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. 2019 Jul 19;10(1):3236.
doi: 10.1038/s41467-019-11137-5.

Molecular retargeting of antibodies converts immune defense against oncolytic viruses into cancer immunotherapy

Julia Niemann  1 , Norman Woller  1 , Jennifer Brooks  1 , Bettina Fleischmann-Mundt  1 , Nikolas T Martin  2   3 , Arnold Kloos  1   4 , Sarah Knocke  1 , Amanda M Ernst  1 , Michael P Manns  1 , Stefan Kubicka  1   5 , Thomas C Wirth  1 , Rita Gerardy-Schahn  2 , Florian Kühnel  6
Affiliations

Molecular retargeting of antibodies converts immune defense against oncolytic viruses into cancer immunotherapy

Julia Niemann et al. Nat Commun. .

Abstract

Virus-neutralizing antibodies are a severe obstacle in oncolytic virotherapy. Here, we present a strategy to convert this unfavorable immune response into an anticancer immunotherapy via molecular retargeting. Application of a bifunctional adapter harboring a tumor-specific ligand and the adenovirus hexon domain DE1 for engaging antiadenoviral antibodies, attenuates tumor growth and prolongs survival in adenovirus-immunized mice. The therapeutic benefit achieved by tumor retargeting of antiviral antibodies is largely due to NK cell-mediated triggering of tumor-directed CD8 T-cells. We further demonstrate that antibody-retargeting (Ab-retargeting) is a feasible method to sensitize tumors to PD-1 immune checkpoint blockade. In therapeutic settings, Ab-retargeting greatly improves the outcome of intratumor application of an oncolytic adenovirus and facilitates long-term survival in treated animals when combined with PD-1 checkpoint inhibition. Tumor-directed retargeting of preexisting or virotherapy-induced antiviral antibodies therefore represents a promising strategy to fully exploit the immunotherapeutic potential of oncolytic virotherapy and checkpoint inhibition.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Design of the bifunctional adapter DE1scFv-pSia for tumor retargeting of antiadenoviral antibodies. a Illustration of the adenovirus capsid protein hexon generated by ExPASy software using the SWISS-Model tool, showing the exposed domains DE1, FG1 and FG2. The domain DE1 is highlighted by a dotted circle b The structure of the adapter molecule DE1scFv-pSia containing the DE1 domain (DE1) provided with a myc/his tag which was linked to an anti-PolySia scFv fragment via a glycine/serine stretch (Linker). c ELISA showing the recognition of purified, immobilized DE1scFv-pSia by IgG in serum of Ad5-naive, or in serum of Ad5-immunized mice pretreated with or without soluble DE1scFv-pSia as competitor (group: Ad5+ + DE1scFv-pSia) to inactivate DE1-specific IgG (n = 3). d Recognition of immobilized DE1scFv-pSia or Ad5-particles by IgG1 and IgG2a in serum of Ad5-naive (n = 3) and Ad5-immunized (Ad5+; n = 4) mice was measured by ELISA. e Binding of DE1scFv-pSia to the polySia-positive human cancer cell lines IMR32 and TE671 and the murine polySia-expressing cancer cells CMT-pSia, MC38-pSia, and B16F10-pSia was measured via flow cytometry. polySia-negative human Panc01 cells were used as negative control. Binding of DE1scFv-pSia to the cell surface was detected using an anti-myc-tag antibody. PolySia expression on the cell surface was measured using the specific antibody mAb735. Two-tailed unpaired t test was used to calculate statistics in c and d: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Error bars refer to standard deviation (SD). Source data are provided as a source data file
Fig. 2
Fig. 2
Ab-retargeting inhibits the growth of subcutaneous and disseminated tumors in Ad5-vaccinated mice. a Illustration showing the experimental time course of tumor establishement in Ad5-immunized or Ad5-naive mice by s.c. injection of PolySia-expressing tumor cells (MC38-pSia, ‚CMT-pSia, or B16F10-pSia as indicated in the Fig. 2b–d) and subsequent treatments with purified DE1scFv-pSia, or saline, respectively. b MC38-pSia tumors were established in Ad5-naive mice (upper panel) or Ad5-immunized mice (lower panel), and treated with DE1scFv-pSia or saline (control). Tumor development (left panel) and survival (right panel) were monitored. Group size: Ad5-naive control, n = 7; Ad5-naive DE1scFv-pSia treated, n = 8; Ad5-immunized control, n = 8; Ad5-immunized DE1scFv-pSia treated, n = 9. Median survival (ms) in days for Ad5-naive control: 30; Ad5-naive DE1scFv-pSia treated: 24; Ad5-immunized control: 21; Ad5-immunized DE1scFv-pSia treated: 26. c Tumor development (left panel) and survival (right panel) were monitored in Ad5-immunized mice bearing CMT-pSia tumors. Group size: n = 8; ms control: 23; ms DE1scFv-pSia: 31. d Tumor development (left panel) and survival (right panel) were monitored in Ad5-immunized mice B16F10-pSia tumor-bearing mice. Group size: n = 5; ms control: 17; ms DE1scFv-pSia: 24. e According to the shown treatment scheme, Ad5-immunized mice were injected i.v. with the lung adenocarcinoma cell line CMT-pSia to establish lung colonies. Mice were then treated i.v. with purified DE1scFv-pSia at the indicated time points or received saline as control. f Representative H/E-stained lung sections of control and DE1scFv-pSia treated individuals at endpoint examination (day 17). g Tumor burden in the lung was calculated based on microscopic investigations of H/E-stained lung sections (n = 4). Log-rank (Mantel–Cox) test was used to calculate survival in b, c, and d and two-tailed unpaired t test was used to calculate statistics in g: *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001. Error bars refer to standard deviation (SD). Source data are provided as a source data file
Fig. 3
Fig. 3
Ab-retargeting reduces intratumor myeloid cells and supports NK cell and CD8 T-cell infiltration. a MC38-pSia cells were used to establish s.c. tumors in Ad5-vaccinated mice. Tumor-bearing mice received i.v. injections of DE1scFv-pSia or saline, and were sacrificed according to the shown schedule. Tumor tissue was examined for infiltration of different leukocyte subsets via flow cytometry. b Frequencies of NK cells (NK1.1+CD49b+) were calculated as percentage of CD45.2+ leukocytes. NK cell activation was determined by surface expression of CD107a. Macrophages (Gr1+F4/80high) and different fractions of myeloid-derived suppressor cells (MDSCs; P1: CD11b+ Gr1high; P2: CD11b+ Gr1int) are shown in c and d, respectively. Group size was in general n = 5 with following exceptions: n = 7 (NK cells/day 10/DE1scFv-pSia); n = 6 (NK cells/day 17/control and DE1scFv-pSia groups); n = 4 (CD107ahigh – NK cells/control). e Frequencies of CD4+ and CD8+ T cells were measured as percentage of CD90.2-positive lymphocytes, and individual ratios of CD8 to CD4 T cells were calculated. Group size: n = 5 (day 10) and n = 6 (day 17). f Splenocytes were prepared from sacrificed mice, incubated with the mutated MC38 peptide Adpgk-R304M (ASMTNMELM), or an irrelevant control peptide, and responding neoantigen-reactive CD8 T cells were identified by intracellular staining for IFNγ (group size: n = 8). Two-tailed unpaired t test was used to calculate statistics: *p ≤ 0.05; **p ≤ 0.01. Error bars refer to standard deviation (SD). Source data are provided as a source data file
Fig. 4
Fig. 4
The therapeutic effect of Ab-retargeting is mediated by NK cell-dependent triggering of CD8 T cells. a According to the procedure illustrated in Fig. 2a, subcutaneous MC38-pSia tumors were established in Ad5-vaccinated mice and treated with i.v. injections of DE1scFv-pSia or saline. In addition, the influence of NK cells, CD8 T cells, and macrophages on treatment success was studied by depletion of the indicated immune cell subsets starting 2 days before first adapter treatment using depleting antibodies α-NK1.1 and α-CD8 for depletions of NK cells and CD8 T cells, respectively, or clodronate liposomes for depletion of macrophages. Tumor development (left) and survival (right) were monitored. Group size n = 5 for all groups, except for the macrophage depleted group (DE1scFv-pSia + clodronate; n = 4); the same control and DE1scFv-pSia group without depletion agent is shown in each plot. b On day 17 after tumor inoculation, splenocytes of control and adapter-treated groups with or without NK cell depletion were prepared and examined for Adpgk-R304M-reactive tumor-specific CD8 T cells (n = 5 per group) by incubation with the corresponding peptide ASMTNMELM as described in Fig. 3f. Log-rank (Mantel–Cox) test was used to calculate survival statistics in a. Two-tailed unpaired t test was used to calculate statistics in b. *p ≤ 0.05; **p ≤ 0.01. Error bars refer to standard deviation (SD). Source data are provided as a source data file
Fig. 5
Fig. 5
Ab-retargeting after intratumor application of an oncolytic adenovirus leads to improved survival. a Subcutaneous MC38-pSia tumors were established in Ad5-naive mice. After tumor formation, animals received a single application of the oncolytic adenovirus hTert-Ad (by either intratumor or intravenous application) followed by a single i.v. administration of DE1scFv-pSia as shown in the treatment scheme. Control animals were treated by i.t. injection of saline. b Tumor development and survival were monitored (group size n = 6, except control group 0.9 % NaCl: n = 7, median survival: 0.9% NaCl 22 days; hTert-Ad i.v. 23.5 days; hTert-Ad i.t. 27.5 days; hTert-Ad i.v. + DE1scFv-pSia 25 days; hTert-Ad i.t. + DE1scFv-pSia 30.5 days). c Tumor-infiltrating immune cells were analyzed on day 8 after start of i.t. virotherapy (n = 4 in all groups). Proportions of NK cells, macrophages, and MDCSs were calculated as percentage of CD45.2-positive tumor-infiltrating leukocytes. CD8 and CD4 T-cell frequencies were calculated as percentage of CD90.2-positive lymphocytes. d Analyses of tumor antigen-specific CD8 T cells in splenocytes 13 days after i.t. virotherapy (0.9% NaCl: n = 5; hTert-Ad: n = 7; hTert-Ad + DE1scFv-pSia: n = 7). To determine neoantigen-specific responses against Adpgk-R304M, splenocytes were stimulated with the peptide ASMTNMELM, or an irrelevant control peptide, and were analyzed by intracellular staining of IFNγ and flow cytometry. Log-rank (Mantel–Cox) test was used to calculate survival statistics in b. Two-tailed unpaired t test was used to calculate statistics in c and d. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Error bars refer to standard deviation (SD). Source data are provided as a source data file
Fig. 6
Fig. 6
Ab-retargeting sensitizes tumors for PD-1 checkpoint inhibition facilitating long-term survival. a Subcutaneous MC38-pSia tumors were established in Ad5-vaccinated mice. Seven days after tumor cell injection, animals were treated with an antagonistic antibody against PD-1 as monotherapy (αPD-1) or in combination with DE1scFv-pSia antibody retargeting (DE1scFv-pSia + αPD-1) or remained untreated (control). b Tumor development and survival (group size: αPD-1: n = 8, ms: 28 days; DE1scFv-pSia + αPD-1: n = 9, ms: 45 days, control: n = 5, ms: 22 days) was monitored. c Blood samples were drawn during treatment on day 13 after start of treatment, and splenocytes were stimulated with the peptide ASMTNMELM to detect CD8 T-cell responses against the Adpgk-R304M neoepitope. Responsiveness of CD8 T cells to peptide stimulation was measured via intracellular IFNγ-staining and flow cytometry. Left panel: αPD-1 vs. DE1scFv-pSia + αPD-1 (n = 9 per group). Right panel: individuals of the DE1scFv-pSia + αPD-1 treatment group were split in those showing tumor progression (n = 6) and tumor-free animals: (n = 3). d Subcutaneous MC38-pSia tumors were established in Ad5-naive mice. Tumor-bearing mice were treated by i.t. application of hTert-Ad as monotherapy with or without i.p. PD-1 immune checkpoint inhibition, and/or antibody-retargeting according to the treatment scheme. e The left panel shows the results of tumor growth monitoring and the right panel survival data (group size n = 5, except the groups 0.9 % NaCl: n = 4 and hTert-Ad: n = 7; median survival: 0.9% NaCl ms = 22; hTert-Ad: ms = 26; hTert-Ad + αPD-1: ms = 36; hTert-Ad + DE1scFv-pSia: ms = 36; hTert-Ad + αPD-1 + DE1scFv-pSia ms = undefined). Log-rank (Mantel–Cox) test was used for survival statistics in b and d. Two-tailed unpaired t test was used for statistics in c. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001. Error bars refer to standard deviation (SD). Source data are provided as a source data file
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