Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC.

CAR T cell therapies face challenges in combating solid tumors due to their single-target approach, which becomes ineffective if the targeted antigen is absent or lost. Universal CAR T cells (UniCAR Ts) provide a promising solution by utilizing molecular tags (linkers), such as biotin conjugated to monoclonal antibodies, enabling them to target a variety of tumor antigens. Recently, we showed that conventional CAR T cells could penetrate the extracellular matrix (ECM) of ADCC-resistant tumors, which forms a barrier to therapeutic antibodies. This finding led us to investigate whether UniCAR T cells, targeted by soluble antibody-derived linkers, could similarly tackle ADCC-resistant tumors where ECM restricts antibody penetration. We engineered UniCAR T cells by incorporating a biotin-binding monomeric streptavidin 2 (mSA2) domain for targeting HER2 via biotinylated trastuzumab (BT). The activation and cytotoxicity of UniCAR T cells in the presence or absence of BT were evaluated in conventional immunoassays. A 3D spheroid coculture was set up to test the capability of UniCAR Ts to access ECM-masked HER2+ cells. For in vivo analysis, we utilized a HER2+ xenograft model in which intravenously administered UniCAR T cells were supplemented with intraperitoneal BT treatments. In vitro, BT-guided UniCAR T cells showed effective activation and distinct anti-tumor response. Upon target recognition, IFNγ secretion correlated with BT concentration. In the presence of BT, UniCAR T cells effectively penetrated HER2+ spheroids and induced cell death in their core regions. In vivo, upon intravenous administration of UniCAR Ts, circulating BT linkers immediately engaged the mSA2 domain and directed effector cells to the HER2+ tumors. However, these co-treated mice died early, possibly due to the lung infiltration of UniCAR T cells that could recognize both native biotin and HER2. Our results suggest that UniCAR T cells guided with soluble linkers present a viable alternative to conventional CAR T cells, especially for patients resistant to antibody therapy and those with solid tumors exhibiting high antigenic variability. Critical to their success, however, is the choice of an appropriate binding domain for the CAR and the corresponding soluble linker, ensuring both efficacy and safety in therapeutic applications.

A Multifunctional Bimetallic Nanoplatform for Synergic Local Hyperthermia and Chemotherapy Targeting HER2-Positive Breast Cancer.

Anti-HER2 (human epidermal growth factor receptor 2) therapies significantly increase the overall survival of patients with HER2-positive breast cancer. Unfortunately, a large fraction of patients may develop primary or acquired resistance. Further, a multidrug combination used to prevent this in the clinic places a significant burden on patients. To address this issue, this work develops a nanotherapeutic platform that incorporates bimetallic gold-silver hollow nanoshells (AuAg HNSs) with exceptional near-infrared (NIR) absorption capability, the small-molecule tyrosine kinase inhibitor pyrotinib (PYR), and Herceptin (HCT). This platform realizes targeted delivery of multiple therapeutic effects, including chemo-and photothermal activities, oxidative stress, and immune response. In vitro assays reveal that the HCT-modified nanoparticles exhibit specific recognition ability and effective internalization by cells. The released PYR inhibit cell proliferation by downregulating HER2 and its associated pathways. NIR laser application induces a photothermal effect and tumor cell apoptosis, whereas an intracellular reactive oxygen species burst amplifies oxidative stress and triggers cancer cell ferroptosis. Importantly, this multimodal therapy also promotes the upregulation of genes related to TNF and NF-κB signaling pathways, enhancing immune activation and immunogenic cell death. In vivo studies confirm a significant reduction in tumor volume after treatment, substantiating the potential effectiveness of these nanocarriers.

Enhanced therapeutic action of Trastuzumab loaded ZnxMn1-xFe2O4 nanoparticles using a pre-treatment step for hyperthermia treatment of HER2+ breast cancer.

In this study, Ferrites (Fe3O4, MnFe2O4, ZnFe2O4) and different stoichiometric ratios of ZnxMn1-xFe2O4 (x = 0.2, 0.4, 0.6, and 0.8) nanoparticles (<15 nm) were synthesized by microwave-assisted method and optimised for hyperthermia studies. The selection of the optimised variant of ferrite i.e. Zn0.4Mn0.6Fe2O4 was found to be the best variant based on VSM (38.14 emu g-1) hyperthermia-based temperature rise (maximum ΔT of 38 °C), SAR and ILP values. Trastuzumab, which is known to bind with HER2 receptors of breast cancer was chemically tethered onto Zn0.4Mn0.6Fe2O4 nanoparticles through EDC/NHS coupling with a loading efficiency of 80%. The attached Trastuzumab aided during the pre-treatment step by aiding in the internalisation of Zn0.4Mn0.6Fe2O4 nanoparticles, with cellular uptake of 11% in SK-BR-3 (cancerous HER2+) cells compared to ∼5% for MDA-MB-231 (cancerous HER2-) and RPE-1 (non-cancerous) cells. In the presence of a hyperthermia trigger for 15 mins, ZnxMn1-xFe2O4 -Trastuzumab formulation had a maximum therapeutic effect by reducing the SK-BR-3 cell viability to 14% without adversely affecting the RPE-1 cells. The mechanism of ZnxMn1-xFe2O4-Trastuzumab combination was examined using an internalisation study, MTT-based viability, proliferation study, and ROS generation assay. By utilizing both Trastuzumab and hyperthermia, we achieve their synergistic anticancer properties while minimizing the drug requirement and reducing any effect on non-cancerous cells.

2-(Benzhydryl sulfinyl)-N-sec-butylacetamide) isolated from fig augmented trastuzumab-triggered phagocytic killing of cancer cells through interface with Fcγ receptor.

The objective of the current study was to extract 2-(benzhydryl sulfinyl)-N-sec-butylacetamide), a novel compound from fig, and then determine its role in enhancing trastuzumab-triggered phagocytic killing of SKOV-3 cancer cells. In this study, Soxhlet was used to extract the compound from the mature and air-dried fig fruits. The production of the isolated extracts was enhanced by using polar and non-polar solvents. Several solvents, such as methanol, ethyl acetate, chloroform, and n-hexane, were used to isolate the effective compound 2-(benzhydryl sulfinyl)-N-sec-butylacetamide) from the organic layer. UV-spectroscopy, FT-IR, 1H-NMR, and 13C-NMR were applied to identify the purified compound. The in vitro and in vivo assays demonstrated that the 2-(benzhydryl sulfinyl)-N-sec-butylacetamide) can increase the activity of the phagocytic cells, via the interaction with FcY receptors, along with trastuzumab, and the pathway can use a model for the therapeutic strategy for effective treatment of ovarian cancer cells.

Trastuzumab-based near-infrared photoimmunotherapy in xenograft mouse of breast cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel form of cancer treatment using conjugates of antibody against overexpressed antigens in cancers and photoabsorber IRDye700DX. HER2 is overexpressed in various cancers, for which molecular targeted therapy such as trastuzumab has been developed. The present study investigated the efficacy potential of HER2-targeted NIR-PIT using trastuzumab-IRDye700DX conjugate (Tra-IR700) in HER2-positive breast cancer. We first examined the reactivity of Tra-IR700 and the cytotoxicity of NIR-PIT in vitro. HER2-positive BT-474 and SK-BR-3 cells and HER2-negative BT-20 cells were used. Tra-IR700 fluorescence was only observed in HER2-positive breast cancer cell lines, and the fluorescence was localized to the cell surface. Furthermore, HER2-positive breast cancer cell lines treated with NIR-PIT showed swelling and blebbing shortly after irradiation, and eventually increased PI-positive dead cells. Next, tumor accumulation of Tra-IR700 and tumor damage by NIR-PIT were examined in vivo. Tra-IR700 was administered intravenously to a xenograft model in which BT-474 cells were implanted subcutaneously in BALB/c nude mice. Tra-IR700 fluorescence was the highest in tumor tissue 1 day after administration, and the fluorescence was localized to the cell membrane of tumor cells. At this time point, NIR-PIT resulted in diffuse necrosis of tumor tissues 1 day after irradiation. These results suggest that NIR-PIT with Tra-IR700 induces a highly selective therapeutic effect in a HER2-positive breast cancer model. NIR-PIT using Tra-IR700 is expected to be a novel treatment for HER2-positive cancers, including breast cancer.

Physicochemical and biological impact of metal-catalyzed oxidation of IgG1 monoclonal antibodies and antibody-drug conjugates via reactive oxygen species.

Biotherapeutics are exposed to common transition metal ions such as Cu(II) and Fe(II) during manufacturing processes and storage. IgG1 biotherapeutics are vulnerable to reactive oxygen species (ROS) generated via the metal-catalyzed oxidation reactions. Exposure to these metal ions can lead to potential changes to structure and function, ultimately influencing efficacy, potency, and potential immunogenicity of the molecules. Here, we stress four biotherapeutics of the IgG1 subclass (trastuzumab, trastuzumab emtansine, anti-NaPi2b, and anti-NaPi2b-vc-MMAE) with two common pharmaceutically relevant metal-induced oxidizing systems, Cu(II)/ ascorbic acid and Fe(II)/ H2O2, and evaluated oxidation, size distribution, carbonylation, Fc effector functions, antibody-dependent cellular cytotoxicity (ADCC) activity, cell anti-proliferation and autophaghic flux. Our study demonstrates that the extent of oxidation was metal ion-dependent and site-specific, leading to decreased FcγRIIIa and FcRn receptor binding and subsequently potentially reduced bioactivity, though antigen binding was not affected to a great extent. In general, the monoclonal antibody (mAb) and corresponding antibody-drug conjugate (ADC) showed similar impacts to product quality when exposed to the same metal ion, either Cu(II) or Fe(II). Our study clearly demonstrates that transition metal ion binding to therapeutic IgG1 mAbs and ADCs is not random and that oxidation products show unique structural and functional ramifications. A critical outcome from this study is our highlighting of key process parameters, route of degradation, especially oxidation (metal catalyzed or via ROS), on the CH1 and Fc region of full-length mAbs and ADCs.Abbreviations: DNPH 2,4-dinitrophenylhydrazine; ADC Antibody drug conjugate; ADCC Antibody-dependent cellular cytotoxicity; CDR Complementary determining region; DTT Dithiothreitol; HMWF high molecular weight form; LC-MS Liquid chromatography-mass spectrometry; LMWF low molecular weight forms; MOA Mechanism of action; MCO Metal-catalyzed oxidation; MetO Methionine sulfoxide; mAbs Monoclonal antibodies; MyBPC Myosin binding protein C; ROS Reactive oxygen species; SEC Size exclusion chromatography.

Potential role of CXCR4 in trastuzumab resistance in breast cancer patients.

Despite the efficacy of trastuzumab in treating HER2-positive breast cancer patients, a significant proportion of patients relapse after treatment. The role of C-X-C chemokine receptor type 4 (CXCR4) in trastuzumab resistance was studied only in cell lines and the underlying mechanisms remain largely unclear. This study investigated the role of CXCR4 in trastuzumab resistance in breast cancer patients and explored the possible underlying mechanisms. The study was performed retrospectively on tissue samples from 62 breast cancer patients including 42 who were treated with trastuzumab and chemotherapy and 20 who received chemotherapy alone in adjuvant setting. Expression levels of CXCR4 and its regulators hypoxia-inducible factor 1-alpha (HIF-1α), tristetraprolin (TTP), human antigen R (HuR), itchy E3 ubiquitin protein ligase (ITCH), miR-302a and miR-494 were determined and their associations with tumor recurrence and disease-free survival were analyzed. In trastuzumab-treated patients, high CXCR4 expression was associated with recurrence and was an independent predictor of progression risk after therapy. CXCR4 correlated positively with its transcriptional regulator, HIF-1α, and negatively with its post-translational regulator, ITCH. HIF-1α, HuR and ITCH were significantly associated with clinical outcome. In chemotherapy-treated patients, neither CXCR4 nor any of its regulators were associated with recurrence or predicted disease progression risk after chemotherapy. In conclusion, this study suggests a potential role for CXCR4 in recurrence after trastuzumab-based therapy in human breast cancer that could be mediated, at least in part, by hypoxia and/or decreased ubiquitination. These findings highlight the potential utility of CXCR4 as a promising target for enhancing trastuzumab therapeutic outcome.

Disrupting Circadian Rhythm via the PER1-HK2 Axis Reverses Trastuzumab Resistance in Gastric Cancer.

Trastuzumab is the only approved targeted drug for first-line treatment of HER2-positive advanced gastric cancer, but the high rate of primary resistance and rapid emergence of secondary resistance limit its clinical benefits. We found that trastuzumab-resistant (TR) gastric cancer cells exhibited high glycolytic activity, which was controlled by hexokinase 2 (HK2)-dependent glycolysis with a circadian pattern [higher at zeitgeber time (ZT) 6, lower at ZT18]. Mechanistically, HK2 circadian oscillation was regulated by a transcriptional complex composed of PPARγ and the core clock gene PER1. In vivo and in vitro experiments demonstrated that silencing PER1 disrupted the circadian rhythm of PER1-HK2 and reversed trastuzumab resistance. Moreover, metformin, which inhibits glycolysis and PER1, combined with trastuzumab at ZT6, significantly improved trastuzumab efficacy in gastric cancer. Collectively, these data introduce the circadian clock into trastuzumab therapy and propose a potentially effective chronotherapy strategy to reverse trastuzumab resistance in gastric cancer. SIGNIFICANCE: In trastuzumab-resistant HER2-positive gastric cancer, glycolysis fluctuates with a circadian oscillation regulated by the BMAL1-CLOCK-PER1-HK2 axis, which can be disrupted with a metformin-based chronotherapy to overcome trastuzumab resistance.

Combination of Near-Infrared Photoimmunotherapy Using Trastuzumab and Small Protein Mimetic for HER2-Positive Breast Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer (IR700Dye) that is activated by near-infrared light irradiation. We previously reported on the use of NIR-PIT with a small protein mimetic, the Affibody molecule (6-7 kDa), instead of a monoclonal antibody. In this study, we investigated a combination of NIR-PIT for HER2-positive breast cancer cells (SK-BR3, MDA-MB361, and JIMT1) with HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate. HER2 Affibody and trastuzumab target different epitopes of the HER2 protein and do not compete. In vitro, the combination of NIR-PIT using both HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate induced necrotic cell death of HER2-positive breast cancer cells without damage to HER2-negative breast cancer cells (MCF7). It was more efficient than NIR-PIT using either the HER2 Affibody-IR700Dye conjugate alone or the trastuzumab-IR700Dye conjugate alone. Additionally, this combination of NIR-PIT was significantly effective against HER2 low-expressing cancer cells, trastuzumab-resistant cells (JIMT1), and brain metastatic cells of breast cancer (MDA-MB361). Furthermore, in vivo imaging exhibited the strong fluorescence intensity of both HER2 Affibody-IR700Dye conjugates and trastuzumab-Alexa488 conjugates in HER2-positive tumor, indicating that both HER2 Affibody and trastuzumab specifically bind to HER2-positive tumors without competing with each other. In conclusion, the combination of NIR-PIT using both HER2 Affibody and trastuzumab expands the targeting scope of NIR-PIT for HER2-positive breast cancer.

Selenium inhibits growth of trastuzumab-resistant human breast cancer cells via downregulation of Akt and beclin-1.

The response rate to treatment with trastuzumab (Tz), a recombinant humanized anti-HER2 monoclonal antibody, is only 12-34% despite demonstrated effectiveness on improving the survival of patients with HER2-positive breast cancers. Selenium has an antitumor effect against cancer cells and can play a cytoprotective role on normal cells. This study investigated the effect of selenium on HER2-positive breast cancer cells and the mechanism in relation to the response of the cells to Tz. HER2-positive breast cancer cell lines, SK-BR-3 as trastuzumab-sensitive cells, and JIMT-1 as Tz-resistant cells were treated with Tz and sodium selenite (selenite). Cell survival rates and expression of Her2, Akt, and autophagy-related proteins, including LC3B and beclin 1, in both cell lines 72 h after treatment were evaluated. Significant cell death was induced at different concentrations of selenite in both cell lines. A combined effect of selenite and Tz at 72 h was similar to or significantly greater than each drug alone. The expression of phosphorylated Akt (p-Akt) was decreased in JIMT-1 after combination treatment compared to that after only Tz treatment, while p-Akt expression was increased in SK-BR-3. The expression of beclin1 increased particularly in JIMT-1 after only Tz treatment and was downregulated by combination treatment. These results showed that combination of Tz and selenite had an antitumor effect in Tz-resistant breast cancer cells through downregulation of phosphorylated Akt and beclin1-related autophagy. Selenite might be a potent drug to treat Tz-resistant breast cancer by several mechanisms.

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.

In Vitro Cytotoxicity of Trastuzumab (Tz) and Se-Trastuzumab (Se-Tz) against the Her/2 Breast Cancer Cell Lines JIMT-1 and BT-474.

Her/2+ breast cancer accounts for ~25% mortality in women and overexpression of Her/2 leads to cell growth and tumor progression. Trastuzumab (Tz) with Taxane is the preferred treatment for Her/2+ patients. However, Tz responsive patients often develop resistance to Tz treatment. Herein, redox selenides (RSe-) were covalently linked to Tz using a selenium (Se)-modified Bolton-Hunter Reagent forming Seleno-Trastuzumab (Se-Tz; ~25 µgSe/mg). Se-Tz was compared to Tz and sodium selenite to assess the viability of JIMT-1 and BT-474 cells. Comparative cell viability was examined by microscopy and assessed by fluorometric/enzymatic assays. Se-Tz and selenite redox cycle producing superoxide (O2•-) are more cytotoxic to Tz resistant JIMT-1 and Tz sensitive BT-474 cells than Tz. The results of conjugating redox selenides to Tz suggest a wider application of this technology to other antibodies and targeting molecules.

Near-infrared photoimmunotherapy (NIR-PIT) on cholangiocarcinoma using a novel catheter device with light emitting diodes.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel therapy for cancers that uses NIR light and antibody-photosensitizer (IR700) conjugates. However, it is difficult to deliver NIR light into the bile duct for cholangiocarcinoma (CCA) from the conventional extracorporeal apparatus. Thus, in this study, we developed a dedicated catheter with light emitting diodes (LEDs) that supersedes conventional external irradiation devices; we investigated the therapeutic effect of NIR-PIT for CCA using the novel catheter. The new catheter was designed to be placed in the bile duct and a temperature sensor was attached to the tip of the catheter to avoid thermal burn. An anti-epidermal growth factor receptor (EGFR) antibody, Panitumumab-IR700 conjugate or anti-human epidermal growth factor receptor type 2 (HER2) antibody, Trastuzumab-IR700 conjugate, was used with EGFR- or HER2-expressing cell lines, respectively. The in vitro efficacy of NIR-PIT was confirmed in cultured cells; the capability of the new catheter for NIR-PIT was then tested in a mouse tumor model. NIR-PIT via the developed catheter treated CCA xenografts in mice. NIR-PIT had an effect in Panitumumab-IR700 conjugate- and Trastuzumab-IR700 conjugate-treated CCA cells that depended on the receptor expression level. Tumor growth was significantly suppressed in mice treated with NIR-PIT using the novel catheter compared with controls (P < .01). NIR-PIT was an effective treatment for EGFR- and HER2-expressing CCA cells, and the novel catheter with mounted LEDs was useful for NIR-PIT of CCA.

African Vegetables (Clerodendrum volibile Leaf and Irvingia gabonensis Seed Extracts) Effectively Mitigate Trastuzumab-Induced Cardiotoxicity in Wistar Rats.

Trastuzumab (TZM) is a humanized monoclonal antibody that has been approved for the clinical management of HER2-positive metastatic breast and gastric cancers but its use is limited by its cumulative dose and off-target cardiotoxicity. Unfortunately, till date, there is no approved antidote to this off-target toxicity. Therefore, an acute study was designed at investigating the protective potential and mechanism(s) of CVE and IGE in TZM-induced cardiotoxicity utilizing cardiac enzyme and oxidative stress markers and histopathological endpoints. 400 mg/kg/day CVE and IGE dissolved in 5% DMSO in sterile water were investigated in Wistar rats injected with 2.25 mg/kg/day/i.p. route of TZM for 7 days, using serum cTnI and LDH, complete lipid profile, cardiac tissue oxidative stress markers assays, and histopathological examination of TZM-intoxicated heart tissue. Results showed that 400 mg/kg/day CVE and IGE profoundly attenuated increases in the serum cTnI and LDH levels but caused no significant alterations in the serum lipids and weight gain pattern in the treated rats. CVE and IGE profoundly attenuated alterations in the cardiac tissue oxidative stress markers' activities while improving TZM-associated cardiac histological lesions. These results suggest that CVE and IGE could be mediating its cardioprotection via antioxidant, free radical scavenging, and antithrombotic mechanisms, thus, highlighting the therapeutic potentials of CVE and IGE in the management of TZM-mediated cardiotoxicity.

Trastuzumab conjugated porphyrin-superparamagnetic iron oxide nanoparticle: A potential PTT-MRI bimodal agent for herceptin positive breast cancer.

BACKGROUND: Theranostic agents can combine photosensitizers and contrast agents into a single unit for photothermal therapy (PTT) and magnetic resonance imaging (MRI). The possibility of treating and diagnosing malignant cancers without any ionizing radiation could become an option. This study investigates the theranostic potential of Fe3O4 nanoparticles (IONs) for the diagnosis and treatment of cancer by developing a single integrated nanoprobe. METHODS: Oleylamin-coated IONs (ION-Ol) were synthesized and surface of the IONs was modified using protoporphyrin (PP) and trastuzumab (TZ) to develop the TZ-conjugated SPION-porphyrin [ION-PP-TZ]. The structure, morphology, size, and cytotoxicity of all samples were investigated using Fourier-transform infrared spectroscopy (FT-IR), Transmission electron microscopy (TEM), X-ray powder diffraction (XRD), WST-1 assay, respectively. In addition to MRI and in vitro laser irradiation (808 nm, 200 mW) to determine the r2 values and photothermal ablation. RESULTS: The sizes of monodispersed nanoparticles were measured in rang 5.74-7.17 nm. No cytotoxicity was observed after incubating MCF 7 cells under various Fe concentrations of nanoparticles and theranostic agents. The transverse relaxation time of the protoporphyrin conjugated to IONs (52.32 mM-1s-1) exceeded that of ION-Ol and TZ-conjugated ION-PP. In addition, the in vitro photothermal ablation of ION-PP-TZ revealed a 74 % MCF 7 cell reduction after 10 min of at the highest Fe concentration (1.00 mg Fe/mL). CONCLUSIONS: In summary, the water-soluble ION-PP-TZ is a promising bimodal agent for the diagnosis and treatment of human epidermal growth factor receptor 2-positive breast cancer cells using a T2 MRI contrast agent and photothermal therapy.

Preclinical evaluation of a novel triple-acting PIM/PI3K/mTOR inhibitor, IBL-302, in breast cancer.

The proviral integration of Moloney virus (PIM) family of protein kinases are overexpressed in many haematological and solid tumours. PIM kinase expression is elevated in PI3K inhibitor-treated breast cancer samples, suggesting a major resistance pathway for PI3K inhibitors in breast cancer, potentially limiting their clinical utility. IBL-302 is a novel molecule that inhibits both PIM and PI3K/AKT/mTOR signalling. We thus evaluated the preclinical activity of IBL-302, in a range of breast cancer models. Our results demonstrate in vitro efficacy of IBL-302 in a range of breast cancer cell lines, including lines with acquired resistance to trastuzumab and lapatinib. IBL-302 demonstrated single-agent, anti-tumour efficacy in suppression of pAKT, pmTOR and pBAD in the SKBR-3, BT-474 and HCC-1954 HER2+/PIK3CA-mutated cell lines. We have also shown the in vivo single-agent efficacy of IBL-302 in the subcutaneous BT-474 and HCC-1954 xenograft model in BALB/c nude mice. The combination of trastuzumab and IBL-302 significantly increased the anti-proliferative effect in HER2+ breast cancer cell line, and matched trastuzumab-resistant line, relative to testing either drug alone. We thus believe that the novel PIM and PI3K/mTOR inhibitor, IBL-302, represents an exciting new potential treatment option for breast cancer, and that it should be considered for clinical investigation.

A novel anti-HER2 antibody GB235 reverses Trastuzumab resistance in HER2-expressing tumor cells in vitro and in vivo.

HER2 overexpression is frequently associated with tumor metastasis and poor prognosis of breast cancer. More evidence indicates that HER3 is involved in HER2-resistant therapies. Combination treatments with two or more different monoclonal antibodies are a promising strategy to overcome resistance to HER2 therapies. We presented a novel fully human HER2-targeted monoclonal antibody, GB235, screened from a phage-display library against the HER2 antigen. GB235 in combination with Trastuzumab overcomes resistance in HER2-positive tumors and results in more sustained inhibition of tumor growth over time. The competition binding assay showed that the epitopes of GB235 do not overlap with those of Pertuzumab and Trastuzumab on HER2. Further HER2 mutagenesis results revealed that the binding epitopes of GB235 were located in the domain III of HER2. The mechanism of action of GB235 in blocking HER2-driven tumors is different from the mechanisms of Trastuzumab or Pertuzumab. GB235 does not affect the heterodimerization of HER2 and HER3, whereas the GB235 combined treatment with Trastuzumab significantly inhibited heregulin-induced HER3 phosphorylation and downstream signaling. Moreover, GB235 in combination with Trastuzumab reversed the resistance to heregulin-induced proliferation in HER2-overexpressing cancer cell lines. GB235 combined with Trastuzumab treatment in xenograft models resulted in improved antitumor activity. Complete tumor suppression was observed in the HER2-positive NCI-N87 xenograft model treated with the combination treatment with GB235 and Trastuzumab. In a Trastuzumab-resistant patient-derived tumor xenograft model GA0060, GB235 plus Trastuzumab reversed the resistance to Trastuzumab monotherapy. Because GB235 showed a different working mechanism with Pertuzumab and Trastuzumab, these agents can be considered complementary therapy against HER2 overexpression tumors.

Calcium signaling affects migration and proliferation differently in individual cancer cells due to nifedipine treatment.

Several papers have reported that calcium channel blocking drugs were associated with increased breast cancer risk and worsened prognosis. One of the most common signs of breast tumors is the presence of small deposits of calcium, known as microcalcifications. Therefore, we studied the effect of dihydropyridine nifedipine on selected calcium transport systems in MDA-MB-231 cells, originating from triple negative breast tumor and JIMT1 cells that represent a model of HER2-positive breast cancer, which possesses amplification of HER2 receptor, but cells do not response to HER2 inhibition treatment with trastuzumab. Also, we compared the effect of nifedipine on colorectal DLD1 and ovarian A2780 cancer cells. Both, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and type 1 sodium calcium exchanger (NCX1) were upregulated due to nifedipine in DLD1 and A2780 cells, but not in breast cancer MDA-MB-231 and JIMT1 cells. On contrary to MDA-MB-231 and JIMT1 cells, in DLD1 and A2780 cells nifedipine induced apoptosis in a concentration-dependent manner. After NCX1 silencing and subsequent treatment with nifedipine, proliferation was decreased in MDA-MB-231, increased in DLD1 cells, and not changed in JIMT1 cells. Silencing of IP3R1 revealed increase in proliferation in DLD1 and JIMT1 cells, but caused decrease in proliferation in MDA-MB-231 cell line after nifedipine treatment. Interestingly, after nifedipine treatment migration was not significantly affected in any of tested cell lines after NCX1 silencing. Due to IP3R1 silencing, significant decrease in migration occurred in MDA-MB-231 cells after nifedipine treatment, but not in other tested cells. These results support different function of the NCX1 and IP3R1 in the invasiveness of various cancer cells due to nifedipine treatment.

Cancer neovasculature-targeted near-infrared photoimmunotherapy (NIR-PIT) for gastric cancer: different mechanisms of phototoxicity compared to cell membrane-targeted NIR-PIT.

BACKGROUND: Near-infrared photoimmunotherapy (NIR-PIT) constitutes a new class of molecular-targeted theranostics utilizing monoclonal antibody (mAb)-photosensitizer conjugates and NIR light. In this study, we developed a new type of NIR-PIT targeting vascular endothelial growth factor receptor 2 (VEGFR-2) expressed on vascular endothelium in an experimental gastric cancer model and evaluated the feasibility by comparing conventional NIR-PIT targeting cancer cell membrane in vitro and in vivo. METHODS: HER2-positive human gastric cancer cells, NCI-N87, were used for the experiments. Anti-HER2 mAb, trastuzumab and anti-VEGFR-2 mAb, DC101 were conjugated to photosensitizer, IR700. Phototoxicity in response to NIR-PIT were investigated in vitro and in vivo. Microvessel densities, as an indicator of angiogenesis, were counted in harvested xenografts after NIR-PIT to elucidate the mechanism. RESULTS: DC101-IR700 did not induce phototoxic effect in vitro because of the absence of expression of VEGFR-2 in NCI-N87 cancer cells. However, it induced an antitumor effect in NCI-N87 xenograft tumors accompanied with damage in tumor neovasculature as determined by decreasing tumor microvessel density, which represents a different mechanism than that of conventional NIR-PIT targeting antigens expressed on the tumor cell membrane. CONCLUSION: We demonstrated a new approach of NIR-PIT utilizing a target on vascular endothelium, such as VEGFR-2, and this treatment might lead to the development of a new therapeutic strategy for human gastric cancer.

Targeting Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor 2 (HER2) Expressing Bladder Cancer Using Combination Photoimmunotherapy (PIT).

Bladder cancer (BC) is heterogeneous and expresses various cell surface targets. Photoimmunotherapy (PIT) involves monoclonal antibodies (MAbs) conjugated to a photoabsorber (PA), IR Dye 700Dx, and then activated by near infra-red light (NIR) to specifically target tumors. We have demonstrated that tumors expressing EGFR can be targeted with PIT. However, PIT may be less effective when a tumor lacks "overwhelming" expression of a single target such as EGFR. We present a combinatorial PIT approach for targeting BC expressing EGFR and HER2, using PA- labeled panitumumab (pan) and trastuzumab (tra), respectively. Human BC tissues and cell lines were analyzed for EGFR and HER2 expression. Efficacy of PA-labeled MAbs singly and in combination was analyzed. About 45% of BC tissues stain for both EGFR and HER2. In vitro, the combination of pan IR700 and tra IR700 with NIR was more efficacious than either agent alone. Tumor xenografts treated with combination PIT showed significant tumor growth retardation. Combination PIT is a promising approach for treating BC with low/moderate expression of surface receptors. In addition, given the molecular heterogeneity of bladder cancer, targeting more than one surface receptor may allow for more effective cell death across different bladder tumors.