HER2 Antibody Research Articles

Conducting Polymer-Coated Carbon Cloth Captures and Releases Extracellular Vesicles by a Rapid and Controlled Redox Process.

Electrochemical techniques offer great opportunities for the capture of chemical and biological entities from complex mixtures and their subsequent release into clean buffers for analysis. Such methods are clean, robust, rapid, and compatible with a wide range of biological fluids. Here, we designed an electrochemically addressable system, based on a conducting terpolymer [P(EDOT-co-EDOTSAc-co-EDOTEG)] coated onto a carbon cloth substrate, to selectively capture and release biological entities using a simple electrochemical redox process. The conducting terpolymer composition was optimized and the terpolymer-coated carbon cloth was extensively characterized using electrochemical analysis, Raman and Fourier transform-infrared spectroscopy, water contact angle analysis, and scanning electron microscopy. The conductive terpolymer possesses a derivative of EDOT with an acetylthiomethyl moiety (EDOTSAc), which is converted into a "free" thiol that then undergoes reversible oxidation/reduction cycles at +1.0 V and -0.8 V (vs Ag/AgCl), respectively. That redox process enables electrochemical capture and on-demand release. We first demonstrated the successful electrochemical capture/release of a fluorescently labeled IgG antibody. The same capture/release procedure was then applied to release extracellular vesicles (EVs), originating from both MCF7 and SKBR3 breast cancer cell line bioreactors. EVs were captured using the substrate-conjugated HER2 antibody which was purified from commercially available trastuzumab. Capture and release of breast cancer EVs using a trastuzumab-derived HER2 antibody has not been reported before (to the best of our knowledge). A rapid (2 min) release at a low potential (-0.8 V) achieved a high release efficiency (>70%) of the captured, HER2+ve, SKBR3 EVs. The developed system and the electrochemical method are efficient and straightforward and have vast potential for the isolation and concentration of various biological targets from large volumes of biological and other (e.g., environmental) samples.

Clinical significance of B7-H3 and HER2 co-expression and therapeutic value of combination treatment in gastric cancer

BackgroundGastric cancer (GC) is a digestive system malignancy. Trastuzumab (a HER2-targeted monoclonal antibody) is an important targeted drug for GC. However, the drug resistance limits its clinical efficacy. B7-H3 was suggested to be a promising target for cancer immunotherapy. This study aimed to investigate the clinical significance of B7-H3 and HER2 co-expression and the therapeutic value of combination treatment in GC. MethodsWe examined the expression of B7-H3 and HER2 in 268 GC patients by immunohistochemistry. Pearson test was used to analyze the correlation between categorical variables. Overall survival was assessed by Kaplan–Meier analysis. All in vitro experiments using HER2-positive GC cells were treated with small interfering RNA targeting B7-H3/HER2 or B7-H3 blocking antibody 3E8/trastuzumab to verify the antitumor efficacy of the combination therapy. GC xenograft mouse models were established to evaluate the in vivo anti-tumor effect of combined therapy. ResultsThere was a significant correlation between B7-H3 and HER2 expression in GC tissues. High co-expression of B7-H3 and HER2 was associated with poor prognosis (P = 0.007) and could be an independent risk factor for survival. In addition, knockdown or targeted therapies of B7-H3/HER2 significantly suppressed cell proliferation, migration, invasion and adhesion in vitro. Trastuzumab combined with 3E8 was significantly effective at reducing mice tumor growth than monotherapy. ConclusionHigh co-expression of B7-H3 and HER2 indicates a poor prognosis, and combination therapy targeting B7-H3 and HER2 could be an immunotherapeutic strategy for GC.

Discordance of positive immunohistochemical results among different primary HER2 antibodies used in breast cancer: A comparison between Histofine® HER2 Kit (MONO) SV2-61γ antibody and Ventana I-VIEW PATHWAY™ HER2 4B5 antibody

IntroductionImmunohistochemical (IHC) testing for human epidermal growth factor receptor 2 (HER2) expression status renders variable scores among different primary antibodies. Materials and methodsWe assessed HER2 expression status by IHC score using two types of kits, i.e., Roche Ventana I-VIEW PATHWAY™ HER2 using the 4B5 antibody that recognizes the intracellular domain (ICD) and Nichirei Biosciences Histofine® HER2 Kit (MONO) using the SV2-61γ antibody that recognizes the extracellular domain (ECD). In addition, we determined the presence or absence of the ECD in samples by Western blotting (WB) and examined its relationship with the IHC score. ResultsOf 101 samples, 44 and 9 samples received a score of 2+/3+ by IHC with 4B5 and SV2-61γ, respectively. A verification by WB in samples with a discrepancy of IHC scores found that samples with the score of 2+/3+ with SV2-61γ showed a significantly higher protein intensity of p185HER2 with the ECD, whereas samples with a score of 2+/3+ with 4B5 but as 0/1+ with SV2-61γ showed a significantly higher protein intensity of p95HER2 without the ECD. ConclusionThe nature of primary antibodies used for IHC and the presence or absence of the ECD in samples may contribute to different positive rates between 4B5 and SV2-61γ.

Abstract 5661: HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers

Abstract HER2 is amplified in about 20% of breast cancers. HER3 is as essential as HER2 for maintaining cell viability in HER2+ breast cancer cells. It is known that inhibition of HER2 tyrosine kinase activity results in upregulation of HER3 transcription and phosphorylation. We sought to identify HER3 binding partners upon pharmacological inhibition of HER2 using neratinib. We immunoprecipitated HER3 using a HER3 antibody from BT474 cells treated ± neratinib. Fmass spectrometry experiments identified non-muscle myosin IIA (NMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To validate the presence of NMIIA, we performed immunoprecipitation experiments in BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes a protein called non-muscle myosin of class II, isoform A (NMIIA). It localizes to actin stress fibers and has been implicated in many cell functions. To confirm the interaction between HER3 and NMIIA we immunoprecipitated NMIIA from BT474 and MDA-MB-453 cells using a NMIIA antibody. Immunoblots indicated that HER3 levels were increased upon inhibition of HER2 with 200 nM neratinib for 24 hours. We next examined overall survival of primary breast cancer patients who have high gene expression for MYH9 from the METABRIC cohort. We observed that patients with high levels of MYH9 have a statistically significant worse overall survival versus patients with low levels of MYH9. Furthermore, we evaluated the overall levels of HER3 and MYH9 mRNA and protein upon treatment with neratinib in BT474 and MDA-MB-453 whole cell lysates. RT-qPCR and immunoblots showed increased HER3 and NM-IIA mRNA and protein levels upon neratinib treatment for 24 hours We examined the affect NMIIA loss has on HER3 signaling. Transduced MDA-MB-453 and BT474 cells with shMYH9 and doxycycline induction demonstrate a reduction in HER3 protein levels compared to cells transduced with a control sequence and doxycycline induction. We observed a concomitant reduction in P-HER3 (Y1289), downstream P-Akt (T308), and P-Erk1/2. In addition, NM-IIA knockdown suppresses cells growth, proliferation, migration, and invasion. In conclusion, there is a bidirectional relationship between NMIIA and HER signaling in HER2+ breast cancer cells. HER2 inhibition increases NMIIA and NMIIA promotes HER3 expression. Loss of NMIIA reduces HER3 protein and concomitant reductions in PI3K/Akt and MAPK signaling. Loss of NMIIA in combination with HER2 inhibition results in reduction in HER2+ breast cancer cell proliferation, growth on matrigel, migration, and invasion. Studies are ongoing to decipher the mechanisms for the bidirectional relationship between NMIIA and HER signaling. Citation Format: Samar M. Alanazi, Rosalin Mishra, Hima Patel, Mary K. Kilroy, Joan T. Garrett. HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers [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 5661.

Abstract 4256: YH012, a novel bispecific anti-HER2 and TROP2 antibody-drug conjugate, exhibits potent antitumor efficacy

Abstract Bispecific antibodies (BsAbs) and antibody-drug conjugates (ADCs) are among the most promising and fastest growing classes of next-generation antibody therapeutics for cancer therapy. The strategy of targeting dual tumor-associated antigens (TAAs) with a BsAb offers several advantages, such as improved efficacy due to synergistic effects, increased target cross-linking and internalization, increased tumor cell specificity and reduced side effects in normal tissues. HER2 is an established therapeutic target for approximately 20% of breast cancers, and its overexpression has been observed in a variety of other solid tumors. Trop-2 is another ideal tumor-associated target overexpressed on a wide variety of solid tumors. Co-expression of HER2 and TROP2 has been found in a number of tumors including gastric, colorectal, bladder and breast cancers. Therefore, co-targeting these TAAs using bispecific antibodies has clinical potential. YH012 is a novel first-in-class bispecific antibody-drug conjugate targeting HER2-TROP2 (HER2-TROP2-ADC), which contains a novel fully human bispecific anti-HER2/TROP2 antibody conjugated with Monomethyl auristatin E (MMAE) via a protease-cleavable linker. The anti-HER2/TROP2 BsAb showed increased endocytosis activity compared with its parental mAbs in tumor cells co-expressing HER2/TROP2, whereas the monovalent HER2 or TROP2 antibodies showed reduced internalization. As expected, YH012 showed increased in vitro potency of cell killing compared to the monovalent HER2/TROP2-ADC. These results suggest that YH012 increases the selectivity of payload delivery demonstrating the beneficial effects of targeting dual TAAs using BsAb-ADC. Moreover, YH012 showed robust anti-tumor efficacy in multiple cell line-derived none-small cell lung cancer (NSCLC) and gastric xenografts. Treatment with YH012 showed sustained tumor growth inhibition, which is more pronounced than the effects of the parental mAb-ADCs and monovalent ADCs. These preclinical data suggest that YH012 could potentially benefit patients with tumors co-expressing HER2 and TROP2 through improved drug selectivity and efficacy. Further investigations on the antitumor activity and safety profile of YH012 are ongoing in dogs. Citation Format: Chengzhang Shang, Liu Yang, Jiyong Zhang, Yanfei Han, Zhuolin Li, Zhenyan Han, Jun Li, Ying Meng, Gao An, Hao Yang, Wenqian An, Lei Chen, John Charpentier. YH012, a novel bispecific anti-HER2 and TROP2 antibody-drug conjugate, exhibits potent antitumor efficacy [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 4256.

Abstract 2552: Effects of liver endothelium on pancreatic cancer growth and metabolism

Abstract Introduction: ~90% metastatic pancreatic ductal adenocarcinoma (mPDAC) are found in the liver, and 5-year survival rate for patients with mPDAC is only at 3%. Therefore, novel therapeutic strategies are urgently needed. A growing body of evidence suggest PDAC rely on mitochondrial function (oxidative phosphorylation, OXPHOS) for survival. However, PDAC liver metastases have been reported to have higher levels of glucose uptake compared to primary tumors suggesting that PDAC liver metastases may rely on glycolytic metabolism. Therefore, the relative metabolic and cellular profiles of primary and metastatic PDAC (mPDAC) remain unclear and the involved regulatory pathway(s) have not been elucidated. Our previous studies showed that liver endothelial cells (ECs) secreted soluble factors to promote the survival of colon cancer cells in a paracrine fashion. The influence of the liver EC microenvironment on mPDAC growth and metabolism has not been elucidated. In this study, we investigate the paracrine effects of liver ECs on the survival and metabolic profiles of PDAC and elucidate the involved mechanism(s). Methods: Primary liver ECs were isolated from non-neoplastic liver. Conditioned medium (CM) from liver ECs were collected and then applied to PDAC cells, with CM from PDAC as control CM. Effects of CM on PDAC cell proliferation were measured by the MTT assay. Changes in phosphorylation of receptor tyrosine kinases (RTK) between PDAC CM and EC CM treated PADC cells were determined by a Phospho-RTK Array kit and then validated by Western blotting. Effects of EC CM on PDAC metabolism was assessed by ATP production with CellTiter-Glo and oxygen consumption rate with Agilent Seahorse Mito Stress Assay. Results: Compared to PDAC CM, liver EC CM promoted proliferation in different PDAC cells. We found that human epidermal growth factor receptor 3 (HER3, also known asERBB3) was only expressed and activated in BxPC-3 cells (HER3+ve), in which the HER3-AKT signaling pathway was activated by EC CM. Furthermore, blocking HER3 activation with a humanized HER3 antibody, seribantumab, significantly blocked EC CM-induced AKT activation and cell proliferation. Moreover, depletion of neuregulin (NRG) from EC CM attenuated HER3-AKT activation and indicated that EC-secreted NRG might play a role in promoting PDAC growth. Furthermore, EC CM decreased the levels of ATP production and O2 consumption in PDAC cells, suggesting that EC cells reprogram PDAC metabolism away from OXPHOS mitochondrial metabolism. Conclusions: Liver EC-secreted factors promoted PDAC growth in vitro and in vivo, and HER3 was expressed in a subset of PDAC cells and mediated EC-induced proliferation. Moreover, EC reprogramed PDAC metabolism towards glycolysis, and inhibiting the activation of HER3 shifted PDAC metabolism to OXPHOS. Our findings provide a new insight to develop new combination of HER antibody and OXPHOS inhibitor for treating patients with HER3+ve mPDAC. Citation Format: Wei Zhang, Michel'le Wright, Moeez Rathore, Mehrdad Zarei, Jordan Winter, Rui Wang. Effects of liver endothelium on pancreatic cancer growth and metabolism [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 2552.

Abstract 4263: A novel pegylated biparatopic antibody-drug conjugate (pb-adc) targeting cancers with low HER2+ expression

Abstract ADCs have demonstrated much improved target selectivity and efficacy comparing to the nonspecific cytotoxic small molecule drug. The goal of ADC technology is to resolve the toxicity issue inherent in potent small molecule drugs. Yet, the main problem facing current ADC technology is still a toxicity issue, evidenced by 9 out of 12 FDA approved ADCs carrying black box warning labels and many failed clinical programs due to their toxicities. Many factors contribute to the ADC’s toxicity including but not limited to random conjugation of payloads to the antibody, premature release of payload, Fc related toxicity, on-target/off-tumor toxicity etc. In this study, we developed a novel pegylated BsAb-ADC formed by site-specific conjugating a fusion protein of two scFvs that target at two different epitopes of Her2, with PEG-MMAE, a pegylated cytotoxic payload MMAE. As expected, the compound is homogeneous without any Fc related toxicity. Although the compound showed lower affinity to Her2 positive cells, it demonstrated higher in vitro cytotoxicity than T-DM1, especially for Her2 low expressing cell lines when its DAR is 4. Furthermore, the compound was comparable to or better than T-DM1 or DS-8201 in inhibiting xenograft tumor models (including Her2+ low expressing models) even when DAR is only 2. The compound also demonstrated efficient internalization without detectable efflux, suggesting improved endosome and lysosome trafficking, most probably due to the lack of Fc promoted efflux mechanism and formation of complex between biparatopic Her2 antibody and Her2. In addition, unlike T-DM1 and DS-8201, the compound did not generate cytotoxicity to megakaryocyte cell line Dami, and is less toxic to the non-cancerous HaCaT cells than T-DM1 or DS-8201. The compound is stable with no detectable release of the payload when incubating with human Plasma for a week at 37°C. The circulation T1/2 of the compound in rat is about 30 hours. In conclusion, our pegylated BsAb-ADC compound demonstrated improved efficacy with better safety profile than T-DM1 and DS-8201. Citation Format: Shumin Liu, Shuqiang Yin, Weidong Lyu, Yang Lei, Qiudong Zhuo, Zibin Wu, Shuangyu Tan, Liling Zheng, Yu (Yvonne) Wen, David Wu. A novel pegylated biparatopic antibody-drug conjugate (pb-adc) targeting cancers with low HER2+ expression [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 4263.

Abstract 5517: Addition of PD-1 antibody camrelizumab overcame resistance to trastuzumab plus chemotherapy in a HER2-positive, metastatic gallbladder cancer patient

Abstract HER2 amplification/overexpression is a common driver in a variety of cancers including gallbladder cancer (GBC). For patients with metastatic GBC, chemotherapy remains the standard of care with limited efficacy. The combination of HER2 antibody trastuzumab plus chemotherapy is the frontline treatment option for patients with HER2-positive breast/gastric cancer. Recently, this regime also showed antitumor activity in HER2-positive GBC. However, resistance to this regime represents a clinical challenge. Camrelizumab is a novel PD-1 antibody approved for Hodgkin lymphoma and hepatocellular carcinoma in China. Here we presented a HER2-positive metastatic GBC patient who was refractory to trastuzumab plus chemotherapy but experienced significant clinical benefit after the addition of camrelizumab. A 69-year-old male GBC patient was found disease progression in the lung six month after operation, staged IV (T3N0M1). Immunohistochemical (IHC) staining was positive for HER2 (3+) and negative for PD-L1 in both primary and pulmonary lesions. Chemotherapy including S-1 plus gemcitabine, as well as oxaliplatin plus paclitaxel consecutive failed, with a new lesion discovered in the liver. Genomic profiling with a multi-gene NGS panel testing (Onco Panscan࣪, Genetron Health) of the pulmonary lesion showed the presence of TP53 S241Y, ARID2 R273*, EGFR E872K mutations, HER2 amplification (fold change, 8.7), and high tumor mutational burden (TMB 10.33). He was treated with trastuzumab in combination with afatinib and capecitabine. Unfortunately, progressive disease in the lung and brain was observed after two cycles. Due to progression of disease and high TMB of the pulmonary biopsy, also based on the reported activity of PD-1 antibody plus trastuzumab in trastuzumab-resistant, advanced HER2-positive breast cancer, a triplet regime of camrelizumab, trastuzumab, and oxaliplatin was administered. The lesions of the lung, liver, and brain completely regressed after five cycles. From cycle 8 of maintance regime, an anti-angiogenic agent apatinib was added to treat reactive cutaneous capillary endothelial proliferation (RCCEP), the most common immune-related adverse event (irAE) related to camrelizumab, with complete regression after four months. Currently, the patient remains in remission. Our case highlights the potential of immunotherapy in combination with HER2-targeted therapy in HER2-positive GBC. We also demonstrated that immune-related adverse events (irAEs) associated with camrelizumab can be managed with an anti-VEGF agent apatinib. This case not only highlights the importance of irAE management in patients treated with camrelizumab, but also demonstrates the potential of PD-1 antibody plus trastuzumab in HER2-positive GBCs who have developed resistance to chemotherapy and trastuzumab-based targeted therapy. Citation Format: Dong Yan, Xiaomo Li, Li Wang, Yurong Cheng, Si Liu. Addition of PD-1 antibody camrelizumab overcame resistance to trastuzumab plus chemotherapy in a HER2-positive, metastatic gallbladder cancer patient [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 5517.

Pyrotinib in combination with metronomic oral vinorelbine in patients with HER2-positive advanced breast cancer who had failed prior trastuzumab-based therapy: A single-center, single-arm, prospective phase 2 study.

1033 Background: In 15% to 30% of breast cancers, human epidermal growth factor receptor 2 (HER2) is overexpressed, this is related to aggressive disease and poor prognosis. Although important clinical benefits for patients have been achieved by the use of the HER2 antibody trastuzumab, 50% to 74% of patients with metastatic disease have no response to treatment, and approximately 75% progress within one year. The purpose of this study was to evaluate the efficacy and safety of oral pyrotinib in combination with oral metronomic vinorelbine in patients with HER2-positive advanced breast cancer who had failed prior trastuzumab-based therapy. Methods: This prospective phase 2 study enrolled patients aged 18-75 years with HER2-positive advanced breast cancer who had failed prior trastuzumab-based therapy, and had an Eastern Cooperative Oncology Group performance score of 0-2. Patients received pyrotinib 400 mg once daily and vinorelbine 40 mg once on Monday, Wednesday, Friday of each week until disease progression or unacceptable toxicity. Both pyrotinib and vinorelbine were orally administered 30 min after meals. The primary endpoint was progression-free survival (PFS). The secondary endpoints were objective response rate (ORR), disease remission rate (DCR), overall survival (OS), quality of life (QoL) and safety (CTCAE 5.0). The follow-up of this study is ongoing, but enrolment is closed. This study is registered on Clinical Trials.gov, number NCT04903652. Results: Between Oct 21, 2019, and Jan 21, 2022, 36 patients were enrolled and all of them were included in the intent-to-treat (ITT) population. 20 of 36 patients had disease progress or death. Median follow-up was 16.23 months. The median PFS (mPFS) was 14.23 months (95% CI 8.13-20.33). The ORR and DCR were 38.9% and 83.3%, respectively. The median OS was not reached. Grade 3 adverse events (AEs) occurred in 17 of 36 patients, the most common were diarrhea 27.8% and stomachache 5.6%. No grade 4 AEs were observed. Conclusions: Pyrotinib combined with metronomic oral vinorelbine showed promising efficacy and manageable safety in patients with HER2-positive advanced breast cancer who had failed prior trastuzumab-based therapy. This study might represent a potential treatment option for these patients. Clinical trial information: NCT04903652..

Trastuzumab Deruxtecan versus Trastuzumab Emtansine for Breast Cancer

BackgroundTrastuzumab emtansine is the current standard treatment for patients with human epidermal growth factor receptor 2 (HER2)–positive metastatic breast cancer whose disease progresses after treatment with a combination of anti-HER2 antibodies and a taxane.MethodsWe conducted a phase 3, multicenter, open-label, randomized trial to compare the efficacy and safety of trastuzumab deruxtecan (a HER2 antibody–drug conjugate) with those of trastuzumab emtansine in patients with HER2-positive metastatic breast cancer previously treated with trastuzumab and a taxane. The primary end point was progression-free survival (as determined by blinded independent central review); secondary end points included overall survival, objective response, and safety.ResultsAmong 524 randomly assigned patients, the percentage of those who were alive without disease progression at 12 months was 75.8% (95% confidence interval [CI], 69.8 to 80.7) with trastuzumab deruxtecan and 34.1% (95% CI, 27.7 to 40.5) with trastuzumab emtansine (hazard ratio for progression or death from any cause, 0.28; 95% CI, 0.22 to 0.37; P<0.001). The percentage of patients who were alive at 12 months was 94.1% (95% CI, 90.3 to 96.4) with trastuzumab deruxtecan and 85.9% (95% CI, 80.9 to 89.7) with trastuzumab emtansine (hazard ratio for death, 0.55; 95% CI, 0.36 to 0.86; prespecified significance boundary not reached). An overall response (a complete or partial response) occurred in 79.7% (95% CI, 74.3 to 84.4) of the patients who received trastuzumab deruxtecan and in 34.2% (95% CI, 28.5 to 40.3) of those who received trastuzumab emtansine. The incidence of drug-related adverse events of any grade was 98.1% with trastuzumab deruxtecan and 86.6% with trastuzumab emtansine, and the incidence of drug-related adverse events of grade 3 or 4 was 45.1% and 39.8%, respectively. Adjudicated drug-related interstitial lung disease or pneumonitis occurred in 10.5% of the patients in the trastuzumab deruxtecan group and in 1.9% of those in the trastuzumab emtansine group; none of these events were of grade 4 or 5.ConclusionsAmong patients with HER2-positive metastatic breast cancer previously treated with trastuzumab and a taxane, the risk of disease progression or death was lower among those who received trastuzumab deruxtecan than among those who received trastuzumab emtansine. Treatment with trastuzumab deruxtecan was associated with interstitial lung disease and pneumonitis. (Funded by Daiichi Sankyo and AstraZeneca; DESTINY-Breast03 ClinicalTrials.gov number, NCT03529110.)

Liver Endothelium Promotes HER3-Mediated Cell Survival in Colorectal Cancer with Wild-Type and Mutant KRAS.

This body of work highlighted a potential strategy of using HER3 antibodies in combination with standard chemotherapy agents for treating patients with either KRAS wild-type or KRAS mutant metastatic colorectal cancer.

Targeted Nanoparticle for Co‐delivery of HER2 siRNA and a Taxane to Mirror the Standard Treatment of HER2+ Breast Cancer: Efficacy in Breast Tumor and Brain Metastasis (Small 11/2022)

Breast Cancer Treatment In article number 2107550, Wassana Yantasee and co-workers develop polymer-decorated mesoporous silica nanoparticles for HER2-positive breast cancer treatment. This patented versatile platform can co-deliver multiple therapeutic classes to ensure that they reach the target cells at the same time to fully realize their synergy. Illustrated herein are nanoparticles with three drug cargos: HER2 antibody, HER2 siRNA, and chemotherapeutic docetaxel.

Abstract P5-10-05: HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers

Abstract HER2 is amplified in about 20% of breast cancers. HER3 is as essential as HER2 for maintaining cell viability in HER2+ breast cancer cells. Inhibition of HER2 tyrosine kinase activity results in upregulation of HER3 transcription and phosphorylation. We sought to identify HER3 binding partners upon pharmacological inhibition of HER2 using the irreversible pan HER inhibitor neratinib. We immunoaffinity-purified HER3 from HER2+ BT474 cells treated ± neratinib. Following immunoprecipitation using a HER3 antibody, binding partners were released under reducing conditions. Mass spectrometry experiments identified non-muscle myosin IIA (NMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To validate the presence of NMIIA, we performed immunoprecipitation experiments in HER2+ BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes the protein NMIIA. NMIIA localizes to actin stress fibers and has been implicated in regulation of cell contractility and stress fiber organization.To test if there is an interaction between HER3 and NMIIA, we immunoprecipitated NMIIA from BT474 and MDA-MB-453 cells using an NMIIA antibody. The products were analyzed using immunoblots. The results indicated that HER3 levels were increased upon inhibition of HER2 with 200 nM neratinib for 24 hours.We next examined long term overall survival of primary breast cancer patients who have high and low levels of gene expression for MYH9 from the METABRIC cohort. We observed that patients with high levels of MYH9 have a statistically significant worse overall survival versus patients who express low levels of MYH9.We next sought to examine overall levels of HER3 and MYH9 mRNA and protein upon treatment with neratinib in HER2+ breast cancer whole cell lysates. We observed that mRNA and protein levels of both HER3 and MYH9 increased upon HER2 inhibition with 24 hours of neratinib treatment. To further investigate the role of NMIIA, we knocked out NMIIA in BT474 and MDA-MB-453 using inducible lentiviral shRNA targeting MYH9. This system utilizes the Tet-On 3G induction system and is a highly controlled system that consists of an inducible RNA polymerase II promoter. In the presence of doxycycline, the TRE3G promoter is bound and activated by the constitutively expressed Tet-On 3G trans-activator protein. Non-targeting shRNA was used as control and cells were selected in 1-2 ug/ml puromycin. Western blot analysis demonstrated that cells infected with lentiviral shRNA targeting MYH9 treated with doxycycline had reduced protein expression of NMIIA. BT474 and MDA-MB-453 transduced with shMYH9 were used to perform growth, migration, and invasion assays. The results indicated that MYH9 knockdown + neratinib treatment suppresses BT474 and MDA-MB-453 growth, migration, and invasion. Currently, in vivo xenograft experiments examining BT474 and MDA-MB-453 tumor growth ±NMIIA shRNA in the presence or absence of neratinib treatment are underway. In conclusion, we have identified that NMIIA mRNA and protein levels are increased in the presence of neratinib treatment. Our data reveals that HER2 inhibition increases NMIIA to promote HER2+ breast cancer growth. Citation Format: Samar M Alanazi, Rosalin Mishra, Hima Patel, Long Yuan, Mary Kate Kilroy, Joan T Garrett. HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers [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 P5-10-05.

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.

Abstract P2-13-19: Statin modulation of antibody drug conjugate activity in breast cancer models and patients

Abstract Background: Membrane HER2 expression levels strongly influence the activity of anti-HER2 therapies directed at the HER2 extracellular domain (1,2). Tumoral cell-surface caveolin-1 (CAV-1) regulates receptor tyrosine kinase membrane trafficking mechanisms (1,3). In xenograft models, CAV-1 can be modulated with cholesterol-depleting drugs, such as statins. We hypothesized that preclinical and clinical use of statins might influence the expression of CAV-1 and thereby affect the efficacy of anti-HER2 antibody-drug conjugates (ADCs) in breast cancer. Methods: Preclinically, statins were given alone and in combination with HER2 antibody drug conjugates such as T-DM1 to assess HER2 levels, drug uptake, and antitumor efficacy in HER2-positive cancer models. Mice received an intravenous injection of T-DM1, oral doses of lovastatin, or a combination of T-DM1 and lovastatin, for 5 weeks. Clinically, we performed retrospective analyses of HER2-positive MBC patients at MSKCC who received T-DM1 and consented to molecular profiling and clinical data abstraction. Progression-free survival (PFS) on T-DM1 was estimated using Kaplan Meyer methods and compared using the log rank test. Univariable and multivariable Cox proportional hazards models were constructed using relevant clinical covariates. Results: Tumor models with high levels of CAV-1 exhibit low HER2 density on the cell surface and show low T-DM1 targeted immunoPET binding when compared with CAV-1-low tumors. Mechanistic studies showed that CAV-1 depletion in HER2-positive BC cells temporally stabilizes HER2 on the surface and delays T-DM1 recycling. In preclinical BC models, CAV-1 depletion induced by synthetic oligonucleotides or statins enhances T-DM1 binding and efficacy in tumors with incomplete HER2 membranous reactivity. For clinical analyses, a total of 164 patients who received T-DM1 were included in the final analysis. Twenty-one (12.8%) of these patients were recorded to be taking statins concurrently with T-DM1, as part of their routine clinical care. Compared with patients with no recorded statin use, patients receiving statins were older on average (median age 58 vs. 50 years, p = 0.007), but did not differ in their race, histologic breast cancer type, ER status, or T-DM1 treatment line. Median PFS on T-DM1 in the overall cohort was 5.5 months. Median PFS in patients who received statins was 14 months vs. 5.4 months in patients who had no recorded statin use (p = 0.1). In univariable Cox analysis, the association between statin use and PFS did not reach statistical significance (p=0.119). In a multivariable analysis including age, ER status, treatment line, and breast cancer histology, no variable reached statistical significance. However, statin use had the greatest observed degree of association with PFS (p = 0.17). Conclusions: Statins modulate surface HER2 levels and T-DM1 efficacy preclinically via CAV-1. Although only a numerical difference in outcomes was observed in the MSKCC cohort, the effect appeared meaningful and therefore assessment in larger patient cohorts is now underway. Based on the results of these forthcoming analyses, prospective trials may be justified that integrate these well-tolerated and low-cost agents into HER2 ADC treatment regimens.

Abstract OT1-02-03: Trastuzumab deruxtecan (T-DXd; DS-8201) vs trastuzumab emtansine (T-DM1) in high-risk patients with HER2-positive, residual invasive early breast cancer after neoadjuvant therapy: A randomized, phase 3 trial (DESTINY-Breast05)

Abstract Background Preoperative chemotherapy in combination with trastuzumab and pertuzumab is a preferred regimen for treating patients (pts) with HER2-positive, invasive, early breast cancer (BC). Pts who have received such treatment but still have residual invasive disease in the breast or lymph nodes at surgery are at greater risk for disease recurrence or death than those with a pathological complete response. The antibody-drug conjugate (ADC) T-DM1 is approved as a postneoadjuvant treatment for pts with residual invasive disease (in the breast and/or axillary nodes) after optimal neoadjuvant chemotherapy and trastuzumab (or trastuzumab with pertuzumab). T-DXd is a potent HER2-targeted ADC with a humanized HER2 antibody attached to a membrane-permeable topoisomerase I inhibitor payload by a cleavable tetrapeptide-based linker and a drug-to-antibody ratio of ≈8. T-DXd is approved globally for the treatment of adult pts with HER2-positive, unresectable or metastatic BC who have received ≥2 prior anti-HER2-based regimens in the metastatic setting or had prior chemotherapy and are refractory to or intolerant of standard treatments. These approvals have been supported by results from DESTINY-Breast01, an open-label, international, multicenter, phase 2 study of T-DXd in patients with HER2-positive metastatic BC. In an updated data cutoff (June 8, 2020), T-DXd demonstrated an objective response rate (ORR) of 61.4% (113/184 pts) and a duration of response of 20.8 months in pts with HER2-positive (IHC 3+ or ISH+), unresectable or metastatic BC previously treated with T-DM1 (Modi et al. Cancer Res. 2021;81[4 suppl]:PD3-06). Yet, further unmet need exists in patients who do not achieve pathologic complete response to neoadjuvant treatment, as these patients have increased risk of recurrence. Here, we describe a randomized phase 3 trial evaluating T-DXd vs T-DM1 as postneoadjuvant treatment for high-risk pts with HER2-positive primary BC who have residual invasive disease following neoadjuvant therapy. Study Description DESTINY-Breast05 is a multicenter, open-label, randomized, phase 3 trial comparing the efficacy and safety of T-DXd with those of T-DM1 in pts with HER2-positive (IHC 3+ or ISH+, centrally confirmed on pretreatment biopsy), invasive BC with pathologic evidence of residual invasive disease in the breast or axillary lymph nodes after neoadjuvant therapy. Additionally, pts must have a higher residual risk for recurrence, following standard T-DM1, defined as either presenting with inoperable disease (clinical stages T4, N0-3, M0 or T1-3, N2-3, M0) or operable BC at presentation (clinical stages T1-3, N0-1, M0) with axillary node-positive disease after neoadjuvant chemotherapy and anti-HER2 treatment. Approximately 1600 pts will be randomly assigned (1:1) to T-DXd or T-DM1 from ≈ 400 sites globally. Randomization is stratified by operative status at presentation, hormone receptor status, pathologic nodal status following neoadjuvant therapy, and type of HER2-targeted neoadjuvant therapy (single vs dual). T-DXd 5.4 mg/kg or T-DM1 3.6 mg/kg will be administered intravenously once every 3 weeks for 14 cycles. Invasive disease-free survival based on investigator assessment is the primary efficacy endpoint. Secondary endpoints are overall survival, disease-free survival, distant recurrence-free interval, and brain metastasis-free interval. The pharmacokinetics of T-DXd, biomarkers, and health-related quality of life will also be evaluated (NCT04622319). Citation Format: Charles E Geyer, Jr, Michael Untch, Aleix Prat, Priya Rastogi, Naoki Niikura, Elton Mathias, Lee Anne McLean, Yibin Wang, Sibylle Loibl. Trastuzumab deruxtecan (T-DXd; DS-8201) vs trastuzumab emtansine (T-DM1) in high-risk patients with HER2-positive, residual invasive early breast cancer after neoadjuvant therapy: A randomized, phase 3 trial (DESTINY-Breast05) [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 OT1-02-03.

The status of HER2 in colorectal carcinoma and the relation of HER2 with prognostic parameters and MSI.

Colorectal cancer (CRC) is the third most commonly occurring cancer in men and the second most common cancer in women. Despite advances in surgical techniques and chemotherapeutic regimens, CRC continues to be one of the main causes of cancer-related deaths in the world. The aim of the study was to evaluate the immunohistochemical expression of human epidermal growth factor receptor 2 (HER2) and the relationship between HER2, clinicopathological parameters, and microsatellite instability (MSI). Two hundred and forty resected CRCs at our institution between 2016 and 2019 were included in the study. Tumors were re-evaluated and classified according to the World Health Organization. Tissue macroarray techniques were used to generate tissue samples. HER2 antibody was performed using the automated system. HER2 antibody was score 3 positive in only 5 cases. Score 2 was observed in 13 cases, score 1 in 27, score 0 in 195. All of the HER2-positive cases were metastatic. All of them were tubular adenocarcinoma. HER2 positive cases were well and moderately differentiated. Four of the HER2 positive cases were T3 stage, and one was T4. A total of 2.1% of CRCs were positive for HER2 antibody. There was a positive correlation between HER2 and distant metastasis. There was no significant relationship between MSI, other prognostic parameters, and HER2.

Role of ADCC, CDC, and CDCC in Vaccine-Mediated Protection against Her2 Mammary Carcinogenesis.

Amplification or mutation of the Her2 oncoantigen in human mammary glands leads to the development of an aggressive breast carcinoma. Several features of this breast carcinoma are reproduced in mammary carcinomas that spontaneously arise in female transgenic mice bearing the activated rat Her2 oncogene under transcriptional control of the mouse mammary tumor virus promoter-BALB-neuT (neuT) mice. We previously demonstrated that carcinoma progression in neuT mice can be prevented by DNA vaccination with RHuT, a plasmid coding for a chimeric rat/human Her2 protein. RHuT vaccination exerts an antitumor effect, mostly mediated by the induction of a strong anti-rat Her2 antibody response. IgG induced by RHuT vaccine mainly acts by blocking Her2 signaling, thus impairing cell cycle progression and inducing apoptosis of cancer cells, but other indirect effector mechanisms could be involved in the antibody-mediated protection. The recruitment of cells with perforin-dependent cytotoxic activity, able to perform antibody-dependent cellular cytotoxicity, has already been investigated. Less is known about the role of the complement system in sustaining antitumor response through complement-dependent cytotoxicity and cellular cytotoxicity in vaccinated mice. This work highlights that the weight of such mechanisms in RHuT-induced cancer protection is different in transplantable versus autochthonous Her2+ tumor models. These results may shed new light on the effector mechanisms involved in antibody-dependent anti-cancer responses, which might be exploited to ameliorate the therapy of Her2+ breast cancer.

Trastuzumab Deruxtecan in HER2-Mutant Non–Small-Cell Lung Cancer

BackgroundHuman epidermal growth factor receptor 2 (HER2)–targeted therapies have not been approved for patients with non–small-cell lung cancer (NSCLC). The efficacy and safety of trastuzumab deruxtecan (formerly DS-8201), a HER2 antibody–drug conjugate, in patients with HER2-mutant NSCLC have not been investigated extensively.MethodsWe conducted a multicenter, international, phase 2 study in which trastuzumab deruxtecan (6.4 mg per kilogram of body weight) was administered to patients who had metastatic HER2-mutant NSCLC that was refractory to standard treatment. The primary outcome was objective response as assessed by independent central review. Secondary outcomes included the duration of response, progression-free survival, overall survival, and safety. Biomarkers of HER2 alterations were assessed.ResultsA total of 91 patients were enrolled. The median duration of follow-up was 13.1 months (range, 0.7 to 29.1). Centrally confirmed objective response occurred in 55% of the patients (95% confidence interval [CI], 44 to 65). The median duration of response was 9.3 months (95% CI, 5.7 to 14.7). Median progression-free survival was 8.2 months (95% CI, 6.0 to 11.9), and median overall survival was 17.8 months (95% CI, 13.8 to 22.1). The safety profile was generally consistent with those from previous studies; grade 3 or higher drug-related adverse events occurred in 46% of patients, the most common event being neutropenia (in 19%). Adjudicated drug-related interstitial lung disease occurred in 26% of patients and resulted in death in 2 patients. Responses were observed across different HER2 mutation subtypes, as well as in patients with no detectable HER2 expression or HER2 amplification.ConclusionsTrastuzumab deruxtecan showed durable anticancer activity in patients with previously treated HER2-mutant NSCLC. The safety profile included interstitial lung disease that was fatal in two cases. Observed toxic effects were generally consistent with those in previously reported studies. (Funded by Daiichi Sankyo and AstraZeneca; DESTINY-Lung01 ClinicalTrials.gov number, NCT03505710.)

Profile of Histopathological Type and Molecular Subtypes of Mammary Cancer of DMBA-induced Rat and its Relevancy to Human Breast Cancer

BACKGROUND: Animal models with mammary cancer that closely mimic human breast cancer for treatment development purposes are still required. Induction of 7,12-dimethylbenzanthracene (DMBA) to rats shows the histopathological features and mammary cancer characterization similar to humans. Examinations of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 expressions are crucial in deciding the treatment and prognosis of breast cancer. AIM: This research aimed to view histopathology images of mammary glands and expressions of ER, PR, Ki67, and HER2 of DMBA-induced rats. METHODS: After 1-week adaptation, 11 5-weeks-old female rats were induced with 20 mg/kg body weight (BW) of DMBA 2 times a week for 5 weeks. On week 29, nodules taken from the mammary gland were examined for hematoxylin-eosin staining and immunohistochemistry with p63, ER, PR, HER2, and Ki67 antibodies. The grading score used the Nottingham Grading System and molecular classifications based on St. Gallen 2013. RESULTS: Six rats had nodules, but the histopathologic features of one nodule showed normal mammary gland without cancer. The histopathological type of mammary cancer was cribriform carcinoma, comedo carcinoma, lipid-rich carcinoma, adenocarcinoma squamous, and adenomyepithelioma. Histopathological grading showed 60% of grade 3 and 40% of grade 2. P63 expression showed 60% positive and 40% negative. The frequency of ER, PR, HER2, and Ki67 of five nodules showed positivity: 40%, 60%, 60%, and 60%, respectively. Molecular subtypes of Luminal A, B, HER2, and triple-negative were 0%, 60%, 20%, and 20%, respectively. CONCLUSION: Histopathological features and molecular subtype of mammary cancer on rats induced with 20 mg/kg BW of DMBA showed similarity to human breast cancer.