Anti-HER2 VHH Targeted Fluorescent Liposome as Bimodal Nanoparticle for Drug Delivery and Optical Imaging.

OBJECTIVES: The aim of this study was to formulate fluorescent-labeled targeted immunoliposome to visualize the delivery and distribution of drugs in real-time. METHODS: In this study, fluorescent-labeled liposomes were decorated with anti-HER2 VHH or Herceptin to improve the monitoring of intracellular drug delivery and tumor cell tracking with minimal side effects. The conjugation efficiency of antibodies was analyzed by SDS-PAGE silver staining. In addition, the physicochemical characterization of liposomes was performed using DLS and TEM. Finally, confocal microscopy visualized nanoparticles in the target cells. RESULTS: Quantitative and qualitative methods characterized the intracellular uptake of 110±10 nm particles with near 70% conjugation efficiency. In addition, live-cell trafficking during hours of incubation was monitored by wide-field microscopy imaging. The results show that the fluorescent- labeled nanoparticles can specifically bind to HER2-positive breast cancer with minimal off-target delivery. CONCLUSION: These nanoparticles can have several applications in personalized medicine, especially drug delivery and real-time visualization of cancer therapy. Moreover, this method also can be applied in the targeted delivery of contrast agents in imaging and thermotherapy.

Immuno-hyperthermia effected by antibody-conjugated nanoparticles selectively targets and eradicates individual cancer cells.

Hyperthermia has been used for cancer therapy for a long period of time, but has shown limited clinical efficacy. Induction-heating hyperthermia using the combination of magnetic nanoparticles (MNPs) and an alternating magnetic field (AMF), termed magnetic hyperthermia (MHT), has previously shown efficacy in an orthotopic mouse model of disseminated gastric cancer. In the present study, superparamagnetic iron oxide nanoparticles (SPIONs), a type of MNP, were conjugated with an anti-HER2 antibody, trastuzumab and termed anti-HER2-antibody-linked SPION nanoparticles (anti-HER2 SPIONs). Anti-HER2 SPIONs selectively targeted HER2-expressing cancer cells co-cultured along with normal fibroblasts and HER2-negative cancer cells and caused apoptosis only in the HER2-expressing individual cancer cells. The results of the present study show proof-of-concept of a novel hyperthermia technology, immuno-MHT for selective cancer therapy, that targets individual cancer cells.Abbreviations: AMF: alternating magnetic field; DDW: double distilled water; DMEM: Dulbecco's Modified Eagle's; Medium; f: frequency; FBS: fetal bovine serum; FITC: Fluorescein isothiocyanate; GFP: green fluorescent protein; H: amplitude; Hsp: heat shock protein; MHT: magnetic hyperthermia; MNPs: magnetic nanoparticles; PI: propidium iodide; RFP: red fluorescent protein; SPION: superparamagnetic iron oxide (Fe3O4) nanoparticle.

Switchable CAR-T Cells Outperformed Traditional Antibody-Redirected Therapeutics Targeting Breast Cancers.

Various antibody-redirected immunotherapeutic approaches, including antibody-drug conjugates (ADCs), bispecific antibodies (bsAbs), and chimeric antigen receptor-T (CAR-T) cells, have been devised to produce specific activity against various cancer types. Using genetically encoded unnatural amino acids, we generated a homogeneous Her2-targeted ADC, a T cell-redirected bsAb, and a FITC-modified antibody capable of redirecting anti-FITC CAR-T (switchable CAR-T; sCAR-T) cells to target different Her2-expressing breast cancers. sCAR-T cells showed activity against Her2-expressing tumor cells comparable to that of conventional anti-Her2 CAR-T cells and superior to that of ADC- and bsAb-based approaches. To prevent antigen escape, we designed bispecific sCAR-T cells targeting both the Her2 receptor and IGF1R, which showed an overall improved activity against cancer cells with low Her2 expression. This study increases our understanding of various explored cancer therapeutics and underscores the efficient application of sCAR-T cells as a promising therapeutic option for breast cancer patients with low or heterogeneous antigen expression.

Generation and testing of engineered multimeric Fabs of trastuzumab.

Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules. Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E. coli and purified. Multimeric Fabs were generated by either disulfide bridge formation or by using MTG-sensitive peptide linkers. Binding to receptor was assessed by ELISA and SPR methods. Internalization and growth inhibition assays were performed on BT-474 and SKBR3 Her2+ cells. Fabs were successfully produced and dimerized or trimerized using MTG and suitably designed peptide linkers. Site-specific derivatizations with fluorophores were similarly achieved. The monomeric, dimeric and trimeric variants bind the receptor with affinities similar or superior to the full antibody. Fab and Fab2 are rapidly internalized in Her2+ cells and exhibit growth inhibition abilities similar to the full antibody. Altogether, the data show that the recombinant Fabs can be produced in E. coli and converted into multimeric variants by MTG-based bioconjugation. Similar approaches are extendable to the introduction of cytotoxic payloads for the generation of novel Antibody Drug Conjugates.

Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer.

Hormone receptor (HR)+ breast cancer (BC) causes most BC-related deaths, calling for improved therapeutic approaches. Despite expectations, immune checkpoint blockers (ICBs) are poorly active in patients with HR+ BC, in part reflecting the lack of preclinical models that recapitulate disease progression in immunocompetent hosts. We demonstrate that mammary tumors driven by medroxyprogesterone acetate (M) and 7,12-dimethylbenz[a]anthracene (D) recapitulate several key features of human luminal B HR+HER2- BC, including limited immune infiltration and poor sensitivity to ICBs. M/D-driven oncogenesis is accelerated by immune defects, demonstrating that M/D-driven tumors are under immunosurveillance. Safe nutritional measures including nicotinamide (NAM) supplementation efficiently delay M/D-driven oncogenesis by reactivating immunosurveillance. NAM also mediates immunotherapeutic effects against established M/D-driven and transplantable BC, largely reflecting increased type I interferon secretion by malignant cells and direct stimulation of immune effector cells. Our findings identify NAM as a potential strategy for the prevention and treatment of HR+ BC.

Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody.

Systemic cytokine release and on-target/off-tumor toxicity to normal tissues are the main adverse effects limiting the clinical utility of T cell-redirecting therapies. This study was designed to determine how binding affinity for CD3 and tumor target HER2 impact the efficacy and nonclinical safety of anti-HER2/CD3 T cell-dependent antibodies (TDBs). Affinity was found to be a major determinant for the overall tolerability. Higher affinity for CD3 associated with rapidly elevated peripheral cytokine concentrations, weight loss in mice, and poor tolerability in cynomolgus monkeys. A TDB with lower CD3 affinity was better tolerated in cynomolgus monkeys compared with a higher CD3-affinity TDB. In contrast to tolerability, T cell binding affinity had only limited impact on in vitro and in vivo antitumor activity. High affinity for HER2 was critical for the tumor-killing activity of anti-HER2/CD3 TDBs, but higher HER2 affinity also associated with a more severe toxicity profile, including cytokine release and damage to HER2-expressing tissues. The tolerability of the anti-HER2/CD3 was improved by implementing a dose-fractionation strategy. Fine-tuning the affinities for both the tumor target and CD3 is likely a valuable strategy for achieving maximal therapeutic index of CD3 bispecific antibodies.

Near-Infrared Photoimmunotherapy Using a Small Protein Mimetic for HER2-Overexpressing Breast Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a new and promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer which is activated by near-infrared light irradiation, causing cell death. We investigated NIR-PIT using a small protein mimetic (6-7 kDa), Affibody molecules, instead of a monoclonal antibody for HER2-overexpressing cancer. Because of its small size, the Affibody has rapid clearance, high imaging contrast, and good tumor penetration. Due to the small size of the Affibodies, which can cross the blood-brain barrier, NIR-PIT using Affibodies has the potential to extend the target cancer of NIR-PIT, including brain metastases. In vitro, NIR-PIT using HER2 Affibody-IR700Dye conjugates induced the selective destruction of HER2-overexpressing breast cancer cells without damage to control cells having low level expression of HER2. HER2-overexpressing cancer cells showed necrotic cell death and their viability maintained at low levels, even 5 days after NIR-PIT. In contrast, treatment with high concentration of HER2 Affibody-IR700Dye conjugate alone or irradiation with high dose of NIR light alone was without effect on cell viability. Affibody and IR700Dye are currently used clinically, and therefore, we would expect the current formulation to be safely and quickly transitioned into clinical trials.

Photothermal and gene therapy combined with immunotherapy to gastric cancer by the gold nanoshell-based system.

BACKGROUND: The gastric cancer is the second most malignant tumor in the world. HER-2 is one of the key targets for the gastric cancer therapy. Anti-HER-2 antibodies like trastuzumab, exhibits the satisfactory therapeutic effect in clinical. However, the drug resistance problem limits its application. METHOD: In this study, we develop a gold nanoshell (Gold Nanoshell) drug carrier for delivery and selective photo-thermal release of genes which target HER-2 and immunologic adjuvant CPG sequence in gastric tumor cells. The drug delivery system generated a multidimensional treatment strategy which includes gene-, immune- and photothermal-therapy. RESULTS: The whole gold nanoshell drug delivery system exhibits the well gene transduction ability and combined treatment effect. Both in vitro and in vivo results demonstrate the multiple therapeutic effects of the drug delivery system is better than the monotherapy. CONCLUSIONS: This study indicates the multiple combined therapy based on the gold nanoshell system would be a promising translational treatment for gastric cancer.

Near-Infrared Photochemoimmunotherapy by Photoactivatable Bifunctional Antibody-Drug Conjugates Targeting Human Epidermal Growth Factor Receptor 2 Positive Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a new class of molecular targeted cancer therapy based on antibody-photoabsorber conjugates and NIR light irradiation. Recent studies have shown effective tumor control, including that of human epidermal growth factor receptor 2 (HER2)-positive cancer, by selective molecular targeting with NIR-PIT. However, the depth of NIR light penetration limits its use. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent maytansinoid DM1. Here, we developed bifunctional antibody-drug-photoabsorber conjugates, T-DM1-IR700, that can work as both NIR-PIT and chemoimmunotherapy agents. We evaluated the feasibility of T-DM1-IR700-mediated NIR light irradiation by comparing the in vitro and in vivo cytotoxic efficacy of trastuzumab-IR700 (T-IR700)-mediated NIR light irradiation in HER2-expressing cells. T-IR700 and T-DM1-IR700 showed almost identical binding to HER2 in vitro and in vivo. Owing to the presence of internalized DM1 in the target cells, NIR-PIT using T-DM1-IR700 tended to induce greater cytotoxicity than that of NIR-PIT using T-IR700 in vitro. In vivo NIR-PIT using T-DM1-IR700 did not show a superior antitumor effect to NIR-PIT using T-IR700 in subcutaneous small-tumor models, which could receive sufficient NIR light. In contrast, NIR-PIT using T-DM1-IR700 tended to reduce the tumor volume and showed significant prolonged survival compared to NIR-PIT using T-IR700 in large-tumor models that could not receive sufficient NIR light. We successfully developed a T-DM1-IR700 conjugate that has a similar immunoreactivity to the parental antibody with increased cytotoxicity due to DM1 and potential as a new NIR-PIT agent for targeting tumors that are large and inaccessible to sufficient NIR light irradiation to activate the photoabsorber IR700.

Fabrication, characterization, and biological evaluation of anti-HER2 indocyanine green-doxorubicin-encapsulated PEG-b-PLGA copolymeric nanoparticles for targeted photochemotherapy of breast cancer cells.

In this study, we aimed to develop anti-human epidermal growth factor receptor 2 (HER2) indocyanine green (ICG)-doxorubicin (DOX)-encapsulated polyethylene glycol-poly(lactic-co-glycolic acid) diblock copolymeric nanoparticles (HIDPPNPs) to explore the co-administration of phototherapy and chemotherapy for HER2-overexpressing breast cancer, a highly aggressive and medicine-resistant breast carcinoma. The HIDPPNPs were fabricated using a solvent evaporation technique followed by carbodiimide-mediated antibody conjugation on the nanoparticle surface. Compared with freely dissolved ICG, the HIDPPNPs conferred enhanced thermal stability to the entrapped ICG, were able to generate a hyperthermia effect at concentrations ≥1 µM ICG equivalent and provided increased production of singlet oxygen under 808-nm laser irradiation with an intensity of 6 W/cm2. Furthermore, the uptake efficiency of the HIDPPNPs in MDA-MB-453/HER2(+) cells was approximately 2-fold higher than that in MCF7/HER2(-) cells, demonstrating that the HIDPPNPs specifically target HER2-expressing cells. Based on the viability analysis, the HIDPPNPs exhibited effective cytotoxicity upon light exposure (808 nm; 6 W/cm2), and the resulting cell death rate was even higher than that caused by using twice amount of encapsulated DOX or ICG alone. These results indicate that the developed HIDPPNPs may serve as a feasible tool for use in anti-HER2 breast cancer therapy with reduced chemotoxicity.

Sorafenib combined with HER-2 targeted vaccination can promote effective T cell immunity in vivo.

The tumor microenvironment (TME) is established and maintained through complex interactions between tumor cells and host stromal elements. Therefore, therapies that target multiple cellular components of the tumor may be most effective. Sorafenib, a multi-kinase inhibitor, alters signaling pathways in both tumor cells and host stromal cells. Thus, we explored the potential immune-modulating effects of sorafenib in a murine HER-2-(neu) overexpressing breast tumor model alone and in combination with a HER-2 targeted granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting vaccine (3T3neuGM). In vitro, sorafenib inhibited the growth of HER-2 overexpressing NT2.5 tumor cells, inducing apoptosis. Sorafenib also interfered with ERK MAPK, p38 MAPK, and STAT3 signaling, as well as cyclin D expression, but did not affect HER-2 or AKT signaling. In vivo, single agent sorafenib disrupted the tumor-associated vasculature and induced tumor cell apoptosis, effectively inducing the regression of established NT2.5 tumors in immune competent FVB/N mice. Immune depletion studies demonstrated that both CD4+ and CD8+ T cells were required for tumor regression. Sorafenib treatment did not impact the rate of tumor clearance induced by vaccination with 3T3neuGM in tumor-bearing FVB/N mice relative to either sorafenib treatment or vaccination alone. In vivo studies further demonstrated that sorafenib enhanced the accumulation of both CD4+ and CD8+ T cells into the TME of vaccinated mice. Together, these findings suggest that GM-CSF-secreting cellular immunotherapy may be integrated with sorafenib without impairing vaccine-based immune responses.

Immunogenic cancer cell death selectively induced by near infrared photoimmunotherapy initiates host tumor immunity.

Immunogenic cell death (ICD) is a form of cell death that activates an adaptive immune response against dead-cell-associated antigens. Cancer cells killed via ICD can elicit antitumor immunity. ICD is efficiently induced by near-infrared photo-immunotherapy (NIR-PIT) that selectively kills target-cells on which antibody-photoabsorber conjugates bind and are activated by NIR light exposure. Advanced live cell microscopies showed that NIR-PIT caused rapid and irreversible damage to the cell membrane function leading to swelling and bursting, releasing intracellular components due to the influx of water into the cell. The process also induces relocation of ICD bio markers including calreticulin, Hsp70 and Hsp90 to the cell surface and the rapid release of immunogenic signals including ATP and HMGB1 followed by maturation of immature dendritic cells. Thus, NIR-PIT is a therapy that kills tumor cells by ICD, eliciting a host immune response against tumor.

Near-infrared photothermal therapy of Prussian-blue-functionalized lanthanide-ion-doped inorganic/plasmonic multifunctional nanostructures for the selective targeting of HER2-expressing breast cancer cells.

We report the synthesis, characterization, and application of Prussian blue (PB) functionalized CaMoO4:Eu@SiO2@Au nanorod hybrid nanoparticles (HNPs), with multimodal capabilities such as fluorescence imaging, surface-enhanced Raman spectroscopy (SERS) detection and photothermal therapy (PTT). The average size of CaMoO4:Eu@SiO2 NPs was 206 nm. The HNPs are highly dispersible in water for several weeks without settling and show a strong absorption in the near-infrared region, overlapping with the PB absorption between 600 nm and 1000 nm and the surface plasmon resonance of Au nanorods around 800 nm. Upon 808 nm laser excitation, HNPs show hyperthermia temperature (∼43 °C). Moreover, PB-functionalized NPs can be used in clinical trials for the treatment of radioactive exposure, and PB acts as a Raman reporter molecule (2152 cm-1 characteristic peak) with good biosafety and stability in the human body. In addition, coating the surface of CaMoO4:Eu NPs with both SiO2 and Au nanorods increases the biocompatibility of the HNPs. Furthermore, the PTT efficiency of human epidermal growth factor receptor 2 (HER2) antibody-conjugated HNPs on MDA-MB-435 cancerous cells was significantly higher than that of hepatocyte cells (noncancerous). This is due to the greater uptake of HNPs on cancerous cells than on noncancerous cells. Together, this study shows the potential applications of these HNPs in fluorescence imaging, SERS detection, and PTT functionalities with good photostability and biocompatibility.

Combination photoimmunotherapy with monoclonal antibodies recognizing different epitopes of human epidermal growth factor receptor 2: an assessment of phototherapeutic effect based on fluorescence molecular imaging.

Photoimmunotherapy is a new class of molecular targeted cancer therapy based on a monoclonal antibody (mAb) conjugated to a photosensitizer and irradiation with near-infrared (NIR) light for both imaging and therapy. Here, we sought to determine the feasibility of combining photoimmunotherapy using conjugates of human epidermal growth factor receptor 2 (HER2)-specific mAb-photosensitizer IR700, trastuzumab-IR700 and pertuzumab-IR700. HER2-expressing and non-expressing cells were treated with mAb-IR700 conjugates and irradiated with NIR light. Fluorescence imaging and cytotoxic effects were examined in cultured HER2-expressng cancer cell lines and in a mouse tumor xenograft model. Trastuzumab-IR700 and pertuzumab-IR700 could specifically bind to HER2 without competing, and the combination treatment of both agents yielded stronger HER2-specific IR700 fluorescence signals than with the treatment with either agent singly. A cytotoxicity assay showed that the combination treatment of both trastuzumab-IR700 and pertuzumab-IR700 followed by NIR light irradiation induced stronger cytotoxic effect than with treatment of either agent plus NIR light irradiation. Furthermore, the phototoxic and cytotoxic effects of mAb depended on HER2-specific IR700 signal intensities. Consistent with in vitro studies, in xenograft tumor models also, IR700 fluorescence imaging-guided NIR light irradiation after the combination treatment of trastuzumab-IR700 and pertuzumab-IR700 led to stronger antitumor effects than by treatment with either agent followed by NIR light irradiation. In conclusion, fluorescence molecular imaging can facilitate the assessment of treatment outcomes of molecular targeted photoimmunotherapy, which holds great potential in facilitating better outcomes in cancer patients.

Molecular targeted photoimmunotherapy for HER2-positive human gastric cancer in combination with chemotherapy results in improved treatment outcomes through different cytotoxic mechanisms.

BACKGROUND: Photoimmunotherapy (PIT) is a novel type of molecular optical imaging-guided cancer phototherapy based on a monoclonal antibody conjugated to a photosensitizer, IR700, in combination with near-infrared (NIR) light. PIT rapidly causes target-specific cell death by inducing cell membrane damages and appears to be highly effective; however, we have previously demonstrated that tumor recurrences were eventually seen in PIT-treated mice, likely owing to inhomogeneous mAb-IR700 conjugate distribution in the tumor, thus limiting the effectiveness of PIT as a monotherapy. Here, we examined the effects of human epidermal growth factor-2 (HER2)-targeted PIT in combination with 5-fluorouracil (5-FU) compared to PIT alone for HER2-expressing human gastric cancer cells. METHODS: NCI-N87 cells, HER2-positive human gastric cancer cells, were used for the experiments. Trastuzumab, a monoclonal antibody directed against HER2, was conjugated to IR700. To assess the short-term cytotoxicity and examine the apoptotic effects upon addition of 5-FU in vitro, we performed LIVE/DEAD and caspase-3 activity assays. Additionally, to explore the effects on long-term growth inhibition, trypan blue dye exclusion assay was performed. NCI-N87 tumor xenograft models were prepared for in vivo treatment studies and the tumor-bearing mice were randomized into various treatment groups. RESULTS: Compared to PIT alone, the combination of HER2-targeted PIT and 5-FU rapidly induced significant cytotoxicity in both the short-term and long-term cytotoxicity assays. While both 5-FU and/or trastuzumab-IR700 conjugate treatment induced an increase in caspase-3 activity, there was no additional increase in caspase-3 activity upon NIR light irradiation after incubation with 5-FU and/or trastuzumab-IR700. The combination of HER2-targeted PIT and 5-FU resulted in greater and longer tumor growth inhibition than PIT monotherapy in vivo. This combined effect of PIT and 5-FU is likely owing to their different mechanisms of inducing tumor cell death, namely necrotic membrane damage by PIT and apoptotic cell death by 5-FU and trastuzumab. CONCLUSIONS: PIT in combination with 5-FU resulted in enhanced antitumor effects compared to PIT alone for HER2-expressing human gastric cancer in vitro and in vivo. This combination photoimmunochemotherapy represents a practical method for treating human gastric cancer and should be investigated further in the clinical setting.

Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting.

In radioimmunotherapy, the contrast between tumor and normal tissue can be improved by using a pretargeting strategy with a primary targeting agent, which is conjugated to a recognition tag, and a secondary radiolabeled molecule binding specifically to the recognition tag. The secondary molecule is injected after the targeting agent has accumulated in the tumor and is designed to have a favorable biodistribution profile, with fast clearance from blood and low uptake in normal tissues. In this study, we have designed and evaluated two complementary peptide nucleic acid (PNA)-based probes for specific and high-affinity association in vivo. An anti-HER2 Affibody-PNA chimera, Z(HER2:342)-SR-HP1, was produced by a semisynthetic approach using sortase A catalyzed ligation of a recombinantly produced Affibody molecule to a PNA-based HP1-probe assembled using solid-phase chemistry. A complementary HP2 probe carrying a DOTA chelator and a tyrosine for dual radiolabeling was prepared by solid-phase synthesis. Circular dichroism (CD) spectroscopy and UV thermal melts showed that the probes can hybridize to form a structured duplex with a very high melting temperature (T(m)), both in HP1:HP2 and in Z(HER2:342)-SR-HP1:HP2 (T(m) = 86-88 °C), and the high binding affinity between Z(HER2:342)-SR-HP1 and HP2 was confirmed in a surface plasmon resonance (SPR)-based binding study. Following a moderately fast association (1.7 × 10(5) M(-1) s(-1)), the dissociation of the probes was extremely slow and <5% dissociation was observed after 17 h. The equilibrium dissociation constant (K(D)) for Z(HER2:342)-SR-HP1:HP2 binding to HER2 was estimated by SPR to be 212 pM, suggesting that the conjugation to PNA does not impair Affibody binding to HER2. The biodistribution profiles of (111)In- and (125)I-labeled HP2 were measured in NMRI mice, showing very fast blood clearance rates and low accumulation of radioactivity in kidneys and other organs. The measured radioactivity in blood was 0.63 ± 0.15 and 0.41 ± 0.15%ID/g for (125)I- and (111)In-HP2, respectively, at 1 h p.i., and at 4 h p.i., the kidney accumulation of radioactivity was 0.17 ± 0.04%ID/g for (125)I-HP2 and 3.83 ± 0.39%ID/g for (111)In-HP2. Taken together, the results suggest that a PNA-based system has suitable biophysical and in vivo properties and is a promising approach for pretargeting of Affibody molecules.

Near infrared photoimmunotherapy for lung metastases.

Lung metastases are a leading cause of cancer related deaths; nonetheless current treatments are limited. Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of intravenously injected antibodies that target tumors with the toxicity induced by photosensitizers activated by NIR-light. Herein, we demonstrate the efficacy of NIR-PIT in a mouse model of lung metastases. Experiments were conducted with a HER2, luciferase and GFP expressing cell line (3T3/HER2-luc-GFP). An antibody-photosensitizer conjugate (APC) consisting of trastuzumab and a phthalocyanine dye, IRDye700DX, was synthesized. In vitro NIR-PIT-induced cytotoxicity was light dose dependent. With 3D culture, repeated NIR-PIT could eradicate entire spheroids. In vivo anti-tumor effects of NIR-PIT included significant reductions in both tumor volume (p = 0.0141 vs. APC) and bioluminescence image (BLI) (p = 0.0086 vs. APC) in the flank model, and prolonged survival (p < 0.0001). BLI demonstrated a significant reduction in lung metastases volume (p = 0.0117 vs. APC). Multiple NIR-PIT doses significantly prolonged survival in the lung metastasis model (p < 0.0001). These results suggested that NIR-PIT is a potential new therapy for the local control of lung metastases.

Near infrared photoimmunotherapy prevents lung cancer metastases in a murine model.

Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of intravenously injected antibodies with the acute toxicity induced by photosensitizers after exposure to NIR-light. Herein, we evaluate the efficacy of NIR-PIT in preventing lung metastases in a mouse model. Lung is one of the most common sites for developing metastases, but it also has the deepest tissue light penetration. Thus, lung is the ideal site for treating early metastases by using a light-based strategy. In vitro NIR-PIT cytotoxicity was assessed with dead cell staining, luciferase activity, and a decrease in cytoplasmic GFP fluorescence in 3T3/HER2-luc-GFP cells incubated with an anti-HER2 antibody photosensitizer conjugate. Cell-specific killing was demonstrated in mixed 2D/3D cell cultures of 3T3/HER2-luc-GFP (target) and 3T3-RFP (non-target) cells. In vivo NIR-PIT was performed in the left lung in a mouse model of lung metastases, and the number of metastasis nodules, tumor fluorescence, and luciferase activity were all evaluated. All three evaluations demonstrated that the NIR-PIT-treated lung had significant reductions in metastatic disease (*p < 0.0001, Mann-Whitney U-test) and that NIR-PIT did not damage non-target tumors or normal lung tissue. Thus, NIR-PIT can specifically prevent early metastases and is a promising anti-metastatic therapy.

Ado-trastuzumab emtansine: a HER2-positive targeted antibody-drug conjugate.

OBJECTIVE: To review the pharmacology, pharmacokinetics, efficacy, adverse effects, drug-drug interactions, dosage and administration, and formulary considerations for ado-trastuzumab emtansine. DATA SOURCES: Sources of information were identified through a PubMed search (1966 to June 2014) using the key terms ado-trastuzumab emtansine, trastuzumab-DM1, trastuzumab-MCC-DM1, and T-DM1. Other information was obtained from clinicaltrials.gov, product labeling, and press releases. STUDY SELECTION AND DATA EXTRACTION: All English-language clinical trials and abstracts evaluating ado-trastuzumab emtansine in humans were reviewed for inclusion. DATA SYNTHESIS: Overexpression or amplification of human epidermal growth factor receptor 2 (HER2) occurs in approximately 20% of breast cancers and is associated with more aggressive tumors and poorer prognosis in the absence of treatment. Although effective therapies for the initial management of HER2-positive metastatic breast cancer (MBC) exist, many patients will experience disease progression. Most second-line therapies are associated with either significant toxicities or limited improvements in overall survival (OS). Ado-trastuzumab emtansine is a HER2-positive directed antibody drug conjugate (ADC) approved in February 2013. In phase III clinical trials comparing the efficacy and safety of ado-trastuzumab emtansine with lapatinib-capecitabine or physician's choice, ado-trastuzumab emtansine had a better tolerability profile and improved progression-free survival compared with lapatinib-capecitabine or physician's choice and increased OS compared with lapatinib-capecitabine. CONCLUSION: Ado-trastuzumab emtansine is a novel ADC effective for HER2-positive MBC in patients previously treated with trastuzumab, lapatinib, and a taxane. Further studies will determine its use in the adjuvant and neoadjuvant setting and in combination with pertuzumab.

Immuno nanoparticles integrated electrical control of targeted cancer cell development using whole cell bioelectronic device.

Electrical properties of cells determine most of the cellular functions, particularly ones which occur in the cell's membrane. Manipulation of these electrical properties may provide a powerful electrotherapy option for the treatment of cancer as cancerous cells have been shown to be more electronegative than normal proliferating cells. Previously, we used an electrical impedance sensing system (EIS) to explore the responses of cancerous SKOV3 cells and normal HUVEC cells to low intensity (<2 V/cm) AC electric fields, determining that the optimal frequency for SKOV3 proliferation arrest was 200 kHz, without harming the non-cancerous HUVECs. In this study, to determine if these effects are cell type dependant, human breast adenocarcinoma cells (MCF7) were subjected to a range of frequencies (50 kHz-2 MHz) similar to the previously tested SKOV3. For the MCF7, an optimal frequency of 100 kHz was determined using the EIS, indicating a higher sensitivity towards the applied field. Further experiments specifically targeting the two types of cancer cells using HER2 antibody functionalized gold nanoparticles (HER2-AuNPs) were performed to determine if enhanced electric field strength can be induced via the application of nanoparticles, consequently leading to the killing of the cancerous cells without affecting non cancerous HUVECs and MCF10a providing a platform for the development of a non-invasive cancer treatment without any harmful side effects. The EIS was used to monitor the real-time consequences on cellular viability and a noticeable decrease in the growth profile of the MCF7 was observed with the application of the HER2-AuNPs and the electric fields indicating specific inhibitory effects on dividing cells in culture. To further understand the effects of the externally applied field to the cells, an Annexin V/EthD-III assay was performed to determine the cell death mechanism indicating apoptosis. The zeta potential of the SKOV3 and the MCF7 before and after incorporation of the HER2-AuNPs was also obtained indicating a decrease in zeta potential with the incorporation of the nanoparticles. The outcome of this research will improve our fundamental understanding of the behavior of cancer cells and define optimal parameters of electrotherapy for clinical and drug delivery applications.