HER2 Extracellular Domain Research Articles

HER2-related biomarkers predict clinical outcomes with trastuzumab deruxtecan treatment in patients with HER2-expressing metastatic colorectal cancer: biomarker analyses of DESTINY-CRC01

DESTINY-CRC01 (NCT03384940) was a multicentre, open-label, phase 2 study that investigated the safety and efficacy of trastuzumab deruxtecan (T-DXd) in patients with human epidermal growth factor receptor 2 (HER2)-expressing metastatic colorectal cancer (CRC). The present exploratory biomarker analysis aims to investigate relationships between biomarkers and clinical outcomes in patients with HER2-positive (immunohistochemistry [IHC] 3+ or IHC 2+ and in situ hybridization [ISH] positive) Cohort A (N = 53) of DESTINY-CRC01. Higher levels of HER2 biomarkers in baseline tissue and liquid biopsies, including HER2 status (IHC/ISH), HER2/CEP17 ratio, HER2 ISH signals, HER2 H-score, plasma HER2 (ERBB2) amplification status, HER2 adjusted plasma copy number, and HER2 extracellular domain correlate with antitumor activity (indicated by objective response rate, progression-free survival, and overall survival) of T-DXd. Baseline circulating tumor DNA (ctDNA) analysis suggests antitumor activity of T-DXd in patients who had baseline activating RAS, PIK3CA, or HER2 mutations detected in ctDNA.

Selective targeting of oncogenic hotspot mutations of the HER2 extracellular domain.

Oncogenic mutations in the extracellular domain (ECD) of cell-surface receptors could serve as tumor-specific antigens that are accessible to antibody therapeutics. Such mutations have been identified in receptor tyrosine kinases including HER2. However, it is challenging to selectively target a point mutant, while sparing the wild-type protein. Here we developed antibodies selective to HER2 S310F and S310Y, the two most common oncogenic mutations in the HER2 ECD, via combinatorial library screening and structure-guided design. Cryogenic-electron microscopy structures of the HER2 S310F homodimer and an antibody bound to HER2 S310F revealed that these antibodies recognize the mutations in a manner that mimics the dimerization arm of HER2 and thus inhibit HER2 dimerization. These antibodies as T cell engagers selectively killed a HER2 S310F-driven cancer cell line in vitro, and in vivo as a xenograft. These results validate HER2 ECD mutations as actionable therapeutic targets and offer promising candidates toward clinical development.

Identification of monoclonal antibodies from naive antibody phage-display libraries for protein detection in formalin-fixed paraffin-embedded tissues

Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3–4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.

HER2-CD3-Fc Bispecific Antibody-Encoding mRNA Delivered by Lipid Nanoparticles Suppresses HER2-Positive Tumor Growth.

The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor and tumor-associated antigen abnormally expressed in various types of cancer, including breast, ovarian, and gastric cancer. HER2 overexpression is highly correlated with increased tumor aggressiveness, poorer prognosis, and shorter overall survival. Consequently, multiple HER2-targeted therapies have been developed and approved; however, only a subset of patients benefit from these treatments, and relapses are common. More potent and durable HER2-targeted therapies are desperately needed for patients with HER2-positive cancers. In this study, we developed a lipid nanoparticle (LNP)-based therapy formulated with mRNA encoding a novel HER2-CD3-Fc bispecific antibody (bsAb) for HER2-positive cancers. The LNPs efficiently transfected various types of cells, such as HEK293S, SKOV-3, and A1847, leading to robust and sustained secretion of the HER2-CD3-Fc bsAb with high binding affinity to both HER2 and CD3. The bsAb induced potent T-cell-directed cytotoxicity, along with secretion of IFN-λ, TNF-α, and granzyme B, against various types of HER2-positive tumor cells in vitro, including A549, NCI-H460, SKOV-3, A1847, SKBR3, and MDA-MB-231. The bsAb-mediated antitumor effect is highly specific and strictly dependent on its binding to HER2, as evidenced by the gained resistance of A549 and A1847 her2 knockout cells and the acquired sensitivity of mouse 4T1 cells overexpressing the human HER2 extracellular domain (ECD) or epitope-containing subdomain IV to the bsAb-induced T cell cytotoxicity. The bsAb also relies on its binding to CD3 for T-cell recruitment, as ablation of CD3 binding abolished the bsAb's ability to elicit antitumor activity. Importantly, intratumoral injection of the HER2-CD3-Fc mRNA-LNPs triggers a strong antitumor response and completely blocks HER2-positive tumor growth in a mouse xenograft model of human ovarian cancer. These results indicate that the novel HER2-CD3-Fc mRNA-LNP-based therapy has the potential to effectively treat HER2-positive cancer.

Abstract PO4-14-06: Impact of Serum HER2 Extracellular Domain in Metastatic Breast Cancer Patients Treated with Trastuzumab Deruxtecan (T-DXd)

Abstract Background: Trastuzumab deruxtecan (T-DXd) is a standard of care as 2nd line and after prior chemotherapy for HER2 positive and low metastatic breast cancer (MBC), respectively, based on Destiny Breast03 and 04 results. However, biopsies over time during treatment have shown that HER2 expression is variable. The predictive factors of trastuzumab deruxtecan (T-DXd) for MBC with HER2 positive and low are unclear. The HER2 extracellular domain (HER2-ECD) has been confirmed as a prognosis marker and predictive marker of treatment for HER2-positive MBC. Especially, HER2-ECD is considered a promising biomarker in cases where HER2 expression in metastases is unconfirmed. We hypothesized that HER2-ECD could serve as a biomarker for T-DXd. Patients and methods: A retrospective study of consecutively treated patients in a single center between 2019-2023 with HER2-low and HER2-positive MBC was performed using chart review. HER2 status was diagnosed according to 2020 ASCO-CAP guidelines. HER2-ECD high was defined as >15.0 ng/ml. HER2-ECD was collected prior to T-DXd treatment. We compared overall response (ORR), progression-free survival (OFS), overall survival (OS), and disease control rate (DCR) at HER2-ECD high and low groups. Cox regression analyses were performed to assess the ORR. We used the Kaplan-Meier method to estimate the PFS and OS and the log-rank test to compare each treatment group. Results: A total of 41 MBC patients were included in this study. Patients with HER-positive and HER2-low were 34 and 7, respectively. Among HER2 positive (n=34), HER2-ECD high and low are 14 (41%) and 20 (59%) patients, respectively. Among HER2 low (n=7), HER2-ECD high and low are 3 (43%) and 4 (57%) patients, respectively. Twenty-six patients received T-DM1 prior to T-DXd treatment for MBC with HER2 positive, but no patients received CPT-11 before T-DXd for MBC with HER2 positive and low. The ORR of T-DXd was 89% and 52% in the HER2-ECD high and low groups, respectively (p< 0.001). All 6 cases with CR were HER2-ECD high. Among HER2 low group (n=7), ORR of T-DXd was 67% (2/3) and 25% (1/4) in HER2-ECD high and low groups, respectively. The median PFS in the HER2-ECD high group showed longer than the HER2-ECD low group, 21.8 vs. 8.0 months (HR 0.31; 95%CI, 0.13-0.78, p=0.012). Although there were no significant differences in OS, HER2 ECD high group tended to show longer OS (HR 0.23; 95%CI, 0.04-1.16, p=0.07). The DCR was 100% in the HER2 ECD high group and 87% in the low group. Conclusion: T-DXd showed significantly better response and prolonged PFS in the group with high HER2-ECD. HER2-ECD has the potential to become a biomarker for T-DXd. Further study is warranted to assess the HER2-ECD as a biomarker, especially for HER2 low MBC patients treated with T-DXd. Table. Association between the response and the HER2 status or HER2-ECD status Citation Format: Kazuki Nozawa, Maho Kusudo, Nari Kureyama, Akira Nakakami, Rie Komaki, Yuka Endo, Ayumi Kataoka, Haruru Kotani, Akiyo Yoshimura, Masaya Hattori, Masataka Sawaki, Hiroji Iwata. Impact of Serum HER2 Extracellular Domain in Metastatic Breast Cancer Patients Treated with Trastuzumab Deruxtecan (T-DXd) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-06.

Abstract PO5-04-02: Combination of trastuzumab competing and non-competing fully human internalizing anti-Her2 monoclonal antibodies drug conjugates increases their inhibitory effect on the growth of HER2 positive metastatic breast cancer cells

Abstract The human epidermal growth factor receptor (HER) family of receptors plays a key role in proliferation stimulator for several human cancers. In particular, HER2 is overexpressed in 15-30% of breast cancers with or without gene amplification with prognostic and predictive implications. HER2 overexpression is associated with shorter disease-free and overall survivals, resistance to hormonal agents and increased risk of brain metastasis. Trastuzumab is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 extracellular domain and blocks its signaling. More recently, two antibody drug conjugates using trastuzumab have been approved: Ado-Trastuzumab-Emtansine (Kadcyla) consisting of trastuzumab conjugated to the drug mertansine DM1 and fam-trastuzumab-deruxtecan-nhki (Enhertu to deliver the topoisomerase inhibitor deruxtecan. We are reporting here the development of fully human internalizing anti-HER2 antibodies with distinct epitopes on the HER2protein which compete or not with trastuzumab for binding to HER2. These monoclonal antibodies have been developed by immunizing fully human (TC-Mab mouse) mice with recombinant HER2 protein. After production of hybridoma secreting fully human immunoglobulins, the screening process included competition with trastuzumab for binding to Her2 by enzyme linked immunoassay and Octet epitope binning and affinity determination as well as internalization assay. Several internalizing antibodies able to compete or not with trastuzumab and with high affinity (Kd ranging from 10−9 M to 10−12 M) were selected. Their ability to deliver a cytotoxic payload in HER2 overexpressing breast cancer cells such as SKBR3, BT474 and AU565 was next investigated. Two groups of antibodies: one competing with trastuzumab and the other one non-competing were further characterized. Data related to these antibodies’ biochemical characteristics as well as their ability to inhibit proliferation in vitro and in vivo in mouse xenografts studies will be presented here. In conclusion, the use of fully human mice to develop monoclonal antibodies provides a powerful and attractive approach to develop fully human monoclonal antibodies against cancer targets by-passing the need for humanization and affinity maturation of antibodies. This work is supported by 5R44CA224718 SBIR grant from the National Cancer Institute. Citation Format: Ginette Serrero, Binbin Yue, Jianping Dong, Jun Hayashi. Combination of trastuzumab competing and non-competing fully human internalizing anti-Her2 monoclonal antibodies drug conjugates increases their inhibitory effect on the growth of HER2 positive metastatic breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-04-02.

Exploratory analysis of serum HER2 extracellular domain for HER2 positive gastric cancer treated with SOX plus trastuzumab.

The aim of this study was to explore the clinical utility of serum HER2 extracellular domain (sHER2 ECD) using data from a clinical trial evaluating trastuzumab combined S-1 plus oxaliplatin (SOX) in HER2 positive gastric cancer. sHER2 ECD were prospectively measured at baseline and subsequent treatment courses. Based on each quantile point of baseline sHER2 ECD levels and its early changes, patients were divided into two groups and compared clinical outcomes. 43 patients were enrolled, and 17 patients (39.5%) were positive for baseline sHER2 ECD. Higher baseline sHER2 ECD levels tended to have lower hazard ratios (HRs). When divided into two groups by baseline sHER2 ECD of 19.1ng/ml, median progression-free survival (PFS) and overall survival (OS) was longer in the higher group (mPFS: 16.8 vs 8.7months, p = 0.359. mOS: 35.5 vs 20.6months, p = 0.270), respectively. After initiation of treatment, sHER2 ECD significantly decreased up until the third cycle. Higher reduction rates of sHER2 ECD within 3 cycles also tended to have lower HRs. When divided into two groups by reduction rate of 42.5%, mPFS and mOS was longer in the higher reduced group (mPFS: 17.2 vs 8.7months, p = 0.095. mOS: 65.0 vs 17.8months, p = 0.047), respectively. Furthermore, higher reduction rates could surrogate higher objective response rates (ORR) (ORR: 90% vs 63.2% for 29.5%, p = 0.065. 100% vs 70% for 42.5%, p = 0.085), respectively. Baseline sHER2 ECD levels and its early decline may be useful biomarkers for SOX plus trastuzumab efficacy in HER2 positive gastric cancer.

Study the technical validity and feasibility of serum HER-2/Neu and its correlation with tissue HER-2 status in female breast cancer patients only

Background: Breast cancer is one of the leading causes of death in women worldwide. The future burden of breast cancer is predicted to increase over 3 million new cases and 1 million deaths in 2040. In India, it is being increasingly diagnosed at a younger age as compared to the west. Human epidermal growth factor receptor 2 (HER-2) transmembrane protein, with a molecular weight 185 kDa, plays a key role in cell growth and its overexpression favors cell proliferation by inhibiting apoptosis. HER-2 extracellular domain, released into blood before appearing into tissue, can be easily detected by enzyme-linked immunosorbent assay (ELISA) using serum. HER-2 has many roles in assisting diagnosis, targeted therapy, and evaluating prognosis. Aims and Objectives: The study aimed to monitor the serum HER-2 level and its correlation with tissue HER-2 status in breast cancer patients. Materials and Methods: We performed a prospective study on technical validity and feasibility of serum HER-2 level by ELISA in breast cancer patients and its correlation with tissue HER-2 status by immunohistochemistry (IHC) method. Results: We found the serum ELISA level was raised and within the reference range in all breast cancer patients. A higher level of serum HER-2 level was seen in high tumor stage, high Nottingham grade, and with axillary lymph node metastasis. Serum HER-2 level was significantly higher in IHC score 3+ as compared to score 2+and 1+. Conclusion: This study concluded that serum HER-2 ELISA is a valid and feasible test. Association of serum HER-2 level with tumor stage; Nottingham grade; and axillary nodal metastasis was significant. The correlation between serum HER-2 and tissue HER-2 was found significant. Thus, serum HER-2 may be used as an early diagnostic and prognostic biomarker to guide in clinical practice.

Perspectives for applying Alphaviruses in antitumor therapy

Oncolytic viral therapy is a promising approach for treating tumors. Oncolytic viruses can directly lyse tumor cells and indirectly activate antitumor immunity. Alphaviruses, as oncolytic viruses, are particularly promising agents because they can selectively infect and lyse tumor cells, modulate microtumor environment, elicit immune-mediated lysis of tumor cells, and serve as a platform for transgene delivery. To ensure safety, attenuated strains of Alphaviruses are typically used for genetic engineering, and immunogenic tumor-associated antigens or cytokines are commonly chosen as transgenes. Studies evaluating both in vitro and in vivo oncolytic and immunomodulatory effects of Alphaviruses and vectors based on them have been growing exponentially. Animal models of various tumor types were used to examine the effectiveness of Alphaviruses, including Sindbis, Semliki Forest virus, Geta (strain M1), Venezuelan equine encephalitis virus, and vectors based on them. Additionally, Alphaviruses revealed enhanced antitumor activity while used in combination therapies with other oncolytic viruses. Alphavirus-like replicon particles based on attenuated Venezuelan equine encephalitis virus may serve for transgene delivery to express heterologous proteins at high levels, and induce both humoral and cellular immune responses. An alphaviral vector-based vaccine, encoding the HER2 extracellular and transmembrane domains, has demonstrated safety and efficacy in preclinical mouse models, as well as in phase I clinical trials for advanced breast cancer patients with HER2 overexpression. This vaccine is known to be safe, effective, and capable of inducing T-cell immunity. In this review, we discuss the current progress in preclinical and clinical investigations, as well as the future potential of Alphaviruses for oncolytic virotherapy.

Abstract CT189: A phase I study of BAT1006, a novel anti-HER2 antibody, in patients with locally advanced/metastatic solid tumors

Abstract Purpose: BAT1006 is a HER2 extracellular domain II-targeted monoclonal antibody with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effect aiming for Her2-positive locally advanced/metastatic solid tumors. Previous studies have shown that de-fucosylated antibody binds to FcγRIIIa with increased affinity and can thus trigger FcγRIIIa-mediated effector functions more efficiently than native antibody. BAT1006 was expressed in an FUT8-knockout CHO cell line, resulting in antibody completely devoid of fucose modification. With approximate 5-fold higher level of ADCC effect compared to Pertuzumab, BAT1006 also exhibited potent anti-tumor activity in HER2 positive Calu-3 xenograft mice model. The ongoing phase I, open-label, first-in-human study is evaluating the safety, pharmacokinetics (PK), immunogenicity and preliminary efficacy of BAT1006 to determine the Maximum-Tolerated Dose (MTD) and/or recommended phase II dose (RP2D). Methods: Patients with locally advanced/metastatic solid tumors received escalating doses of BAT1006 (3, 6, 10, 15 mg/kg), administered intravenously (IV) once every three weeks. Patients must have had documented HER2 positivity defined as 3+ by validated immunohistochemistry or amplified on in situ hybridization (ISH). Assessments include archival tumor molecular status, PK, and efficacy by Response Evaluation Criteria in Solid Tumors (RECIST). Results: Fifteen patients [with 2-9 prior lines of therapy] have been treated with BAT1006. All patients had metastatic breast cancer and had received previous HER2-targeted therapies including Trastuzumab, Pertuzumab, T-DM1 and Pyrotinib. No treatment related ≥ Grade 3 AEs have been observed. Of 9 patients at 10mg/kg and 15mg/kg, 1 in 3 patients had CR at 10mg/kg, 2 in 6 patients had PR at 15mg/kg. Conclusion: BAT1006 was well tolerated and showed promising anti-tumor activity in patients with heavily pre-treated HER2-positive cancers. Based on the safety and efficacy results, one additional dose level (20 mg/kg) will be added to the escalation phase and combination therapy with other HER2-based therapy will also be initiated. Citation Format: Xingxing Mei, Weijia Tang, Shuqiang Song, Hao Wang, Chuanqi Cao, Cuiying Feng, Bert E. Thomas, Shengfeng Li, Jin-Chen Yu. A phase I study of BAT1006, a novel anti-HER2 antibody, in patients with locally advanced/metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT189.

Generation of Rat Monoclonal Antibodies Against Human Epidermal Growth Factor Receptor 2

Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor without any known ligands and a member of the epidermal growth factor receptor (EGFR) family. It is a proto-oncogenic protein that, through signaling cascades, promotes cell proliferation and inhibits apoptosis in cancer cells through homo- and heterodimerization with other EGFR family receptors. Since several cancers, including breast cancer, overexpress HER2, it is a target of tumor therapy. Both trastuzumab and pertuzumab are recombinant humanized monoclonal antibodies (mAbs) used in clinical trials that target the extracellular domain (ECD) of HER2. Therefore, it is important to generate antibodies against various ECDs of HER2. In this study, we describe rat mAbs, which were generated against the ECD of human HER2. The human breast cancer cell line SK-BR-3 was subjected to immunofluorescence staining as it expresses HER2, and mAbs can detect both intact and endogenous HER2 within the cell line.

Anti-cancer effect of afatinib, dual inhibitor of HER2 and EGFR, on novel mutation HER2 E401G in models of patient-derived cancer

BackgroundPrecision medicine with gene panel testing based on next-generation sequencing for patients with cancer is being used increasingly in clinical practice. HER2, which encodes the human epidermal growth factor receptor 2 (HER2), is a potentially important driver gene. However, therapeutic strategies aimed at mutations in the HER2 extracellular domain have not been clarified. We therefore investigated the effect of EGFR co-targeted therapy with HER2 on patient-derived cancer models with the HER2 extracellular domain mutation E401G, based on our previous findings that this mutation has an epidermal growth factor receptor (EGFR)-mediated activation mechanism.MethodsWe generated a xenograft (PDX) and a cancer tissue-originated spheroid (CTOS) from a patient’s cancer containing an amplified HER2 E401G mutation. With these platforms, we compared the efficacy of afatinib, a tyrosine kinase inhibitor having anti-HER2 and anti-EGFR activity, with two other therapeutic options: lapatinib, which has similar properties but weaker EGFR inhibition, and trastuzumab plus pertuzumab, for which evidence exists of treatment efficacy against cancers with wild-type HER2 amplification. Similar experiments were also performed with H2170, a cell line with wild-type HER2 amplification, to contrast the characteristics of these drug’s efficacies against HER2 E401G.ResultsWe confirmed that PDX and CTOS retained morphological and immunohistochemical characteristics and HER2 gene profiles of the original tumor. In both PDX and CTOS, afatinib reduced tumor size more than lapatinib or trastuzumab plus pertuzumab. In addition, afatinib treatment resulted in a statistically significant reduction in HER2 copy number at the end of treatment. On the other hand, in H2170 xenografts with wild-type HER2 amplification, trastuzumab plus pertuzumab was most effective.ConclusionsAfatinib, a dual inhibitor of HER2 and EGFR, showed a promising effect on cancers with amplified HER2 E401G, which have an EGFR-mediated activation mechanism. Analysis of the activation mechanisms of mutations and development of therapeutic strategies based on those mechanisms are critical in precision medicine for cancer patients.

Abstract B013: Loss of the extracellular domain of human epidermal growth factor receptor 2 (HER2) p95 isoform is common in large DCIS lesions

Abstract Background/Aim: Testing for human epidermal growth factor receptor 2 (HER2) positivity in women diagnosed with ductal carcinoma in situ (DCIS) is not performed routinely, although the frequency of HER2+ DCIS is at least 30%. HER2+ DCIS lesions may be at higher risk of progression to invasive carcinoma, and specific isoforms of HER2 may lead to different malignant potential. Loss of the HER2 extracellular domain engineered in mouse models demonstrates rapid progression to invasive behavior. Evaluating isoforms of HER2 may aid in classifying DCIS lesions as higher or lower risk, and may provide insight for novel treatment and prevention approaches. Methods: A cohort of high risk DCIS cases were assembled, enriched for the presence of high risk features, including large size (>5 cm) and palpable. Immunohistochemistry (IHC) and FISH were used to identify cases that were HER2 positive. MultiplexIHC was used to detect extracellular and intracellular domains of HER2, with imaging analysis performed to identify HER2 isoforms of HER2+ specimens. Isoforms were characterized as 1) full-length/extracellular domain (ECD) intact, 2) pure/complete loss of ECD (p95 isoform), 3) subclonal populations of both full-length ECD and p95 and 4) gradient/representing partial loss of ECD per cell (reflecting post-translational cleavage). Results: Out of 51 cases, 34 (67%) cases were HER2+. In multiplexIHC analysis for HER2, 51% displayed gradient (partial) or focal (subclonal) loss of the extracellular domain. 33% displayed pure p95 isoform (complete loss of ECD). Only 16% of the cohort displayed full-length HER2 extracellular domain. Conclusions: HER2 expression is much more common in large in situ lesions than in invasive lesions. This suggests that HER2 positivity may play a role in protecting against invasion. Investigation of HER2 Isoform testing using multiplex staining reveals heterogeneity with partial or complete loss of the HER2 extracellular domain being most common. Loss of the extracellular domain of human epidermal growth factor receptor 2 (HER2) p95 isoform is predominant among patients diagnosed with HER2 positive ductal carcinoma in situ (DCIS). Citation Format: Nicole F. Schindler, Alexa Glencer, Hidetoshi Mori, Gillian Hirst, Cristian Maldonado Rodas, Michael Campbell, Laura Esserman, Alexander Borowsky. Loss of the extracellular domain of human epidermal growth factor receptor 2 (HER2) p95 isoform is common in large DCIS lesions [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B013.

Prognostic Value of the Serum HER2 Extracellular Domain Level in Breast Cancer: A Systematic Review and Meta-Analysis.

Simple SummaryWe performed a systematic review and meta-analysis evaluating the predictive value of the serum HER2 extracellular domain (sHER2 ECD) for breast cancer prognosis. Our review investigated 40 studies (12,229 patients) that assessed the impact of the sHER2 ECD levels on breast cancer prognosis and explored the clinical significance of sHER2 ECD levels in different treatment modalities. Our findings indicated that an elevated sHER2 was an unfavorable prognostic factor in breast cancer. More interestingly, sHER2 ECD was found to be a promising biomarker for predicting adverse clinical outcomes of trastuzumab-based treatment but did not affect the efficacy of tyrosine kinase inhibitors. In addition, the baseline cutoff value of sHER2 ECD in different treatment stages of breast cancer remains to be further explored. Overall, our study suggested that sHER2 ECD levels have important prognostic value in breast cancer and may be helpful for clinicians to select the appropriate anti-HER2 therapy for HER2-positive breast cancer, providing more evidence for guiding clinical practice.An elevated serum HER2 extracellular domain is associated with poor prognosis in breast cancer, but the relationship between sHER2 and the efficacy of different modalities remains controversial. Herein, we aimed to evaluate the prognostic value of serum HER2 extracellular domain (sHER2 ECD) in breast cancer and to identify its correlation with the efficacy of different treatment regimens. A systematic search of the PubMed, Embase, Cochrane Library, Web of Science, and Scopus databases was conducted to identify studies exploring the association between HER2 ECD level and clinical outcomes among patients with breast cancer. Using the random effects models, pooled hazard ratios (HRs), and odds ratios (ORs) with 95% confidence intervals (CI), were calculated for progression-free survival (PFS), overall survival (OS), disease-free survival (DFS), and the objective response rate (ORR). Heterogeneity was further evaluated by subgroup and sensitivity analysis. Overall, 40 studies comprising 12,229 patients were included in this systematic review and meta-analysis. Elevated HER2 ECD levels were associated with worse PFS (HR 1.74, 95% CI 1.40–2.17; p < 0.001), and this effect was observed in patients treated with chemotherapy (HR 1.81, 95% CI 1.37–2.39; p < 0.001), endocrine therapy (HR 1.91, 95% CI 1.57–2.32; p < 0.001), and trastuzumab (HR 1.74, 95% CI 1.31–2.30; p < 0.001). However, this association was not present in patients treated with tyrosine kinase inhibitors (TKIs) (HR 1.44, 95% CI 0.85–2.43, p = 0.17). The HRs/ORs for an elevated HER2 ECD level for DFS, OS, and ORR were 2.73 (95% CI 2.17–3.42; p < 0.001), 2.13 (95% CI 1.77–2.57; p < 0.001), and 0.80 (95% CI 0.49–1.31; p = 0.381), respectively. An elevated sHER2 ECD was an unfavorable prognostic factor in breast cancer but did not affect the efficacy of tyrosine kinase inhibitors such as lapatinib. Detection of sHER2 ECD may be helpful for clinicians selecting the appropriate anti-HER2 therapy for patients with HER2-positive breast cancer.

Mixed lineage kinase 3 and CD70 cooperation sensitize trastuzumab-resistant HER2+ breast cancer by ceramide-loaded nanoparticles

Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our invitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer.

MO25-4 Exploratory analysis of HER2 extracellular domain in HER2-positive gastric cancer treated with SOX plus trastuzumab

MO25-4 Exploratory analysis of HER2 extracellular domain in HER2-positive gastric cancer treated with SOX plus trastuzumab

Abstract LB151: Deep learning - based protein therapeutics design to treat HER2+ cancers

Abstract Background: HER2 amplification and activation occurs in approximately 15~25% of all breast cancers and is further indicated in other cancer types with high un-met medical needs, such as cholangiocarcinoma, ovarian and esophageal cancer. Further improvement of current HER2 targeting therapeutics on safety and efficacy remains to be desired. Deep learning - based computational approach, such as AlphaFold, has been successfully applied to the understanding of protein structure. However, its applications to protein therapeutic design remain largely underdeveloped. Method: We have developed an end-to-end protein design engine Apollo, which can be used to design target-binding protein therapeutics in silico. Apollo uses recurrent neural networks (RNNs), transformers and graph convolution neural networks (GCNs) to represent proteins, protein structures and protein-protein interactions. This is further improved by multi-head and hierarchical attention layers. Using generative AI, Apollo can either generate putative binding proteins de novo, or to optimize protein lead molecules. Candidate proteins are evaluated in silico for binding affinity and specificity, secondary and tertiary structure, isoelectric point, solubility, and other drug-like properties. Generated designs are iteratively improved by Apollo via a reinforcement learning loop. Results: Apollo has achieved high prediction accuracy by in silico evaluations, using metrics such as AUC and the confusion matrices. Using Apollo, we then designed mini protein binders of 50~100 aa to target HER2 extracellular domain (ECD). These binders can be produced with high yield and purity using mammalian protein expression systems. From de novo generation, the designed candidates are shown to bind to human HER2 with EC50 in the nanomolar range by ELISA. From optimizations based on lead molecules, the designed candidates have shown improved production yield and thermostability while maintaining binding affinity. Conclusions: We have shown the proof-of-concept success of using machine learning to design protein therapeutics. Citation Format: Ziwei Liang, Tom Murray, Max Bluestone, Chris Zoumadakis, Luke Martin, Alexey Vlasenko, Jeffrey LaFrence, John Lazar, Matthias Denecke, David Longo. Deep learning - based protein therapeutics design to treat HER2+ 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 LB151.

The HERPET study: Imaging HER2 expression in breast cancer with the novel PET tracer [18F]GE-226, a first-in-patient study.

3069 Background: Over-expression the human epidermal growth factor receptor-2 (HER2) is seen in 20% of breast cancers; this is an adverse prognostic factor and used to guide therapy selection. At present HER2 expression can only be determined using biopsy material using immunohistochemistry or fluorescence in situ hybridisation. Heterogeneous expression of HER2 is now being recognised as a cause of treatment resistance but is difficult to characterise. A non-invasive method for determining HER2 expression could have several advantages and help select appropriate therapy for patients. GE-226 is a novel radiolabelled GE-Affibody radioligand which binds to the HER2 receptor with high affinity at a different epitope than trastuzumab. Methods: Patients with locally advanced or metastatic breast cancer were recruited and scanned for 65 mins after iv injection of 200MBq of GE-226 (mean activity injected for each patient 202MBq (range 164-223MBq, mean radiochemical purity 94%) of radioligand, over one bed position for dynamic imaging, followed by a half-body scan. Blood sampling was used to measure metabolism of the tracer. Safety was assessed. HER2-extracellular domain (ECD) domain was measured in blood. Tumoural uptake was quantified by semi-quantitative and fquantitative parameters in HER2 positive and HER2 negative tumours. Patients had routine baseline FDG imaging. Results: Twenty patients completed the study. GE-226 scans were well tolerated. There were no serious adverse events. GE-226 was slowly metabolised into a single metabolite in the liver; 97% of parent remained at 60 minutes post injection (range 82-100). There was a significant difference in tumoural radioligand uptake between biopsy proven HER2 positive and HER2 negative tumoural patients as measured by SUVmean and SUVmax (p &lt; 0.001). Comparing HER2 positive to HER2 negative cases, there was also a significant difference between tumour to normal tissue uptake ratios SUVmean. Heterogeneous uptake was observed in three patients, two with interlesional uptake variation and one with intralesional heterogeneity. Tumoural uptake increased over time. Normal physiological uptake in salivary glands and the thyroid gland was noted. GE-226 was able to differentiate between lymphadenopathy due to sarcoidosis and cancer in one patient and was superior to FDG which had shown widespread uptake in the benign and malignant nodes. Conclusions: [18F]GE-226 imaging is well tolerated and shows promise for imaging of HER2 positive breast cancer. Further studies with this agent are now planned. Clinical trial information: NCT03827317.

Computational and Functional Analyses of HER2 Mutations Reveal Allosteric Activation Mechanisms and Altered Pharmacologic Effects.

This study identified new oncogenic HER2 allosteric mutations, including ECD mutations that share covalent dimerization as a mechanism of oncogenicity, suggesting the need for novel inhibitors to treat HER2-mutant cancers.

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.