Adapter-mediated retargeting of capsid-engineered virions

Biodistribution of therapeutic viruses within the body is subject to manifold interactions of the virions with tissue-resident and circulating blood cells and lymphoid tissue, as well as soluble factors like blood coagulation FX. FX endangers the success of an adenoviral gene vector as it activates the innate immunity in the transduced cells22 and mediates liver uptake36. Therefore, we ablated binding of FX to HAdV5 by mutating the FX binding site on the hexon as recently reported37. Four mutations in the hypervariable region 7 (HVR7) of the hexon protein were introduced (I421G, T423N, E424S and L426Y), resulting in HAdV5HVR7. To enhance the targeting of virions to tumor cells of interest, and reduce virion attachment to endogenous fiber receptors, we made use of the recently developed adapter, which binds to and blocks the viral fiber knob (Fig. 1a)34.

Fig. 1 Retargeting of FX-binding-ablated HAdV5HVR7 to HER2+ and EGFR+ tumor cells. a Overview of the knob-adapter complexes. Knob-binding DARPins are trimerized through SHP and bind the knob in a quasi-covalent manner34. The retargeting module (orange), a target-specific DARPin, allows binding of tumor biomarkers like HER2 or EGFR. Non-targeting (control) DARPins are shown in blue. b Hexon-engineered HAdV5HVR7 infects CAR-expressing SKOV3 cells with similar efficiency as the wt virus, but addition of FX boosted only wt transduction, since HAdV5HVR7does not bind FX. RLU, relative light units; HSPG, heparan sulfate proteoglycan. c, d Knob binding of adapter decreased HAdV5HVR7 viral gene delivery to tumor cells in both c SKOV3.ip and d A431 cells through blocking of CAR interaction. Fusion of a HER2- or EGFR-binding DARPin to the adapter resulted in a ×50, or ×80 increase of tumor cell transduction, respectively. Shown are sample means ± SD from biological replicates ((b) n=4; (c) n = 2; (d) n = 4), two-way ANOVA of log-transformed data, *P < 0.05, **P < 0.01, ****P < 0.0001. e, f Analysis of binding and internalization of Alexa-Fluor 488-labeled HAdV5wt to A431 cells. Viruses were bound to cells at +4 °C for 1 h and were fixed immediately or after subsequent 1 h of incubation at 37 °C. Images shown represent maximum projections of individual confocal stacks. Nuclei (DAPI stain) are blue and virus particles green. e The EGFR-retargeted virus showed increased cell binding in comparison to wt or fiber knob-blocked viruses and bind all over the cell. Pictures from the confocal microscopy are maximal projections of the cells, not slices through one plane, as explained in further detail in Supplementary Fig. 1. Scale bar = 10 µm. f At 4 °C (left), cell binding occurs, while at 37 °C internalization of EGFR-mediated HAdV5wt results in nuclear trafficking of the particles. Nuclei are shown as outlines. Scale bar = 10 µm. g Retargeting of fiber-blocked virus to EGFR increases transduction of A431 tumor cells. A431 cells were imaged by automated fluorescence microscopy. The mean GFP intensity over a DAPI mask was quantified in single cells. In the box-and-whisker plots, center lines show the mean; box limits the 25th and 75th percentiles; whiskers according to Tukey. For each condition between 4 and 9 × 103 cells were analyzed. AU, arbitrary units Full size image

To test whether FX binding was truly ablated, we infected SKOV3 cells, which express heparin sulfate proteoglycans on the cell surface38, with either HAdV5wt or FX-binding-ablated HAdV5HVR7. With the adapter34 tightly bound to the fiber knob, CAR-mediated luciferase transgene expression should be reduced, as only FX-mediated entry was expected to take place. Indeed, blocking of the fiber knob itself reduced transduction of SKOV3 cells by both HAdV5wt and HAdV5HVR7 (Fig. 1b), and in the presence of FX, transgene expression of the fiber knob-blocked HAdV5wt was increased by 100-fold. In contrast, FX had essentially no effect on the transduction of HAdV5HVR7, indicating effective inhibition of FX binding.

We further applied the adapter retargeting strategy34 to the FX-ablated HAdV5HVR7. Depending on the retargeting module, the virus could be retargeted to cancer cells such as A431, expressing EGFR, or SKOV3.ip cells, expressing HER2 (Fig. 1c, d). Confocal microscopy imaging of A431 cells infected with Alexa-Fluor 488-labeled HAdV5wt indicated that virions with normal or adapter-blocked fiber knobs bound inefficiently to these cells (Fig. 1e). In contrast, an EGFR-retargeting adapter led to significant increase of virion binding to cells. Importantly, the modification of the viral entry mechanism by targeting a non-native receptor did not ablate the cell entry and nuclear trafficking of the virus (Fig. 1f and Supplementary Fig. 1). EGFR retargeting enhanced cell binding and subsequently increased the number of internalized particles, which in turn also resulted in higher transgene expression (Fig. 1g). In conclusion, viral gene delivery by FX-ablated HAdV5HVR7 to cancer cell lines can be strongly enhanced with the fiber knob adapter strategy that targets tumor surface biomarkers.

Retargeting improves gene delivery to xenograft tumors

Having shown the potential of FX-ablated retargeted viruses in vitro, we were interested in analyzing their behavior in vivo. Today, most clinical trials of virotherapy have used direct intratumoral delivery of the virion39, and thus we tested this approach first. To gain insight into the retargeting of HAdV5HVR7 in vivo we analyzed viral gene delivery in two subcutaneous xenograft models in immunodeficient Rag1-/- mice (an immunodeficient strain that lacks mature T and B lymphocytes)40, thereby testing the performance of the DARPin adapter. Control experiments in an A431 cell culture indicated that there the adapter is stable in Rag1-/- mice serum, since preincubation of adapter in serum had no significant impact on infection efficacy (Supplementary Fig. 2a).

Upon intratumoral administration into A431 tumor xenografts, an EGFR-specific retargeting adapter increased the payload delivery (luciferase) in the tumor by 20-fold, compared to HAdV5HVR7 with a free fiber knob, and by 34-fold compared to a blocked fiber knob virus (Fig. 2a). At the same time, the binding of the adapter decreased liver targeting by approximately 37-fold compared to the non-targeted HAdV5HVR7. In lung, spleen and kidney, the luciferase signal was lower than in the liver, and the signal was essentially at background levels in kidney and lung when blocking the fiber knob. In the context of viral therapeutic gene delivery, the ratio of expressed payload genes between tumor and liver is of high relevance due to potential off-target side effects of future payloads. In the A431 EGFR+ tumor model, intratumoral application of HAdV5HVR7 thus led to a tumor-to-liver ratio of 50, which was strongly increased to about 7200 by EGFR targeting and inhibition of the CAR uptake pathway, representing a 140-fold gain in specificity (Fig. 2b).

Fig. 2 Retargeting of HAdV5HVR7 increases tumor-specific gene delivery after intratumoral injection. a 1.5 × 106 HAdV5HVR7 particles were injected into subcutaneous EGFR+ A431 tumor xenografts in Rag1-/- mice. Gene delivery was analyzed 48 hpi by luciferase activity, and the values obtained were normalized to total protein amount. The experiment was performed with randomized groups and blinded. Virus alone (free knob) showed significant transgene signal in all analyzed organs other than kidney. Gene delivery to the liver was reduced by blocking the fiber knob with the adapter (blocked knob). EGFR-specific retargeting adapter significantly increased tumor infection (retargeted knob). Background signals from control injections with PBS are indicated by dashed lines (mean, each symbol represents one organ, n = 2–3 mice per group. RLU, relative light units. One-way ANOVA of log-transformed data, *P < 0.05, **P < 0.01, ***P < 0.001). b, c The tumor-to-liver ratio was calculated for each individual mouse. The tumor-to-liver ratio was 50 for the unmodified virus and 7200 for virus retargeted to EGFR (b). In the case of HER2 xenografts, the values were 1200 and 24,600 for unmodified and HER2-retargeted virus, respectively (c). Sc, subcutaneous. Pooled data from repeated independent experiments were used for statistics, and individual experiments are indicated (mean, each symbol represents the ratio of an individual, two-sided, unpaired Welch’s t-test of log-transformed data, *P < 0.05, **P < 0.01) Full size image

A HER2-overexpressing SKOV3.ip xenograft model in Rag1-/- mice showed similar effects. Using the knob-binding adapters, liver targeting was significantly reduced by up to 30-fold (Supplementary Fig. 2b). With the HER2-specific retargeting module, the payload delivery to the tumor was increased by eightfold compared to the fiber knob-blocked virus upon intratumoral virus injection. The signals in kidney and lung were decreased by the adapters essentially to background levels. Also in the HER2+ tumor model, the tumor-to-liver ratio of the delivered viral payload of about 1200 was increased to about 24,600 by HER2-retargeting and fiber knob blocking, a 20-fold change of tumor selectivity (Fig. 2c). Thus, the application of a retargeting and CAR pathway-blocking adapter in combination with FX-ablated HAdV5HVR7 improved the localization upon intratumoral vector administration, being encouraging for future therapeutic strategies.

EGFR retargeting enhances tumor cell-specific gene delivery

Next, we evaluated the cellular specificity of viral gene delivery in A431 tumor xenografts in more detail. Immunohistology for luciferase transgene expression revealed that the untargeted HAdV5HVR7 virus almost exclusively infects murine fibroblasts or fibrocytes after intratumoral administration (Fig. 3). Only an occasional luciferase-positive tumor cell was observed. The fiber knob-blocked virus showed a similar result. In contrast, virus with the EGFR-retargeting adapter resulted in luciferase expression in tumor cells, which were mainly observed as large patches. Nonetheless, also the retargeted virus transduced fibroblasts in the tumor-surrounding stroma. These results show that, even after intratumoral injection, gene delivery with unmodified HAdV5HVR7 is limited to murine stromal cells. The present data reveal the necessity of a tumor-specific targeting adapter module to infect the A431 tumor cells and demonstrate its functionality in vivo. It is worth noting that only in the case of the unmodified HAdV5HVR7, but not for the adapter-bound virions, luciferase-positive hepatocytes could be detected in the liver (Supplementary Fig. 3a). This is in accordance with the observed reduction of liver targeting in the xenograft (Fig. 2a).

Fig. 3 EGFR-retargeting results in gene delivery to A431 tumor cells after IT injection. In situ assessment of viral transgene expression in A431 xenograft tumors by immunohistology. Host fibroblasts are detected based on the expression of vimentin; luciferase staining reflects viral transgene expression. With both free virus and fiber knob-blocked virus, mostly fibroblasts (arrowheads) express the transgene luciferase, both in the tumor and in the surrounding stroma (asterisks). Luciferase-positive neoplastic cells (blue arrows) occur only rarely. In contrast, EGFR-retargeted virus is mainly found in viable tumor cells (blue arrows), although stromal fibroblasts are also luciferase positive (arrowheads). Analysis of tumor sections was conducted in a blinded fashion, and tumor cells were confirmed by their EGFR expression. Scale bar = 50 µm. The lower row provides an overview of the tumor after luciferase staining (N necrotic tumor tissue, T viable tumor tissue); here scale bar = 200 µm Full size image

Adapter reduces liver tropism in systemic injections

Intratumoral administration of therapeutics has limited benefit for the patient with disseminated tumors, as it is not applicable to poorly accessible tumors. We thus explored systemic delivery of HAdV5HVR7, where native particles are rapidly scavenged by the liver41. We investigated the impact of the fiber knob-binding adapters in EGFR+ (A431) and HER2+ (SKOV3.ip) subcutaneous xenograft models with HAdV5HVR7 upon intravenous injection. The retargeting adapter reduced the viral liver tropism around 70- and 20-fold in the A431 and SKOV3.ip xenograft models, respectively (Fig. 4a, b). Similar results were obtained with viruses containing the blocking adapter. This suggests that the knob is playing an active role in liver tropism. Surprisingly, blocking of the fiber knob with the adapter also strongly reduced off-targeting to the kidney. In contrast, the signals in spleen and lung remained unchanged. However, no increase in tumor targeting by the retargeting adapter was detected after intravenous administration, compared with the knob-blocking adapter, although the tumor-to-liver ratio of payload expression was significantly increased by a factor of around 170 in the EGFR+ tumor model and by 25-fold in the HER2+ tumor model (Fig. 4c, d), compared to the naked HAdV5HVR7.

Fig. 4 Adapter reduces off-targeting and increases tumor–liver ratio after intravenous (IV) injection. a, b 3 × 106 HAdV5HVR7 particles were injected in the tail vein of Rag1-/-mice, bearing either EGFR- or HER2-overexpressing subcutaneous tumors. Gene delivery was analyzed 48 hpi by luciferase activity, normalized to total protein amount. a More than 99% of the transgene activity was located in the liver after IV application of the virus alone. The blocking of the fiber knob by either adapter significantly decreased the gene delivery to liver, kidney and lung. Either knob-binding adapter increased the gene delivery to the tumor and the spleen. Background signals from control injections with PBS are indicated by dashed lines (each symbol represents one organ, n=5, two-way ANOVA of log-transformed data, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). b Intravenous injection of blocked, non-targeted HAdV5HVR7, and HER2-targeted HAdV5HVR7 resulted in strong reduction of liver and kidney transduction. A HER2 adapter-mediated targeting of the tumor was absent, since the luciferase activity was not higher than for the blocked virus. Background signals from control injections with PBS are indicated by dashed lines (each symbol represents one organ, n = 4, two-way ANOVA of log-transformed data, ***P < 0.001, ***P < 0.0001). c The ratio of transgene activity between tumor and liver was increased by the EGFR-retargeting adapter by a factor of 170 (each symbol represents the ratio of an individual mouse, two-sided, unpaired Welch’s t-test of log-transformed data, ****P < 0.0001). d The HER2-specific adapter increased the tumor–liver ratio in the SKOV3.ip xenograft by a factor of 25 (each symbol represents the ratio of an individual mouse, two-sided, unpaired Welch’s t-test of log-transformed data, *P < 0.05) Full size image

Design of a capsid-binding shield based on a humanized scFv

We next analyzed viral payload expression in different tumor cell lines in the presence of the hexon-specific antibody 9C1235. As expected, immunoglobulin G-mediated ADIN-based neutralization prevented viral payload delivery in SKBR3, BT474 and A431 cells, when cell entry occurred via an adapter targeting the virion to EGFR or HER2 (Fig. 5a–c). To overcome these limitations, and to protect the virion from undesired interactions with host factors, we designed an artificial protein-based shield around the hexon shell protein of the virion.

Fig. 5 Design of a hexon-binding trimerized scFv with low picomolar affinity to hexon. a–c HAdV5HVR7 retargeted by an adapter to HER2- or EGFR-expressing tumor cells are still susceptible to ADIN by the mAb 9C12 in a concentration-dependent manner. Blocked denotes the blocking adapter without targeting function (mean ± SD, n=2-3, one-way ANOVA of logarithmic data, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Note that the blocked control for c is shown in Fig. 1d. d Humanization of a scFv by CDR grafting on a stable human framework. Model of scFv colored by sequence origin. e For trimerization, the scFv was fused to the phage SHP protein43 and this resulted in stable trimers in solution. The scFv consists of a heavy chain followed by a light chain connected with a glycine–serine linker. Affinity of monovalent and trivalent scFv measured by SPR with immobilized hexon. Monovalent scFv was injected at five different concentrations (1 nM, 3.16 nM, 10 nM, 31.6 nM, and 100 nM). A heterogenous ligand model resulted in two K D s of 12 nM and 550 pM. The trivalent scFv was injected in concentrations of 31.6 pM, 100 pM, 316 pM, 1 nM, and 3.16 nM. Fitting the data with a Langmuir model resulted in an affinity of 10 pM Full size image

The construction of a humanized scFv provided the basic module of the protein shield (Supplementary Fig. 4a). A CDR (complementarity-determining region) graft from the murine hexon-binding mAb 9C12 onto a human scFv framework led to a stable, monomeric scFv (Fig. 5d and Supplementary Fig. 4b)35,42. To increase the affinity of the shielding scFv to multimers of hexons as present in the virion capsid, we constructed a trimeric scFv by fusing a highly stable trimerization domain, SHP of lambdoid phage 2143, to the carboxy-terminus of the scFv (Supplementary Fig. 4c, d). This strategy exploits the various threefold symmetry axes present on the triangular faces of the icosahedral shell. Trimerization led to an increase in affinity from low nM of the monovalent scFv to 10 pM of the trivalent scFv to immobilized hexons (Fig. 5e). In contrast to the monovalent scFv, the trivalent scFv blocked binding of a bivalent mAb to both purified hexon and virions (Supplementary Fig. 5a, b). We therefore conclude that the trivalent scFv provides a high-affinity shield on the virion surface, most likely through avidity effects.

Shield covers virion by binding the domains of a hexon trimer

To evaluate the arrangement of the shield on the icosahedral capsid, the structure of the complex between HAdV5HVR7 and the trimeric scFv shield was determined by single-particle cryo-electron microscopy to a resolution of 7.4 Å (Supplementary Fig. 6). A three-dimensional (3D) reconstruction revealed additional density all around the capsid (Fig. 6a). In the naked capsid, the deep canyons between the inner capsid shell (red) and the trimeric hexons (yellow and green) are clearly visible by applying a color gradient indicating the distance to the center of the virion (Fig. 6b). The shield expanded the diameter of the hybrid particle by approximately 10 nm from 88 to 98 nm (Supplementary Fig. 7a), thereby masking the icosahedral shape of the capsid and yielding a rather spherical object.

Fig. 6 EM structure of shielded virus and crystal structure of hexon–scFv complex. a, b Comparison of EM structure of naked and shielded HAdV5. Color reflects distance to the core (white: <32 nm, red: 32–38 nm, yellow 41 nm, green 43 nm, cyan 46 nm, blue 48 nm). Trivalent shield proteins (green–blue) bind all over the capsid, resulting in a dense cover of viral capsid proteins (red–light green). c High-resolution crystal structure of scFv–hexon complex elucidates the atomic interactions. Both heavy (magenta) and light (cyan) chain of the scFv bind to the tower of a hexon monomer (three different shades of blue, one scFv displayed as surface, others as cartoon), formed mainly by HVR2 and HVR7. The structure also shows few interactions with HVR5. Importantly, all three epitopes in the trimeric hexon were occupied with three scFvs Full size image

To explore atomic details of the hybrid particle we also determined the X-ray crystal structure of the scFv–hexon complex (Fig. 6c and Supplementary Fig. 8). The high-resolution structure of the hexon and a 9C12 antibody fragment unveiled the complex interaction network between the heavy and the light chains of the scFv and different domains of a hexon trimer, especially HVRs 2, 5 and 7. In order to assess possible differences between the crystal and the in situ structure of the scFv–hexon complex, we performed molecular dynamics flexible fitting (MDFF) with the electron microscopy (EM) map of the shielded capsid. During a 1 ns MDFF run we observed only a very minor increase of the cross-correlation between map and structure from an initial value of 0.87 to a plateau value 0.90. This was mostly caused by a slight reorientation of the scFv on the hexon that did not affect epitope binding (Supplementary Fig. 7b). The high similarity between the two structures was also reflected by a root-mean-square deviation of only 1.5 Å (Supplementary Fig. 7c). This is further evidence for the validity of the structure of the complex and underlines the high stability of the trimeric shield and the hexon trimer.

To analyze the stoichiometry and the surface occupancy of the shield on the viral capsid, we projected the complex structure onto 6 asymmetric units (AUs) of one triangular facet of the icosahedral capsid (Fig. 7a, b). Binding of the scFv is influenced by the symmetrical arrangement of the neighboring hexons: each icosahedral facet comprises 72 epitopes of mAb 9C12; at 42 epitopes, the scFvs bind without any clashes. Especially around the threefold axes between hexons, the binding was sufficient for the flexibly linked SHP to be resolved in the EM structure. At 18 epitopes two towers of neighboring hexons are facing each other, resulting in a clash of the respective scFvs. At the 12 epitopes around the pental vertex, the viral surface is slightly tilted, which results in less clash volume. As a result, more additional shield density was observed in the EM structure at these positions.

Fig. 7 Projection of crystal structure explains binding stoichiometry on viral surface. a Structural and b schematic representation of shield occupancy and stoichiometry on viral capsid. One triangular capsid face is formed by three asymmetric units (AU, black outlines, consisting each of one pentameric subunit (yellow) and four hexon trimers (different shades of blue)). Hexons adjacent to the black outline belong to the neighboring facet. For the shielded surface, all scFvs were placed onto the hexons according to the crystal structure. At the threefold symmetry axes between neighboring hexons, a complete trivalent scFv-SHP could bind. The projection of the structure predicted a clash if the tower of two neighboring hexons face each other (orange scFvs or circles). At the vertices close to the penton, the surface is bent, which results in only a minor clash (red scFvs and circles) Full size image

Shield keeps virus infectivity and inhibits neutralizing Abs

We next investigated whether the artificial trimeric protein shield prevented virus neutralization. A single-cell transduction analysis of A549 cells using high-throughput microscopy showed an efficient reduction in the infectivity of the unshielded HAdV5HVR7 when mAb 9C12 was added, consistent with the reported ADIN16. This neutralization was significantly blocked by the shield (Fig. 8a). We next analyzed the effect of the shield in the context of the retargeting adapter. Single-cell transduction analysis of SKOV3.ip cells with HER2-retargeted virus confirmed the efficacy of the shield against antibody-mediated neutralization (Fig. 8b). It has been shown that this ADIN-mediated viral degradation is proteasome dependent16 for HAdV5wt using the CAR pathway. Inhibition of the proteasome with MG132 in HER2-overexpressing SKBR3 cells diminished ADIN-mediated viral degradation, indicating proteasome dependency for HER2-retargeted virions as well (Supplementary Fig. 9a)16. The retargeting of a shielded virus was further studied in a panel of different tumor cell lines (Fig. 8c). The shielded virus efficiently infected EGFR- and HER2-overexpressing cancer cells, demonstrating again that the shield does not sterically interfere with adapter-mediated receptor binding nor does it prevent virion uptake. In A431 cells the payload expression was slightly reduced upon shielding (Supplementary Fig. 9b). The analysis of cell binding and entry with A488-labeled EGFR-retargeted virus particles suggested a reduction in cell binding of the shielded virions compared to the control virions in this instance (Supplementary Fig. 9c, d). The underlying reasons are so far unknown. Importantly, however, the antibody 9C12 did not affect the payload expression from the shielded virus, in contrast to the non-shielded virus (Supplementary Fig. 9b).

Fig. 8 Shielding of HAdV5HVR7 reduces neutralization through immune system. a, b Single-cell analysis of GFP reporter by HT-microscopy indicated more than 95% reduction of gene delivery with HAdV5 in the presence of anti-hexon mAb 9C12. If virus was shielded by the trivalent scFv, HAdV5 infection of a A549 cells or infection of b SKOV3.ip cells was not significantly affected by the 9C12 antibody (mean ± SD, two-sided, unpaired Welch’s t-test, n = 2, *P < 0.05). For each condition, around 3000 cells were analyzed. c Antibody-dependent neutralization of HAdV5HVR7 can be blocked by the trivalent shield as shown by infection assays in A431 (EGFR targeting), SKOV3.ip (HER2-targeting), or BT474 (HER2-targeting) cells (mean ± SD, two-sided, unpaired Welch’s t-test, n=2-3, *P < 0.05, **P < 0.01). d Incubation of virus with wt mouse serum reduced viral gene delivery to 45% compared to Rag1-/- serum (set to 100%), while the shielded virus was only slightly affected by the wt serum (84%) (mean normalized to Rag1-/- ctrl ± SD, two-way ANOVA of normalized data with post hoc Bonferoni, n = 4, *P < 0.05, ****P < 0.0001). e Analysis of whether neutralization by human serum is due to hexon-specific antibodies. Transduction was performed with or without soluble hexon as competitor for such antibodies; presence of 0.88 µM hexon was set to 100%. Unshielded HAdV5HVR7 is neutralized by human serum in the presence of hexon binders, i.e., when they are not removed, but the transduction of the shielded virus is similar in the presence or absence of hexon-specific binders in human serum (mean normalized to hexon-depleted condition ± SD, two-way ANOVA of normalized data, n = 4, **P < 0.01) Full size image

Fig. 9 Shielding with retargeting reduces off-targeting and increases tumor–liver ratio. a 1.5 × 106 HAdV5HVR7 particles were injected into subcutaneous HER2+ SKOV3.ip tumors in Rag1-/- mice. Gene delivery was analyzed 48 hpi by luciferase activity, normalized to total protein amount. Fiber knob blocking of a shielded virus reduces viral gene delivery to the tumor only slightly compared to virus with free knob. In contrast, HER2 retargeting significantly increases gene delivery within the tumor by a factor of around 40 compared to the fiber knob-blocked virus. Shielding in combination with a blocked fiber knob significantly reduces gene delivery to the liver and all other organs, compared to non-modified virus. Presented data are representative for two independent experiments. b Shielding and retargeting significantly increases the tumor-to-liver ratio of gene delivery from ca. 1300 to ca. 1.1-million-fold, an improvement by a factor of ca. 900. Tumor-to-liver ratios of individual mice from two independent experiments are presented. c Retargeting of a shielded virus increased viral gene delivery to the tumor after intratumoral injection in A431 xenografts. In addition, off-targeting to liver and spleen was reduced. d The ratio of transgene activity between tumor and liver was increased from 300 for the unmodified virus to 9200 for the retargeted and shielded virus. Tumor-to-liver ratios of individual mice from two independent experiments are presented. e Intravenous delivery: in comparison to unmodified virus, shielding of an EGFR-targeted virus decreased the gene delivery to the tumor slightly upon intravenous virus injection, but drastically reduced off-targeting to all organs analyzed. f Systemic biodistribution was changed by the shield. The tumor–liver ratio in the A431 xenograft was increased by more than 2500-fold (each symbol represents (a, c, e) one organ analyzed with two-way ANOVA of log-transformed data or (b, d, f) the ratio of an individual with two-sided, unpaired Welch’s t-test of log-transformed data, *P < 0.05, **P < 0.01, ****P < 0.0001). Background signals from control injections with PBS are indicated by dashed lines Full size image

Next, we analyzed the effect of the shield against neutralizing factors other than mAb 9C12. It has been described that preexisting germline IgM antibodies were sufficient to activate the complement cascade and hence inactivate viral particles, especially if FX binding was ablated19. Normal mouse serum indeed resulted in a significant decrease of infectivity to 45%, compared to Ig-deficient Rag1-/- serum in case of the HAdV5HVR7, which was recovered to 84% by the shield (Fig. 8d).

HAdV5 is a common human pathogen with a serum prevalence of 95%44. We thus tested the ability of the shield to block neutralization of HAdV5 by human sera. To assess the impact of anti-hexon antibodies on virus transduction, we depleted anti-hexon antibodies by competition with soluble hexon protein. The transduction of the non-shielded HAdV5HVR7 is reduced to 38% by human serum, compared to the level after depletion of hexon binders. Strikingly, the infectivity of the shielded virus was not reduced by the hexon binders, indicating that immune-epitopes on the hexon were sufficiently blocked by the shield (Fig. 8e).

Shield reduces viral gene delivery to liver and spleen

We then studied the effect of the shield on viral biodistribution in an EGFR and HER2 xenograft model in immunodeficient Rag1-/- mice. In the subcutaneous SKOV3.ip xenograft, the combination of non-targeted fiber knob blocking and shielding of the virus reduced the gene delivery within the tumor by a factor of 10, compared to unmodified HAdV5HVR7 upon intratumoral injection (Fig. 9a). In contrast, the HER2-retargeting adapter significantly increased the tumor-specific gene delivery of the shielded HAdV5HVR7 by approximately 40-fold compared to the fiber knob-blocked shielded virus, or 3-fold better than unmodified HAdV5HVR7.

Importantly, the shielding and fiber knob blocking, independent of the retargeting DARPin, significantly reduced off-targeting to all other tissues to background levels. The increased gene delivery within the tumor and strong reduction in liver off-targeting led to an improved tumor-to-liver ratio of 1.1 million for the HER2-retargeted, shielded virus compared to a ratio of 1300 for the unmodified HAdV5HVR7 upon intratumoral injection (Fig. 9b), and thus about a 1000-fold improvement.

Similar results were achieved in the EGFR-positive subcutaneous A431 xenograft model after intratumoral administration. The combination of shielding and EGFR retargeting resulted in a fivefold increased gene delivery to the tumor (Fig. 9c). At the same time, the off-targeting to the liver and spleen was reduced through the shield by a factor of 260 and 6, respectively. While direct application of HAdV5HVR7 to the subcutaneous tumor led to a tumor-to-liver ratio of 300 of the transgene signal, the shielded EGFR-retargeted HAdV5HVR7 resulted in 9200 times higher payload activity in the tumor, in both cases upon intratumoral injection (Fig. 9d). Immunohistology confirmed that the shielded and retargeted HAdV5HVR7 infects tumor cells in a similar manner to the unshielded, retargeted virion (Supplementary Fig. 3b and Fig. 3).

After intravenous application into tumor-bearing Rag1-/- mice, we could confirm the very strong liver tropism of HAdV5HVR7 virus (Fig. 9e). Strikingly, a shielded, EGFR-retargeted HAdV5HVR7 showed a robust reduction in liver off-targeting by a factor of approximately 14,000. While also the tumor targeting was reduced (×5), the off-targeting to spleen (×11), kidney (×307) and lung (×26) was more strongly decreased. The liver scavenging resulted in a tumor-to-liver ratio of 0.001 for unshielded HAdV5HVR7 (Fig. 9f). However, the shielded and EGFR-retargeted virus showed a tumor-to-liver ratio of about 3, constituting an increase by a factor of more than 2500 (Fig. 9e, f). Similar effects were observed in the HER2 xenograft model upon intravenous administration (Supplementary Fig. 10a, b). Here, the liver targeting was massively decreased upon retargeting and shielding, which led to a tumor-to-liver ratio of 2, a 300-fold improvement compared to untargeted and unshielded HAdV5HVR7.