Nanobody-targeted photodynamic therapy induces significant tumor regression of trastuzumab-resistant HER2-positive breast cancer, after a single treatment session

Abstract

Rationale A substantial number of breast cancer patients with an overexpression of the human epidermal growth factor receptor 2 (HER2) have residual disease after neoadjuvant therapy or become resistant to trastuzumab. Photodynamic therapy (PDT) using nanobodies targeted to HER2 is a promising treatment option for these patients. Here we investigate the in vitro and in vivo antitumor efficacy of HER2-targeted nanobody-photosensitizer (PS) conjugate PDT. Methods Nanobodies targeting HER2 were obtained from phage display selections. Monovalent nanobodies were engineered into a biparatopic construct. The specificity of selected nanobodies was tested in immunofluorescence assays and their affinity was evaluated in binding studies, both performed in a panel of breast cancer cells varying in HER2 expression levels. The selected HER2-targeted nanobodies 1D5 and 1D5-18A12 were conjugated to the photosensitizer IRDye700DX and tested in in vitro PDT assays. Mice bearing orthotopic HCC1954 trastuzumab-resistant tumors with high HER2 expression or MCF-7 tumors with low HER2 expression were intravenously injected with nanobody-PS conjugates. Quantitative fluorescence spectroscopy was performed for the determination of the local pharmacokinetics of the fluorescence conjugates. After nanobody-PS administration, tumors were illuminated to a fluence of 100 J∙cm -2 , with a fluence rate of 50 mW∙cm -2 , and thereafter tumor growth was measured with a follow-up until 30 days. Results The selected nanobodies remained functional after conjugation to the PS, binding specifically and with high affinity to HER2-positive cells. Both nanobody-PS conjugates potently and selectively induced cell death of HER2 overexpressing cells, either sensitive or resistant to trastuzumab, with low nanomolar LD 50 values. In vivo , quantitative fluorescence spectroscopy showed specific accumulation of nanobody-PS conjugates in HCC1954 tumors and indicated 2 h post injection as the most suitable time point to apply light. Nanobody-targeted PDT with 1D5-PS and 1D5-18A12-PS induced significant tumor regression of trastuzumab-resistant high HER2 expressing tumors, whereas in low HER2 expressing tumors only a slight growth delay was observed. Conclusion Nanobody-PS conjugates accumulated selectively in vivo and their fluorescence could be detected through optical imaging. Upon illumination, they selectively induced significant tumor regression of HER2 overexpressing tumors with a single treatment session. Nanobody-targeted PDT is therefore suggested as a new additional treatment for HER2-positive breast cancer, particularly of interest for trastuzumab-resistant HER2-positive breast cancer. Further studies are now needed to assess the value of t...

Nanobody: A Small Antibody with Big Implications for Tumor Therapeutic Strategy

“… 140 The junction which might selectively accumulate in vivo between Anti-HER2 nanobody and PS can give rise to striking inhibitory effect on especially against trastuzumab HER2 + breast carcinoma with a single course of therapy, the tumor extinction was positive dose dependent, which will greatly enhance specifical targeting for photothermal anticancer agents. 141 The combination of molecular imaging and tumor therapy in the connection application of Anti-EGFR univalent or multivalent Nb and traceable PS (IRDye-700DX) is major progress in the clinical application of PDT technology. 142 …”

The Potential of Nanobody-Targeted Photodynamic Therapy to Trigger Immune Responses

“…In the context of PDT, this allows the application of the light shortly after conjugate administration, ensures more extensive tumor damage, and reduces the chances of phototoxicity present in both PDT and antibody-targeted PDT protocols. In vitro, NB-PDT has been reported to be a very specific, selective, and potent approach to kill cancer cells expressing a variety of membrane receptors, such as epidermal growth factor receptor (EGFR) [7,8], human epidermal growth factor receptor 2 (HER2) [12], hepatocyte growth factor receptor (c-Met) [13], and G proteincoupled receptor (GPCR) [14]. Furthermore, NB-PDT has proven very effective in inducing tumor necrosis in an orthotopic mouse model of head and neck cancer expressing EGFR [7], and to induce significant tumor regression in an orthotopic mouse model of breast cancer expressing HER2 [12].…”

“…In vitro, NB-PDT has been reported to be a very specific, selective, and potent approach to kill cancer cells expressing a variety of membrane receptors, such as epidermal growth factor receptor (EGFR) [7,8], human epidermal growth factor receptor 2 (HER2) [12], hepatocyte growth factor receptor (c-Met) [13], and G proteincoupled receptor (GPCR) [14]. Furthermore, NB-PDT has proven very effective in inducing tumor necrosis in an orthotopic mouse model of head and neck cancer expressing EGFR [7], and to induce significant tumor regression in an orthotopic mouse model of breast cancer expressing HER2 [12]. Additionally, similar to the observations with conventional PDT, damage to the tumor vasculature induced by NB-PDT has also been reported [15].Subsequently, we aimed to explore the third component of responses triggered by PDT, i.e., whether NB-PDT can also trigger the immune system, possibly leading to an antitumor response, which would greatly add to the potential that NB-PDT has shown so far.…”

Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy

“…Follow-up studies will aim at the efficacy evaluation of the conjugates in mice with human tumors. In this context, the cancer cell targeted nanobody–PS conjugates are expected to efficiently distribute through tumors and allow light application 1–2 h after intravenous injection [ 19 , 20 ], while these VEGFR2 nanobody–PS conjugates are expected to interact and be taken up by the (mouse) tumor vasculature. Of note, the time point post injection at which light will be applied will be carefully determined, aiming at the most practical and effective protocol.…”

“…In a more recent study, HER2 targeted nanobody–PS conjugates were injected intravenously in HER2-positive breast cancer orthotopic mouse tumor model. Illumination 2 h later induced significant tumor regression after a single nanobody-targeted PDT treatment [ 20 ].…”