, including A20, display poor immunogenicity with high levels of M2 macrophages compared to M1. Considering that M2 macrophage-mediated phagocytosis following anti-CD47 treatment is less prominent than that mediated by M1 macrophages 29, blockade of CD47 would not effectively promote tumor phagocytosis by macrophages in a low-immunogenic tumor microenvironment. Hence, for the anti-CD47 therapeutic effect to be prominent, the existence of macrophages with phagocytic activity in the tumor microenvironment is crucial 17.

To evaluate the combined effect of CD47 suppression with conventional chemotherapy, we investigated the antitumor activity of the CMP-22 peptide in combination with doxorubicin using the CT26 mouse model. CMP-22 alone showed weak antitumor activity when administered at 5 mg/kg for 12 days. This may be attributed to the low immunogenicity of the CT26 model 30, which may inhibit macrophage-mediated phagocytosis of tumor cells. Our results were consistent with other studies where anti-CD47 treatment did not slow tumor growth in CT-26 tumor-bearing mice 15. Interestingly, combination treatment with CMP-22 and doxorubicin resulted in enhanced tumor growth inhibition compared to doxorubicin treatment alone. The CMP-22 peptide also prolonged the survival of tumor-bearing mice compared to either saline-treated mice or the doxorubicin-treated mice. Our findings were consistent with other reports where anti-CD47 treatment showed synergistic activity and enhanced the activity of doxorubicin in CT26 tumor-bearing mice 19, 24. This synergism has never been demonstrated before by peptide-based immune checkpoint inhibition. Certain chemotherapeutic agents such as doxorubicin have shown to induce tumor immunogenic cell death (ICD) 31. ICD is considered a form of regulated cell death that can activate antigen-specific immune response via secretion of signals such as damage-associated molecular patterns (DAMPs) or “eat me” signals. The increased expression of DAMPs allows their interaction with pattern recognition receptors (PRRs) expressed by innate immune cells such as macrophages 32. This eventually will lead to activation and maturation of cancer cells that migrate to lymph nodes. The specific cancer antigens that are expressed as a result of ICD are then presented to T cells (CD4+ and CD8+ T lymphocytes) which activate a specific adaptive immune response against tumor cells 33. It has been shown that the expression of calreticulin is the most important element for ICD activity to induce the immune system 34. However, calreticulin effect of providing “eat me” signal is counterbalanced by the expression of CD47 35. Therefore, CD47 blockade combined with ICD-inducing drug such as doxorubicin may provide synergistic activity and enhance the effect of “eat me” signal by calreticulin 24.

5. Conclusion

In summary, we discovered anti-human CD47 peptides by phage display biopanning that can markedly inhibit the CD47/SIRPα interaction. The discovered anti-CD47 peptides specifically bind to CD47 on tumor cells without inducing hemagglutination. The anti-CD47 peptides enhanced macrophage-mediated phagocytosis of tumor cells. In particular, the CMP-22 peptide enhances preclinical antitumor activity and increases the survival of CT26 tumor-bearing mice in combination with chemotherapy. These anti-CD47 peptides can be considered potential candidates for cancer immunotherapy by blocking the CD47/SIRPα interaction, especially in combination with chemotherapy, to elicit synergistic effects. Moreover, these anti-CD47 peptides can be easily linked to other peptide-based checkpoint inhibitors to form multivalent inhibitors to achieve synergistic effects.

Acknowledgements

We would like to thank Dr. Yang-Xin Fu at UT Southwestern Medical Center for providing MC38-CD47 knockout MC38-CD47KO cell line.

Funding Information

This work was supported in part by the National Institutes of Health (R01CA231099 and R01CA271592).

Footnotes

Conflict of interest disclosure statement:

The authors declare no competing interests.

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