Edited by: Asis Palazon, CIC bioGUNE, Spain
Reviewed by: Alvaro Teijeira, University of Navarra, Spain; Michael Croft, La Jolla Institute for Immunology (LJI), United States
*Correspondence: Luis Alvarez-Vallina,
This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology
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Agonistic monoclonal antibodies (mAbs) targeting the co-stimulatory receptor 4-1BB are among the most effective immunotherapeutic agents across pre-clinical cancer models. However, clinical development of full-length 4-1BB agonistic mAbs, has been hampered by dose-limiting liver toxicity. We have previously developed an EGFR-targeted 4-1BB-agonistic trimerbody (1D8N/CEGa1) that induces potent anti-tumor immunity without systemic toxicity, in immunocompetent mice bearing murine colorectal carcinoma cells expressing human EGFR. Here, we study the impact of human EGFR expression on mouse liver in the toxicity profile of 1D8N/CEGa1. Systemic administration of IgG-based anti-4-1BB agonist resulted in nonspecific immune stimulation and hepatotoxicity in a liver-specific human EGFR-transgenic immunocompetent mouse, whereas in 1D8N/CEGa1-treated mice no such immune-related adverse effects were observed. Collectively, these data support the role of FcγR interactions in the major off-tumor toxicities associated with IgG-based 4-1BB agonists and further validate the safety profile of EGFR-targeted Fc-less 4-1BB-agonistic trimerbodies in systemic cancer immunotherapy protocols.
The success of immune checkpoint blockade using PD-1/PD-L1 and/or CTLA-4 inhibitors has validated the concept of immunomodulating monoclonal antibodies (mAbs) as a powerful therapeutic strategy, but responses are still limited to a minor fraction of cancer patients (
However, off-tumor toxicities have been the major impediment to the clinical development of first-generation IgG-based 4-1BB agonistic mAbs. The fully human IgG4 urelumab caused dose-dependent liver toxicity, including two fatalities (
We have recently described a novel EGFR-targeted Fc-less 4-1BB agonistic trimerbody (1D8N/CEGa1), which is a potent costimulator
C57BL/6 wild-type (WT) female mice and transgenic Alb-Δ654–1186huEGFR (ΔEGFR-tg) (
HEK293 (CRL-1573) cells were obtained from the American Type Culture Collection and mouse CT26 cells (CRL-2638) expressing human EGFR (CT26huEGFR) or infected with the empty vector retrovirus (CT26mock) were provided by Dr M. Rescigno (European Institute of Oncology, Milan) (
Hepatocytes were isolated as previously described following the two-step collagenase perfusion technique followed by isodensity purification in a Percoll gradient (
The 1D8N/CEGa1 trimerbody was produced in stably transfected HEK293 cells (
Purified mouse 4-1BB:hFc chimera (mo4-1BB), mouse EGFR:hFc (moEGFR) and human EGFR:hFc chimera (huEGFR) (all from R&D Systems) were immobilized at 3 µg/ml on Maxisorp ELISA plates (NUNC Brand Products) overnight at 4 °C. After washing and blocking with 200 µl PBS 5% BSA (Merck Life Science), 100 µl of purified 3H3 IgG or 1D8N/CEGa1 trimerbody were added and incubated for 1 hour at room temperature. The wells were washed for three times with PBS 0.05% Tween-20, and 100 µl of anti-FLAG mAb (clone M2; mIgG1; cat#F1804, Merck Life Science) were added for 1 hour incubation at room temperature. The plate was washed as above and 100 µl of HRP-conjugated goat anti-rat IgG or HRP-conjugated goat anti-mouse IgG (both from Merck Life Science) were added to wells previously incubated with 3H3 IgG or 1D8N/CEGa1 trimerbody, respectively. Afterwards, the plate was washed and developed using OPD (Merck Life Science).
All biolayer interferometry was performed on an Octet RED96 (Fortebio). To investigate the binding of 1D8N/CEGa1 to hu-EGFR or moEGFR, 30 nM of huEGFR or moEGFR in fusion with a human Fc region were immobilized onto AHC biosensors (Fortebio) coated with anti-human Fc antibodies for 20 min, in 20 mM HEPES, 150 mM NaCl pH 7.4 buffer (HBS). Then, biosensors were moved into 20 nM 1D8N/CEGa1 in HBS and association was measured for 20 min followed by one hour of dissociation in HBS. To investigate the binding of hu-EGFR or moEGFR in solution to immobilized 1D8N/CEGa1, biosensors coated with mo4-1BB in fusion with a human Fc region were prepared using amine reactive chemistry. Briefly, AR2G biosensors (Fortebio) were activated with s-NHS/EDC, coated with 2 µg mouse 4-1BB per biosensor at pH 6 for 20 min, and quenched with ethanolamine. Then, 10 nM of 1D8N/CEGa1 in HBS was immobilized onto the biosensors for 30 min. Human or moEGFR (50 nM in HBS) was then introduced and allowed to associate for 20 min and dissociate for one hour. In both experiments, a reference biosensor coated and immobilized with the same ligands, but not receiving the experimental analyte proteins, was subtracted from the other sensorgrams prior to data analysis. Data were fit to 1:1 binding models using the Octet Data Analysis software (Fortebio). In the case of moEGFR’s binding to immobilized 1D8N/CEGa1, fitting included only its initial association phase, due to its biphasic binding.
The cell surface expression of EGFR was analyzed on freshly-isolated liver cells from C57BL/6 WT and EGFR-tg mice, and on CT26mock and CT26huEGFR cells after incubation for 30 min with the human EGFR-specific chimeric mouse/human IgG1 cetuximab (Merck KGaA), or the purified 1D8N/CEGa1 trimerbody. After washing, cells were treated with appropriate dilutions of phycoerytrin (PE)-conjugated goat anti-human IgG F(ab′)2 (Fc specific; cat#109-116-097, Jackson Immuno Research), or anti-FLAG mAb (clone M2), and then with PE-conjugated goat anti-mouse IgG F(ab’)2 antibody (cat#115-116-072, Jackson Immuno Research). Samples were analyzed with a MACSQuant Analyzer 10 flow cytometer (MiltenyiBioteh). A minimum of 20,000 events were acquired for each sample and data were evaluated using FCS Express V3 software (De Novo Software).
Eight weeks old C57BL/6 wild-type and ΔEGFR-tg littermates received a weekly i.p. dose of 3H3 IgG or 1D8N/CEGa1 (6 mg/kg) for 3 weeks. Mice were anesthetized and bled on days 0, 7, 14, and 21. To obtain mouse serum, blood was incubated in BD microtainer SST tubes (BD Biosciences), followed by centrifugation. Serum was stored at −20 °C until use. Serum levels of alanine aminotransferase (ALT) were determined at day 14 using Reflotron GPT/ALT strips and the Reflotron plus analyzer (Roche Diagnostics). One week after the last dose of antibodies, mice were euthanized and the liver and spleens, were surgically removed, weighted, and fixed in 10% paraformaldehyde for 48 h. Then fixed tissues were washed and embedded in paraffin. Tissue sections (5 µm) were stained with hematoxylin and eosin. Lymphocyte infiltration in the liver was quantified using the ImageJ software.
Tissue samples were fixed in 10% neutral buffered formalin (4% formaldehyde in solution), paraffin-embedded and cut at 3 μm, mounted in superfrost® plus slides and dried overnight. For different staining methods, slides were deparaffinized in xylene and re-hydrated through a series of graded ethanol until water. Consecutive sections for several immunohistochemistry reactions were perform in an automated immunostaining platform (Ventana Discovery XT, Roche; AS Link, Dako, Agilent). Antigen retrieval was first performed with the appropriate pH buffer, (CC1m, Ventana, Roche; Low pH buffer, Dako, Agilent) and endogenous peroxidase was blocked (peroxide hydrogen at 3%). Then, slides were incubated with the appropriate primary antibody as detailed: rabbit monoclonal anti-EGFR (mouse preferred) (D1P9C, 1/600, Cell Signaling, #71655) and mouse monoclonal anti-huEGFR (EGFR.113, 1/10, Leica, NCL-EGFR). After the primary antibody, slides were incubated with the corresponding visualization systems (OmniMap anti-Rabbit, Ventana, Roche; EnVisionFLEX+Mouse Linker, Dako, Agilent) conjugated with horseradish peroxidase. Immunohistochemical reaction was developed using 3, 30-diaminobenzidine tetrahydrochloride (ChromoMap DAB, Ventana, Roche; FLEX DAB, Dako, Agilent) and nuclei were counterstained with Carazzi’s hematoxylin. Finally, the slides were dehydrated, cleared and mounted with a permanent mounting medium for microscopic evaluation. Positive control sections known to be primary antibody positive were included for each staining run. Whole slides were acquired with a slide scanner (AxioScan Z1, Zeiss).
Statistical analysis was performed using GraphPad Prism Software version 6.0. Data is presented as mean ± SD. Significant differences (
The EGa1 is a well characterized EGFR-specific VHH that was generated from a phage-displayed llama VHH library after immunizing and screening with EGFR-positive human cells (
Biolayer interferometry investigating the binding of 1D8N/CEGa1 to human and mouse EGFR.
Transgenic Alb-Δ654–1186EGFR mice (from now abbreviated as ΔEGFR-tg) are immunocompetent animals expressing an hepatocyte-specific truncated form of the human EGFR that lacks the intracellular catalytic domain (amino acids 654–1186) (
Analysis of human EGFR expression by IHC in liver sections from
We compared the toxicity profile of the 1D8N/CEGa1 trimerbody with that of the well-characterized anti-4-1BB agonistic 3H3 IgG (
Treatment with 1D8N/CEGa1 does not induce toxicity.
In summary, we demonstrated that treatment of ΔEGFR-tg mice with the strong 4-1BB-agonistic 3H3 IgG induced a toxicity profile similar to that observed in WT C57BL/6 mice, with significant immune cell infiltration and systemic inflammation, indicating the suitability of the model to study 4-1BB–related toxicity. In contrast, none of these features were observed in ΔEGFR-tg mice treated with the 1D8N/CEGa1 trimerbody, despite the expression of both huEGFR and moEGFR on the hepatocyte surface, which excludes that the lower affinity of 1D8N/CEGa1 for moEGFR may be responsible for the absence of liver toxicity observed in WT mice. These results further support the role of FcγR interactions in the 4-1BB-agonist-associated immunological abnormalities and organ toxicities (
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The animal study was reviewed and approved by the Animal Experimentation Ethics Committee of the Instituto de Investigaciones Biomédicas Alberto Sols, and the Animal Welfare Division of the Environmental Affairs Council of the Government of Madrid (66/14, 118/19).
MC, JMZ, and LA-V designed and supervised the study. MC, SLH, JM-T, GP-C, PG-G, and AT-G performed the core experiments. MC, GP-C, and JMZ were responsible for the animal experiments. PG-G and JM-T performed IHC analysis. MC, SLH, JM-T, PMPVBEH, AS, IF, LS, JMZ, and LA-V provided scientific feedback, discussed the data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.
This study was supported by grants from the European Union [IACT Project (602262)], the Spanish Ministry of Science and Innovation; the Spanish Ministry of Economy and Competitiveness (SAF2017-89437-P, PID2019-110405RB-100, RTC-2016-5118-1, RTC-2017-5944-1), partially supported by the European Regional Development Fund; the Carlos III Health Institute (PI16/00357), co-founded by the Plan Nacional de Investigación and the European Union; the CRIS Cancer Foundation (FCRIS-IFI-2018), and the Spanish Association Against Cancer (AECC, 19084).
MC is an employee of Leadartis. LA-V and LS are co-founders of Leadartis.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank M. Rescigno for providing the reagents.
The Supplementary Material for this article can be found online at: