HER2-negative Cells Research Articles

Abstract P2-03-10: DNA replication licensing is associated with resistance to CDK4/6 inhibitors in ER-positive breast cancer

Abstract Background: The use of CDK4/6 inhibitors (CDK4/6i) has led to a remarkable progress in the treatment of advanced ER-positive (+) breast cancer (BC). The fact that both CDK4/6i and endocrine therapy (ET) target cell-cycle G1-S transition suggests some overlap of resistance mechanisms. However, clinical variability in response to CDK4/6i in patients progressing on ET suggests the involvement of additional or unique resistance mechanisms. Using bioinformatics analyses of clinical and preclinical data, we now identified genes involved in DNA replication licensing, in particular, excess MCM2 as a new mechanism of resistance to CDK4/6i. Methods: Gene expression data from the neoadjuvant NeoPalAna trial of the CDK4/6i palbociclib (Palbo) and the aromatase inhibitor (AI) anastrozole in ER+ BC was downloaded from Gene Expression Omnibus (GSE93204). Response to AI and Palbo was obtained using the published Ki67 immunohistochemistry data and cutoff on biopsies post treatment. Gene Set Enrichment Analysis comparing tumors with different sensitivity to AI vs. Palbo was performed using the Gene Ontology related to DNA replication, DNA repair, and DNA damage checkpoint. The DNA replication gene set was further sub-assigned by their selective role in origin licensing, firing, elongation, replication repair, and checkpoint regulation. A series of bioinformatics analyses was applied on five ER+/HER2-negative BC cell lines with gene expression and DepMap (from CCLE) and Palbo sensitivity data (from GDSC). Two ER+ BC cell models (MCF7 and T47D) and their estrogen deprivation (mimicking AIs)-resistant (EDR) derivatives were used for further studies including RNA-seq, Palbo sensitivity (data from PMID: 33536276), and chromatin fractionation assays. Results: We found that the DNA replication-associated gene set, comprising > 80% genes outside the Rb-loss gene signature, was significantly enriched in baseline biopsies of Palbo-resistant (PalboR) tumors from patients in the NeoPalAna trial. Of the DNA replication genes, the subset of genes involved in origin licensing were preferentially enriched in PalboR vs. AI-resistant tumors. Similarly, the enrichment of genes involved in replication initiation was also seen in ER+/HER2-negative BC cell lines with a decreased response to Palbo. Notably, these PalboR cell lines showed a reduced vitality to shRNA knockdown of the replication initiation genes compared to randomly selected other genes or the Rb-loss gene set. Based on the modeling of DepMap gene dependency score and Palbo sensitivity, we nominated minichromosome maintenance 2 (MCM2), the key origin licensing factor, as the top gene that is essential for PalboR cell survival. Additionally, using our previously reported MCF7-EDR and T47D-EDR cell models with increased or decreased sensitivity to Palbo compared to their parental lines, respectively, we observed a corresponding decrease or increase in the expression of origin licensing genes and MCM2 in EDR vs. parental lines. Decreased Palbo sensitivity in the EDR cells was associated with sustained MCM2 chromatin loading and reduced expression of genes including the cyclin-dependent kinase inhibitor p21. Ongoing studies investigate whether elevated MCM2 levels confer resistance to CDK4/6i by resolving replication stress. Conclusions: Our strategic bioinformatics analyses reveal that excess DNA replication licensing is associated with CDK4/6i resistance in clinical and preclinical settings upon resistance to estrogen deprivation. Among the replication initiation-associated genes, MCM2 plays a potential role in conferring CDK4/6i resistance via sustaining origin licensing and suppressing p21. Our study provides a new avenue via lens of replication licensing to explore novel mechanisms and therapeutic opportunities in CDK4/6i resistance. Citation Format: Sarmistha Nanda, Martin J. Shea, Rachel Schiff, C. Kent Osborne, Mothaffar Rimawi, Xiaoyong Fu. DNA replication licensing is associated with resistance to CDK4/6 inhibitors in ER-positive breast cancer [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 P2-03-10.

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.

Molecular characterization and potential therapeutic roles of miR125a in HER-2 positive gastric cancer.

miR-125a-3p could have a role in gastric cancer by targeting HER2. This study aimed to investigate the expression pattern of miR-125a-3p, identify the expression level of its target gene in gastric carcinoma, and test its effect in HER-2 positive gastric carcinoma cells. The levels of miR-125a-3p in both cancer and noncancer tissues were measured by using Quantitative real-time polymerase chain in 70 gastric carcinomas. Immunohistochemical study was used to measure the expression of HER2 protein in these carcinomas. In addition, the level of expression of this miRNA is correlated to different pathological and clinical parameters. The effects of miR-125a-3p alone and in combination with 5-FU (fluorouracil) on the growth of HER2 positive (NUGC4) and HER2 negative (ECC10) gastric carcinoma cells were also analyzed by in vitro studies. Most gastric cancer tissues samples showed downregulation of miR-125a-3p (84%) when compared to their noncancer tissues. Significant correlations of downregulation of miR-125a-3p with cancer recurrence and pathological staging of gastric carcinoma (P = 0. 02 and 0.02, respectively) were noted. HER2 protein expression correlated significantly and inversely with miR-125a-3p expression (P < 0.05). A reduction in cell growth rate was noted significantly in miR-125a-3p transfected gastric carcinoma cells when 5-FU was added to them in comparison to other control cells (P < 0.01). When both gastric carcinoma cell lines were transfected with miR-125a-3p, a significantly higher growth inhibition percentage in HER2 positive (NUGC4) cell line was seen in comparison to the HER2 negative (ECC10) cells (P < 0.01). miR-125a-3p plays a significant role in the pathogenesis of gastric carcinoma. Therapeutic transfection of miR-125a-3p in HER2 positive gastric cancer cells resulted in reduced cell proliferation and potentiate the effect of 5-FU.

ICG-Dimeric Her2-Specific Affibody Conjugates for Tumor Imaging and Photothermal Therapy for Her2-Positive Tumors.

Human epidermal growth factor receptor 2 (Her2) is abundantly expressed in various solid tumors. The Her2-specific Affibody (ZHer2:2891) has been clinically tested in patients with Her2-positive breast cancer and is regarded as an ideal drug carrier for tumor diagnosis and targeted treatment. Indocyanine green (ICG) can be used as a photosensitizer for photothermal therapy (PTT), in addition to fluorescent dyes for tumor imaging. In this study, a dimeric Her2-specific Affibody (ZHer2) based on ZHer2:2891 was prepared using the E. coli expression system and then coupled to ICG through an N-hydroxysuccinimide (NHS) ester reactive group to construct a novel bifunctional protein drug (named ICG-ZHer2) for tumor diagnosis and PTT. In vitro, ICG-ZHer2-mediated PTT selectively and efficiently killed Her2-positive BT-474 and SKOV-3 tumor cells rather than Her2-negative HeLa tumor cells. In vivo, ICG-ZHer2 specifically accumulated in Her2-positive SKOV-3 tumor grafts rather than Her2-negative HeLa tumor grafts; high-contrast tumor optical images were obtained. However, Her2-negative HeLa tumor grafts were not detected. More importantly, ICG-ZHer2-mediated PTT exhibited a significantly enhanced antitumor effect in mice bearing SKOV-3 tumor grafts owing to the good photothermal properties of ICG-ZHer2. Of note, ICG-ZHer2 did not exhibit acute toxicity in mice during short-term treatment. Overall, our findings indicate that ICG-ZHer2 is a promising bifunctional drug for Her2-positive tumor diagnosis and PTT.

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 1day 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 1day 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.

1705P HER2 intratumoral genetic and non-genetic heterogeneity in metastatic colorectal cancer

HER2-positive metastatic colorectal cancer (mCRC) is a distinct subtype for which HER2-targeted therapy is established. HER2 intratumoral heterogeneity (ITH), containing two types of heterogeneity–genetic heterogeneity (GH, a mixture of HER2 positive and negative tumor cells) and non-genetic heterogeneity (NGH, a mixture of HER2 positive tumor cells and amplified HER2 gene tumor cells without HER2 protein expression)– is a predictor of response to HER2-targeted treatment in breast cancer (BC). Here, using samples with mCRC, we evaluated the diagnostic accuracy of the Gene-Protein Assay (GPA), an assay assessing HER2 ITH by a combined HER2 immunohistochemistry (IHC) and bright-field HER2 in situ hybridization (ISH) on the same tissue section, and the frequency of HER2 ITH and heterogeneous patterns.

Cell Proliferation Receptor-Enhanced 3D High-Throughput Screening Model for Optimized Drug Efficacy Evaluation in Breast Cancer Cells.

A higher correlation of epidermal growth factor receptor (EGFR)-targeting drugs has been reported with the use of the cell proliferation receptor-enhanced three-dimensional high-throughput screening model (CPRE 3D-HTS model) compared with two-dimensional (2D) cell-based HTS. A greater expression of differential human EGFR 2 (HER2) protein between HER2-positive and HER2-negative cell lines was observed in breast cancer (BC) cell lines cultured using the CPRE 3D-HTS model compared with 2D-cultured cells. When using 2D-cultured cells, properties such as the expression of the cell proliferation receptor are lost as the cells attach to the bottom of the well plate. In an effort to solve this problem, the CPRE 3D-HTS model expressing high cell proliferation receptors was optimized by the selection of alginate as the extracellular matrix. Results from the use of the CPRE 3D-HTS model showed higher drug resistance with increased expression of drug resistance-related proteins. Of particular interest, a higher correlation of HER2-targeted drugs was observed with the use of the CPRE 3D-HTS model. In order to validate this higher correlation of target drugs observed in the CPRE 3D-HTS model, the results of Western blot analysis and high content imaging analysis were analyzed, which confirmed that 3D-cultured BC cell lines showed a greater difference in the expression of HER2-positive and HER2-negative BC cell lines than 2D-cultured cells. Thus, the use of CPRE 3D-HTS using a 384-pillar plate resulted in increased accuracy when screening HER2-targeted drugs in BC, and it is a very useful platform for analyzing the efficacy of targeted drugs by enhancing the expression of HER2.

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.

SNHG6 203 RNA May be Involved in the Cell Cycle Progression in HER2-Negative Breast Cancer Cells

Background & Objective: Long noncoding RNAs (lncRNAs) as challenging molecules are more known compared to those in the last decade. These transcripts have been validated for carcinogenesis in many types of tissue. Functions of lncRNAs in cancer induction include cell cycle, epithelial to mesenchymal transition progression, apoptosis inhibition, cell migration, and invasion stimulation . LncRNA small nucleolar host (SNHG6) have been proven as an oncogenic transcript in many types of cancer.Methods: RNA extraction was performed for 47 breast specimens in patients with cancer and cDNAs were synthesized. Relative expression of target variants was determined by qPCR and calculated based on the ΔΔCt method. SNHG6 203 was cloned into pcDNA 3.1+ vector for overexpression in MCF7 (HER2-) and SK-BR3 (HER2+) cells. The cell cycle progression of transfected cells was assessed by flow cytometry. Cell migration ability of transfected cells was evaluated by the scratch method and Image J software. Finally, cell viability was assessed by the MTT method.Results: Among four splice variants of SNHG6 (202, 203, 204, and 207), SNHG6 203 was proved as an overexpressed splice variant in breast cancer tumors. This transcript was expressed in HER2-negative breast tumors more frequently than in the positive ones. Overexpression of this variant in target cells resulted in cell cycle progression of MCF7 as HER2-negative cells. Moreover, the overexpression of SNHG6 203 led to a lower migration ability of MCF7 cells and a non-significant reduction of their viability as HER2-negative breast cancer cells.

Sex and gender disparities in patients with advanced gastroesophageal adenocarcinoma: data from the AGAMENON-SEOM registry

BackgroundRecommendations for research articles include the use of the term sex when reporting biological factors and gender for identities or psychosocial or cultural factors. There is an increasing awareness of incorporating the effect of sex and gender on cancer outcomes. Thus, these types of analyses for advanced gastroesophageal adenocarcinoma are relevant.Patients and methodsPatients with advanced gastroesophageal adenocarcinoma from the Spanish AGAMENON-SEOM registry treated with first-line combination chemotherapy were selected. Epidemiology, characteristics of the disease, treatment selection, and results were examined according to sex.ResultsThis analysis included 3274 advanced gastroesophageal adenocarcinoma patients treated with combination chemotherapy between 2008 and 2021: 2313 (70.7%) men and 961 (29.3%) women. Tumors in females were more frequently HER2-negative (67.8% versus 60.8%; P < 0.0001), grade 3 (45.4% versus 36.8%; P < 0.001), diffuse (43.3% versus 26.5%; P < 0.0001), and signet ring cell histology (40.5 versus 23.9%; P < 0.0001). Peritoneal spread was more common in women (58.6% versus 38.9%; P < 0.0001), while liver burden was lower (58.9% versus 71.1%; P < 0.0001). There were no significant differences in treatment recommendation. Treatment doses, density, and duration were comparable between sexes. Women experienced more diarrhea (46% versus 37%; P < 0.0001), neutropenia (51% versus 43%; P < 0.0001), and anemia (62% versus 57%; P < 0.0001). After a median 59.6-month follow-up [95% confidence interval (CI) 54.5-70.8], there were no statistically significant differences between the sexes in progression-free survival [6.21 months (95% CI 5.8-6.5 months) versus 6.08 months (95% CI 5.8-6.3 months); log-rank test, χ2 = 0.1, 1 df, P = 0.8] or in overall survival [10.6 months (95% CI 9.8-11.1 months) versus 10.9 months (95% CI 10.4-11.4 months); log-rank test: χ2 = 0.6, 1 df, P = 0.5].ConclusionThis sex analysis of patients with advanced gastroesophageal adenocarcinoma from the AGAMENON-SEOM registry receiving first-line polychemotherapy found no differences in survival. Although women had worse prognostic histopathology, metastatic disease pattern, and greater toxicity, treatment allocation and compliance were equivalent.

Using American Type Culture Collection Cell Lines to Evaluate Interlaboratory Variables for Estrogen Receptor and Human Epidermal Growth Factor Receptor 2 Immunostaining.

Most laboratories currently use patient tissues for validating immunohistochemical stains. To explore advantages of using cell lines with known antigenicity as a validation method. Five American Type Culture Collection (ATCC) cell lines with known negative, low positive, and moderate to strong estrogen receptor (ER) expression as well as negative, equivocal, and positive human epidermal growth factor receptor 2 (HER2) expression were cultured and made into cell blocks. One block from each cell line was fixed in formalin and another in ethanol before cell block preparation. Two sets of paired unstained slides from each block were sent to 10 different laboratories for HER2 and ER staining to be stained on runs from different days according to each laboratory's defined protocol. The 10 study participants evaluated 40 slides in a blinded fashion. For ER expression, all 80 interpretations for the ER strong and moderate positive cell lines had the target ER-positive result, and 74 of 80 ER-negative cell lines (92.5%) had agreement with the intended negative result. The ER low positive cell line showed varied but positive expression among all observers. The HER2 (3+)-positive cell lines yielded a target interpretation of 3+ in 65 of 80 interpretations (81.2%). For the HER2-negative cell line 69 of 78 interpretations (88.5%) were consistent with the target response (0 or 1+). No significant variation was observed between the ethanol- and non-ethanol-exposed cell lines, or between runs by the same laboratory. Variation from target results clustered within laboratories. This study indicates that variability between laboratories can be identified by using cell lines for quantitative or semiquantitative immunohistochemistry when using cultured cell lines of known antigenicity. These cell lines could potentially play a role in aiding anatomic pathology laboratories in validating immunohistochemistry tests for formalin- and ethanol-fixed tissues.

Abstract P2-13-33: Preclinical development of DHES0815A: A HER2-directed antibody-drug conjugate comprised of a reduced potency PBD dimer linked to a domain I binding HER2 antibody

Abstract Background: Many HER2-directed antibody-drug conjugates (ADCs) in early development employ trastuzumab linked to tubulin binding agents as the cytotoxic drug. DHES0815A is an ADC consisting of a THIOMABTM humanized IgG1 anti-HER2 monoclonal antibody (hu7C2), which binds domain 1 of the HER2 ECD, conjugated via a disulfide linker to PBD-monoamide (MA), a DNA mono-alkylating agent. The reduced potency of the PBD-MA payload compared to PBD dimers and the stability of the conjugation site and linker were designed to improve tolerability, whereas the binding of hu7C2 to a HER2 epitope distinct from trastuzumab and pertuzumab allows combination with existing HER2 therapies. . Methods: Modifications at one imine in the PBD (pyrrolobenzodiazepine) dimer produced mono-alkylating PBDs that were assessed for DNA binding, permeability and cell potency. Each modified PBD was conjugated to hu7C2 LC K149C via a disulfide linker and assessed in HER2-positive models in vitro and in vivo. Safety studies were performed in female and male cynomolgus monkey treated every 3 weeks X 5, with a 7-week follow-up. Assessments included hematology, clinical chemistry, anti-drug antibody, standard clinical, neurological and ophthalmic exams and histopathology. Results: Our goal was design of a reduced potency PBD dimer ADC to achieve dosing in the linear PK range for optimal exposure, efficacy and safety. PBD dimers contain 2 reactive imines that bis-alkylate and crosslink DNA. Mono-alkylating PBD dimers were assessed in DNA binding, potency and permeability assays to select the optimal mono-alkylator, PBD-MA, for further investigation. hu7C2-disulfide-PBD-MA, or DHES0815A, was potent across HER2-positive breast and gastric cancer models in vitro and in vivo, with little effect on HER2-negative tumors or normal primary cells in vitro. DHES0815A was more potent than T-DM1 in HER2-positive and HER2-low breast and gastric cancer PDX models and demonstrated combination activity with standard of care agents T-DM1 or docetaxel. Mechanistically, treatment of HER2-positive cells with DHES0815A resulted in late S-phase cell cycle arrest; induction of DNA damage markers γH2AX, phospho-p53, phospho-CHK2, induction of apoptosis, and bystander activity. Pharmacokinetic analysis showed dose-proportional PK, with linear biphasic PK &amp;gt;/= 4mg/kg in cynomolgus monkey. Safety studies in cynomolgus monkey treated with 4, 8 or 12 mg/kg demonstrated that findings were non-adverse, monitorable and manageable. All doses were tolerated with few clinical observations and minimal to mild clinical pathology changes. Target organs were skin, bone marrow, lung and eye. The HNSTD (highest non-severely toxic dose) was 12 mg/kg. This dose enabled a starting dose of 0.6 mg/kg for phase 1 testing. Conclusions: DHES0815A is a HER2-directed ADC comprised of a domain I binding antibody linked to a reduced potency PBD dimer, PBD-MA, via a reducible disulfide linker. The antibody does not interfere with trastuzumab or pertuzumab binding, allowing combination with existing HER2 therapies. The reduced potency payload allows dosing in the linear PK range, supporting our hypothesis. DHES0815A demonstrated dose-dependent anti-tumor efficacy in multiple HER2+ breast and gastric cancer models, and showed efficacy in HER2-low models, where T-DM1 was inactive. Enhanced anti-tumor activity was observed combining DHES0815A with T-DM1, trastuzumab/pertuzumab or docetaxel. Studies in cynomolgus monkey demonstrated a safety profile compatible with initiating a phase 1 clinical trial. The phase 1 data will be presented in a separate presentation. Citation Format: Gail Lewis Phillips, Guangmin Li, Jun Guo, Shang-Fan Yu, Genee Lee, Donglu Zhang, Peter Dragovich, Thomas Pillow, Binqing Wei, M. Violet Lee, Ola Saad, Shab Masih, Douglas Leipold, Lisa Crocker, Melissa Schutten. Preclinical development of DHES0815A: A HER2-directed antibody-drug conjugate comprised of a reduced potency PBD dimer linked to a domain I binding HER2 antibody [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-33.

SNHG6 203 RNA May be Involved in the Cell Cycle Progression in HER2-Negative Breast Cancer Cells.

Background & Objective:Long noncoding RNAs (lncRNAs) as challenging molecules are more known than those in the last decade. These transcripts have been validated for carcinogenesis in many types of tissue. Functions of lncRNAs in cancer induction include cell cycle, epithelial to mesenchymal transition progression, apoptosis inhibition, cell migration, and invasion stimulation. LncRNA small nucleolar host gene 6 (SNHG6) have been proven as an oncogenic transcript in many types of cancer. Methods:RNA extraction was performed for 47 breast specimens in patients with cancer and cDNAs were synthesized. Relative expression of target variants was determined by qPCR and calculated based on the ΔΔCt method. SNHG6 203 was cloned into pcDNA 3.1+ vector for overexpression in MCF7 (HER2-) and SK-BR3 (HER2+) cells. The cell cycle progression of transfected cells was assessed by flow cytometry. Cell migration ability of transfected cells was evaluated by the scratch method and Image J software. Finally, cell viability was assessed by the MTT method.Results:Among four splice variants of SNHG6 (202, 203, 204, and 207), SNHG6 203 was proved as an overexpressed splice variant in breast tumors. This transcript was expressed in HER2-negative breast tumors more frequently than in the positive ones. Overexpression of this variant in target cells resulted in cell cycle progression of MCF7 as HER2-negative cells. Moreover, the overexpression of SNHG6 203 led to lower migration ability of MCF7 cells and a non-significant reduction of their viability as HER2-negative breast cancer cells. Conclusion:Our results revealed that SNHG6 203 may be involved in the carcinogenesis of HER2-negative breast cancers via cell cycle progression.

Abstract P062: The CL2A-SN38 linker-payload system conjugated to trastuzumab results in improved cellular cytotoxicity over time relative to T-DM1

Abstract Background The approved antibody-drug conjugate (ADC) sacituzumab-govitecan (SG) that exploits the topoisomerase I inhibitor SN38, represents a substantial advance in the ADC field. SN38 is the first payload with low nanomolar cytotoxicity. This deviates from current design principles, which utilize ultracytotoxic payloads. The CL2A linker has a short non-cleavable seven polyethylene glycol segment that enables a higher number of hydrophobic SN38 molecules to be attached to reduced cysteines. CL2A is also designed to permit the release of SN38 in an acidic environment via a pH-sensitive benzyl carbonate bond to SN38’s lactone ring. There is no cathepsin B cleavage site. Key to the performance of SG is the SN38 release half-life of 1-2 days. This also deviates from very stable linker systems that require full ADC internalization and lysosomal processing for payload release. As a result of these novel ADC properties, SG is given in higher and repeated doses, which results in slow release at the tumor site and enhanced uptake by target cancer cells and reduced toxicity on healthy cells due to low potency SN38 alone. Our goal is to further evaluate the CL2A-SN38 system when conjugated to trastuzumab. Material and methods Trastuzumab was conjugated to CL2A-SN38 to reduced cysteines (T-SN38). Monitoring the changes in DAR over time was performed to determine the stability of T-SN38 in PBS at pH values of 7.4, 5.72, and 4.58, to mimic circulating blood, early endosomes, and lysosomes, respectively. Cytotoxicity assays for 72 h on HER2-positive ovarian cancer SKOV3.ip and HER2-negative CHO cells were performed and compared to the approved anti-HER2 ADC trastuzumab-emtansine (T-DM1). Cytotoxicity assays were also performed between T-SN38 and T-DM1 at their respective IC50 values and the percent of cell survival determined daily from 24-96 h. Results T-SN38 was generated with a DAR of 6.97. Surprisingly, the half-life of SN38 release in acidic pH was extremely slow (t1/2=~120 h) and at the final evaluated time point (120 h) 69% of SN38 remained conjugated to trastuzumab. As anticipated, as a function of concentration T-DM1 was significantly more potent than T-SN38 (IC50=0.0013 vs 0.0832 mg/mL). However, when cytotoxicity was evaluated as a function of time T-SN38 switched to the more potent ADC at the late time points. T-SN38 killed SKOV3.ip cells 36% more effectively than T-DM1 at 96 h. Conclusions Our data from SN38 release experiments in acidic buffer suggest that pH-induced cleavage of the CL2A linker is not a contributable release mechanism of SN38 when bound to trastuzumab and is counterintuitive for what is currently described for SG. However, traceless release of hydrophobic SN38 capable of traversing cell membranes is only possible by hydrolysis of the benzyl carbonate bond. Our cytotoxicity studies over time suggest that pH-dependent release does occur. Further investigations are ongoing to more precisely determine the contribution of the carbonate bond for SN38 release and cytotoxic potency. Citation Format: Hardeep Singh, Victor Jeffrey Leyton. The CL2A-SN38 linker-payload system conjugated to trastuzumab results in improved cellular cytotoxicity over time relative to T-DM1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P062.

Targeting HER2 protein in individual cells using ICP-MS detection and its potential as prognostic and predictive breast cancer biomarker

The human epidermal growth factor receptor 2 (HER2) is a transmembrane protein that has become one of the most specific prognostic and predictive biomarker of breast cancer. Its early detection is key for optimizing the patient clinical outcome. This work is focused on the detection of HER2 in individual cells using an antibody containing lutetium (Lu) as reporter group that is monitored by introducing the individual cells into the inductively coupled plasma mass spectrometer (ICP-MS). This Lu-containing antibody probe is used to label different breast cancer cell lines considered HER2 negative (MDA-MB-231) and positive (SKBR-3 and BT-474). Optimizations regarding the amount of the probe necessary to ensure complete labelling reactions are conducted in the different cell models. Concentrations in the range of 0.006 fg Lu/cell and 0.030 fg Lu/cell could be found in the HER2 negative and HER2 positive cells, respectively. In addition, the selectivity of the labelling reaction is tested by using two different metal-containing antibody probes for HER2 (containing Lu) and for transferrin receptor 1 (containing Nd), respectively, within the same cell population. Finally, the methodology is applied to the targeting of HER2 positive cells in complex cell mixtures containing variable amounts of BT-474 and MDA-MB-231 cells. The obtained results showed the excellent capabilities of the proposed strategy to discriminate among cell populations. This finding could help for scoring HER2 positive tumors improving existing technologies.

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

ABSTRACT 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.

Comparative Evaluation of Engineered Polypeptide Scaffolds in HER2-Targeting Magnetic Nanocarrier Delivery.

Targeted drug delivery is one of the most intriguing and challenging issues in modern biomedicine. For active targeting, full-size IgG molecules (150 kDa) are usually used. Recent studies have revealed that small artificial polypeptide scaffolds such as DARPins (14 kDa) and affibodies (8 kDa) are much more promising tools for drug delivery due to their small size, artificial nature, low immunogenicity, and many other properties. However, there is no comparative information on the targeting abilities of scaffold polypeptides, which should be taken into account when developing drug delivery systems (DDSs). The present work is the first comprehensive study on the comparison of the effectiveness of different HER2-targeting proteins within the architecture of nanoparticles. Namely, we synthesized trimodal nanoparticles: magnetic, fluorescent, and directed toward HER2 oncomarker on cancer cells. The magnetic particles (MPs) were covalently modified with (i) full-size IgG, 150 kDa, (ii) DARPin_G3, 14 kDa, and (iii) affibody ZHER2:342, 8 kDa. We showed that the number of DARPin_G3 and affibody ZHER2:342 molecules conjugated to the nanoparticle surface are 10 and 40 times higher, respectively, than the corresponding value for trastuzumab. Using the methods of magnetic particle quantification (MPQ)-cytometry and confocal microscopy, we showed that all types of the obtained magnetic conjugates specifically labeled HER2-overexpressing cells. Namely, we demonstrated that particle binding to HER2-positive cells is 1113 ± 39 fg/cell for MP*trastuzumab, 1431 ± 186 fg/cell for MP*ZHER2:342, and 625±21 fg/cell for MP*DARPin_G3, which are 2.77, 2.75, and 2.30 times higher than the corresponding values for control HER2-negative cells. Thus, we showed that the smallest HER2-recognizing polypeptide affibody ZHER2:342 is more effective in terms of specificity and selectivity in nanoparticle-mediated cell labeling.

Visualization of Intratumor Pharmacokinetics of [fam-] Trastuzumab Deruxtecan (DS-8201a) in HER2 Heterogeneous Model Using Phosphor-integrated Dots Imaging Analysis

Abstract Purpose: We assessed the intratumor pharmacokinetics of [fam-] trastuzumab deruxtecan, T-DXd (known as DS-8201a), a novel HER2-targeted antibody–drug conjugate, using phosphor-integrated dots (PID)-imaging analysis to elucidate its pharmacologic mechanism. Experimental Design: We used two mouse xenograft models administered T-DXd at the concentration of 4 mg/kg: (i) a heterogeneous model in which HER2-positive and HER2-negative cell lines were mixed, and (ii) a homogeneous model in which both cell types were transplanted separately into the same mouse. PID imaging involved immunostaining using novel high-intensity fluorescent nanoparticles. The distribution of T-DXd was assessed by PID imaging targeting the parent antibody, trastuzumab, and the payload, DXd, in serial frozen sections, respectively. Results: After T-DXd administration in the heterogeneous model, HER2 expression tended to decrease in a time-dependent manner. The distribution of trastuzumab and DXd was observed by PID imaging along the HER2-positive area throughout the observation period. A detailed comparison of the PID distribution between trastuzumab and DXd showed that trastuzumab matched almost perfectly with the HER2-positive area. In contrast, DXd exhibited widespread distribution in the surrounding HER2-negative area as well. In the HER2-negative tumor of the homogeneous model, the PID distribution of trastuzumab and DXd remained extremely low throughout the observation period. Conclusions: Our results suggest that T-DXd is distributed to tumor tissues via trastuzumab in a HER2-dependent manner and then to adjacent HER2-negative areas. We successfully visualized the intratumor distribution of T-DXd and its mechanism of action, the so-called “bystander effect.”

Au@Pt Core-Shell Nanoparticle Bioconjugates for the Therapy of HER2+ Breast Cancer and Hepatocellular Carcinoma. Model Studies on the Applicability of 193mPt and 195mPt Radionuclides in Auger Electron Therapy.

193mPt and 195mPt radionuclides are therapeutically attractive Auger electron emitters with notably high Auger electron yield per decay. The present paper summarizes the first step of research on the applications of core-shell (Au@Pt) nanoparticles for electron Auger therapy of HER2+ (human epidermal growth factor receptor 2) breast cancer and hepatocellular carcinoma. Gold nanoparticles (30 nm) were synthesized covered with a platinum shell at high efficiency (>80%) and were further evaluated for in vitro studies such as binding affinity, internalization and cytotoxicity. To find the mechanism(s) responsible for platinum cytotoxicity in HepG2 cells, the platinum concentration in isolated cell nuclei and cytoplasm was determined using ICP-MS (inductively coupled plasma mass spectrometry). Lack of platinum in cell nuclei suggests that the cytotoxic effect is associated with the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Studies carried out on the SKOV-3 cell line with the use of a synthesized targeting bioconjugate (Au@Pt-PEG-trastuzumab) revealed a high affinity of this preparation to HER2+ cells, its internalization, its placement in the perinuclear area and partial intranuclear location. The specific binding for HER2 negative cells, MDA-MB-231, was negligible and Au@Pt-PEG-trastuzumab did not enter these cells. The results obtained are promising and warrant future investigation of Auger electron therapy using 193mPt and 195mPt based radiopharmaceuticals.

Trastuzumab deruxtecan for the treatment of HER2-positive advanced gastric cancer: a clinical perspective.

Human epidermal growth factor receptor 2 (HER2)-positive gastric cancer is a subtype for which new drugs and specific treatment strategies should be developed. Trastuzumab deruxtecan (T-DXd) is a novel HER2-targeted antibody-drug conjugate containing topoisomerase I inhibitor as a payload. In the randomized phase 2 study (DESTINY-Gastric01) for HER2-positive advanced gastric or gastroesophageal junction cancer (AGC), patients treated with T-DXd showed a significantly higher response rate compared with the chemotherapy of physician's choice, associated with remarkably prolonged progression-free and overall survival. T-DXd also exhibits anti-tumor activity to HER2-negative tumor cells close to HER2-positive cells (so-called bystander killing effect). T-DXd was effective even for HER2-low expressing breast and gastric cancer in several clinical studies. Taking advantage of these strong points and synergism with other cytotoxic, molecular-targeted and immunological agents, it is expected that T-DXd will bring further progression in treatment both for strongly and weakly HER2 positive AGC in various treatment settings including perioperative chemotherapy.