stm.sciencemag.org/cgi/content/full/12/564/eabb2311/DC1

Supplementary Materials for

Immunocytokines are a promising immunotherapeutic approach

against glioblastoma

Tobias Weiss, Emanuele Puca, Manuela Silginer, Teresa Hemmerle, Shila Pazahr, Andrea Bink, Michael Weller, Dario Neri*, Patrick Roth*

*Corresponding author. Email: patrick.roth@usz.ch (P.R.); neri@pharma.ethz.ch (D.N.)

Published 7 October 2020, Sci. Transl. Med. 12, eabb2311 (2020)

DOI: 10.1126/scitranslmed.abb2311

The PDF file includes:

Fig. S1. Amino acid sequence of L19 immunocytokines. Fig. S2. Biological activity assessments of molecules based on IL2, TNF, or IL12. Fig. S3. Target antigen distribution in human glioblastoma samples. Fig. S4. Body weight curves of mice treated with immunocytokines. Fig. S5. Amino acid sequence of KSF fusion proteins. Fig. S6. Survival of GL-261 glioma-bearing mice treated with immunocytokines upon in vivo depletion of CD4 or CD8 T cells. Fig. S7. Sequential MRI assessments and effect of CD4 or CD8 T cell depletion in SMA-560 glioma-bearing mice upon immunocytokine treatment. Fig. S8. Combination of L19-mTNF and L19-mIL12 with local irradiation and temozolomide. Fig. S9. Tumor-infiltrating immune cells in glioma-bearing mice treated with KSF fusion proteins and immune cells and cytokines from peripheral blood upon immunocytokine treatment. Fig. S10. Overview of study treatment and patients treated with L19-TNF. Fig. S11. Toxicity overview of patients treated with L19-TNF.

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/564/eabb2311/DC1)

Data file S1 (Microsoft Excel format). Primary data.

Fig. S1. Amino acid sequence of L19 immunocytokines. The amino acid sequences of L19-IL2, L19-mTNF, and

L19-mIL12, including leader sequences and linker, are shown as cloned into the expression vector. A key to

the colors is shown above each sequence.

Fig. S2. Biological activity assessments of molecules based on IL2, TNF, or IL12. Left: CTLL-2 cells were

treated with increasing concentrations of the indicated molecules and proliferation was measured after 48 h.

Center: SMA-560 or GL-261 cells were exposed to increasing concentrations of the indicated molecules and

viability was measured after 24 h. Right: NK-92 cells were exposed to increasing concentrations of the

indicated molecules and IFN- released into the supernatant after 24 h was measured by ELISA. rIL2,

recombinant IL2; rhTNF, recombinant human TNF; mTNF, mouse TNF; rhIL12, recombinant human IL12;

mIL12, mouse IL12. Data are presented as mean and standard deviations from 1 of 3 experiments.

Fig. S3. Target antigen distribution in human glioblastoma samples. Quantification of EDB across 81

human glioblastoma samples. Each dot represents a tumor. Median and standard deviation are indicated with

horizontal lines.

Fig. S4. Body weight curves of mice treated with immunocytokines. Mice bearing GL-261 or SMA-560

tumors were treated intravenously with 50 µg L19, 50 µg L19-IL2, 4 µg L19-mTNF, or 12 µg L19-mIL12 at

days 5 and 10 after tumor cell implantation. Body weight was monitored every other day. Data for 4 mice per

group are shown as mean ± SD.

Fig. S5. Amino acid sequence of KSF fusion proteins. The amino acid sequences of KSF-IL2, KSF-mTNF,

and KSF-IL12, including leader sequences and linker, are shown as cloned into the expression vector. A key

to the colors is shown above each sequence.

Fig. S6. Survival of GL-261 glioma-bearing mice treated with immunocytokines upon in vivo depletion

of CD4 or CD8 T cells. GL-261 glioma-bearing mice were treated with 50 µg L19, 4 µg L19-mTNF, or 12

µg L19-mIL12 at days 5 and 10 after tumor implantation. One day before tumor cell inoculation and on days

2, 5, 8, and 11, the animals received intravenous injections of depleting antibodies to eliminate CD4 or CD8 T

cells or received injections of matched isotype control. There were 6 mice per condition. The upper panel

shows CD4 or CD8 T cells detected in the peripheral blood at day 16 after tumor cell implantation. Statistical

significance for T cells was determined by two-tailed Student’s t-tests (**p < 0.01). The lower panel shows

Kaplan-Meier plots. P values for survival differences were calculated with log-rank test (*p < 0.05; **p <

0.01).

Fig. S7. Sequential MRI assessments and effect of CD4 or CD8 T cell depletion in SMA-560 glioma-

bearing mice upon immunocytokine treatment. (A) SMA-560 tumor-bearing mice were treated with a

single intravenous injection of L19, L19-mTNF, or L19-mIL12 at day 11 after tumor implantation. MRI scans

were acquired before injection on day 11 and after 72 h. MRI scans from 3 mice per group are shown and

median and SD of tumor volume is plotted in the lower panel. (B) SMA-560 glioma-bearing mice were

treated with L19, L19-mTNF, or L19-mIL12 at days 5 and 10 after tumor implantation. One day before tumor

inoculation and on days 2, 5, 8, and 11 the animals received intravenous injections of depleting anti-CD4 or

anti-CD8 antibodies or received injections of matched isotype control. There were 6 mice per condition. The

upper panel shows CD4 or CD8 T cells detected in the peripheral blood at day 16 after tumor cell

implantation. Statistical significance for T cells was determined by two-tailed Student’s t-tests (**p < 0.01).

The lower panel shows Kaplan-Meier plots. P values were calculated with log-rank test (*p < 0.05; **p <

0.01).

Fig. S8. Combination of L19-mTNF and L19-mIL12 with local irradiation and temozolomide. GL-261

tumor-bearing mice were treated intravenously with 4 µg L19-mTNF or 12 µg L19-mIL12 at days 5 and 10

after tumor cell implantation. As indicated, mice were also treated with a single local irradiation with 6 Gy at

day 10 after tumor cell implantation and temozolomide (TMZ) for five consecutive days starting at day 7 after

tumor cell implantation. Survival data are presented as Kaplan-Meier plots. P values were calculated with log-

rank test (**p < 0.01 compared to L19 control, and ++p <0.01 compared to single treatment with the indicated

immunocytokine).

Fig. S9. Tumor-infiltrating immune cells in glioma-bearing mice treated with KSF fusion proteins and

immune cells and cytokines from peripheral blood upon immunocytokine treatment. (A) GL-261 tumor-

bearing mice were treated intravenously with KSF, KSF-IL2, KSF-mTNF, or KSF-mIL12 at days 5 and 10

after tumor cell implantation. At day 14 after tumor implantation, brains were isolated and tumor-infiltrating

immune cells were analyzed by immunofluorescence. Representative images are shown. 7 mice were in each

treatment group. (B) GL-261 (left) or SMA-560 (right) tumor-bearing mice were treated with L19, L19-

mTNF, or L19-mIL12 at days 5 and 10 after tumor cell implantation. At day 14 after implantation, immune

cells were analyzed from peripheral blood by flow cytometry. Data are from 4 mice per group. (C) GL-261

tumor-bearing mice were treated as in B. At 14 days after tumor cell implantation, cytokines in peripheral

blood were detected by ELISA. Data are from 3 mice per group.

Fig. S10. Overview of study treatment and patients treated with L19-TNF. (A) Study scheme

(NCT03779230). (B) Clinical characteristics of 3 patients treated with 10 µg/kg L19-TNF. IDH-1 encodes

isocitrate dehydrogenase.

Fig. S11. Toxicity overview of patients treated with L19-TNF. (A) Total number of adverse events (AEs)

or serious adverse events (SAEs) from 3 patients treated with L19-TNF. (B) Course of hematological

parameters, serum creatinine, and serum liver function tests over the first treatment cycle of patients treated

with 10 µg/kg L19-TNF. All days are from the first treatment cycle and indicated either with D#. ALT,

alanine transaminase; AST, aspartate transaminase; GGT, gamma-glutamyltransferase; WBC, white blood

cells; DLT, dose-limiting toxicities.