An IL-2-grafted antibody immunotherapy with potent efficacy against metastatic cancer

Modified interleukin-2 (IL-2) formulations are being tested in cancer patients. However, IL-2 immunotherapy damages IL-2 receptor (IL-2R)-positive endothelial cells and stimulates IL-2Rα (CD25)-expressing lymphocytes that curtail anti-tumor responses. A first generation of IL-2Rβ (CD122)-biased IL-2s addressed some of these drawbacks. Here, we present a second-generation CD122-biased IL-2, developed by splitting and permanently grafting unmutated human IL-2 (hIL-2) to its antigen-binding groove on the anti-hIL-2 monoclonal antibody NARA1, thereby generating NARA1leukin. In comparison to hIL-2/NARA1 complexes, NARA1leukin shows a longer in vivo half-life, completely avoids association with CD25, and more potently stimulates CD8+ T and natural killer cells. These effects result in strong anti-tumor responses in various pre-clinical cancer models, whereby NARA1leukin consistently surpasses the efficacy of hIL-2/NARA1 complexes in controlling metastatic disease. Collectively, NARA1leukin is a CD122-biased single-molecule construct based on unmutated hIL-2 with potent efficacy against advanced malignancies.


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Sample size was calculated with R studio using data from pilot or previous experiments with interleukin-2 complexes (Arenas-Ramirez, 2016, Sci Transl. Med.). As measured parameters are continuous values, power.anova.test was used with an alpha error of 5% (0.05) and a beta error of 20% (Power = 80%). For certain experimental setups with high variability and/or modest treatment effects, sample size was calculated to be higher.
In Fig. 6h-i, one mouse in the hIL-2/NARA1 group was excluded as it reached euthanasia criteria before the end of experiment. In Supp. Fig. 4 a,b one mice in hIL-2/NARA1 (0.5 ug) group was excluded as the lung cells were lost during processing and no events were detectable in flow cytometry. In Supp. Fig. 2, ALT and AST values were not included from hemolytic blood samples.
All experiments were repeated independently and on different days at least two times. Replications showed the same trend and pooled data are displayed throughout the manuscript. Three replication experiments of the intravenous B16-F10 (pulmonary) model (two repetitions of Mice were assigned to the respective treatment groups in a randomized fashion. For in vitro experiments using human or mouse cells same sample was equally distributed to all groups.
Tumor measurements, metastatic nodule counting and survival assessment were done by a blinded technician or researcher. Other analyses performed by using flow cytometry or ELISA was done unblinded as the data collection was quantitative and unbiased by nature.
Information on all antibodies used in this study are provided in Supplementary Table 1. Antibodies were validated by respective manufacturers for flow cytometry by staining of human or mouse primary cells or cell lines. Validation statements as well as references from the literature can be found on the manufacturers' websites. The reactivity of the antibodies are indicated with "m" for mouse and "h" for human. Antibodies that do not have an indication are reported to be cross-reactive for mouse and human e.g. pSTAT5, TOX, Ki67. Antibodies were not validated by/for other techniques than flow cytometry.
Only cells derived from the original aliquots were used within the study. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Instrument Software Cell population abundance The initial vial of cells were expanded to establish a stock, which was tested negative for mycoplasma. Only cells from this stock were used in this study.
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C57Bl/6J mice were purchased from Charles River Laboratories. Balb/c (JAX Stock No:006584) mice were obtained from the Jackson Laboratory and bred in house. Female mice were used for experiments at 2-3 months of age.
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Animal experiments received prior approval by the veterinary office of the Canton of Zurich (License numbers 142/2017 and 246/2016) and were conducted in accordance with Swiss Federal and Cantonal Law.
Anonymized buffy coats were obtained from the Swiss Blood Bank Zurich. Characteristic data on the donors is not available Anonymized buffy coats were obtained from the Swiss Blood Bank Zurich.
The "Fundamental research project for phenotypical and functional characterization of different leukocyte subsets in healthy and diseased individuals" (PFCL-1, BASEC no. 2016-01440) project has been reviewed and approved by the Kantonale Ethikkommission Zurich and has been carried out in accordance with principles enunciated in the current version of the Declaration of Helsinki, the guidelines of Good Clinical Practice, and Swiss legal requirements.
Single cell suspensions of lymph nodes and spleens were prepared according to standard protocols. Tumors were cut into small pieces, and incubated in 10 ml dissociation buffer (RPMI, 5% FCS, 10 ug/ml DNAse I (Sigma-Aldrich), and 200 U/ml collagenase type I (Thermo Fisher Scientific) for 60 min at 37°C and shaking with 25 rpm. Cell suspensions were then passed through a 70 m cell strainer. In experiments with intradermally implanted tumors, after one wash a Percoll (40% and 70%; GE Healthcare) gradient centrifugation was performed. Lungs were passed through a 70 m cell strainer followed by Percoll gradient centrifugation. All cell suspensions were stained for flow cytometry analysis using flow cytometry buffer (PBS with 2% FCS, 2 mM EDTA) and fluorochrome-conjugated antibodies for at least 20 min at 4°C. A list of used antibodies is provided in Supplementary  Table 1. Intracellular staining with Ki67 and Foxp3 was performed following manufacturer's instructions (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, ThermoFisher). For STAT5 phosphorylation analysis, freshly isolated mouse splenocytes or human PBMCs were incubated for 15 min at 37°C with indicated formulations. Cells were directly fixed with Fix Buffer I (BD) for 10 min at 37°C followed by permeabilization with Perm Buffer III (BD Phosflow™) and staining with fluorochrome-conjugated antibodies in flow cytometry buffer. For in vivo pSTAT5 spleens were directly fixed in Lyse/Fix Buffer (BD Phosflow™) followed by permeabilization with Perm Buffer III (BD Phosflow) for 1h and staining with fluorochrome-conjugated antibodies .
Samples were acquired with a BD LSR II flow cytometer (BD Biosciences).
Data was collected and analysed by BD FACS DIVA and BD FlowJo 10.
Cell sorting was not used in this study.