Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer (PS) and has been used to treat head and neck cancers. Yet, common PS lack tumor specificity, which leads to collateral damage to normal tissues. Targeted delivery of PS via antibodies has pre-clinically improved tumor selectivity. However, antibodies have long half-lives and relatively poor tissue penetration, which could limit therapeutic efficacy and lead to long photosensitivity. Here, in this feasibility study, we evaluate at the pre-clinical level a recently introduced format of targeted PDT, which employs nanobodies as targeting agents and a water-soluble PS (IRDye700DX) that is traceable through optical imaging. In vitro, the PS solely binds to cells and induces phototoxicity on cells overexpressing the epidermal growth factor receptor (EGFR), when conjugated to the EGFR targeted nanobodies. To investigate whether this new format of targeted PDT is capable of inducing selective tumor cell death in vivo, PDT was applied on an orthotopic mouse tumor model with illumination at 1h post-injection of the nanobody-PS conjugates, as selected from quantitative fluorescence spectroscopy measurements. In parallel, and as a reference, PDT was applied with an antibody-PS conjugate, with illumination performed 24h post-injection. Importantly, EGFR targeted nanobody-PS conjugates led to extensive tumor necrosis (approx. 90%) and almost no toxicity in healthy tissues, as observed through histology 24h after PDT. Overall, results show that these EGFR targeted nanobody-PS conjugates are selective and able to induce tumor cell death in vivo. Additional studies are now needed to assess the full potential of this approach to improving PDT.

Background: Photoimmunodetection, in which monoclonal antibodies [mAbs] are labeled with fluorescent dyes, might have clinical potential for early detection and characterization of cancer. For this purpose, the dye should be coupled in an inert way to mAb. In this study, different equivalents of IRDye800CW, a near-infrared fluorescent dye, were coupled to ⁸⁹Zr-labeled cetuximab and bevacizumab, and conjugates were evaluated in biodistribution studies. Radiolabeled mAbs were used to allow accurate quantification for assessment of the number of dye groups that can be coupled to mAbs without affecting their biological properties. Methods: ⁸⁹Zr-cetuximab and ⁸⁹Zr-bevacizumab, containing 0.5 ⁸⁹Zr-desferal group per mAb molecule, were incubated with 1 to 10 eq IRDye800CW at pH 8.5 for 2 h at 35°C, and ⁸⁹Zr-mAb-IRDye800CW conjugates were purified by a PD10 column using 0.9% NaCl as eluent. HPLC analysis at 780 nm was used to assess conjugation efficiency. In vitro stability measurements were performed in storage buffer (0.9% NaCl or PBS) at 4°C and 37°C and human serum at 37°C. ⁸⁹Zr-mAb-IRDye800CW conjugates and ⁸⁹Zr-mAb conjugates (as reference) were administered to nude mice bearing A431 (cetuximab) or FaDu (bevacizumab) xenografts, and biodistribution was assessed at 24 to 72 h after injection. Results: Conjugation efficiency of IRDye800CW to ⁸⁹Zr-mAbs was approximately 50%; on an average, 0.5 to 5 eq IRDye800CW was conjugated. All conjugates showed optimal immunoreactivity and were > 95% stable in storage buffer at 4°C and 37°C and human serum at 37°C for at least 96 h. In biodistribution studies with ⁸⁹Zr-cetuximab-IRDye800CW, enhanced blood clearance with concomitant decreased tumor uptake and increased liver uptake was observed at 24 to 72 h post-injection when 2 or more eq of dye had been coupled to mAb. No significant alteration of biodistribution was observed 24 to 48 h after injection when 1 eq of dye had been coupled. ⁸⁹Zr-bevacizumab-IRDye800CW showed a similar tendency, with an impaired biodistribution when 2 eq of dye had been coupled to mAb. Conclusion: Usage of ⁸⁹Zr-mAbs allows accurate quantification of the biodistribution of mAbs labeled with different equivalents of IRDye800CW. Alteration of biodistribution was observed when more than 1 eq of IRDye800CW was coupled to mAbs.

SARS-CoV-2 causes acute respiratory disease, but many patients also experience neurological complications. Neuropathological changes with pronounced neuroinflammation have been described in individuals after lethal COVID-19, as well as in the CSF of hospitalized patients with neurological complications. To assess whether neuropathological changes can occur after a SARS-CoV-2 infection, leading to mild-to-moderate disease, we investigated the brains of four rhesus and four cynomolgus macaques after pulmonary disease and without overt clinical symptoms. Postmortem analysis demonstrated the infiltration of T-cells and activated microglia in the parenchyma of all infected animals, even in the absence of viral antigen or RNA. Moreover, intracellular α-synuclein aggregates were found in the brains of both macaque species. The heterogeneity of these manifestations in the brains indicates the virus’ neuropathological potential and should be considered a warning for long-term health risks, following SARS-CoV-2 infection.

Safe and effective coronavirus disease–19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1–skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen.

The post-acute phase of SARS-CoV-2 infection was investigated in rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). During the acute phase of infection, SARS-CoV-2 was shed via the nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, computed tomography (CT) and positron emission tomography (PET)-CT revealed pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, such as conjunctiva, cervical, and mesenteric lymph nodes. However, 5–6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans.