Background CD73 is an ecto-enzyme that is involved in the conversion of pro-inflammatory extracellular ATP (eATP) excreted by cancer cells under stress to anti-inflammatory adenosine (ADO). A broad variety of solid cancer types was shown to exploit CD73 overexpression as a suppressive immune checkpoint. Consequently, CD73-antagonistic antibodies, most notably oleclumab, are currently evaluated in several multicenter trials for clinical applicability. However, the efficacy of conventional monospecific CD73-inhibiting antibodies may be limited due to on-target/off-tumor binding to CD73 on normal cells. Therefore, a novel approach that more selectively directs CD73 immune checkpoint inhibition towards cancer cells is warranted.
Methods To address this issue, we constructed a novel tetravalent bispecific antibody (bsAb), designated bsAb CD73xEGFR. Subsequently, the anticancer activities of bsAb CD73xEGFR were evaluated using in vitro and in vivo tumor models.
Results In vitro treatment of various carcinoma cell types with bsAb CD73xEGFR potently inhibited the enzyme activity of CD73 (~71%) in an EGFR-directed manner. In this process, bsAb CD73xEGFR induced rapid internalization of antigen/antibody complexes, which resulted in a prolonged concurrent displacement of both CD73 and EGFR from the cancer cell surface. In addition, bsAb CD73xEGFR sensitized cancer to the cytotoxic activity of various chemotherapeutic agents and potently inhibited the proliferative/migratory capacity (~40%) of cancer cells. Unexpectedly, we uncovered that treatment of carcinoma cells with oleclumab appeared to enhance several pro-oncogenic features, including upregulation and phosphorylation of EGFR, tumor cell proliferation (~20%), and resistance towards cytotoxic agents and ionizing radiation (~39%). Importantly, in a tumor model using immunocompetent BALB/c mice inoculated with syngeneic CD73pos/EGFRpos CT26 cancer cells, treatment with bsAb CD73xEGFR outperformed oleclumab (65% vs 31% tumor volume reduction). Compared with oleclumab, treatment with bsAb CD73xEGFR enhanced the intratumoral presence of CD8pos T cells and M1 macrophages.
Conclusions BsAb CD73xEGFR outperforms oleclumab as it inhibits the CD73/ADO immune checkpoint in an EGFR-directed manner and concurrently counteracts several oncogenic activities of EGFR and CD73. Therefore, bsAb CD73xEGFR may be of significant clinical potential for various forms of difficult-to-treat solid cancer types.
- immune checkpoint inhibitors
- tumor microenvironment
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Inhibition of the CD73 immune checkpoint has been hailed as a promising alternate or complementary approach in cancer immunotherapy. However, recent midterm clinical trial reports indicate that, as a single treatment modality, the efficacy of CD73 antagonistic antibody oleclumab remains modest at best.
WHAT THIS STUDY ADDS
A novel tetravalent bispecific antibody (bsAb) CD73xEGFR is presented that allows to inhibit the CD73/adenosine immune checkpoint on cancer cells in an EGFR-directed manner. Its mode-of-action involves the rapid co-internalization and prolonged concurrent displacement of CD73 and EGFR from the cancer cell surface. BsAb CD73xEGFR showed potent capacity to reinvigorate the anticancer activities of adenosine-suppressed cytotoxic T cells and concurrently counteracted cancer cell-surface CD73-mediated and EGFR-mediated pro-oncogenic activities. Of note, we uncovered that identical treatment of carcinoma cells with CD73-antagonistic antibody oleclumab appeared to enhance several pro-oncogenic features, including the upregulation and phosphorylation of EGFR, enhancement of tumor cell proliferation, and promotion of resistance towards chemotherapeutic agents and ionizing radiation.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
The uncovered negative attributes of CD73 antagonistic antibody oleclumab can have major ramifications for its future application in cancer treatment. Therefore, alternate approaches that inhibit the CD73 immune checkpoint in a more tumor-selective manner while counteracting the pro-oncogenic activity of EGFR and CD73 appear warranted. In this respect, the multiple and more tumor-selective anticancer activities of bsAb CD73xEGFR may be of particular promise.
Immunotherapy has significantly contributed to the therapeutic armamentarium currently available to patients with cancer with advanced disease. In particular, antagonistic antibodies towards immune checkpoints programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) have strongly improved therapeutic outcome, although only for a relatively small subgroup of these patients. Apparently, the majority of patients with cancer harbor malignant cells that exploit alternate and/or additional immunosuppressive checkpoints to achieve immune evasion. Consequently, there is an unmet need for novel inhibitors that can selectively block such alternate immune checkpoints. In this respect, antagonistic antibodies that can selectively inhibit the CD73 immune checkpoint, such as oleclumab, appear to be of clinical promise.1 2
CD73 is a cell surface-expressed enzyme that is key in maintaining immune system homeostasis by the stepwise hydrolysis of the autocrine and paracrine danger signals conveyed by extracellular ATP (eATP) into anti-inflammatory adenosine (ADO). Infection, tissue injury, ischemia, and metabolic stress are known to result in a sharp elevation of eATP release at the site of such lesion(s), where it serves to initiate pro-inflammatory immune responses by attracting and activating various types of immune cells. These pro-inflammatory responses are appropriately locally counterbalanced by the concerted action of cell surface-anchored ectonucleotides CD39 and CD73, which sequentially convert eATP via AMP to ADO. In this process, the enzyme activity of CD73 is the rate-limiting step in catalyzing the conversion of AMP to ADO. This catalysis results in a rapid local increase of ADO levels, which engages the immunosuppressive actions of ADO receptors on various, locally present immune cells, thereby providing a self-limiting mechanism for a timely and localized resolution of immune responses.1
Due to intrinsic high metabolic stress, numerous cancer cell types excrete remarkably high levels of pro-inflammatory eATP, which they rapidly convert into anti-inflammatory ADO by concurrently overexpressing CD73. Subsequently, diffusion of tumor-produced ADO molecules results in the formation of a potent immunosuppressive “halo” that acts not only locally in the tumor microenvironment, but also outside of the tumor site. Consequently, a halo of ADO molecules chronically suppresses the anticancer immune response, which promotes the induction of immune tolerance, immune escape, and subsequent cancer progression.3
Preclinical studies have demonstrated that CD73-inhibiting antibodies may be used to overcome this form of tumor-induced immunosuppression in a broad variety of cancer types, including various carcinomas. In particular, oleclumab (MEDI9447), a recombinant fully human antibody, selectively binds to CD73 and subsequently inhibits its enzyme activity.4 Currently, several multicenter trials are ongoing in patients with advanced solid malignancies to evaluate the clinical potential of oleclumab. However, the efficacy of a conventional monospecific CD73-inhibiting antibody like oleclumab is anticipated to be hampered by the “on-target/off-tumor” binding to a vast surplus of CD73 molecules present on normal cells and tissues,5 potentially precluding its sufficient accumulation at the tumor site(s). Moreover, CD73-inhibiting antibodies may induce a generalized (re-)activation of T cells, potentially leading to autoimmune-related adverse events, analogous to what is observed for other immune checkpoint inhibitors.
Intriguingly, we uncovered that in vitro treatment of carcinoma cells with oleclumab induced upregulation and phosphorylation of oncogenic proteins EGFR and HER2, which coincided with a marked enhancement of cancer cell proliferation and resistance towards cytotoxic regimes.
To address these issues, we constructed a novel tetravalent bispecific antibody (bsAb), designated “bsAb CD73xEGFR”, that was engineered to inhibit cancer cell surface-expressed CD73 in an EGFR-directed manner. Our results demonstrate that treatment with bsAb CD73xEGFR reduced the outgrowth of inoculated syngeneic tumors in immunocompetent mice, which coincided with a marked increase in tumor-infiltration by CD8pos effector T cells and activated macrophages. Moreover, bsAb CD73xEGFR sensitized cancer to the cytotoxic activity of various chemotherapeutic agents and that of ionizing radiation in vitro and potently inhibited the proliferative and migratory capacity of cancer cells in vivo.
Antibodies and reagents
Antibodies: Fluorescein isothiocyanate (FITC)-labeled-anti-CD73 (clone-MM07, Sino Biological), Allophycocyanin (APC)-labeled-anti-EGFR (clone-528, Santa Cruz), FITC-labeled-annexin-V (ImmunoTools), goat-anti-human-IgG-APC (SouthernBiotech), sheep-anti-human-CD73 (R&D), rabbit-anti-human-EGFR (Abcam), rabbit-anti-human-HER2 (R&D), rabbit-anti-human/mouse-β-Actin (Abcam), rabbit-anti-sheep-IgG (Thermo Fisher), goat-anti-rabbit-IgG (Dako), anti-CD4 (ab183685, Abcam), anti-CD8 (98941, Cell Signaling Technology), anti-FoxP3 (ab99964; Abcam), anti-F4/80 (ab240946, Abcam), anti-CD206 (ab64693, Abcam), anti-CD11c (ab52632; Abcam).
Reagents/proteins: VivoGlo (Promega), Carboxyfluorescein succinimidyl ester (CFSE), CFSE-Far Red (Thermo Fisher), Vybrant DiD, DiO, propidium iodide (PI), fluorescent caspase 3/8–488 probe (Biotium), adenosine 5'-(α,β-methylene)diphosphate (APCP, Sigma), anti-human Fc saporin-6 toxin-labeled Fab (Fab-ZAP, Advanced Targeting Systems), CypHer5E (Fisher Scientific), AMP (Sigma-Aldrich), T-cell activation/expansion beads (Miltenyi Biotec), recombinant soluble human-CD73 (s.hCD73) (Abcam), interferon (IFN)-γ-ELISA (eBioscience), colorimetric malachite green-based inorganic phosphate (Pi) (ab65622, Abcam).
Cell lines and transfectants
Cell lines CHO-K1, SK-BR-3, 4T1, FaDu, H292, OvCAR3, H322, PC3M, A375m, A2058, SK-MEL-28, MDA-MB-231, DLD1, CT26, and HEK293AD were obtained from the American Type Culture Collection (Manassas, Virginia, USA). A549, A549.EGFR-KO,6 H1650, and H1650 EGFR-KO were kindly provided by Professor Dr H J Haisma. H292-luc cancer cells were purchased from Cellomics Technology (SC-1087).
Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 or Dulbecco's Modified Eagle Medium (DMEM) (Lonza), supplemented with 10% fetal calf serum (Thermo Fisher) at 37°C in a humidified 5% CO2 atmosphere. CHO-K1 cells were cultured in GMEM (FirstLink), supplemented with 5% dialyzed fetal bovine serum (Sigma-Aldrich).
CHO.CD73 cells stably expressing human CD73 were generated by lipofection (FuGENE-HD, Promega) of a plasmid containing complementary DNA (cDNA) encoding human CD73 (OriGene). Likewise, CHO.EGFR cells were generated by lipofection of a plasmid containing cDNA encoding human EGFR (Sino Biological).
CD73-knockout (KO) cells were generated using CRISPR-Cas9 by transfection of pSpCas9 BB-2A-GFP (PX458) plasmid (Addgene plasmid #48138) containing CD73-targeting sgRNA 5’-GCAGCACGTTGGGTTCGGCG-3’.7 Likewise, EGFR-KO cells were generated by transfection of pSpCas9 BB-2A-GFP (PX458) plasmid containing EGFR-targeting sgRNA 5’-GAGTAACAAGCTCAGGCAGT-3’ (GenScript).
Construction of bsAb CD73xEGFR-IgG2 silent
DNA fragments encoding scFvCD73 and VHH-EGFR were generated by commercial gene synthesis service (GenScript) based on published VH and VL sequence data from oleclumab4 and EGFR-directed camelid single-domain antibody fragment NRC-sdAb028,8 respectively. For construction of bsAb CD73xEGFR-IgG2 silent and controls, we refer to the supplementary documentation.
Production of recombinant bsAbs
BsAbs were produced using the Expi293 Expression System (Thermo Fisher) and purified using ӒKTA-Start chromatography system as described previously by our group.9
Assessment dual binding activity of bsAb CD73xEGFR
Cancer cells were incubated at given concentration with bsAbs CD73xEGFR or controls at 4°C for 45 min. Subsequently, cells were re-incubated with APC-labeled secondary anti-human-Ig-antibody at 4°C for 45 min. Binding data of the respective bsAbs were acquired using Guava easyCyte 6/2L Flow Cytometer (Merck Millipore) and analyzed by GuavaSoft V.3.2 software.
Similar, competitive binding of bsAb CD73xEGFR (1 µg/mL) was assessed in the presence of either recombinant soluble human CD73 (s.CD73), EGFR-competing bsAb MockxEGFR, or a combination thereof (each 10 µg) at 4°C for 20 min and evaluated by flow cytometry essentially as described above.
Assessment of internalization of bsAb CD73xEGFR/antigen complexes
Cancer were incubated with increasing concentrations (0.01–10 µg/mL) bsAb CD73xEGFR (or appropriate controls) at 37°C for 24 hours, after which residual CD73-cell and EGFR-cell-surface exposure was assessed using anti-CD73 mAbMM07 and anti-EGFR mAb528, which bind to non-overlapping epitopes on CD73 and EGFR, respectively.
Cancer cells were incubated with bsAb CD73xEGFR‐pHAb (CypHer5E) or controls (1 µg/mL) in the presence (or absence) of EGFR‐competing bsAb MockxEGFR (10 µg/mL) at 37°C for 0 min, 10 min, 60 min, 240 min, or 360 min. CypHer5E is a non-toxic, pH-sensitive dye that is non-fluorescent at basic pH (extracellular: culture medium) and fluorescent at acidic pH (intracellular: endosomes, lysosomes). Internalization of pHAb‐labeled bsAbs by cancer cells was evaluated by flow-cytometry.
Cancer cells were incubated with bsAb CD73xEGFR or controls (1 µg/mL) in the presence of anti-human Fc saporin-6 toxin-labeled Fab (Fab-ZAP, Advanced Targeting Systems). Fab-ZAP is a goat anti-human IgG (H+L) monovalent Fab antibody fragment conjugated with the pro-apoptotic ribosome-inactivating protein saporin. The pro-apoptotic activity of the “Fab-ZAP” agent strictly depends on its internalization. Apoptotic cancer cell death was evaluated after 24 hours by flow-cytometry using annexin-V/PI staining.
Assessment capacity of bsAb CD73xEGFR to inhibit the enzyme activity of CD73
Pi formed during CD73-mediated hydrolysis of AMP to ADO was measured using a colorimetric malachite green-based Pi assay as described previously by our group.9 Additionally, we refer to the online supplemental files 1; 3.
Assessment capacity of bsAb CD73xEGFR to restore proliferation capacity of ADO-suppressed T cells
CFSE-labeled peripheral blood mononuclear cells (PBMCs) were activated by addition of T-cell activation/expansion beads in a bead-to-cell ratio of 1:1. Subsequently, PBMCs were cultured in medium supplemented (or not) with AMP (100 µM) and incubated with bsAb CD73xEGFR or controls (1 µg/mL) for 5 days. T-cell proliferation was analyzed by CFSE dilution using flow-cytometry.
Similarly, proliferation of CFSE-Far Red-labeled PBMCs evaluated using live cell imaging was performed as described previously by our group.9
Assessment capacity of bsAb CD73xEGFR to restore anticancer activity of ADO-suppressed T cells in vitro
PBMCs were cultured in a medium supplemented with AMP (100 µM) and incubated with increasing concentrations (0.01–10 µg/mL) bsAb CD73xEGFR or controls for 24 hours. Next, the T cells present were stimulated and re-directed to kill epithelial cell adhesion molecule (EpCAM)-expressing PC3M prostate cancer cells and evaluated using live cell imaging as described previously by our group.9
Assessment capacity of bsAb CD73xEGFR to restore anticancer activity of ADO-suppressed T cells in vivo
Syngeneic tumors were established by subcutaneous injection of 5×105 CT26 cells suspended in 0.1 mL of phosphate buffered saline (PBS) into the right flanks of 4–6 weeks old female BALB/c mice (Janvier, France). Animals were randomized using RandoMice into five groups of nine mice each based on body weight (total=45 animals. Sample size was determined using G*Power tool). BsAb CD73xEGFR and controls were administered by intraperitoneal (I.P.) injection at 7.5 mg/kg on days 4, 7, 10, and 14. Tumor volume was assessed by caliper measurements. Mice were euthanized using cardiac puncture, followed by cervical dislocation after 21 days. Tumor and organs and processed for multiplexed immunofluorescence or histology.
Multiplexed immunofluorescence to assess tumor-infiltrating leukocytes
Multiplexed immunofluorescence to evaluate T cell and macrophage populations in tumors resected from immunocompetent mice was performed as described previously.10 Additional information is provided in the online supplemental file 3.
Assessment inhibitory effect of bsAb CD73xEGFR on proliferation of cancer cells in vitro
Cancer cells were seeded into an E-plate 16 (ACEA Biosciences) and incubated with bsAb CD73xEGFR or controls (1 µg/mL) at 37°C for 60 hours. Cell proliferation was monitored using the xCELLigence RTCA instrument (ACEA Biosciences).
Cancer cells were seeded into a 96-well culture plate and incubated with bsAb CD73xEGFR or controls (1 µg/mL) at 37°C. Live cell imaging technology (IncuCyte) was used to follow proliferation of cancer cells by taking pictures at 4× magnification every 4 hours for 3 days. Confluence (%) was measured using IncuCyte software 2019B.
Assessment inhibitory effect of bsAb CD73xEGFR on proliferative capacity of cancer cells in vivo
Tumors were established by subcutaneous injection of 1×106 H292-Luc cells suspended in 0.1 mL of PBS into the right flanks of a 4−6 weeks old female athymic nude mouse (Crl:NU(NCr)-Foxn1nu, Charles River, Germany). Animals were randomized using RandoMice into five groups of nine mice each based on body weight (total=45 animals. Sample size was determined using G*Power tool). BsAb CD73xEGFR or controls were administered via I.P. injection at 7.5 mg/kg on days 7, 10 and 17. Bioluminescent imaging (IVIS Spectrum, 75 mg/kg VivoGlo; Promega) and caliper measurements were performed to evaluate tumor growth. Mice were euthanized using cardiac puncture, followed by cervical dislocation after 28 days.
Assessment inhibitory effect of bsAb CD73xEGFR on migratory capacity of cancer cells
Cancer cells were seeded in 24-well plates, equipped with a stopper, in the presence of bsAb CD73xEGFR or controls (1 µg/mL) at 37°C for 24 hours. Subsequently, the stopper was removed and Live Cell Imaging technology (IncuCyte) was used to assess the treatment effect on the migratory capacity of cancer cells by taking pictures at 4× magnification every 0.5 hours for 3 days. Closure of the standardized cell-free zone (%) was measured using IncuCyte software 2019B.
Assessment capacity of bsAb CD73xEGFR to sensitize cancer cells towards chemotherapeutic agents and radiation
Cancer cells were seeded into a 96-well culture plate and incubated with bsAb CD73xEGFR or controls (1 µg/mL) with (or without) 5FU (50 µg/mL), taxol (50 nM), cisplatin (1 µg/mL), or doxorubicin (50 nM) at 37°C. Live Cell Imaging technology (IncuCyte) was used to assess treatment effect on proliferative capacity on cancer cells by taking pictures at 4× magnification every 6 hours for 6 days. Confluence (%) was measured using IncuCyte software 2019B.
Cancer cells were seeded into a 6-well culture plate and incubated with increasing concentrations (0.01–10 µg/mL) of bsAb CD73xEGFR or controls at 37°C for 14 days. Subsequently, cell colonies were washed with PBS and then stained with crystal violet. Colony number and size were analyzed using ImageJ software.
Cancer cells were seeded into a 6-well culture plate, incubated with bsAb CD73xEGFR or controls (1 µg/mL), irradiated with an increasing dose of radiation (0.5–2 Gy), and then cultured at 37°C for 14 days. Subsequently, cell colonies were washed, stained, and analyzed as described above. Irradiation (0.59 Gy/min) was performed using a 137Ce source (IBL637 Cesium-137 γ-ray machine).
Statement animal experiments
All procedures using animals were in accordance with Dutch Act on Animal Experimentation and approved by the Institutional Animal Welfare Committee at the Rijksuniversiteit Groningen/University Medical Center Groningen (AVD1050020209544). EMP was aware of group allocation at the different stages of the experiment (during allocation, conduct of the experiment, outcome assessment, and data analysis). Additionally, we did not adjust for confounding since randomization was successful.
Statistical analysis was done by (multiple) t-test, one-way analysis of variance followed by Tukey post hoc test, or (non-)linear regression, as indicated using Prism software. Data was pooled (n-number and technical repeats are specified in the figure legends) for analysis. P value<0.05 was defined as a statistically significant difference. Where indicated; ns=p>0.05; *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.
bsAb CD73xEGFR has dual binding specificity for CD73 and EGFR
BsAb CD73xEGFR was constructed in the bispecific taFv-Fc format11 equipped with two identical CD73-directed scFv antibody fragments derived from oleclumab and two identical EGFR-directed camelid single-domain antibody fragments8 (online supplemental files 1; 3). Dual binding activity of bsAb CD73xEGFR was confirmed using Chinese hamster ovary (CHO) cells transfected with either human CD73 or human EGFR. BsAb CD73xEGFR bound dose-dependently to both CHO.CD73 and CHO.EGFR cells, and not to parental CHO cells (figure 1A). Binding of bsAb CD73xEGFR towards SK-BR-3 breast cancer cells was partly reduced in the presence of soluble human CD73 (s.CD73), whereas antagonistic EGFR bsAb MockxEGFR strongly inhibited the binding. Of note, binding of bsAb CD73xEGFR was fully abrogated in combined presence of s.CD73 and bsAb MockxEGFR (figure 1B), indicating bsAb CD73xEGFR selectively and simultaneously binds to CD73 and EGFR. Importantly, binding levels of bsAb CD73xEGFR towards a panel of four CD73pos/EGFRpos cell lines closely correlated with their respective expression levels for EGFR (figure 1C, online supplemental file 3). Moreover, bsAb CD73xEGFR showed reduced binding activity towards EGFR-KO/CD73pos cell lines (figure 1C). Additionally, bsAb CD73xEGFR showed capacity to simultaneously bind to CD73 present on one cell type and EGFR present on another cell type when both cell types are in close proximity. In particular, bsAb CD73xEGFR cellularly bridged FaDu cancer cells (DiO-labeled) and CHO.CD73 cells (DiD-labeled), as was evident from a marked increase in DiOpos/DiDpos cell clusters detected by flow cytometry (online supplemental file 3).
bsAb CD73xEGFR induces rapid co-internalization and subsequent prolonged displacement of both CD73 and EGFR from the cancer cell surface
Treatment of CD73pos/EGFRpos H292 cancer cells with bsAb CD73xEGFR resulted in a concurrent and dose-dependent cancer cell surface displacement of CD73 and EGFR. At a concentration of 1 µg/mL, treatment with bsAb CD73xEGFR reduced cell surface exposure of CD73 and EGFR by 81% and 73%, respectively (figure 2A,B, online supplemental file 3), which persisted for up to 96 hours (figure 2C,D). Comparable prolonged displacement of CD73 and EGFR was detected when primary patient-derived ovarian and colon cancer cells were treated with bsAb CD73xEGFR (figure 2E). Internalization of bsAb CD73xEGFR/antigen complexes occurred after 10–60 min, which was abolished in presence of saturating amounts of EGFR-competing bsAb MockxEGFR (figure 2F). Likewise, “piggybacking” of Fab-ZAP toxin on internalizing bsAb CD73xEGFR resulted in saporin-mediated apoptotic cell death (figure 2G). Analogously, treatment of CD73pos/EGFRpos cancer cells with bsAb CD73xEGFR significantly reduced CD73 expression levels and attenuated EGFR tyrosine phosphorylation (online supplemental file 3).
bsAb CD73xEGFR internalizes cancer cell surface-exposed CD73 in an EGFR-directed manner
BsAb CD73xEGFR showed a marked capacity to displace CD73 from CD73pos/EGFRpos cancer cells, whereas only minimal CD73 displacement was observed when treating CD73pos/EGFRneg cancer cells (online supplemental file 3). Analogously, bsAb CD73xEGFR essentially failed to internalize CD73 when treating A549 EGFR-KO and H1650 EGFR-KO cells (online supplemental file 3). Importantly, treatment of a series of 10 individual CD73pos/EGFRpos cancer cell line types with bsAb CD73xEGFR demonstrated a positive linear dependance (R2=0.89) between residual CD73 and EGFR cell surface-expression levels, whereas identical treatment with oleclumab failed to do so (R2=0.0003) (figure 2H). Again, this indicated that on treatment with bsAb CD73xEGFR, cancer cell surface-expressed CD73 and EGFR are subject to antibody-mediated co-internalization.
bsAb CD73xEGFR potently inhibits the CD73 enzyme activity in an EGFR-directed manner
Treatment of various carcinomas with bsAb CD73xEGFR dose-dependently inhibited the CD73 enzyme activity (figure 3A, online supplemental file 3) and in this respect significantly outperformed oleclumab in three out of three cancer cell lines (group average; 71% vs 52%, respectively) (figure 3B) and in eight out of nine primary patient-derived carcinoma cells (group average; 55% vs 40%, respectively) (figure 3C). The respective IC50 values of bsAb CD73xEGFR calculated for six cell lines ranged from 0.001 to 0.038 µg/mL, whereas those of oleclumab ranged from 0.005 to 0.563 µg/mL (online supplemental file 3). Additionally, inhibition of CD73 enzyme activity of H929 cancer cells by bsAb CD73xEGFR was dose-dependently decreased in presence of the EGFR-competing bsAb MockxEGFR (figure 3D). The CD73 enzyme activity of A549 EGFR-KO cancer cells was only marginally reduced on treatment with bsAb CD73xEGFR (figure 3E).
bsAb CD73xEGFR overcomes ADO-mediated suppression of T-cell proliferation
When activated CFSE-labeled T cells were subjected to AMP, which is locally enzymatically converted to ADO by CD73, their proliferation capacity was significantly repressed. Importantly, treatment with bsAb CD73xEGFR or oleclumab fully abrogated the ADO-mediated inhibition of T-cell proliferation, as can be appreciated from the successive dilution of the CFSE-dye (figure 4A). These results corroborated the increase in activated T-cell cluster size (figure 4B).
bsAb CD73xEGFR restores anticancer activity of ADO-suppressed cytotoxic T cells
When cytotoxic T cells were subjected to AMP and subsequently redirected to kill EpCAM-expressing cancer cells using EpCAM-directed/CD3-agonistic bsAb BIS-1, induction of cancer cell death, evident from high caspase-3/8 activation levels in target cells, significantly dropped (figure 4C,D, online supplemental file 3). Importantly, treatment with bsAb CD73xEGFR dose-dependently restored the capacity of these ADO-suppressed T cells to eliminate cancer cells (figure 4E). These results corroborate ELISA data quantifying the restored capacity of cytotoxic T cells to secrete IFN-γ (figure 4F).
bsAb CD73xEGFR increases the tumor-infiltrating capacity of leukocytes in immunocompetent mice
Subsequently, the apparent effect of in vitro treatment with bsAb CD73xEGFR on the anticancer activities of cytotoxic T cells was evaluated in vivo using BALB/c mice inoculated with CT26 cancer cells (in vitro data CT26; online supplemental file 3). At 4, 7, 10, and 14 days after inoculation, animals were treated I.P. with 7.5 mg/kg bsAb CD73xEGFR, oleclumab, or controls (figure 5A). In vivo treatment with bsAb CD73xEGFR significantly reduced tumor volume (65%) and tumor wet-weight (77%), thereby markedly outperforming oleclumab (31% reduction in tumor volume) (figure 5B,C, online supplemental file 3). Importantly, tumors procured from animals treated with bsAb CD73xEGFR showed increased infiltration of CD4pos and CD8pos lymphocytes and a decreased infiltration of FoxP3pos lymphocytes (figure 5D–G, online supplemental file 3). Moreover, in these tumors an increased infiltration of F4/80pos (total macrophage) and CD11cpos (M1 macrophage) cells, and a decreased infiltration of CD206pos (M2 macrophage) cells was observed (figure 5H–K, online supplemental file 3). In contrast, tumors procured from animals treated with oleclumab only demonstrated an increased infiltration with CD8pos lymphocytes and a decreased infiltration of total and M2 macrophages. Importantly, animals treated with bsAb CD73xEGFR appeared to show no signs of systemic toxicity in vital organs (online supplemental file 3).
bsAb CD73xEGFR inhibits proliferative and migratory capacity of cancer cells in vitro
Both enzymatic and non-enzymatic attributes of CD73 overexpression were reported to be implicated in enhancement of cancer cell proliferative/migratory capacity and resistance to chemo/radiotherapy.12 In this respect, it is noteworthy that treatment with bsAb CD73xEGFR decreased the proliferative capacity of carcinoma cell line types (four out of four) by ~40% (figure 6A,B, online supplemental file 3). Additionally, treatment with bsAb CD73xEGFR significantly reduced the colony forming capacity of cancer cells both in number and size (figure 6C–E). In this respect, IC50 values calculated for bsAb CD73xEGFR ranged from 0.16 to 0.65 µg/mL, which was superior compared with oleclumab (IC50 9.38–24.33 µg/mL) in three out of three cell lines tested (online supplemental file 3). Representative light-microscopic pictures shown in figure 6F indicate that treatment of cancer cells with bsAb CD73xEGFR almost fully abrogated their migratory capacity. These results corroborate the observed delayed closure of the cell-free zone over time on treatment with bsAb CD73xEGFR (figure 6G).
Unexpectedly, in vitro treatment of various carcinoma cell line types with oleclumab (or small-molecule CD73-inhibitor APCP) appeared to rather promote than inhibit cancer cell growth (figure 6B). Moreover, treatment with oleclumab enhanced the migration capacity of cancer cells by ~20% (figure 6G). These results may be explained by the fact that in vitro treatment with oleclumab enhanced (both total and phosphorylated) EGFR and HER2 levels in these cancer cells (online supplemental file 3). Of note, oleclumab-induced upregulation of cell-surface expressed EGFR is also apparent in figure 2H in 8 out of 10 cell lines evaluated.
bsAb CD73xEGFR inhibits the proliferative capacity of xenografted cancer cells in immunodeficient mice
Subsequently, the apparent opposing effect of in vitro treatment with bsAb CD73xEGFR versus oleclumab treatment on the proliferative capacity of cancer cells was evaluated in vivo using athymic nude mice inoculated with H292-Luc2 cells. At 7, 10, and 17 days after inoculation, animals were treated I.P. with 7.5 mg/mL bsAb CD73xEGFR, oleclumab, or controls (figure 6H). Compared with treatment with isotype control, in vivo treatment with bsAb CD73xEGFR inhibited tumor growth by 56% (figure 6I,J, online supplemental file 3). In comparison, the volume of tumors that developed in animals treated with oleclumab or bsAb CD73xMock increased by 24% and 40%, respectively.
bsAb CD73xEGFR sensitizes cancer cells towards treatment with chemotherapeutic agents and ionizing radiation
In vitro co-treatment with bsAb CD73xEGFR sensitized cancer cells towards the cytotoxic activity of cisplatin, doxorubicin, taxol, and 5FU by ~1-fold, 0.45-fold, 0.74-fold, and 0.4-fold, respectively (figure 7A,B, online supplemental file 3). In contrast, treatment with oleclumab enhanced resistance of cancer cells towards cisplatin, doxorubicin, and 5FU by ~2-fold, 1.8-fold, and 1.44-fold, respectively.
Similarly, co-treatment with bsAb CD73xEGFR enhanced sensitivity of cancer cells towards cytotoxicity induced by ionizing radiation (figure 7C). In this respect, IC50 values calculated for bsAb CD73xEGFR treatment decreased from 1.35 Gy to 0.536 Gy (figure 7E). In contrast, identical treatment with oleclumab enhanced resistance towards ionizing radiation up to 39% at 2 Gy (figure 7C), whereas calculated IC50 values increased from 1.35 Gy to 3.77 Gy (figure 7E). Importantly, treatment of CD73-KO cancer cells with oleclumab did not change their resistance towards ionizing radiation (figure 7D).
Antagonistic CD73 antibodies like oleclumab4 appear useful to overcome the inhibitory activity of the CD73/ADO immune checkpoint in a broad variety of cancer types. Currently, several multicenter trials are ongoing to evaluate the clinical potential of oleclumab as an immune checkpoint inhibitor in patients with advanced solid malignancies, including various carcinomas. Unfortunately, recent midterm clinical trial reports indicate that, as a single treatment modality, the efficacy of oleclumab remains modest at best.13–16 Possibly, monospecific antagonistic CD73 antibodies like oleclumab suffer from “on-target/off-tumor” binding to CD73 molecules present on normal cells,5 which limits CD73-inhibitory activity at the tumor site(s).
Here, we report on the construction and preclinical evaluation of bsAb CD73xEGFR that was engineered to inhibit cancer cell surface-expressed CD73 in an EGFR-directed manner. We selected EGFR for this purpose as it is a well-established tumor-associated cell surface target antigen that is frequently mutated and/or overexpressed by various difficult-to-treat solid malignancies.17 18 Moreover, many malignancies were shown to selectively co-overexpress CD73 and EGFR.19–21 BsAb CD73xEGFR is equipped with two identical scFv antibody fragments derived from oleclumab and two identical EGFR-directed nanobodies. Of note, these antibody domains bind to their respective human—and mouse orthologues with similar affinities. This strategy allows for preclinical evaluation of bsAb CD73xEGFR in both human and murine tumor model systems. To exclude antibody-dependent cellular cytotoxicity (ADCC)-mediated antitumor effects during such evaluation, we equipped bsAb CD73xEGFR with an IgG2-silent Fc domain with nullified effector function.22
Importantly, compared with oleclumab, bsAb CD73xEGFR showed superior antagonistic activity towards cancer cells that co-overexpress CD73 and EGFR. This superiority is most likely attributable to the enhanced avidity associated with the tetravalent format of bsAb CD73xEGFR. Previously, we reported on similar attributes for tetravalent bsAbs that were designed to block immune checkpoints PD-L123 24 and CD4725 26 in a more tumor-selective manner. More recently, we reported on tetravalent bsAb CD73xEpCAM, which allowed to potently inhibit CD73 exposed on carcinoma-derived exosomes, whereas oleclumab showed no or very limited capacity to do so.9
Intriguingly, in vitro treatment of CD73pos/EGFRpos cancer cells, both various cell lines and primary patient-derived cancer cell types, with bsAb CD73xEGFR induced the rapid internalization of bsAb/antigen complexes, resulting in the prolonged and concurrent displacement of CD73 and EGFR from the cancer cell surface for up to 96 hours. Of note, the internalization of oleclumab (labeled with CypHer5E) is higher than that of bsAb CD73xMock. This may be explained by the fact that CypHer5E NHS-Ester-based labeling may be more efficient for a conventional antibody like oleclumab than for the bispecific taFv-Fc format of our bsAbs. Although not investigated here, such difference may result in higher dye-to-antibody ratio for oleclumab.
The bsAb CD73xEGFR-induced internalization of cancer cell surface-exposed CD73 resulted in a similarly prolonged incapacity of these cancer cells to convert extracellular AMP to ADO. Importantly, in vitro treatment of ADO-suppressed cytotoxic T cells with bsAb CD73xEGFR potently reinvigorated their anticancer activities. Moreover, compared with controls, treatment with bsAb CD73xEGFR of immunocompetent BALB/c mice (7.5 mg/kg) inoculated with syngeneic CT26 colorectal carcinoma cells, resulted in an average reduction of 65% in tumor size development, whereas identical treatment with oleclumab reduced tumor size by only 31%. Importantly, the in vivo application of bsAb CD73xEGFR increased the presence of tumor-infiltrating CD4pos T cells,—CD8pos T cells, and—macrophages by 38%, 52%, and 82%, respectively. These results appear favorable compared with those reported by Hay et al4 using essentially the same mouse tumor model in which treatment with oleclumab (at 10 mg/kg) resulted in a reduction in tumor size of 57% (at 16 days) and an increase in tumor-infiltrating CD4pos T cells,—CD8pos T cells, and—macrophages by only 15%, 25%, and 20%, respectively. Of note, our in vitro data confirms that bsAb CD73xEGFR has potent capacity to inhibit the enzyme activity of mCD73 on CT26 cells. However, treatment of CT26 cancer cells with bsAb CD73xEGFR does not induce co-internalization of mCD73 and mEGFR. The reason for this difference between human and murine cancer cells remains to be determined.
Several reports indicated that overexpression of CD73 is implicated in the enhancement of various oncogenic attributes of cancer cells, including increased cell proliferation and migration capacity.12 To evaluate whether bsAb CD73xEGFR has capacity to counteract the oncogenic attributes of CD73 overexpression, we treated immunodeficient mice inoculated with EGFR-overexpressing H292-luc tumor cells with bsAb CD73xEGFR and assessed its treatment effect on tumor outgrowth. Indeed, treatment with bsAb CD73xEGFR potently reduced the size of xenografted tumors. In contrast, and rather unexpectedly, identical treatment with oleclumab (or bsAb CD73xMock) appeared to enhance the growth capacity of xenografted H292-luc carcinoma cells. Intriguingly, in vitro treatment of H292-luc cells with oleclumab also resulted in enhancement of tumor growth, which coincided with enhanced expression—and phosphorylation levels of oncogenic proteins EGFR and HER2 in these cancer cells. In this respect, our observations appear to contradict with those mentioned in some previous reports.12 27 28 Apparently, apart from its ADO-producing activity, cancer cell-exposed CD73 appears to serve as a signaling transduction molecule that can deliver opposing intracellular signals. Because CD73 is linked to the cell surface by a glycosylphosphatidylinositol (GPI) anchor, the transduction of such signals is probably indirectly mediated by a lateral interaction with juxtaposed transmembrane molecules.29 Depending on the particular membrane context, CD73 signaling may thus be coupled to either their tyrosine kinase or phosphatase activities (or possibly both). Similarly, it has been previously reported that CD73 present on CD8pos T cells can act as a co-stimulatory signal by laterally interacting with receptor-linked protein tyrosine phosphatase CD45RC.30 We speculate that cancer cell-expressed CD73 may have analogous capacity to modulate oncogenic activities of EGFR and HER2 in a direct or indirect manner. The underlying mechanism by which oleclumab treatment promoted tumor growth and enhanced expression and phosphorylation levels of EGFR and HER2 in our model systems remains to be elucidated by follow-up studies.
Previously, it was reported that patients with cancer treated with chemotherapeutic agents, such as carboplatin, gemcitabine, and paclitaxel, show enhanced CD73 expression levels on cancer cells,31 which correlate with adopting a multidrug-resistance phenotype.32 33 To evaluate whether bsAb CD73xEGFR has capacity to counteract multidrug-resistance, we treated cancer cells in vitro with bsAb CD73xEGFR and assessed its treatment effect on the sensitivity of these cells towards cytotoxicity induced by various chemotherapeutic agents. In this respect, it is encouraging that bsAb CD73xEGFR sensitized cancer cells towards the cytotoxicity of cisplatin, doxorubicin, taxol and 5FU, respectively. Surprisingly, combined treatment of cancer cells with bsAb CD73xMock and bsAb MockxEGFR or oleclumab and cetuximab did not have similar effects as treatment with bsAb CD73xEGFR alone. This suggests that bsAb CD73xEGFR sensitizes cancer cells towards the cytotoxicity of chemotherapeutic agents through the concurrent removal of (juxtaposed) CD73 and EGFR molecules from the cell surface.
Analogously, in vitro treatment with bsAb CD73xEGFR enhanced the sensitivity of H292 non-small-lung cancer cells towards radiation-induced cytotoxicity up to ~40%. In contrast, identical treatment with oleclumab reduced sensitivity of these cancer cells towards ionizing radiation and increased the IC50 value from 1.35 Gy to 3.77 Gy. The latter observation appears to corroborate with those of Dietrich et al34 who reported that inhibition of the enzyme activity of CD73 by small-molecule CD73-inhibitor APCP promoted the proliferative capacity of irradiated cancer cells, thereby enhancing their radiation-resistance, due to the absence of (locally) accumulated ADO. This suggests that CD73-produced ADO potentiates radiation-induced cell death in certain cancer types. Of note, in our study, treatment of H292 cancer cells with bsAb CD73xMock did not increase their resistance to radiation. This may be due to the fact that, compared with oleclumab, bsAb CD73xMock has a somewhat lower capacity to inhibit the enzyme activity of CD73. Whether the observed oleclumab-induced enhancement to radiation also applies to other cancer cell lines/types remains to be evaluated in follow-up studies.
In conclusion, bsAb CD73xEGFR appears to be a promising approach to overcome the inhibitory activity of the CD73/ADO immune checkpoint in a broad variety of cancer types. In particular, it allows to inhibit the CD73 enzyme activity in a more tumor-selective manner, while simultaneously counteracting pro-oncogenic activities of CD73 and EGFR. Therefore, bsAb CD73xEGFR may be of significant clinical potential for various forms of difficult-to-treat solid cancer types.
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
Patient consent for publication
Contributors Conceptualization: EMP and WH. Methodology: EMP, DFS, HZ, and WH. Investigation: EMP, DFS, XK, XX, IB, APvW, and MAJMH. Visualization: EMP. Funding acquisition: WH. Writing (original text): EMP and WH. Writing (review and editing): All authors. Guarantor: WH.
Funding This work was supported by the Dutch Cancer Society (project numbers 11464 and 6986, to WH).
Competing interests No, there are no competing interests.
Provenance and peer review Not commissioned; externally peer reviewed.
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