HER2-overexpressing Cells Research Articles

Abstract PD18-11: Dose-Expansion Study of Trastuzumab Deruxtecan as Monotherapy or Combined With Pertuzumab in Patients With Metastatic Human Epidermal Growth Factor Receptor 2-Positive (HER2+) Breast Cancer in DESTINY-Breast07 (DB-07)

Abstract Background: In trials of HER2+ metastatic breast cancer (mBC), trastuzumab deruxtecan (T-DXd) monotherapy showed durable efficacy (DESTINY-Breast01) and significantly prolonged progression-free survival vs trastuzumab emtansine (DESTINY-Breast03). T-DXd is approved in the US for patients with HER2+ unresectable/mBC who received ≥1 prior anti-HER2–based treatment (tx) in the metastatic or neo-/adjuvant setting and recommended for approval in the EU as 2nd-line tx. Preclinical data suggest that T-DXd used in combination with other anticancer tx may lead to improved efficacy. The purpose of DB-07 is to assess the safety and efficacy of T-DXd alone or with other anticancer tx for patients with HER2+ mBC. Here we report preliminary data from the DB-07 dose-expansion phase for T-DXd monotherapy and T-DXd + pertuzumab (P) as 1st-line (1L) tx in mBC. Methods: DB-07 (NCT04538742) is an ongoing, phase 1b/2, 2-part (part 1: dose finding; part 2: dose expansion), modular, open-label trial of T-DXd alone or with other anticancer tx in patients with HER2+ mBC. In part 2, patients in module (mod) 0 received T-DXd 5.4 mg/kg every 3 weeks (Q3W) and in mod 2, T-DXd 5.4 mg/kg + P 420 mg Q3W (loading dose: 840 mg), the recommended phase 2 dose. Patients in these mods must be mBC tx naive. For part 2, the primary objective is to assess safety and tolerability. A secondary objective is to assess the objective response rate (ORR) per local investigators by Response Evaluation Criteria In Solid Tumors v1.1. We report results for patients randomized before Oct 13, 2021 to mods 0 and 2 of part 2 (data cutoff [DCO]: Mar 4, 2022); recruitment is ongoing. Based on the distinct mechanism of action of T-DXd and P, we conducted preclinical studies with the drugs in HER2-overexpressing cell lines to elucidate their potential synergies. To assess the effects on T-DXd internalization, live cell imaging was performed using pH-dependent fluorescently labeled T-DXd. To assess the effects on HER2 signaling, total and p-HER2 levels and downstream substrates were evaluated by immunoblot. Results: 23 patients were enrolled in the T-DXd monotherapy mod; 20 (87.0%) were receiving tx and 3 (13.0%) discontinued tx (withdrawal by patient, n=2; adverse event [AE], n=1) by DCO. 22 patients were enrolled in the T-DXd + P mod; 20 (90.9%) were receiving tx and 2 (9.1%) discontinued tx (AE, n=1; disease progression, n=1) by DCO. All patients experienced AEs (Table); 1 patient in each mod died. The unconfirmed ORR (80% CI) with T-DXd monotherapy and T-DXd + P was 82.6% (68.2%-92.2%) and 77.3% (61.9%-88.5%), respectively; updated data will be presented. Preclinical studies showed that T-DXd was more rapidly and effectively internalized in combination with P than when administered alone. Immunoblotting of cell lysates showed a greater reduction in total HER2 and HER2 signaling in response to combination tx than with T-DXd or P alone. Discussion: In summary, 1L T-DXd monotherapy and T-DXd + P safety profiles and antitumor activity were consistent with those previously reported for T-DXd. Mature data in these mods are awaited, and other T-DXd combinations are being investigated in additional mods. Preclinical studies showed the potential for P to induce greater internalization of T-DXd and inhibition of HER2-driven signaling. These results support investigation of T-DXd in larger ongoing trials (eg, NCT04784715). Table 1: Summary of treatment duration and safety data Citation Format: Erika Hamilton, Komal Jhaveri, Sherene Loi, Carey Anders, Peter Schmid, Konstantin Penkov, Elena Artamonova, Lyudmila Zhukova, Daniil L. Stroyakovsky, Dinesh Chandra Doval, Rafael Villanueva, Flavia Michelini, Sarat Chandarlapaty, Matt Wilson, Sarice R. Boston, Adam Konpa, Shoubhik Mondal, Fabrice Andre. Dose-Expansion Study of Trastuzumab Deruxtecan as Monotherapy or Combined With Pertuzumab in Patients With Metastatic Human Epidermal Growth Factor Receptor 2-Positive (HER2+) Breast Cancer in DESTINY-Breast07 (DB-07) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD18-11.

Design and construction of scFv-PE35KDEL as a novel immunotoxin against human epidermal growth factor receptor 2 for cancer therapy.

Human epidermal growth factor receptor 2 (HER2) is overexpressed on the surface of some kinds of cancer cells including breast cancer. In this study, we designed and produced a novel immunotoxin consisting anti-HER2 single-chain Fv (scFv) from pertuzumab and a modified form of Pseudomonas exotoxin (PE35KDEL). The three-dimensional (3D) structure of the fusion protein (anti-HER IT) was predicted by MODELLER 9.23 and its interaction with HER2 receptor was assessed using HADDOCK web server. Anti-HER2 IT, anti-HER2 scFv, and PE35KDEL proteins were expressed by Escherichia coli BL21 (DE3). After purification of the proteins using Ni2+ affinity chromatography and refolding through dialysis, the cytotoxicity of proteins against breast cancer cell lines was examined by MTT assay. In-silico studies showed that (EAAAK)2 linker can efficiently prevent the formation of salt bridges between two functional domains and the constructed fusion protein has a high affinity to HER2 receptor. The optimum condition of anti-HER2 IT expression was 25°C and 1mM IPTG. The protein was successfully purified and refolded by dialysis with a final yield of 45.7mg per 1 L of bacterial culture. The cytotoxicity results showed that anti-HER2 IT was much more toxic on HER2-overexpressing cells, BT-474 (IC50 ~ 95nM) compared with HER2-negative cells, MDA-MB-23 (IC50 ˃ 200nM). This novel immunotoxin has the potential to be applied as a therapeutic candidate for HER2-targeted cancer therapy. However further in vitro and in vivo evaluations are still required to confirm the efficacy and safety of this protein.

Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages.

Basal-like breast cancer (BLBC) has a clinically aggressive nature. It is prevalent in young women and is known to often relapse rapidly. To date, the molecular mechanisms regarding the aggressiveness of BLBC have not been fully understood. In the present study, mechanisms of aggressiveness of BLBC involving EGFR and/or HER2 expression and interactions between tumor and tumor-associated macrophages (TAMs) were explored. The prognosis of breast cancer patients who underwent surgery at Samsung Medical Center was analyzed. It was found that the co-expression of EGFR and HER2 was associated with a worse prognosis. Therefore, we generated EGFR-positive BLBC cells with stable HER2 overexpression and analyzed the profile of secretory cytokines. Chemokine (C-C motif) ligand 2 (CCL2) expression was increased in HER2-overexpressed BLBC cells. Recombinant human CCL2 treatment augmented the motility of TAMs. In addition, the conditioned culture media of HER2-overexpressed BLBC cells increased the motility of TAMs. Furthermore, activation of TAMs by CCL2 or the conditioned culture media of HER2-overexpressed cells resulted in the production of pro-inflammatory cytokines, such as IL-8 and IL-1β. These observations reveal that CCL2 derived from EGFR and HER2 co-expressed BLBC cells can lead to increased TAM recruitment and the induction of IL-8 and IL-1β from recruited TAMs, triggering the tumorigenesis of breast cancer with the expression of both EGFR and HER2. Our findings demonstrate that EGFR+ and HER2+ BLBC aggressiveness is partially mediated through the interaction between BLBC and TAMs recruited by CCL2.

Two-Step Targeted Drug Delivery via Proteinaceous Barnase-Barstar Interface and Doxorubicin-Loaded Nano-PLGA Outperforms One-Step Strategy for Targeted Delivery to HER2-Overexpressing Cells.

Nanoparticle-based chemotherapy is considered to be an effective approach to cancer diagnostics and therapy in modern biomedicine. However, efficient tumor targeting remains a great challenge due to the lack of specificity, selectivity, and high dosage of chemotherapeutic drugs required. A two-step targeted drug delivery strategy (DDS), involving cancer cell pre-targeting, first with a first nontoxic module and subsequent targeting with a second complementary toxic module, is a solution for decreasing doses for administration and lowering systemic toxicity. To prove two-step DDS efficiency, we performed a direct comparison of one-step and two-step DDS based on chemotherapy loaded PLGA nanoparticles and barnase*barstar interface. Namely, we developed and thoroughly characterized the two-step targeting strategy of HER2-overexpressing cancer cells. The first targeting block consists of anti-HER2 scaffold polypeptide DARPin9_29 fused with barstar. Barstar exhibits an extremely effective binding to ribonuclease barnase with Kaff = 1014 M-1, thus making the barnase*barstar protein pair one of the strongest known protein*protein complexes. A therapeutic PLGA-based nanocarrier coupled to barnase was used as a second targeting block. The PLGA nanoparticles were loaded with diagnostic dye, Nile Blue, and a chemotherapeutic drug, doxorubicin. We showed that the two-step DDS increases the performance of chemotherapy-loaded nanocarriers: IC50 of doxorubicin delivered via two-step DDS was more than 100 times lower than that for one-step DDS: IC50 = 43 ± 3 nM for two-step DDS vs. IC50 = 4972 ± 1965 nM for one-step DDS. The obtained results demonstrate the significant efficiency of two-step DDS over the classical one-step one. We believe that the obtained data will significantly change the direction of research in developing targeted anti-cancer drugs and promote the creation of new generation cancer treatment strategies.

Network Pharmacological Analysis Identifies the Curcumin Analog CCA-1.1 Targeting Mitosis Regulatory Process in HER2-Positive Breast Cancer

Recent studies present that the CCA-1.1 (a curcumin derivative) impedes the proliferation of breast cancer cells (luminal, HER2-overexpressed, and TNBC cells). Currently, we analyze the possible target of action of CCA-1.1, particularly in breast cancer cells with HER2 amplification using bioinformatics analysis. The differentially expressed genes (DEGs) of HER2-positive breast cancer were retrieved from TCGA-BRCA data (via UALCAN). We used three web-based tools (Swiss Target Prediction, BindingDB, and TargetNet) to predict the potential target of CCA-1.1 using the SMILE-similarity feature. The functional annotation and network enrichment were processed in WebGestalt. The alteration of selected genes was observed in CBioPortal. The protein-protein interaction (PPI) network was constructed in STRING, then ranked based on the degree score using Cytohubba feature in Cytoscape. The survival analysis from the hub-gene was collected in GEPIA2 with selection only for HER2-positive breast cancer cases. The correlation between the hub genes and tumor-infiltrating immune markers was determined using TIMER web tools. The pathway network analysis highlighted the cell cycle regulation in mitosis as affected signaling amid the putative CCA-1.1 targets. We denoted eight potential genes that could be responsible for inhibiting mitosis regulation upon CCA-1.1 treatment, including AURKA, AURKB, PLK1, TPX2, KIF11, MELK, CDK1, and CHEK1. Several of the potential markers (AURKB, AURKA, CDK1, and CHEK1) revealed to be correlated with the immune cells’ infiltration markers. CCA-1.1 might regulate mitosis to induce cell cycle arrest and lead to cell death. The predicted targets of CCA-1.11 gave insight into the potency of CCA-1.1 to be with immunotherapy. Further validation of the data presented in the study is essentially needed to develop CCA-1.1 for breast cancer.

Synthesis and in vivo evaluation of [11C]tucatinib for HER2-targeted PET imaging

Tucatinib is a selective human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration (FDA) in April 2020 for HER2-positive lesions in metastatic breast cancer patients, including CNS metastases. In this article, we attempted to develop the first small molecule, blood–brain-barrier (BBB) penetrant HER2 PET imaging probe based on tucatinib. [11C]tucatinib was synthesized via a Stille-coupling from the respective trimethylstannyl precursor and its biodistribution was evaluated in NMRI nude mice bearing HER2-overexpressing human ovarian cancer cells (SKOV-3). No significant tumor accumulation was observed despite its high affinity for HER-2 receptors (IC50 = 6.9 nM). High liver and intestinal uptake indicate that [11C]tucatinib is too lipophilic to be used as a tumor targeting PET tracer. Therefore, chemical modifications of [11C]tucatinib are needed to increase the polarity for tumor imaging. Tucatinib as an FDA approved drug is still an interesting platform to develop the first small molecule HER2-selective PET tracer. The study highlights the differences between a drug, which needs to be effective, and an imaging agent, which is dependent on contrast.

Modular Site-Specific Conjugation of Nanobodies Using Two Co-Associating Tags.

The homogeneous labeling of antibodies and their fragments is a critical step for the generation of robust probes used in immuno-detection applications. To date, numerous chemical, genetic and peptide-based site-specific coupling methods have been developed. Among these methods, co-assembling peptide-tags is one of the most straightforward and versatile solutions. Here, we describe site-specific labeling of nanobodies through the use of two co-associating peptides tags, E3 and K3, originating from the tetramerization domain of p53. These E3 and K3-tags provide a simple and robust method for associating stoichiometric amount of VHH and fluorescent probes, either fluorescent proteins or fluorochromes, at specific positions. As a proof of concept, a nanobody targeting the human epidermal growth factor receptor 2 (HER2), the nano-HER2 was genetically fused to the E3 and associated with different fluorescent K3-derivates. Entities were produced separately in Escherichia coli in soluble forms at high yields and co-assembled in vitro. These molecular probes present high binding specificity on HER2-overexpressing cells in flow-cytometry with relative binding constants in the low nanomolar range and are stable enough to stain HER2-receptor on living cells followed detection using fluorescent confocal microscopy. Altogether, our results demonstrate that the non-covalent conjugation method using these two co-associating peptides can be easily implemented for the modular engineering of molecular probes for cell immuno-staining.

Novel Small Multilamellar Liposomes Containing Large Quantities of Peptide Nucleic Acid Selectively Kill Breast Cancer Cells.

Simple SummaryWe present, for the first time, the preparation of small (60–90 nm in diameter) liposomes containing extremely large amounts (~8000 molecules per vesicle) of short, cytosine-rich peptide nucleic acid. The outer surface of liposomes wasfunctionalized with scaffold molecules specific to tumor-associated antigen overexpressing in breast cancer. We have shown that targeted liposomesspecifically interact with cancer cells and reduce their viability in sub-nanomolar concentrations. The results presented here can be widely used in cancer therapy based on cytosine-rich PNA oligonucleotides.Peptide nucleic acid (PNA) may be used in various biomedical applications; however, these are currently limited, due to its low solubility in aqueous solutions. In this study, a methodology to overcome this limitation is demonstrated, as well as the effect of PNA on cell viability. We show that extruding a mixture of natural phospholipids and short (6–22 bases), cytosine-rich PNA through a 100 nm pore size membrane under mild acidic conditions resulted in the formation of small (60–90 nm in diameter) multilamellar vesicles (SMVs) comprising several (3–5) concentric lipid membranes. The PNA molecules, being positively charged under acidic conditions (due to protonation of cytosine bases in the sequence), bind electrostatically to negatively charged phospholipid membranes. The large membrane surface area allowed the encapsulation of thousands of PNA molecules in the vesicle. SMVs were conjugated with the designed ankyrin repeat protein (DARPin_9-29), which interacts with human epidermal growth factor receptor 2 (HER2), overexpressed in human breast cancer. The conjugate was shown to enter HER2-overexpressing cells by receptor-mediated endocytosis. PNA molecules, released from lysosomes, aggregate in the cytoplasm into micron-sized particles, which interfere with normal cell functioning, causing cell death. The ability of DARPin-functionalized SMVs to specifically deliver large quantities of PNA to cancer cells opens a new promising avenue for cancer therapy.

Quantifying the Effects of Combination Trastuzumab and Radiation Therapy in Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer.

Simple SummaryIn vitro, combination trastuzumab and single-dose radiotherapy induces an additive effect. In vivo, combination trastuzumab and single-dose radiotherapy induces greater tumor kill than a higher single dose of radiotherapy, suggesting that combination therapy can be considered to achieve a similar reduction in tumor burden as traditional radiotherapy, with fewer adverse effects.Background: Trastuzumab induces cell cycle arrest in HER2-overexpressing cells and demonstrates potential in radiosensitizing cancer cells. The purpose of this study is to quantify combination trastuzumab and radiotherapy to determine their synergy. Methods: In vitro, HER2+ cancer cells were treated with trastuzumab, radiation, or their combination, and imaged to evaluate treatment kinetics. In vivo, HER2+ tumor-bearing mice were treated with trastuzumab and radiation, and assessed longitudinally. An additional cohort was treated and sacrificed to quantify CD45, CD31, α-SMA, and hypoxia. Results: The interaction index revealed the additive effects of trastuzumab and radiation in vitro in HER2+ cell lines. Furthermore, the results revealed significant differences in tumor response when treated with radiation (p < 0.001); however, no difference was seen in the combination groups when trastuzumab was added to radiotherapy (p = 0.56). Histology revealed increases in CD45 staining in tumors receiving trastuzumab (p < 0.05), indicating potential increases in immune infiltration. Conclusions: The in vitro results showed the additive effect of combination trastuzumab and radiotherapy. The in vivo results showed the potential to achieve similar efficacy of radiotherapy with a reduced dose when combined with trastuzumab. If trastuzumab and low-dose radiotherapy induce greater tumor kill than a higher dose of radiotherapy, combination therapy can achieve a similar reduction in tumor burden.

Improvement of specific aiming of X-ray radiotherapy on HER2-overexpressing cancerous cell lines by targeted delivery of silver nanoparticle

Human epidermal growth factor receptor 2 (HER2) as a member of epidermal growth factor receptor (EGFR) family is expressed in various kinds of malignant cells at a level higher than normal cells. Therefore, HER2 could be consider as a target for specific chemotherapy and selective ablation of cancerous cells. ZHER2 affibody as an engineered peptide with great affinity for HER2 receptor could be employed for specific targeting of cancer cells with high HER2 expression level. On the other hand, X-ray radiotherapy is one of the most prevalent approaches in cancer therapy and nanoparticles with high atomic number could enhance its efficiency. As an outcome, targeted delivery of these nanoparticles can increase intensity and focus of X-ray radiation on cancerous cells. In the current in vitro study, silver nanoparticles (AgNPs) in conjugations with ZHER2 affibody were investigated for enhancement of X-ray radiation therapy of HER2-positive cancerous cells originated from various kinds of malignancies. Cell lines with high expression of HER2 (SK-BR-3, HN-5, SK-OV-3) and also MCF-7 cell line with normal expression of HER2 were irradiated with X-ray in the presence of various concentrations of AgNPs or AgNP-ZHER2 conjugates. Morphology and size of prepared AgNPs and AgNP-ZHER2 were examined by atomic force microscopy (AFM) and dynamic light scattering (DLS). Success of conjugation was confirmed by UV–Vis spectroscopy and viability of treated cells was assessed by MTT assay. Our results showed that while sensitivity of four examined cell lines to AgNPs, AgNP-ZHER2, and X-ray radiation is different, but presence of AgNP-ZHER2 conjugate could significantly enhance efficiency of X-ray radiation in ablation of HER2-overexpressed cells at in vitro conditions.

Blockade of exosome release alters HER2 trafficking to the plasma membrane and gives a boost to Trastuzumab.

Exosomal HER2 has been evidenced to interfere with antibody-induced anti-tumor effects. However, whether the blockade of HER2+ exosomes release would affect antibody-mediated tumor inhibition has yet to be investigated. Exosomes derived from BT-474, SK-BR3 and SK-OV3 (HER2-overexpressing tumor cells) and MDA-MB-231 cells (HER2 negative) were purified and characterized by bicinchoninic acid (BCA) assay, western blotting and Transmission electron microscopy (TEM). Inhibition of exosome release was achieved by neutral sphingomyelinase-2 (nSMase-2) inhibitor, GW4869. The effects of exosome blockade on the anti-proliferative effects, apoptosis induction, and antibody-mediated cellular cytotoxicity (ADCC) activity of Trastuzumab were examined using MTT, flow cytometry, and LDH release assays. Also, the effects of exosome inhibition on the surface expression and endocytosis/internalization of HER2 were studied by flow cytometry. Purified exosomes derived from HER2 overexpressing cancer cells were positive for HER2 protein. Blockade of exosome release was able to significantly improve apoptosis induction, anti-proliferative and ADCC responses of Trastuzumab dose dependently. The pretreatment of Trastuzumab/purified NK cells, but not PBMCs, with HER2+ exosomes could also decrease the ADCC effects of Trastuzumab. Exosome inhibition also remarkably downregulated surface HER2 levels in a time-dependent manner, but does not affect its endocytosis/internalization. Based on our findings, HER2+ exosomes may benefit tumor progression by dually suppressing Trastuzumab-induced tumor growth inhibition and cytotoxicity of NK cells. It seems that concomitant blocking of exosome release might be an effective approach for improving the therapeutic effects of Trastuzumab, and potentially other HER2-directed mAbs. In addition, the exosome secretion pathway possibly contributes to the HER2 trafficking to plasma membrane, since the blockade of exosome secretion decreased surface HER2 levels.

Human Blood Serum Inhibits Ductal Carcinoma Cells BT474 Growth and Modulates Effect of HER2 Inhibition.

Trastuzumab, a HER2-targeted antibody, is widely used for targeted therapy of HER2-positive breast cancer (BC) patients; yet, not all of them respond to this treatment. We investigated here whether trastuzumab activity on the growth of HER2-overexpressing BT474 cells may interfere with human peripheral blood endogenous factors. Among 33 individual BC patient blood samples supplemented to the media, BT474 sensitivity to trastuzumab varied up to 14 times. In the absence of trastuzumab, human peripheral blood serum samples could inhibit growth of BT474, and this effect varied ~10 times for 50 individual samples. In turn, the epidermal growth factor (EGF) suppressed the trastuzumab effect on BT474 cell growth. Trastuzumab treatment increased the proportion of BT474 cells in the G0/G1 phases of cell cycle, while simultaneous addition of EGF decreased it, yet not to the control level. We used RNA sequencing profiling of gene expression to elucidate the molecular mechanisms involved in EGF- and human-sera-mediated attenuation of the trastuzumab effect on BT474 cell growth. Bioinformatic analysis of the molecular profiles suggested that trastuzumab acts similarly to the inhibition of PI3K/Akt/mTOR signaling axis, and the mechanism of EGF suppression of trastuzumab activity may be associated with parallel activation of PKC and transcriptional factors ETV1-ETV5.

In vivo study of the immune response to bioengineered spider silk spheres

Bioengineered MS1 silk is derived from major ampullate spidroin 1 (MaSp1) from the spider Nephila clavipes. The MS1 silk was functionalized with the H2.1 peptide to target Her2-overexpressing cancer cells. The immunogenic potential of drug carriers made from MS1-type silks was investigated. The silk spheres were administered to healthy mice, and then (i) the phenotypes of the immune cells that infiltrated the Matrigel plugs containing spheres (implanted subcutaneously), (ii) the presence of silk-specific antibodies (after two intravenous injections of the spheres), (iii) the splenocyte phenotypes and their activity after restimulation ex vivo in terms of proliferation and cytokine secretion (after single intravenous injection of the spheres) were analyzed. Although the immunogenicity of MS1 particles was minor, the H2.1MS1 spheres attracted higher levels of B lymphocytes, induced a higher anti-silk antibody titer, and, after ex vivo restimulation, caused the activation of splenocytes to proliferate and express more IFN-γ and IL-10 compared with the PBS and MS1 groups. Although the H2.1MS1 spheres triggered a certain degree of an immunological response, multiple injections (up to six times) neither hampered the carrier-dependent specific drug delivery nor induced toxicity, as previously indicated in a mouse breast cancer model. Both findings indicate that a drug delivery system based on MS1-type silk has great potential for the treatment of cancer and other conditions.

Synergistic Cytotoxic and Antimigratory Effect of Brazilein and Doxorubicin on HER2-Overexpressing Cells.

The present research aims to report cytotoxic and antimigratory activities of the oxidized form of brazilin, i.e., brazilein, and the effects of the combination of brazilein-doxorubicin on MCF-7/HER2 cells. The MTT assay was conducted to test the cytotoxic activity, while flow cytometry with PI and PI-annexin V staining were respectively performed for cell cycle and apoptosis analyses. Migration and invasion analyses were assessed via Boyden chamber assay, while HER2, Rac1, p120, MMP2, and MMP9 protein levels were determined by immunoblotting and gelatin zymography. Molecular docking of ligands with HER2, Src, PI3Kα, PI3KΔ, and PI3Kγ proteins was evaluated using MOE 2010. The MTT assay showed that the IC50 value of brazilein against MCF-7/HER2 cells was 51 ± 2.1 µM. Moreover, brazilein and its combination with doxorubicin-induced G2/M accumulation and apoptosis. Combination of brazilein-doxorubicin inhibited cell migration and tended to decrease HER2, Rac1, p120, MMP2, and MMP9 protein expression levels. Based on our molecular docking study, the docking score of brazilein with PI3Kγ is comparable to that of the native ligand. Taken together, a combination of brazilein-doxorubicin exhibited synergistic cytotoxic and antimigratory effects on MCF-7/HER2 cells.<br />.

Unravelling the Potential of Lutetium-177 Labeled Pertuzumab: Targeting HER 2 Receptors for Theranostic Applications

<h2>ABSTRACT</h2><h3>Purpose</h3> Pertuzumab, a humanized antibody against HER2 receptors has shown promising results in combination with trastuzumab and docetaxel for the neoadjuvant treatment of patients with HER2-positive breast cancer. The aim of the present study was to demonstrate the potential of <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab for targeting HER2 positive tumors. Comparative studies with <b>[</b>¹⁷⁷Lu]-Lu-DTPA-trastuzumab and synergism in presence of cold trastuzumab was also envisaged. <h3>Methods & Materials</h3> Pertuzumab and trastuzumab were conjugated with p-NCS-CHX-A''-DTPA and radiolabeled with Lutetium-177. The stability of the formulations was assessed and <i>in-vitro</i> binding parameters (immunoreactivity, saturation and competitive binding) were examined in HER2 overexpressing and HER2 negative cell lines. SPECT imaging studies were performed in SCID mice bearing HER2 positive tumors. <h3>Results</h3> <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab and trastuzumab could be prepared with >95% radiochemical purity (% RCP). <i>In-vitro</i> binding studies revealed high affinity and specificity of the formulations towards HER2 receptors. Specific tumor uptake of <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab in HER2 positive tumors was observed by SPECT imaging studies in tumor bearing mice, comparable to <b>[</b>¹⁷⁷Lu]-Lu-DTPA-trastuzumab. Significant retention of the formulations was observed in the tumor for up to 120 h. The studies also showed enhanced uptake of <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab in the presence of cold trastuzumab in HER2 positive tumors. <h3>Conclusions</h3> The present study revealed significant uptake of both ¹⁷⁷Lu labeled pertuzumab and trastuzumab in HER2 positive tumors. Further studies are warranted to determine efficacy of these formulations against tumor growth for confirming their potential as effective radioimmunotheranostic agents. Increase in uptake of radiolabeled pertuzumab in presence cold trastuzumab provides an avenue for more effective radionuclide imaging and therapy of HER2 positive tumors.

Discovery and evaluation of cytisine N-isoflavones as novel EGFR/HER2 dual inhibitors

Aberrant signaling of EGFR (ErbB) family members, in particular epidermal growth factor receptor (EGFR) and human epidermal growth factor 2 (HER2), is associated with the occurrence and development of many types of human malignancies (e.g., breast, lung, and gastric cancers), and dual targeting of EGFR/HER2 by small-molecular inhibitors has proven to be an effective therapeutic approach for treating these cancers. Herein we extracted and isolated from the medicinal plant Sophora alopecuroides L. a new natural product, dubbed Cytisine N-methylene-(4′,7-dihydroxy-3′-methoxy)-isoflavone (CNI1) that features a unique molecular framework. Our biochemical kinase assay suggested that one of its derivative CNI3 exhibited the best, micromolar (μM) inhibition activities against the EGFR (IC50 of 1.1 μM; Ki of 0.6 μM) and HER2 (IC50 of 3.5 μM; Ki of 1.8 μM) kinases. By contrast, another derivative CNI4 was most potent in inhibiting the EGFR-overexpressing A431 cancer cell line (IC50 of 45.5 μM) and the HER2-overexpressing BT-474 cancer cell line (IC50 of 32.9 μM), while the respective cellular activities of Lapatinib (a marketed drug) were 24.9 and 20.3 μM under the same assay condition. Moreover, both CNI3 and CNI4 showed desirable anti-metastatic efficacy in another two breast cancer models (viz., MDA-MB-231 and 4T1). In addition, we explored the inhibitory mechanisms of the CNIs against EGFR and HER2 by molecular dynamics simulation and revealed a novel mode of action that engages the cytisine and chromone moieties in CNIs. By combining structure- and ligand-based analysis, we further rationally engineered a new CNI compound that exhibits considerably improved cytotoxicity against both types of A431 and BT-474 cancer cells. Our study demonstrates the CNI compounds as a new class of EGFR/HER2 dual inhibitors and paves a way for their further development.

Potent anti-tumor immune response and tumor growth inhibition induced by HER2 subdomain fusion protein in a mouse tumor model.

Several approaches have so far been employed to establish anti-tumor immunity by targeting HER2 protein. Active immunization with recombinant HER2 subdomains has previously been demonstrated to induce potent immune response and tumor growth inhibition. In the present study, we investigated the immunogenicity and tumor inhibitory effect of a fusion protein consisting of human HER2 extracellular subdomain (ECD-DI + II) together with T-helper cell epitopes of Tetanus toxin (p2 and p30). BALB/c mice were immunized with two recombinant proteins (DI + II and p2p30-DI + II) emulsified in 4 different adjuvants. Anti-DI + II antibody response, cytokine profile, frequency of splenic CD25+FOXP3+ regulatory T cells (Tregs) and CD8+CD107a+ cytotoxic T lymphocytes (CTLs) were assessed in the immunized mice. To assess the anti-tumor effect, the immunized mice were subcutaneously challenged with HER2-overexpressing tumor cells and the tumor growth was determined. Both recombinant proteins were able to induce comparable levels of ECD-DI + II-specific antibodies. Immunization with p2p30-DI + II resulted in a significant increase in the level of Interferon-gamma (IFN-γ) secretion compared to DI + II protein and significantly higher frequency of CTLs and lower frequency of Tregs. The number of mice that remained tumor-free until day 120 was significantly higher in p2p30-DI + II vaccinated groups. Our data suggest that the p2p30-DI + II fusion protein together with CpG adjuvant induces more potent anti-tumor immune responses in a mouse tumor model. Accordingly, this formulation might be considered as a potential immunotherapeutic approach in HER2+ cancers.

Abstract 2112: Chimeric antigen receptor macrophages (CAR-M) sensitize solid tumors to anti-PD1 immunotherapy

Abstract Despite the remarkable efficacy achieved by CAR-T cell therapy in hematologic malignancies, achieving efficacy against solid tumors has been challenging. We previously developed human CAR-M and demonstrated that adoptive cell transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival1. Given that CAR-M are M1-polarized macrophages with the potential to remodel the tumor microenvironment (TME) and act as professional antigen presenting cells, we developed an immunocompetent animal model to evaluate the interaction of CAR-M with the endogenous immune system and to study the combinatorial approach of CAR-M with blockade of the PD1/PDL1 T cell checkpoint axis. Murine bone marrow-derived macrophages were engineered to express an anti-HER2 CAR using the chimeric adenoviral vector Ad5f35. In addition to efficient gene delivery, Ad5f35 transduction promoted a pro-inflammatory (M1) phenotype in murine macrophages. Anti-HER2 CAR-M, but not control macrophages, phagocytosed and killed HER2-overexpressing tumor cell lines. CAR-M induced MHC-I expression on tumor cells and enhanced the cytotoxicity of CD8+ T cells. To evaluate the safety and efficacy of CAR-M therapy, immunocompetent mice were engrafted with HER2+ tumors and treated with syngeneic HER2-CAR or untransduced (UTD) macrophages. CAR-M treated mice showed significant tumor control and improved survival compared to control groups. Analysis of the TME showed increased intratumoral immune infiltration - as well as an increase in T cell responsiveness to tumor-associated antigens, indicating enhanced epitope spreading. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with anti-PD1 in tumors resistant to anti-PD1 monotherapy and found that the combination further reprogrammed the TME, significantly enhanced tumor control, and improved overall survival compared to monotherapy with either agent. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse in a HER2-negative rechallenge model, indicating long-term anti-tumor immunity. Finally, the combination of CAR-M with anti-PD1 did not trigger sustained elevations of serum analytes associated with cytokine release syndrome (CRS) and was well tolerated across numerous safety assessments. These results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors. The anti-HER2 CAR-M, CT-0508, is under evaluation in a phase I clinical trial for patients with HER2 overexpressing solid tumors. Citation Format: Stefano Pierini, Rashid Gabbasov, Alison Worth, Ilyssa Ramos, Daniel Blumenthal, Yumi Ohtani, Linara Gabitova, Michael Ball, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Chimeric antigen receptor macrophages (CAR-M) sensitize solid tumors to anti-PD1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2112.

Newly identified breast luminal progenitor and gestational stem cell populations likely give rise to HER2-overexpressing and basal-like breast cancers

Breast Cancer (BrC) is a common malignancy with genetically diverse subtypes. There is evidence that specific BrC subtypes originate from particular normal mammary cell populations. However, the cell populations that give rise to most BrC subtypes are unidentified. Several human breast scRNAseq datasets are available. In this research, we utilized a robust human scRNAseq dataset to identify population-specific marker genes and then identified the expression of these marker genes in specific BrC subtypes. In humans, several BrC subtypes, HER2-enriched, basal-like, and triple-negative (TN), are more common in women who have had children. This observation suggests that cell populations that originate during pregnancy give rise to these BrCs. The current human datasets have few normal parous samples, so we supplemented this research with mouse datasets, which contain mammary cells from various developmental stages. This research identified two novel normal breast cell populations that may be the origin of the basal-like and HER2-overexpressing subtypes, respectively. A stem cell-like population, SC, that expresses gestation-specific genes has similar gene expression patterns to basal-like BrCs. A novel luminal progenitor cell population and HER2-overexpressing BrCs are marked by S100A7, S100A8, and S100A9 expression. We bolstered our findings by examining SC gene expression in TN BrC scRNAseq datasets and S100A7-A9 gene expression in BrC cell lines. We discovered that several potential cancer stem cell populations highly express most of the SC genes in TN BrCs and confirmed S100A8 and A9 overexpression in a HER2-overexpressing BrC cell line. In summary, normal SC and the novel luminal progenitor cell population likely give rise to basal-like and HER2-overexpressing BrCs, respectively. Characterizing these normal cell populations may facilitate a better understanding of specific BrCs subtypes.

Photothermal Therapy with HER2-Targeted Silver Nanoparticles Leading to Cancer Remission.

Nanoparticles exhibiting the localized surface plasmon resonance (LSPR) phenomenon are promising tools for diagnostics and cancer treatment. Among widely used metal nanoparticles, silver nanoparticles (Ag NPs) possess the strongest light scattering and surface plasmon strength. However, the therapeutic potential of Ag NPs has until now been underestimated. Here we show targeted photothermal therapy of solid tumors with 35 nm HER2-targeted Ag NPs, which were produced by the green synthesis using an aqueous extract of Lavandula angustifolia Mill. Light irradiation tests demonstrated effective hyperthermic properties of these NPs, namely heating by 10 °C in 10 min. To mediate targeted cancer therapy, Ag NPs were conjugated to the scaffold polypeptide, affibody ZHER2:342, which recognizes a clinically relevant oncomarker HER2. The conjugation was mediated by the PEG linker to obtain Ag-PEG-HER2 nanoparticles. Flow cytometry tests showed that Ag-PEG-HER2 particles successfully bind to HER2-overexpressing cells with a specificity comparable to that of full-size anti-HER2 IgGs. A confocal microscopy study showed efficient internalization of Ag-PEG-HER2 into cells in less than 2 h of incubation. Cytotoxicity assays demonstrated effective cell death upon exposure to Ag-PEG-HER2 and irradiation, caused by the production of reactive oxygen species. Xenograft tumor therapy with Ag-PEG-HER2 particles in vivo resulted in full primary tumor regression and the prevention of metastatic spread. Thus, for the first time, we have shown that HER2-directed plasmonic Ag nanoparticles are effective sensitizers for targeted photothermal oncotherapy.