Photodynamic therapy is a treatment involving three key components: a photosensitiser, light, and tissue oxygen. It can be used as a stand-alone modality or in combination with surgery, radiotherapy, chemotherapy, or anti-angiogenic therapy  for cancer. However, ntPDT can cause severe side effects due to the poor selectivity of photosensitisers [18, 19]. To overcome the poor selectivity and improve the effect, targeting of the photosensitisers with antibody and ligand has been tested [20–24, 34–36], with the hope of specifically targeting the diseased cells and then internalising the photosensitisers into the target cells.
In this paper, we target the receptor TF for the development of a novel fVII-targeted verteporfin PDT. The reason for targeting TF is based on its over-expression in many types of cancer cells, including solid cancers [1–6, 12, 25, 37] and leukaemia [38–40], and very importantly its selective expression in pathological neovascular endothelial cells in cancer [1, 2, 4], choroidal neovasculature [7, 8] and endometriosis  but not on normal VECs [1, 2, 4, 7, 8]. Garen and Hu have targeted TF using its natural ligand fVII for the development of Icon (fVII/IgG1 Fc) immunotherapy [2–4], which showed dramatic efficacy for treatment of several types of cancer [2–5], wMD [7, 8] and endometriosis  in animal models. The results in Figure 2B confirmed that TF expression was only detected on VEGF-stimulated angiogenic HUVECs but not on unstimulated quiescent normal HUVECs. The results in Figure 2B also showed that fVII-containing mouse Icon could bind to angiogenic HUVECs but not normal resting HUVECs, providing the basis and rationale that fVII-tPDT that could selectively kill angiogenic VECs but have no side effects on unstimulated HUVECs (Figure 3D), whereas ntPDT could not distinguish angiogenic VECs from normal VECs for killing (Figure 3E). The reasons for using fVII instead of making antibodies to TF are that: (i) the affinity (Kda) of fVII to TF (10-12 M)  is far higher than that of antibodies to TF (10-8 to 10-9 M)[41, 42], and (ii) therapeutic and diagnostic fVII-containing proteins can be made in human sequence by DNA recombinant technologies for future clinical trials and usages in murine sequence for tests in preclinical studies.
To develop fVII-tPDT, we first tested and conjugated dimeric Icon protein (fVII/IgG1 Fc, molecular weight ~210 kDa, unpublished data) with various photosensitisers in our preliminary studies. For better penetration into tumour tissues, we decided to make a smaller monomeric fVII protein fused with S-tag  and His-tag (molecular weight = 52,800 daltons, Figure 1B). Both the S-tag and His-tag can be used for purification and detection. In addition, since the conjugation of photosensitisers to targeting antibodies or ligands could possibly reduce or even abolish the binding activity of targeting proteins, Alan Garen and Zhiwei Hu proposed to use S protein/S peptide  as a delivery system for the development of targeted PDT, in which S peptide is synthesised with the targeting protein molecules, e.g., mfVII in this case, by a recombinant DNA technique and then commercially available S protein is conjugated with the photosensitisers (unpublished data). Since conjugation of VP to mfVII did not affect the fVII binding activities to cancer cells (Figure 1D-1E), it was not necessary to use the S protein/S peptide system as the two components delivery system for the development of VP tPDT in this paper. Nevertheless, this S protein/S peptide system could be useful for those targeting molecules if their binding activities are reduced or abolished after direct conjugation with photosensitisers for tPDT.
Visudyne is approved for the treatment of wet macular degeneration in clinical settings and is being tested as ntPDT for cancer in preclinical studies . In our pilot studies, we first conjugated fVII protein with Visudyne using EDC, which had been used in combination with NHS (N-hydroxysuccinimide) to conjugate a scFv antibody to SnCe6 . We observed that free Visudyne could not be separated from the conjugated dye by Sephadex G50 spin columns. This indicated that the other components in the liposomal formulation of VP could interfere with the separation of unconjugated VP from the conjugated dye. Therefore, it was necessary to extract the active component of benzoporphyrin derivative (BPD-MA) from Visudyne, which is referred to here as VP. When using the extracted VP, free VP was held in the size exclusion Sephadex G50 spin columns when PBS buffer alone was added to the EDC-activated VP reaction (PBS-VP control in Figure 1C). This was the reason for the lack of a Q-band absorbance peak in the PBS-VP control after being separated by the spin column (Figure 1C). A better source of VP for the conjugation reaction, of course, is a chemically synthesised pure formulation of VP (BPD-MA), which was not available to us at the time when we carried out the experiments reported here.
Nevertheless, VP either extracted from Visudyne or synthesised in pure formulation has been targeted with homing peptide for VEGF receptor or single chain Fv antibodies for treatment of wMD  and cancer [34, 36], respectively. Although those previous papers used a different conjugation procedure, the molar ratio of VP to scFv was 14.1:1 [34, 36], which is similar to the ratios of VP to mfVII (13.1 ± 2.6:1, mean ± SD from 15 separate conjugation reactions). However, Bhatti et al. noticed that there was 30% of non-covalent binding VP present in the final conjugates , possibly due to inefficient separation of free dye by the use of a dialysis procedure. Considering that the molecular weight of mfVII protein (52.8 kDa) is bigger than that of scFv (30 kDa) , our results of VP-to-fVII ratios were not surprising. Bhatti et al. also found that the IC50 of VP (1.4 μM) in anti-HER2 C6.5 scFv-targeted VP PDT was about four-fold less than that (5.4 μM) in free VP PDT for the HER 2+ SKOV-3 ovarian cancer line, indicating that scFv targeting improved the effect of VP PDT for that cancer line in vitro by about four fold. Their observations were similar to our results, although the EC50 of VP in fVII-tPDT (0.25 μM for human breast cancer MDA-MB-231 and 0.33 μM for murine breast cancer EMT6 in Figure 4A-4B) was about one fifth or one third of that (1.4 μM) in the previous paper . In vivo studies were not done with scFv-VP or free VP PDT in the previous papers [34, 36]. In this paper we showed that fVII-tPDT had a significantly stronger effect than ntPDT for the treatment of breast cancer in mice (Figure 6A). The reason that ntPDT did not have the effect in vivo but had the effect in vitro is probably because liposomal components had been removed, so that the transportation of liposome-free VP to the tumour mass by intravenous injection was not as efficient as that of the liposomal formulation of Visudyne, although there was no problem for transportation because VP was directly incubated with the test cells. Further increasing the VP concentration in the fVII-VP conjugate did not further increase the effect of fVII-tPDT in the current setting, probably because fVII even at the lower concentration had reached the saturation binding to the tumour vasculature. Therefore, it is possible to get stronger effect by increasing the laser light fluence (but not VP concentration) in future experiments. Taking the in vitro and in vivo results reported in this paper together with the major findings in our recent paper on fVII-tPDT for wMD , we conclude that this novel fVII-targeted VP PDT improves the selectivity and efficacy of free VP PDT for the treatment of breast cancer and wMD and induces apoptosis and necrosis as the underlining mechanisms of action.
For future clinical trials, we have constructed human fVII proteins with a lys341Ala mutation, similarly by recombinant DNA technology, for fVII-tPDT for patients with cancer or wMD. To establish additional proof of principle, our laboratory has developed and tested another fVII-tPDT using another potent photosensitiser, Sn(IV) chlorin e6, and the results showed that fVII-targeted SnCe6 PDT was more selective and also safe for the treatment of breast and lung cancer in vitro and in vivo in mice (Hu et al. Breast Cancer Research and Treatment. In press).